NANOPHARMACEUTICALS

EXPECTATIONS AND REALITIES OF MULTIFUNCTIONAL DRUG DELIVERY SYSTEMS VOLUME 1

Edited by

RANJITA SHEGOKAR, PhD Capnomed GmbH, Zimmern, Germany Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

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Hend Abd-Allah Department of Pharmaceutics and and Health Sciences, Universidad del Rosario, Industrial Pharmacy, Faculty of Pharmacy, Ain Bogota, DC, Colombia Shams University, Egypt Riham I. El-Gogary Department of Pharmaceutics Mona Abdel-Mottaleb Department of Pharmaceu- and Industrial Pharmacy, Faculty of Pharmacy, tics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Egypt Ain Shams University, Egypt; PEPITE EA4267, N.B. Jadav Centre for Pharmaceutical Engineering Univ. Bourgogne Franche-Comté, Besançon, France Sciences, Faculty of Life Sciences, University of Annis Catur Adi Faculty of Health, University of Bradford, Bradford, United Kingdom Airlangga, Surabaya, Indonesia AnCelka B. Kovacevic Department of Pharmaceu- Mukta Agrawal Rungta College of Pharmaceutical tical Technology, Institute of Pharmacy, Faculty of Sciences and Research, Bhilai, Chhattisgarh, India Biological Sciences, Friedrich-Schiller University Amit Alexander Rungta College of Pharmaceutical Jena, Jena, Germany Sciences and Research, Bhilai, Chhattisgarh, India Atsarina Larasati Research Center for Nanosciences Mahavir Bhupal Chougule Translational Bio- and Nanotechnology, Bandung Institute of Tech- pharma Engineering Nanodelivery Research Labo- nology, Bandung, Indonesia ratory, Department of Pharmaceutics and Drug Peter Mattei CAS, a Division of the American Delivery, School of Pharmacy, University of Missis- Chemical Society, Columbus, OH, United States sippi, University, MS, United States; Pii Center for Maha Nasr Department of Pharmaceutics and In- Pharmaceutical Technology, Research Institute of dustrial Pharmacy, Faculty of Pharmacy, Ain Pharmaceutical Sciences, University of Mississippi, Shams University, Egypt University, MS, United States; National Center A. Paradkar Centre for Pharmaceutical Engineering for Natural Products Research, Research Institute Sciences, Faculty of Life Sciences, University of of Pharmaceutical Sciences, University of Missis- Bradford, Bradford, United Kingdom sippi, University, MS, United States Heni Rachmawati School of Pharmacy, Bandung Juan Bueno Research Center of Bioprospecting Institute of Technology, Bandung, Indonesia; and Biotechnology for Biodiversity Foundation Research Center for Nanosciences and Nanotech- (BIOLABB), Armenia, Quindío, Colombia nology, Bandung Institute of Technology, Bandung, J.R. Campos Department of Pharmaceutical Tech- Indonesia nology, Faculty of Pharmacy, University of Coim- Kobra Rostamizadeh Zanjan Pharmaceutical Nano- bra (FFUC), Polo das Ciências da Saude, Coimbra, technology Research Center, Zanjan University of Portugal Medical Sciences, Zanjan, Iran; Center for Pharma- Anne Marie Clark CAS, a Division of the American ceutical Biotechnology and Nanomedicine, North- Chemical Society, Columbus, OH, United States eastern University, Boston, MA, United States Diana Diaz-Arévalo Molecular Biology and Immu- A. Santini Department of Pharmacy, University of nology Department, Fundacion Instituto de Inmu- Napoli “Federico II”, Napoli, Italy nología de Colombia-FIDIC, School of Medicine

vii viii Contributors

Shailendra Saraf University Institute of Pharmacy, E.B. Souto Department of Pharmaceutical Technol- Pt. Ravishankar Shukla University, Raipur, Chhat- ogy, Faculty of Pharmacy, University of Coimbra tisgarh, India (FFUC), Polo das Ciências da Saude, Coimbra, Swarnlata Saraf University Institute of Pharmacy, Portugal; CEB - Centre of Biological Engineering, Pt. Ravishankar Shukla University, Raipur, Chhat- University of Minho, Braga, Portugal tisgarh, India Asur Srinivasan CAS, a Division of the American P. Severino Universidade Tiradentes (Unit), Aracaju, Chemical Society, Columbus, OH, United States Sergipe, Brazil; Instituto de Tecnologia e Pesquisa, Amanda Starling-Windhof CAS, a Division of the Laboratorio de Nanotecnologia e Nanomedicina American Chemical Society, Columbus, OH, (LNMed), Aracaju, Sergipe, Brazil; Tiradentes United States Institute, Dorchester, United States Tina Tomeo CAS, a Division of the American Ranjita Shegokar Capnomed GmbH, Zimmern, Chemical Society, Columbus, OH, United States Germany Vladimir P. Torchilin Center for Pharmaceutical A.M. Silva School of Biology and Environment, Uni- Biotechnology and Nanomedicine, Northeastern versity of Tras-os-Montes e Alto Douro (UTAD), Vila University, Boston, MA, United States Real, Portugal; Centre for Research and Technology Mingtao Zeng Center of Emphasis in Infectious Dis- of Agro-Environmental and Biological Sciences eases, Department of Molecular and Translational (CITAB), University of Tras-os-Montes e Alto Douro Medicine, Paul L. Foster School of Medicine, Texas (UTAD), Vila Real, Portugal Tech University Health Sciences Center El Paso, El S.B. Souto Department of Endocrinology, S. Joao~ Paso, TX, United States Hospital, Alameda Prof. Hernani^ Monteiro, Porto, Portugal Preface

The book series titled Expectations and Real- (4) establish collaborations between academic ities of Multifunctional Drug Delivery Systems scientists, and industrial and clinical covers several important topics on drug-delivery researchers. systems, regulatory requirements, clinical studies, Innovative cutting-edge developments in intellectual properties trends, new advances, micro-nanotechnology offer new ways of pre- manufacturing challenges, etc. written by leading venting and treating diseases like cancer, ma- industry and academic experts. Overall, the laria, HIV/AIDS, tuberculosis, and many more. chapters published in this series reflect the broad- The application of micro-nanoparticles in drug ness of nanopharmaceuticals, microparticles, delivery, diagnostics, and imaging is vast. other drug carriers and the importance of the Hence, Volume 1: Nanopharmaceuticals,in respective quality, regulatory, clinical, GMP scale the book series mainly reviews advances in up, and regulatory registration aspects. drug delivery area via targeted therapy with This series is destined to fill the knowledge improved drug efficiency at a lower dose, trans- gap through information sharing and with orga- portation of the drug across physiological bar- nized research compilation between diverse riers as well as reduced drug-related toxicity. areas of pharma, medicine, clinical, regulatory One of the contributions by Campos et al. practices, and academics. (Chapter 1) discusses the influence of physico- Expectations and Realities of Multifunctional Drug chemical factors affecting long-term stability, Delivery Systems is divided into four volumes: release and toxicological profiles of solid lipid Volume 1: Nanopharmaceuticals nanoparticles. This chapter also reviews the Volume 2: Delivery of Drugs importance of composition and administration Volume 3: Drug Delivery Trends routes studied for lipid nanocarrier systems. Volume 4: Drug Delivery Aspects In another manuscript by Rachmawati et al. The specific objectives of this book series are (Chapter 2), the authors highlight the current to: status of drug delivery development for herbal (1) provide a platform to discuss opportunities bioactives. Along with various mucosal bio- and challenges in development of carriers, the authors describe biokinetic and nanomedicine and other drug-delivery clinical translation challenges with herbal deliv- systems; ery and limitations with regulatory procedures. (2) discuss current and future market trends; In this chapter herbal nanocarriers like lipid (3) facilitate insight sharing within various nanoparticles, nanosuspensions etc. are areas of expertise; and discussed in detail.

ix x Preface

Chapter 3 by Rostamizadeh et al. describes promising although some limitations like stability the development of polymeric micelles for multi- and cytotoxicity needs to be overcome. ple drug delivery in oncology. The co-loading of Chapter 8 by Kovacevic discusses delivery of two or more drugs is possible using polymeric poorly soluble and low-permeable drugs via micelles. The authors describe the types of lipid nanocarriers. An overview of available polymers employed, preparation methods, and mechanistic studies in model and in real cell characterization techniques for such carrier membranes for better understanding of cell systems. Furthermore, wider applications like internalization processes is provided. chemotherapeutic delivery, stimuli-responsive The chapter by Alexander et al. (Chapter 9) drug delivery, and targeted-drug delivery via reviews approaches for effective brain drug deliv- such carriers is discussed in detail. On the other ery using nasal mucosa. This route can deliver hand, hyaluronic acid nanoparticles are being drugs effectively at target sites with improved widely explored in nanomedicines. The promising therapeutic performance of drugs. In addition, nanocarriers to deliver drugs in conjugated, self- the authors explain limitations of this drug deliv- assembled, or in nanocomplex form are discussed ery route and regulatory market approval in the book chapter by Nasr et al. in Chapter 4. The challenges. authors confirm that the current research Starling-Windhof et al. (Chapter 10) address shows impressive research findings in areas like industry and technology trends in the intellec- osteoarthritis, tissue engineering, cancer target- tual property (IP) landscape of pharmaceutical ing, theranostic applications, and so on, which drug delivery over 3 decades. It is fascinating are under further exploration by industry. to see the global picture; it makes scientists The work by Jadav and Paradkar (Chapter 5) aware of the trends and special interests of is aimed at discussing widely studied drug specific geographical regions or markets. delivery systems in academics and in industry, This chapter provides information on IP trends i.e. solid dispersions. Various aspects like classi- in oral, topical, and parenteral drug delivery fication of solid dispersions, their formulation area. Guidance on emerging trends and optimization, processing and physicochemical IP-monitoring strategies are also presented. characterization are reviewed in this chapter. In summary, I am sure this book volume and Chapter 6 by Bueno highlights the impor- the complete book series will provide you great tance of understanding nanotoxicity at early insights in areas of micro-nanomedicines, drug stages of development. Although nanocarriers delivery sciences, new trends, and regulatory have shown promising results in delivering aspects. drugs at target sites or locations, the accumula- O. Farokhzad, R. Langer, and the National tion of nanoparticles at cellular and tissue Cancer Institute are gratefully acknowledged for levels in excess causes toxicity. Current literature the book cover image, which represents the po- contains very limited information on this topic. tential of nanopharmaceuticals in targeting drug This chapter reviews various aspects of nanotox- molecules. This photograph presented on cover icity and provides information on key concepts page captures interactions of surface-modified for evaluation of the toxicity. polymeric nanoparticles with prostate cancer The topic presented by Diaz-Arévalo et al. cellsdit is an ideal example for drug targeting. (Chapter 7) describes the systemic review of All the efforts of experts, scientists, and nanoparticles-based vaccine development. Initial authors are highly acknowledged for sharing their results of nanocarriers like liposomes, virus like knowledge, ideas, and insights about the topic. particles, metallic and nonmetallic particles, and Ranjita Shegokar, PhD polymeric nanoparticles in vaccine therapy are Editor CHAPTER 1

Solid lipid nanoparticles (SLN): prediction of toxicity, metabolism, fate and physicochemical properties J.R. Campos1, P. Severino2,3,4, A. Santini5, A.M. Silva6,7, Ranjita Shegokar8, S.B. Souto9, E.B. Souto1,10 1Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), Polo das Ci^encias da Saude, Coimbra, Portugal; 2Universidade Tiradentes (Unit), Aracaju, Sergipe, Brazil; 3Instituto de Tecnologia e Pesquisa, Laboratorio de Nanotecnologia e Nanomedicina (LNMed), Aracaju, Sergipe, Brazil; 4Tiradentes Institute, Dorchester, United States; 5Department of Pharmacy, University of Napoli “Federico II”, Napoli, Italy; 6School of Biology and Environment, University of Tras-os-Montes e Alto Douro (UTAD), Vila Real, Portugal; 7Centre for Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Tras-os-Montes e Alto Douro (UTAD), Vila Real, Portugal; 8Capnomed GmbH, Zimmern, Germany; 9Department of Endocrinology, S. Jo~ao Hospital, Alameda Prof. Hern^ani Monteiro, Porto, Portugal; 10CEB - Centre of Biological Engineering, University of Minho, Braga, Portugal

1. Introduction formulations [2]. Various controlled drug deliv- ery systems like polymer-based controlled- Solid lipid nanoparticles (SLNs) gained release systems, hydrogels, as well as nano- greater attention as a drug delivery system and microparticles have been introduced in when in 1991 Muller€ developed them [1]. This recent years in order to improve solubility, sta- promising drug carrier system is at the interface bility and bioavailability of poorly water-soluble in the preexisting lipid systems (emulsions and drugs. In this context, lipid nanoparticles offer liposomes) and polymeric nanoparticle systems. attractive and ideal properties for drug or gene Lipid nanoparticles, known as SLNs or nano- delivery. These particles (either composed of structured lipid carriers (NLCs), have specific solid lipids only in SLNs, or of a blend of solid features of structure and composition, showing and liquid lipids in NLCs) stabilized with surfac- benefits in comparison to conventional tants have the advantages of other colloidal

Nanopharmaceuticals https://doi.org/10.1016/B978-0-12-817778-5.00001-4 1 © 2020 Elsevier Inc. All rights reserved. 2 1. Solid lipid nanoparticles (SLN) particles (polymeric nanoparticles, fat emul- Recognized as Safe (GRAS) excipients [4,6]. sions, and liposomes) by overcoming their limi- SLNs are biodegradable (fulfilling the require- tations [3,4]. Taking account their polymeric ments of preclinical safety) and are also stable in and lipid raw materials, several modifications blood, with prolonged lifetime in the bloodstream of drug delivery systems have been proposed [13e15]. In addition, compared to liposomes, to increase the bioavailability of loaded drugs. SLNs exhibit high encapsulation efficiency, stabil- SLNs are made of a solid lipid matrix and a sur- ity against light and oxygen, do not need organic factant layer and they can load poorly water- solvents for their preparation, and have high soluble drugs, delivering them at defined rates drug-loading capacity (mainly for lipophilic com- and with improved bioavailability [5]. These pounds) [12,15,16]. On the other hand, SLNs have colloidal drug delivery systems protect the also limitations, mainly attributed to the risk of 0 drug against chemical degradation and modify polymorphic transitions (from a to b ,andfrom 0 its release profile since the drug is entrapped in b to b) which causes stability challenges during the solid lipid matrix [6,7]. These nanoparticles administration or storage, resulting in drug of spherical shape have a mean size of expulsion from the particles and eventual particle 40e1000 nm [8,9]. The lipid matrix is composed size increase [6,10,17].Thesedisadvantagesare of a solid lipid (or a mixture of solid and liquid related to the crystallization behavior and lipid lipids) in a 0.1%e30% (w/w) concentration matrix’s polymorphic transitions, which depend dispersed in aqueous medium, and their stability on the type of lipids used for the production of is ensured by the presence of a surfactant in a SLNs [6]. There are various methods described 0.5%e5% (w/w) concentration [8] and can be in the literature to produce SLNs based on used for lipophilic or hydrophilic drugs [9].Tri- solidified emulsion technologies: high shear ho- glycerides (tristearin), sterols (cholesterol), par- mogenization and ultrasound, high pressure tial glycids (glyceryl monostearate), fatty acids (hot and cold) homogenization, oil/water (O/ (stearic acid), as well as waxes (cetylpalmitate) W) and water/oil/water (W/O/W) microemul- are especially used as lipids in the SLNs. In these sions, as well as solvent evaporation [10]. systems emulsifiers and polymers are used as These techniques interfere with various character- stabilizers in order to avoid aggregation of the istics of the particles, mainly in morphology. Ac- particles. Examples of stabilizers are bile salts cording to the literature, the most commonly (e.g., taurodeoxycholate), lecithins, and copoly- applied methods are those that use high pressure mers of polyoxyethylene and polyoxypropylene homogenizers (HPH). Muller€ and Luck devel- (Poloxamer) [10]. oped the HPH technique (European Patent No. It is clear that lipid nanocarriers are ideal for 0605497) for obtaining nanoemulsions for large- sensitive bioactive compounds. SLNs exhibit scale parenteral nutrition [10].Therearediverse biocompatibility, matrix with lipophilic nature kinds of equipment with various sizes, prices as protecting active compounds of chemical degra- well as different capacities. The different equip- dation, drug targeting, controlled release profile, ments work by pulling the liquid in high pressure and high drug payload [9,12]. Moreover, they (100e2000 bar) through a narrow piston (nano- are suitable for industrial production mainly meter scale), which is accelerated over a small because they are easy to scale up, are stable under distance at a high speed (over 1000 km/h). This sterilization conditions, and they have the advan- fluid is subjected to high stress, disrupting the tage of being non-toxic or of very low toxicity, macroscopic oil droplets by cavitation forces because of their composition in Generally and thus generating the nanodroplets [10].In 2. Toxicity profiling 3 the hot process, the hot preemulsion is passed been explored for drug delivery using lipid through the hot homogenizer to obtain nanoe- nanoparticles. Components of lipid nanopar- mulsions, which are then cooled down in order ticles (lipids and surfactants) determine the to solidify and crystallize the hot inner liquid product quality, its physicochemical properties, phase to obtain SLNs. In the cold process, the as well as the administration route [4] (Table drug is firstly ground milled in a mortar mill 1.1). In this chapter, the concept behind the with the solid lipid at room temperature, and SLNs and their physicochemical properties, then the obtained powder is dispersed in an pharmacokinetics, and biopharmaceutics and aqueous surfactant solution, which is then sub- their toxicological testing are discussed. jected to HPH. The influence of the type of homogenizer, pressure, and number of cycles employed, and the temperature used to obtain 2. Toxicity profiling the ideal particle size, have been intensively stud- ied. Depending on the type of lipid, it is possible SLNs are known to be stable in aqueous to use lipid concentrations above 40% and obtain dispersion, allows the encapsulation of hydro- the particle size distribution in a low range philic and lipophilic drugs, are adaptable to (polydispersity index <0.2) [18]. To obtain SLNs several administration routes, can modify the from microemulsions, Gasco and collaborators release profile and avoid their adverse effects developed a technique that has been modified (protecting the drug from undesirable interac- by numerous researchers. In this technique, tions or directing it to its target) [30]. However, SLNs are produced by diluting a hot oil-in-water their toxicological profile has to be very well (O/W) microemulsion in high volume of cold- characterized in vitro before any pre-clinical water (0e4oC). The internal phase of this microe- and clinical studies [31]. There is a relationship mulsion is composed of low-melting lipids and, between the size of the particles and their when in contact with the cold water, they suffer toxicity, as the lower the size, the higher the sur- crystallization and form SLNs [18e20]. The type face area and the higher the reactivity. A pre- of lipids used to make the microemulsion, the requisite to be marketable is the GRAS status preparation parameters (stirring and tempera- of the excipients of SLNs [32], but additional ture), rate of microemulsion’sadditioninthe nanotoxicological studies are needed to allow cold water, volume of water, and the technique the understanding of the effect of nanoparticles used to remove the excess water, all affect the in the body [14,33,34]. Nanotoxicology helps in characteristics of obtained nanoparticles [18]. identifying the SLN formulations to be selected This technique is therefore difficult to scale up. for preclinical studies by evaluating their safety, Marengo has developed a device that processes tolerance, and cytotoxicity. 100 mL of microemulsion and can produce Preclinical toxicological studies allow to SLNs of mean size below 100 nm [20]. determine the concentration of substances that The biomedical applications of lipid nanopar- cause toxic effects, and allow identification of ticles are manyfold. Indeed, they can be used as target organs predisposed to these effects. Pre- drug and gene carriers, and as contrast agents clinical safety tests require the appropriate selec- for imaging analysis [21]. In recent years, tion of the animal species, age, physiological different administration routes (e.g., oral, paren- status, the administration route, dosage form as teral, dermal, pulmonary, rectal, ocular) have well as the treatment regime. Preclinical TABLE 1.1 Commonly used lipids in the composition of SLNs/NLCs.

Therapeutic Name Chemical structure Surfactant System Drug application References

Tristearin Dihexadecyl SLNs e Pulmonary [22] phosphate delivery PEGs SLNs e Oral delivery [23]

Glyceryl Mixture of Tween SLNs ee [24] monostearate 80 and Span 80

Stearic acid Omega-3 PUFA SLNs Resveratrol Oral [25] delivery Cystamine SLNs ee [26] Tween 80 NLCs ee [27] Cholesterol Pluronic F-68 NLCs Simvastatin Oral [28] delivery

Vitamin E O D-a-tocopheryl NLCs Rapamycin Ocular [29] n O polyethylene glycol delivery O R O succinate (TPGS) O O 3. Physicochemical properties 5 toxicological evaluation of a new compound can nanoparticle-based drug therapies available is provide information about acute, subacute, sub- still very limited [8]. chronic, and chronic toxicity. Before any preclin- ical study, cytotoxicity assessment is carried out 3. Physicochemical properties in cell culture, also allowing the study of the interaction between nanoparticles and cells. The use of primary cells (isolated directly from The characterization of SLNs usually re- the animals) provides more realistic toxicological quires the determination of the particle size results. On the other hand, in vivo studies eval- and zeta potential, morphological evaluation, uate the organism as a whole. Therefore, in vivo determination of the loading capacity and fi studies are relevant to determine the location encapsulation ef ciency, kinetics of drug and concentration of the drug in the tissues, and release, as well as the nanoparticles over systemic toxicity [35]. Systems with large quan- time and storage temperature. tities of surfactants (e.g., nanoemulsions) have higher risk of exhibiting cytotoxicity, since these 3.1 Encapsulation parameters agents interact with cell membranes composed of phospholipids. Likewise, their production re- Determination of the amount of drug associ- quires the use of organic solvents, which may ated with the nanoparticles is a crucial parameter also increase the risk of toxicological events if for the characterization of SLNs. This task is poorly removed. Although precise determina- however difficult because of the small size of fi tion of the toxicity can only be quanti ed the particles, which compromises the separation in vivo, there are several in vitro toxicological of the free drug fraction from the associated frac- tests that provide preliminary information tion. Ultracentrifugation is the most commonly [10,14,36]. In vitro studies have shown that used separation procedure, after which the < SLNs are acceptable at concentrations 1mg/ non-loaded drug is quantified in the superna- mL (total lipids), and with particle diameter tant. From the difference between total drug > 500 nm can be less tolerated, which can be and the drug found in the supernatant, the explained by their aggregation. It was also drug concentration associated with nanostruc- shown that the stabilized formulations tures is calculated [37,38]. Ultrafiltration can be composed of several surfactants are less biocom- coupled to the process of ultracentrifugation. In patible in comparison to those based on one sur- this approach, a membrane (100 kDa) is used to factant only. For polysorbate 80 and poloxamer separate the aqueous phase from the nanopar- 188, two surfactants mostly used in SLNs formu- ticles. Although the free drug concentration in lations, enough evidence has been found to this technique is determined in the ultrafiltrate, determine their safety [14]. It is clear that the the drug fraction associated with the nanostruc- fi knowledge of the toxicological pro le of any ma- tures is also found by the difference between the terial and the biocompatibility of the drug deliv- total and free concentrations [39]. Encapsulation ery systems are crucial for the implementation of efficiency (EE%) and loading capacity (LC%) are drug therapies, but the information of determined using the following equations [40]:

Amount ðmgÞ of loaded drug determined experimentaly EEð%Þ¼ 100 Theoretical amount of drug ðmgÞ in formulation 6 1. Solid lipid nanoparticles (SLN)

Amount ðmgÞ of loaded drug determined experimentaly LCð%Þ¼ 100 Theoretical amount of lipid ðmgÞ in formulation

3.2 Particle size 3.3 Zeta potential

Size and polydispersity index are parameters Zeta potential is another parameter used to that indicate the stability of the nanoparticles. evaluate the long-term stability of the particles An optimized SLN formulation should exhibit and its assessment is instrumental for the physi- a mean particle size less than 1 mm, together cochemical characterization of the nanoparticles' with a small polydispersity index. Several pa- dispersion. When determining the zeta potential, rameters affect the particle size and polydisper- it is possible to understand the interactions sity, e.g., composition of the formulation between the particle and the drug. This param- (mixture of surfactants, lipid structural proper- eter shows the surface electrical charge of the ties, and also incorporated drug), methods and particles, which is modified by changes in the conditions of the production (time, temperature, interface with the dispersing medium since there stirring velocity, pressure, etc) [41]. There is a is a dissociation of functional groups on the par- relationship between the proportion of surfac- ticle' surface and adsorption of ionic species of tant/lipid and the particle size, i.e., the greater the aqueous dispersion medium [39]. The mea- the concentration of surfactant, the smaller the surement of this parameter allows to clarify the particle size [42]. The temperature used in the stability of the formulation during its storage HPH technique is another parameter that inter- time. The higher the zeta potential, the higher feres with the particle size. In the production of the electrostatic repulsion between the particles, SLNs and NLCs, the lipid phase is heated up resulting in reduction of the risk of particles' to a temperature higher than the melting point aggregation. According to literature, a zeta of the solid lipid. The final step is the cooling potential higher than the 30 mV modulus gua- down of the systems so that the lipid recrystal- rantees the stability of SLNs [45]. However, there lizes to solidify the lipid droplets [43]. Homoge- are SLN formulations with zeta potential below nization at low temperatures (i.e., below the |30 mV| that remain stable over time due to melting point of the solid lipid) favors the in- the stereochemical stability offered by surfac- crease of particle size, but temperatures near tants [46]. Changing a formulation by varying the melting point improve the homogenization the concentration of its components, allows and lower particle sizes can be obtained. Simi- understanding which of those whether larly, the hot HPH procedure produces particles contribute to the stereochemical stability or to with lower mean size and polydispersity than the electrostatic stability [47]. Differential scan- the cold procedure [41]. Generally, the particle ning calorimetry, based on the study of the peaks size increases when lipids of long fatty acid of SLN formulation recorded on the thermo- chains are employed. The use of mixtures of gram, allows assessing if the drug is loaded short- and long-chain fatty acids may therefore within the particles and eventually its contribu- reduce the mean particle size and polydispersity tion to the surface electrical charge of SLNs [48]. index of SLNs/NLCs [4,44]. 3. Physicochemical properties 7

3.4 Particle morphology DSC analysis, a melting point depression is usu- ally observed when transforming the bulk lipid To learn about the shape and size of nanopar- into lipid nanoparticles [48,52]. ticles, scanning (SEM) and transmission electron microscopy (TEM) are commonly used [50]. Atomic force microscopy (ATM) is another tech- 3.6 Stability of formulations and release nique that gives information with high resolu- tion in three dimensions at the nanometer scale profile (even surface details at the atomic level), and is The stability of the loaded drug within lipid also used to understand the surface morphology nanoparticles is dependent on the chemical of nanoparticles [39]. composition of the lipid matrix (e.g., type of lipid, surfactant) and production procedure. 3.5 Differential scanning calorimetry Upon storage, triglycerides undergo polymor- phic changes that may result in drug expulsion The physical and chemical changes of a sam- from the lipid nanoparticles [53]. Surfactants ple can be measured as a function of its temper- also play a relevant role on the crystallization ature; differential scanning calorimetry (DSC) behavior of the lipid nanoparticles, i.e., quantifies the loss or heat gain resulting from whether the recrystallization of the particles these changes. There are two types of DSC in- occurs onto their surface or within the lipid struments: (1) the power compensate DSC, core [54]. The long-term stability of lipid nano- which is made of two separate ovens; and (2) particles over storage is therefore dependent the heat-flux DSC, which has only an oven that on the lipid and surfactant composition of the heats up the reference and sample pans. The particles and of the production procedure sample and reference receive the heat through [55]. The presence of imperfections in the lipid the sample pan, which is placed in a disc that matrix offers higher capacity to accommodate is the main source of heat. Both differential drug molecules, improving the loading capac- heat flow and sample temperature are moni- ity of the particles [19,56].Toachieveamodi- tored, and the calorimetric sensitivity is main- fied release profile of the loaded drug, the tained by the software linearization of the cell particles should however retain the drug dur- calibration [52]. The endothermic processes, i.e., ing storage until their administration. To those that absorb heat, include fusion (melting), increase the encapsulation efficiency, the use boiling, sublimation, vaporization, desolvation, of mixed lipids (having fatty acids with as well as solid-solid transitions. The crucial different liquid and solid chain lengths) is usu- exothermic process, i.e., the one that releases en- ally recommended. These blends create small ergy, is crystallization. These analyzes can be liquid reservoirs inside the particles e as hap- used to identify materials, investigate their pu- pens in the NLCs e delaying the polymorphic rity, polymorphism or solvation, analyze quanti- changes over storage [18, 53, 57]. The encapsu- tative and qualitative degradation, aging, glass lation efficiency of lipophilic drugs is also transition temperature, as well as their affinity higher in SLNs and NLCs than hydrophilic to other substances. DSC is useful to obtain drugs. To load these latter in lipid matrices, information about the degree of crystallinity of other strategies are needed such as the devel- the lipid matrix and polymorphic behaviour. In opment of insoluble conjugates between the 8 1. Solid lipid nanoparticles (SLN) lipid and the drug by a covalent bonding [58, 4. Administration routes and drug 59]. Hydrophilic drugs might show higher bioavailability risk to be partitioned to the aqueous phase dur- ing the production of SLNs and create a drug- Pharmaceutical nanotechnology comes up as a enriched shell model. Indeed, upon cooling of strategy to improve the bioavailability of poorly- the dispersions, the lipid solidifies first, crystal- water soluble drugs, enhancing their therapeutic lizes and forms the cores in which the hydro- effectiveness and reducing the risk of adverse re- philic drug will be precipitating onto their actions [64e67].SLNshavebeenextensively surface. This type of SLNs (drug-enriched shell exploited as an interesting approach to improve model) does not exhibit a modified release pro- the drug’s bioavailability in particular those of file but rather a fast release attributed to the class II and IV of the biopharmaceutical classifica- presence of drug onto the surface of the tion system (BCS) [64, 67]. Indeed, due to their SLNs. For drugs that solidify first (e.g., of lipid composition, they may act as absorption en- melting point higher than that of the solid hancers [67]. On the other hand, surfactants sur- lipid) or for lipophilic drugs, a drug enriched rounding the particles besides ensuring their core model can be produced, which exhibits a steric stability in aqueous dispersion, they induce modified release profile [17].Typicalmethods specific surface-chemical properties and may also used for assessment of the in vitro release are modulate the biopharmaceutical profile. For the the dialysis and the static or dynamic Franz selection of the best surfactant, several parameters diffusion methods. The assays can be designed have to be taken into account e.g., hydrophilic- so that isotonicity and pH value can be lipophilic balance (HLB) values, their effect on adjusted to mimic the intended administration the lipid polymorphism and on the particle size. route, and can also include the effect of protein The HLB values for the stabilization of oil-in- adsorption, plasma compatibility, whole blood water dispersions vary between 8e18 [68].The compatibility and sterilisation. It is known that right choice of the surfactant minimizes the in a physiological environment, proteins can risk of production of particles’ aggregates which bind the surfaces of nanoparticles, forming a may compromise the stability of the dispersion nanoparticleeprotein complex that influences in vitro and its performance in vivo [45]. the biological response. Nanoparticles can be incubated with bulk serum, plasma and also with solutions of individual proteins, in order 4.1 Topical and dermal routes to evaluate which physical properties are responsible for the protein binding onto their The administration of drugs through the skin surface [60], such as the type of polymer used may contribute to increase the drug’s bioavail- to stabilize the particles [61,62]. Freeze-drying ability as it overcomes the first-pass meta- may also be used to enhance the long-term bolism. This administration route reduces the stability of SLNs and NLCs. Lipid in inter/intra-patient variation and increases nanoparticles can be transformed in a dry patient compliance. However, it is also associ- product to improve their physicochemical ated with interactions of drug and/or excipients stability [63]. with skin that may cause irritation [69].The 4. Administration routes and drug bioavailability 9 loading of drugs within lipid nanoparticles can corneum lipid film. Indeed, these nanoparticles minimize the risk of skin irritation and aller- create a protective lipid film onto the skin upon genic reactions, by preventing direct contact their topical application, promoting skin hydra- between the drug and the skin and by control- tion [76]. ling the drug release through the skin e [14,70 72]. Besides, as they are composed of 4.2 Ocular delivery biocompatible and physiological lipids of GRAS status, SLNs and NLCs exhibit low risk For the treatment of eye diseases, direct ocular of acute and/or chronic toxicity [10]. Most of instillation is the most accepted approach by pa- the surfactants used in the production of SLNs tients. Lipid nanoparticles have also gained inter- and NLCs are already used in topical pharma- est as drug carriers for this administration route, ceutical or cosmetic formulations. To further due to their biocompatibility with the ocular tis- reduce the risk of irritation, the surfactants sues, mucoadhesiveness and modified-release should be non-ionic, while polyethoxylated sur- profile [79e81]. Conventional ophthalmic solu- factants should also be avoided tions have low precorneal retention time. Lipid [14,42,49,73e75]. There is evidence that most nanoparticles increase the retention time of ocular ofthePEG-freesurfactantshaveshowntostabi- drugs, improving their bioavailability [82].Tobe lize lipid nanoparticles without the need of suitable for ocular instillation, lipid nanoparticles cosurfactants. In addition, when compared to should be of small particle size to avoid blurred the PEG-containing counterparts, the PEG-free vision and discomfort [83]. To avoid damage of surfactants have been shown to require less con- the corneal tissues, inflammation and immuno- centration of surfactant to obtain small and uni- logic reactions, the formulations also need to form particle size [76].Themethodsusedto exhibit sterility, isotonicity and pH between evaluate the skin irritation include typical in 7e9. Toxicity of the formulations that could alter vitro test methods based on the reconstructed the corneal epithelial integrity or disrupt the tis- human epidermis (RhE) and also in vivo animal sue, resulting in deficient drug delivery into eye tests. In vivo experiments are more useful but (which is not their aim) also need to be consid- their cost, tight regulation, and ethical issues to- ered [84]. The Draize rabbit eye test is routinely wards the promotion of reduction, reuse, and used to evaluate eye irritation. This test was recycling imply the need to develop improved developed with the aim to predict human eye irri- in vitro tests [77]. New strategies to decrease tation of pharmaceutic and cosmetic products. Its skin irritation are based on the use of controlled drawbacks are the ethical issues associated with release systems, i.e., creating a gradual drug de- the use of animals, its costs and the number of livery that prevents the accumulation of high variables of the test [85]. concentrations of drug in the skin, which are usually responsible for this skin irritation, and 4.3 Oral administration also increase drug deposition in the piloseba- ceous unit, which reduces the dose frequency Oral delivery is painless and is easy for self- and risk of adverse events [78]. Lipid nanopar- administration. This administration route has ticles have additional advantages as they can high patient compliance and is appropriate for prevent and even reduce skin irritation by the outpatients. All these advantages make it the reinforcement and repair of the stratum most accepted drug administration route [86]. 10 1. Solid lipid nanoparticles (SLN)

However, the gastrointestinal tract has chemical inefficient activity in treating the diseases [91]. and enzymatic barriers that limit the effective- Many oral SLN tests in cell-based and animal ness of oral drug delivery, and also show low studies are described in the literature, however, permeability for several drugs [87]. By opti- their clinical trials are still limited due to their mizing the formulations, it is possible to amelio- cost, as well as the unknown side effects (they rate their efficiency and bioavailability, have to be investigated first). From the commer- promoting the therapeutic potency and reducing cial point of view, to be viable the nanoparticles side effects. Lipid nanoparticles may be used as have to show 5-fold-improved oral bioavail- absorption enhancers through the gastrointes- ability or other convincing benefits [91]. tinal tract to improve the oral bioavailability of several drugs [88]. SLNs developed for oral 4.4 Parenteral administration administration have been shown to enhance and control drug delivery, mainly due to the spe- Until now, typical SLN components have not fi fi ci c characteristics of the surface modi cation, yet been used in parenteral formulations, except increased permeation of the gastrointestinal medium-chain triglycerides (MCTs) and poly- tract, as well as resistance against degradation. sorbate 80 [92]. Usually, the final suspension The solid state of the matrix can protect chemi- for injection contains solid particles, which cally unstable drugs and promote the drug resi- means that its safety profile will have to be dence onto the site of absorption. SLNs have confirmed. It is known that the parenteral shown low cytotoxicity against mammalian cells dispersion should not show any solid particles and high tolerance in vivo. They can be further visible to the naked eye. The admissible limits formulated in classical dosage forms e.g., cap- of solid particles of size below 50, 25, and sules, tablets or pellets for oral administration 10 mm are not yet defined, and are still open to [89, 90]. To improve the formulation stability, discussion, and there is no available regulation SLN conversion into powders for further pro- of particles with size below 1 mm. SLNs have cessing into solid dosage forms have also been shown intensity with a diameter below 1 mm proposed. Shegokar et al. investigated the possi- and, in some cases, below 200 nm, but it is neces- bility of enhancing the long-term stability of sary to take into account that they have a ten- nanoparticles with freeze-drying studies, con- dency to aggregate over time (especially when verting SLNs in a dry product and observing exposed to high ionic strength environment or that have good size distribution, which also proteins) [14,93,94]. The key parameters to be proves that SLNs are a suitable drug delivery evaluated for parenteral application include un- system [63]. There is still a demand to design derstanding of plasma drug profile, fate of nano- and develop new SLNs to obtain a viable release particles, and acute and chronic dose toxicity. fi pro le, which depends on the drugs selected for Some studies have been published on bare and these formulations as different drugs have surface-coated nanoparticles for parenteral different physicochemical characteristics and application, lab to large scale, targeting, toxicity also different interactions with nanoparticles. studies, solid product conversion, phagocytic fi The release pro le from solid dosage forms uptake studies, cytotoxicity studies, as well as needs to be critically controlled; a burst drug organ distribution studies. For example, Shego- release can be linked to toxicity problems kar et al. evaluated the potential of lipid nano- whereas a slow drug release that can cause particles for active delivery of an antiretroviral 5. Conclusions 11 drug to lymphatic tissues. SLNs were developed various routes, including dermal, ocular, and taking account of various physicochemical oral, with the dermal route the safest. Lipid parameters, e.g., appearance, particle size, poly- nanoparticles are composed of biocompatible dispersity index, as well as zeta potential. and biodegradable lipids, with a melting point Authors investigated the targeting potential of above 40 C to ensure solid status at room and these SLNs through ex vivo cellular uptake also at body temperatures. SLN production is studies, showing an enhanced uptake in compar- based on the incorporation of the drug in the ison to pure drug, and the lymphatic drug levels melted lipid and then mixed with the aqueous and organ distribution studies also showed the surfactant solution, and they can be made by efficiency of these nanoparticles for prolonged high energy techniques (e.g., ultrasound residence. The study demonstrated that these methods and supercritical fluid technologies) or lipid carriers can be used for effective and tar- low-energy techniques (e.g., solvent emulsifica- geted drug delivery, enhance the therapeutic tion evaporation, coacervation, microemulsion safety and decrease collateral effects [95,96]. technique, and phase-inversion temperature method). Lipid nanoparticles protect the drug 4.5 Nasal and pulmonary delivery against chemical degradation and achieve controlled drug release, once the drug is entrap- Lipid nanoparticles are also a new approach ped in a biocompatible lipid core surrounded by for controlled drug delivery to lungs. There are asurfactantattheoutersurface.Melttempera- various studies of SLN formulations with the ture and crystallinity index as well as the selec- aim of administration by inhalation. Many that tion of excipients and SLN dose are their most have been developed for delivery of antifungal important features. They need to be evaluated and antimicrobial drugs have proved efficacy for final product stability, their thermal in vivo. Nasal administration is commonly used behavior during storage, as well as their safety. to deliver drugs to the central nervous system Nanoparticles have physicochemical properties (CNS) in a noninvasive way, thereby providing that give them exceptional biological activity, higher patients' compliance. SLNs for intranasal with their toxicological profile dependent on administration are gaining more attention lately these properties, mainly particle size and size but there are still few reports about their safety; distribution, as well as zeta potential. This is adverse effects have been observed in crucial in toxicological studies because it allows laboratory animals, probably due to assessing their toxic effects, identifying routes encapsulated drugs, inappropriate SLN doses of exposure as well as predicting the risks of used for these studies, or faulty selection of their synthesis or use. The potential toxicity excipients [14]. and the biocompatibility of drug delivery for- mulations are crucial for the implementation of drug therapies. Although the toxicity mecha- 5. Conclusions nisms of nanoparticles have been well studied, the available information about nanoparticle- SLNs and NLCs are the most studied lipid- based drug therapies is still very limited. In based drug delivery systems, having potential summary, the development of SLN and NLC to deliver drugs and also nutrients for several formulations continues to grow, with many pat- administration routes due to their biocompati- ents created worldwide. Toxicological testing bility, low toxicity, high-loading capacity, slow documents that lipid nanoparticles are safe release rate, and high stability. They are in devel- drug carriers for the various administration opment as drug carriers for administration by routes. 12 1. Solid lipid nanoparticles (SLN)

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Role of nanocarriers and their surface modification in targeting delivery of bioactive compounds Heni Rachmawati1,2, Atsarina Larasati2, Annis Catur Adi3, Ranjita Shegokar4 1School of Pharmacy, Bandung Institute of Technology, Bandung, Indonesia; 2Research Center for Nanosciences and Nanotechnology, Bandung Institute of Technology, Bandung, Indonesia; 3Faculty of Health, University of Airlangga, Surabaya, Indonesia; 4Capnomed GmbH, Zimmern, Germany

1. Bioactive compounds as promising are listed in various databases [1]. The therapeutic agents continuing interest in exploring health benefits of plants is evidence of the safety or minimal Apart from their typical other uses, plants are adverse events of herbal medicines, a part of a potential source for medicinal purposes. The the nature sync belief. A few of the species that exploration of therapeutic functions of contain important antioxidants to cure a wide substances present in plants has been done since range of diseases and have proven safe for long before prehistoric age. The practical reasons long-term use are described in this chapter, of traditional ways of medicine using plant including Curcuma sp, Zingiber sp, Silybum maria- materials for a wide range of human ailments num L, Gnetum gnemon, and Physalis angulate. are increase in population, insufficient drug supplies, prohibitive cost of treatments, adverse 1.1 Curcuma sp effects of several synthetic drugs, and develop- ment of resistance to currently used agents for Curcuma genus is classified under the family communicable diseases. Zingiberaceae. The genus consists of more than According to the World Health Organization, 80 species [2]. They grow abundantly in Asia, around 80% of people worldwide depend on including Southeast Asia, China, India, New medicinal plants, in some cases for their primary Guinea, and Northern Australia [3].Allcurcuma health care, and around 21,000 plant species sp have similar flower spikes that arise from the show potential benefits for human health and top of the pseudo stem or sometimes on a

Nanopharmaceuticals https://doi.org/10.1016/B978-0-12-817778-5.00002-6 17 © 2020 Elsevier Inc. All rights reserved. 18 2. Role of nanocarriers and their surface modification in targeting delivery of bioactive compounds separate stem directly from the rhizome. They differ mainly in the inner part of rhizomes, which vary in color, i.e., white, cream, yellow, orange, blue, bluish-green, and black [3]. In the family of curcuma, particularly the rhizomes show economic potential both for food and med- icine. For many years, the rhizomes of curcuma have been used for food additives, spice, and condiments. More importantly, the potential therapeutic value of curcuma’s rhizomes are reported elsewhere to cure various pathological conditions, including cancer [4e11]. Various species of Curcuma have been studied and reported such as Curcuma amada Roxb, Curcuma aromatica, Curcuma caesia Roxb, Curcuma longa, Curcuma manga, Curcuma purpurascens, Curcuma xanthorriza, and Curcuma zedoria. Plants belonging to the genus Curcuma are gaining importance worldwide and are an interesting topic for investigation and exploration. The plants contain bioactive molecules with various biological activities such as antiinflammation, antimicrobe, anti hypercholesterolemia, anti- rheumatic, antivirus, antifibrosis, antivenomous, antihepatotoxicity, antidiabetes, anticancer, and gastroprotector (Fig. 2.1). The common phytoconstituents found in Cur- FIGURE 2.1 Chemical structure of xanthorrhizol (1), cur- cuma are phenols, flavonoids, alkaloids, terpe- cumin (2), demethoxycurcumin (3), bisdemethoxycurcumin (4). noids, tannins, saponins, steroids, glycosides. Among other phytoconstituents in Curcuma, Ginger originated in Southeast Asia. It has xanthorrizhol and curcuminoid are two impor- been cultivated for thousands of years as a spice tant substances showing similar bioactivities as well as for medicinal purposes in countries and many health-promoting benefits, are being like India, China, Nigeria, Indonesia, widely explored for therapeutic purposes [12]. Bangladesh, Thailand, Philippines, and Jamaica. It is also grown in Australia, Fiji, Brazil, Sierra Leone, Japan, United Kingdom, United States, 1.2 Zingiber officinale and Saudi Arabia. Ginger is an herbaceous rhizo- matous perennial, reaching up to 90 cm in height Ginger, scientifically known as Zingiber offici- under cultivation. It is widely used in a variety of nale Roscoe, belonging to family Zingiberaceae, foods because of its nutritional value and is one of the most important plants with several flavoring compounds. Ginger rhizomes are a medicinal, nutritional, and ethnomedical values, rich source of carbohydrates, vitamins, minerals, and hence explored extensively all over the and iron. Phytochemical analysis describes the world as a spice, flavoring agent, and herbal content of ginger rhizome with a variety of phar- medicine [13]. macological effects. Z. officinale is reported to 1. Bioactive compounds as promising therapeutic agents 19 contain essential oils, phenolic compounds, silydianin, and silychristin. Silibinin is the most flavonoids, carbohydrates, proteins, alkaloids, active of the three, and is largely responsible glycosides, saponins, steroids, terpenoids, and for the hepatoprotective benefits attributed to tannins as the major phytochemical groups silymarin (Fig. 2.3). [13,14]. Ginger possesses its characteristic organ- oleptic properties due to two classes of constitu- 1.4 Gnetum gnemon ents: the odor and the flavor, determined by the constituents of its steam-volatile oil. The volatile Gnetum gnemon L. (Gg) or melinjo is a plant oil consists mainly of the mono- and sesquiter- growing abundantly in tropical areas such as penes; camphene, b-phellandrene, curcumene, Southeast Asia. This plant belongs to the genus cineole, geranyl acetate, terphineol, terpenes, of Gnetum (Gnetaceae family) [21,22]. The plant borneol, geraniol, limonene, b-elemene, is a small- to medium-sized evergreen tree with zingiberol, linalool, a-zingiberene, b-sesquiphel- nearly conical crown and a stature of 10e15 m. landrene, b-bisabolene, zingiberenol, and a- Zin- The stem is well branched and possesses a cylin- giberol are the principal aroma-contributing drical bole and a diameter up to 40 cm. Leaves components of ginger rhizome [14] (Fig. 2.2). are petiolate, ovate-oblong, or elliptical, 10e20 cm long and 4e7 cm broad, reticulately 1.3 Silybum marianum L veined, glabrous and shiny, dark green, apex acute to subacuminate, margins entire, base Silybum marianum (L.) Gaertn, whose com- acute and phylotaxy opposite; young leaves are mon name is milk thistle, is an edible plant reddish purple. Inflorescences are borne on belonging to the Asteraceae family [15,16]. Milk young shoots and older branches. thistle is a native of the Mediterranean region and has also spread to East Asia, Europe, Australia, and the Americas. Since the first cen- tury, milk thistle has been used medicinally [17e20]. Its flowers, leaves, and roots have been used in the European diet as vegetable, and its achene is used as a coffee. It is considered as a spinach substitute. In 1968, a flavonolignan complex in milk thistle fruit was identified and isolated. The major bioactive substituent present in the plant is the flavonoid complex silymarin, which is about 80% of the extract. This complex was found to be responsible for the medicinal effects of the silymarin complex and is made up of three parts: silibinin (also called silybin),

FIGURE 2.3 Chemical structure of silibinin (1), silydianin FIGURE 2.2 Chemical structure of zingiberol. (2), and silychristin (3). 20 2. Role of nanocarriers and their surface modification in targeting delivery of bioactive compounds

G. gnemon is one of the most studied wild referred to as cape gooseberry or cut leaf ground plants. The plants from the family of Gnetaceae cherry. Various phytochemicals including flavo- have been used as traditional medicines for noids, alkaloids, and plant steroids known as many years. Several bioactive phytoconstituents physalins (A and B), with anolides and secoste- present in G. gnemon, such as saponins, tannins, roids, which have never been reported before, and Melinjo seeds contain abundant resveratrol are present in this plant [27e30]. P. angulata L. (stilbenoid), mainly in the form of dimers (gnetin has been explored in recent clinical research, C). Gnetin C has been explored for more than 10 with preliminary evidence demonstrating it as years, principally by Japanese scientists. Well- an effective immune stimulant, toxic to documented clinical data is already available numerous types of cancer like leukemia, and and even more studies are continuing to emerge shown to have antimicrobial properties (Fig. 2.5). [21e24]. It has been reported that stilbenoids from melinjo showed strong antioxidant activity leading to various biological effects such as anti- inflammatory, antiaging, antihyperuricemia, 2. Complexity of bioactive compounds antimicrobe, anticardiovascular, and anticancer [25] (Fig. 2.4). Most bioactive substances isolated from plants are either in the form of crude extract, fraction, and single compound show poor 1.5 Physalis angulata bioavailability, hence have low-therapeutic outcome, resulting from the challenging physi- Physalis angulata L. is an annual herb indige- cochemical properties, instability issues, or nous to tropical areas like Africa, Asia, and extensive in vivo response. Bioavailability and South America including the Amazon [26].The solubility are key issues that have emerged plant grows up to 1 m high, plant parts are as top technical concerns in drug formulation smooth and glabrescent, it bears small, yellow- and delivery even for bioactive compounds. colored flowers, and produces small, light yellowish- orange, edible fruit sometimes

FIGURE 2.4 Chemical structure of resveratrol (1) and FIGURE 2.5 Chemical structure of physalin A (1) and gnetin C (2). physalin B (2). 3. Biological barriers 21

In addition, stability of the active agents both organism’s survival. Different types of biological physically and chemically also contribute signif- barriers are described next to present the critical icantly during formulation development. The parameters on the therapeutic failures as well as main challenges faced by pharmaceutical com- to find the appropriate strategies or solutions by panies in drug formulation as described in which a bioactive can be delivered successfully many reports [31e36] are safety (c.79%), appro- in vivo to exert desired clinical effects. priate therapeutic and delivery profiles (61%), and bioavailability (57%). In oral solid dosage forms, the top three formulation challenges 3.1 Physical barriers are bioavailability (41%), stability (37%), and For most therapeutics, reaching the targets of solubility (35%). action require penetration across tissues and/or The fact that bioactive compounds are chal- entry within cells. The design of strategies to lenging during formulation and delivery has control the transport of therapeutic compounds been described in several reports [31e36]. through these physiological barriers has become As discussed in the first part of introduction, an imperative and a challenging need in the several potential bioactive compounds exhibit a quest for better therapeutics. The physical bar- lack of pharmaceutical properties like low solu- riers for drugs entering systemic circulation are bility and permeability, sensitivity toward the membrane, a biological architecture of a external factors (humidity, light, and tempera- membrane border, like a single-layer or multi- ture), as well as presystemic degradation upon layer cell lining. This also includes lining of entering the body. All these challenges have endothelial cells of blood vessels, stratum led to unsuccessful therapy in the clinic using corneum of skin, lining of epithelial cells of bioactive components. various mucosa, BBB, blood ocular barrier, and the mucus covering the epithelial mucosa 3. Biological barriers (Fig. 2.6).

A major challenge in the drug delivery area is 3.2 Biochemical barriers how to transport the active agents across the bio- logical system entering the blood circulation and The biochemical barriers that can reduce the reaching the target of action to demonstrate the therapeutic function of a drug are the metabo- biological effects. Several biological barriers lizing enzymes, transporters, and efflux pumps. include the bloodebrain barrier (BBB), the small Drug-metabolizing enzymes are present in intestine, nasal, skin, and the mucosa. Biological almost all parts of the body where the drug is barriers are designed naturally with the aim to passing through, such as gastrointestinal tract, protect the organism from foreign materials respiratory system, ocular mucosa, in the blood that can damage homeostasis and physiologic circulation, and other entry points of drugs to function and eventually can threaten the the systemic compartment.

FIGURE 2.6 The physical barrier illustration of drug absorption: (A) lining endothelial cells, (B) stratum corneum, (C) lining epithelial cells, (D) mucosal membrane. 22 2. Role of nanocarriers and their surface modification in targeting delivery of bioactive compounds

Drug transporters are the proteins that are various other properties like surface coating, present in many organs (liver, lung, kidney, type of matrix used, and timely delivery, can intestine, brain, skin, blood vessels, and others). help to deliver a drug at a target site. The suc- Naturally, the proteins play important roles for cessful outcome of the nanocarrier to help the traffic between organs and elimination of drugs bioactive compounds show their effect upon and foreign compounds. Their function some- reaching the target site of action is also deter- how is beneficial, but they may also display mined by the in vivo behavior of the nanocarrier, deleterious effects that do not allow drugs to in particular in the phase of biological membrane enter the target organ to show their effects. passage to reach the systemic circulation, during In the BBB, drug transporters and drug metabo- traveling in the circulation to reach the target site lizing enzymes are also present to control the and after reaching the target. access to the brain and local concentration of Various nanoparticles are being studied for endobiotics and xenobiotics. delivery of synthetic as well as bioactive drugs. Efflux pumps demonstrate resistance to cyto- In this section, different drug delivery systems toxic drugs, hence also act as a barrier for drug explored for bioactive drug delivery are dis- delivery. By their nature, efflux pumps are trans- cussed (Table 2.1). port proteins involved in the extrusion of toxic Different types of nanocarrier systems are substrates from cells into the external environ- developed based on the characteristic of the ment. These membrane proteins function as a bioactive compounds as well as the aim of their pump that can decrease the intracellular accu- delivery target. Table 2.2 presents various nano- mulation of drug, leading to the ineffective systems along with their method of preparation. drug therapy. For better drug delivery, the key to understanding how these pumps operate involves the determination of the structures of 4.1 Lipid-based nanocarrier systems representative pumps and the elucidation of Considering the complex in vivo barrier sys- the conformational changes that accompany tem in which the drugs are protected to be able drug translocation. to survive for therapeutic presentation, various strategies in the area of formulation and delivery either to target the drug at specific site or to con- 4. Nanocarrier: a strategy to overcome trol the drug release have recently been reported biological barriers and described [31e36]. The former focuses more on the use of the excipients, the composition, and A primary reason for drug failing to demon- the manufacturing process, while the later em- strate its effect is the biology underlying the mo- phasizes the drug-carrier constructs. The lecular-, cellular-, and tissue-level barriers, following discussion reviews the typically used which makes the delivery process extremely drug delivery systems in bioactive delivery that complex. Therefore, to bypass the limitations are effective in overcoming the biological bar- regarding the biology of conventional drug riers, thereby improving the drug therapeutic in- delivery, it might be best to improve on the dex. In this category, we selected liposomes, concepts and approaches that are efficient and nanoemulsions, and lipid nanoparticles; other effective [37,38]. forms of nanoparticles are still under research One way to overcome barrier challenges and have not yet reached the market like the could be formation of effective drug delivery. above ones, hence we decided to discuss only Nanoparticles, due to their smaller size and the relevant ones. 4. Nanocarrier: a strategy to overcome biological barriers 23

TABLE 2.1 Bioactive-loaded nanocarrier systems and the potential medical promise.

Challenges in Added value of Class Category Compound Desired functionality formulation nanoemulsions

Flavonoid Flavanols EGCG, catechin, Free-radical scavenging, Catechins generally - Increased epicatechin anticancer, decreasing exhibit high water storage cholesterol level in solubility. Partition in the stability of blood, preventing lipophilic core is easily arterial sclerosis, improved by adding degradable thrombosis, heart 1-dodecanol catechins attacks, reducing fat and sugar uptake Flavonols Quercetin, Free-radical scavenging, The scarce solubility of - Improved kaempferol, fighting the effects of most flavonols in oil phase thermo- and myricetin aging and inflammation, requires the addition of photostability downregulating or amphiphilic molecules in - Enhanced suppressing the lipophilic core delivery inflammatory pathways and functions

Flavones Apigenin, Antimutagenic, Supersaturated flavones - Improved luteolin, rutin, antiinvasive, and in oil phases easily form physical tangeretin antiproliferative agent crystals, requiring the stability addition of compounds, - Bioaccessibility such as soy protein isolates, to slow down the crystallization process Flavanones Naringenin, Antiinflammatory, Flavanones exhibit poor - Increased hesperidin anticarcinogenic, water solubility. Oil and release of hepatoprotective, and emulsifier are added to compounds antilipid peroxidation ensure maximum loading - Improved the and stability in the bioavailability nanoemulsion Isoflavones Daidzein, Protection against Incorporation in oil Improved the genistein, hormone-related droplets should be permeation puerarin disorders, menopausal promoted by suitable through epidermal symptoms, heart disease, emulsifiers (i.e., lecithin) layers and osteoporosis Nonflavonoids Hydroxybenzoic Gallic acid, Antiproliferative and Complexation with Increased acids ellagic acid, antioxidant phospholipids is bioaccessibility, p-hydroxybenzoic required to increase reduced acid physical stability in interaction with nanoemulsions the food matrix Hydroxycinnamic Cinnamic acid, Free-radical scavenging, The solubility of some Improved acids coumaric acid, preventing cell damage phenolic acids is very biological activity, ferulic acid, by ultraviolet light, low in aqueous permeability, the caffeic acid antiaging, decreasing solutions, but they can absence of blood glucose easily be adsorbed at cytotoxic effect oil/water interface of nanoemulsions

(Continued) 24 2. Role of nanocarriers and their surface modification in targeting delivery of bioactive compounds

TABLE 2.1 Bioactive-loaded nanocarrier systems and the potential medical promise.dcont'd

Challenges in Added value of Class Category Compound Desired functionality formulation nanoemulsions

Stilbenes Trans-resveratrol Anticancer, Poor solubility in water - Improved IV- antiinflammation, and in oil requires either light stability lowering low-density high amounts of - Enhanced cell lipoproteins (bad surfactants or a permeation cholesterol), fighting combination of lipophilic the plaque buildup and hydrophilic leading to Alzheimer emulsifiers disease, preventing insulin resistance Curcuminoids Curcumin Antiinflammatory, Poor solubility in water - Improved cell antioxidant, boosting and in oil requires the use uptake and brain-derived of amphiphilic molecules anticancer neurotrophic factor, such as lecithin in the lipid activity lowering the risk of phase - Enhanced food heart disease, anticancer, compatibility fighting age-related and reduced chronic disease impact on sensory properties Carotenoids Carotenes b-Carotene, Antioxidant, free-radical Light and oxidative Improved physical lycopene scavenging, decreasing instability requires the stability, control of the risk of cardiovascular use of biopolymers in lipolysis, and disease, oral cavity, and nanoemulsions release lung cancers formulation Xanthophylls Lutein, Preventing macular Limited solubility and Improved zeaxanthin degeneration, promoting dissolution requires bioavailability normal visual functions, complex formulation strengthening eye tissue, supporting visual acuity and brain function

Among various nanocarriers developed to (e.g., solid lipid nanoparticle, nano/microemul- transport the active substances, liposomes, sion, SEDDS/SMEDDS/SNEDDS, nanocapsule, lipid-based nanocarriers, and nanoemulsions liposome), the power of the carrier to load the are more promising because of their diversity, active substance, and the capability to improve favorable biocompatibility, and specific the drug therapeutic index. Therefore, types of functionality. lipid are very important for successful delivery In the case of lipid nanoparticles, a majority of of the bioactive compounds loaded in the nano- lipids excipients are derived from dietary oils/ carrier systems. fats, which offers advantages in terms of biode- 4.1.1 Liposomes gradability and the ability to penetrate the bio- logical membrane barriers. The selection of Liposomes are tiny bilayer vesicles with lipid by considering the chemical structure and spherical shape that can be formed from choles- properties determines the type of nanocarriers terol and/or natural inert phospholipids. 4. Nanocarrier: a strategy to overcome biological barriers 25

TABLE 2.2 Various nanosystems for effective bioactive compound delivery.

Bioactive Nanosystem Technique Composition compound Size (nm) References

Nanocapsules Ionic pregelation/ Chitosan, alginate Naringenin 200e300 [39] coacervation Ionotropic polyelectrolyte Alginate/chitosan/pluronic Curcumin 100e120 [40] pregelation

Ionotropic polyelectrolyte Alginate/chitosan Vitamin B2 86e200 [41] pregelation

Nanohydrogels Physical self-assembly b-lactoglobulin and u-3 fatty acids 100 [42] low methoxyl pectin Temperature- and pH- b-lactoglobulin Epigallocate- 7e10 [43] induced gelation chin-3-gallate Temperature-induced b-Lactoglobulin/hen ɑ-Tocopherol e [44] gelation egg white protein/alginate Temperature-induced b-lactoglobulin Curcumin 142 [45] gelation Nanoemulsions High-pressure Corn oil, Tween 20, Curcumin 119.5e152.9 [46] homogenization SDS, and DTAB Solvent displacement þ Hexane, Tween 20 b-carotene 9e280 [47] ultraturrax Melt-homogenization Medium-chain triglycerides, Curcumin 130e205 [48] trimyristin, and tristearin Solid lipid Microemulsification Polysorbate 80/soy lecithin Curcumin 134.6 [49] nanoparticles High-pressure Soy lecithin/sodium Curcumin 100e110 [50] homogenization glycocholate/glycerol Micelles Self-assembly Casein b-Carotene 80 [51] b-casein Curcumin e [52]

The bilayers consist of hydrophobic and hydro- Liposomes have demonstrated the potential philic compartments that enable a wide range benefit to improve the bioactivity of various of drugs to encapsulate, hence are a promising natural compounds with lack in pharmaceutical system for drug delivery. The type of lipids properties [74]. Bonechi et al. described the composing liposomes determine the surface improved therapeutics of various plant-derived charge, size, and the method of liposome prepa- phenolic compounds after they were incorpo- ration. The surface rigidity and fluidity of rated into liposomes [75]. As the natural liposomes are influenced using additional agents compounds are appreciated for their broad like cholesterol. The choice of liposome prepara- spectrum activities, this makes them more tion method is dependent on a variety of factors appropriate to interfere in multifactorial dis- like lipid composition and size for in vivo drug eases, such as cancer. As compared to synthetic delivery, etc. compounds, natural bioactive compounds 26 2. Role of nanocarriers and their surface modification in targeting delivery of bioactive compounds generally have better safety profiles, and are well The role of TPGS-coated resveratrol lipo- accepted by the public. Despite these factors, the somes (RSV-TPGS-Lipo) was investigated by use of bioactive compounds poses a number of Vijayakumar et al. to improve pharmacokinetic challenges that need to be overcome for better parameters and brain-targeted delivery of RSV establishment as clinically effective therapeutic after intravenous (i.v.) administration in rats. agents. They also studied passive brain targeting effi- Currently, there is a huge lack of human cacy and hemocompatibility of RSV-TPGS-Lipo clinical trials that address their absorption, for revealing their potential and safe i.v. admin- distribution, metabolism, and excretion in rela- istration for treatment of glioma. In vitro study tion to efficacy. As reported, various compounds demonstrated that RSVTPGS-Lipo 2 showed that indicated poor bioavailability as well as higher cytotoxicity due to presence of TPGS, being unstable and prone to degradation or which is known for its cytotoxic potential. The oxidation, such as resveratrol (logP 3.4), thymol liposomes were highly concentrated in the cyto- (logP 3.4), caffeic acid (logP 1.5), caffeic acid phe- plasm of the cells, which is a major site of action nethyl ester (logP 3.9), carvacol (logP 3.4), and of RSV for its anticancer activity. This finding carvacrol disodiumphosphate (logP 2.0), have confirms the promise of liposomal approach to been successfully formulated into liposomal encapsulate a potential natural anticancer systems, resulting in better properties for each compound with high cell selectivity [77]. compound [75]. In line with the reports of Coimbra et al., 4.1.2 Nanoemulsions Bonechi et al. [75] described liposome formula- Among lipid-based nanocarrier systems, the tions made by a saturated phosphatidyl-choline nanoemulsion is most commonly used system (DPPC) and cholesterol (or its positively charged for delivery of various bioactive compounds. derivative, DC-CHOL) to optimize the loading This is because of its easy preparation at both of a rigid hydrophobic molecule such as resvera- laboratory and industry production scale, and trol. They demonstrated the safe use of the the unique emulsion characteristics compared systems on stabilized cell lines (mouse fibroblast to other nanocarrier systems. The flexibilities of NIH-3T3 and human astrocytes U373-MG). nanoemulsions are promising to create novel Recent reports by Cavalcanti et al. [76] carrier systems with advanced potential benefits describe the successful liposomal formulation as drug transporters. on new compounds of natural origin that exhibit Nanoemulsions can also be used as building antimicrobial activity: usnic acid (UA), a second- blocks for other types of structures, such as filled ary lichen metabolite; and b-lapachone (b-lap), a hydrogels. Filled hydrogels are designed to naphthoquinone derived from lapachol encapsulate, protect, and control the release of extracted from Tabebuia avellanedae bark. bioactive components by changing their dimen- These molecules exhibit proven antimicrobial sions, internal composition, or structure. Colloi- activity, however, show low water solubility dosomes or microclusters are other structures and high toxicity. Liposomes containing b-lap that can be derived from nanoemulsions. A col- (b-lap-lipo) or usnic acid (UA-lipo) were loidosome consists of a large central particle prepared by the thin lipid film hydration method with smaller particles adsorbed to its surface, followed by sonication and were able to improve whereas a microcluster consists of a number of the antimicrobial activity of vancomycin in the smaller particles held together by attractive treatment of methicillin-resistant Staphylococcus forces. These structures are created from nanoe- aureus. mulsions in order to alter the rheological, optical, 4. Nanocarrier: a strategy to overcome biological barriers 27 or stability properties of materials or for The lutein-loaded nanocarriers prepared with controlled-release applications as well. high-pressure homogenization have the mean A very recent study reported the potential use particle size of about 150 nm to maximum of nanoemulsion for thymol, an essential oil 350 nm. The penetration study in in vitro system component of plants, and Quillaja saponin, a using cellulose membrane demonstrated that glycoside surfactant of the Quillaja tree [78]. lutein loaded in nanoemulsion passed the mem- Thymol(2-isopropyl-5-methylphenol), a major brane with highest percentage of 60% after 24 h, essential oil component of plants from the Lam- as compared to NLCs. Permeation study using iaceae family, possesses the phenolic hydroxyl fresh pig ear skin showed no or very little lutein group that contributes to its antimicrobial activ- loaded in NLCs was absorbed to systemic ity. Thymol has been classified as a generally circulation. recognized as safe (GRAS) by the U.S Food and Another potential promise of nanoemulsion Drug Administration in its use as a food addi- as bioactive carrier was demonstrated by Shofia tive. Its application has been widely reported in et al. on brown seaweed against colon cancer cell the medical, food, and agricultural fields. lines HCT 116 [80]. For centuries, seaweeds have However, like other compounds, the main chal- been used as a food throughout Asia, and lenge is low water solubility, which limits its because of its high nutrient content seaweeds application in aqueous medium, as well as being are of high pharmaceutical interest. Polysaccha- unstable physically and chemically in the pres- rides from brown seaweeds have been reported ence of oxygen, light, and temperature. All of to have potent bioactive functions like antiin- these things eventually limit its biological activ- flammatory activity, antioxidant activity, and ity and its efficiency. antiproliferative effect on various cancer cells. Kumari et al. reported thymol nanoemulsion Nanoemulsions and NLCs were developed to prepared by sonication method is able to overcome the instability and bioavailability improve the antibacterial effect of the thymol problem of exopolysaccharides extracted from against bacterial pustule disease and growth brown seaweed (Sargassum longifolium). Nanoe- promoting action on soybean [78]. The 50-min mulsions were prepared using essential oil (or- sonication of mixture of thymol and saponin in ange oil) and biosurfactants (Span 80 and the ratio of 6:1 (w/v) in deionized water resulted Pluronic L81) by high shear stirring followed in nanoglobules with the size of about 250 nm. by ultrasonication method at room temperature. The nanoemulsion with concentration of 0.01% Combination of two different surfactants aimed e0.06% inhibited the growth of Xanthomonas axo- to maintain the synergistic effect on emulsion nopodis. In addition, the nanoemulsion of thymol stability. also lowered the disease severity and increased In other work, encapsulation of curcumin into percent efficacy of disease control of bacterial nanoemulsion system was established using pustule in soybean caused by X. axonopodis pv low- and high-energy techniques [81e83]. Incor- glycine. poration of curcumin, a phenolic substance Other reports described the benefit of lipid- present in Curcuma sp, improved the pharma- based nanocarrier, i.e., nanoemulsion and nano- ceutics property as well as the chemical stability structured lipid carriers (NLCs) to improve solu- of this yellow compound leading to a more bility as well as the stability of lutein against UV flexible formulation development. Some reports light [79]. Lutein, a natural carotenoid, has regarding the use of nanoemulsion for strong antioxidant property, which can protect bioactive delivery are also presented in Tables 2.2 skin from the damage due to photo irradiation. and 2.3. 28 2. Role of nanocarriers and their surface modification in targeting delivery of bioactive compounds

TABLE 2.3 The materials-forming nanosystem for bioactive compound encapsulation.

Encapsulated nutrient Type of delivery system Main ingredients References

Vitamin B2 Nanoparticle Alginate and chitosan [41] Vitamin D3 Nanoparticle Carboxymethyl chitosan and soy protein [53] Rutin Nanocomplex Sodium caseinate and pectin [54]

EPA/DHA Nanoparticle Sodium caseinate and gum arabic [55]

Poly-L-lysine LbL nanocapsule Chitosan and fucoidan [56] Glycomacropeptide LbL nanocapsule Chitosan and alginate [57] Quercetin SLN-nanostructured lipid MCT as oil phase, Tween 80, lecithin and [58] carriers/ span 20 as emulsifiers lipid nanoemulsions (LNE) Quercetin Nanoparticle Chitosan and lecithin [59] Curcumin Nanoemulsions MCT as oil phase and WPC and Tween 80 as [60] emulsifiers b-Carotene Micelle Casein [51] b-Carotene Nanoemulsions Tween 20 [47,61] Resveratrol Multilamellar liposome Lecithin [62] Curcumin Interpenetrating polymeric Gelatin [63] network nanogel Curcumin Core-shell biopolymer Zein (core) and pectin (shell) [64] nanoparticle Chlorogenic acid Nanoparticle Chitosan [65] Folic acid Nanocomplex b-Lactoglobulin and sodium alginate [66] Resveratrol, rutin, Nanosuspensions and Hydrophilic surfactants [67e73] hesperidin, smart crystals hesperetin, curcumin, quercetin, apigenin

4.1.3 Solid lipid nanoparticles across biological membrane, protection of the Solid lipid nanoparticles (SLNs) were first labile molecules against external factors, as well fi described in 1991, as an alternative carrier struc- as modi cation of the release of the active ture to previously developed colloidal carriers molecules. such as emulsions, liposomes, and micelle. SLN Various bioactive compounds loaded in SLNs is a colloidal nanocarrier with size in the range show better performance as compared to their of 50e1000 nm composed by lipid. Like other free form, such as lutein, rutin, curcumin, Q10, e lipid-based carrier systems, SLNs offer potential and others as shown in Table 2.4 [67 73,79]. benefit to improve the bioavailability of bioac- A recent report describes the development of tive compounds through various mechanisms Hibiscus rosa-sinensis extract loaded in SLNs us- including enhanced solubility, permeability ing glycerol monostearate or beeswax as lipids 6. Cellular uptake capability of bioactive-loaded nanocarrier system 29 for antidepressants. The SLNs were prepared The choice of GRAS excipients at least will mini- with emulsion-quenching technique resulting mize risk use of the nanoconstruct for human in nanoparticles of w175 nm with better antide- health. pressant activity in vivo as compared to native Safety consideration is made not only crude extract [84]. SLNs have higher physico- focusing on humans but also on the environ- chemical stability and protect the labile drugs ment. Several studies must be performed prior from degradation. In addition, the production to human application ranging from in vitro could be done on large scale, which makes this toxicity tests to in vivo acute, subchronic, and nanocarrier system more attractive as compared chronic evaluations. As we previously reported, to other lipid-based systems. Other benefits of nanoemulsion composed from castor oil, Cremo- SLNs are that the matrix is solid, which protects phor RH 40, and PEG 400 is safe upon incubation the drug from chemical degradation, and the on various types of cell line as well as orally crystallization of product causes efficient encap- administered to Webster mice [94]. The nanoe- sulation and release of drugs, enabling use of mulsion was successfully used to carry potent SLNs for various routes of administration. phytochemicals such as curcumin [81e83] for Table 2.4 summarizes the studies demon- various studies both in vitro and in vivo. strating the potential benefits of various bioac- The reports on safety data of several bioactive tive molecules loaded on SLNs/NLCs using compounds loaded in various types of nanocar- different techniques of preparation. rier system are presented in Table 2.5.

5. Safe-by-design bioactive-loaded 6. Cellular uptake capability of bioactive- nanocarrier system development loaded nanocarrier system

Safe-by-design is a concept that is well estab- There are two main mechanisms of transport lished in fields like building, nuclear technology, that affect nanoparticles: transcellular and para- water treatment, health facilities, and occupa- cellular routes [102]. Transcellular transport is a tional health and safety. It describes safety process by which the cells of a tissue utilize a measures for the prevention of accidents, ill- mechanism of transport through the cell nesses, or environmental damage that are [102,103], whereas paracellular transport is a applied during the design stage of a facility, process that occurs due to free passage or phys- process, practice, material, or product. Applica- ical effect that passes through extracellular tion of this concept requires comprehensive spaces [104]. knowledge questioning of what properties Although these mechanisms are common to make a nanomaterial or nanoproduct safe. many molecules, their role in nanoparticle Nanomaterials are distinguished by their phys- uptake is lesser known [105]. Nanomaterials ical properties such as optic, magnetic, electric, may enter cells and be readily transported mechanic, solubility, and others, which are better through a number of different mechanisms, compared to those of the materials formed. The including endocytosis, active transport, and mechanisms of occurrence of nanotoxicity may facilitated diffusion [106e108]. The potential of be the result of various factors: the composition, a nanoparticle to be transported through any the particle size and shape, solubility, ability for one of these mechanisms is inherently and aggregation, surface reactivity and production of primarily influenced by size, surface charge, reactive oxygen species, as well as the route of and reactivity [109]. Positively charged nanoma- administration. The first is most important terials will capitalize on the negatively charged when the nanocarrier system will be developed. tight junction filaments between cells, increasing 30 2. Role of nanocarriers and their surface modification in targeting delivery of bioactive compounds

TABLE 2.4 The advantages of lipid nanoparticles (SLNs/NLCs) to improve different bioactive molecules.

Production Bioactives Formulation/preparation method Route Advantages References

D-limonene Palm oil soy lecithin hot HPH Oral/food • Increased [85] antimicrobial activity Curcumin Hydrogenated soya HPH Oral • Increased cellular [86] phosphatidylcholine, distearoyl, uptake and phosphatidylethanolamine, • Reduced cytotoxicity cholesterol, and triolein Vitamin A Glyceryl bebenate Ultraturax Transdermal • Increased skin [87] delivery permeation • Improved release rate Lutein Solid lipid or mixture of solid HPH Topical • Increased drug [79] and delivery stability against UV liquid • Controls drug release to the skin • Better permeability Leonotis leonurus TagoCare, Cutina CP, Miglyol HPH Oral • Increased [88] bioavailability Rosmarinic acid Witepsol-Carnauba wax Ultrasonication Oral • Increased stability [89] against gastrointestinal degradation • Improved drug delivery

Cryptotanshinone Glyceril monostearate e soy Sonication-HPH Oral • Bioavailability [90] lecithin Enhancement Astaxanthin Oleic acid, Glyceryl behenate, Melt emulsification- Oral • Improved [91] Tween-80, sonication physical stability Lecithin Green tea extract Cetyl palmitate, Glyceryl Modified HPH Oral • Improved antioxidant [92] stearate, vegetable oil, activity Tween-20/80 • Increased antimicrobial activity Lycopene Eumulgin SG, orange wax, HPH Oral • Enhanced chemical [93] rice bran oil stability • Improved antioxidant activity their transport through paracellular mechanisms such as clathrin-mediated endocytosis in the [110]. Various nano-based approaches have oral mucosa [105]. been applied to enhance the uptake of drugs Lu et al. described the correlation between with poor bioavailability or aqueous solubility nanoemulsion structure and cellular uptake of b [111,112]. The nanomaterials are readily trans- encapsulated -carotene in vitro using CaCo2 ported via highly specific cellular mechanisms, cells [113]. The aim of nanoencapsulation of the TABLE 2.5 Bioactive-loaded nanocarrier systems and the safety confirmation data.

Materials and Methods Model used Size (nm) Dose tested Assay Cytotoxicity results References In vitro studies

SLN

Curcumin/trimyristin and emulsifiers soy Caco-2 and 100 10 3.125 mg/mL, 2 h NADPH production No cytotoxic effect neither on [50] lecithin, sodium glycocholate and poloxamer HT29-MTX using a colorimetric Caco-2 nor on HT29-MTX 407 (high-pressure homogenization) cells (75:25) assap (CellTiter 96) b-Carotene (BC)/SC, WPI, SPI) Caco-2 cells 75 (SC)/90 10 mg BC/mL, 10 MTT Low toxicity at about 10 mg BC/ [95] (homogenization-evaporation) (SPI) times diluted or more, mL (73%e92% of CV) and 48 h insignificant when diluted 1 time or more (CV > 95%) Nanoliposomes

Lactoferrin/PC/cholesterol/Tween 80 Caco-2 <100 1, 5, and 10 mg/mL MTT, ROS detection, Mitochondrial activity reduction; [96] (reverse-phase evaporation) and apoptosis CV decreased (at 5 and 10 mg/ induction (AO/EB mL); ROS increased (5 mg/mL) staining); LDH Nanoemulsions

Resveratrol/soy lecithin/GMO/sugar ester Caco-2 cells 128e235 Nanoemulsion XTT assay and Formulations do not cause any [97] mixture/peanut oil/polysorbate Tween 20 dilution (1:10, 1:50 and confocal laser harm to the cells (high-pressure homogenization) 1:100), 24, 28, and 72 h scanning microscopy Curcumin/b-lactoglobulin complexes Caco-2 cells 50e200 20e400 mg/mL, 24 h MTT Curcumin concentrations were not [98] emulsifier WPI (freeze-drying of nanoemulsion) toxic to the cells at 100 mg/mL Polymer and SLN

GLP-1/PLGA, Witepsol E85 lipid and porous Caco-2 and 200 1.5, 3.75, 0.5 and CellTiter-Glo CV above 80%, with HT29-MTX [99] silicon (Psi)/chitosan coated (solvent HT29-MTX 15 mg mL, 3 and 12 h luminescence assay cells, the nanoparticles showed emulsification-evaporation method) cells less toxicity compared to Caco-2 cells at the same point Rosmarinic acid (RA) Lymphocyte 900 0.15 mg/mL MTT The SLN is safe when loaded with [89] cell moderate concentrations of RA, without in vitro genotoxicity Peptide-polysaccharide nanoparticles

Epigallocatechin-3-gallate/genipin-crosslinked Human <300 12.5e200 mg/mL, Trypan blue dye Naked CPP-CS NPs cross-linked [100] caseinophosphopeptide-chitosan (CPP-CS) gastric 24, 36 and 48 h exclusion test with genipin did not show BGC823 cells cytotoxicity In vivo studies

Hydrogel nanoparticles

Curcumin/HPMC/PVP (solvent emulsion- Holtzman rats 100 2000 mg/kg, Acute and subacute No toxicity [101] evaporation technique and free-drying) 14 days toxicity 32 2. Role of nanocarriers and their surface modification in targeting delivery of bioactive compounds bioactive b-carotene was to improve the cellular have been engineered to target difficult-to-treat uptake of the compound. Enhanced cellular diseases like tumors and disease sites that have uptake is also considered as one of the potential permeable vasculature, allowing easy delivery mechanisms of improved bioavailability of of payload. Specific targeting and reduced clear- bioactive nutrients, because uptake of nanopar- ance increases the therapeutic index, which ticles by digestive tract mucosa via mucosa- consequently lowers the dose required for associated lymphatic tissues is possible. efficacy. Nanoencapsulationinanemulsionthatcan Nanodrug carriers can increase the bioavail- also significantly improve the cellular uptake ability of the drug, including natural drugs at of encapsulated molecules. These important the target site, reduce the frequency of adminis- findings demonstrated that cellular uptake of tration, and reach sites that are otherwise inac- encapsulated BC is dependent on particle size cessible. In order to be useful in drug delivery, and interfacial structure (emulsifiers). the nanocarrier must possess very important The influence of surfactants such as Tween 80, characteristics. Functionalization or surface sodium dodecyl sulfate, and sodium caseinate modification of the bioactive-loaded nanomate- on physicochemical, morphological, and cellular rials is often done to obtain unique and specific uptake properties of lutein nanodispersions was properties in the biological system, i.e., long reported by Tan et al. [114]. The surfactants used systemic circulation, organ or cell selectivity to for that study showed different stabilizing mech- improve their efficacy and lower the adverse anisms on lutein nanodispersions. The lutein effects. nanodispersions in this study were produced us- The first attempt at developing modified ing the solvent displacement method and dis- nanocarrier for active drug targeting was pro- played good physicochemical properties. Their posed in the 19th century by the scientist Paul interesting findings demonstrated how the Elrich. Since then, more attempts were done to different types of surfactants could affect the improve the therapeutic value of the nanocarrier, characteristics of the lutein nanodispersions pro- which also helps to load bioactive compounds as duced. As well, the utilization of a small- presented in Table 2.6. molecule electrostatic surfactant, such as sodium Other promising modification was also dodecyl sulfate, could be useful in producing demonstrated by SLNs [115]. The conventional lutein nanodispersions with small particle sizes SLNs have several advantages, although there (of less than 100 nm) and high zeta potential is a challenge to oral delivery of bioactive values. compounds such as burst release of the loaded compounds in the stomach at a lower pH of about 1e3. To solve this matter, SLNs are sub- 7. Surface modification of nanocarriers jected to surface modification to improve the therapeutic benefit. Surface-modified SLNs can Nanocarriers for drug delivery systems offer be constructed using heparin, albumin, polyeth- advantages that are desirable for therapeutics. ylene glycol, and polysaccharides. Chitosan is Drug nanocarriers also have the ability to also used as it is highly degradable and has improve the pharmacokinetics and increase bio- lower immunogenicity, thus it is suitable for distribution of therapeutic agents at the target controlled oral delivery of the bioactive organs that result in improved efficacy and compounds under various pH conditions. reduction of adverse effects. The nanocarriers 9. Challenges of bioactive-loaded nanocarrier to clinical translation 33

TABLE 2.6 Surface modification-functionalization of nanocarrier to improve the nanosystem properties.

Carrier system Surface modification Bioactive compound Benefit References Nanoemulsion

Solid lipid N-carboxymethyl Curcumin To inhibit the rapid release of [116] nanoparticle chitosan (NCC) curcumin coating in acidic environment and enhance the bioavailability Solid lipid Chitosan-coated Resveratrol, caffeic acid, To enhance the delivery [117,118] nanoparticle ferulic acid Solid lipid Trimethyl chitosan- Curcumin, resveratrol To enhance the bioavailability [117,119] nanoparticle coated Solid lipid N-trimethyl chitosan- Resveratrol To enhance the bioavailability [120] nanoparticle g-palmitic acid Liposome PEGylated Coenzyme Q10 Long-circulating liposomal [121] delivery systems

8. Biokinetic profile of bioactive-loaded 9. Challenges of bioactive-loaded nanocarriers nanocarrier to clinical translation

The first step in demonstrating the efficacy of The nanocarrier intended for clinical applica- the bioactive-loaded nanocarrier when given tions should use materials safe as pharmaceu- through oral route is that it must pass through tical excipients and its formulation should have the gastrointestinal (GIT) barrier. After the inges- good manufacture processes with scale-up abil- tion process, there are some barriers to overcome ity. The challenge is to design safe, approvable, before bioactive compounds can reach the sys- and easily scaled-up production, intellectual temic circulation in an active form. The bioavail- property, government regulations, and overall ability of these compounds can be impaired due cost-effectiveness in comparison to current to various physicochemical and physiological therapies. phenomena such as restricted liberation from The design of bioactive-loaded nanocarrier delivery matrices, insufficient gastric residence systems must consider several aspects such as time, low solubility in gastrointestinal fluids, route of administration, complexity in formula- formation of insoluble complexes with other tion design, final dosage form for human use, components in the GIT, low permeability across biocompatibility and biodegradability, and the mucus layer or epithelium cells, and/or mo- pharmaceutical stability (physical and chemical). lecular transformations/chemical instability in In addition, the pre- and clinical evaluations are the GIT. also important. Considerations for preclinical These phenomena can cause a large percent- evaluation include validation and standardiza- age of compounds to remain unabsorbed and tion of assays for early detection of toxicity, eval- be excreted out of the body. Tables 2.7 and 2.8 uation in appropriate animal models of disease, present the in vivo bioaccessibility and bioavail- and adequate understanding of in vivo behavior ability of different types of nanocarrier systems such as cellular and molecular interactions, phar- loading bioactive compounds. macokinetic profile, and the pharmacodynamics 34 2. Role of nanocarriers and their surface modification in targeting delivery of bioactive compounds

TABLE 2.7 Various bioactive-loaded nanocarrier systems and their bioaccessibility.

Nutrient Delivery system Details Composition/testing details References b-Carotene Nanoemulsions Encapsulation active 3.1% (dispersed in MCT) [122] 35.6% (nanoencapsulated) Lycopene Nanoemulsions Size 0.01% (Unemulsified) [123]

0.53% (size: 150 nm) 0.77% (size: 69 nm) Quercetin SLM nanostructured lipid Lipid nanocarrier 39.7% (SLN) [58] carrier (NLC), LNE 52.7% (NLC) 58.4% (LNE) b-Carotene Nanoemulsions Carrier oil z66% (LCT) [124]

z2% (MCT) z0% (orange oil) Curcumin Nanoemulsions Surfactant 16.4% (Tween 20) [56] 17.7% (SDS) 1.2% (DTAB) Resveratrol Biopolymer nanoparticles (BnPs) Nanoencapsulation, z73% (BnP) [125] and complexes (BC) nanocarrier z70% (BC) z60% (free) Curcumin Nanosuspension Active þ surfactant 99% active content [73] 100e500 nm Apigenin 99.5% active content [72] 210e450 nm of the bioactive compound loaded in the nano- important for bioactive components to reach carrier. Currently, many aspects are still under market success (Table 2.9). exploratory phase and need collaboration and willingness of different expert areas. Clinical evaluation for commercialization 9.1 Patents on herbal nanoparticles for demands simplification of the development breast cancer pathways from invention to the market. To minimize time and expense, evaluation of The interest in nanoproduct developments is safety/toxicity in humans both acute and also reflected by several patents: Liang et al. chronic, the evaluation of therapeutic efficacy [139] developed nano-micelles, and Zale et al. in patients, as well as the optimal clinical trial [140] developed polymeric nanoparticles of design are key essentials. vinca alkaloids (vindesine, vinorelbine vincris- In addition, quality and constant supply of tine and vinblastine) using PEG for anticancer herbal extracts or raw material are equally applications. These preparations have good 9. Challenges of bioactive-loaded nanocarrier to clinical translation 35

TABLE 2.8 List of various bioactive-loaded nanocarrier systems and their bioavailability.

Compound Formulation type Animal model Bioavailability Dose References

Folic acid Zein nanoparticles Rats 2-fold higher than the 1 mg/kg [127] free form ɑ-Tocopherol Nanoemulsion Male Wistar rats, 2.6-fold increase 30 mg/kg [128] 210 10 g Lycopene Lipid-based solid Pigs, female landrace, 2.4-fold higher than the 50 mg [129] (lipophilic dispersion (LBSD) 13e15 kg commercial product carotenoid) Lycovit (in gelatin beadlets)

Lutein PLGA nanoparticle Male Fischer Increased pharmacokinetic 10 mg/kg [130] 344 rats, 238 g parameters, such as Cmax (54.5-fold) and AUC (77.6-fold) than the free form

Lutein Low-molecular-weight Swiss albino mice, Postprandial lutein level 200 mm [131] chitosan 25 2g in the plasma 54.5% higher than control Silymarin Lipid nanoparticles Beagle dogs Higher bioavailability than 8 mg/kg [132] (15 2 kg) their lipolysate counterparts Curcumin Lauryl sulfated chitosan SD rats, 220e250 g 48.79-fold more than free 10 mg/kg [133] form Curcumin Phosphatidylcholine- Albino Wistar rats, 130-fold increase oral 50 mg/kg [134] maltodextrin-based 200e250 g bioavailability hydrophilic lipopolysaccharide Diosgenin in Liquid crystal Male Wistar rats 6.2-fold more than 2 mL/kg [135] wild yam (glyceryl monooleate þ (200e250 g) free form (Dioscorea villosa) b-cyclodextrin) Capsaicin (PVP)/sodium cholate/ Male SD rats 2.42-fold more than 90 mg/kg [136] phospholipid mixed free form polymeric micelles

Apigenin Supercritical antisolvent Male Spraguee Absolute bioavailability 50 mg/kg [137] process Dawley rats (230 increased e270 g) from 2.0% for free coarse powder to 6.9% for nanocrystal Resveratrol Solid nanoparticles Wistar male rats 8-fold increase than 20 mg/kg [138] suspension

Acquired with permission from Ref. Nallamuthu I, Khanum F, Fathima SJ, Patil MM, Anand T. Enhanced nutrient delivery through nanoencapsulation techniques: the current trend in food industry, Book: nutrient delivery, Elsevier September 2016, 619. 36 2. Role of nanocarriers and their surface modification in targeting delivery of bioactive compounds

TABLE 2.9 Commercial nanoherbals in the market.

Category/ Product name Company Ingredient Administration route

LycoVit BASF, Germany Carotenoid lycopene Nutraceutical/oral Taiji ring Limited Hebei, China Nano-selenium rich black tea Food/oral Nanotea Shenzhen Become Extract tea, selenium Food/oral food Industry & Trade Co., Ltd. Canola active oil Shemenm Haifa, Israel Phytosterols, Fortified fruit juice High Vive.com., USA Fortified vitamin, lycopene, Food/oral theanine, and sun-active iron

Nanoceuticals Slim RBC Lifesciences, Assorted flavors Nutritional food/oral Shake rving, USA NanoSlim NanoSlim, Canada Lagerstroemia speciosa L., cha’ de Nutritional food/oral bugre extract, green coffee extract and thallus powder and corosolic acid Oat Nutritional Drink Toddler Health, Assorted flavors Nutritional food/oral Los Angeles, USA “Tip-Top” Up Bread Enfield, Australia Tuna fish oil Nutritional food/oral NanoResveratrol Life Enhancement, USA Solid triglyceride, phosphatidylcholine Nutritional food/oral delivery Nutri-Nano CoQ-10 3.1x Solgar, USA CoQ10, natural oils Cosmetic/dermal Softgels NovaSOL capsule Aquaniva Darmstadt, e Nutritional food/oral Germany

Spray for Life Health Plus International, Vitamin Nutraceutical/oral Inc., USA Vitamin Supplements “Daily boost” Jamba Juice Hawaii, USA Vitamin or bioactive components Nutraceutical/oral “Color emulsion” Wild Flavors, Inc, USA b-Carotenal, apo-carotenal, or paprika Food/ora “Nano-silver” (NS) A-DO Global Col., Ltd, Silver Food/oral South Korea Cutanova Cream Dr. Rimpler, Germany Q10, polypeptide, hibiscus extract, Cosmetic/dermal Nano Repair Q10 ginger extract, ketosugar Intensive Serum Dr. Rimpler, Germany Q10, polypeptide, mafane extract Cosmetic/dermal NanoRepair Q10 Cutanova Cream NanoVital Dr. Rimpler, Germany Q10, TiO2, polypeptide, ursolic acid, Cosmetic/dermal Q10 oleanolic acid, sunflower seed extract References 37 stability, improved drug distribution, increased particular, for difficult to treat diseases that effectiveness, and decreased toxicity and have require safe long-term therapy with low cost. shown efficiency in the clinical treatment of breast cancer. Ringas et al. [141] provided a method of treat- References ing breast cancer by administering phospho- valproic acid, phosphor-ibuprofen, phosphor- [1] Ata A. International conference on primary health sulindac or their pharmaceutically acceptable care, declaration of Alma-Ata. WHO Chron 1978;32: e salt, together with bioavailability enhancers 428 30. [2] Larsen K. Distribution patterns and diversity centres like cimetidine and curcumin in the form of solid of Zingiberaceae in SE Asia. Biol Skr 2005;55:219e28. lipid nanoparticles, liposomes, or polymer [3] Sirirugsa P, Larsen K, Maknoi C. The genus Curcuma molecules. Another invention to watch is L. (Zingiberaceae): distribution and classification with encapsulation a physiologically effective dose reference to species diversity in Thailand. Gard Bull e of triterpene glycoside or triterpene complex (Singap) 2007;59:203 20. [4] Ramachandran C, Lollett IV, Escalon E, Quirin KW, nanoparticles in liposomes or exosomes that Melnick SJ. Anticancer potential and mechanism of exhibit preventive or therapeutic activity in action of mango ginger (Curcuma amada Roxb.) super- breast cancer [142]. critical CO2 extract in human glioblastoma cells. J Evid Based Complement Altern Med 2015;20: 109e19. 10. Conclusion [5] Li Y, Shi X, Zhang J, Zhang X, Martin RCG, et al. He- patic protection and anticancer activity of Curcuma:a potential chemo preventive strategy against hepato- Bioactive compounds derived from various cellular carcinoma. Int J Oncol 2014;44:505e13. plants are promising therapeutic agents in the [6] Sahu B, Kenwat R, Chandrakar S. Medicinal value of Curcuma cassia future due to safety concerns, being environmen- Roxb: an overview. UK J Pharma Bio- sci 2016;4:69e74. tally friendly, and the availability of plenty of [7] Shukla DP, Shah KP, Rawal RM, Jain NK. Anticancer resources. Despite the lack of pharmaceutical and cytotoxic potential of turmeric (Curcuma longa), properties of most natural products, there is neem (Azadirachta indica), tulasi (Ocimum sanctum) and ginger (Zingiber officinale) extracts on HeLa cell great interest in the application of new technol- e ogy for better formulation and delivery. line. Int J Life Sci Sci Res 2016;2:309 15. [8] Bavarsad K, Riahi MM, Saadat S, Barreto G, Atkin SL, Different types of nanocarrier systems offer Sahebkar A. Protective effects of curcumin against potential pockets to pack bioactive compounds ischemia-reperfusion injury in the liver. Pharmacol that lack pharmaceutical properties such as low Res 2018;141:53e62. solubility, permeability, and stabilitydthree [9] L Hadem K, Sharan RN, Kma L. Inhibitory potential main parameters determining therapeutic of methanolic extracts of Aristolochia tagala and Cur- fi cuma caesia on hepatocellular carcinoma induced by success. Speci c structures of these nanosystems diethylnitrosamine in BALB/c mice. J Carcinog can be modified on their surface to obtain certain 2014;13:7. properties, such as for selective distribution and [10] Hong GW, Hong SL, Lee GS, Yaacob H, Malek SN, control release, to make them suitable carriers in et al. Non-aqueous extracts of Curcuma mangga rhi- bioactive delivery. By considering the target of zomes induced cell death in human colorectal adeno- carcinoma cell line (HT29) via induction of apoptosis therapy, an appropriate nanosystem can be and cell cycle arrest at G0/G1 phase. Asian Pac J developed using unique material forming Trop Med 2016;9:8e18. nanosystem and manufacturing technology. [11] Sanatombi R, Sanatombi K. Nutritional value, phyto- As well, the initiation of clinical studies of these chemical composition, and biological activities of bioactive-loaded nanosystems provides edible Curcuma species: a review. Int J Food Prop 2017;20:S2668e87. optimism for better therapeutic outcomes, in 38 2. Role of nanocarriers and their surface modification in targeting delivery of bioactive compounds

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Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy Kobra Rostamizadeh1,2, Vladimir P. Torchilin2 1Zanjan Pharmaceutical Nanotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran; 2Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA, United States

1. Introduction pathways including defects in cell signaling pathways and induces changes in the surround- Cancer, after heart disease, ranks as the ing stroma and immunoresponses. Thus, target- second-most illness-related cause of death ing a specific pathway with a single worldwide with a growing mortality number chemotherapeutic agent is less successful in and incidence. Cancer is one of the most chal- eradication of cancerous cells [2]. To date, lenging diseases to treat because of its great improvement of the therapeutic effect of chemo- diversity and complexity. To date, chemo- therapy has been directed particularly toward therapy has been used most widely as an effi- combination approaches, particularly codelivery cient and successful method in clinical practice. nanoplatforms that attack cancerous cells via However, it has not provided adequate thera- multiple pathways. The general principle of peutic efficacy and lacks complete effectiveness. combination chemotherapy involves the simul- The major issue is that most chemotherapeutic taneous administration of two or more selected agents have poor solubility and often exhibit drugs with nonoverlapping toxicities and dis- other deficiencies including low bioavailability, similar mechanisms of action that inhibit multi- rapid blood/renal clearance, and nonspecific tar- drug resistance phenomena [3e5]. geting with significant undesirable side effects Combination therapy can overcome the toxicity on healthy tissues [1]. Nonspecific bio- problems of single-drug therapies by targeting distribution is another hurdle that limits the multiple-signaling pathways. Some combination localization of drugs at the tumor site and conse- therapy regimens have been established for quently generates demands for higher doses, cancer therapy and clinical practices that have which in turn leads to significant nonspecific demonstrated synergistic effects (a greater effect toxicity. Above all, the major problem is that than the sum of the separately applied individ- cancer progresses via a wide range of different ual drugs) and less systemic toxicity [6]. Besides

Nanopharmaceuticals https://doi.org/10.1016/B978-0-12-817778-5.00003-8 45 © 2020 Elsevier Inc. All rights reserved. 46 3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy that, combination regimens can offer improve- concentrations of each drug, and manipulation ment in the treatment results, although there of sequential drug release. are still unsuccessful outcomes and significant To date, considerable efforts have been made side effects observed due to low drug bioavail- to develop nanoparticulate codelivery systems ability and nonuniform biodistribution. In addi- for combination chemotherapy [11,12]. Various tion, to take advantage of possible synergy with nanocarriers have been investigated, including multiple drugs, specific molar ratios at the tumor lipid nanoparticles [13,14], liposomes [15,16], site are often needed, which are hard to obtain dendrimers [17,18], and polymeric nanoparticles by conventional administration methods due to [19,20]. Among these, increasing attention has differences in injection schedules, pharmacoki- been paid to polymeric nanoparticles due to their netic properties, metabolism, and nonuniform potential to carry both hydrophobic and hydro- biodistribution of drugs [7]. philic drugs, their controlled drug release One strategy to address this issue and deliver characteristics, low toxicity, high stability, and drugs to the tumor site at the desired molar ratio long circulation time, all of which ultimately is to merge nanotechnology with pharmacology enhance drug accumulation in the tumor target. and take advantage of nanoscale structures as Recently more attention has been given to poly- vehicles to carry multiple drugs, tune drug meric micelles as drug delivery systems with release, and modify the biodistribution and respect to the unique properties for codelivery pharmacokinetics of chemotherapeutic agents of drugs. [8]. This approach refers to codelivery systems. Codelivery systems can not only regulate the dosages and the ratio of different chemothera- 2. Micelles, principles and peutic agents at the tumor site but also improve characterization the efficiency of anticancer drugs through enhanced water solubility of hydrophobic mole- Polymeric micelles, first developed by cules, low toxicity, and high stability that Kataoka and colleagues in the late 1980s and prolongs circulation time in blood and enhances early 1990s, have spontaneously self- accumulation in tumor tissues. Moreover, assembling nanoscale core-shell architectures further enhancement of therapeutic efficacy can consisting of a hydrophobic core domain be achieved by taking advantage of stimuli- surrounded by a hydrophilic shell (Fig. 3.1). responsive drug delivery systems equipped Generally, the size of polymeric micelles is with various targeting moieties that can reduce 10e200 nm depending on the length of hydro- nonspecific delivery [9,10]. phobic and hydrophilic segments, temperature, An ideal carrier for codelivery should have and the concentration of polymer. Polymeric the potential for encapsulation of both hydro- micelles are composed mainly of amphiphilic phobic and hydrophilic drugs. Platforms for copolymers where polyethylene glycol (PEG) is codelivery systems should be designed so that the most common domain of the hydrophilic they carry traditional chemotherapeutics and shell. Other hydrophilic polymers like poly(N- agents such as siRNA in the same delivery vinyl-2-pyrrolidone) [21], poly(N-) pNIPAM vehicle. Although many efforts have been [22,23], and poly(acrylic acid) [24,25] are among made with nanotechnology-based codelivery the most widely used polymers as the hydrophil- systems, there are several challenges, which ic segment. The hydrophobic core-forming mainly include encapsulation of drugs with segment typically consists of poly(L-aspartate) various solubilities and physicochemical proper- [26,27], poly(propylene oxide) (PPO) [28,29], ties, targeting tumor tissues, regulation of the poly(esters) such as (PDLLA) [30,31], 2. Micelles, principles and characterization 47

FIGURE 3.1 Illustration of polymeric micelles. From Stern T, Kaner I, Laser Zer N, Shoval H, Dror D, Manevitch Z, et al. Rigidity of polymer micelles affects interactions with tumor cells. J Controlled Release. 2017;257:40e50 with permission. hydrophobic poly(ε-caprolactone) (PCL) [32,33], selectively penetrate cancerous tissues. Addi- copolymers of lactic acid and glycolic acids tionally, for reduction of the elimination by the (PLGA), and poly(ε-caprolactone) [34,35]. Hav- reticuloendothelial system (RES) and kidney, ing both hydrophilic and hydrophobic domains the optimum size for polymeric micelles is about in one platform is perhaps the most attractive 100e150 nm [36]. feature of polymeric micelles. This allows car- The hydrophilic shell of polymeric micelles rying a variety of therapeutic agents ranging also exhibits stealth properties, particularly in from those that are practically insoluble to highly cases when PEG serves as the hydrophilic part water-soluble drugs. Their remarkably low crit- of the shell of the micelles structure, avoids opso- ical micelle concentration (CMC) (on the order nization of micelles by the RES, and prolongs of 10 6-10 7 M) is another major feature of poly- circulation time of polymeric micelles in body meric micelles as drug delivery systems that en- fluids. These characteristics, as well as the biode- ables them to maintain structural integrity even gradability and functionality of polymeric at a very low concentration when once diluted micelles, have made them promising candidates in body fluids. Polymeric micelles can protect a for therapeutic delivery of desirable pharmaceu- drug payload against degradation by various en- tical formulations to tumors. zymes or metabolisms. The small size of poly- fi meric micelles also allows targeting of speci c 2.1 Preparation methods pathological cancerous sites through the passive targeting phenomena known as the enhanced There are three current methods for prepara- permeation and retention (EPR) effect. The EPR tion of polymeric micelles, including effect relies on greater capillary endothelial cell emulsification-solvent evaporation, solvent gaps within tumor tissues (in the range of displacement, and salting out. In the e 60 800 nm) compared to normal tissues, which emulsification-solvent evaporation method, an enables the smaller polymeric micelles to aqueous solution containing an appropriate 48 3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy surfactant like sodium cholate, or polyvinyl the solution, amphiphilic copolymers disperse alcohol is dispersed in a polymer solution pre- only within the solution and show no tendency pared using a volatile water immiscible solvent to form micelles. The CMC is one of the most like ethyl alcohol or chloroform to prepare an important properties as it indicates the kinetic emulsion using shear force methods, i.e., homog- stability of polymeric micelles [44]. Spectrofluo- enization, stirring, or probe sonication [38]. rometry by pyrene and light scattering are Eventually, the solvent is evaporated, and poly- among the most popular techniques used to meric micelles are obtained as dispersed nano- measure the CMC. particles in water, which can be collected by Static or dynamic light scattering (DLS)/ centrifugation. Generally, a single emulsion photon correlation spectroscopy is an intensively (water in oil, W/O) or double emulsion (W/O/ used method for evaluation of the size (hydrody- W) is used for trapping hydrophobic and hydro- namic diameter) and polydispersity index (PdI) philic drugs, respectively. With solvent evapora- of micellar structures [45]. In fact, DLS works tion, or a solution-casting technique, a thin based on the intensity distribution of relaxation uniform film containing copolymer and drug is times and the time dependence of the light inten- left on the surface of the substrate after the evap- sity fluctuations to obtain the diffusion coeffi- oration of solvent [39]. Next, the hydration of the cient of nanoparticles, which is then used to film by aqueous solution forms polymeric calculate hydrodynamic diameter (RH) of nano- micelles. Solvent diffusion, also referred to as particles using the StokeseEinstein equation. the nanoprecipitation or solvent displacement The PdI indicates the polydispersity of poly- method, is used extensively to encapsulate meric micelles in terms of particle size on the hydrophobic drugs [40]. In this method, a water scale of 0e1. The PdI values of 0 and 1 indicate miscible solvent like acetone is used to dissolve a monodisperse solution and the highest solu- polymer and drug. Due to the miscibility of tion polydispersity, respectively. Various types solvents, there is no need to use surfactant or of electron microscopy, including transmission high shear force to obtain nanoscale materials. electron microscopy, scanning electron micro- Salting out is also used to load hydrophobic scopy, and atomic force microscopy, are widely drugs [41]. In this method, a W/O emulsion is used for determination of micellar morphology first prepared using water immiscible solvents, and size. The stability of polymeric micelles, followed by addition of a salting out agent either thermodynamic or kinetic, is another crit- such as sodium chloride, magnesium acetate, ical parameter [46]. Thermodynamic stability or magnesium chloride to make the solvent indicates how the system acts as micelles when insoluble by saturation of the solution and in equilibrium. Thermodynamic stability of formation of polymeric micelles. polymeric micelles depends mainly on the surface charge, the zeta potential, and the steric hindrance provided by surfactants. Kinetic 2.2 Characterization techniques stability indicates the rate at which the micelles The suitability of polymeric micelles as drug disassemble when the copolymer concentration delivery systems is evaluated with respect to is lower than the CMC. Several factors affect properties including particle size, surface charge, the in vitro and in vivo stability of polymeric and stability [42,43]. The CMC is the concentra- micelles, including the strength of hydrophobic tion of copolymer in a solution above which interactions between hydrophobic segments of copolymers self-assemble to form micelles. polymeric micelles and the size of the hydrophil- Below the CMC, due to low surface tension of ic part of the copolymer. DLS is the most popular 3. Polymeric micelles for codelivery of chemotherapeutics 49 method to measure zeta potential. DLS uses a the factors that influence drug-loading effi- phase analysis approach to determine the ciency. However, hydrophobic drugs represent surface charge of the nanoparticles. the majority of those loaded into micelles. There are some reports on encapsulation of hydrophilic drugs into micelles, particularly by the double- 2.3 Drug-loading methods emulsion method [50].

One of the most desirable features of poly- meric micelles is their relatively high drug- 3. Polymeric micelles for codelivery of loading capacity. The loading efficiency for a chemotherapeutics micellar carrier is defined as the amount of incor- porated drug per micelle. Generally, different Increasing attention is now being paid to the drug-loading strategies for polymeric micelles use of polymeric micelles for codelivery of ther- fi can be classi ed in three main categories, apeutic agents in cancer therapy [51,52]. Mainly, including: these codelivery systems are used to overcome L physical entrapment multidrug resistance, which is perhaps the L chemical conjugation most challenging issue in chemotherapy. They L polyionic complexation methods. also show a high potential for codelivery of chemotherapeutic agents for synergistic therapy The physical entrapment method, the and mitigation of side effects. They have also simplest, has been used widely to incorporate been employed as stimuli-responsive codelivery different drugs, particularly hydrophobic drugs, vehicles for codelivery of therapeutics using fi using emulsi cation or dialysis [47]. In this different therapeutic approaches. In the method, hydrophobic interactions like van der remainder of this chapter, different aspects of Waals forces and hydrogen bonding determine polymeric micelles as codelivery platforms drug loading. In the chemical conjugation with emphasis on applications are explored. approach, a strong covalent bond is formed between the polymer backbone and the drug [48]. In this case, the amount of incorporated 3.1 Codelivery of chemotherapeutics to drug is limited to the number of functional overcome multidrug resistance groups in the polymer backbone, and the rate of drug release is dependent on the rate of the Multidrug resistance (MDR) remains a major bond cleavage. Finally, the ionic complexation challenge for successful treatment of cancer in method is preferred for loading species able to the clinic. MDR is defined as the resistance of form strong electrostatic interactions with tumor cells toward a broad range of chemother- charged polymers [49]. The compatibility apeutic agents by inactivation of the drug or by between the drug and the copolymer play a pumping it from tumor cells. Different mecha- determinant role in loading efficiency. In addi- nisms have been proposed for MDR, including tion to the size and type of the core and corona increased drug efflux mediated by the overex- forming copolymer, the stability of polymeric pressed MDR-related transporters, the increased micelles in aqueous medium, molecular weight capability of DNA repair, dysfunctional of the copolymer, the type and concentration of apoptosis, and activation of prosurvival path- the drug and copolymer, the method of drug- ways (Fig. 3.2). However, MDR is a very loading, and the ratio of organic to aqueous solu- complex phenomenon, and no single mechanism tion, as well as their order of addition are among of resistance is likely. It usually happens as the 50 3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy

FIGURE 3.2 Cellular changes occurring in MDR cancer cells in comparison to normal cells. MDR cancer cells show: (1) Increased extracellular acidity, (2) Change in cell membrane lipid composition resulting in thicker, less fluid, and less perme- able membranes, (3) Overexpression of drug efflux pumps and cell surface receptors like EGFR, (4) Increased intracellular alka- linity, (5) Increase in cytoplasmic vesicle number and volume, (6) Increased intravesicular acidity, (7) Overexpression of ion/ drug transporters on intracellular vesicles, (8) Modulated levels of detoxifying and drug activating enzymes, (9) Modulated levels of importins, (10) Overexpression of exportins, and (11) Increased mutations and altered gene expression levels. From Singh MS, Tammam SN, Shetab Boushehri MA, Lamprecht A. MDR in cancer: Addressing the underlying cellular alterations with the use of nanocarriers. Pharmacol Res. 2017;126:2e30 with permission. result of the action of a combination of several the mass ratio of chemotherapeutic agent and mechanisms. MDR inhibitor is a determinant parameter that Various strategies have been adapted to over- has been used to achieve the highest therapeutic come MDR, including the use of monoclonal efficiency [51]. Polymeric micelles have been antibodies against P-glycoprotein (P-gp), used to deliver different bioactive agents simul- ATP-binding cassette transporter inhibitors, taneously, including chemotherapy agents and and inactivation of MDR-associated gene expres- inhibitors, at an optimized ratio to the site of sion using small interfering (si) RNAs. Inhibitors interest. The MTT assay has been widely used have only limited indications for clinical usage to obtain the optimal mass of drugs for the best due mainly to their association with chemother- synergistic composition. For instance, using the apeutics and high toxicities. On the other hand, MTT assay of micelles composed of 3. Polymeric micelles for codelivery of chemotherapeutics 51 poly(ethylene oxide)-blockepoly (propylene substances acting at small doses without cyto- oxide)-blockepoly(ε-caprolactone) (PEOePPOe toxicity toward cancerous cells, which promote PCL), and the payload with different mass ratios synergism of the effects of chemotherapeutic of docetaxel (DTX) and chloroquine (CQ), an agents when both anticancer drugs and chemo- autophagy inhibitor, the highest synergetic anti- sensitizer are delivered to the same cellular loca- cancer effect was obtained for the DTX/ tion. Lower toxicity and reduced side effects are CQ ¼ 0.8/0.2 [28]. It has been shown that the the most important differences of these formula- synergetic therapeutic efficacy of codelivery sys- tions compared to combination chemotherapy tems containing chemotherapeutic agents and with two anticancer drugs. P-gp inhibitors [58] inhibitors in a single nanoformulation can be and autophagy inhibitors [59] are two of the related to the increase in the cellular uptake most widely used chemosensitizers. and prolonged drug retention in the cytoplasm. Overexpression of P-gp is likely the main Codelivery of rapamycin with piperine as mechanism of MDR, which is also referred to chemosensitizer through Poly(D,L-lactide-co-gly- either as MDR1 or ATP-binding cassette subfam- colide) (PLGA) micelles increased the uptake ily B member 1 (ABCB1). P-gp is an ATP- and bioavailability of the rapamycin by about dependent efflux pump that pumps foreign 4.8-fold [54]. Indeed, the use of nanoformula- substances out of cells. Most of the widely used tions for codelivery of different therapeutics is chemotherapeutic agents such as the taxanes a promising method to obtain synergistic effects and anthracyclines are classified as substrates and eliminate most of the shortcomings of of P-gp transporters. Inhibition of the function chemotherapy due to MDR. Two strategies that of P-gp is one strategy used to reverse MDR have been used to reverse MDR with a micellar and sensitize tumor cells to conventional chemo- codelivery system, including codelivery of therapeutics. Currently, despite considerable chemotherapeutics with a chemosensitizer and efforts that have been devoted to the develop- codelivery of chemotherapeutics with downre- ment of P-gp inhibitors, there is no P-gp inhibitor gulating gene agents, will be discussed in the approved for clinic use. This is due mainly to the following sections. nonspecific action of P-gp inhibitors on other molecular targets and their nonselectivity for 3.1.1 Codelivery of chemotherapeutics and tumorigenic P-gp, which can result in remark- chemosensitizers able toxicity. Recently, nanocarriers, particularly Generally, multiple mechanisms are involved polymeric micelles, have been used to deliver in any single MDR phenotype. To overcome P-gp inhibitors into tumors cells with high MDR, based on the mechanisms involved in its efficiency (Table 3.1) [60]. The uptake of nanocar- development, a combination of two or more riers by MDR cells occurs through nonspecific drugs are used to target multiple oncogenetic endocytosis, leading to high intracellular accu- pathways. For example, combinational therapy mulation of drug. In addition, the nanocarriers using P-gp inhibitors, tyrosine kinase inhibitors, can be modified with a variety of targeting or proapoptotic agents enhanced the cytotoxicity moieties such as ligands or antibodies to of formulations [55,56]. To improve therapeutic improve their specific uptake by tumor cells. efficiency of drugs, additional nanoformulations The manipulation of surface charge of micelles have emerged that target more than one MDR can also be used to facilitate nanocarrier internal- mechanism. The codelivery of small molecule ization into cancerous cells. Micelles consisting chemotherapeutic drugs and chemotherapy sen- of poly(ethylene glycol)-block-poly(L-lysine) sitizers to reverse MDR has attracted growing (PEG-b-PLL) block copolymers with negative attention [57]. Chemosensitizers are chemical charge in plasma (pH 7.4) have a prolonged 52 3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy

TABLE 3.1 Polymeric codelivery micelles used to overcome MDR.

Polymer used Drug 1 Drug 2 Study type Refs

PHis-PLA-PEG-PLA-PHis/ Curcumin Pluronic L61 MCF-7/ADR xenograft mice model [9] Pluronic F127 unimers PEG-PLA Cyclosporin Gefitinib NSCLC xenograft- BALB/c nude mouse model [70] A BDP Docetaxel Silibinin 4T1 xenograft mice model [71] PEOePPOePCL/TPGS Docetaxel Chloroquine MCF-7 and MCF -7/ADR cell lines [28] Drug conjugated PEG Dox Curcumin HepG2 xenograft BALB/c nude mice model [72] (SMA)- ADH Dox Disulfiram MCF-7/ADR xenograft mice model [62]

PLGA Dox Chloroquine A549 cells and A549/Taxol cells [73] PEO-b-PCL Dox MDR-1 siRNA Athymic mice bearing MDA-MB-435/LCC6MDR1- [74] resistant tumors

NSCePLLePA Dox P-gp siRNA HepG2/ADM xenograft Athymic nude mice model [75] PLGA Dox MDR1 targeting MCF-7/ADR cell line [76] siRNA PDP-PDHA Dox shSur MCF-7/ADR xenograft mice model [77] PFeDP Dox PTX MCF-7/ADR xenograft mouse model [78] PLGA MDR1 BCL2 siRNA Resistant SKOV3-TR and A2780-CP20 human [79] ovarian cancer cells PLGA PTX Tetrandrine via A2780/PTX cell line [13] irgd peptide PEG2k-Fmoc-NLG PTX NLG919, an 4T1.2 xenograft BALB/c mice model [80] indoleamine 2,3-dioxygenase PEOz-PLA PTX Honokiol MDA-MB-231-luc-GFP xenograft nude mice model [81]

PEG-b-PLL PTX Disulfram MCF-7 cell line [61] PLGA Rapamycin Piperine Healthy SD rats [54] PEG-PE PTX Curcumin DU145Xenograft BALB/C nude mice model [82]

PEG-PLA (poly(ethylene glycol)-b-poly(L-lactide)); PEG (poly(ethylene glycol)), poly(D,L-lactide-co-glycolide) (PLGA) and poly(L-histidine) (PHis-PLA-PEG-PLA-PHis); PLGA (poly(D,L-lactide-co-glycolide)); BDP (polyethylene glycol-block-poly[(1,4-butanediol)-diacrylate- b-N,N-diisopropylethylenediamine]); PEOePPOePCL/TPGS(poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ε-caprolactone) and D-a-tocopheryl poly(ethylene glycol)); PEOz-PLA(poly(2-ethyl-2-oxazoline)-poly(D,L-lactide)); PEG-b-PLL (poly(ethylene glycol)-block- poly(L-lysine) block copolymer); (SMA)-ADH (poly(styrene-co-maleic anhydride) (SMA) derivative with adipic dihydrazide); Poly(lactic- co-glycolic acid) and alpha-tocopherol polyethylene glycol 1000 succinate; PEO-b-PCL (poly(ethylene oxide)-block-poly(ε-caprolactone) block copolymers); NSCePLLePA (N-succinyl chitosanepoly-L-lysineepalmitic acid); PDP-PDHA (poly[(1,4-butanediol) diacrylateb-5- polyethylenimine]); Polyethylene GlycolePhosphatidylethanolamine PEG-PE; Pluronic P105 and Pluronic F127 PFeDP; poly(ethylene oxide)- blockepoly(propylene oxide)-blockepoly(ε-caprolactone) PEOePPOePCLPHis-PLA-PEG-PLA-Phis) poly (L-histidine)-poly (D,L-lactide)- polyethyleneglycol-poly (D,L-lactide)-poly (L-histidine); PEG-PLA (poly(ethylene glycol)-b-poly(L-lactide)); BDP (polyethylene glycol-block-poly [(1,4-butanediol)-diacrylate-b-N,N-diisopropylethylenediamine]); PEOePPOePCL/TPGS(poly(ethylene oxide)-block-poly(propylene oxide)- block-poly(ε-caprolactone) and D-a-tocopheryl poly(ethylene glycol)); PEG (poly(ethylene glycol)); (SMA)- ADH (poly(styrene-co-maleic anhydride) (SMA) derivative with adipic dihydrazide); PLGA (poly(D,L-lactide-co-glycolide)); PEO-b-PCL (poly(ethylene oxide)-block-poly(ε- caprolactone) block copolymers); NSCePLLePA (N-succinyl chitosanepoly-L-lysineepalmitic acid); PDP-PDHA (poly[(1,4-butanediol) diacrylateb-5- polyethylenimine]); PFeDP Pluronic P105 and Pluronic F127; PEOz-PLA(poly(2-ethyl-2-oxazoline)-poly(D,L-lactide)); PEG-b-PLL (poly(ethylene glycol)-block-poly(L-lysine) block copolymer); PEG-PE (polyethylene glycolephosphatidylethanolamine). 3. Polymeric micelles for codelivery of chemotherapeutics 53 circulation time, while its more positive charge 3.1.2 Codelivery of chemotherapeutics and when exposed to the weak acid environment of downregulating gene agents e tumor tissue (pH 6.5 6.8) facilitates their uptake Inhibition of the expression of the MDR-1 gene by cancerous cells. Huo et al. [61] have shown (rather than its function) is another strategy to that grafting paclitaxel (PTX) to the side chain prevent/reduce drug efflux. Use of siRNA is a fi of L-lysine and encapsulation of disul ram, a highly promising technology for downregulation fi P-gp inhibitor, in micelles signi cantly increases of gene expression, particularly for cancer ther- the cellular internalization and cytotoxicity of apy [66]. Recently, several siRNA therapeutic for- PTX against MCF-7/ADR cells. Encapsulation mulations have been studied in clinical trials and fi of disul ram into doxorubicin (Dox)-conjugated successfully exhibited gene silencing of various poly(styrene-co-maleic anhydride) (SMA) deriv- oncogenes [67]. SiRNA has also been designed ative with adipic dihydrazide (ADH) micelles to silence MDR-1 gene expression in hopes of also exhibited very high-antitumor activity downregulating the expression of P-gp genes with almost complete inhibition of tumor [68]. However, because of its instability and growth [62]. Codelivery of verapamil and toxicity, the success of this approach depends vincristine in poly(D,L-lactide-co-glycolide acid) on the development of safe and efficacious deliv- (PLGA) micelles also demonstrated higher ery systems that deliver siRNA in a safe and drug accumulation in MCF-7/ADR cells in selective manner into cancerous cells. Micelles comparison to free vincristine/verapamil combi- composed of novel poly(ethylene oxide)- nations [63]. Tariquidar is also a P-gp inhibitor modified poly(beta-amino ester) (PEO-PbAE) fi that exhibits signi cant tumor growth inhibition. and PEO-modified poly(ε-caprolactone) (PEO- Biotin-functionalized nanoparticles coencapsu- PCL) nanoparticles showed high potential for de- lated with both PTX and tariquidar showed livery of MDR-1-silencing siRNA and anticancer high potential for inhibition of tumor growth in drugs such as PTX [69]. Generally, the a mouse MDR model compared to free PTX at high positive charge of micelles represents a the same dose [64]. Drug resistance was partially favorable property that enables them to form abolished using polymeric micelles composed of complexes with negatively charged siRNA by PEG-PLA coencapsulated with a chemosensi- electrostatic interaction. Cationic PLGA micelles tizer, cyclosporin A (CsA), and the anticancer (PLGA decorated with didodecyl dimethylam- fi drug, ge tinib [65]. Cyclosporin A is a P-gp monium bromide (a cationic surfactant), effi- inhibitor that inactivates the STAT3/Bcl-2 ciently coencapsulated siRNA and Dox at a signaling pathway. Coadministration of CsA specific high ratio of micelles-to-siRNA. Survivin fi and ge tinib encapsulated in polymeric micelles is an inhibitor of apoptosis family member over- fi demonstrated the high potency of ge tinib in expressed in drug resistance that inhibits cell fi nonge tinib-resistant cells as well as in primarily apoptosis. Survivin-targeting shRNA (shSur) colo fi and secondarily ge tinib-resistant cells, appar- aded with Dox into amphiphilic poly (b-amino ently due to increased apoptosis and impaired ester), poly[(1,4-butanediol)-diacrylate-b-5-poly proliferation shown in nonsmall cell lung ethylenimine]-block-poly[(1,4-butanediol)-diacry fi cancers. Together, these results con rm that late-b-5-hydroxy amylamine] (PDP-PDHA) codelivery of P-gp inhibitors and anticancer micelles increased Dox accumulation, and down- drugs is a promising approach to overcome regulated 57.7% of the survivin expression, MDR. 54 3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy resulting in 80.8% cell apoptosis and cell cycle trials. Although combinational therapy provides changes in MCF-7/ADR cells [69].Italso the opportunity for an additional and synergetic increased the accumulation of Dox and shSur in therapeutic effect with low side effects, there are the tumor tissue by 10.4- and 20.2-fold, respec- some issues associated with combinational ther- tively, resulting in a remarkable inhibition of tu- apy that must be considered, including cross- mor growth of 95.9%. resistance and toxicity of drug combinations. Although siRNA technology has become a To avoid these problems, the choice of synergis- promising approach to chemotherapy, these tic drug pairs according to their mechanism of reports confirm that the methods developed action, and administration of drugs following are based on only partial reverse MDR by optimization of dosing schedules, are of great silencing the expression of the genes encoding importance. Until recently, clinical experiences P-gp, i.e., MDR-1 and Survivin. On the other were used to test for synergistic drug combina- hand, siRNA makes a significant difference tions, which are time-, labor-, and cost- between the IC50 values of chemotherapeutic intensive. Lately, high-throughput screening agents in MDR cancer cells compared to sensi- (HTS) has become a relatively quick and tive cell lines. The in vivo studies have clearly low-cost technique used to choose synergistic shown their capacity to slow the rate of tumor drug pairs with less harm to patients [83]. growth but not to ultimately decrease the tumor Recently, using machine learning techniques volume. for the description of the large synergistic space [84], accurate predictive models have been generated by leveraging the available HTS 3.2 Codelivery of chemotherapeutics to synergy data. Table 3.2 presents different achieve synergistic effects by methods polymeric codelivery systems with synergic other than effects on MDR properties in cancer treatment. Dox and PTX are one of the most-studied Recently, combination therapy has been paired drugs for codelivery systems. Their shown to be a promising regimen for cancer different mechanisms of action on cancer cells treatment of those with historically poor prog- can result in high and synergistic therapeutic ef- nosis of cancers treated by monotherapy that fects. Dox binds to DNA and prevents nucleic are diverse, complex, and heterogeneous in acid synthesis, while PTX’s usefulness is based nature. The coadministration of several types of on promotion of microtubule assembly from therapy has resulted in remarkable synergetic ef- tubulin dimers and stabilization of microtubules fects (namely “1 þ 1 > 2”), in which the effect is through inhibition of depolymerization. Duong more potent than any individual therapy or their et al. [85] studied codelivery of Dox and PTX in theoretical combination. Given the different micellar formulations with two different mechanisms involved in a cancer’s progression, approaches. They compared the effect of codeliv- it is reasonable to attack cancerous cells with ery of two single-drug-loaded micelles with combinations of drugs with different mechanism dual-drug-loaded micelles. The results of actions in the hope of a greater effectiveness. confirmed a synergistic effect for both methods. Administration of drug combinations instead of With codelivery of single-drug-loaded micelles, a single drug also reduces host toxicity and it would be easier to control different drug com- adverse side effects due to the lower dosage of bination ratios for specific treatments. However, drugs typically used in the combinational ther- there is less control of a designated ratio at the apy regimens. There have been several combina- site of the tumor. Using dual-drug-loaded torial therapeutic regimens tested in clinical micelles provides the opportunity to maintain 3. Polymeric micelles for codelivery of chemotherapeutics 55

TABLE 3.2 Codelivery systems by polymeric micelles to achieve synergetic effect in cancer therapy.

Micelle composition Drug 1 Drug 2 Study type Refs

PEG-FTS Curcumin FTS 4T1 xenograft syngeneic mouse model [89] DTX-PEG-GEM Docetaxel GEM DMBA-induced breast cancer model [86] PEGePLLePLLeu Docetaxel siRNA MCF-7 xenograft nude mice (87)

PLFCL Dox Dasatinib 4T1.2 xenograft female BALB/c mice model [88] PLGA-PEG, PLGA-PEG-FOL and Dox PTX Human oral cavity carcinoma KB cell line [85] PLGAPEG-TAT

HA-VES Dox Curcumin 4T1 xenograft mice model [90] PCLeSSeCTS-GA Dox Curcumin HepG2 and HUVEC cells [91] PEG-PLA Sildenafil (Viagra) Crizotinib MCF-7 cells [92] mPEG-b-P(Glu)-b-P(Phe) PTX Cisplatin A549 xenograft Balb/C nude mice model [93]

FTS (trans-farnesylthiosalicylic acid); GEM (Gemcitabine); PEGePLLePLLeu (poly(ethylene glycol)-b-poly(L-lysine)-bpoly(L-leucine)); PLFCL a ε e e fi (PEG5000-lysyl-( -Fmoc- -Cbz-lysine)2); HA-VES (hyaluronic acid-vitamin E succinate); PCL SS CTS-GA (glycyrrhetinic acid-modi ed chitosan-cystamine-poly(ε-caprolactone)) mPEG-b-P(Glu)-b-P(Phe) (methoxy poly(ethylene glycol)-b-poly(L-glutamic acid)-b-poly(L- phenylalanine)). the initial drug ratio in vivo. However, further estimation, and hemolytic toxicity. Docetaxel optimization to change the ratio of the two drugs has also been used with siRNA for combined for specific treatment would be needed. cancer therapy [87]. A micelle system based on Considering their different mechanism of the triblock copolymers poly(ethylene glycol)- action and nonoverlapping toxicity, docetaxel b-poly-L-lysine-b-poly-L-leucine (PEG-PLL- and gemcitabine represent another candidate PLLeu), with PLLeu as the hydrophobic core, drug pair for codelivery systems. However, PLL as the cationic shell, and PEG as the hydro- these drugs have difference in hydrophilicity/ philic corona, was used to encapsulate the nega- hydrophobicity properties, and encounter a tively charged siRNA via electrostatic serious problem when loaded into a single nano- interactions and the hydrophobic docetaxel in formulation. Docetaxel is highly hydrophobic. the core segment of the micelles via hydrophobic Gemcitabine is a highly hydrophilic drug and associations. The codelivery of these micelles is quickly metabolized into an inactive metabo- revealed a significant effect on the tumor growth 0 0 0 lite, 2 -deoxy-2 ,2 -difluorouridine. To address in a xenograft model, resulting in an animal this issue, they were conjugated to PEG to form survival of 40% at 27 days for the docetaxel an amphiphilic molecule with the capability for nanoparticles plus siRNA/nanoparticles self-assembly [86]. A 4.8-fold higher AUC compared to 100% for the DTX-siRNA nanopar- (0-N) for gemcitabine compared to Gemzar ticles group. was the most important feature of the resultant A superior antitumor effect of Dox and dasa- codelivery micelle system. Lower hepato- and tinib (Das) in tumor suppression has also been nephrotoxicity was another advantage of using reported using the PEGylated peptidic nanocar- these micelles. This was confirmed by both histo- rier, PEG5000-lysyl-(a-Fmoc-ε-Cbz-lysine) pathological sections, biochemical marker level (PLFCL) micellar system against a variety of 56 3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy cancer cells including colon, breast, and prostate disease progression but survival. Dox, in spite [88]. Administration of Dox and Das at a dose of of its well-established use for cancer treatment, 5 mg/kg produced a 95% inhibition of tumor has serious life-threatening side effects, particu- growth in a murine breast cancer model. larly dose-related myocardial toxicity with Regardless of drug type, the drug ratio is also increasing total lifetime doses at and above important for the synergetic effect. Duong et al. 400 mg/m2. Amongst the different mechanisms [85] used Calcusyn software to find the suitable suggested for doxorubicin hydrochloride- dose combination of Dox and PTX as free drug. induced cardiomyopathy, it is clear that free- To confirm the combined synergistic effect quan- oxygen radicals formed during the redox cycling titatively, the CI values of all Dox/PTX combina- of the quinoloneesemiquinolone ring of the tions (1/0.1, 1/0.2, 1/1, 0.2/1, and 0.1/1) were doxorubicin hydrochloride is a determining simulated as a function of the cell viability parameter. Thus, codelivery of Dox with an from 5% to 98%. They found that a decrease in agent capable of acting against this redox cycling PTX in the Dox-based combinations reduced might help to mitigate doxorubicin the CI values. For the PTX-based combinations, hydrochloride-induced cardiotoxicity. Some nat- the CI values increased with the decrease in the ural products with remarkable free radical scav- added Dox. A higher antagonistic interaction enging properties and potent chemosensitizing (CI >1) and higher synergistic interaction properties mitigate doxorubicin hydrochloridee (CI <1) of drugs was observed in the PTX- induced cardiotoxicity while maintaining and/ based combinations (Dox/PTX 0.2/1 and 0.1/ or improving its potency against cancer cells. 1) and in the Dox-based combinations (Dox/ For example, codelivery of resveratrol and cur- PTX 1/0.2). The combination of the free drugs cumin at a molar ratio of 5:1 in F127 micelles Dox/PTX at molar ratio of 1/0.2 showed a syn- (mRC) coadministered with doxorubicin hydro- ergistic therapeutic effect compared to the treat- chloride showed cardioprotective effects. This ment of a free single drug, Dox or PTX, which was attributed to scavenging of free radicals was used to prepare a micellar codelivery system with a synergistic effect on SKOV-3 cells and of two single drugs (Dox/PTX). antagonistic effect in H9C2 cells, most probably through chemosensitization [95].

3.3 Codelivery of chemotherapeutics to mitigate side effects 4. Stimuli-responsive codelivery of polymeric micelles Current chemotherapy has significantly increased the survival rate of patients with can- Conventional chemotherapy treatments suf- cer. However, some chemotherapeutic agents fer from the lack of specificity, which exposes have very harsh side effects during the treatment healthy cells to toxic chemotherapeutic agents and even after several years. Side effects may as much as to cancerous cells. Moreover, the cause a disruption in the treatment schedule fact that low concentration of drugs at the tumor and/or a reduction in dosing or even lead to a site significantly decreases the therapeutic effi- treatment’s discontinuation. Simultaneous deliv- ciency of treatment and leads to the highest ery of some protective agents as well as anti- frequency and dosing of drugs and harsher cancer drugs may mitigate the side effects side effects, suggests an urgent need to generate associated with chemotherapy [94]. Moreover, efficient, targeted drug delivery systems for codelivery of chemotherapeutic drugs with efficient chemotherapeutics. The most important some protective agents would impact not only feature of the newly generated drug delivery 4. Stimuli-responsive codelivery of polymeric micelles 57 systems, referred to as targeted drug delivery There are many reports on codelivery systems systems, is the ability to selectively deliver drugs of targeted polymeric micelles that release the to the tumor site. Targeted drug delivery payload drugs in response to a stimulus in the systems are designed to selectively deliver ther- tumor tissue, such as a reductive environment, apeutic agents to a site of action, accumulate at low pH, and high temperature (Table 3.3). the tumor, and subsequently reduce exposure Considering the long circulating time of micelles, of healthy cells. Since targeted drug delivery which usually comes with small size and stealth systems release the payload drug upon exposure properties, it is desirable to avoid drug leakage to specific stimuli, they are also termed stimuli- within the circulation and have triggered drug responsive, intelligent, or smart drug delivery release as much as possible. Different parameters systems. Targeted drug delivery systems are considered to control the rate of drug release also classified as either “passive targeting” or from micellar structures include micelle stability “active targeting” systems. Passive targeting sys- (either thermodynamic or kinetic), the rate of tems take advantage of the differences between drug diffusion, partition coefficient, and in the the environment of tumor tissue and normal case of biodegradable polymers, the rate of tissue, including the pathophysiological charac- biodegradation. Table 3.3 summarizes some teristics of tumor vessels, pH, temperature, and polymeric codelivery micelles used as targeted external stimuli such as a magnetic field or heat drug delivery systems. as a trigger for drug release. Passive targeting has been somewhat successful in improving the 4.1 pH-sensitive codelivery systems efficacy of chemotherapy, but it suffers from several limitations because of the variation in Among the applied stimuli, acidic pH is the the microenvironment of tumor tissue at most commonly used internal trigger for the different stages of tumor progression and within selective release of anticancer drugs because of different types of tumors. To overcome these the relatively acidic pH of cancer tissues in shortcomings of passive targeted drug delivery both primary and metastasized tumors systems, an active targeting approach has been (6.5e7.2), which is lower than the extracellular used to improve the performance of targeted pH of normal tissue and blood (pH 7.4). More- drug delivery systems. In this approach, various over, micelles experience an even more acidic affinity ligands like aptamers, antibodies, pep- condition following their uptake via endocytosis tides, and small molecules are used to direct and formation of endosomes and lysosomes drug delivery systems toward tumors cells via with pH values of 5.0e6.0 and 4.0e5.0, respec- specific receptors overexpressed on the cell tively. These differences of pH between healthy surface by tumor cells. After binding to the cell and cancerous cells make the pH value a suitable surface, they are often internalized by receptor- stimulus for targeted drug release. For instance, mediated endocytosis followed by the formation Dox has been conjugated via acid-sensitive link- of an endosome from which they release the age to a Pluronic F127- chitosan (F127-CS) poly- drug payload due to a weak acidic environment mer to form a self-assembling and pH-sensitive or enzymes. A variety of codelivery systems, polymeric micelle system for codelivery of Dox including polymerosomes, liposomes, polymeric and PTX [97]. The hydrophobic nature of PTX micelles, dendrimers, and lipid-based nanostruc- allows efficient entrapment via hydrophobic tures, have been developed that enhance drug interactions into the core segment of the micelles. accumulation at the tumor site using passive or The pH sensitivity of micelles was confirmed by active targeting strategies [96]. increase in the release rate of Dox and PTX 58 3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy

TABLE 3.3 Polymeric codelivery micelles used as stimuli-responsive drug delivery systems.

Micelle composition Drug 1 Drug 2 Animal model or cell line Stimuli Refs

PEG-PLGAeSSeDTX Docetaxel Verapamil Healthy male Wistar rats Redox-sensitive [99] SHRss Dox microRNA-34a DU145 Xenograft BALB/C nude Redox-sensitive (82) mice model DTDAP Dox PTX B16 xenograft mice model Redox-sensitive [107] PEG-PAsp(AED)- Dox siRNA HepG2 and HUVEC cells Redox-sensitive [91] PDPA mPEG-PAsp-NI Dox Chlorin e6 (Ce6) 4T1 xenograft mice model Hypoxia-and singlet [108] oxygen responsive ACeCSePpIX Dox Apatinib MCF-7/ADR xenograft mice Light-sensitive [109] model F127-CS Dox PTX Healthy male Wistar rats pH-sensitive [97] mPEGeSSeDOX Dox PTX B16 xenograft melanoma mice Redox-sensitive [110] model pHPMA Dox Axitinib A549 xenograft mice model Dual-pH responsive [111] PAP MDR-1 Survivin-targeting MCF-7/ADR xenograft Balb/c Redox-sensitive [112] RNA nude mice model PEG-pp-PEI-PE PTX siRNA A549 xenograft nude mice model Metalloproteinase 2 [113] (MMP2)-sensitive mPEGeSSeC18 PTX Dasatinib MCF-7 and MCF-7/ADR cell line Redox-sensitive (98) LDLeNSCeSSeUA PTX Lipoprotein and MCF-7 xenograft mice model Dual redox and pH [114] siRNA sensitive PEOz-PLA PTX Honokiol MDA-MB-231-luc-GFP xenograft pH-sensitive [81] nude mice model

(PEG-PLGAeSSeDTX) poly (ethylene glycol)-poly (D,L-lactide-co-glycolide) -docetaxel; (SHRss) poly(L-arginine)-poly(L-histidine)-stearoyl; (DTDAP) D-A-tocopherol polyethylene glycol 1000 succinate; (HA-VES) hyaluronic acid-vitamin E succinate; (PEG-PAsp(AED)-PDPA) poly(2- 0 (diisopropyl amino)ethyl methacrylate) and poly(N-(2,2 -dithiobis(ethylamine)) aspartamide) and poly(ethylene glycol); (mPEG-PAsp-NI) nitroimidazole-bearing methoxy poly(ethylene glycol)-co-poly(aspartic acid); (ACeCSePpIX) acetylated-chondroitin sulfate-protoporphyrin; (F127-CS) pluronic f127-chitosan polymer; (HPMA) N-(2-hydroxypropyl) methacrylamide; (PAP) poly[bis(2-hydroxylethyl)-disulfide- diacrylate-b-tetraethylenepentamine]; (PEG-pp-PEI-PE) polyethylene glycol -peptide -polyethylenimine - 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine; (PEG-PE) polyethylene glycol-phosphatidyl ethanolamine; (LDLeNSCeSSeUA) N-succinyl chitosanecystamineeurocanic acid; (PEOZ-PLA) poly(2-ethyl-2-oxazoline)-poly(D,L-lactide). during in vitro drug release studies. The pH 4.2 Redox-sensitive codelivery systems decrease facilitated the cleavage of an acid- liable cis-aconityl bond. In an in vivo pharmaco- The higher reducibility potential of tumor kinetic study of PTX-loaded F127-CS-Dox mi- cells compared to healthy cells has become a celles in rats, the area under the plasma popular stimulus for targeted intracellular drug delivery systems. The reducing environment of concentration time curve (AUC0eN) values of tumor cells is controlled mainly by the reduc- PTX and Dox were 3.97- and 4.38- fold higher þ in comparison to those for a PTX plus Dox solu- tion/oxidation states of NADPH/NADP and tion [97]. glutathione (GSH, GSH/GSSG). In tumor cells, 4. Stimuli-responsive codelivery of polymeric micelles 59 a high concentration of GSH compared to Taxol, and SS-PNPs and CC-PDNPs. A codeliv- NADPH provides a reductive microenviron- ery system composed of a redox-sensitive ment. In fact, GSH, through the formation and mPEG-PLGAeSSeDTX conjugate possessed cleavage of disulfide bonds and the reaction the ability to carry the P-gp inhibitor verapamil with excess reactive oxygen species (ROS) plays (VRP) and Docetaxel (mPEG-PLGAeSSeDTX/ a determinant role with respect to the VRP (PP-SS-DTX/VRP)) and overcome MDR intracellular-reducing potential. The concentra- [99]. Fig. 3.3 illustrates schematically the mech- tion of GSH determines the extent of the cellular anism of action of the redox-sensitive micelles reducing environment. The intracellular concen- of PP-SS-DTX/VRP. These findings indicate tration of GSH in normal cells is 10 mM, whereas that PP-SS-DTX/VRP micelles inhibit P-gp in the concentration in tumor tissues is increased at MCF-7/ADR cells because of verapamil’shigh least four times and is even higher in some efficiency in lowering the efflux activity of P-gp. multidrug-resistant tumors. The intercellular- reducing potential of tumors can serve as a trigger in drug delivery systems known as redox-sensitive nanocarriers. Since redox- sensitive drug delivery systems tend to degrade and release their payload only in tumor cells due to the reducing environment, they show little toxicity toward normal cells, leading to low asso- ciated side effects of such chemotherapeutics. In addition, a high concentration of GSH in tumor cells allows rapidly release of drug payload upon exposure to the reducing-intracellular environment. PEGSS-C18 copolymer has been used to form redox-sensitive polymeric micelles for codeliv- ery of PTX and dasatinib (SS-PDNPs) [98].The disulfide bonds present in the backbone of the SS-PDNPs are reduced when internalized by tu- mor cells with a high-reducing environment. Consequently, degradation of the amphiphilic polymer structure results in quick disassembly of the micelles and fast drug release. The anti- cancer effect of various reduction-sensitive micelles, including blank redox-sensitive micelles (referred to as SS-NPs) and redox- nonsensitivemicelles(referredtoasCC-NPs), PTX-loaded redox-sensitive micelles (referred to as SS-PNP), PTX and dasatinib coloaded FIGURE 3.3 Schematic illustration of the combination of redox-sensitive micelles (referred to as SS- DTX and VRP by using a redox-responsive micelle to PDNP), and redox-nonsensitive micelles overcome MDR in cancer cells. From Guo Y, He W, Yang S, Zhao D, Li Z, Luan Y. Co-delivery of docetaxel and verapamil (referred to as CC-PDNP) showed a 5.6-fold by reduction-sensitive PEG-PLGA-SS-DTX conjugate micelles higher apoptosis incidence for MCF-7/ADR to reverse the multi-drug resistance of breast cancer. Colloids cells treated with SS-PDNPs, compared to Surf B Biointerfaces. 2017; 151:119e27. with permission. 60 3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy

5. Targeted codelivery of polymeric 10%) of the total covalently bound cell- micelles associated cisplatin reached the DNA fraction [106]. These barriers markedly decrease the effi- The use of polymeric micelles for targeted cacy of some chemotherapeutic drugs, such as drug delivery is a promising strategy to improve Dox, which acts via DNA injury. To solve this the efficacy of pharmaceuticals in chemotherapy. problem, nuclear-targeted vehicles that facilitate The principle of targeted drug delivery is based drug translocation into the nucleus have been on nanoparticles acting as carriers for pharma- explored. Generally, nuclear-targeted molecules ceutical agents, with a targeting molecule such like transactivator of transcription (TAT) have as antibody or ligands attached to the surface been used to modify the surface of drug delivery of the nanoparticles, which then directs them to systems. TAT is a cell-penetrating peptide and the receptors/antigens on the surface of tumor has the ability to quickly enter almost all living cells. To achieve the maximum therapeutic effi- cells. It actively delivers proteins, DNA, and ciency, it is very important to find a suitable nanoparticles into the nucleus. Micelles consist- target and potent drug. In addition, however, ing of grafted poly-(N-3-carbobenzyloxy-lysine) the type of carrier plays an important role in (CPCL) with TAT-chitosan decorated on the the success of targeted drug delivery systems. surface have been used as a codelivery system This high drug-loading capacity and the possi- for p53 and Dox into the nucleus of cancer cells bility of surface modification of micelles make [106]. Using confocal laser scanning microscopy, them promising candidates for targeted drug the vector delivered more TAT-CPCL into the delivery systems. Polymeric micelles have also nucleus than did CPCL alone. The TAT- been used as codelivery agents in development modified vector served as a highly efficient of targeted drug delivery systems for chemo- gene and drug codelivery system with high therapy. A variety of targeting moieties have gene transfection efficiency and a high anti- been used to design micellar-based targeted cancer effect leading to low viability in HeLa drug delivery systems [100e105]. cells [106]. An MTT cell viability assay clearly The nucleus has been a very important site to confirmed that TAT-CPCL/p53/Dox micelles target and improve the therapeutic efficiency of possessed a higher cytotoxicity compared to cancer treatment. To date, drug delivery systems the blank TAT-CPCL’s negligible cytotoxicity. have been designed mainly to enable drug deliv- ery at endo/lysosomal sites of cancer cells. To reach the nucleus, in these systems, drugs 6. Application of polymeric micelles in need to escape from endo/lysosomal compart- codelivery for multiple therapies ments and overcome a variety of intracellular resistance mechanisms, including drug meta- Due to the diversity, and complexity of bolism and detoxification, overexpression of cancer, the integration of two or more therapies drug efflux pumps (e.g., P-gp), drug seques- with different therapeutic mechanisms of action tering to acidic compartments, and drug deacti- in one nanoplatform (e.g., chemotherapy and vation. Only a small portion of any drug is photothermal therapy) seems a promising strat- likely to reach the cytosol with eventual delivery egy for significant enhancement of the therapeu- to the nucleus, particularly in drug-resistant tic efficacy and a better long-term prognosis. cells. For instance, it was found that most of Codelivery nanoplatforms provide the opportu- the cisplatin delivered to the cytoplasm bound nity to integrate chemotherapy with multiple to protein and only a small portion (less than therapeutic approaches. Treatment of tumors 6. Application of polymeric micelles in codelivery for multiple therapies 61 with multiple approaches has attracted a great immune system’s activity against cancerous deal of attention because of their success in cells. One strategy is to prohibit cancer cells overcoming the challenge of tumor heterogene- from expressing inhibitory receptors referred to ity, reversing MDR, and achieving additive or as immune checkpoints, or to inhibit cancerous synergistic anticancer effects (Table 3.4). cell expression of immune inhibitory ligands. Despite the high potential for immunotherapy in cancer treatment, the overall clinical success 6.1 Chemo-immunotherapy of this regimen is still far from an acceptable sit- uation. This is probably because the efficacy of The function of the immune system is a deter- this treatment is closely associated with preexist- minant parameter in cancer’s initiation and ing antitumor immune responses. By initiating growth. Cancer cells have the potential to an antitumor immune response, chemotherapy escape, inactivate, or overpower the immune agents could improve the efficiency of immuno- system. Immunotherapy is now a well- therapy. Chemo-immunotherapy combines established clinical regimen that boosts the immunotherapy and chemotherapy. Recently,

TABLE 3.4 Polymeric micelles in codelivery for multiple therapies.

Micelle Animal model or cell Therapeutic composition Drug 1 Drug 2 line approach Refs

Heparin-D-a-tocopheryl Dox Toll-like receptor 7 4T1 xenograft BALB/c Chemo- [115] succinate agonist imiquimod mice immunotherapy (IMQ) POEG-b-PMBC Dox Immune checkpoint 4T1.2 xenograft Chemo-immunotherapy [115] inhibitor NLG919 mice model F127/PDPP Dox poly(dithienyl- HeLa cells Chemo-phototherapy [116] diketopyrrolopyrrole) PCL-ss-PEG-ss-PCL and Dox Indocyanine green BEL-7404 xenograft Chemo-photothermal [117] PCL-acetal-PEG nude mice model therapy mPEG-S-S-C16 Dox Semiconducting HepG2 xenograft Chemo-photothermal [118] polymer nude therapy dots PCPDTBT mice model dots (Pdots) Porphyrin-based SN-38 - HT-29 xenograft Chemo-phototherapy [119] telodendrimer mice model PEG-b-PHEA GW627368X Gold nanorod Sarcoma S180 Photothermal therapy [120] Cayman xenograft mice chemical model PEG-PLA PTX CaWO4 (CWO) HN31 xenograft mice Chemo-radio therapy [121] model

0 POEG-b-PMBC (poly(oligo(ethylene glycol) methacrylate) -b-poly N,N -(t-butyoxycarbonyl)cystamine); F127/PDPP (PluronicF127/ poly(dithienyl-diketopyrrolopyrrole)); PCL-ss-PEG-ss-PCL and PCL-acetal-PEG (poly(ε-caprolactone)-ss-poly(ethylene glycol)-ss-poly(ε- caprolactone) and poly(ε-caprolactone)-acetal-poly(ethylene glycol)); mPEG-S-S-C16 (monomethoxy-poly(ethylene glycol)-S-S-hexadecyl); SN- 38 (7-thyl-10-hydroxycamptothecin), PEG-b-PHEA (Poly(ethylene glycol) monomethy ether, poly 2-hydroxyethyl acrylate); PEG-PLA(poly (ethylene glycol)-poly(lactic acid)). 62 3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy there have been a few reports on the application tumor tissues through the tumor interstitial of polymeric micelles for concurrent codelivery matrix [124]. On the other hand, the lack of selec- of chemotherapeutic and immunotheraputic tivity of anti-VEGF agents and free cytotoxic agents. Chen et al. [80,122] used a dual- drugs can also result in significant side effects functional, immunostimulatory nanomicellar (e.g., hypertension, and thrombotic events). carrier composed of a NLG919-conjugated PEG Thus, there is an apparent need for targeted with a Fmoc linker (PEG2k-Fmoc-NLG) as an codelivery of anti-VEGF agents with common indoleamine 2,3-dioxygenase (IDO) inhibitor chemotherapeutic drugs. For instance, a dual- for delivery of PTX. They showed that the code- pH responsive cross-linked micelle for two-step livery immune-chemotherapy they developed release of the small molecule receptor tyrosine led to a significantly improved antitumor kinase inhibitor, axitinib (AXI), which acts by response in both breast cancer and mouse mela- an antiangiogenesis effect and a cytotoxic agent, noma models. Polymeric micelles have also been Dox, has been reported [111]. A pH-sensitive used for simultaneous delivery of Dox and hydrazone linkage was used to conjugate Dox NLG919 for the treatment of leukemia [123]. to amphiphilic N-(2-hydroxypropyl) methacry- The Dox-loaded micelles self-assembled from a lamide (HPMA) to take advantage of the EPR PEG-Fmoc-NLG conjugate showed cytotoxicity of micelles in tumors and accumulate selectively comparable to that of free Dox. In vivo studies at the tumor site. In a related study, axitinib was showed significant improvement in antitumor also encapsulated into the core segment of self- activity for the Dox/PEG-Fmoc-NLG group assembling micelles [111]. In addition, to pro- compared to Doxil or the free Dox group in an long the micelles, circulation time, and increase A20 lymphoma mouse model. their stability, the micelles were cross-linked through benzoiceimine bonds, which are trig- gered to break at lower extracellular pH (pH 6.2 Chemo-angiogenic therapy 6.5) and enable delivery of the axitinib and Dox selectively to the tumor site. Axitinib interacts Angiogenesis, defined as the growth of new with tyrosine kinase receptor located on the cell blood vessels from a preexisting vasculature, membrane for vasculature modulation plays a crucial role in the progression of cancer (Fig. 3.4). After nanoparticle uptake by cancer because the proliferation and metastatic spread cells, exposure of micelles to the more acidic of cancer cells strongly depends on an abundant environment of the lysosome (pH 5.0) results in supply of oxygen and nutrients, and the removal the hydrolysis of hydrazone linkages, which in of metabolites. Thus, the inhibition of angiogen- turn releases the conjugated Dox and induces esis represents a promising approach to limita- cell death. The in vivo antitumor effect of dual- tion of access to oxygen and nutrients and drug-loaded cross-linked micelles (DA-CM) sensitization of tumor cells to chemotherapy. showed the highest tumor growth inhibition The combination of antibodies against endothe- (88.38%) with no apparent body weight loss. lial growth factor (anti-VEGF) and cytotoxic Such a high growth inhibition might be therapeutic agents has been used intensively in explained by the synergistic effect of axitinib the clinic for cancer treatment. The synergy of and Dox delivered by the cross-linked micelles. treatment might be explored by the high Free-Dox and single-Dox-loaded cross-linked apoptosis rate of the cancer cells, which increases micelles (D-CM) resulted in 49.16% and 67.23% blood flow in tumor sites after tumor vascular suppression of tumor growth, indicating the sig- normalization consequently promotes the pene- nificance of the EPR effect for tumor-targeted tration of nano-vehicles into the deepest part of delivery. 6. Application of polymeric micelles in codelivery for multiple therapies 63

pH6.5 pH5.0 Tumor microenvironment Endo-Iysosome

nucleus Iysosome

HPMA Axitinib Doxorubicin β -sitosterol

FIGURE 3.4 Illustration of dual-pH responsive micellar platform for codelivery of axitinib (AXI) and doxorubicin (Dox) based on HPMA copolymers. From Xu X, Li L, Zhou Z, Sun W, Huang Y. Dual-pH responsive micelle platform for codelivery of axi- tinib and doxorubicin. Int J Pharm. 2016;507(1):50e60 with permission.

6.3 Chemo-photothermal therapy for a targeted synergistic chemo-photothermal strategy [125,126]. Aptamer (Apt)-polydop- Photothermal therapy uses electromagnetic amine (pD) and its derivatives are well known radiation, including visible light, NIR, radio for their effective photothermal properties. pD- frequency, and microwaves, to stimulate a functionalized CA-(PCL-ran-PLA) micelles photosensitizer that converts this energy into fi have been developed for effective delivery of heat. Minimal invasiveness and high speci city DTX and improved therapeutic efficacy [127]. with minimal toxicity are the most important The results demonstrated that DTX-loaded features of photothermal therapy. Recently, inte- Apt-pD-CA-(PCL-ran-PLA) micelles had gration of photothermal therapy with chemo- high-therapeutic efficacy that was due to its therapy has been considered a useful technique synergistic chemo-photothermal properties. 64 3. Polymeric nanomicelles as versatile tool for multidrug delivery in chemotherapy 6.4 Chemo-radiotherapy 6.5 Chemo-enzyme prodrug therapy

A radiosensitizer, or a radiosensitizing agent, Chemo-enzyme prodrug therapy (EPT) has is a pharmacologic agent that potentiates the recently been introduced as a therapeutic toxicity of radiation therapy. Radiation therapy approach to improve specificity and anticancer uses various external energy sources such as efficiency of chemotherapeutics [132]. EPT con- X-rays and protons to shrink tumors by disrupt- sisting of a foreign prodrug-activating enzyme ing their DNA. Some radiosensitizers are and a nontoxic prodrug are delivered to the directly toxic by themselves, while others show tumor cells using various targeting approaches. toxicity only on exposure to radiation. The prodrug is then activated and converted A different mechanism has been proposed for into a potent pharmacologic agent through enzy- radiosensitizing agents. For instance, the mecha- matic activity. The time interval between nism of action of PTX as a radiosensitizer enzyme and prodrug treatment is the most chal- involves the inhibition of cell cycle progression lenging step. Too short a time interval means the at a radiosensitive phase (G2/M). The 17-(allyla- prodrug will be activated by the enzyme while in mino)-17-demethoxygeldanamycin (17-AAG) the circulation with decreased drug concentra- augments radiosensitization by inhibiting the tion at tumor sites and enhanced side effects. function of heat shock protein 90 (Hsp-90) On the other hand, too long a time interval [128]. Rapamycin (RAP) radiosensitizes cancer means the prodrug-activating enzymes are elim- cells by inhibiting mTOR, which is downstream inated by the immune cells of the body and of the PI3K-Akt survival pathway [129]. A sensi- consequently cannot activate prodrug into a tivity enhancement ratio is used to compare the potent cytotoxin. Therefore, delivery of pro- potency of radiosensitizing agents in chemo- drugs and enzymes simultaneously to tumor radiotherapy [130].Itisdefined as the ratio of sites with a single nanoplatform is important. radiosensitivity of radiation-treated cells to The combination of 3-indoleacetic acid (IAA) radiosensitivity of drug combined with and horseradish peroxidase (HRP), which is radiation-treated cells. Tomoda et al. [131] stud- highly cytotoxic to mammalian cells, is one of ied a codelivery micellar system, referred to as the most studied enzymeeprodrug for anti- Triolimus, containing a variety of radiosensitiz- cancer therapy. IAA stimulates cell division ing species including PTX, 17-allylamino-17- and promotes cell differentiation. HRP is a demethoxygeldanamycin (17-AAG), and rapa- widely studied heme containing an enzyme mycin used to investigate the corresponding with the ability to convert the auxin IAA into potential of different radiosensitization agents cytotoxins via catalysis. Neither IAA nor HRP on A549 cells and the suppression of tumor is cytotoxic at clinically used concentrations. growth with their combinations in an A549 xeno- But when combined, HRP can activate IAA to graft mouse model. The radiosensitizing effects produce a series of free radicals, which can were as follows: PTX alone > (PTX and RAP) initiate the tumor cell killing process and >Triolimus> (PTX and 17-AAG) > (17-AAG apoptosis. Among the different chemical bonds and RAP). However, Triolimus showed less used to prepare prodrug, esters are the most acute toxicity compared to PTX alone or radia- common linkages. They are readily hydrolyzed tion alone, indicating the high potency of poly- by esterases widely distributed in the body to meric micelles as carriers for codelivery of facilitate drug release. Ethyl 3-indoleacetate radiosensitizing and chemotherapeutic agents. (EIA) is a hydrophobic prodrug that is quickly References 65 hydrolyzed by the double action of esterases and systems for combinational therapy. Despite the low-pH conditions of tumors to produce the high potential and variety of codelivery IAA. Nanomicelles formed by self-assembly of systems developed, there are still issues to be the amphiphilic copolymer PEG-PAsp(-AED)- addressed. Optimization of the dose ratio of CA have been used for codelivery of HRP and different therapeutic agents is still a big chal- EIA [133]. To enhance the targeted delivery of lenge for generation of synergetic effects. There nanomicelles, a reductive-sensitive disulfide remains a need to control the level of drug bond has also been added to the polymeric payload to maintain dose ratios. To minimize micelle structure to initiate the drug and enzyme toxicity, the design of new methods to find the release once the micelle has been taken up by best pairs of drugs for concurrent delivery tumor cells [133]. As a result, IAA activated by wouldbeofmorethanalittleinterest.Thepos- HRP produces an abundance of ROS, offering sibility of payload release in a controlled great potential for cancer treatment. manner and the need for optimum dosing must be taken into consideration in order to have a highly efficient codelivery system. 7. Conclusions Development of such codelivery systems will ultimately lead toward more effective therapies The codelivery of chemotherapeutic drugs forcancertreatment. represents a promising strategy to achieve high Acknowledgments efficacy in cancer therapy. 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Nanoparticulate systems for wound healing Maha Nasr1, Riham I. El-Gogary1, Hend Abd-Allah1, Mona Abdel-Mottaleb1,2 1Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Egypt; 2PEPITE EA4267, Univ. Bourgogne Franche-Comte, Besançon, France

1. Introduction which is wounds attributed to different causes, in order to accelerate wound healing to over- Wounds attributed to injuries, traumas, and come the increased risk of infection accompa- diseases are increasing in incidence with time, nying the delayed healing process [5]. creating a financial burden for patients and the Nanoparticles were reported to penetrate the health care system [1]. The process of wound stratum corneum and reach the deep skin healing involves four stages: hemostatic phase, epidermal and dermal layers owing to one or inflammatory phase, proliferative phase, and several combined possible mechanisms: their remodeling phase (Fig. 4.1). As shown in the small size, their deformable properties, their figure, the wound healing process involves an lipidic nature, or their lipid-fluidizing nature inflammatory stage, which would benefit from [6e9]. One of the commonly researched areas is the delivery of antiinflammatory drugs to over- the use of nanoparticles for wound healing, in come excessive tissue destruction and necrosis, which nanoparticles were loaded with several as well as a proliferative phase that involves therapeutic molecules, or themselves exhibited the formation of new blood vessels, hence would wound healing properties without drug loading. benefit from the delivery of angiogenesis and Therefore, the aim of this chapter is to highlight collagen-promoting therapeutic molecules. the most commonly used nanoparticles with Topical delivery of pharmaceuticals is currently emphasis on lipid-based systems (vesicular, the mainstay of therapy in wound treatment, emulsions, and solid matrix based) as represen- however, topical delivery is hampered by the tatives of organic nanoparticles and gold nano- barrier nature of the stratum corneum. particles, silver nanoparticles, and metal oxide Nanoparticle-based delivery has recently nanoparticles as representatives of inorganic emerged as a very promising approach for treat- nanoparticles. ment of dermatological diseases [2e4], among

Nanopharmaceuticals https://doi.org/10.1016/B978-0-12-817778-5.00004-X 73 © 2020 Elsevier Inc. All rights reserved. 74 4. Nanoparticulate systems for wound healing

Injury - Day 1 Day 1 - 4 Day 4 - 21 Day 21 - Months

Hemostasis Inflammation Proliferation Remodeling • Reduced blood flow • Neutrophil infiltration • Reepithelialization • Collagen remodeling • Platelet aggregation • Monocyte recruitment • Granulation tissue formation • Apoptosis of fibroblasts • Inflammation initiation • Reduction of scar • Macrophage differentiation • Angiogenesis • Lymphocyte infiltration

FIGURE 4.1 The four stages implicated in wound healing. Abstracted with permission from Das S, Baker AB. Biomaterials and nanotherapeutics for enhancing skin wound healing. Front Bioeng Biotechnol 2016;4:82.

2. Lipid-based nanoparticles phospholipids, with no other additives such as surfactants or penetration enhancers, and hence Lipidic nanoparticles are those comprised of they could be referred to as first-generation ves- one or more lipidic/oily substance in their icles. Liposomes owe their popularity to their composition. Nanoparticles can penetrate the phospholipid content, which is biocompatible skin through several mechanisms, as demon- with skin lipids [8]. Liposomes act as reservoirs strated in Fig. 4.2. Lipidic nanoparticles were allowing controlled release of drugs, but they reported to increase skin hydration, and allow are mainly confined to the skin’s upper layers. rearrangement of skin cells, which may either Despite their inflexible nature, many papers induce fusion with the nanoparticles or enhance reported their effectiveness as topical delivery their permeation across skin layers [5]. The most systems. Incorporation of liposomes within commonly reported categories of lipid-based hydrogels was reported to aid the process of particles are either vesicular in nature such as wound healing by the semiocclusive nature of liposomes, penetration enhancer vesicles, etho- the latter, which promotes angiogenesis and somes and transfersomes, emulsion-based in allows the growth of granulation tissue [10]. nature (nanoemulsion and microemulsions), or Moreover, functionalization of gauzes with lipo- contain a solid lipid matrix such as solid lipid somes was reported to offer high biocompati- nanoparticles (SLNs) and nanostructured lipid bility with human skin fibroblasts, and was carriers (NLCs). reported to be the best vehicle for incorporation within gauzes [11]. Table 4.1 summarizes some of the promising uses of liposomes for wound 2.1 Liposomes healing, in which the described vesicles are Liposomes are the most commonly reported devoid of any penetration enhancer/surfactant vesicular systems, and the term “liposomes” content. implies that they are only formulated using 2. Lipid-based nanoparticles 75

Hair shaft

1 2 3

Langerhans Cell Stratum Corneum

Stratum Granulosum Epidermis Stratum Spinosum

Stratum Basale

Sebaceous Melanocyte Nanoparticles Hair Gland Follicle

Dermis

Dermal Fibroblast Dendritic Cell Vasaculature Vasaculature

Hypodermis

FIGURE 4.2 Mechanisms of nanoparticles penetration across the skin: (1) Transfollicular route, (2) Intracellular route, (3) Intercellular route. Abstracted with permission from Palmer BC, DeLouise LA. Nanoparticle-enabled transdermal drug delivery systems for enhanced dose control and tissue targeting. Molecules 2016; 21(12):E1719.

TABLE 4.1 Liposomal formulations used for wound healing.

Status of Active Formulation and composition investigation ingredient Observations/Advantages References

Liposomes prepared from In vitro/ Curcumin Sustainment of drug release for 24 h, and better [12] phosphatidylcholine and In vivo wound diameter reduction on the 14th day cholesterol postinduction compared to the unencapsulated form, verified for better skin penetration using fluorescence imaging Liposomes prepared from In vitro/ Danggui The extract-loaded liposomes placed in a [13] phosphatidylcholine and In vivo Buxue thermosensitive gel significantly improved cholesterol extract collagen synthesis and angiogenesis, hence improving wound healing compared to blank vehicleetreated and model control groups Liposomes prepared from In vitro Substance P The chitosan-coated liposomes provided [14] lecithin and cholesterol, and neuropeptide significant reduction in cellular gap closure of coated with chitosan HaCaT cells, delineating the system as a promising topical carrier for further in vivo wound healing experiments Liposomes prepared from In vitro/ Glycyl-l- The liposomal encapsulated tripeptide displayed [15] (1,2-dioleoyl-sn-glycero-3- In vivo histidyl-L- better angiogenic effect and shortened wound phosphocholine), lysine healing time compared to the free peptide 1,2-dioleoyl-sn-glycero-3- 0 phospho-(1 -rac-glycero1) sodium salt] and cholesterol

(Continued) 76 4. Nanoparticulate systems for wound healing

TABLE 4.1 Liposomal formulations used for wound healing.dcont'd

Status of Active Formulation and composition investigation ingredient Observations/Advantages References

Liposomes prepared from In vitro Quercetin Sustainment of drug release for 24 h [16] phosphatidylcholine and cholesterol Liposomes prepared from In vitro recombinant Enhanced skin permeation of the human [17] egg lecithin human epithelial growth factor when encapsulated in on a sucrose-based powder epidermal liposomes compared to its free form growth factor (rhEGF) Liposomes prepared from In vitro/ Basic The wound healing effect of bFGF liposomes was [18] soybean In vivo fibroblast dose dependent, demonstrating faster collagen lecithin and cholesterol growth generation with prolonged effect caused by factor bFGF encapsulation Liposomes prepared from In vitro/ Epidermal Liposomal encapsulated EGF demonstrated [19] dipalmitoyl- In vivo growth superior wound healing effect compared to mere phosphatidylcholine and factor (EGF) EGF administration and the silverdine ointment, cholesterol with enhanced epidermal thickness Undisclosed phospholipid In vivo Polyvinyl- Enhanced tissue repair property of liposomes [20] type pyrrolidone led to faster wound healing for the liposomal iodine hydrogel group loaded with polyvinyl- pyrrolidone iodine Liposomes prepared from In vitro/ Buflomedil Accelerated wound closure around days 9 and [21] lecithin and In vivo 13 in normal and ischemic wounds, respectively, phosphatidylserine compared to untreated control

2.2 Penetration enhancer vesicles for delivering active ingredients with PEVs for wound healing purposes. Penetration enhancer vesicles (PEVs) are the same as liposomes, with the exception that they contain additional penetration enhancers in their 2.3 Ethosomes composition such as polyethylene glycol, labra- sol, and transcutol. Compared to conventional Ethosomes are identical in their structure to vesicles, the penetration enhancers content liposomes, in the sense that they contain phos- causes additional disordering of the skin [22], pholipids as the bilayer forming agent, in addi- hence they can be categorized as flexible vesicles tion to ethanol, which acts as a skin penetration that can facilitate dermal penetration and allow enhancer [26], hence they are categorized as flex- better fibroblast cell uptake. Table 4.2 summa- ible vesicles. Only a few papers reported the use rizes some of the reported successful attempts of ethanol as topical vesicular treatment of 2. Lipid-based nanoparticles 77

TABLE 4.2 Penetration enhancer containing vesicles used for wound healing.

Formulation and Penetration Status of Active composition enhancer used investigation ingredient Observations/Advantages References

PEVs prepared from Sorbitol In vitro Baicalin The modified sorbitol PEVs [23] soybean lecithin and tween formulation displayed better skin 80 (a penetration enhancer accumulation in all layers, provided emodified formulation higher proliferative stimulation of version of transfersomes) fibroblasts, and provided better skin protection compared to transfersomes, and were proven more superior in promoting cellular- based wound closure PEVs prepared from Polyethylene In vitro/ Madecassoside Vesicles exhibited higher deposited [5] egg yolk lecithin and glycol In vivo amount of the drug in the skin cholesterol (PEG 1500) compared to its solution form, and enhanced wound healing rate accompanied with reduced scar formation by day 12 in wound healing animal model PEVs prepared from Ethylene In vitro/ Polyphenolic PEVs encapsulating the [24] soybean phospholipids glycol In vivo phytocomplex extracted polyphenols resulted from Fraxinus in significant reduction in edema angustifolia with concomitant decrease in myeloperoxidase activity compared to unencapsulated extracts, with normal skin appearance in animal inflammatory model

PEVs prepared from Polyethylene In vitro/ Quercetin PEVs-encapsulated quercetin [25] phospholipids mixture glycol In vivo exhibited significant reduction (PEG 400) in tissue damage with significant tissue regeneration manifested by increase in collagen fibers compared to unencapsulated drug PEVs prepared from Polyethylene In vitro/ Quercetin and PEVs loaded with quercetin and [22] soybean phospholipids glycol In vivo curcumin curcumin exhibited better (PEG 400) antiinflammatory activity and more significant dermal deposition of drugs, with superior effect encountered with curcumin compared to quercetin vesicles in animal inflammatory model wounds, probably owing to their ethanolic con- to topically applied ethosomal erythromycin, tent, which might be an irritant to open wounds. hence delineating the importance of the vesicular A study conducted by Godin et al. [27] reported structure in promotion of skin penetration. that the topical application of hydroalcoholic In a study reported by Partoazar et al. [28], solution loaded with erythromycin was not as ethosomal curcumin was found to significantly effective as a wound healing modality compared inhibit gram-positive and gram-negative 78 4. Nanoparticulate systems for wound healing bacteria isolated from wounds compared to the animal model compared to the nonencapsulated unencapsulated curcumin, and to significantly form. In addition, when transfersomes prepared reduce the wound area in a second-degree from phosphatidylcholine, cholesterol-gellan, burn wound model in rats owing to its strong and tween 80 were loaded with baicalin, they reepithelization, angiogenesis, collagen synthe- were shown to exhibit complete skin healing in sis, and granulation tissue promotion potential. inflammatory animal model compared to beta- Moreover, an ethosomal gel loaded with Sesa- methasone cream [33]. Transfersomes composed mum indicum seed extract was proven to exhibit of phosphatidylcholine and one of either tween significantly higher wound contraction percent- 20, 40, 60, or 80 and loaded with tocopherol age by the 16th day in rat excision model were prepared by Caddeo et al. [34], in which compared to povidone iodine ointment, corre- the authors proved the superiority of tween 80 lating with high collagen synthesis inferred transfersomes in providing skin accumulation from the significantly higher hydroxyproline of tocopherol, with faster HaCaT cell layer levels achieved with the ethosomal form [29]. regeneration and complete wound closure in Finally, an ethosomal formulation loaded with 3T3 cells compared to other transfersomes. silver sulfadiazine displayed faster wound heal- Moreover Lei et al. [35], proved that transferso- ing process cascade compared to the silver sulfa- mal gel prepared from phosphatidylcholine diazine gel or a marketed product, and was and tween 80/span 80 as edge activator mixture found to exhibit higher wound contraction in an atopic dermatitis-induced animal model percentage [30]. managed to deposit tacrolimus into the deep skin layers with significant suppression of inflammation, and the associated wounds 2.4 Transfersomes managed to grow a scab after 7 days compared to ointment and liposomal gel groups, confirm- Transfersomes are another example of modi- ing the superiority of transfersomes. fied vesicular generation, in which a surfactant (edge activator) is incorporated within the phos- pholipid bilayer, conferring flexibility to the 2.5 Nanoemulsions vesicles, and consequently enhancing their skin delivery potential [31]. Like PEVs and etho- Nanoemulsions are nano-lipid-based soft somes, transfersomes can be considered as flex- delivery systems, composed of water, oil, and ible vesicles. surfactant/cosurfactant [36]. Owing to their Regarding wound healing Chhibber, Kaur, biocompatible and safe components, several and Kaur [32] conducted an interesting study studies were reported in the literature for their in which they encapsulated bacteriophages use as wound healing therapies. Microemulsions rather than drugs (Staphylococcus aureus lytic are also often referred to as nanoemulsions, but phages MR-5 and MR-10 in transfersomes they contain more surfactant/cosurfactant composed of phosphatidylcholine, cholesterol, concentration and are prepared using a delicate tween 80, and stearylamine to overcome the ratio of oil/water/surfactant components, decreased persistence of the phages at the place mostly using the water dilution method [9]. of the wound. Results showed that the Since microemulsions exhibit nanometer-size transfersomal-encapsulated phages exhibited range, they will be referred to as nanoemulsions significant enhancement of their wound persis- in this work. tence, accompanied with better wound closure The oily phase of the nanoemulsion was and faster healing rate in a diabetic wound reported to influence its wound healing 2. Lipid-based nanoparticles 79 potential, since it affects the release of the ingre- Quercetin-loaded SLNs accelerated the heal- dients from the oily core [37], and the inclusion ing of wounds compared to quercetin alone of actives within nano/microemulsion formula- [46]. SLNs also managed to create a combination tions was shown to exhibit enhanced wound therapy loaded with a peptide LL37 and an elas- healing potential. Cinnamon oil nanoemulsion tase inhibitor Serpin A1 that accelerated wound was found to be more effective than the oil alone healing in cellular experiments [47], and another in accelerating wound healing in animals [38]. combination therapy of curcumin and ampi- Moreover, nanoemulsions can contain more cillin, which also accelerated wound healing, than one active ingredient, in which Shanmu- but in animal model [48]. When tetrahydrocur- gapriya et al. [39] loaded astaxanthin and alpha cumin was encapsulated in SLNs gel, it tocopherol in nanoemulsion formulation that displayed faster wound healing compared to managed to improve closure of cells in scratch the unencapsulated form [49], and similarly, wound healing cellular assay compared to con- the topically applied SLN-loaded astragaloside trol, delineating this nanoemulsion system as a IV displayed significantly higher wound closure potentially promising topical carrier. Similarly, percent compared to the unencapsulated phenytoin nanoemulsions improved cellular form [50]. closure in scratch assay compared to the free Regarding NLCs, those prepared using olive phenytoin [40]. Nanoemulsion hydrogels were oil and loaded with eucalyptus oil caused signif- also loaded with growth factor combinations, icant reduction in wound areas of rats [51]. displaying better skin permeability compared A comprehensive research reported by Gainza to the unencapsulated form, and were delineated et al. [52] studied several wound healing param- as promising systems worthy of experimentation eters in porcine full-thickness excision wound as wound healing systems [41]. In addition Cao model, in which they compared the effectiveness et al. [42], prepared benzalkonium chloride of NLCs-encapsulated recombinant human nanoemulsion, and proved their antiinflamma- epidermal growth factor with the free form, tory activity and therapeutic efficacy in skin and reported that even when smaller dose of abrasion wound model compared to control. the growth factor was administered in NLCs Moreover, fusidic acid loaded in nanoemulsion form, it exhibited better wound healing in terms gel accelerated wound healing in animals of healing wounds percentage, epithelization, compared to the marketed cream of the collagen formation, lower inflammation than drug [43]. the higher dose administered of the free form, attributed to its protection of the latter against enzymatic degradation. Another interesting 2.6 Solid lipid nanoparticles/ study by Alalaiwe [53] reported that loading of Nanostructured lipid carriers oxacillin antibiotic within cationic NLCs enhanced the bactericidal activity of the anti- Among the popular lipidic delivery systems biotic against methicillin-resistant S. aureus are solid lipid nanoparticles and nanostructured MRSA, and resulted in almost complete treat- lipid carriers. Both systems are composed of ment of the abscesses, with significant reduction solid lipid matrix, with NLCs additionally con- of water loss from infected wound compared to taining oil in their matrices [44]. Both systems mere antibiotic administration, accompanied can be used on their own or can be incorporated with much less inflammation of the wound. within additional dressings [45]. 80 4. Nanoparticulate systems for wound healing

3. Inorganic nanoparticles sulfadiazine cream. Nowadays, several forms of silver-based dressings are currently available Among the various types of inorganic nano- commercially, either as fibers or polymeric scaf- particles, metallic nanoparticles such as silver folds impregnated or coated with Ag salt or (Ag), gold (Au), selenium (Se), and copper (Cu) metallic Ag in nanoparticulate form. Recently, nanoparticles, as well as zinc oxide (ZnO), iron it has been proposed that silver performs its anti- oxide (Fe2O3), and titanium dioxide (TiO2) nano- bacterial activity through interacting with the particles have attracted significant attention bacterial cell wall, altering the bacterial DNA, lately in the area of wound healing, due to the and blocking its respiratory pathways, resulting proven biomedical applications of their metallic in its death. Clinically, some studies have content, in addition to the delivery ability and confirmed their safety for patients while others unique properties of nanoparticles that reduce have raised concerns about their cytotoxicity on the cytotoxicity of the metals and increase their fibroblasts and keratinocytes [56]. stability and therapeutic efficacy [54,55]. There- Silver nanoparticles (NPs) (AgNPs) have been fore, the next sections of this chapter summarize shown to possess unusual physical, chemical, studies conducted on the different inorganic and biological properties. Silver nanoparticles (especially metallic) nanoparticles for wound have been reported to possess antibacterial, anti- healing and regeneration. fungal, antiviral, antiinflammatory, antiangio- genesis, and antiplatelet activity. Nanoparticles have many advantages, such as possessing a 3.1 Silver (Ag) nanoparticles large surface-to-volume ratio resulting in high reactivity, in addition to the ability of NPs to sus- Different forms of silver (both metallic and tain the release of silver, hence prolonging its ionic) have long been used for wound healing effect and minimizing its toxicity [57]. AgNPs applications due to their broad-spectrum antimi- have been used extensively as antimicrobial crobial effect, antiinflammatory traits, and agents against different pathogens, especially wound healing promoting properties. The against dermal pathogens, including S. aureus, importance of silver was displayed thousands Pseudomonas aeruginosa, and Streptococcus pyro- of years ago when the therapeutic applications gens, as well as the methicillin- and oxacillin- of silver powder started to be discovered. The resistant S. aureus (MRSA and ORSA) [58]. The importance of silver nitrate was identified mechanism of action of AgNPs doesn’t differ around the 17th and 18th centuries, in which it greatly from that of Ag ion or metal. The pres- was applied clinically for treatment of skin ulcers ence of Ag in a nanoparticulate form results in and wounds, and was used as a disinfecting better contact with the cell membrane of micro- agent for wounds during the World War I era. organisms. Moreover, it provides better interac- The use of silver salts declined consequent to tion with sulfur present in cell membrane the introduction of antibiotics in 1940 due to proteins as well as cellular DNA that contains the cost and doubtful toxicity of the metal. The phosphorus. This is followed by the interaction interest in the use of silver for wounds was of NPs with the respiratory chain and cell divi- regained following the emergence of bacterial sion. Moreover, AgNPs were also reported to antibiotic resistance [54]. This resulted in the improve tensile properties of repaired skin by development of silver dressings and their com- influencing collagen alignment [59]. mercial use for burns and wounds. Silver was Tian et al. [60] compared the antibacterial applied to burns, either in the form of impreg- property of silver nanoparticles (at a lower nated dressing or as the benchmark silver amount) to 1% silver sulfadiazine cream both 3. Inorganic nanoparticles 81 grafted on a dressing, and compared their effi- exhibited better cytocompatibility, faster and cacy in healing of wounds. Two types of wounds more complete healing of the wounds. were induced, one formed by thermal injury as Regarding toxicity of AgNPs, recent studies an acute wound model and another diabetic demonstrated their possible toxic effects on wound as a chronic wound model in mice. The human fibroblasts and keratinocytes through animals treated with silver nanoparticles decreasing mitochondrial function, shrinking of showed higher healing rates in both acute and cells, as well as production of reactive oxygen diabetic wound models with almost 11 days species (ROS), which was found to be both par- difference in the case of acute wounds. More- ticle size and concentration dependent [56]. over, healed skin after treatment with AgNPs Another group monitored mitochondrial func- showed the most resemblance to normal skin tionality in human fibroblasts, and they found in comparison to that treated with sulfadiazine signs of reduced activity with lack of apoptosis cream and control. For confirmation, the antibac- or cell death signs. This reduction occurred terial and antiinflammatory activity of both temporarily as a cell protective mechanism formulations was studied throughout the against AgNPs and didn’t affect the cell viability. wounds’ healing process and compared. Silver Reproliferation of mitochondria would occur nanoparticles inhibited bacterial growth for once the silver was removed, resulting in the 7 days post injury while silver sulfadiazine renewal of the dermal tissue in vivo [55,56]. group showed bacterial growth after 3 days. By The concept of surface modification or coating monitoring the level of inflammatory mediators of metallic nanoparticles, especially AgNPs, has throughout the healing process, they found been recently introduced with the aim of that the level of some mediators as IL-6 mRNA improving the biocompatibility and decreasing was significantly lower in the wound areas the toxicity of Ag ions through delaying their treated with AgNPs. Several studies showed release. Table 4.3 displays some examples for that the therapeutic effects of AgNPs (in suspen- coated AgNPs. Keletemur et al. [63] coated 0 sion form) depended on important nanoparticu- AgNPs with oligonucleotide [5 eHSe (CH2)6- late features, including particle size (surface area TAATGCTGAAGG-3] and compared their activ- and energy), particle shape (catalytic activity), ity to uncoated AgNPs on an in vivo mouse particle concentration (therapeutic index), and wound model. Surface functionalization of particle charge (oligodynamic quality) [55,58]. AgNPs resulted in acceleration of the prolifera- Biosynthetic methods of AgNPs have been tive phase of wound healing through faster recently investigated as an alternative to chemi- deposition of fibroblasts and collagen in the cal and physical ones. Sundaramoorthi et al. wound, hence achieving more rapid wound [61] investigated the potential usefulness of healing. Similar results were obtained by Im Aspergillus niger in the production of AgNPs et al. [64] upon modifying the AgNPs with chon- extracellularly, and evaluated their wound heal- droitin sulfate and acharan sulfate. Several other ing activity using both excision and thermal studies coated AgNPs with different polymers wound models. Results confirmed the superior and proved the efficacy of the modified system wound healing activity of AgNPs synthesized in enhancing their antimicrobial effect, healing from A. niger compared to silver nitrate. Synthe- and regeneration of wounds besides decreasing sis of AgNPs from plant origin has been also the toxicity of Ag. The utilized polymers were reported by Dhapte et al. [62] where they synthe- chitosan/poly(vinyl alcohol) nanofibers [65], sized AgNPs from Bryonialaciniosa leaf extract hyaluronan [66], and pectin [67]. Pallavicini and tested their wound healing efficacy in et al. [67] utilized pectin for dual purposes, as a comparison to sulfadiazine cream. The NPs reducing agent and as a coating material for 82 4. Nanoparticulate systems for wound healing

TABLE 4.3 Surface-coated silver nanoparticles for wound healing.

Coating material Model Wound healing Source 0 Oligonucleotide ([5 eHSe (CH2)6- BALB/C mice excision Faster fibroblasts and collagen deposition [63] TAATGCTGAAGG-3]) wound model associated with faster wound healing Chondroitin sulfate and acharan Male ICR mice incisional Enhanced wound healing activity [64] sulfate wound model Hyaluronan Nondiabetic and diabetic rat Enhanced antibacterial activity and wound [66] models healing activity Chitosan/poly(vinyl alcohol) Male spragueeDawley rats Enhanced cytokines and collagen production, [65] incisional wound model and accelerated wound healing Pectin In vitro wound healing Enhanced antibacterial activity, fibroblasts [67] model proliferation, and wound healing activity

AgNPs. The association of AgNPs with pectin wound dressing enhanced both its antibacterial greatly enhanced their antibacterial activity and wound healing abilities. Singh and Singh against both Escherichia coli (gram negative) [57] prepared AgNPs embedded in chitin mem- and Staphylococcus epidermidis (gram positive) branes and tested their antimicrobial wound bacteria. Moreover, it enhanced the wound heal- healing ability. They found that 100 ppm AgNPs ing activity through accelerating fibroblasts in chitin membranes showed promising antimi- proliferation. crobial activity against common wound Regarding antimicrobial dressings, they were pathogens. found to decrease the risk of multiple infections and provide a favorable environment for pro- moting the normal healing process. Nanosilver 3.2 Gold (Au) nanoparticles dressings possess unique features for wound management due to their antimicrobial effects Besides the various known therapeutic appli- in controlling infection and inflammation, ability cations of gold nanoparticles in tumor therapy to balance moisture content in the wound and and diagnosis, the use of gold nanoparticles manage epithelial regeneration. Nowadays, (AuNPs) in wound healing, alone or with other natural biomaterials (collagen, gelatin, chitosan, antioxidants, has been investigated owing to its fibroin, and keratin) and their derivatives are antioxidant properties and its skin penetration used as constructing materials for biodegradable capabilities through interaction with skin lipids wound dressing [54]. One of the most advanta- and opening of the stratum corneum [59,69]. geous biodegradable polymers used in the fabri- The combination of AuNPs with two antioxi- cation of wound dressings is chitosan. AgNPs in dants, epigallocatechin gallate (EGCG) and chitosan dressing was synthesized by Lu et al. a-lipoic acid (ALA), by Leu et al. [69] showed [68] and its wound healing activity was accelerated wound healing on diabetic mouse compared to sulfadiazine dressing and plain wound through promoting the proliferation unloaded chitosan dressing. AgNPs-chitosan and migration of dermal fibroblasts and kerati- dressing showed 21% and 15% acceleration in nocytes. Complete wound healing in the group wound healing rates compared to sulfadiazine treated with the AuNPs/antioxidants combina- and mere chitosan dressing, respectively, and tion was exhibited by the seventh day after hence, the incorporation of NPs in the mentioned injury. By further investigation to elucidate the 3. Inorganic nanoparticles 83 mechanism of wound healing of the system, it 3.3 Metal oxide nanoparticles was found that AuNPs/antioxidants combina- tion accelerated wound healing through exhibit- Some studies investigated the use of metal ing antiinflammatory and antioxidant effects. oxides as zinc oxide (ZnO), titanium dioxide Significant increase of vascular endothelial cell (TiO2), and iron oxide for wound healing pur- growth factor and angiopoietin-1 protein expres- poses. Therefore, examples of the therapeutic sion was shown after 7 days, whereas CD68 potential of these nanoparticles will be provided protein expression decreased and Cu/Zn in the following section. superoxide dismutase increased significantly in 3.3.1 Zinc oxide nanoparticles the wound area in the group treated with the combination compared to other groups, thus Zinc oxide NPs (ZnONPs) are incorporated explaining the significant antiinflammatory and into a variety of wound healing skin coatings antioxidant effects of the combination of AuNPs due to their proven antimicrobial and/or anti- with other antioxidants. Similarly, Huang et al. fungal properties, which becomes even more [70] reported high acceleration in wound healing pronounced in the NPs rather than microparti- in diabetic mice wound model upon combining cles forming due to the higher surface-to-volume AuNPs with EGCG and applying the mixture ratio of the former. through topical gas injection using the GNT For control of postoperative wounds, GoldMed Liquid Drug Delivery. As similarly ZnONPs were fabricated and impregnated in encountered with the other authors, a significant cefazolin nanofiber and compared with individ- increase of the vascular endothelial cell growth ual components. The combination achieved high factor on day 7 and the Cu/Zn superoxide entrapment efficiency and sustained release dismutase expression from day 3 to day 7 was behavior for ZnO. Higher antimicrobial activity reported. Moreover, a significant increase in was achieved with ZnONPs and cefazolin com- collagen I, III and hyaluronic acid protein bination of 1:1 w/w. A conducted in vivo study expression was detected in the wound area after on Wistar rats showed higher wound healing 7 days. rate in the group receiving the ZnONPs/cefazo- Recently, AuNPs alone were used as antioxi- lin combination compared to plain cefazolin and dant to overcome the oxidative stress generated ZnONPs-loaded dressings, which was related to during wound healing process delineated by their enhanced cell adhesion, epithelial migra- the increased ROS production in the wound tion, hence faster and more efficient collagen area during photobiomodulation therapy synthesis [72]. Raguvaran et al. [73] synthesized (PBMT), which is a technique that depends on ZnONPs and embedded them in a biodegrad- light application to stimulate cellular function, able matrix with a known reported wound heal- cellular migration, angiogenesis, and enhance ing activity (sodium alginate-gum acacia quality of wound. Typically, ROS is generated hydrogel matrix) with the aim of decreasing as an active by-product during the healing pro- toxicity and increasing efficacy of ZnONPs. The cess, especially with PBMT, which if not embedded NPs showed significant antibacterial restricted would damage DNA, RNA, protein effect on P. aeruginosa and Bacillus cereus at lower and inhibit growth. The application of AuNPs concentration than ZnONPs alone. with powerful antioxidant effects in this case resulted in significant increase in wound healing 3.3.2 Iron oxide nanoparticles rate owing to enhanced epithelialization, The application of different forms of iron NPs collagen deposition, and fast vascularization in wound healing has also been recently [71]. reported. Moradi et al. [74] attempted a triple 84 4. Nanoparticulate systems for wound healing combination approach toward better manage- post injury compared to more than 18 days in ment of acute wounds, aiming for faster healing other groups treated with miR-146a or cerium and better skin characteristics after healing. They oxide nanoparticles alone. In addition the healed combined the PBMT laser therapy with the skin in the cerium oxide nanoparticles-miR146a- application of curcumin-bound iron oxide treated group was more tensile and elastic with (Fe3O4) superparamagnetic NPs, and compared improved biomechanical properties (increased the antibacterial and in vivo wound healing maximum load and modulus), suggesting their activity to other groups (control group, curcu- promising nature. min suspension group, laser only group). Results showed significantly higher antibacterial and 3.3.4 Titanium dioxide nanoparticles wound closure rate in addition to better skin strength after healing with the curcumin-bound Titanium dioxide (TiO2) has several applica- iron oxide NPs. tions in drug, cosmetics, and pharmaceutical fields owing to its therapeutic effects, safety, 3.3.3 Cerium oxide nanoparticles and corrosion resistance. It is applied pharma- fi Cerium oxide nanoparticles were reported to ceutically in the form of nanotube lms to sup- possess autoregenerative and radical- port bone and stem cells, prevent bacterial scavenging properties, which eliminate oxida- adhesion, and stop hemorrhage. TiO2NPs have tive stress in the wound area by scavenging the been applied in the area of wound healing either excess of ROS [75]. Moreover, cerium oxide alone or in conjugation with AgNPs due to their nanoparticles were reported to induce hydroxyl- germicidal and antimicrobial activities. More- proline and collagen production, resulting in over, as previously mentioned, the NPs provide increased wound tensile strength and reduced slow release of the metal, achieving better con- wound closure time activity [76]. Recently, trol of the microorganisms and better wound Rather et al. [77] fabricated cerium oxide NPs healing results). Archana et al. [78] combined functionalized with polycaprolactone (PCL)- TiO2NPs of previously reported antimicrobial, fl gelatin nanofiber (PGNPNF) by electrospinning antiin ammatory, and wound healing capabil- and evaluated their antioxidative and prolifera- ities with the biocompatible polymers chitosan tive potentials. The PGNPNF exhibited strong and PVP. The in vivo wound closure rates of antioxidant property, which was evidenced by open excision wounds in albino rat model were fi the strong ROS scavenging potential measured signi cantly higher in case of the TiO2/chito- by fluorescence microscopy. Consequently, the san/PVP combination compared to conven- viability and proliferation of cells increased by tional gauze, soframycin skin ointment, and three-fold. Moreover, it has been recently chitosan-treated groups with 100% complete reported that diabetic wound healing is usually wound closure after 16 days whereas other fi impaired due to increased inflammation and groups required signi cantly longer periods. decreased expression of the regulatory micro- Similar results were obtained by Javanmardi RNA (miR-146a), which is a key regulator of et al. [79] who synthesized TiO2/gelatin compos- fi inflammatory response. Therefore, a new study ite and compared its wound healing ef cacy on by Zgheib et al. [75] conjugated miR-146a to burn models in male albino rats to different cerium oxide nanoparticles and tested its antiin- groups (control group, group receiving silver flammatory, antioxidant, and wound healing sulfadiazine, and group receiving gelatin-based efficacy in diabetic wound model. The group ointment). The best results were achieved with treated with 100 ng of the nanoparticles in the TiO2/gelatin combination, hence delineating conjunction with miR146a showed faster healing the composite as promising wound healing rate, where wounds were fully closed at day 14 modality. 3. Inorganic nanoparticles 85

3.4 Copper (Cu) nanoparticles Owing to the several advantages of the biode- gradable polymer chitosan, it has been exten- Copper was reported to exhibit a powerful sively investigated as a dressing material on wound healing activity, owing to its antimicro- fl wounds and burns with different nanosystems. bial, antiin ammatory, immune boosting, angio- Besides its biocompatibility and biodegrad- genesis enhancing, and antioxidant properties. ’ ability, chitosan possesses an inherent antimicro- The copper s antioxidant activity is exhibited bial property that made it an excellent polymer by acting as cofactor for enzymes such as super- for wound dressings, especially when combined oxide dismutase and cytochrome oxidase. It aug- with drugs or NPs for wound healing. Recently, ments immunity by stimulating the production Jayaramudu et al. [82] synthesized two types of of interleukin-2, and stimulates angiogenesis chitosan-capped copper nanocomposites, which through induction of vascular endothelial showed good antibacterial activity elicited by growth factor (VEGF) expression. For better large inhibition zones, with better activity activity in wound healing, its presentation in against gram-negative E.coli. compared to nanoform as copper nanoparticles (CNPs) gram-positive Bacillus microorganism. would provide more catalytic activity and better bioavailability. Newly emerging approaches have been tried to minimize the toxicity of 3.5 Silicon nanoparticles CNPs and increase their efficacy [80]. One of them was to find safer methods of production Silicon is an abundant trace element in the hu- of these nanoparticles such as biosynthesis man body, which was reported to exhibit a from microorganisms and green synthesis from controversial effect on skin, bone, and blood ves- plants. Moreover, coating of the nanoparticles sels. Silicon-based formulations, such as gels, with biocompatible and biodegradable polymers dressings, bioactive glass ointment, and silica has been reported as an alternative approach. gel fiber fleeces, have been reported to be effec- CNPs were biosynthesized from P. aeruginosa tive in wound healing. It was proposed that and their wound healing activity was tested they act as both excellent dressing medium by in vivo on rat excision wound model. The pace providing favorable environment for healing of wound healing was faster in case of CNPs and also perform a crucial action in healing than control and native copper group with 92% through being released from the dressing, and healing rate achieved in only 10 days in CNPs reaching the dermal and epidermal layers. It treated group [80]. Coating and stabilization of was assumed that they work by increasing CNPs was tried by other authors using different epidermal and dermal fibroblasts proliferation biodegradable polymers. Xiao et al. [81] stabi- through enhancing the expression of b-FGF lized CNPs by folic acid and assessed its cytotox- fibroblasts growth factor through the release of icity on cells and wound healing on splinted orthosilicic acid Si(OH)4 molecule. Therefore, excisional dermal wound model in diabetic the formulation of silicon in the form of silicon mice. Lower cytotoxicity and enhanced cell NPs would provide a slow release of the active migration was recorded in folic acidestabilized molecule in addition to being more easily inter- CNPs group, which was attributed to the slower nalized, thus providing more efficient control release of copper ions from the composite. More- of the wound [83]. over, folic acidestabilized CNPs composite Furthermore, the loading of silicon nanopar- enhanced angiogenesis, collagen deposition, ticles with drugs or bioactive molecules with and reepithelialization, consequently resulting the aim of increasing the efficacy of wound heal- in faster wound closure rates. ing has been tried. A recent study loaded 86 4. Nanoparticulate systems for wound healing

Flightless I (Flii) siRNA into porous silicon nano- antioxidant, antiproliferative, and antibiofilm particles, which is an actin remodeling protein properties of selenium NPs. Rostami et al. [86] that increases in wounds and is responsible for studied the in vivo wound healing activity of wound progression. The siRNA of this protein selenium NPs prepared using chitosan as modi- is responsible for silencing the protein, hence fier and stabilizer on rat wound excision model, interfering with wound progression and pro- in which the animal group treated with sele- moting wound healing. The loading of siRNA nium/chitosan NPs showed significantly higher in NPs is expected to overcome the drawbacks rate of new blood vessels formation and fibro- of applying this molecule alone, such as its blasts proliferation, hence higher wound healing inability to cross cell membranes, its sensitivity, rate. and degradation by the endogenous nuclease enzyme. NPs would also provide sustained release of the siRNA, providing an additional 4. Conclusions advantage for its nanoencapsulation. For better control of the release and augmented escape Nanoparticles proved to be a versatile plat- from endogenous siRNA degrading enzymes, form for wound healing purposes. Whether the system was coated with a chitosan layer, they were organic or inorganic in nature, their and the in vivo wound healing potential of the unique properties cause them to overcome the prepared system on acute excisional wounds problems of conventional treatment modalities was tested in comparison to siRNA alone and and induce improved treatment outcome. With siRNA-unloaded NPs. Significant reduction in the advancements in the discovery of functional wound area (20% after 6 or 7 days) was observed materials, futuristic studies on composite nano- with the siRNA NPs system compared to other particles customized for wound healing are groups and control group. Therefore it was expected to increase, and to eventually replace concluded that the proposed chitosan-coated the conventional therapies. siRNA-silicon NPs can effectively deliver a suffi- cient dose of siRNA to the wound to induce wound closure and healing [84]. References [1] Das S, Baker AB. Biomaterials and nanotherapeutics for enhancing skin wound healing. Front Bioeng Bio- 3.6 Selenium nanoparticles technol 2016;4:82. [2] Amer SS, Nasr M, Mamdouh W, Sammour O. Insights on the use of nanocarriers for acne alleviation. Curr Selenium is an essential trace element, with Drug Deliv 2019;16(1):18e25. several reported medical applications such as [3] Bseiso EA, Nasr M, Sammour O, Abd El Gawad NA. prevention of cardiovascular diseases, cancer, Recent advances in topical formulation carriers of anti- hypercholesterolemia, and diabetes. Its recently fungal agents. Indian J Dermatol Venereol Leprol 2015; e proposed use in wound healing is attributed to 81(5):457 63. [4] Hatem S, Nasr M, Elkheshen SA, Geneidi AS. Recent its inherent antioxidant property, in addition to advances in antioxidant cosmeceutical topical being a central constituent of many antioxidant delivery. Curr Drug Deliv 2018a;15(7):953e64. enzymes and vitamins, which makes it an excel- [5] Li Z, Liu M, Wang H, Du S. Increased cutaneous lent candidate for use in wound healing prepara- wound healing effect of biodegradable liposomes con- tions when formulated as NPs [85]. Selenium taining madecassoside: preparation optimization, in vitro dermal permeation and in vivo bioevaluation. NPs were synthesized from Streptomyces minuti- Int J Nanomed 2016a;11:2995e3007. scleroticus M10A62 bacteria ,which was isolated [6] Bsieso EA, Nasr M, Moftah NH, Sammour OA, Abd El from magnesite mine and proved the Gawad NA. Could nanovesicles containing a References 87

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Solid dispersions: technologies used and future outlook N.B. Jadav, A. Paradkar Centre for Pharmaceutical Engineering Sciences, Faculty of Life Sciences, University of Bradford, Bradford, United Kingdom

1. Introduction To enhance the dissolution profile of poorly soluble APIs, various approaches are being Over the past 2 decades, due to the develop- investigated in drug research and development ment of fast throughput screening and combina- stage such as salt formation, particle size reduc- torial screening tools, most of the pharmaceutical tion, prodrug design, complexation reaction, mi- development sectors are facing a major challenge cro/nano emulsions, solid-lipid nanoparticle with dissolution and solubility performance of generation, and solid dispersion (SD), which poorly soluble active pharmaceutical ingredients seem to be promising strategies to increase the (APIs). According to the Biopharmaceutics Clas- dissolution profile of hydrophobic drug entities. sification System (BCS), a drug moiety is catego- Numerous studies are being conducted using the rized as a poorly soluble drug when the highest above-mentioned approaches, and among them dose of the drug is not soluble in 250 mL of SD strategy has displayed various advantages aqueous medium over wide pH range at 37 C. in enhancing the dissolution and solubility pro- The BCS has categorized these drug molecules file of poorly water-soluble drugs. SDs are in Class II (see Fig. 5.1). These drug molecules defined as the dispersion consisting of one or retain poor aqueous solubility and good perme- more API moiety dispersed in inert carrier ability according to the pH of the gastrointestinal matrix prepared using solvent, solvent-melting fluids. These drugs, due to poor solubility and method, or melting method [3]. In the SD, the dissolution rate, have bioavailability perfor- API entity can be dispersed as a separate parti- mance that is hindered. Therefore, one of the cle, crystalline, semicrystalline, or amorphous major challenges in pharmaceutical drug discov- form. The carrier entities are inert molecules in ery and development is to find out different the form of amorphous or crystalline state. SD novel ways to enhance the solubility and disso- has various advantages like molecular level par- lution rate of these drugs [1,2]. ticle size reduction, change in the API crystalline form to amorphous form, drug polymorphism,

Nanopharmaceuticals https://doi.org/10.1016/B978-0-12-817778-5.00005-1 91 © 2020 Elsevier Inc. All rights reserved. 92 5. Solid dispersions: technologies used and future outlook

TABLE 5.1 Marketed products based on solid dispersion technique [4].

Dosage Product Model drug Carrier type form

Certican Everolimus HPMC Tablet Cesamet Nabilone PVP Tablet Gris-PEG Griseofulvin PEG Tablet Isoptin Verapamil HPC/HPMC Tablet SR-E FIGURE 5.1 fi Biopharmaceutics classi cation system. Nivadil Nivaldipine HPMC Tablet as well as enhanced wettability and porosity. Rezulin Troglitazone HPMC Tablet Despite a wide range of advantages, the pres- Kaletra Lopinavir, PVPVA Tablet ence of SD in the development of pharmaceutical ritonavir formulations failed in achieving expected market Intelence Etravirine HPMC Tablet success; as of now, only limited products are available using this concept (Table 5.1). Zelboraf Vemurafenib HPMCAS Tablet This disappointing outcome is due to scalabil- Incivek Telaprevir HPMCAS-M Tablet ity issue, as physicochemical instability is Crestor Rosuvastatin HPMC Tablet observed during processing or storage, leading to phase separation and phase transformation Afeditab Nifedipine Poloxamer/ Tablet CR PVP from amorphous to crystalline state. Therefore, fi to overcome such limitations of SDs, in-depth Fenoglide Feno brate PEG Tablet knowledge and understanding on physical and Prograf Tacrolimus HPMC Capsule chemical properties of API and carrier, process- Sporanox Itraconazole HPMC Capsule ing and characterization techniques and mecha- nism of SD or matrix formation are required to ensure the preparation of marketable and 2.1 First generation productive SDs. The first-generation SDs are referred to as crystalline SDs, in which crystalline API is 2. Classification of solid dispersions dispersed in a crystalline carrier like urea, lactose, etc., to form a eutectic or monotectic fi SDs are classified based on the physical state mixture. Pharmaceutical SD was rst generated of the carrier and API entity and composition. by Sekiguchi and Obi, who dispersed crystalline sulphathiazole in crystalline urea (carrier) to A. Chemical composition form eutectic or monotectic mixture [5].In fi a. rst generation eutectic mixture, the melting point of the mixture b. second generation is below the melting point of individual compo- c. third generation nents (API and carrier), whereas in a monotectic d. fourth generation mixture the melting points of API and carrier fi e. fth generation (Fig. 5.2) molecule stay constant. Among both, eutectic B. Physical state solid mixture displayed enhanced dissolution a. crystalline SDs rate as both the drug and carrier molecule crys- b. amorphous SDs tallize during a cooling phase, which results in 2. Classification of solid dispersions 93

FIGURE 5.2 Classification of solid dispersions based on composition and their properties. a well-dispersed drug-carrier matrix. Addition- used as a crystalline carrier because of its solubi- ally, the processing temperature for the eutectic lity in water and various organic solvents; it is mixture is much lower than that of the monotec- suitable for solvent-assisted SD techniques. tic mixture. Phase separation or phase crystalli- Whereas sugar-based carriers are less soluble in zation is observed in case of the eutectic water and retain high melting temperatures, mixture if the mixture composition of API and which limit their use in melt and solvent-based carrier is not exactly similar to the eutectic SD techniques. Okonogi et al. investigated the mixture. Very limited studies are available that change in dissolution rate of ofloxacin by prepar- display the exact composition of eutectic ing SD with urea and mannitol. Both urea- and mixture [6]. mannitol-based SDs displayed enhanced disso- In drug-carrier complex, if the drug molecule lution rate; in comparison to ofloxacin-urea, SD is present in crystalline state as a separate mole- displayed higher dissolution rate compared to cule then the SD is called as crystalline solid ofloxacin-mannitol SD. Based on thermal anal- solution; if the drug molecule replaces or substi- ysis and X-ray diffraction studies, the author tutes the crystal structure of carrier molecule, it is concluded that urea-based SD retains the higher referred to as substituted crystalline solid solu- capability to reduce the crystallinity of drug tion; or if it occupies the internal structural compared to mannitol [7]. spaces in the crystal lattice of the carrier, then it is called an interstitial crystalline SD. Most 2.2 Second generation commonly used crystalline carriers in the first generation SDs are sugars such as sorbitol, In the second-generation SDs, polymer mate- mannitol, etc. and urea. Of them, urea is often rials are generally the amorphous carriers to 94 5. Solid dispersions: technologies used and future outlook generate SDs. Based on the physical state of the polymer in most of the melting techniques or drug, the SDs can be classified as amorphous very suitable polymer when the drug molecule SD and amorphous solid solutions (glass solu- is sensitive to high temperatures. In contrast to tions). In amorphous solid solution, both the PEG is PVP, which retains high melting temper- drug and carrier material are completely ature with relatively high glass transition tem- miscible with each other, resulting in formation perature, so it is used in solvent-based SD of a homogenous phase of amorphous liquid so- techniques. Except for crospovidone, all the lution. In amorphous SD, the drug molecule is above-listed polymers are soluble in most of dispersed in the carrier matrix, forming two the acceptable organic solvents, therefore, SDs separate phases; these amorphous suspensions can be prepared using solvent-assisted tech- are formed when the drug molecule has limited niques. These polymers can also improve the solubility with the carrier due to its chemical wettability of the drug molecule and enhance nature or due to the extremely high melting the drug release rate. Crospovidone, however, point. In amorphous SD, the drug particles are is not soluble in water, as when brought in con- suspended or dispersed in very small size tact with water it swells immediately, leading to (molecular, amorphous, or crystalline particles), enhanced drug release rate. The chain length or and due to forced solubility of the drug in this molecular weight of these polymers often con- carrier, they exist in a supersaturated state. trols the aqueous solubility and viscosity of the When these amorphous SDs are dissolved in polymers. Polymers with high molecular weight the aqueous solution, the amorphous carrier retain less aqueous solubility and high viscosity, can either enhance the wettability or dispersibil- which prevents the recrystallization of the drug ity of the drug or even inhibit the precipitation of during preparation, storage, and dissolution. the drug from the carrier matrix. Thus due to the However, with such high-molecular-weight above properties of amorphous carrier, fast polymers (PVP), the dispersion of drug in the dissolution rate of an amorphous carrier in water polymer matrix is hindered, leading to delay in enhances the drug solubility and release rate [8]. the dissolution rate of the drug when the SDs Based on their origin, amorphous polymers are dispersed in water. On the other hand, if are classified as synthetic polymers and natural we select polymers with low molecular weight polymers. The synthetic polymers include poly- (PEG), this can make the SD very soft and sticky ethylene glycol (PEG), crospovidone (PVP-CL), in nature, which challenges the further process- polyvinylpyrrolidone-co-vinyl acetate (PVPVA), ing steps like making tablets. There are various povidone (PVP), and poly(methyl acrylate). The factors that need to be considered while prepar- natural polymer includes hydroxyl-propyl meth- ing second-generation SDs, such as thermal ylcellulose (HPMC), methylcellulose, hydroxy- properties of drug and polymer, polymorphic propyl cellulose (HPC), hydroxyl-propyl properties of the drug, and most important, in- methylcellulose phthalates (HPMCP), hydroxy- teractions between the drug and carrier mole- propylmethylcellulose acetate succinate cule. There are few examples where the drug (HPMCAS), sugar glass (sucrose, trehalose, molecule interacts with the polymer matrix inulin), and starch (potato starch and corn through hydrogen bonding and effective plasti- starch) [9]. Among all the polymers listed above, cizing property of the polymer. Karavas and PEG, PVP, and HPMC are the most widely used. group reported this interaction when they inves- PEG is a semicrystalline structure with low tigated the SD of felodipine with different types melting point (below 65 C) regardless of its of PVP and PEG carrier molecule [10]. The Four- molecular weight. Therefore, due to its low ier transform infrared spectroscopy (FTIR) data melting nature, PEG is a widely accepted revealed the formation of hydrogen bonds 2. Classification of solid dispersions 95 between felodipine (FELO) and both the poly- affect negatively due to precipitation or recrys- mer materials. The felodipine drug moiety was tallization of the drug during the processing present in the form of crystalline microparticles stage or during storage. This recrystallization in FELO-PEG SD, and in FELO-PVP SD complex will reduce the drug concentration, which has the drug particles are dispersed in the form of been shown in both in vitro and in vivo studies. amorphous nanoparticles; this is due to the pres- Thus, to further increase the stability of the ence of weak hydrogen-bonding interactions in second-generation SDs, certain surface active FELO-PEG SD compared to strong hydrogen- agents are added in third-generation SDs. In bonding interactions in FELO-PVP SD. Kohri this case, surfactants are used to increase the bio- and team investigated the dissolution and solu- pharmaceutical performance of the saturated bility profile in vitro and in vivo for albendazole SDs. Apart from increasing the dissolution pro- SDs formulations with HPMC and HPMCP. In file by adding surfactants, the physical/chemical both cases, he observed enhanced dissolution stability and wettability of drugs in polymer ma- and bioavailability compared to physical mix- trix is also enhanced. The most commonly used tures. Therefore, these cellulose-based natural surfactants employed are poloxamer, gelucire, polymers are widely used in various marketed inutec, Compritol 888 ATO, Soluplus, sodium SD products [11]. lauryl sulfate (SLS), Tween 80, polyethylene hy- Some polymers are used to prevent the release drogenated castor oil, and sucrose laurate. of drug in the stomach and further release in the Poloxamer has been used for a long time as a car- intestinal region; these polymers are enteric rier in SD and soluplus has been used in recent polymers like HPMCP, HPMCAS, etc. A major- years due to its superior performance in hot ity of the drugs are released in the stomach melt extrusion processes. The drug molecules region and absorbed in the small intestinal in such carrier material exist as crystalline micro- region, and to enhance the in vivo bioavailability particles or in an amorphous state based on the of drug some gastric resistant polymers are used. drugecarrier ratio. Ali et al. prepared SD of Zhang et al. have previously reported the use of ibuprofen and ketoprofen by using two different HPMCE5 polymer carrier for immediate release grades of Poloxamer 407 and 188. FTIR data and HPMCP55 and HPMCAS for enteric release revealed the disruption of ibuprofen and keto- of SD of fenofibrate (FB). The group has reported profen dimer structure, forming concomitant that there was a significant improvement in the hydrogen bonding with the carrier molecule. in vivo bioavailability of FB-HPMCAS granules When the drugecarrier is processed in 1:2 M ra- in comparison with HPMCE5 granules [12]. tios the drug is present in amorphous form, and in higher drug load the drug molecule coexists as amorphous and crystalline phase [13]. In another 2.3 Third generation report, Van der Mooter et al. introduced a novel carrier called Inutec SP1 (produced by the reac- In the third generation, SD surfactants (sur- tion between the polyfructose backbone and iso- face active agents) or self-emulsifiers are intro- cyanate), which displayed a potential increase in duced as carriers or additives into the SDs to the drug release rate [14]. further enhance the stability of the SDs by pre- venting precipitation or recrystallization of drugs from polymer matrix during processing 2.4 Fourth generation or during storage. In second-generation SDs, because of forced solubility the drug molecule Controlled-release SDs (CRSDs) are included is present in the supersaturated state, it may in fourth-generation SDs. This is generally for 96 5. Solid dispersions: technologies used and future outlook drug molecules with low water solubility or drugs with short biological shelf life. CRSD fi systems are designed to ful ll two prominent (3) requirements: enhancement of solubility and achievement of extended release of the drug. In these systems, when poorly soluble drug moiety is dispersed in water-soluble polymer matrix, its solubility is enhanced, and when dispersed in (4) water-insoluble polymers they swell upon con- (2) tact with water to release the drug in a controlled Drug release (1) manner [15]. This CRSD can promote the controlled release of drug from polymer matrix for longer duration, which in turn enhances the patient compliance, prevents multiple dosing, (0) decreased side effects, and promotes the pro- longed therapeutic activity of poorly soluble drug Time entities. Most conventionally used polymer car- FIGURE 5.3 Comparison of dissolution profile of solid riers for CRSD are carboxyvinylpolymer (Carbo- dispersion in all four generations in comparison with pure pol), ethyl cellulose (EC), hydroxypropylcellulose API. (0) pure API, (1) first-generation, (2) second- (HPC), poly(ethylene oxide) (PEO), and Eudragit generation, (3) third-generation, and (4) fourth-generation solid dispersion [4]. RS, RL. These polymers are poorly soluble mate- rials, which upon contact with water start swelling pure drug. In the second-generation SD one slowly, which delivers the entrapped drug mole- can see faster and increased dissolution rate cule in a prolonged period of time. Based on the compared to the first-generation SD. In third- type of the drug release, these CRSDs are catego- generation SD, where surfactants are added, rized into two basic mechanisms, diffusion and we can observe faster and increased dissolution erosion. Ohara et al. reported the sustained- rate compared to the second-generation SD, release SD dissolution of indomethacin and EC and we can also attain lower precipitation rate and HPMC in 1:1 M ratio. It was investigated and extent of supersaturation rate. Finally, in that at lower pH hydrophobic interactions occur fourth-generation SDs there is enhanced zero- between indomethacin and EC, which strongly order dissolution profile as controlled-drug delayed the release rate of the drug. Trans and release takes place. his group prepared the PEO-based CRSD of ace- clofenac by adding gelucire 44/14 poloxamer as 2.5 Fifth generation surfactant carrier and sodium carbonate as pH modifier. Due to the swelling property of PEO Recently, fifth-generation SDs are increas- and the presence of surfactant and pH modifier, ingly being reported. These SDs are generated the dissolution rate of the drug was reduced signif- to enhance the functional performance of the icantly compared to pure drug [16]. Fig. 5.3 repre- final product by generating nano- and micron- sents the dissolution profile of a drug in all the four size structures [17]. This fifth-generation SD generations of SDs. came into focus due to multidisciplinary In the first-generation crystalline SD, we can approaches including SD, polymer science, fluid see the increased dissolution rate compared to dynamics, nanotechnology, and nano-science. In 3. Structure-based classification of solid dispersion 97 previously mentioned fourth-generation SDs, 3. Structure-based classification of solid CRSDs are prepared where the drug plasma con- dispersion centration in the body is maintained for a longer period of time; with this formulation, initially Based on the structure or molecular state, the when the SD is administered into the body this SDs are classified as solid solutions, glass solu- often results in a burst release of the drug parti- tions or glass suspensions, and eutectic mixture. cles present on the surface. Due to burst-release mechanism, the drug is immediately available, causing rapid action followed by a controlled 3.1 Solid solutions release for a longer period of time. Therefore, to control or predict the burst mechanism is diffi- Herein, SD is the mixture of drug and carrier cult and this burst mechanism can adversely molecule; in this type, based on the miscibility affect the therapeutic properties of the formula- of drug-carrier complex they are categorized as tions developed. To overcome this issue, the a continuous or discontinuous solid solution. In fifth-generation SDs are being developed. Due continuous solid solution, a drug can be added to the advancement and development of in any proportion with the carrier molecule as different nanotechnologies, both “top-down” the miscibility and molecular interactions and “bottom-up” methods have generated between the drug and carrier are stronger than different varieties of nanoparticles that can be individual components. In discontinuous solid used in varied drug delivery systems, including solution, the miscibility of each component is fifth-generation SDs. The complex nano-sized limited in the solid solvent. Based on the molec- structures developed are core-shell, Janus, and ular size, the solid solutions are categorized as triple-layered structures containing both organic substitutional and interstitial solid solutions. In and inorganic materials; all these nano-sized substituted SDs the solute molecules replace or architectures are frequently used in pharmaceu- substitute in the crystal lattice structure of tical drug delivery systems. In core-shell nano- solvent, whereas in interstitial solid solution structures, both the core and shell can have the solute molecules are present in the interstitial different compositions of the drug, polymeric gaps or spaces of the solvent component. material to control the release of drug when Fig. 5.4A,B represent the schematic molecular administered. In all the generations, from first arrangement of substitutional and interstitial to fourth, the drug release is based on the phys- solid solutions. icochemical properties of the drug and carrier fi molecule, whereas fth-generation SDs provide 3.2 Glass solution programmed and controlled functional perfor- mance of both drug and carrier molecule used In this glass solution, the drug molecule is to produce nano-structures of SDs. completely dissolved in the glassy solvent to

FIGURE 5.4 Schematic structure of (A) Substitutional and (B) Interstitial solid solutions. 98 5. Solid dispersions: technologies used and future outlook

All Melt dispersed or dissolved in this carrier layer and if the viscosity of this layer is high, then the

TB drug diffusion through this concentrated layer TA is the challenge and the limiting factor is the Liquid diffusion of this concentrated layer into the bulk solutiondthis kind of drug release mecha- Melt + A Melt + B Solid nism is referred to as carrier-controlled release. In drug-controlled release mechanism, the drug E is either insoluble or sparingly soluble in the concentrated carrier layer; in this case, the disso- Crystals A + B Temperature (in °C) lution profile depends on the status of the drug A Composition (molecular % or weight %) B particles, like polymorphic form, particle size, and drug solubility in water. Ideally in every FIGURE 5.5 Phase diagram of eutectic mixture: A-drug, SD, these two drug release mechanisms occur B-carrier, and E-Eutectic point [19]. simultaneously as the drug may be less soluble or entrapped in the concentrated layer of the generate homogenous phase or the drug mole- carrier. Various scientific studies are being cule is suspended on the surface of glass solvent. reported that explain the correlation between the dissolution rate and concentration ratios of 3.3 Eutectic mixtures drug carrier in SDs. The dissolution rate of SD with a higher concentration of carrier molecule In a eutectic mixture, when the drug and is enhanced as the drug is completely dispersed carrier molecule are mixed with each other and or as an amorphous state to form homogenous heated, they melt at a single melting temperature drug carrier complex. In such SDs, the mecha- lower than individual component melting nism involved is drug-controlled mechanism. points. Fig. 5.5 represents the phase diagram In carrier-controlled release mechanism, the explaining the melting temperatures and phys- carrier forms a thick concentrate layer that acts ical state of individual components and eutectic as a barrier for the drug to diffuse through the mixture. In the figure, we can observe that the layer. Karavas and group reported these two melting temperature of the mixture is lower mechanisms by preparing an SD of felodipine than the individual component melting points. and two types of carriers, PVP and PEG [20]. This temperature points (E) of the drug and They proposed that at low drug concentrations carrier molecules are homogeneously dispersed the drug diffusion through the polymer layer is to form eutectic mixture [18]. carrier-controlled drug release and at high drug concentrations it is drug-controlled release mechanism. Therefore, to enhance the dissolu- 4. Drug release mechanism from SDs tion profile of the drug in SDs, apart from considering the polymorphic state or solubility Based on the drug release from carrier matrix, of drug, drug release mechanism, carrier viscos- the mechanisms in SDs are categorized into two ity, carrier-drug solubility, drug-carrier concen- types: carrier-controlled release and drug- tration, and swelling or gelling property of controlled release. When SD is dispersed in the carrier need to be considered [9]. aqueous medium, the hydrophilic carrier readily The above two mechanisms are commonly absorbs water or dissolves in water to form a observed mechanisms in first-, second-, concentrated (gel) layer of carrier. If the drug is and third-generation SDs. Whereas in the 6. Challenges in SD development 99 fourth-generation SDs, which are controlled The interactions between the drug and the release SDs, the release mechanisms are based polymer matrix inhibit the drug from agglomer- on diffusion and erosion of drug from the car- ation, and the drug concentration in the polymer rier matrix. The miscibility of drug and carrier matrix is in the supersaturated state, which plays an important role to determine whether further enhances the dissolution rate. the release mechanism is through diffusion or Solid orals (tablets, capsules) are the most through erosion. When the drug molecule is accepted dosage forms, and SD formulations completely miscible or well dispersed in the can be easily formulated into solid oral dosage polymer matrix, then the release of drug forms compared to other techniques like salt through the polymer matrix is via diffusion formation, emulsions, or suspensions. mechanism; if the drug is not miscible or not Most of the drugs present in SDs are in the well dispersed in the polymer matrix (present form of amorphous or semicrystalline state, in two different phases), then the release mech- which eventually enhances the bioavailability anism is through erosion. and dissolution rate as less energy is required to break the crystal lattice compared to crystal- 5. Applications of SDs line drug particles. Furthermore, in SDs the drug particles are either dispersed in the carrier matrix or entrapped in the carrier matrix, thus In SD formulations, the drug particles are in gaining further advantages due to swelling, hy- molecular size as they are molecularly dispersed drophilic, stabilizing, and solubilizing properties in the carrier matrix. Whereas in other conven- of carrier molecules. tional techniques, for size reduction the particle SDs are highly porous materials (mainly SDs size is in micron level (2e5 mm); there are few produced using solvent evaporation technique), sophisticated nano-sizing approaches, and they which retain higher specific surface area, which are very time-consuming, less stable (require helps to attain higher dissolution rate compared stabilizers), and expensive. The dissolution to powder blend. Zang et al. while preparing the profile of particles generated from conventional SD of felodipine using film freeze-drying tech- size-reduction technologies is hindered as the nique, have observed a 40 times increase in micron-sized particles are less stable, undergo dissolution rate of felodipine compared to agglomeration during formulation development conventional crystalline powder material [12]. and during storage [21]. In SDs, as the drug molecule is present in higher saturated conditions, they can get 6. Challenges in SD development absorbed immediately in the dissolution medium and enhance the drug release profile. Despite various advantages of SDs, very few In SD formulations, the drug particles are either marketed products based on SD technique are present in the amorphous state (when available. Even though SD enhances the drug completely miscible with polymer matrix) or dissolution profile for poorly soluble drug nano-sized crystalline particles (when less entities, it also has a major limiting factor of miscible in polymer matrix), which are thermo- stability during processing and storage. During dynamically unstable and readily get solubilized processing and storage of SDs, the drug mole- in the dissolution medium. Thus, even they cules that are dispersed in the carrier matrix as undergo agglomeration if the particle size of semicrystalline or amorphous particles undergo agglomerates is in submicron size (<1 mm), recrystallization into crystalline state, which which have much less effect on dissolution leads to a decrease in bioavailability. As the profile and release rate. 100 5. Solid dispersions: technologies used and future outlook drug molecule is present in the supersaturated Selection of suitable SD techniques is a prom- state in carrier matrix under the influence of inent challenge identified by researchers and external stresses like heat, humidity, shear while manufacturing companies. In the melting tech- cutting or packaging enables the drug molecule nique, drug and carrier molecules undergo ther- to undergo structural mobility and attain its mal degradation. In the solvent evaporation stable crystalline form and recrystallize out of method, the presence of residual solvent in the the complex. During recrystallization, initially structural complexity of SD is the major under the influence of external stress, the crystal- challenge. Recrystallization of drugs in solidifi- line nuclei of the drug is generated, which is cation technique is a major drawback. Due to further followed by crystal growth. Therefore, these physical, chemical, and thermal instabil- molecular mobility of the drug is the key factor ities, the drug dissolution rate is hindered, low that needs to be considered to attain physical in vivo ein vitro correlation and scalability and chemical stability of SD. issues. Molecular mobility in the drug chemical Effects of processing conditions also need to structure can occur in two ways: be considered. Ayenew et al. reported the effect of compression force during tableting on the (a) Global mobility (a-relaxation)dwhere the miscibility of naproxen-PVP K25 SDs. The FTIR molecular structure completely undergoes studies revealed that the compression pressure rotation or structural translation. above 565.05 MPa leads to phase separation. (b) Local mobility (b-relaxation)dwhere only This is when 30%e40% w/w of naproxen con- part of the molecular structure undergoes centration is compressed during table processing movement, and this occurs for a shorter and the miscibility of the amorphous drug and period of time than global mobility [22].In carrier material is lost due to weakening of the the presence of moisture, the crystallization hydrogen bond interaction between the drug is often rapid, as the water brings down the carrier material, resulting in amorphous- T of amorphous material due to plasticizing g amorphous phase separation [25]. effect, therefore resulting in an increase in Various screening studies, practical theories, molecular mobility. When the temperature and models are being developed by research of the SD increases, this external stress (heat) groups globally to enhance the physicochemical also causes molecular mobility. The stability and performance of SDs. Some theories temperature range (50 C below the are focused on the selection of starting materials, transition temperature Tg) effect of some on processing techniques, and some on a-relaxation is completely negligible or designing surfactants (additives) to enhance the absent, therefore the b-relaxation molecular stability. mobility causes instability in the amorphous Selection of polymer and drug moiety is the SD. Therefore, to attain physical and first important step to consider in attaining chemical stability of the SDs, processing and desired stability of SDs. Polymers generally storage temperature and humidity have high glass transition temperature (T ) conditions needed to be considered and g compared to drug molecule, so this polymer ma- optimized. terial will increase the Tg of the miscible drug- Other important factors that hinder the polymer complex and inhibit the molecular commercial performance of the SD are the poor mobility of the drug. Some polymer materials scalability and uneconomical processing and are hygroscopic in nature, so when exposed to manufacturing conditions [23,24]. moisture they will absorb water and this 7. Techniques for solid dispersions generation 101 increases the drug mobility due to plasticizing used nonionic surfactant (Tween 60) and anionic effect. Selected polymers need to be completely surfactants (SLS, dioctyl sulfosuccinate [DSS]) to miscible with the drug by generating strong prepare the SD of ketoprofen with PEG-15000. hydrogen bonding between the drug and poly- The authors stated that in the absence of surfac- mer. This hydrogen bond force is the main force tant the dissolution rate of ketoprofen was that will prevent the drug from recrystallization enhanced, but in presence of surfactant the during processing and storage conditions. dissolution rate was further enhanced compared Therefore, the selection of nonhygroscopic poly- to pure drug. This further increase in dissolution mer, polymer with high Tg and polymer having rate in the presence of surfactant is due to strong hydrogen-bonding interactions with increase wettability and decrease in surface drugs, is one of the strategies to increase the tension on the drug. The surfactant concentra- stability of the drug in SDs [11]. Vansanthavada tion in the medium is below the critical micellar et al. investigated the correlation between the concentration, and the degree of crystallinity of solubility and phase separation kinetics of SDs the ternary SD (drug-polymer-surfactant) is far in griseofulvin-PVP and indoprofen-PVP. They below the degree of crystallinity of binary SD studied the solubility and phase separation (drug-polymer). They have proposed that the kinetics by performing accelerated stability at effect of surfactants depends on the type of sur- 40 C/70%RH for 3 months. Using FTIR as the factants used, the anionic surfactant is more analytical tool, they reported that griseofulvin effective than nonionic surfactants due to higher did not show any solid-state miscibility with hydrophilic properties of anionic surfactants PVP, whereas indoprofen displayed 13%w/w compared to nonionic surfactants [26]. solid-state miscibility with PVP. The higher miscibility of indoprofen was justified as the hydrogen-bonding interactions between indo- 7. Techniques for solid dispersions profen and PVP. Therefore, the authors generation concluded that the physical-phase separation be- tween the drug and polymer is proportional to Apart from developing strategies to enhance drug-polymer interactions and concentration of the stability of SDs, the scientists also focused drug in the SD. on developing novel techniques to prepare SD Surface active agents or surfactants are added based on the targeted formulation design, phys- externally as additives to SDs to enhance their ical and chemical properties of starting mate- stability. Due to the amphiphilic property of sur- rials. To date, all the techniques developed are factant, it enhances the miscibility of the drug in based on the solidification of the liquid system. the carrier matrix and reduces the chances of Therefore, based on the working fluid types recrystallization upon processing and storage. and their properties, the technologies can be When melting technique is used for the prepara- sorted into three categories (see Fig. 5.6): tion of SDs, these surfactants can reduce the pro- cessing melting temperature due to its (1) Solvent method: includes spray drying, plasticizing property. These surfactants, when lyophilization, coprecipitation, used in solvent-based techniques, can get electrospinning, vacuum drying, super- dissolved in the wide range of organic solvents critical fluid technology, and fluidized bed- and act as a solubilizing or wetting agent to coating technologies. enhance the solubility of drug and carrier mate- (2) Melting method: includes fusion, hot melt rials and prevent precipitation. This effect was extrusion, and melt agglomeration investigated by Mura et al. where the authors techniques. 102 5. Solid dispersions: technologies used and future outlook 7.1 Solvent evaporation method

Solvent evaporation method is one of the most commonly used techniques for preparation of SD of poorly soluble drug components. This technique was implemented for the first time in 1965 by Tachibana and Nakamura for prepara- tion of SD of beta-carotene (API molecule) using PVP as carrier matrix. The authors dissolved these two moieties (drug and carrier) using chlo- roform as a common organic solvent, and further, on solvent evaporation, SDs of beta- carotene and PVP complex were generated. FIGURE 5.6 This solvent-based SD technique is based on Manufacturing techniques of solid two major principles: (1) preparation of homog- dispersions. enous solution of drug and carrier matrix by dis- solving them in a suitable volatile organic (3) Melt-solvent methods: includes both the solvent system, (2) second step is solvent evapo- aspects stated above. ration step where the solvent is removed from the solution under constant external agitation Table 5.2 represents the overview of different resulting in the formation of SD mass. As the techniques applied to generate SDs of poorly sol- temperature required for this method to form uble drug molecules. SD is very low, it gains its importance over

TABLE 5.2 Overview of different technologies used to generate solid dispersions of poorly soluble drugs (ES: elec- trospinning) [17].

Applied energy Products

Main AdditiveTechnology Feed format Drug form Size

Mechanical e Ball milling Powder/Liquid Crystalline/Amorphous Nano/Micro

Thermal Spraying Solution Amorphous/Crystalline Micro Thermal Fluidized bed coating Solution Amorphous/Crystalline Micro Thermal Mechanical Fusion Solid Amorphous/Crystalline Macro Electrostatic e Solvent ES Solution Amorphous Nano Mechanical Melt extrusion Solid Amorphous/Crystalline Macro e Freeze drying Solution Amorphous/Crystalline Macro

Thermal Elevated T ES Solution Amorphous Micro Thermal Melt ES Melt Amorphous Micro Thermal Electrospray Solution Amorphous Micro Microwave Mechanical Microwave Solution Amorphous Macro Thermal/Mechanical Microwave Melt Amorphous Macro 7. Techniques for solid dispersions generation 103 melting method in preparing SDs of heat- organic solvent. Kumar et al. used a solvent sensitive drug and carrier molecules and carriers mixture of the dioxane-butanol-water mixture molecules with high melting temperatures can to prepare amorphous SD of opioid molecules also be used with low melting drug molecules [29]. Another disadvantage of these techniques as they are dissolved in common volatile organic is the cost of processing to remove the residual solvent system. Thus, the main advantage of this organic solvent; specialized instruments are technique is that it prevents thermal decomposi- required, which inhibits the scalability of this tion of drug and carrier maintaining its thera- technique. peutic performance [5,14,27]. An important Like melting technique, solvent evaporation aspect of this technique is the sufficient solubility method also determines the physical state of of drug and carrier in the selected solvent sys- the SD. Rapid evaporation techniques are tem. As the carrier molecule is hydrophilic and preferred to generate amorphous SD. There are the drug molecule is hydrophobic in nature, various techniques developed to enhance the sol- finding the suitable solvent is sometimes very vent evaporation process and generate stable difficult. Most commonly used nontoxic organic SDs such as: solvents are ethanol, methanol, acetone, ethyl ac- 7.1.1 Spray drying etate, acetone, methyl chloride, methanol-water mixture, etc. Sometimes to further enhance the Spray drying technique is an efficient and solubility in an organic solvent, surfactants like scalable drying technology used in pharmaceu- Tween-80 or SLS are used, but in very small tical and food industries. In this technology, the quantity so that the crystalline structure of the solvent evaporation occurs within seconds, carrier molecule is not altered. In 1966, solution resulting in the fast transformation of drug- evaporation SD technique was implemented by polymer solution into drug-polymer particles. Mayersohn and Gibaldi in preparing SD of gris- In this technique, the API-carrier molecules are eofulvin and PVP polymer using chloroform as solubilized or suspended in the common solvent solvent system. Upon SD, the dissolution rate or solvent mixture, forming homogenous solu- of griseofulvin was enhanced by 11X compared tion or suspension, respectively. Further, this to its physical mixture or pure drug molecule solution or suspension is passed through the pre- [28]. Since then this technique has been imple- heated nozzle via a pump system and atomized mented to enhance the solubility and dissolution into droplets. Due to the large surface area of rate of various poorly soluble, thermolabile these fine droplets when atomized into hot air drugs like azithromycin, flurbiprofen, cilostazol, stream, rapid solvent evaporation occurs, result- diclofenac, abetic acid, piroxicam, indomethacin, ing in the formation of fine particles with loratadine, efavirenz, and tectorigenin. enhanced solubility and dissolution rate The only limitation of this technique is the (Fig. 5.7). This rapid evaporation time is enough presence of residual solvent even after solvent for the generation of amorphous SDs, but some- evaporation step. This residual solvent can cause times based on the solubility of the drug in the toxicity, and if the solvent system is water mole- solvent system, crystalline drug-polymer parti- cules, it may cause plasticizing effect by reducing cles are also generated. The size of the particle the Tg of the SD causing phase separation over a mass can be optimized by controlling some period of time or during stability. Therefore, to process parameters like input-output tempera- overcome or to reduce the issue of residual ture, feed rate, atomization, and air pressure. solvent in the SD mass, certain researchers This spray drying process gained its importance used solvent mixture consisting of water and in processing various SDs due to its continuous organic solvent instead of using pure 100% manufacturing process, low cost, high yield, 104 5. Solid dispersions: technologies used and future outlook

peristaltic pump through an atomizer or nozzle. Drying Atomizer This atomizer can be pressure nozzle, rotatory gas nozzle, or two-fluid nozzle; the atomization pro- Liquid feed cess occurs due to pressure, centrifugal or kinetic Drying energy, respectively. Through this nozzle, fine chamber liquid droplets are generated in the chamber Exhaust gas wherein the presence of hot air stream sudden solvent evaporation occurs, causing the transfor- Cyclone mation of liquid droplets into solid particles. The solid mass is separated from the air stream Dry particles in the cyclone and collected in the collection collector chamber below. The spray drying process can FIGURE 5.7 Schematic representation of spray drying be used for liquid solutions, suspensions, process [23]. emulsions, slurries, and pastes or melts. The spray drying process allows two configu- and efficient molecular dispersion of drug in the rations: open-loop or closed-loop configuration. polymer matrix. Commercially available spray- In open-loop configuration, air is used as the dried based SD products are Incivek and Inte- drying gas and the gas is not circulated; it is lence [30]. This spray drying technique is used the most commonly used configuration as it is to enhance the solubility and bioavailability pro- cost effective and stable. In closed-loop configu- file of various poorly soluble and thermolabile ration, inert gas like nitrogen is used as drying drugs like valsartan, spironolactone, nilotinib, gas and is recirculated; it is generally used to pre- and artemether [31]. Herbrink et al. have used vent explosions caused due to the mixing of this spray drying technique to prepare the SD gases, and it is used when oxygen-sensitive ma- of poorly soluble nilotinib with Soluplus (poly- terials are processed. The flow of the drying gas mer) in 1:7 wt./wt. ratio. They have reported is categorized into two types based on the direc- that SD of nilotinib displayed an increase in sol- tion of flow: (1) cocurrent flow, and (2) counter- ubility profile of the drug by 630-fold compared current flow (Fig. 5.8). to pure drug [21]. Similarly, Pawar et al. in In cocurrent flow, the direction of the drying another study reported the preparation of SD gas is in the same direction of the atomization of artemether: Soluplus in 1:3 wt./wt. ratio liquid flow; this type of gas flow is useful for increased drug release rate by 4.1-fold higher heat-sensitive materials and the air temperature than the pure drug (20%) [31]. is low. In counter-current flow, the drying gas The spray drying process represented in flow direction is in the opposite direction to at- Fig. 5.7 consists of four fundamental steps: (1) at- omization liquid flow; in this case, the liquid omization of the drug-carrier liquid solution/ droplets come in contact with hot air. In this sce- suspension, (2) drying process: where the liquid nario, counter-current flow directions cannot be feed is sprayed into the drying chamber, (3) par- used for heat-sensitive materials. To obtain a ticle generation: upon solvent evaporation fine desired stable solid product by using spray particles are generated in the chamber and drying process, different parameters like process cyclone, and (4) collection and separation of parameters, properties of liquid feed, and config- dried particles into the collector from the drying uration and design of equipment need to be gas. Firstly, homogenous solution or suspension considered. Faster feed rate, higher nozzle diam- of drug and carrier is prepared, and then this eter, and higher drug concentration in the liquid liquid mass is fed into the drying chamber by a feed lead to the formation of larger particles. 7. Techniques for solid dispersions generation 105

any major changes or modifications. The yield (A)Atomization (B) Atomization percentage of the products is also very high. Using the spray drying process one can use Drying different physical states of liquid feed such as gas solutions, pastes, emulsions, and suspensions. It is widely used as a suitable technique to pro- cess thermolabile ingredients, high melting poly- mers, and poorly soluble actives. The decomposition of active ingredients is very min- Drying imal as the exposure time of the liquid droplets gas with the hot air stream is very minimal. This technique became more attractive to the researchers to generate encapsulated stable FIGURE 5.8 Schematic diagram of (A) cocurrent flow drugs with polymeric nanoparticles, microparti- and (B)counter-current flow against liquid atomization flow cles, or microcomposites. The flow properties of direction [23]. the powders generated by this technique are high, so they can be used for inhalation drug Whereas higher atomization pressure, low delivery systems, tableting processes or for nozzle diameter, and high surface tension render filling capsules. smaller particles. The porosity of the dried solid Regardless of the various advantages of spray mass depends on the rate of evaporation of the drying technique, there are certain limitations solvent system; a higher rate of evaporation gen- while using this technique. Percentage of prod- erates highly porous materials due to a shorter uct yield is the major factor that needs to be time for the droplet shrinkage. Table 5.3 repre- considered carefully; the yield percentage sents the major parameters that need to be depends on the scale of the spray dryer. In a considered while working with the spray drying large-scale process, the yield percentage is high process. because the fraction lost in the increasing pro- Spray drying technique is widely used in the duction volume is less compared to the small- chemical, pharmaceutical, food, cosmetic, and scale spray dryer. Generally, the low yield materials industries. This technique has gained percentage is because of the material sticking to in importance in both lab-scale and industrial the walls of the drying chamber; if the exhaust setup because it is scalable, continuous process, pressure is not sufficient, particles less than less costly, and a single-step process without two-micron size may escape through the

TABLE 5.3 Spray drying adjustable parameters [23].

Equipment design Process parameters Liquid feed

Geometry of atomizer Pressure Concentration of API: Carrier Cocurrent or counter-current flow Gas inlet/outlet temperature Boiling point of solvent Mixed flow Drying rate Viscosity of liquid feed

Drying gas used Density Feed rate Surface tension Percent aspiration 106 5. Solid dispersions: technologies used and future outlook exhaust. However, in a large-scale process, 3 h, the solution was freeze-dried for over 24 h certain air filters are used to prevent the loss of to generate SDs of a nevirapine-dextran complex micron-sized small particles from escaping with higher dissolution rate compared to SDs through exhaust. generated using kneading technique. Altamimi et al. had previously reported the application 7.1.2 Vacuum drying and rotatory evaporation of lyophilization technique to generate SDs In vacuum drying and rotatory evaporation with enhanced physicochemical and in vitro technique, moderate heating is involved so that properties. They generated the SDs of nifedipine the drug and carrier molecules are homoge- and sulfamethoxazole by using Soluplus and neously dissolved in the solvent, and on further PEG 6000 as carrier molecules, respectively, vacuum drying solid mass is generated. In this and reported that SDs of both the API molecules technique, to prevent the degradation of the display enhanced dissolution and solubility drug and carrier material moderate temperature properties compared to SDs generated using conditions are applied. Once the vacuum drying conventional technique [33]. This technique process is completed, the resultant solid mass is was later used by S. Kaur et al. to prepare SDs stored in the vacuum desiccator for complete of anticancer drug exemestane with phospho- removal of residual solvent. Although this lipid/sodium deoxycholate as carrier molecules; process is quite straight forward, its processing their investigation reports reveal that the absorp- is tedious and during the drying step the chances tive transport and dissolution rate of SD of the of phase separation or recrystallization are high. anticancer drug was 4.6-fold greater in compari- This technique lacks in scalability due to process son to pure drug [24]. Furthermore, Singh et al. time and the presence of residual solvent [32]. in 2011, prepared certain selected SDs by using the minimum amount of cyclohexanol as a sol- 7.1.3 Freeze drying (lyophilization) vent. They found that upon lyophilization of Lyophilization or freeze-drying technique of different SDs, porous, fluffy, and lightweight the drug and carrier complex is not subjected solid materials were generated. The selected SD to thermal stress during the formation of SD. solution in cyclohexanol was freeze-dried by This is an alternative route to solvent evapora- sending the solution into the inner surface layer tion technique in which the drug and carrier ma- of a rotating cold flask in 50 C methanol terial is dissolved in the suitable solvent and then bath. Once the certain thickness of the material this solution in the presence of liquid nitrogen is was solidified on the surface of the flask, for frozen to form lyophilized SD. This technique is the removal of residual solvent, the solvent more suitable for thermolabile drug and carrier was further sublimated under pressure in the materials, which are unstable in the liquid state lyophilization apparatus at 8e10 mmHg but stable in the dry state for the prolonged stor- at 75 C [34]. age period. This technique further resolves the Apart from the various advantages of the problem of phase separation as soon as the solu- lyophilization technique, there are various limi- tion is vitrified. Lokamatha et al., in 2011, used tations as well: (1) high cost of preparations of this lyophilization technique to prepare SDs of SDs, (2) lengthy processing time is required for low-soluble nevirapine API molecule with novel the completed removal of residual solvent, (3) se- dextran carrier material at different concentra- lection of common solvent for both the drug and tions ranging from the drug:carrier 3:1 and 1:1 carrier molecule is a difficult task, as most of the w/w ratio. Initially, the SD solution of drug organic solvents retain low freezing temperature and carrier was prepared using water as the and do not stay frozen during freezing or subli- solvent system. After mixing the solution for mation step, (4) the sample temperature during 7. Techniques for solid dispersions generation 107 freezing process has to be maintained below the particles generated using the SFD process exhibit Tg of the frozen concentration fraction for a higher chemical and physical stability compared longer time period, therefore longer time is to conventional lyophilization process. required when low temperature is required for This property was investigated by Van the materials to undergo solidification, and Drooge et al., prepared SDs using SFD tech- (5) sufficient vapor pressure is required for the nique; authors observed higher physicochemical solvent during the lyophilization process. stability compared to conventional lyophiliza- tion process, especially in higher supersaturation 7.1.4 Cryogenic processing concentration of drug in carrier matrix [35]. The cryogenic process is similar to the lyoph- In comparison to other conventional tradi- ilization process, but the freezing rate is rapid in tional techniques, the cryogenic process has the cryogenic process compared to the lyophili- gained importance in preparing SDs of thermo- zation process. There are two technologies devel- labile components, as spray drying and melt oped based on the cryogenic process: spray- methods are not always suitable for thermo- freeze-drying (SFD) and ultra-rapid freezing labile components. As SFD and URF techniques (URF). In the URF process, cryogenic solid are operated under subambient temperature substrates with thermal conductivity between conditions the degradation of the materials is 10 and 20 mK are used. In this technique, the minimal, and as the liquid particles are rapidly solution mixture of drug and carrier is applied cooled the percent crystallinity is very minimal to the cryogenic substrate and further frozen. in comparison with traditional techniques. The frozen particles are separated from the 7.1.5 Supercritical fluid technology substrate and further lyophilized for the removal of residual solvent. As the super-fast cooling Supercritical fluid (SCF) technology was process is involved, the resultant solid products introduced in the late 1980s and early 1990s. are amorphous in nature as due to rapid cooling The technology is referred to as “supercritical” the nucleation of the crystalline API molecule is as the substance used in this technique is in its prevented. In the SFD cryogenic process, the supercritical state. The supercritical state is drug-carrier solution is sprayed on to the liquid achieved when the processing temperature and nitrogen or supercooled air stream, resulting in pressure conditions are above the critical point. the generation of solid SD material. When the Previously, in 1897, Hanny and Hogarth drug dispersed in carrier solution is sprayed reported that the solid materials processed by through the nozzle, liquid droplets rapidly turn SCF technology display narrow particle size into frozen droplets when brought in contact distribution (nano and micron size) without con- with supercooled air or liquid nitrogen stream, taining any residual solvent [36]. This technol- and then these frozen droplets are further lyoph- ogy is referred to as novel green technology as ilized for removal of residual solvent. SFD no organic solvents are used while processing allows the generation of nano- and microparti- materials; the most commonly used solvent is cles without subjecting to external stress or carbon dioxide (CO2) in its supercritical state mechanical forces, preventing the degradation due to its lower critical temperature and pres- of API and carrier materials. Due to the higher sure conditions. The basic principle of this tech- surface area of the liquid droplets with direct nology is that the drug and carrier molecules contact with cooling stream, rapid vitrification are dissolved or dispersed in the supercritical occurs, preventing phase separation or genera- solvent (CO2) at critical temperature and pres- ¼ e tion of crystalline drug nuclei. Apart from sure conditions (Tc 30 35 C and ¼ e preventing the phase separation issue, the Pc 70 75 bar) and sprayed through the nozzle 108 5. Solid dispersions: technologies used and future outlook into the expansion or collecting chamber at supercritical drug-carrier solution, precipitation lower pressure conditions. Due to the rapid or particle generation occurs once the solution expansion of the materials, change in density is added to antisolvent. In this technique, the ma- and increase in solvent power causes rapid jor drawback is the use of large volumes of nucleation of the drug and carrier molecules to organic solvent as antisolvent and the presence occur, resulting in the formation of SDs with of fractions of residual solvent in the final SDs. desired particle size distribution in a very short Adeli et al. in 2016, used this SAS technique to time period. SCF technology, due to its favorable generate SD with enhanced dissolution rate of operating conditions (lower temperature, high- low-soluble irbesartan API molecule with polox- dissolving property), gained tremendous impor- amer 407 (carrier molecule) in 1:1 w/w ratio. As tance in generation of SDs and various other a result, the SD generated displayed 14-fold in- pharmaceutical formulations with desired parti- crease in the dissolution profile of irbesartan cle size distribution. Apart from the lower critical SD compared to pure drug [39]. This SAS tech- temperature and pressure conditions, the use of nique was further used to increase the biological nontoxic, nonflammable, and inexpensive sol- activity of apigenin by preparing nanocrystals of vents (CO2) made this technology attractive in apigenin. In drug and formulation development processing industries. At supercritical state, the processes, supercritical fluid technology is a CO2 gases act as the solubilizing agent with potentially scalable green technique used to high diffusibility, low viscosity, and low surface enhance the biological activity, dissolution, and tension enables the user to precisely control the solubility rate of various poorly soluble drug solubility of various drugs and carrier molecules moieties; the only drawback of this technique is [19,37,38]. To date, scientists have used this not all the drugs and carrier molecules are solu- principle of SCF technology to develop various ble in the supercritical solvent systems. techniques of particle generation, such as super- 7.1.6 Coprecipitation method critical antisolvent technique (SAS), rapid expan- sion from supercritical solution (RESS), solution In coprecipitation (CP) technique particles are enhanced dispersion by SCF (SEDS), precipita- generated based on the principle of antisolvency. tion from gas saturated solutions (PGSS), precip- In this technique, first, the carrier molecule is itation with compressed fluid antisolvent (PCA), dissolved in the suitable organic solvent and to and gas antisolvent technique (GAS). In RESS this solution drug is incorporated under stirring technique the drug and carrier molecules are dis- conditions, forming a homogenous solution solved in the supercritical fluid and sprayed containing drug and carrier moieties. Finally, to through the nozzle into the expansion vessel at generate particles, to this solution water (which lower pressure conditions, resulting in the gener- acts as antisolvent) is added dropwise under stir- ation of SDs of drug and carrier complex. This ring conditions, which results in precipitate for- technique is wide because the processing time mation. The resultant suspension is filtered and is less, and no organic solvents are used in this washed to remove residual solvent and then technique. In the supercritical antisolvent tech- dried. This CP technique is suitable for prepara- nique, organic solvents are used as antisolvent tion of SDs of poorly soluble drugs and drugs to generate particles. In this technique the drug with a high melting point, which are difficult and carrier molecule is dissolved in the super- to process using melting or other solvent critical solvent to form supercritical solution, methods. In this CP process, the resultant pow- then this solution at lower pressure conditions der material is called a “microprecipitated” is added to the antisolvent in a controlled bulk powder containing both drug and polymer manner. Due to supersaturated conditions of materials. Most commonly used polymer 7. Techniques for solid dispersions generation 109 materials in the CP process retain pH-dependent 7.1.7 Electrostatic spinning solubility, including HPMC, HPMCAS, polyme- Electrospinning technique came into focus thacrylates, cellulose acetate phthalate, polyvi- due to multidisciplinary research that was con- nyl phthalate, and polymethylmethacrylate ducted between nanotechnology, pharmaceu- [40]. The selection of solvent systems is also tical science, and fluid dynamic sciences. This very important as the common solvent should technique is considered as a combination of have higher solvency power so that it can nanotechnology and SD technology. In this tech- dissolve both the drug and carrier materials. nology, with the drug and the carrier, the mole- The most commonly used solvent systems for cule is dissolved in the suitable common solvent poorly soluble drugs, high melting drugs, and to form a homogenous solution or homogenous long-chained-polymer matrix are dimethylaceta- melt solution; this drug-polymer solution is mide, N-methyl-pyrrolidone, and dimethylfor- delivered through the millimeter-scale nozzle mamide. This CP process is scalable and a to generate amorphous SDs in the form of widely practiced process due to its suitability nano- or microfibers. In this technology, high to a wide range of drug and carrier materials voltage between 5 and 30 kV is applied to the and due to the absence of residual solvent as it tip of the nozzle or needle to induce electrostatic is washed by water during the washing step. charge to the drug-polymer solution. The region fi The ionic properties of polymer matrix at speci c between the spinneret and collector vessels is pH conditions promote the precipitation of drug also electrically charged with a fixed voltage, and polymer materials in the form of SD solid and when the electric forces dominate the sur- mass. A commercial product in the market pre- face tension of the feeding solution at the air pared by this technique is Zelboraf [41].Itis interface, polymer jets are ejected. The solution, very important to consider that the solvent and after coming in contact with air under fixed antisolvent system used in the coprecipitation electric voltage, due to the instability caused process can act as the plasticizer, resulting in between the surface tension of the solution and an increase in molecular mobility and crystalli- electric voltage coils spirally, and as the solution zation. Therefore, monitoring or optimizing comes out from the nozzle through the electric precipitation process, selection of a suitable field the solvent evaporates, resulting in genera- solvent, and the antisolvent system are very tion of micron or submicron-sized fibers on the fi crucial factors to achieve required nal formula- spinning mandrel or collector base. The collected tion or SD. Dong et al. [4] previously reported fibers can be used directly as an oral dosage form the comparison between the hot-melt extrusion by incorporating it into the capsule or further process and coprecipitation process in gener- processing, like milling. Though the electrospin- ating SDs. They selected BCS class II drugs and ning process seems to be quite a straightforward HPMCAS as a carrier molecule to generate SD. process, it involves a very critical correlation Based on the X-ray diffraction data, both the between rheology, fluid dynamics, and electro- techniques generated amorphous SDs but the dynamics. This process is comprised of four CP-processed SDs displayed faster dissolution steps: (1) formation of the Taylor core near the rate compared to melt extrudates. This is due tip of the spinneret, (2) ejection of polymer jet fi to higher porosity, the speci c surface area of from the cone when the surface tension of the particles generated from the CP process solution is dominated by electric field, (3) the compared to extrudates. 110 5. Solid dispersions: technologies used and future outlook

FIGURE 5.9 Schematic representation of single-fluid electrospinning and electrospraying and their applications [17]. twinning or bending of the polymer jet, and but due to repulsion forces (Fc) between the adja- (4) collection of solid fibers onto the collector. cent filaments caused due to coiling slowdowns, Fig. 5.9 represents the schematic process and the process and the third forces that enhance the fl applications of single- uid electrospinning and process is Coulombs repulsive forces (Fc). Due to electrospraying. these three forces the thinning of the filaments The spinning process is initiated when the occurs, causing quick evaporation of the solvent electric force applied to the solution overcomes and generating thin amorphous SD fiber the surface tension of the solution resulting in materials with enhanced surface area. the ejection of the thin liquid jet on to the collec- Electrospraying technique is like the electro- tor surface. The semivertical angle of the Taylor spinning process. In electrospraying process cone formed at the tip of the nozzle is in the charged drug-polymer droplets are generated range of 32 degrees < q < 46 degrees, the angle from the spinneret instead of fibers, resulting in of defection depends on the surface tension of the formation of spherical-shaped amorphous the solution and electrostatic applied. When we SD particles. While processing the drug- use insoluble polymer materials using coaxial polymer solution with low viscosity, we can electrospinning technique (here a concentric observe both spinning and spraying process spinneret is used where one fluid is nested or taking place. Due to lower entanglement of the inserted into other) the semivertical angle of fibers, the solution from the spinneret forms Taylor cone lies in the range of q ¼ 57 degrees. small spherical beads onto the fiber materials; During electrospinning through the capillary this beading on the fiber filaments is referred to nozzle, the interaction between the solution as the hybrid combination of nanofibers and and inner wall of the spinneret nozzle wall is microparticles. In the case of electrospray, also responsible to influence the theta angle of low-viscosity solutions are selected. We can Taylor cone. When the bulk material of the solu- observe in Fig. 5.9 that near the spinneret tip in tion comes in contact with the air stream, it electrospraying process homogenously undergoes solvent evaporation because of insta- dispersed droplets are generated initially, which bility caused due to the influence of a series of eventually under the influence of Coulombic electric forces. The attraction forces between forces (Fe) quickly shrink, causing sudden sol- the two electrodes (E) propagates the solution vent evaporation and generation of amorphous through the spinneret onto the collector surface, particles. If the solvent mass is not evaporated 7. Techniques for solid dispersions generation 111 effectively, then thin films are observed on the pellets are coated by spraying the drug- collector; therefore, both the electrospinning polymer solution in the fluid-bed coater. In this and electrospraying techniques are similar in technique homogenous solution of drug and car- terms of time and working principle [42]. rier matrix is produced by dissolving in a suit- Different process and environmental factors able common solvent; later this solution like solution feed rate, electric field strength, mixture is sprayed on the surface of nonpareil the surface tension of the solution, drug- pellets in the coating chamber. The hot air stream polymer solution dielectric constant, the distance in the coating chamber enables the solvent to between nozzle tip to collector, humidity, tem- evaporate and solution to coprecipitate on the perature, and velocity of air in the chamber surface of nonpareil pellets, generating amor- need to be considered to generate required diam- phous SDs. The final product collected can eter and morphology of filaments. Most directly be used for further tableting process or commonly used hydrophilic polymer materials for filling into the capsules without further pro- to generate amorphous SD filaments of poorly cessing. Sun et al. have reported the enhanced soluble drug moieties are PVP, HPMC, PVA, in vivo bioavailability of fenofibrate and sily- and PEO. However, for controlled release of marin SD prepared using fluid-bed coating tech- drug molecules, hydrophobic polymer materials nique. The air stream flow and temperature are are also been investigated. By using these hydro- important factors that need to be considered phobic polymer materials as carrier matrix, for complete removal of the solvent system, as advanced amorphous SD formulations or any residual solvent may act as a plasticizer dosage forms can be developed using electro- enabling the generation of crystalline nuclei spinning technology. This electrospinning that hinder the dissolution and bioavailability method gained its importance in generating of final SD formulations [45]. sustained or controlled release biomedicines; due to filament high surface area per volume there is rapid evaporation of solvent taking place 7.2 Melting method that generates stable amorphous SDs, and due to high surface energy the dissolution or drug Sekiguchi and Obi in 1961 developed the release profile is also significantly enhanced. melting methods. This melt technology is also Yu et al. in 2013, used the combination of electro- termed as fusion technology; it is called “melt” spraying and electrospinning for generation of when the two starting materials used are crystal- biphasic-controlled release amorphous SD fibers line in nature; otherwise, this technique is [43]. J. S. Pamudji et al. have prepared the SD commonly termed as “fusion method.” Fusion/ nanofibers of ketoprofen and PVA in 1:1 w/w melt method is the technique where two solid ration using the electrospinning process. To materials, i.e., drug and carrier molecule, are date, very few articles have explained the influ- melted or fused together at high shear and tem- ence of different additive materials to the drug- perature conditions to generate well-dispersed polymer solution during electrospinning [44]. solid melt mixture. The melting temperature is They reported that the dissolution profile of just above the eutectic point, which is the lowest ketoprofen nano fibers is significantly enhanced possible melting temperature point of the compared to pure drug. mixture. Then, this melt liquid mixture is further cooled rapidly, forming solid materials using 7.1.8 Fluid-bed coating different techniques like ice bath cooling with This technique is very similar to spray drying continuous agitation, immersion in the liquid ni- technique, except in this technique nonpareil trogen, forming layers on the steel surface cooled 112 5. Solid dispersions: technologies used and future outlook with ice, and then placed in a Petri dish inside 7.2.1 Hot melt extrusion the desiccator. The resultant solid mass is further Hot melt extrusion (HME) is the continuous crushed, milled, and pulverized to generate green-processing technology where the solid desired particle size distribution or it is pro- mixture of drug and carrier molecules are mixed, cessed through injection molding to develop melted in eutectic melting temperature and pres- suitable dosage forms [46]. Sekiguchi et al. sure conditions to form homogenous or hetero- prepared the SDs of sulfathiazole API molecule geneous melt phase; this melt phase is further using different carrier materials like urea, nico- passed through the orifice to generated extru- tinamide, nicotinic acid, ascorbic acid, succini- dates. HME technology attracted the attention mide, and acetamide. The melt mixture was of various research groups and pharmaceutical further cooled to generate dry solid mass by industries due to its scalability and applicability using an agitating ice bath [4]. to generate stable amorphous materials [8,48]. The main advantages of fusion methods are This technology was very frequently used in its simplicity of processing and economy of the polymer chemistry and processing industry, processing technology. This technique is more but recently in the 1980s this technology was suitable for thermolability and prevents oxida- introduced into the field of pharmaceutical tion of drug or carrier materials. Short process- development to enhance the solubility or disso- ing time of melting method at high lution profile of poorly soluble drug moieties temperatures makes this technique suitable for by generating amorphous SDs. HME is referred various thermolabile active molecules, and to as green technology as only temperature and when this process is used or blanketed with inert pressure are the processing factors and no gas then this method prevents the oxidation of organic solvent is used to generate the SDs, drug and carrier molecules. Apart from various therefore, preventing the issue of residual advantages of the melting method, it contains solvent content. In this technology, the solid some serious limitations. Firstly, very few pairs mixture containing the drug, one or two poly- of drug and carrier material are compatible mers, sometimes even plasticizer, and pH with each other to form homogenous single modify the mixture, which is heated, melted, amorphous phase during the heating step, but homogenized, and extruded in the form of tab- if the drug and carrier materials are incompatible lets, pellets, or rods, or further milled with other with each other, then inhomogeneous solid ma- additives to generated final formulation. Due to terials are formed. To overcome this issue, the presence of intense shear conditions like tem- certain surfactants or third carrier materials are perature and pressure, the drug molecules get added to enhance the miscibility of the drug in dispersed into the molten polymer matrix, and the carrier matrix. Secondly, during the cooling due to mixing, homogenous dispersion is step, the drug-carrier miscibility reduces, lead- formed. In HME process, the solid mass is trans- ing to crystallization of the drug molecule. To formed into a semisolid melt phase or viscous overcome this issue, a fast-cooling step is used, mass due to intense temperature and pressure as upon slow cooling crystalline nuclei are gener- conditions. To reduce the processing tempera- ated and upon fast cooling amorphous material ture and pressure conditions for high-melting is formed. Thirdly, due to use of high tempera- drug and polymer molecules, plasticizers are ture and shear conditions, in fusion method added; due to the addition of plasticizer, the Tg various drug and carrier materials undergo of the mixture is reduced to lower melting thermal degradation during processing [47]. 7. Techniques for solid dispersions generation 113 temperatures. The melting step of the materials Not all the polymer materials are suitable for in the HME process is mainly based on the phys- the HME process. In order to select the suitable ical, chemical properties of drugs and polymer polymer material, Hansen solubility parameters and also rheological properties of polymers. can be used to determine the drug-polymer The hot melt extruder can be divided into four miscibility. Drug molecules with high miscibility zones: (1) Feed: Input materials are placed in the in polymer matrix result in the generation of feed either as a physical mixture of the drug and highly amorphous SD with enhanced drug polymer or are placed separately in the feed; dissolution profile. Polymer needs to act as a sol- (2) Melting zone: Predominately, this screw orien- vent or cosolvent, should retain higher solubiliz- tation in the zone is made up of only conveying ing or wetting property, show weak or strong elements. Temperature for this zone should be molecular interactions with drug molecule, offer kept according to the melt of the physical a wide variety of release profiles, and have a mixture of the drug and polymer; (3) Mixing wide range of glass transition temperatures (Tg). Zone: This is the most important zone. The type Despite the use of high-shear and temperature of SD formed is dependent on the uniform mix- conditions, HME process has considerable ad- ing of the melt. Screw configuration of this zone vantages such as (1) lower residence time where consists of only the kneading element (Fig. 5.10). the drug and carrier are at elevated temperature The orientation of the kneading elements gov- in the extruder, preventing the degradation of erns the type and texture of the SD product; molecules such as thermolabile drug moieties; (4) Homogenous Discharge: If a uniform SD melt (2) compared to other melting methods or con- mixture or eutectic mixture is formed in the mix- ventional solvent methods, this HME process is ing zone, then discharge zone is responsible for a continuous, green process that is efficient, conveying of the melt. The screw configuration easily scalable, and generates end products of this zone consists of only metering elements. with high thermodynamic stability; and (3) appli- Additionally, this zone is responsible for cability to a wide variety of drug delivery sys- uniformly passing the mix through a die to tems such as sustained-release tablets, produce the extrudates (Fig. 5.11). controlled-release tablets, immediate-release

FIGURE 5.10 Schematic diagram of twin screw extruder [49]. 114 5. Solid dispersions: technologies used and future outlook

CONVEYING MIXING

Feed Screws ++++++++ +

30° 60° 90° 30 deg Forward +++++ ++ Kneading elements Conveying element 60 deg Forward ++++ +++++ (A) (B) 90 deg Alternate Zero ++++++++

conveying staggered mixing 60 deg Reverse - - - - (C) (D) ++++++

Reverse Feed Screw ------++ dispersive mixing discharge

FIGURE 5.11 Configuration of twin screws (top left), screw elements (right), and mixing zones (bottom left) [50]. tablets, ophthalmic ocular inserts, amorphous molten mass to pass through the extrusion chan- granules, pellets, and transdermal delivery sys- nel continuously, resulting in a decrease in resi- tems. HME also has several disadvantages, dence time (approximately by 2 min) and including (1) due to high local temperature and preventing the drug and carrier degradation pressure conditions, processing low-melting due to thermal stress. This technique is suitable APIs or thermolabile carrier materials may result for drug molecules sensitive to oxidation and in degradation; and (2) similar to the fusion pro- hydrolysis as moisture is removed during the cess, the selection of drug-polymer pair with heating process [51]. high miscibility is a challenge. The most 7.2.2 Melt agglomeration commonly used polymer carrier materials in the HME process are HPMC, HPMCAS, PVP, Generally, amorphous SDs prepared using PEO, PEG, and PVP-vinyl acetate. SD of fenofi- melting method are not applicable for the large brate with Eudragit E100 or PVP-vinyl acetate pharmaceutical industry because the end prod- S630 copolymer was prepared using HME tech- uct generated from the melting method is nology to enhance the drug dissolution profile hard, soft and sticky material containing poor and bioavailability. Even though artemisinin is flow properties, compressibility, which hinders thermolabile in nature, SD of artemisinin with its application in preparing tablet dosage Soluplus can be produced by processing below forms. Thus to overcome this major limiting 110 C using HME process. Recently, Meltrex, factor of melting method, an alternative tech- an advanced technology based on HME process, nique, melt agglomeration, was developed. was developed, which displayed higher success Melt agglomeration technique works on the rate in generating amorphous SDs with various principle of the melting method with a rotatory commercial products such as Isotip, Novir, processor and the carrier acts as a binder to Rezulin, Cesamet and Kaletra. generate amorphous SDs. In this process the Meltrex is the advanced version of the HME mixture is prepared in three different routes: process that is patented under the name of SD heating the mixture of drug, carrier, and excip- processing. This design works on the principle ient together with the processing temperature of HME containing two twin-screw extruders, just above the melting point of the carrier; addi- two independent hoppers that convey the tion of molten carrier to the heated mixture of 7. Techniques for solid dispersions generation 115 drug and excipient; and addition of molten car- immersion of neighboring nuclei molecules [52]. rier containing the drug to the heated excipients. In both mechanisms the crystallinity, particle In all the above-mentioned processing, a rotatory size distribution of the agglomerates is controlled processor is used to generate homogenous by processing conditions like rotatory speed and dispersion of drug, molten carrier, and excipi- physicochemical properties of the binder mate- ents. In this technique, as the rotatory processor rial. Sea et al., in 2003, implemented melt agglom- is used solid mixtures are not subjected to high- eration process to prepare SD of diazepam using shear conditions (as in HME process) and the lactose monohydrate as a binder and PEG 3000 as temperature and other processing conditions an excipient. They observed an increased dissolu- can be easily controlled; a higher binder can tion rate of diazepam at lower drug concentra- also be used to generate highly stable amor- tions due to a higher degree of molecular phous SD. In this process uniform dispersion of dispersion compared to pure drug [4]. the particles is generated; it is a green process 7.2.3 KinetiSol technique as the usage of solvent is inhibited, and thermo- labile drugs can be used without causing any KinetiSol is a recently developed process to thermal degradation of the solid materials. Two prepare SD considering certain limitations of different modes of melt agglomeration take hot melt extrusion and spray drying process. place: coalescence and immersion (see Fig. 5.12). It is very suitable for drug molecules with poor In agglomeration by distribution mechanism, solubility in organic solvents and retains a high the binder material in its molten state binds on melting point. This technique is based on fusion the surface of the primary nonmeltable particles method where the drug, carrier, and excipient and upon further distribution agglomeration of mixtures are melted rapidly by exerting heat particles takes place via coalescence between the generated from high-shear and frictional forces two neighboring wetted particles. In immersion between the particles without subjecting the mechanism, the nonmeltable particles are particles to external heat conditions. In this immersed on the surface of the molten binder technique a compounded instrument is custom and agglomerates are formed due to the designed to meet the requirements for

FIGURE 5.12 Modes of agglomeration process. 116 5. Solid dispersions: technologies used and future outlook pharmaceutical processing. This design consists and solvent-based methods is that the treatment of rotating shafts with varied designs and temperature and mixing time are lower than in several types of blades facing outwards from melting and solvent methods, respectively, mak- the shaft; during processing, due to shear gener- ing this technique more suitable for thermolabile ated because of rotation of shaft and frictional drug molecules. Due to the mixing of drug solu- forces between the particles, the product temper- tion into the molten carrier matrix, molecular ature increases causing the materials to melt dispersion of drug occurs into the carrier matrix, within the vessel. Once the desired temperature enhancing the miscibility of the drug in the car- conditions and physical state of the material rier matrix [4]. are reached, the molten mass is immediately separated from the heating vessel and subjected to cooling. Generally, the processing time in this 8. Future outlook technique is around 20e30 s and the residence time where the materials are subjected to Due to the increase in the number of drug can- elevated temperature is for 5s before cooling didates with poor water solubility, pharmaceu- the molten mass. This thermokinetic motion of tical research and development groups the particles inside the vessel is called KinetiSol diverted their interest in developing novel mech- dispersion. In this process, the drug and carrier anisms and processing technologies to overcome materials are rapidly melted and the drug is the major challenges in a wide variety of drug molecularly dispersed into the carrier matrix, candidates. Among them, preparation of amor- generating single-phase amorphous SDs. phous SD strategy has gained a great amount The feasibility studies on this technique and of interest due to its capability to enhance the sol- design were performed by LaFontaine et al., ubility and dissolution rate of poorly soluble who selected a model drug, ritonavir, with drug entities. Even though SD is considered as very low solubility, thermolabile in nature, and the most effective technique to generate amor- low rheological property, and polyvinyl alcohol phous materials, there are very few products in as a carrier molecule to prepare amorphous the market that are prepared using SD tech- SDs. This technique is a semicontinuous batch nique. This commercial failure is due to certain process where 1000 kg/h commercial batches issues related to preparation technique, stability can be processed to generate SD products [4]. during storage, and formulation development. But in recent years due to the development of novel carrier entities, technologies, advanced 7.3 Melting-solvent method characterization tools, and additives, there is new hopes to develop more SD products in the In this melting-solvent method, the solvent is future. Recently postulated advances or devel- used to dissolve the drug molecule and it is opments in SDs are: addition of certain surfac- mixed with the molten carrier material to form tants, additives (surface property modifiers, pH a homogenous molecular dispersion of drug modifier super-disintegrants); design and devel- into the carrier matrix. After mixing, the opment of novel carriers; development of dispersed solution is left for the solvent to evap- advanced analytical tools for characterization orate and form a solidified mass. As both solvent of physical and chemical properties of starting system and melting step are involved in this materials; and end products and step-by-step melting-solvent method, it is referred to as a investigation of various thermodynamic mecha- combination of melting and solvent method. nisms of synthesis process, formulation, dissolu- The advantage of this technique over melting tion, and storage. To date, continuous Abbreviations 117 investigations are being conducted to elucidate microscopy (AFM), etc., are used to analyze different mechanisms and clinical performance various properties of SDs. By using HDSC, of SDs. In recent years, stable SDs have been RHC, and chip calorimetry these advanced tech- prepared using novel carriers, or even using nologies overcome the shortcomings (slow heat- more than one carrier to prevent the recrystalli- ing and cooling rate, unwanted thermal zation and to design the release kinetics of degradation of materials) of using conventional drug molecules fulfilling patient requirement. DSC analyzer. NTA and LTA analyzers are Some of the novel carrier materials used are Sol- used to understand the thermal behavior and uplus, Pluronic, Gelucire, and Inulin. The impact miscibility of the drug in the carrier matrix. of the third carrier in the binary SD was investi- AFM is the high-resolution microscopic tech- gated by Al-Obaidi et al., who used poly [N-(2- nique used to investigate the structural, spatial hydroxypropyl) methacrylate] (PHPMA) as the arrangement and interactions of drug and carrier third polymer in drug-PVP binary SD. They re- molecules in SDs. Various other advanced ported that PHPMA interacts with various crys- analytical techniques are being investigated talline API molecules like eofulvin, phenindione, and developed to gain in-depth knowledge and progesterone through hydrogen bonding related to thermodynamic properties and mech- where PVP cannot; they observed that the anisms of SD formation. ternary SD takes longer time to recrystallize compared to binary SD. This study concluded that the addition of a third carrier that retains 9. Conclusion the capability to form hydrogen bond with API molecule can enhance the stability and also the In this chapter, we have included the classifi- solubility of the drug molecule [53]. cation of SDs based on carrier molecule and In large-scale process, KinetiSol dispersing structural arrangement of drug and carrier com- (KSD) is a well-established, novel high-shear ponents, mechanism of SD, current challenges of mixing process to generate stable SDs. As dis- SD, and current techniques and developed cussed earlier, in this KSD process the drug procedures to overcome the challenges, limita- and carrier materials are fused together with tions of SDs, and different techniques used to the help of rotating blades (kinetic energy) and generate stable SDs. Critical attributes of the thermal energy (due to friction between the par- techniques and their applications and challenges ticles and the instrument surface) without are discussed in this chapter. In-depth under- applying any external heating source. Various standing regarding the physicochemical state of investigations are underway to generate contin- SDs generated using different techniques is uous and batch processing techniques to explained, as well as elucidating the challenges produce stable and advanced SD drug and dissolution mechanisms of various poorly formulations. soluble drugs. This will help to overcome the Recently, various advanced analytical or char- scalability issues with SD techniques and poor acterization techniques are being developed to bioavailability of various drug components. access various physical and chemical properties of starting materials and end SD formulation. Some advanced techniques like high- Abbreviations performance diffraction scanning calorimetry (HDSC), Project Rapid Heat/Cool (RHC), chip AFM Atomic force microscopy calorimetry, nanothermal analysis (NTA), local- API Active pharmaceutical ingredient ized thermal analysis (LTA), atomic force BCS Biopharmaceutics Classification System 118 5. Solid dispersions: technologies used and future outlook

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Nanotoxicity: the impact of increasing drug bioavailability Juan Bueno Research Center of Bioprospecting and Biotechnology for Biodiversity Foundation (BIOLABB), Armenia, Quindío, Colombia

Medicine is to although the possibility of toxicity is still present preserve health and cure diseases arising from causes [2]; adverse reactions of nanoformulations can be affecting the body and producing symptoms established by cell viability, reactive oxygen Beware that the warming drugs are such as those species (ROS) level, and gene/protein expres- which was selected and tried sion. In this search for increased drug-loading Medical Poem (“Al-Urjuzah Fi Al-Tibb”) efficiency (DLE) [3], the implementation of inter- of Ibn Sina (Avicenna, 980e1037) national regulations is necessary by agencies including the U.S. Food and Drug Administra- tion (FDA), Organization for Economic 1. Introduction Co-operation and Development (OECD), Euro- pean Medicine Agency (EMA), Environmental Nanomedicines are currently the fastest revo- Protection Agency (EPA), National Institute for lutions in drug delivery. Nanoformulations have Occupational Safety and Health (NIOSH), and been developed based in liposomes, niosomes, World Health Organization (WHO) [4,108]. polymer-based micelles, polymersomes, dendri- Also the design and implementation of pre- meric nanostructures, and nanoparticles struc- clinical evaluation platforms for nanomedicines tures as drug delivery systems (DDSs) [1]; also, is an important topic to develop. Due to the with the discovery of the enhanced permeability interference that occurs in comparison with the and retention (EPR) effect, research into the current platforms, this requires combining development of new nanodrugs has increased in vitro models with adsorption, distribution, for a wide range of tumors. This technology metabolism, and elimination (ADME) studies has not only increased the body’s drug distribu- [5], Equally, the inclusion of a standard risk anal- tion rates, but new bioactive and less invasive ysis will be of great importance in which the formulations have also been implemented, magnitude of risk is equal to the magnitude of

Nanopharmaceuticals https://doi.org/10.1016/B978-0-12-817778-5.00006-3 121 © 2020 Elsevier Inc. All rights reserved. 122 6. Nanotoxicity: the impact of increasing drug bioavailability the hazard measurement (dose response) with as skin and inhalation [7]. It has also been the goal of determining the nanoformulation possible to achieve a decrease in the number of DDS toxicity [6]. doses due to the increase in the half-life of the The main objective of this chapter is to medication, which improves the adherence to provide the tools for the construction of an algo- treatments [8]. In this order of ideas, nanopar- rithm for the evaluation of toxicity both for nano- ticles absorption spectrum is very high due to medicines and for nanomaterials, with the small size of the nanoparticles, which is application in the industry, that will mitigate between 100 and 500 nm (and below 100 nm); the possible alterations that the massive use of consequently, they can enter the human body nanotechnology can bring to the future. through three routes: direct injection, inhalation, and orally [9], from there the nanoparticles pass to the bloodstream, are distributed in the organs 2. Nanomedicines, absorption and coupled to proteins, and then eliminated theranostics through the lymphatic pathway in breast milk, urine, and feces (Fig. 6.1) [10,11]. Although these Nanotechnology has managed to increase the facts have been proposed to design nanoformu- absorption of drugs, thus improving their lations that avoid the phenomenon of the first bioavailability. Also with the use of nanoformu- step, thus decreasing the hepatoxicity when lations it is possible to use new routes of passing from the intestine to the lymphatic administration to reach therapeutic levels, such vessels [12], it is necessary to continue

FIGURE 6.1 Pharmacokinetics of nanoparticles. 2. Nanomedicines, absorption and theranostics 123 considering the risk of the accumulation of nano- Due to the increase in research in theranostic materials in different tissues that can alter the pa- nanoparticles capable of carrying out treatment tient’s biological systems due to DNA alteration. and diagnosis with the same formulation, and Therefore, because of their lymphatic transport, in which its structure and composition are immunotoxicity is a topic of analysis in the different compared to other nanomedicines, it case of nanomedicines [13]. Additionally, after becomes a priority to reduce the lack of under- the absorption of the nanomaterials used as standing and knowledge about the pharmacoki- nanocarriers, in new drug design there may be netics of these nanoparticles, as well as to report the risk of an unwanted accumulation of the the presence of their toxic potential, in order to nanomaterials within the human body, so it is develop innovative solutions that allow the use important to emphasize the importance of the of these promising approaches without present- design of nanoformulations that allow the meta- ing adverse effects [15]. Equally, it is also neces- bolism and excretion of these compounds. It is sary to determine the environmental toxicity in also a priority to establish the pharmacokinetic the different ecosystems of the nanocomposites, phenomenon for nanocarriers in the same way especially of the engineered nanomaterials as for drug molecules (Fig. 6.2) [14].

FIGURE 6.2 Pharmacokinetic-pharmacodynamic process for drug molecules inside the human body. 124 6. Nanotoxicity: the impact of increasing drug bioavailability

(ENMs), as well as the effects of their accumula- compounds in industry, as well as in hospitals tion in the ecological niches [16]. and homes where these are applied [20]. This is how finally each nanocomposite must contain the following information for its use [21]: 3. Risk assessment process - Possible danger identified by its use. - Dose response effect and toxic potential The design of a nano-surveillance structure, in determined. which both the general community and industry - Evaluation of the consequences after can evaluate and determine the effects on the exposure to the product. environment and public health of the ENMs, - Characterization of risk. depends on several aspects, including [17]: - Implementation of harmonized methods of activity and toxicity assessment for ENMs. 4. In vitro toxicity - Implementation of a structure for data collection and sharing. Oxidative stress, cytotoxicity, and inflamma- - Recruitment of the users and analysts of the tion are the most common adverse effects of data to allow integration of the information exposure to nanomaterials [22]. As a result, of the studies in the field with those of the mutagenicity, immunotoxicity, nephrotoxicity, laboratory and the reports of the and neurotoxicity are present after prolonged communities. exposure to ENMs [23,24]. In particular, nano- materials associated with heavy metals, which Also, the initial data included for the use of are due to their high exposure, can produce car- ENMs should contain the following information diovascular disorders as well as neuronal and [18]: renal damage [25]; it has also been shown that - Implementation and design of ENM the toxic effect of nanoparticles can be synergistic formulation standards (physical and beyond the chemical nature of the nanocompo- chemical characterization, presence of sites involved [26]. Likewise, nanomaterials aerosols or suspensions). with theranostic potential represent, due to their - In vitro testing data of ENM (activity and capacity to form nanostructures, a source of toxicity). danger that must be evaluated at their cost - In vivo testing data of ENM (activity and benefit for the development of new diagnostic toxicity). tools [26a]. Therefore, it is necessary to imple- - Positive reports of mutagenicity, ment standardized protocols to evaluate the carcinogenicity, immunotoxicity, and in vitro toxicity of nanomaterials in order to pre- reproductive toxicity. dict the possible risks and damages to human health and ecosystems [27,28]. The preclinical Furthermore, these structures do not become evaluation of nanocomposites should be based functional without the active participation of on the concept of the correlation between cellular governments in having effective governance uptake and intracellular persistence, that sepa- mechanisms to determine the toxicity and de- rates the nanoparticles into four categories, gree of exposure to nanomaterials [19]. In this namely [29]: way it becomes very important to categorize the nanoparticles by their degree of toxicity - Category I: This category includes the and environmental risk, in order to know the combination of low cellular uptake with low precautions that must be taken with these intracellular persistence. 5. In vitro assay interferences 125

- Category II: In this topic a high cellular Finally, in vitro studies are needed to deter- uptake is contemplated, but the intracellular mine the toxicokinetics (ADME), which are persistence is low. used to determine the systemic availability of - Category III: Although cellular uptake is low the evaluated products prior to use in animals in this category, intracellular persistence and thus reduce the number of specimens to be occurs to a greater degree. used [35,36]. The evolution toward a quantifica- - Category IV: This item contemplates the tion of drug metabolism based on a nonanimal highest risk where uptake is high, as well as model will lead to the use of microsomes as intracellular persistence. well as the implementation of cellular systems for evaluating in vitro metabolism that promise Several aspects should be determined in the great predictive value [37]. laboratory prior to in vivo testing of nanomateri- als, including complement activation and innate immunity, platelet aggregation, the presence of 5. In vitro assay interferences destruction of blood cells, damage caused by oxidative stress, cell integrity after exposure, In this way, for a correct relation of in vitro activation of phagocytosis processes as well as toxicity, it is possible to have standardized eval- damage to the genetic material, and the onset uation protocols with high precision; therefore, of both acute and chronic inflammation [30,31]. is important to consider that nanomaterials Therefore, any process of in vitro evaluation of have physicochemical characteristics that can toxicity must take into account the production alter the biological tests to which they are of ROS, cytotoxicity in target organs, immuno- subjected, such as their ionic charge that is toxicity, and genotoxicity as risk predictive capable of changing the results of colorimetric criteria [32,33]. Equally, a rational model of assays using tetrazolium dyes such as MTT in vitro toxicity evaluation has been proposed and XTT by altering the absorbance and chang- by Jain et al. [34], where it is possible to identify ing redox potential [38]. As well, lactate dehy- the following phases of the evaluation of in vitro drogenase (LDH) assay, which is used to toxicity of any compound: determine the membrane cell integrity, can be - Identification of possible target organs of affected in nanoparticles’ evaluation [39,40]. toxicity. The vast majority of these interferences with - Design, characterization, and implementation in vitro tests are due to the optical, oxidative, of a reproducible and predictive in vitro and fluorescent properties of nanoparticles [41]; system for toxicity evaluation. also the size, chemical composition, coverage, - Design of controlled and comparative toxicity and capacity agglomeration are critical factors studies with different concentrations and involved with these tests (cytotoxicity, oxidative exposure times. stress, and proinflammatory response) [42].Soit - Selection and implementation of a set of is recommended to evaluate the presence of robust, reproducible, and, to a greater extent, interference before beginning any toxicity evalu- automatable cytotoxicity tests. ation involving nanomaterials and to use alter- - Measurement and statistical comparison of native methods such as flow cytometry to carry the cytotoxicity of novel compounds. out the tests [43,44]; in spite of this, the correct - Elucidation and determination of the toxicity inclusion of control spikes is necessary for an mechanism. adequate and unambiguous reading of the cell - Development of validation and corroboration populations involved in cytometry [45], and it studies both intra- and inter-laboratory. is also necessary to remember that before each 126 6. Nanotoxicity: the impact of increasing drug bioavailability evaluation, all nanomaterials to be tested must can be expected with their medical, so to avoid be characterized, in order to predict possible possible immunotoxicity it is necessary to deter- interference [46]. mine the use of nanoformulation and its route of Another factor that must also be considered is administration, in order to know if it will be in the synergistic combination of nanoparticles contact with cells and elements of the immune with physical agents such as light, heat, and system of the host [53]. This is how nanoparticles radiation, so the different tests must be adjusted interact with both the cells of innate and adap- depending on the model and the activity to be tive immunity, producing immunomodulation evaluated [47]. and through oxidative stress inducing inflamma- tion [54]. In addition, nanoparticles when injected into the bloodstream acquire a protein 6. Genotoxicity corona capable of triggering an uncontrolled im- mune response [55]. Thus, the evaluation of Is important to take into account that nano- immunotoxicity includes interactions with blood materials can induce genotoxicity in two ways: elements in hemolysis, coagulation, complement a direct one that occurs when entering the activation, cytokines modulation, and protein nucleus of the cell and causing direct damage binding assays; likewise, the accumulation in to DNA, and an indirect one derived by genetic the phagocytic system and its interference with damage mediated by ROS [48]. Likewise, the phagocytosis must be determined, as well as greatest amount of information about the geno- the presence of immunomodulation in response toxicology of nanoparticles has been obtained to antigens [56]. It is also necessary to determine by the in vivo comet assay, because the antimi- which assays have the highest in vitroein vivo crobial activity of the compounds can alter correlation in order to select the safest nanocom- assays with microorganisms such as in the posites in a rigorous preclinical evaluation [57]. Ames test, which is how in this type of test it In the same way, the selection of animal models has been possible to determine that the direct predictive of immunotoxicity in vivo is of crucial toxicity on the genetic material depending on importance, which depends on the intensity of the electronic properties of the nanocomposites the immune response and its comparison [49,50]. Likewise, in environmental genotoxicity between different species (rodents, dogs, rats, tests, the presence of chromosomal aberrations and nonhuman primates) [58]. has been observed in exposed plants, as well as the formation of micronuclei and the reduction of the mitotic index [51]. On the other hand, in 8. Dermal toxicity order to determine the indirect genotoxicity mediated by ROS, in vivo methods have been The different chemical substances can be used with hemocytes of Drosophila melanogaster absorbed by the skin through the following in which the intracellular concentration of reac- routes [59]: tive species can be determined to determine - Intercellularly whether oxidative stress is the causal agent of - Intracellularly DNA injury [52]. - Absorbed through sweat glands - Using the pathway of the hair follicles 7. Immunotoxicity Although the toxicity of the nanoparticles administered by the intravenous route is greater The interaction of nanomaterials with the im- than by the dermal route, it is necessary to mune system is one of the adverse effects that monitor the different alterations that can occur 12. Prediction in vivo of human response to nanomaterials 127 in the different strata of the skin [60]. In this way, with cell death due to the alteration of the mito- transdermal formulations of nanodrugs have chondrial bioenergetics of nanoparticles, which been implemented to take advantage of the few decrease oxidative phosphorylation and swell adverse effects compared to systemic adminis- mitochondria [72]. Finally, the evaluation of tration [61]. Despite this, a thinning of the skin, mitochondrial function both in vitro and inflammation and increase in the Langerhans in vivo may be a useful tool to predict liver cells has been observed during cutaneous expo- toxicity to nanocomposites and thus control their sure to nanoparticles [62], so adverse effects dose and administration [73,74]. continue to be of concern especially in cosmetic preparations in which nanomaterials can enter the bloodstream, causing organ and tissue 11. Brain toxicity toxicity. Therefore, greater analysis of the penetration of these compounds is needed [63]. Prolonged exposure to nanomaterials has been shown to produce neuronal toxicity and be able to cross both the brain and the placental 9. Nephrotoxicity blood barriers [75,76], with the unpredictable fact that the nanoparticle protein corona evolves In this order of ideas, it has been shown that and changes drastically when crossing the brain nanomaterials, especially those containing blood barrier [77]. Thus, it has been reported that heavy metals, can enter the interior of the renal nanoparticles can cause dysfunction of the cell and induce toxicity [64]. This toxicity is central nervous system by inducing oxidative believed to be linked to oxidative stress induced stress and an activation of microglia cells [78]. by nanoparticles [65]; this stress produces DNA In summary, neurotoxicity can lead to cellular damage in kidney, causing tissue necrosis [66]. apoptosis, as well as to autophagy and neuroin- In this way, to reduce the toxicity of nanopar- flammation, affecting the blood brain barrier ticles, it has been proposed to combine their [79]. Likewise, nanomaterials can affect brain administration with antioxidant molecules to microvascular endothelial cells, producing cell achieve a protective effect on the organs that death, necrosis, and inflammation [80]. Likewise, may be affected [67]. Therefore this redox nano- in vivo tests have been able to show a decrease in particle field will be a promising application of spatial learning and memory capacity, as well as nanomedicines in the development of new affect the hippocampus [81]. Also, the ability of drug delivery systems [68]. nano-objects to inhibit ionic transport channels in neurons has been reported, altering mem- brane potential and synaptic transmission [82]. 10. Liver toxicity Therefore, it is necessary to establish the cost benefit and the scope of the use of nanomaterials Likewise, other tissues are affected by oxida- in diseases of the central nervous system, to tive stress and inflammation induced by nano- determine the potential final benefit for patients. particles, including liver [69]. But it is important to bear in mind that this toxicity exists as fundamental role of mitochondrial oxidative 12. Prediction in vivo of human response stress [70]. Therefore, mitochondrial dysfunction to nanomaterials must be a matter of concern when designing platforms for the evaluation of liver toxicity Considering the previous sections, it is impor- [71]. Additionally, this dysfunction is associated tant to take into account that the correct and 128 6. Nanotoxicity: the impact of increasing drug bioavailability exact prediction of the in vivo response to the medicines and prevent the appearance of re- different toxic agents depends on the implemen- lapses [88], which makes the monitoring of the tation of preclinical models of high reproduc- biodistribution of nanomedicines when they ibility and precision [83]. In this way, if the enter the human body and its tissues of funda- nanomaterials are taken as xenobiotic com- mental importance in order to establish the pos- pounds, they should be studied in vivo with spe- sibilities of toxicity [89]. In this way, the ADME/ cific toxicity parameters that include [84]: Tox should take into consideration the chemical modifications induced by the nanoparticles that - Acute toxicity studies increase the absorption, distribution, and meta- - Subchronic toxicity studies bolism of the drugs; these modifications increase - Chronic toxicity studies the accumulation of nanomaterials in the tissues, - Pyrogenic test so it is necessary to develop studies that provide - Genotoxicity tests (mammalian in vivo information on this approach [90]. To evaluate chromosomal aberrations assay, the biodistribution of nanoparticles, it is possible micronucleus test, comet assay) to use imaging techniques such as magnetic - Carcinogenic studies resonance imaging, computed tomography, - Embryotoxicity studies and positron emission tomography [109]. - Immunotoxicity studies As well, it would be necessary assess the cyto- - Hematological studies chrome P450 (CYP) inhibition of nanomaterials - Biochemical parameters (general profile, lipid for predicting drug interactions and inhibition profile, renal profile, and hepatic profile) of the human ether-a-go-go-related gene - Histopathological studies (HERG) to determine the potential for producing - Behavioral studies cardiac arrhythmias [91]. In this order of steps, the OECD is following these tests as protocols to assess the acute toxicity of nanomaterials [85]: 14. Organ-on-chip systems - Oral toxicity test Organ-on-chip systems are microfluidic - Ocular irritation devices that have been proposed as an in vitro - Dermal irritation model that can predict human pharmacokinetics - Lethal dose 50 (LD50) in vivo, as well as the possibility of adverse reac- Likewise, the implementation of mitochon- tions to new formulations for lung, kidney, liver, drial oxidative stress and cellular consumption and heart [92]. This system is considered a choice tests should be implemented as predictors of by the European Union Reference Laboratory for toxicity and primary screening [86]. Finally, the Alternatives to Animal Testing for their high use of computational methods to predict the predictive value [93]. toxicity of nanomaterials will be of great help to select nanoparticles, as well as bioactive nano- structures of low toxicity [87]. 15. Whole-animal models

An interesting whole-animal model to eval- 13. ADME model uate the toxicity of nanomaterials is the use of Caenorhabditis elegans for nanotoxicity testing Currently, nanoformulations are a promising [94]. Another interesting whole-animal model alternative to increase the pharmacokinetics of with the ability to detect low levels of toxicity References 129 is Galleria mellonella larvae, which is considered toxicity depends on the size and shape of the to be a significant complement to cytotoxicity nanomaterials, an interesting alternative is the assays [95]. Equally, zebrafish model is an search for new configurations to avoid inflam- important functional assay for determining mation and oxidative stress; in this regard, nano- ecotoxicity of nanoparticles and determining its particle spherical shape has presented promising environmental hazards [96]. results [104]. Also it is necessary to determine the interactive toxicity of nanomaterials with other molecules, which can happen in the presence of 16. Regulation of nanotechnology lipopolysaccharides and bisphenol A [105]. products In addition, is important to know the possible effects on the epigenetic regulation of nanomate- The most accepted regulations are the Euro- rials, because they can alter the function of his- pean Union Registration, Evaluation, Authorisa- tones and therefore the transcription of DNA tion and Restriction of Chemicals regulations; [106]. Equally, the developments in nonmamma- these regulations considers the physiologically lian in vivo models will be very useful in the based kinetic (PBK) models and dynamic (PBD) future as alternative protocols in the evaluation models for evaluations of nanomaterials [97]. of nanosafety of new medicines and diagnostic Also PBK models have been approved by the methods [107]. OECD, FDA, EMA, and Ministry of Health in Japan for the evaluation of the risks and dangers of nanoparticles [98]. In PBK model, the human References body is divided into structures known as com- partments, to which medicines are distributed [1] Koopaei NN, Abdollahi M. Opportunities and obsta- cles to the development of nanopharmaceuticals for and from which they are metabolized; thanks human use. Daru 2016;24:23. to this tool it is possible by means of differential [2] Deray G. Amphotericin B nephrotoxicity. equations to determine the ADME process of the J Antimicrob Chemother 2002;49(1):37e42. substances inside the organism [99]. As well, [3] Thakur M, Pandey S, Mewada A, Patil V, Khade M, fl PBK can be useful to adjust the dosimetry of Goshi E, Sharon M. Antibiotic conjugated uorescent carbon dots as a theranostic agent for controlled drug the different nanoformulations for a correct release, bioimaging, and enhanced antimicrobial in vitroein vivo correlation [100]. activity. J Drug Deliv 2014. 282193. [4] Bawa R, Audette GF, Rubinstein I. Handbook of clin- ical nanomedicine: nanoparticles, imaging, therapy, and clinical applications. Pan Stanford; 2016. 17. Conclusions and perspectives [5] Finch G, Havel H, Analoui M, Barton RW, Diwan AR, Hennessy M, et al. Nanomedicine drug development: The toxicity of nanomedicines is becoming a a scientific symposium entitled “Charting a roadmap ” e growing concern that must be assumed to to commercialization . AAPS J 2014;16(4):698 704. [6] Shatkin JA. Nanotechnology: health and environ- reduce possible adverse events [101]. In this or- mental risks. CRC Press; 2017. der of ideas, the evaluation of toxicity should [7] Onoue S, Yamada S, Chan HK. Nanodrugs: pharma- be reconciled with the new tendencies in looking cokinetics and safety. Int J Nanomed 2014;9:1025e37. for trials that are an alternative to the use of an- [8] Khan MS, Roberts MS. Challenges and innovations of imals [102]. This is how adequate standards drug delivery in older age. Adv Drug Deliv Rev 2018; 135:3e38. should be developed to implement translational [9] Rizvi SA, Saleh AM. Applications of nanoparticle sys- medicine projects capable of bringing nanomedi- tems in drug delivery technology. Saudi Pharm J 2018; cine to its safe use in clinical practice [103]. As the 26(1):64e70. 130 6. Nanotoxicity: the impact of increasing drug bioavailability

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Nanoparticle-based vaccines: opportunities and limitations Diana Diaz-Arevalo1, Mingtao Zeng2 1Molecular Biology and Immunology Department, Fundacion Instituto de Inmunología de Colombia- FIDIC, School of Medicine and Health Sciences, Universidad del Rosario, Bogota, DC, Colombia; 2Center of Emphasis in Infectious Diseases, Department of Molecular and Translational Medicine, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, TX, United States

1. Chapter outline effective in the distribution of medications to target specific organs and control drug delivery. 1.1 Introduction These drug carriers not only transport drugs to sites of cancer or other target diseased organs, Nanomedicine has seen increased interest by preventing damage to healthy cells, but also pro- the medical community in recent years, as it tect the drug from degradation [3]. Some of the has enabled modification of engineering devices metal nanoparticles (e.g., gold, silver, or PLGA) for delivery to and interaction with cell environ- themselves may have dual functions, working ments. In particular, this technology has enabled both as carriers and as targeted delivery systems advances in the design of delivery systems for using the membranes of cancer cells, which drugs and vaccines [1]. Nanoparticles (NPs) are contain tumor-associated markers. In a recent now being engineered from different biodegrad- study, Fang et al. used a membrane from mouse able materials, including natural and synthetic melanoma cancer cells in the outer layer of polymers (poly[lactic-co-glycolic] acid (PLGA) PLGA nanoparticles. These NPs were stable and polylactic acid (PLA)), metals (gold, copper and persisted in the structure during cellular oxide, aluminum oxide, zinc oxide, iron oxide, endocytosis, activating the maturation of den- and silver), or lipids (phosphatidylserine, dritic cells (DCs) and presenting especially to phosphatidylcholine, and cholesterol) [2]. T cells that have TCR binding to gp100 and NPs and microparticles (MPs) have been inducing the production of IFN-g. Moreover, widely used to deliver drugs, especially the authors found that the PLGA covering mem- cytotoxic drugs or immune-suppression treat- brane had receptors that allow interaction with ments for transplantation. These NPs are

Nanopharmaceuticals https://doi.org/10.1016/B978-0-12-817778-5.00007-5 135 © 2020 Elsevier Inc. All rights reserved. 136 7. Nanoparticle-based vaccines: opportunities and limitations cancer cells and delivery of the drug [4,5]. delivery system (Table 7.1). The combination of Furthermore, the NPs were used to deliver drugs the vaccine with the adjuvant or delivery system to the specific site of organ transplantation to should be safe, stable, and have the ability to prevent rejection. Systemic immunosuppression induce long-lived memory B and T cell is a high risk for recipients of organ transplanta- responses, preferably with a single dose and a tion, as it uses high doses of immunosuppression maximum of two doses and be free from strict agents that make them more susceptible to infec- storage requirements [9]. DNA and RNA vac- tions and in some cases cause death. Previous cines are safe but need a second boost with studies found that diabetic mice were trans- recombinant protein or DNA from another planted with islets into the eye to prevent islet vector. NPs have become an alternative to target- graft immune rejection in vivo using rapamycin ing vaccine delivery to immune cells, improving MPs. The islets transplanted with the immuno- vaccine efficacy with slow release, easy antigen suppressed drug MPs survived more than uptake, and induction of humoral and cellular 1 month compared with the control (empty responses [8]. MPs), which rejected the islets in the second NPs have played an important role in the week [6]. activation of antigen-presenting cells (APCs), Work in last few decades has increased our especially DCs, which may determine vaccine ef- knowledge of infectious diseases and the mecha- ficacy. Although there is some cytotoxic effect of nisms of evasion of immune responses. Howev- the NPs [10,11], the risk is low compared with er, new variants of antibiotic-resistant the benefits of vaccine delivery [12]. In this chap- pathogenic microorganisms have emerged and ter, we will summarize the different nanocarrier- are becoming a challenge for designing new vac- based vaccine formulations that achieve the cines and adjuvants. Until now vaccines have desired host immunity against infectious been developed from live-attenuated microor- diseases and cancer, and at the end, we will ganisms or killed pathogens (first-generation discuss on the limitations of the respective vaccines) [7], DNA vaccines (third-generation carriers. vaccines), subunit vaccines [8], and synthetic peptides (second-generation vaccines). The last 1.2 Immune responses after vaccination three vaccine types eliminate the risk of devel- oping the disease, but they must be used in DCs are specialized APCs that coordinate the conjunction with an adequate adjuvant or innate and adaptive immune responses to

TABLE 7.1 Types of vaccines.

Vaccine Constituent Examples

Live-attenuated vaccines Whole pathogen Measles, mumps, rubella, rotavirus, smallpox, chickenpox, yellow fever

Inactivated vaccines Whole pathogen Rabies, flu, polio, hepatitis A Subunit, recombinant, polysaccharide, Part of the pathogen Hepatitis B, whooping cough, Hib (Haemophilus and conjugate vaccines influenzae type b) disease, human papillomavirus, shingles, meningococcal disease, pneumococcal disease Toxoid vaccines Toxin Diphtheria, tetanus Future of vaccines DNA Research studies 1. Chapter outline 137 induce “talking” by chemokines to start the and type of NP that can enter through receptors, defense against infectious diseases. There are such as Toll-like receptors (TLRs), pinocytosis, three subtypes: plasmacytoid, myeloid, and phagocytosis, or special receptor targets. The follicular DCs. Depending on the subtype of entry of DNA vaccines using different types of DC, the cytokine profile will be different, and NPs is shown in Fig. 7.1 [5]. the responses may be protective or not. The interaction and delivery of antigens and adju- vants to DCs is a research priority, in order to 1.3 Types of NPs used to deliver optimize the humoral and cellular vaccine vaccines responses [13]. Follicular DCs are the key to acti- € Until now there have been various types of vating naive T and B cells to initiate the adaptive NPs, composed of gold, dendrimers, carbon, immune response and the induction of long- polymers, and liposomes, that have been used lived memory cells. Lymphocyte activation is to deliver vaccines. All can stimulate the produc- started after recognition of the antigen by the tion of cytokines and antibody responses T cell receptor (TCR) and B cell receptor (BCR). [15e19]. The cargo is not limited to vaccines, The recognition of a specific antigen is associated and it is possible to add adjuvants and immune with reorganization of costimulatory surface stimulatory molecules, including silica and proteins such as CD40 ligand, CD28, CD4, or iron, TLR agonist, and cytokines, to improve CD8 in T cells and CD40, CD80, and CD86 in immunogenicity [20,21]. Several vaccines have APCs. The early molecular events that underlie been tested on the different types of NPs the formation of the synapse are highly coordi- (Table 7.2). nated and tightly controlled. The B cells spread over the antigen- APC- C3 or FcR or CR, rapidly, preventing antigen internalization and phagocy- 1.4 Liposomes tosis by DCs and their posterior presentation to MHC class I or class II. Before the antigen is Liposomes are the second-most common type processed and presented along with MHC class of NP, and they self-assemble in water under II molecules to the TCR, antigeneMHC com- special conditions. They are composed of lipids, € þ plexes mediate the recruitment of naive CD4 which have a hydrophilic head and a hydropho- T helper cells. The DCs present the antigene bic tail that maintain hydrophilic inner and outer MHC class II complex to the TCR, and the membranes, in lamellar lipid bilayers or in T cells differentiate into one of the subtypes of multilayers that simulate vesicles found within þ CD4 T helper cells (Th). The interaction of B cells [22]. Liposomes can induce cellular cell MHC class IIeantigen complexes with the responses or humoral responses, depending on þ TCR of CD4 T cells is necessary for full B cell the charge, size, and lipid composition. A previ- activation and production of antibodies [14]. ous study showed that charge had the main role The DCs are the principal target for delivery of in activation of the cellular or humoral immune the vaccine and hence for NP, although other responses. The authors’ approach was to investi- APCs, such as macrophages, B cells, and lung gate the importance of the antigeneliposome epithelial cells, can present the antigen to interaction in immunogenicity and depot T cells. Unlike DCs, macrophages induce high formation. They used subunit antigens Ag85Be lysosomal activity after phagocytosis to enhance ESAT-6 (pI ¼ 4.9) from TB, and “CTH1” antigen presentation and thus effector immune (pI ¼ 9.0), from Chlamydia vaccines and a responses. Nanotechnology has been improving model antigen, lysozyme (pI ¼ 11). The injection antigen delivery, depending on the size, charge, of cationic dimethyldioctadecylammonium 138 7. Nanoparticle-based vaccines: opportunities and limitations

FIGURE 7.1 Advantages of NPs for vaccine design. (A) Various combinations of adjuvants and antigens can be formulated using NP platforms such as liposomes, emulsions, nanogels, and other substances. (B) Nanovaccines can access the lymphatic drainage system for lymph node delivery while protecting their cargoes from environmental degradation. Once at the lymph nodes, the nanocarriers deliver their cargoes to APCs for immune processing. (C) Nanovaccine properties can be tuned to efficiently deliver their cargoes for maximum immune activation. For example, NPs can be modified to target specific subsets of immune cells. They can also be delivered to specific intracellular compartments, where receptors for immune pathways can be triggered. From Kroll AV, Jiang Y, Zhou J, Holay M, Fang RH, Zhang L. Biomimetic nanoparticle vaccines for cancer therapy. Adv Biosyst 2019:1800219 with permission.

TABLE 7.2 List of antigens delivered by nanocarriers for the treatment of different diseases in vaccine research.

Antigen Nanocarrier used Disease

Against Bacterial Infection Antigenic protein Poly(D,L-lactic-co-glycolic acid) Anthrax nanospheres DNA encoding T cell epitopes of Chitosan nanoparticle Tuberculosis Esat-6 and FL Mycobacterium lipids Chitosan nanoparticle Tuberculosis Polysaccharides Liposomes Pneumonia Bacterial toxic and parasitic protein Liposomes Cholera and malaria

Fusion protein Liposomes Helicobacter pylori infection Antigenic protein Nanoemulsion Cystic fibrosis Antigenic protein Nanoemulsion Anthrax Mycobacterium fusion protein Liposome Tuberculosis 1. Chapter outline 139

TABLE 7.2 List of antigens delivered by nanocarriers for the treatment of different diseases in vaccine research.dcont'd

Antigen Nanocarrier used Disease

Against Viral Infection Antigenic protein Chitosan nanoparticles Hepatitis B Viral protein Gold nanoparticles Foot and mouth disease Membrane protein Gold nanoparticles Influenza Viral plasmid DNA Gold nanoparticles HIV Tetanus toxoid Poly(D,L-lactic-co-glycolic acid) nanospheres Tetanus

Hepatitis B surface antigen Poly(D,L-lactic-co-glycolic acid) nanospheres Hepatitis B Hepatitis B surface antigen Alginate-coated chitosan nanoparticle Hepatitis B Live virus vaccine Chitosan nanoparticles Newcastle disease Capsid protein VLPs Norwalk virus infection Capsid protein VLPs Norwalk virus infection Influenza virus structural protein VLPs Influenza

Nucleocapsid protein VLPs Hepatitis Fusion protein VLPs Human papilloma virus Multiple proteins VLPs Rotavirus Virus proteins VLPs Blue tongue virus Enveloped single protein VLPs HIV Viral protein Polypeptide nanoparticles Corona virus for severe acute respiratory syndrome (SARS) Against Parasitic Infection Merozoite surface protein Iron oxide nanoparticles Malaria Epitope of Plasmodium berghei circumsporozoite Polypeptide nanoparticles Rodent malarial parasitic protein. infection

Surface protein from Eimeria falciformis ISCOMs Diarrhea sporozoites

From Pati R, Shevtsov M, Sonawane A. Nanoparticle vaccines against infectious diseases. Front Immunol 2018;9. adapted with permission.

0 bromide (DDA): trehalose 6,6 -dibehenate (TDB) site of immunization, possibly mediated by liposomes with associated Ag85BeESAT-6 form depot formation. These liposomes induce strong a deposit at the site of injection. By contrast, the T cell proliferation and differentiation through anion disappears immediately. The cationic lipo- Th1 and Th17 responses, although the authors somes induce the infiltration of monocytes at the could not find DCs. However, DCs are known 140 7. Nanoparticle-based vaccines: opportunities and limitations

FIGURE 7.2 Immune responses in mice 3 weeks after the last of three immunizations with 2 mg of Ag85BeESAT-6 alone (white) or in combination with DSPCeTDB (dashed) or DDAeTDB (gray). (A) IFN-g responses in the spleen. (B) Frequency of CD44high T cells in response to each of eight possible cytokine subsets of IFN-g, IL-2, and/or TNF-a. (C) IL-5 responses in the spleen. (D) IL-17 responses in the spleen. Adapted from Henriksen-Lacey M, Christensen D, Bramwell VW, Lindenstrøm T, Agger EM, Andersen P, et al. Liposomal cationic charge and antigen adsorption are important properties for the efficient deposition of antigen at the injection site and ability of the vaccine to induce a CMI response. J Control Release 2010;145(2):102e8 with permission. to migrate to the secondary lymph nodes to acti- Th17, or Tfh immune responses. Cationic lipo- vate the T and B cells, which the authors did not somes can increase the uptake of subunit analyze in this manuscript [23]. Previous studies vaccines as synthetic peptides and recombinant have shown that DDA induces proinflammatory proteins; this can be explained by the interaction cytokines and chemokines after infiltration of between positively charged liposome with the monocytes and macrophages in vivo [24].One APCs that have a negative charge in their mem- of the advantages of liposomes is mimicking brane [26,27]. Another modification is the properties of the pathogens, inducing humoral pH-sensitive fusogenic liposomes, which are and cellular immune responses. The antigen’s stable in neutral pH 7.4, but in acidic conditions, presentation to APCs depends on the membrane the antigen is released and presented by MHC characteristics, size, and ligand-receptor bind- class I or II. These liposomes containing phos- ing. Liposomes could induce Th2 responses if phatidylethanolamine and amphiphilic stabi- the lipids are unsaturated whereas saturated lizers allow PE-containing liposomes to form lipids promoted Th1-type immune responses aggregates, due to the poor hydration of their [25,26]. Liposomes could be modified according headgroups, which can explain their high to the needs of delivery to produce Th1, Th2, affinity to adhere to cell membranes [28,29]. 1. Chapter outline 141

Another study designed a unique structure responses. However, the CSP part of RTS, S is composed of inter-bilayer-crosslinked multila- an antigenic polymorphism, and T cell epitopes mellar vesicles (ICMVs), which is stable in the present on the CS protein that are incorporated extracellular environment but rapidly released into the vaccine are also polymorphic. To in endosomes/lysosomes, thereby enhancing address this problem, the newest generation of vaccine immune responses. The ICMVs carried this vaccine is coformulated with Matrix-M, the antigen OVA mixed with the adjuvant mono- a saponin-based liposomal adjuvant [32]. How- phosphoryl lipid A (MPLA). This mixture ampli- ever, the vaccine protection against fied vaccine responses and upregulated the P. falciparum has not been achieved due to the costimulatory cells on splenic and bone marrow short-lived memory cells and the evasion mech- DCs. In addition, splenic DCs incubated with the anisms of the parasite [33]. The first malaria þ OVA vaccine MPLA triggered proliferation of vaccine was tested in 1967 in animals using € þ naive OT-1 CD8 T cells in vitro, suggesting Plasmodium bergheis radiation-attenuated sporo- that the ICMVs enhanced cross-presentation of zoites, with a 100% of protection [34]. Hoffman the antigen. The same results were obtained et al. reported in 2002 that 10 human volunteers in vivo after the vaccination of mice with the were vaccinated with irradiated sporozoites of mixture, and the ICMVs elicited robust antibody P. falciparum strain NF54, and all of them were titers that were w1000-fold greater than simple protected between 2 to 9 weeks [35]. This vaccine liposomes. These results could be attributed to will be used to vaccinate 360,000 children a year activation enhancement of DCs and antigen in three African countries (Ghana, Malawi, and cross presentation [30]. The first use of liposomes Kenya). The vaccine could prevent 4 in 10 cases as a delivery system for a malaria vaccine was in according to previous clinical trials. the 1980s. Ballou et al. synthetized peptides derived from the repetitive region of the circum- sporozoite (CS) protein of Plasmodium falciparum 1.5 Virus-like particles sporozoites. The synthetic peptides were conju- gated to keyhole limpet hemocyanin (KLH) Virus-like particles (VLPs) are composed of a proteins and were incorporated into liposomes. self-assembling viral membrane maintaining Immunized mice and rabbits produced anti- viral surface proteins. VLPs can be modified to bodies against the repeat region of the protein express additional proteins of other microorgan- with biologic activity correlated with protection isms, which could be engineered by fusion of the [31]. RTS, S is the only vaccine against malaria proteins with membrane antigens or by endoge- that is in phase three clinical trials in Africa. nous expression of other antigens [36]. Gardasil The RTS, S vaccine has the central repeat region is a four-component VLP-type vaccine specific of CSP, and T cell epitopes localized in the against HPV. It contains the L1 major capsid C-terminal region are fused to hepatitis B surface protein of HPV types 6, 11, 16, and 18 and is antigen (HBsAg) and expressed in Saccharomyces administered along with an aluminum adjuvant. cerevisiae yeast. This virus-like particle (VLP) This vaccine was administered to 1158 women vaccine also contains MPLA and QS-21, which and was followed up for incidence of persistent þ enhances humoral and CD4 T cell responses associated infection for 35 months. The efficacy in the first 4 years, with a level of protection of was 100% for preventing clinical disease. 18%e36%. Protection decreased rapidly there- The HPV combination vaccine was immuno- after, with negative efficacy in some children. genic, inducing the production of long-lived an- The problem is not RTS, S nor the adjuvant tibodies [37]. However, some problems with the system, as both induce potent immune vaccine in teenage girls were reported. 142 7. Nanoparticle-based vaccines: opportunities and limitations

The adverse events included dermatologic/ systems [40]. PLGA is approved for human use mucosa-allergic reaction (25%), rash (22%), and by the US Food and Drug Administration local/injection-site reaction (20%). However, (FDA) and European Medicines Agency some serious adverse events following immuni- (EMA), while PLGA has been used to deliver zation were reported (7.5% of reports), including drugs for long periods. DCs play an important two incidents of anaphylaxis, two seizures, one role in the activation of adaptive immune incident of thrombocytopenia, and one death responses, in which B cells and T cells activate [38]. and differentiate into subpopulations that deter- mine which humoral or cellular immune 1.6 Metal and nonmetal inorganic NPs responses eliminate the microorganism. Cruz et al. studied the delivery of NPs and MPs to Inorganic metal NPs are frequently used in DCs to create a biocompatible and biodegrad- drug delivery and bioimaging, especially in able slow-release vaccine and effectively target e treating cancer patients. DNA vaccines are the cells. The NPs and MPs (PLGA PEG) were e more stable and protected from degradation loaded with tetanus toxoid peptide FITC linked e when carried by a gold, silica, or silver NP deliv- to a Lys Lys cathepsin cleavage site and an anti- ery system. The covalent attachment of Chito6 to DC-SIGN antibody. The DCs targeted PLGA- GNPs increases the NP molecular weight, based vaccine NPs but not MPs. The NPs were fi enhancing DNA binding and stability without ef ciently taken up with the help of anti-DC- compromising DNA release and transfer. SIGN antibodies and induced proliferation of Chito6eGNPeDNA (HBsAg) complexes induce T cells (Fig. 7.3). However, the MPs were taken effective antibody and T cell responses after im- up for all APCs, including the DCs (Fig. 7.4). munization of BALB/c mice. By contrast, naked New research is needed to understand the DNA-primed HBsAg induces antibodies after a biology, antigen processing, and presentation series of four immunizations with 10 mg of needed to induce long-lived memory B and þ naked DNA. HBsAg-specific CD8 T cells T cells [41]. eliminated P815/BALB target cells that had Botulism is a lethal neuroparalytic disease been sensitized with an HBsAg CTL epitope produced by Clostridium botulinum toxins peptide in vitro. These chimeric NPs, employing (A-H). Ruwona et al. demonstrated that cationic a minimal amount of DNA, induce effective im- PLGA NPs can carry plasmid DNA encoding the mune responses when compared with naked BoNT heavy-chain (Hc) fragment and that its DNA [39]. product is nontoxic. Immunized mice produced high titers of antibodies after 5 to 9 weeks (Fig. 7.5). 1.7 Polymeric NPs After four immunizations, specific IgG1 had decreased, but IgG2a had increased, compared Biodegradable polymers are of significant with the first immunization. After challenge, interest in the delivery of drugs and vaccines 100% of the mice vaccinated with PLGAe against infectious diseases. These polymers pVax/opt-BoNT/C-Hc50 survived, while only consist of either natural or synthetic monomers 80% of the mice immunized with naked plasmid that are biodegradable, are nonimmunogenic, were protected (Fig. 7.6) [17]. have low cytotoxicity, and are easy to Modifications of polymeric NPs have been prepare. There are several polymers, such as chi- used to deliver synthetic peptides or recombinant tosan, PLGA, polyethylene glycol (PEG), poly- proteins to dendritic cells and macrophages. caprolactone, and dextran used as delivery 1. Chapter outline 143

FIGURE 7.3 Uptake of PLGA MPs with FITCeTT peptide by DCs results in antigen presentation. DCs were incubated with FITCeTT containing MPs (green; light gray in printed version) for 1 h to confirm uptake by human DCs. Cells were analyzed by confocal laser scanning microscopy. The cell surface was visualized by MHC class II staining (blue; white in print version). The image represents the middle focal plane of the DCs, with the iris set at 2 nm (A). Presentation of PLGA-encapsulated FITCeTT peptide was studied by culturing DCs for 18 h in culture medium alone, medium supplemented with empty PLGA MPs (PLGA), or with 0.1 mg of FITCeTT peptide encapsulated within PLGA MPs (PLGA TT). In addition, DCs were pulsed with 1 mM TT peptide as a positive control for antigen presentation (TT). Subsequently, autologous TT-responsive peripheral blood lymphocytes were added. After 3 days, cellular responses were assessed in a proliferation assay. Data are mean proliferation indices SD relative to medium control for experiments performed in triplicate. Significant differences from medium control according to ANOVA and Dunnett’s test:*P < .01. From Cruz LJ, Tacken PJ, Fokkink R, Joosten B, Stuart MC, Albericio F, et al. Targeted PLGA nano-but not microparticles specifically deliver antigen to human dendritic cells via DC-SIGN in vitro. J Control Release 2010;144(2):118e26 with permission.

Salvador et al., modified PLGA MS, 50:50 1.8 NP-investing companies and clinical lactideeglicolide ratio, to produce cationic NPs trials using polyethylene imine (PEI), and BSA as anti- gen. In addition, the NPs were modified encap- The interest of companies in nanoparticle- sulating monophosphoryl lipid A (MPLA) or based vaccine delivery dramatically increased polyinosinic-polycytidilic acid poly(I:C) and in the last decades. Pevion Biotech Ltd., a Swiss a-galactosyl ceramide to address their adjuvant company founded in 2002, developed the effect. Coumarin 6 was incorporated into the virosome-based technology platforms to make fi oily phase for the preparation of fluorescent ef cient and safe prophylactic/therapeutic NPs to evaluate in vitro assays. The NPs were vaccine candidates. For Candida albicans that tested in vitro using monocytes and dendritic can cause vulvovaginal mucosal infections, cells, and mice were immunized with the Pevion used the aspartyl-proteinase (Sap2), different NP modifications to determine the im- which is an immunodominant antigen and viru- mune responses in vivo. The cationic NPs lence factor; this recombinant protein was showed a noticeable enhancement of their assembled with virosomes (PEV7). The evalua- uptake by the monocytes, MDCs, and PDCs in tion in mouse model and the initial clinical trial comparison to the classical NPs. In addition, on women showed that this candidate vaccine, the cationic NPs increased immunostimulatory intravaginally administered, has a therapeutic effect since they induced the production of potential for the treatment of recurrent candidi- antibodies and Th1 cell responses producing asis [42]. In addition, this company tested a IFN-g [12]. malaria vaccine in Africa. This vaccine had 144 7. Nanoparticle-based vaccines: opportunities and limitations

FIGURE 7.4 Antibodies were introduced on the surface of PLGA NPs and MPs. The morphology of NPs (A, left panel) and MPs (A, right panel) with PEGelipids was analyzed by scanning electron microscopy. The presence of the antibodies on the PLGA particle surface was confirmed by flow cytometry. The NPs and MPs were stained with fluorescence-labeled secondary antibodies and analyzed on a flow cytometer (B). PLGA MPs were mounted on glass slides and analyzed by confocal laser scanning microscopy to visualize antibodies present on the particle surface. FITCeTT peptide was detected as a green (light gray in print version) fluorescent ring surrounding the PLGA particles (see also Fig. 7.2A). Antibodies on the particle surface were detected by secondary antibody staining with Alexa 647-labeled anti-human IgG. The images represent the middle focal plane of particles and show split channels of the FITC signal (FITCeTT), the Alexa 647 signal (antibody), and a merged image showing the antibody in red (dark gray in print version) and the FITCeTT peptide in green (white in print version) (C). From Cruz LJ, Tacken PJ, Fokkink R, Joosten B, Stuart MC, Albericio F, et al. Targeted PLGA nano-but not microparticles specifically deliver antigen to human dendritic cells via DC-SIGN in vitro. J Control Release 2010;144(2):118e26 with permission. combination of FFM ME-TRAPþPEV3A (AMA- Novavax is developing respiratory syncytial 1) and was tested in phase I/IIa clinical trials. virus (RSV) F nanoparticle vaccine with Although the vaccine did not show sterile aluminum. The purpose of the reported study protection, it induced responses on blood stage was to evaluate the safety and efficacy of mater- parasites and lower rates of parasite growth in nally transferred antibodies in preventing RSV in human volunteers vaccinated with PEV3A, infants. The vaccinated healthy third-trimester compared to unvaccinated controls [43]. GlaxoS- pregnant women showed significant protection mithKline plc is working to improve the cofor- to newborn children from RSV challenge and mulated RTS, S vaccine using Matrix-M, a reduced pulmonary inflammation, and the saponin-based liposomal adjuvant [32]. vaccine was safe and effective for maternal and 1. Chapter outline 145

FIGURE 7.5 Serum anti-BoNT/C-Hc50 IgG (A), IgG1 (B), and IgG2a (C) induced by intramuscular immunization of mice with pVax/opt-BoNT/C-Hc50 (20 mg/mouse), alone (pBoNT/C) or coated on cationic PLGA NPs (pBoNT/C-NP). SKH-1 Elite mice (n ¼ 5) were dosed in weeks 0, 2, 4, and 8. Control mice received PBS only. Blood samples were collected in week 5 (day 35) and week 9 (day 63). Data are mean SD (n ¼ 5). *P < .05 compared with control group, #P < .05 compared with pBoNT/ x C only, and p < .05, day 35 versus day 63. Ruwona TB, Xu H, Li J, Diaz-Arevalo D, Kumar A, Zeng M, et al. Induction of protective neutralizing antibody responses against botulinum neurotoxin serotype C using plasmid carried by PLGA nanoparticles. Hum Vaccines Immunother 2016;12(5):1188e92 with permission.

The antibody responses are able to neutralize against both homologous and heterologous strains [45]. The nanoparticles have been used in recent years in only a few of advanced clinical trials. The vaccine against RSV infection has three studies using RSV F nanoparticle vaccine with aluminum in phase 2 and 3 trials. The first clin- ical study of RSV was RSV F Dose-Ranging Study in Women, which started October 2013 and finished May 2016. The study showed that FIGURE 7.6 all formulations were well tolerated, without Protective immunity against BoNT/C chal- treatment-related serious adverse events. Anti- lenge in immunized mice. SKH-1 Elite mice (n ¼ 5) were dosed in weeks 0, 2, 4, and 8 with pVax/opt-BoNT/C-Hc50 bodies anti-F IgG and palivizumab-competitive plasmid (20 mg per mouse), plasmid alone (pBoNT/C), or antibody responses were correlated and plasmid coated onto PLGA NPs (pBoNT/CeNP), with con- increased after both doses, while microneutrali- trol mice receiving PBS only and challenged in week 12 fi zation assays increased signi cantly after the with 100 MLD50 of BoNT/C toxin. Ruwona TB, Xu H, Li first dose, then plateaued [46]. The second study J, Diaz-Arevalo D, Kumar A, Zeng M, et al. Induction of protective neutralizing antibody responses against botulinum neurotoxin was RSV F Vaccine Maternal Immunization serotype C using plasmid carried by PLGA nanoparticles. Hum Study in Healthy Third-trimester Pregnant Vaccines Immunother 2016;12(5):1188e92 with permission. Women, which started September 2014 and fi adult vaccination [44]. Furthermore, this nished June 2017. This study demonstrated company is working in recombinant trivalent that the vaccine is safe to infants and pregnant nanoparticle influenza vaccine with matrix women. The third clinical study is in phase three fi M-1. The vaccine can induce responses to one trials and will nish in July 2019. The vaccine is or more conserved HA head and stem epitopes. safe and effective for infants and pregnant 146 7. Nanoparticle-based vaccines: opportunities and limitations women [44]. The study “Evaluation of the Safety bilayers and may interact with sensitive organs and Immunogenicity of a Recombinant Trivalent [48]. The clearance and excretion of NPs is medi- Nanoparticle Influenza Vaccine with Matrix M-1 ated through the mononuclear phagocytic Adjuvant (NanoFlu)” was used to test protection system, the renal urinary system, and by biliary in people older than age 60, but no results have clearance. Macrophages phagocytize the NPs been publicly released. The other study is the and keep them in the secondary lymph organs phase two trial entitled “Dose and Formulation and/or liver using the Mertk (Mer) receptor Confirmation of Quad-NIV in Older Adults.” (the same receptor tyrosine kinase family as In this study, 1375 subjects were randomized to Axl and Tyro-3), which is responsible for pro- seven treatment groups to receive the vaccine moting apoptotic cell engulfment and supports or an active comparator. Table 7.3 summarizes platelet aggregation and clot stability in vivo the clinical trials [47]: phase IeIV clinical [49]. In addition, Kupffer cells, which are liver trials/vaccines/nanoparticles. macrophages, sequester 100-nm nanoparticles [50]. When the particles are 5 nm in diameter, they are rapidly cleared from the circulatory sys- 1.9 Nanocytotoxicity tem via renal filtration [51]. On the other hand, some studies have shown that metallic NPs Engineered NPs have revolutionized the (MeNPs) have effects on innate immunity. Spe- delivery of drugs, monoclonal antibodies, and cifically, in vitro assays showed that MeNPs vaccines to target cells. NPs have been designed have some cytotoxicity and genotoxicity and from metals and nonmetals, polymeric mate- interfere with cytokine production and gene rials, lipids, VLPs, and bioceramics, giving expression due to receptor modifications. The distinctive physicochemical and electrical char- response of the innate immune system to threats acteristics that specifically interact with a is mediated by inflammation and the production targeted cell or organ. However, NPs can enter of cytokines, chemokines, free radicals (nitric easily into the human body by crossing lipid oxide) [52], and other reactive oxygen species

TABLE 7.3 Phase I and III clinical trials with vaccines delivery by nanoparticles.

ClinicalTrials.gov Title Infectious diseases identifier: Phase

Evaluation of the safety and immunogenicity of Influenza NCT03293498 I and II a recombinant trivalent nanoparticle influenza vaccine with matrix M-1 adjuvant (NanoFlu) Dose and formulation confirmation of Influenza NCT03658629 II Quad-NIV in older adults RSV F doseeranging study in women Respiratory syncytial virus NCT01960686 II infections RSV F vaccine maternal immunization study in Respiratory syncytial virus NCT02247726 II healthy infections third-trimester pregnant women.

A study to determine the safety and efficacy Respiratory syncytial virus NCT02624947 III of the RSV F vaccine to protect infants via infections maternal immunization 2. Conclusion 147

(ROS). In this process, there is an accumulation However, the NPs (PLGA) had been modified of oxidized glutathione (GSSG), generating to release antigens slowly to immunize only stress from the proinflammatory signal once and to increase the efficiency of protective (involving TLRs) on the MeNPs and causing adaptive immune responses. Nevertheless, intra- cell death and cancer [53]. Other studies have nasal immunization with NPs had been reported shown that adenovirus VLPs combine with a to cause lung injury through oxidative stress, gene to control Gelsinger’s ammonia metabolism which induced the production of cytotoxic (encoding ornithine transcarbamylase), which cellular responses and inflammatory cytokines. invades all the organs and induces severe reac- Another problem with the NPs is the aggrega- tions that can lead to death [54]. Other severe tion that may block the blood vessels in the side reactions have been reported as carcinogen- host. Modifications of polymeric NPs with PEI esis or germline alterations observed in animal have been used to prevent the recognition to models, in which the VLPs can become widely other cells such as epithelial cells in the lung. dispersed in the body, and the viral vector could Other technologies could be used as the single- end up in the ovaries and testes [55]. Eudragit is chain fragment region of antibodies binding to a drug delivery polymer: poly (ethyl acrylate-co- NPs, which recognizes the specific receptor on methyl methacrylate-cotrimethylammonioethyl dendritic cells or macrophages that may deliver methacrylate chloride) 1:2:0.1. It has been the antigens to target organ. Other modifications reported that using NR8383 macrophages cocul- in the NPs have been used as PLGA covered tured with NP/ERS, the NPs were close to the with PEG (PLGAePEG) and formulated with inner membrane of the mitochondria, as tetanus toxoid peptideeFITC linked to a Lyse observed by transmission electronic microscopy. Lys cathepsin cleavage site and an anti-DC- The authors also analyzed genes responsible for SIGN antibody, which help the delivery to DCs mitochondrial function by microarray and found but not macrophages, with enhanced efficient that Opa1 was reduced in expression. This gene proliferation of T cells [56]. Negatively charged helps to maintain network morphology and NPs prevent the activation of the immune dynamics and in the regulation of the signaling system and induction of immunotolerance, pathways for cell death, and NP/ERS-treated therefore preventing the effect of exacerbated cells reduced glutathione (GSH), stimulating inflammatory responses. Nanoparticles can also ROS production. Due to unbalancing of be engineered to be porous to increase the diffu- oxidanteantioxidant homeostasis and changes sion of intracellular proteases, resulting in earlier of proteins implied in activation and autophagy, processing of APCs and presentation T cells [57]. the mitochondria decay through phagophore In addition, some NPs carrying antigen and and autophagosome formation, and mitophagy adjuvant in the same NP are less efficient to can occur [11]. induce the immune response than the ones The NPs are characterized for their size with adjuvant separated from antigens in (<100 nm) with greater surface area per mass different NPs. This may because of the compared with larger-sized particles of the competing effect of coactivation in several same chemistry causing NPs more active biolog- pathways [58]. ically. The physicochemical properties of the material could increase the uptake by the antigen-presenting cells, but they can go to other 2. 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[45] Smith G, Liu Y, Flyer D, Massare MJ, Zhou B, Patel N, [52] Stuehr DJ, Nathan CF. Nitric oxide. A macrophage et al. Novel hemagglutinin nanoparticle influenza vac- product responsible for cytostasis and respiratory inhi- cine with Matrix-MÔ adjuvant induces hemagglutina- bition in tumor target cells. J Exp Med 1989;169(5): tion inhibition, neutralizing, and protective responses 1543e55. Epub 1989/05/01. in ferrets against homologous and drifted A (H3N2) [53] Petrarca C, Clemente E, Amato V, Pedata P, Sabbioni E, subtypes. Vaccine 2017;35(40):5366e72. Bernardini G, et al. Engineered metal based nanopar- [46] August A, Glenn GM, Kpamegan E, Hickman SP, ticles and innate immunity. Clin Mol Allergy 2015; Jani D, Lu H, et al. A Phase 2 randomized, observer- 13(1):13. Epub 2015/07/17. blind, placebo-controlled, dose-ranging trial of [54] Marshall E. Gene therapy death prompts review of aluminum-adjuvanted respiratory syncytial virus F adenovirus vector. Science 1999;286(5448):2244e5. particle vaccine formulations in healthy women of [55] Boyce N. Trial halted after gene shows up in semen. childbearing age. Vaccine 2017;35(30):3749e59. Nature Publishing Group; 2001. [47] Medicine NUSNLo. Clinical trials. Gov. [56] Cruz JC, Pfromm PH, Tomich JM, Rezac ME. Confor- [48] Pourmand A, Abdollahi M. Current opinion on mational changes and catalytic competency of hydro- nanotoxicology. Daru 2012;20(1):95. Epub 2013/01/29. lases adsorbing on fumed silica nanoparticles: II. [49] Sather S, Kenyon KD, Lefkowitz JB, Liang X, Secondary structure. Colloids Surf B Biointerfaces Varnum BC, Henson PM, et al. A soluble form of the 2010;81(1):1e10. Epub 2010/07/20. Mer receptor tyrosine kinase inhibits macrophage [57] Bookstaver ML, Tsai SJ, Bromberg JS, Jewell CM. clearance of apoptotic cells and platelet aggregation. Improving vaccine and immunotherapy design using Blood 2007;109(3):1026e33. Epub 2006/10/19. biomaterials. Trends Immunol 2018;39(2):135e50. [50] Devadasu VR, Bhardwaj V, Kumar MN. Can contro- [58] Mohsen MO, Gomes AC, Cabral-Miranda G, versial nanotechnology promise drug delivery? Chem Krueger CC, Leoratti FM, Stein JV, et al. Delivering ad- Rev 2013;113(3):1686e735. Epub 2013/01/02. juvants and antigens in separate nanoparticles elimi- [51] Tang S, Chen M, Zheng N. Sub-10-nm Pd nanosheets nates the need of physical linkage for effective with renal clearance for efficient near-infrared photo- vaccination. J Control Release 2017;251:92e100. thermal cancer therapy. Small 2014;10(15):3139e44. Epub 2014/04/15. CHAPTER 8

Lipid nanocarriers for delivery of poorly soluble and poorly permeable drugs AnCelka B. Kovacevic Department of Pharmaceutical Technology, Institute of Pharmacy, Faculty of Biological Sciences, Friedrich-Schiller University Jena, Jena, Germany

1. Introduction delivery technologies for poorly soluble compounds. It is estimated that about 40% of new drug can- In the broadest sense, approaches to overcome didates in the development pipelines of origi- the poor aqueous solubility of drug candidates nator companies and approximately 60% of new comprise chemical and physical modifications. chemical entities coming directly from chemical Among chemical modifications the most synthesis are poorly soluble. Therefore, most of commonly used are pH adjustment and prodrug these drug candidates exhibit a poor oral design, whereas physical modifications comprise bioavailability and fail to meet appropriate modification of the solid state of the drugs (forma- absorption, distribution, metabolism, and excre- tion of polymorphs, hydrates, salts, cocrystals, tion (ADME) properties, because poor solubility conversion of the crystalline form of the drug to is generally correlated with poor dissolution ve- the amorphous one), particle size reduction locity. If the dissolution velocity is too low, suffi- technologies, the use of cosolvents, complexation ciently high blood levels cannot be achieved. As a with cyclodextrins, hydrotropy, preparation of result of that, the bioavailability from conven- solid dispersions/coprecipitates, and formulation tional formulations (e.g., tablets) may be of surfactant- and lipid-based drug delivery unacceptable [1]. Consequently today research systems. All these strategies can be used alone or focuses on the development of more effective in combination and offer panel options for formu- and versatile approaches that can make poorly lators to address the challenges related to poorly soluble compounds sufficiently bioavailable [2]. soluble drugs. Each approach has its applicability Simultaneously, regulatory authorities (e.g., U.S. and constraints and hence comprehensive physi- Food and Drug Administration (FDA), European cochemical characterization of drug candidates is Medicines Agency (EMA)) are developing new needed to provide the key for a successful guidelines to deal with special delivery technolo- development [3]. gies and quality requirements related to drug

Nanopharmaceuticals https://doi.org/10.1016/B978-0-12-817778-5.00008-7 151 © 2020 Elsevier Inc. All rights reserved. 152 8. Lipid nanocarriers for delivery of poorly soluble and poorly permeable drugs

Some authors suggested that when devel- 2. Biopharmaceutics Classification oping formulation strategies for poorly water Systemdimplications for drug delivery soluble drugs it is useful to distinguish between compounds whose solubility is mainly limited Biopharmaceutics Classification System by their solid state properties (colloquially called (BCS) is a scientific classification of a drug sub- “brick dust” molecules) and substances with stance based on its aqueous solubility and intes- solvation-limited solubility (“grease balls”). tinal permeability that correlates in vitro Solid stateelimited solubility results from dissolution and in vivo bioavailability of drug strong intermolecular bonds within a tightly products [14,15]. When combined with in vitro packed crystalline lattice, indicated by a rather dissolution characteristics of the drug product, high melting point of the compound BCS takes into account two major factors: solu- (e.g., >200 C). In contrast, “grease ball”elike bility and intestinal permeability, which govern drug substances have high lipophilicity (re- the rate and extent of oral drug absorption from flected by a high logP/logD value >3) that ham- solid dosage forms and, ultimately, its bioavail- pers interaction with water molecules. ability [16].Duetothisreason,BCSisafunda- Compounds that combine both unfavorable mental tool in the drug development, properties also exist (e.g., levothyroxine with a especially in the development of oral drug logP of 4.6 and melting point of 235 C) [4]. products [3]. BCS implies that aqueous solubil- “Grease-ball”elike substances can often be ity and membrane permeability are two major formulated using lipid-based approaches, factors limiting drug absorption [14].Adrug whereas “brick dust” molecules usually require is considered to be highly soluble when the formulationinamodified solid state [5]. highest dose strength is soluble in 250 mL water Nanocarriers represent a more recent option or less over a pH range from 1 to 7.5 at for the formulation of poorly soluble drugs that 37 0.5 C. In contrast, if the dose to solubility can involve both chemical and physical modifi- ratio is above 250 mL, the drug is considered cations. They can be found in the sections as poorly soluble [17]. describing prodrug design, small drug particles, A highly permeable drug is defined as a drug surfactant- and lipid-based formulations [6]. where the extent of absorption (including intesti- Among them, lipid nanocarriers, representing nal and liver first-pass metabolism) is greater around 4% of the commercially available drug than 90% of the dose administered. Absorption products in the United States, United Kingdom, is, thus, taken to be the transport of the drug into and Japan, are particularly interesting because the first cell, tissue, or interstitial fluid through of their wide diversity, favorable biocompati- the tight junctions between the intestinal epithelial bility, and specific functionality. cells. The 90% fraction absorbed defines the lower This chapter aims to provide a concise com- limit for a highly permeable drug [14].Lipinski’s pendium of current knowledge on lipid nanocar- rule of five predicts that poor absorption or riers for oral drug delivery and their key poor permeation is more likely for molecules application area, i.e., solubility and permeability that have more than five hydrogen bond donors, enhancement, as well as targeted drug delivery, more than 10 hydrogen bond acceptors, molecu- except liposomes, where a vast majority of liter- lar mass above 500 Da, and octanol-water parti- ature data already exists [7e13]. tion coefficient above 5 [17]. 3. Classification and composition of lipid nanocarriers for oral drug delivery 153

Classification systems such as the BCS and solubility, low metabolism), the formulation Lipinski’s rule of five are useful particularly at may require the presence of lipid-based excipi- the initial screening stage, but they also have ents that facilitate permeation in addition to solu- limitations. While determining the fundamental bilization [20]. physicochemical properties of the drug, such as solubility and lipophilicity, are central to any oral formulation strategy, it is important not to 3. Classification and composition of lipid focus solely on these parameters. The interaction nanocarriers for oral drug delivery between a drug and the gastrointestinal (GI) environment, or the biopharmaceutical properties A large variety of lipid formulations can be of the drug, requires close consideration. Conse- used to improve the oral absorption of lipophilic quently, aqueous solubility and/or octanol- drugs, ranging in complexity from the least com- fi water partition coef cient alone are unlikely to plex e.g., one-excipient formulations such as fi be suf cient for identifying the suitability of a lipid lipid solutions to more complex systems encom- formulations, as they do not adequately predict passing vesicular and nonvesicular lipid nano- potential in vivo (i.e., physiological) effects. The carriers [21e24]. fl complex physiological factors that in uence the The range of excipients used in lipid nanocar- rate of drug presentation at the intestinal surface, riers for oral drug delivery is broad and can be fi transmembrane transport process, and nally categorized into three groups: lipids, (co)surfac- release into the systemic circulation are also essen- tants, and (co)solvents (Table 8.1). Since the effi- tial and cannot be taken in isolation. Wu and Benet ciency of oral absorption of the drug compounds [18] reassessed the application of the BCS as a from the lipid-based formulations depends on means of predicting drug disposition. A review many formulation-related parameters, only fi e of the drugs classi ed in classes I IV by the very specific pharmaceutical excipient combina- BCS highlighted that drugs in classes I and II tions will lead to an efficient system. The param- were metabolized and eliminated, in contrast to eters to be considered in the selection of drugs in classes III and IV that were eliminated excipients for oral lipid formulations are safety fi fi unchanged. On this basis, a modi ed classi ca- potential, dispersibility, digestibility, stability, tion system, namely the Biopharmaceutics Drug capacity to dissolve/solubilize poorly soluble fi Disposition Classi cation System (BDDCS) was drugs, and price. proposed. Under this system, the extent of meta- The frequently chosen components of the bolism (or major route of drug elimination) lipid phase are vegetable oils, predominantly would replace membrane permeability as a classi- composed of both saturated and unsaturated fi cation criterion. The proposed advantages of the medium-chain and long-chain fatty acids. Edible fi BDDCS include making drug classi cation easier oils that could represent the logical and by avoiding the ambiguity that exists with perme- preferred lipid excipient choice are not ability, as metabolism may be easier assessed frequently used because of their poor ability to experimentally, thus reducing the number of dissolve large amounts of lipophilic drugs. In or- drugs listed in more than one category [19].By der to decrease the number of double bonds and fi ade nition, a type II BDDCS drug (low solubil- confer resistance to oxidative degradation, ity, intense metabolism) needs solubility hydrogenated oils and modified vegetable oils enhancers. For a type IV BDDCS drug (low have been widely used since these excipients TABLE 8.1 The most frequent excipients in lipid nanocarriers for oral drug delivery [5,26,35e37].

Solvents/ Carrier type Lipids Surfactants/Cosurfactants Cosolvents

Microemulsions Long-chain triglycerides Polyoxyethylene-polyoxypropylene block copolymers Ethanol Nanoemulsions (olive oil, corn oil, soybean oil, (poloxamer 407, poloxamer 188) Propylene Ò Ò SEDDSs castor oil, sesame oil, peanut oil, rapeseed oil) Polyoxylglycerides (Labrasol , Labrafil M-2125CS, glycol Ò Ò Ò SMEDDs Hydrogenated vegetable oils (castor oil, soybean oil, Labrafil M1944 CS, Labrafac Hydrophile, Labrafac PEG Ò Ò SNEDDSs cotton oil, palm oil, corn oil) CM6 BM290, Labrafac CM10, Labrafil WL 2609 BS, Glycerol Ò Ò Ò Medium-chain mono- and diglycerides Labrafac CM 10, Tagat TO, Gelucire 44/14, Ò Ò Ò Ò Medium-chain triglycerides (Miglyol 810, Miglyol Cremophor RH40, Cremophor EL) Ò Ò 812, Captex 255, Captex 300) Mono- and diglycerides (glyceryl monocaprylate, glyceryl Fatty acids (oleic acid, palmitic acid, soy fatty acids) monooleate, glyceryl monostearate, glyceryl monolinoleate) Fatty acid esters (e.g., ethyl oleate, glyceryl dioleate) Propylene glycol fatty acid esters (propylene glycol Other lipids (dyacetylated monoglycerides, dicaprylate/dicaprate, propylene glycol monocaprylate TM D-a-tocopherol, beeswax) (Capryol 90), propylene glycol monolaurate (LauroglycolTM 90)) Ò Ò Ò Sorbitane fatty acid esters (Span 20, Span 40, Span 60, Ò Span 80) Polyethoxylated sorbitane fatty acid esters (polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80) Ò Ò Ò Polyoxyethylene alkyl ethers (Brij 35, Brij 56, Brij 78) Macrogol fatty acid esters (macrogol-8 stearate, macrogol- 40 stearate) Other excipients (D-a-tocopherol-polyethyleneglycol-1000 succinate (TPGS), Tyloxapol, sodium deoxycholate, Ò Ò Transcutol HP, Triton X-100) Lipid Monoglycerides (glyceryl monostearate, glyceryl Ionic surfactants (sodium cholate, sodium glycocholate, nanoparticles hydroxystearate, glyceryl behenate) sodium taurocholate, sodium taurodeoxycholate, sodium Diglycerides (glyceryl palmitostearate, glyceryl oleate, sodium dodecyl sulfate, cetylpyridinium chloride) dibehenate) Nonionic surfactants (polysorbate 20, polysorbate 60, Ò Ò Ò Ò Triglycerides (glyceryl tricaprylate, glyceryl tricaprate, polysorbate 80, Span 20, Span 80, Span 85, Brij 78, Ò Ò glyceryl trilaurate, glyceryl trimyristate, glyceryl Tego Care 450, Cremophor EL) tripalmitate, glyceryl tristearate, glyceryl tribehenate) Amphoteric surfactants (egg phosphatidylcholine (Lipoid E Polyoxylglycerides (polyethylene glycol monostearate) PC S), soy phosphatidylcholine (Lipoid S 100, Lipoid SP C), Hard fats (stearic acid, palmitic acid, behenic acid) hydrogenated egg phosphatidylcholine (Lipoid E PC-3), Fatty alcohol (octadecyl alcohol) hydrogenated soy phosphatidylcholine (Lipoid S PC-3, Ò Ò Steroids (cholesterol) Phospholipon 80H, Phospholipon 90H), egg Waxes (cetyl palmitate, beeswax, carnauba wax) phospholipid (Lipoid E 80, Lipoid E 80 S), soy phospholipid Fatty acids (dodecanoic acid, myristic acid, palmitic (Lipoid S 75)) acid, stearic acid) Polymeric surfactants (Tyloxapol, poloxamer 188, Ò Liquid lipids (soybean oil, medium chain triglycerides poloxamer 407, poloxamine 908, Solutol HS15, polyvinyl Ò (Miglyol 812), oleic acid, vitamin E, squalene, isopropyl alcohol) myristate, oleoyl macrogol-6 glycerides) 4. Types of lipid nanocarriers for oral drug delivery 155 form good emulsification systems with a large glycol, and glycerol. They enable the dissolution of number of surfactants approved for oral admin- large quantities of either the hydrophilic surfac- istration and exhibit better solubility potential tant or the drug in the lipid base. These solvents than edible oils [25e28]. The panel of solid lipids can even act as cosurfactants in microemulsion used in lipid nanocarriers is very broad, systems [26]. including triglycerides, partial glycerides, fatty acids, steroids, waxes, and fatty alcohols [20]. 4. Types of lipid nanocarriers for oral drug Safety is a major determining factor in delivery choosing a surfactant. The surfactants of natural origin are preferred since they are considered 4.1 Vesicular lipid nanocarriers safer than the synthetic one. However, these excipients have a limited self-emulsification Vesicles are water-filled colloidal particles. The capacity [29]. Nonionic surfactants are less toxic walls of these capsules consist of amphiphilic than ionic ones, but they may lead to reversible molecules in a bilayer conformation. In an excess changes in the permeability of the intestinal of water, these amphiphilic molecules can form lumen [27]. one (unilamellar vesicles) or more (multilamellar The concentration of the surfactants should be vesicles) concentric bilayers [38].Hydrophilic properly determined, because high concentra- drugs can be entrapped into the internal aqueous tions may cause gastrointestinal tract (GIT) irri- compartment, whereas amphiphilic, lipophilic, tation, whereas low concentrations negatively and charged hydrophilic drugs can be associated influence the size of the droplets/particles. In with the vesicle bilayer by hydrophobic and/or principle, increasing the surfactant concentration electrostatic interactions [39]. leads to droplets with smaller mean size. This In 1998 deformable vesicles consisting mainly could be explained by the stabilization of the of nonionic bilayer forming surfactants called oil droplets as a result of the localization of niosomes were introduced [40,41]. They are the surfactant molecules at the oil/water inter- structurally analogous to liposomes, but the syn- face [30]. On the other hand, in some cases, the thetic surfactants used have advantages over mean droplet size may increase with increasing phospholipids in that they are significantly less surfactant concentrations [31e33]. This phenom- costly and have higher chemical stability than enon could be attributed to the interfacial their naturally occurring phospholipid counter- disruption elicited by enhanced water penetra- parts [42]. Niosomes are obtained on the hydra- tion into the oil droplets mediated by the tion of synthetic nonionic surfactants (mainly of increased surfactant concentration and leading alkyl or dialkyl polyglycerol ether class), with or to the ejection of oil droplets into the aqueous without incorporation of cholesterol or other phase [34]. lipids. Addition of cholesterol provides rigidity Various pharmaceutically acceptable surfac- to the bilayer leading to the formation of less tants can be found in lipid formulations permeable niosomes. They are similar to lipo- Ò Ò including Cremophor EL, Cremophor RH40, somes in functionality and also increase the Ò Cremophor RH60, polysorbate 20, polysorbate bioavailability of the drug and reduce the clear- 80, D-a-tocopherol polyethylene glycol 1000 suc- ance like liposomes [43]. To overcome the low, Ò Ò cinate (TPGS), Span 20, various Labrafils , Lab- variable oral bioavailability of carvedilol, Arzani Ò Ò rasol , and Gelucires (Table 8.1) [35]. et al.,[44] prepared three different niosomal for- The most frequent lipid soluble/miscible mulations: plain niosomes, bile salt (sodium cosolvents in lipid-based formulations are poly- cholate or sodium taurocholate)-enriched nio- ethylene glycol 400 (PEG 400), ethanol, propylene somes, and charged niosomes (negative and 156 8. Lipid nanocarriers for delivery of poorly soluble and poorly permeable drugs positive charged). All three formulations its oral bioavailability. In vivo absorption study enhanced the oral absorption of carvedilol. Phar- in rats indicated that the powder precursor of macokinetic studies showed that incorporation cubosomes improved the bioavailability of drug into niosomes helped increase plasma of tamoxifen citrate in terms of rate and extent levels of carvedilol compared to a suspension. of absorption compared to plain powder drug. Moreover, the incorporation of bile salts into ves- Pharmacosomes are amphiphilic phospho- icles gave enhanced intestinal absorption of car- lipid complexes that contain phospholipid and vedilol. The results of chylomicron flow blocking both positive and negative charge, water loving experiments suggest a major contribution of the and fat loving active ingredient or phytocon- lymphatic route to the enhanced oral absorption stituent. In pharmacosomes, the lipid and active of carvedilol conferred by niosomes [44]. constituent have conjugated by electron pair The use of special lipids, such as diether lipids sharing and electrostatic forces or by formation or fully saturated bipolar tetraether lipids of hydrogen bond with lipid [51]. They are stable (extracted from Archaeobacteria or synthetic and more bioavailable drug delivery systems archaeal lipid) results in the formation of vesicles with low interfacial tension between the system in the nano range named archeosomes. They and the GI fluid, thereby facilitating were reported to be highly stable as compared the membrane, tissue, or cell wall transfer in to conventional liposomes in various conditions the organism. Therefore, drug formulation as like high temperature, acidic or alkaline pH, pharmacosomes may be a potential approach and oxidative stress. It was also described that to improve solubility and to minimize the GIT they prevent degradation by bile salts and toxicity of some drugs (e.g., diclofenac, ketopro- lipases and help to control drug release [45]. fen, rosuvastatin). In the pharmacosomes the In presence of polar solvents, the hydrophobic drug is reversibly bonded chemically with region of amphiphilic molecules self-assembles the lipids and thus shows not only better loading into an array of thermodynamically stable liquid capacity than also better stability than in lipo- crystalline phases with lengths on the nanometer somes. Depending upon the chemical structure scale. One example is a bicontinuous cubic liquid of the drug-lipid complex they may exist as crystalline phase. One of the important proper- ultrafine vesicular, micellar, or hexagonal aggre- ties these cubic phases have is their ability to gates [52e55]. To improve the water solubility of be dispersed into the particles stabilized with diclofenac, Semalty et al. [56], have been pre- surfactants, termed as cubosomes [46]. There- pared and evaluated pharmacosomes. Physico- fore, cubosomes consist of folded lipid bilayers chemical investigations showed that diclofenac curved in three-dimensional space with inter- formed a stoichiometric complex with phospho- woven water channels. The complex amphi- lipid in pharmacosomes with improved solubil- pathic structure of the cubosome makes ity and dissolution profile, as compared to incorporation of hydrophilic (in the aqueous diclofenac powder. channels), hydrophobic (in the lipid bilayers) and amphiphilic drugs (at the bilayer-water 4.2 Nonvesicular lipid nanocarriers interface) possible [47]. Cubosomes offer a unique possibility of encapsulating and protect- Microemulsions are isotropic, transparent, ing protein and peptide drugs from degradation thermodynamically stable colloidal systems con- [48,49]. Nasr and Dawoud [50] developed sisting of the water phase, an oil phase, and suffi- sorbitol-based powder precursor of cubosomes cient concentration of surfactant in combination loaded with tamoxifen citrate as a model of with an appropriate cosurfactant. From drug poorly water soluble drug, aimed at enhancing delivery perspective microemulsions are 4. Types of lipid nanocarriers for oral drug delivery 157 attractive due to the existence of microdomains of incorporated surfactant necessary to promote different polarity within the same single-phase self-emulsification, which can occasionally be solution, which can facilitate solubilization of problematic concerning local or systemic toxicity, either hydrophilic or lipophilic materials. More- particularly when it is necessary to administer over, the existence of microstructure organized multiple dosage units for a single dose [26]. on the level below 100 nm provides a large inter- Nanoemulsions are defined as a thermody- face for solubilization of drug molecules [57e60]. namically unstable colloidal dispersion of two To improve the solubility and bioavailability immiscible liquids, with one liquid dispersed as of poorly water-soluble biphenyl dimethyl dicar- small spherical droplets in another liquid [64]. boxylate, the drug used in treating liver diseases, Darunavir is a second-generation synthetic pep- Kim et al. [61] prepared a premicroemulsion tidomimetic protease inhibitor, designed for the concentrate and evaluated its physicochemical treatment of human immunodeficiency virus properties and the pharmacokinetic parameters infection with resistance to other available pro- in comparison to 0.5% calcium carboxymethylcel- tease inhibitors. Desai and Thakkar [65] aimed lulose suspension containing the same drug. The to formulate darunavir-loaded nanoemulsion drug solubility in premicroemulsion concentrate to increase oral drug bioavailability and brain improved compared to the drug suspension or uptake. In comparison to drug nanosuspension, drug powder alone. Area under the curve asignificant enhancement in the bioavailability (AUC) and the mean maximum plasma level of of darunavir was observed after in vivo admin- biphenyl dimethyl dicarboxylate after oral istration of drug-loaded nanoemulsions in male administration of premicroemulsion concentrate Wistar rats. Improvement in bioavailability in rats were 5- and 9.8-fold higher, respectively, relative to suspension can be attributed to than those of drug within the suspension. These many factors, which, either in combination or results demonstrate that premicroemulsion alone, contribute for favored absorption. These greatly enhances the bioavailability of the drug, factors are (a) the presence of the lipophilic possibly due to the increase in solubility and drug in the solution or in small emulsion drop- immediate dispersion of biphenyl dimethyl lets that eliminates the dissolution step and dicarboxylate in the GIT. Nazar et al. [62] keeps drug in a dissolved state during transport reported enhanced solubility of piroxicam in to the unstirred water layer of the GIT mem- castor oil/Tween 80/ethanol/phosphate buffer brane, (b) lymphatic transport through intesti- microemulsion. nal transcellular pathways, (c) modulation of Nandi et al. [63] examined the solubility of two the P-gp efflux pumps, (d) modulation of model drugs, progesterone and indomethacin, in CYP450 enzymes function at the intestinal re- isopropyl myristate microemulsion system con- gion, and (e) improvement in the drug absorp- taining alkanols and cyclodextrins. Isopropyl tionbythepresenceofTween80assurfactant myristate-based microemulsion systems alone in the formulation. The results of this study could increase the solubility of progesterone and also envisage favored absorption of darunavir indomethacin up to 3300-fold and 500-fold, from nanoemulsion due to the presence of respectively, compared to water. However, the long-chain triglyceride in soya bean oil that en- addition of cyclodextrins to the microemulsion hances lipoprotein synthesis and subsequent systems did not show a synergistic effect in lymphatic absorption [66,67]. increasing the solubility for the two selected Singh and Vingkar [68] formulated primaquine- model drugs. loaded nanoemulsion for oral delivery and One of the challenges in the formulation of reported improved oral drug bioavailability and microemulsions is the high concentrations of higher levels of the drug in the liver. Evaluation 158 8. Lipid nanocarriers for delivery of poorly soluble and poorly permeable drugs of antimalarial activity showed that primaquine spontaneously emulsify and form coarse oil/wa- nanoemulsion could display effective therapeutic ter (o/w) emulsions (droplet size usually above activity at a lower dose level than the conventional 250 nm) [26,71]. At a given temperature, self- dose. emulsification occurs when the entropy change Silymarin, the drug approved for the treatment that favors dispersion is greater than the energy of various liver disorders, possesses poor oral required to increase the surface area of the emul- bioavailability due to rapid hepatic metabolism, sion [72]. Because of this property, they are poor solubility in water, and erratic absorption. considered as good candidates for oral delivery Nagi et al. [69]. developed silymarin-loaded of hydrophobic drugs with adequate oil solubil- nanoemulsion that resulted in improved in vitro ity. The certain limitations ascribed to the drug release, significant improvement in pharma- SEDDSs are low drug-loading capacity, risk of cokinetic parameters, and enhanced bioavail- drug precipitation, chemical instability, incom- ability compared to marketed silymarin patibility of the volatile components of the nanosuspension. formulation with gelatin capsule shell that can Although the nanoemulsions improve the lead to drug precipitation at storage, drug absorption of hydrophobic drugs in GIT, their leakage, portability, high production costs, and use in oral delivery may be limited. Due to their problems in storage [73e75]. lipid composition or consumption of a higher Self-microemulsifying drug delivery systems volume to achieve the necessary therapeutic con- (SMEDDSs) or microemulsion preconcentrates centration for certain drugs that have limited are defined as physically stable isotropic mix- solubility in the pharmaceutically acceptable tures of oils (up to 20%), surfactants (HLB > lipophilic solvents, these carriers may have 11) (20%e50%), cosolvents (20%e50%), and poor palatability. Nanoemulsions have high drug. They are classified as type IIIb lipid formu- water content, and hence cannot be delivered lations for the oral drug delivery, according to through soft gelatin, hard gelatin, or hydroxy- the classification system proposed by Pouton propylmethylcellulose capsules. The water [70]. On initial dilution of SMEDDSs with content within nanoemulsion may promote aqueous media, o/w microemulsion may form, hydrolysis and/or precipitation of certain drugs which on further dilution may then undergo on long-term storage, which could affect their phase inversion to either a water/oil (w/o) or utility. Moreover, the physical stability of water/oil/water (w/o/w) microemulsion [60]. nanoemulsions may be affected by pH and tem- SMEDDSs are distinguished from SEDDSs by perature, parameters that change upon delivery emulsion droplet size produced on dilution. of nanoemulsions to the patients. This led to Typically, the size of the droplets produced by the development of their preconcentrates, which dilution of SEDDSs is above 250 nm. Upon generated much academic and industrial interest dispersing in water, SMEDDSs form a trans- in recent years [9]. parent microemulsion with droplets usually Self-emulsifying drug delivery systems smaller than 100 nm, depending on the excipient (SEDDSs) are classified as type II lipid formula- selection and relative composition of the formu- tions for oral delivery, according to the lipid lation. When compared with emulsions, which formulation classification system proposed by are sensitive and metastable dispersed forms, Pouton [70]. They are described as isotropic mix- SMEDDSs are physically stable formulations tures of oils (40%e80%), lipophilic surfactant that are easy to manufacture [76]. An additional (HLB < 12) (20%e60%), and a solubilized reason for the increasing use of SMEDDSs for the drug. Upon mild agitation followed by dilution delivery of poorly soluble drugs is the fact that in aqueous media, such as GIT fluids, they these systems are presented in the form of 4. Types of lipid nanocarriers for oral drug delivery 159 preconcentrated solution. Hence, the dissolution thermodynamically stable, anhydrous mixture step required for solid crystalline compounds of oil, surfactant, cosurfactant, and drug. After shall be avoided [30,77]. Of particular interest oral administration upon gentle agitation or to the pharmaceutical industry is also the fact digestive motility of the aqueous fluid in the that such “preconcentrates” can be packaged GIT, they rapidly disperse and spontaneously into soft and hard gelatin (or hydroxypropyl- form o/w nanoemulsions (usually droplet size methylcellulose) capsules allowing precise and between 100 and to 200 nm) [82,83]. Therefore, convenient dosing [26]. The optimum concentra- SNEDDSs have the advantage in possessing tions of lipids, surfactants, and cosurfactants higher solubilization capacity than simple necessary to promote self-emulsification in micellar solutions, leading to the incorporation SMEDDSs are determined by the construction of poor water-soluble pharmaceuticals inside of a pseudo-ternary phase diagram, whereas the oil phase [84]. The advantages of SNEDDSs experimental design can be used to further opti- are the significantly reduced energy required for mize these systems [78]. their preparation, physical stability upon storage, To improve the solubility and release rate and easy manufacture on a large scale [25,85]. of resveratrol, Bolko et al. [79] formulated To improve the peroral bioavailability of SMEDDSs. Overall, incorporation of resveratrol nystatin, Kassem et al. [86] developed and opti- in SMEDDSs resulted in improved solubility mized nystatin-loaded SNEDDSs and evaluated (over 23-fold) and dissolution rate compared to in vitro and in vivo performance of the formula- crystalline resveratrol. tion. The release patterns revealed that nystatin Hwang et al. [80] developed and character- release was significantly higher from SNEDDSs ized SMEDDSs containing a fixed-dose combina- compared to plain drug suspension under the tion of atorvastatin and ezetimibe. In vitro same conditions. Drug release from suspension dissolution studies showed that the SMEDDSs did not exceed 47% after 48 h, whereas 98% of had higher initial dissolution rates for both the drug was released from SNEDDSs after 48 drugs compared to the marketed products. h. The faster drug release from SNEDDSs is More importantly, ezetimibe had a significantly attributed to the spontaneous formation of nano- increased dissolution profile in distilled water emulsion due to low surface free energy at the and acetate buffer (pH ¼ 4), implying enhanced oil-water interface, which causes immediate solu- bioavailability. To improve solubility and bilization of drug in the release medium. During absorption of an immunosuppressive drug and emulsification with water, the nanoemulsion P-gp substrate, sirolimus, SMEDDSs containing components (oil, surfactant, and cosurfactant) a novel P-gp inhibitor, honokiol, were prepared. effectively swell and this decreases the globule It was shown that the absorption rate constant, size, which leads to increase in surface area and the effective permeability coefficients of siroli- decrease of surface free energy, thus eventually mus, in situ intestinal absorption, and the increasing the drug release rate [87].Invitro apparent permeability coefficients of sirolimus and in vivo evaluations against Candida albicans in Caco-2 cells were significantly enhanced by depicted promoted antifungal efficacy of selected Cremophor EL-based SMEDDSs with honokiol nystatin-loaded SNEDDSs compared to mar- as compared with those of SMEDDSs without keted and plain nystatin suspensions. The results honokiol [81]. indicate that nystatin-loaded SNEDDSs, with Self-nanoemulsifying drug-delivery systems enhanced solubilization and nanosizing, have (SNEDDSs) are the technological advances of the potential to improve the absorption of the the SEDDSs that have been described in the drug and increase its oral antifungal efficacy. literature as homogenous, transparent, 160 8. Lipid nanocarriers for delivery of poorly soluble and poorly permeable drugs

Solid self-emulsifying drug delivery systems in a formulation containing TPGS as surfactant (S-SEDDSs) provide an effective alternative to and solidifier [91]. the liquid SEDDSs for formulating drugs with Inugala et al. [92] investigated the potential of poor aqueous solubility. They consist of a disper- S-SNEDDSs and liquid SNEDDSs in improving sion of the drug in an inert excipient matrix, the dissolution and oral bioavailability of daru- where the drug could exist in either the finely navir. In vitro dissolution studies indicated divided crystalline, solubilized, or amorphous faster dissolution of darunavir from the devel- states or a mixture thereof [88]. S-SEDDSs are oped S-SNEDDSs with three-times greater formulated by incorporation of liquid or semi- mean dissolution rate compared to pure drug. solid self-emulsifying ingredients into powder Dissolution test showed higher drug release or nanoparticles by different solidification tech- over 24 h from liquid SNEDDSs containing dar- niques (e.g., spray drying, adsorption to solid unavir compared to pure darunavir dispersion. carriers, melt granulation, melt extrusion) where Such a pattern of drug release from liquid the powders or nanoparticles refer to self- SNEDDSs by carrying the entrapped drug in emulsifying nanoparticles, dry emulsions, and the form of fine emulsion droplets to the site of solid dispersions that can be further processed absorption is advantageous in increasing into other solid self-emulsifying dosage forms bioavailability, by enhancing the release of the or filled into capsules [89]. Since S-SEDDSs are poorly water-soluble drug [24]. In the same solid at room temperature, they can be exploited study liquid SNEDDSs were transformed into Ò into various dosage forms that are solid with solid SNEDDSs using Neusilin US2 as adsor- self-emulsifying properties, like capsules, solid bent. The comparative dissolution profiles of dispersions, pellets, tablets, microspheres, nano- pure darunavir and darunavir in S-SNEDDSs, particles, suppositories, implants, and dry emul- performed in 0.1 N HCl (pH 1.2) as dissolution sions. S-SEDDSs are more desirable than medium, showed faster drug release vis-a-vis conventional liquid SEDDSs, which are normally pure drug. S-SNEDDSs showed better dissolu- prepared either as a liquid or encapsulated in tion performance with higher mean dissolution soft gelatin capsules [3]. They have been widely rate and lower mean dissolution time versus studied for the enhancement of solubility and pure drug signifying rapid drug release from dissolution of various poorly soluble drugs. the prepared S-SNEDDSs. Rapid drug release Agarwal et al. [90] prepared powdered self- from the S-SNEDDSs may be due to the low emulsified lipid formulation of meloxicam by surface free energy of the self-emulsifying simple trituration of liquid SEDDSs with the systems, which favors rapid emulsification by solid adsorbent (1:1 mixture of silicon dioxide the quick establishment of an interface between and magnesium aluminum silicate). The dissolution medium and oil [31]. Further powdered SEDDSs showed higher bioavail- improved dissolution is also credited by (a) the Ò ability in beagle dogs compared to commercially greater surface area of the Neusilin US2 with available tablets. high porosity which allows quick entrance of In the study of Kanaujia et al. [91], S-SEDDSs release medium into the pores and rapid of fenofibrate was formulated by solidification of emulsification, (b) small size of the globules, the molten solution of the oily phase, surfactant, (c) transformation of darunavir physical state cosurfactant, and drug mixture with a polymer from low water-soluble crystalline form to an (PEG 6000). S-SEDDSs with 10% (w/w) fenofi- amorphous or disordered crystalline form. brate showed as much as a 20-fold increase in In vivo oral bioavailability studies conducted the dissolution profile compared to fenofibrate in Wistar rats by comparing the pharmacokinetic 4. Types of lipid nanocarriers for oral drug delivery 161 profiles of darunavir from pure darunavir, liquid These numerous studies confirm that S- SNEDDSs, and S-SNEDDSs revealed signifi- SEDDSs can substantially improve the solubility, cantly higher serum drug profiles from both dissolution rate, and bioavailability of drugs. liquid SNEDDSs and S-SNEDDSs compared to They can be also regarded as a cost-effective pure drug. The peak drug concentrations of approach for the preparation of various solid liquid SNEDDSs and S-SNEDDSs were approxi- oral dosage forms of poorly soluble drugs over- mately 2- and 2.5-fold higher, respectively, than coming the disadvantages of conventional liquid the peak drug concentrations of pure drug. SEDDSs concurrently. However, certain aspects However, the time to reach the peak remained of S-SEDDSs such as oxidation of vegetable oils, constant, which indicates that the transformation physical aging associated with glycerides, strong of emulsion from liquid SNEDDSs and adsorption and physical interaction of the drug, S-SNEDDSs was spontaneous. Similarly, other and excipients that may cause retarded or incom- pharmacokinetic parameters such as half-life, plete drug release from the S-SEDDSs must be mean residence time, and AUC were also found considered while formulating future S-SEDDSs to be significantly higher from both liquid [93,94]. SNEDDSs and S-SNEDDSs concerning pure daru- To overcome such difficulties there has been navir. However, liquid SNEDDSs showed a an increasing focus on the application of super- greater initial rate of absorption compared to saturatable SNEDDSs [95,96]. Supersaturable pure darunavir and S-SNEDDSs, which might be SNEDDSs contain a water-soluble polymeric due to the liquid state of these carriers. Overall, precipitation inhibitor (PPI) in the typical these results corroborate the improved rate and SNEDDSs. The PPI retards excessive drug pre- extent of the drug absorption from developed S- cipitation following dilution and maintains a SNEDDSs. Improvement in the oral bioavail- temporary supersaturated state [97]. Conse- ability of darunavir from S-SNEDDSs can be quently, the generation and stabilization of intra- attributed to many factors like presence of the lipo- luminal supersaturation can provide an efficient philic drug in solution or in small emulsion glob- solution for the enhanced oral bioavailability of ules that eliminates the dissolution step and lipophilic drugs [98]. Further, it is important to keeps the drug in a dissolved state during trans- have these liquid supersaturable formulations port to the unstirred water layer of the GIT mem- as a solid dosage form having higher stability, brane and lymphatic transport through intestinal better transportability, simple and cost-effective transcellular pathways. Also, the vehicles used in manufacturing, and improved therapeutic suc- the formulation modulate the P-gp efflux pumps cess owing to the better patient compliance and/or function of CYP450 enzymes at the intes- [99e101]. tine region and improve the drug absorption. Dash et al. [102] aimed to develop solid super- In vivo pharmacokinetic studies showed saturable SNEDDSs of glipizide that could improved oral bioavailability, which might be generate a supersaturated state by retarding the due to the collective mechanism of nanoemulsion precipitation of solubilized drug. Comparative formation with greater surface area, improved release studies indicated that there was signifi- drug dissolution and release, transcellular and cantly faster glipizide release from the solid paracellular absorption, P-gp modulation poten- supersaturable SNEDDSs compared to solid Ò tial of excipients, reduced CYP450 metabolism in SNEDDSs, Glucotrol (uncoated tablets), and the gut enterocytes, and lymphatic bypass via the pure drug. Further, it was indicated that Peyer’s patches, which protects drugs from hepat- self-emulsifying systems need less free energy to ic first-pass metabolism [92]. form an emulsion due to the spontaneous 162 8. Lipid nanocarriers for delivery of poorly soluble and poorly permeable drugs formation of the interface between oil droplets advantages, like better physical stability, and water. This, in turn, decreases the droplet improved biocompatibility, and biodegradability, size leading to immediate solubilization of drug ability to provide controlled drug release, pro- in the dissolution medium [99].Thegeneration duction at large scales without using organic sol- of nanoemulsion following dilution of solid vents [109]. Lipid nanoparticles can also enhance supersaturable SNEDDSs resulted in a large sur- the solubility of poorly soluble drugs, which can face area for enhanced glipizide solubilization contribute to better drug bioavailability. Another and dissolution. As was evident from the results advantage of lipid nanoparticles is the lipid pro- of scanning electron microscopy, the drug is tection of the drug rapidly metabolized in vivo molecularly dissolved within the matrix of solid from chemical as well as enzymatic degradation, supersaturable SNEDDSs and a large surface thereby delaying the in vivo metabolism. By area might have generated that improves the incorporation into the lipid nanoparticles, the wettability and in vitro drug release [89]. The drug can be embedded into a solid lipid matrix, microporous surface of the solid supersaturable which reduces its exposure to enzymatic degra- SNEDDSs might have created channels for infil- dation following absorption [110]. tration of dissolution media to facilitate disper- The first generation of the lipid nanoparticles, sion of nanoemulsion [101]. Solid supersaturable solid lipid nanoparticles (SLNs) was developed SNEDDSs witnessed an enhanced in vitro release, at the beginning of the 1990s by replacing the in vivo absorption, and therapeutic activity of gli- liquid lipid (oil) of an o/w emulsion by a solid pizide as compared to the pure drug. This might lipid or a blend of solid lipids, i.e., the lipid parti- be attributed to the multiprocess mechanism of cle matrix being solid at both room and body tem- SNEDDSs, both physical and biological: perature. SLNs are composed of 0.1% (w/w) to (a) higher solubilization of drug in the GIT milieu 30% (w/w) solid lipid dispersed in an aqueous [103,104], (b) enhanced drug absorption by medium and if necessary stabilized with prefer- lymphatic transport [105,106], (c) enhanced ably 0.5% (w/w) to 5% (w/w) surfactant. The mucosal permeability due to disruption of the mean size of SLNs is in the submicron range, lipid bilayer by surfactant [99], (d) enhanced from about 40 to 1000 nm [111]. The lipid particle drug absorption through P-gp inhibitory effect matrix remains solid at both room and body tem- Ò of the surfactant (Solutol HS15) [107],and perature allowing sustained drug release. The (e) reduction in CYP 450 mediated metabolism drug may be incorporated into the SLNs in [101,106]. Moreover, the in vitro release, in vivo different ways, i.e., into a homogeneous matrix, absorption, and therapeutic efficacy of solid into shells, or as a lipid-coated core. supersaturable SNEDDSs were found to be Hu et al. [110] have developed all-trans significantly higher than the solid SNEDDSs. retinoic acid (ATRA)-loaded SLNs and carried Hence, it can be concluded that the overall out detailed studies of the solubilization and improved performance of solid supersaturable improving the oral absorption of ATRA. In vitro SNEDDSs lies in its precipitation resistance release tests have confirmed that the release rate nature, compared to classical SNEDDSs [102]. from SLNs is significantly faster compared to The interest toward lipid nanoparticles as a ATRA solution. An increase in saturation solubi- novel particle technology has been increasing in lity and, consequently, the drug release rate al- recent years because of their potential to act as lows it to reach high concentrations in the GIT. alternative carriers to conventional carriers, i.e., Moreover, the oral bioavailability of ATRA in emulsions, liposomes, polymeric micro- and SLNs was compared with that in a solution to nanoparticles [108].Comparedtoconventional assess the absorption enhancement of SLNs. The carriers, lipid nanoparticles possess several authors showed that ATRA absorption was 4. Types of lipid nanocarriers for oral drug delivery 163 enhanced significantly by employing SLNs. Also, be increased up to 95% [115]. The mixture of solid the in vivo behavior of an ATRA emulsion was and liquid lipids leads to the creation of the less- assessed. The effect of surfactant on the oral ab- ordered inner structure. The drug molecules can sorption of ATRA was also studied with SLNs. thus be accommodated in between the lipid An interesting result was that the surfactants layers and/or fatty acid chains and a higher used for stabilization of these nanosystems active drug loading of the particles can be contributed to an increase in the permeability of achieved. Thus, NLCs are considered as a smarter the intestinal membrane or improved affinity generation of the lipid nanoparticles than SLNs. between lipid particles and the intestinal mem- Depending on the method of preparation and brane. Some particles may be taken up into the the composition of the lipid blend, NLCs with lymphatic organs and eventually enter the sys- different structures can be obtained, i.e., the temic circulation [112,113].SLNscontainingPlur- imperfect, amorphous, and multiple type. NLCs onic F68 prolong the absorption time when can achieve passive targeting by altering particle compared to SLNs containing Tween 80 or size and active targeting by modification of ATRA solution. This may be because SLNs con- proper materials [116,117]. taining pluronic F68 exhibit high adhesion and One of the most widely exploited routes of remain for a longer time in the GIT. On increasing administration for NLCs has been the oral route Ò the amount of Tween 80 and pluronic F68, it [118e121]. Due to the increased drug-loading seems that there was not only a reduction in the capacity of NLCs, most of these studies have particle size but also an improvement in ATRA been focused on the ability of NLCs to improve absorption. Moreover, it has shown that soy leci- the bioavailability of poorly water-soluble drugs Ò thin with either Tween 80 or pluronic F68 (classified as class II or IV according to BCS). moderately inhibits P-gp, which contributed to While increasing highly lipophilic drug’s improved oral bioavailability of ATRA. From bioavailability, NLCs exhibit a prolonged resi- in vivo pharmacokinetic data, the authors dence time in the GIT compared to other lipid concluded that SLNs improved the bioavail- formulations, and present a different release ability of ATRA significantly compared to mechanism that can be modulated by tuning ATRA solution and the amount of surfactant the lipids contained within their matrix. also enhanced the oral absorption of ATRA [110]. Beloqui et al. [122] evaluated the potential of Despite all advantages, some common prob- NLCs to enhance the oral bioavailability of lems are associated with SLNs including drug saquinavir, a BCS class IV drug, and P-gp sub- expulsion, low drug-loading capacity, risk of strate, and studied NLCs transport mechanisms gelation, and drug leakage during storage across the intestinal barrier. Three NLC formula- caused by lipid polymorphism [114]. tions differing in the mean particle size (165, 247, To overcome the disadvantages of the SLNs, at and 1000 nm, formulation A, B, and C, respec- the end of the 1990s, nanostructured lipid carriers tively) and surfactant contents were evaluated. (NLCs), the second generation of the lipid nano- All three formulations significantly increased particles, was developed. NLCs are obtained by permeability of saquinavir across Caco-2 cell controlled mixing of solid lipids and spatially monolayers when compared to drug in suspen- incompatible liquid lipids, leading to special sion, although formulation B did it to a higher nanostructures with improved properties for extent compared to formulations A and C. drug loading. To obtain the blends for the matrix These findings highlight the importance of the of the particles, solid lipids are mixed with liquid composition of NLCs designed, as well as their lipids (oils), preferably in a ratio of 70:30 up to physicochemical properties (size and surface 99.9:0.1. The overall solid content of NLCs could characteristics) toward oral bioavailability 164 8. Lipid nanocarriers for delivery of poorly soluble and poorly permeable drugs enhancement, and the ability of NLCs to circum- microemulsions, and luteolin-loaded suspen- vent the P-gp drug efflux [122]. sions were compared after they were adminis- To enhance the bioavailability of baicalin after tered to rats by oral gavage. Both NLCs and oral administration, Luan et al. [109] developed microemulsions improved the oral bioavail- NLCs by the emulsion-evaporation and low ability of luteolin, but microemulsions exhibited temperature-solidification method. The results a higher capacity. Both carrier systems had demonstrated that incorporating baicalin into similar properties in the in vitro release and NLCs could improve the oral absorption and in situ intestinal absorption studies. However, enhance the bioavailability significantly. The in vitro lipolysis showed that microemulsions pharmacokinetic study in rats showed that are digested faster during the initial stage and the AUC and mean residence times of baicalin- the concentration of solubilized drug in the loaded NLCs were greater than those of baicalin digestion media is higher than that associated suspension. The fact indicated that baicalin- with luteolin-loaded NLCs, which may loaded NLCs could improve the bioavailability contribute to faster absorption and higher of baicalin markedly following oral administra- bioavailability [125]. tion [109]. Several factors could be the reasons In 2014, smartLipids particles were devel- for the absorption enhancement of baicalin by oped as the third generation of the lipid NLC [123,124]. First, the small size of the nano- nanoparticles after SLNs and NLCs. Their parti- particles increased the solubility, dissolution, cle matrix consists of up to 10 different lipids, and bioavailability according to NoyeseWhitney forming a highly chaotic structure with many equation. Second, the excellent adhesion of the imperfections and much higher drug-loading nanoparticles to biological surfaces could increase capacity. Due to the very different spatial forms drug residence time on the GIT surface, which of the various lipid molecules, polymorphic tran- contributed to the sufficient uptake of baicalin. sitions during storage are minimized or can be Third, nanoparticles had good absorption in the completely avoided [126]. intestinal lymphatic or lymph nodes. All in all, baicalin-loaded NLCs increased the absorption of baicalin in the GI wall, thus improving its 5. Solubility and permeability oral bioavailability [109]. enhancement strategies by lipid Liu et al. [125] evaluated the efficiency of nanocarriers NLCs and microemulsions in bioavailability enhancement of luteolin, based on the investi- There are three primary mechanisms by which gation of two key processes that occur during lipids and lipophilic excipients affect drug ab- the traverse of NLCs and microemulsions along sorption, disposition, and bioavailability after the intestinal tract: the solubilization process oral administration. These are the alteration of and the intestinal permeability process. the composition and character of the intestinal Sustained release profiles were observed for milieu, the recruitment of intestinal lymphatic both luteolin-loaded NLCs and microemul- drug transport, and the interaction with sions. This can increase the circulation time of enterocyte-based transport processes [127]. luteolin, leading to prolonged drug residence The ability of lipid formulations to facilitate time in the systemic circulation and better oral absorption of poorly soluble drugs has been bioavailability. To assess further the ability of thoroughly documented in the literature [67,88]. the nanoparticulate carriers to improve oral Lipid nanocarriers have the obvious advantage bioavailability, the pharmacokinetic parameters of increasing the surface area for drug dissolution of luteolin-loaded NLCs, luteolin-loaded compared to simply solution/suspension of the 5. Solubility and permeability enhancement strategies by lipid nanocarriers 165 drug in the lipids. Since droplets/particles sur- activity [23,129]. Additionally, after localization face is inversely proportional to diameter, smaller of lipid nanocarriers within the GIT, the fate of particles/droplets with their associated, greater the loaded drugs is determined by the processes surface are thought to facilitate dissolution, of dispersion and subsequent digestion process resulting in more rapid and uniform drug release [127,130]. Apart from that, lipids are degraded [128]. Thus, for lipophilic drugs that exhibit disso- by gut enzymes, creating micelles. The incorpo- lution rate-limited absorption, lipid nanocarriers rated drugs are likely to be solubilized within mi- may offer an improvement in the rate and extent celles or micelle-like structures, which are formed of absorption and hence result in more reproduc- by the product of lipid digestion to the nanocar- ible blood-time profiles [26]. riers with endogenous ingredients, such as bile The positive influence of the lipid formulations salts and phospholipids. Solubility enhancement on the drug absorption can be ascribed to the occurs as the lipid matrix (containing the dis- prevention of drug precipitation on intestinal solved drug) is incorporated into these micelles. dilution, enhancement of the membrane perme- Upon lipid degradation, the drug is released ability, inhibition of the efflux transporter mecha- and absorbed [127,131] (Fig. 8.1). nism, reduction of CYP enzymes activity, Due to the structural characteristics of the sur- enhancement in the chylomicron production and factants, i.e., the presence of lipophilic and hydro- lymphatic transport [26,67,127]. Their mecha- philic domains in the structure, they tend to nisms of action also include stimulation of biliary partition between the lipid and protein domains and pancreatic secretions, prolongation of GIT in the cell membrane. Surfactants are known to residence time, stimulation of lymphatic trans- be permeability enhancers because of the effect port, enhancement in the intestinal wall perme- on tight junction, i.e., interaction of the surfactant ability, and reduction of metabolism and efflux head groups with lipid bilayers or modification of

Liver Stomach

Gallbladder Undispersed formulation Common Dispersed bile duct droplets Endogenous Lipase BS/PL micelles Pancreas Co-lipase Small intestine

Stomach

Absorption

FIGURE 8.1 Lipid digestion and drug solubilization in small intestine. From Porter CJ, Pouton CW, Cuine JF, Charman WN. Enhancing intestinal drug solubilisation using lipidbased delivery systems. Adv Drug Deliv Rev 2008; 60(6):673e91. Originally adapted from Porter CJH, Trevaskis NL, Charman WN, Lipids and lipid-based formulations: optimizing the oral delivery of lipophilic drugs, Nat Rev Drug Discov 2007;6(3): 231e48. 166 8. Lipid nanocarriers for delivery of poorly soluble and poorly permeable drugs the hydrogen bonding and ionic forces. Thus, the that arises from the nanosized droplets provide surfactant molecules increase the permeability by a large interfacial area for pancreatic lipase to hy- disturbing the cell membrane [132]. Moreover, drolyze triglycerides and, consequently, to form proper selection of the surfactant can inhibit P- mixed micelles that promotes solubilization of gp efflux mechanism and hence increase oral the lipophilic drugs in the intestinal aqueous bioavailability. For example, the incorporation environment and their absorption [103].Further- Ò of Tween 80 in the formulation of nanoemulsion more, S(M)EDDSs show a prolonged residence is reported to inhibit the efflux mechanism and time on mucosal membranes [138] and could enhance the oral bioavailability [133]. reach greater mucosal surfaces, resulting in a For the drugs that undergo extensive hepatic comparatively higher drug uptake [26,139]. metabolism, nanoemulsions have been reported Potential features of SNEDDSs in enhancing to enhance the bioavailability by avoiding portal oral bioavailability of lipophilic drugs consist circulation and being absorbed via the lymphatic of facilitating transcellular and paracellular ab- route [134]. sorption, reducing cytochrome P450 (CYP450) The advantages of microemulsions in oral metabolism in the gut enterocytes, promoting delivery of poorly soluble drugs include good lymphatic transport via Peyer’s patches, and adherence to the GIT membranes as a result of protection of drugs from hepatic first-pass meta- small droplet size, drug protection against enzy- bolism [82,85,99,140]. matic hydrolysis, and the potential for enhanced The absorption-enhancing effect of orally absorption due to a surfactant-induced changes administered lipid nanoparticles may be attrib- in the membrane fluidity and increment in uted to the adhesion of the particles to the gut permeability [125,135]. wall. The adhesion of the particles to the mucus S(M)EDDSs can reduce the limitation of slow can improve the residence time and contact of and incomplete dissolution of poorly soluble the drug with the underlying epithelium, thus drugs and facilitate the formation of solubilized increasing the concentration gradient [141] phase from which absorption might occur [76]. (Fig. 8.2). Also, the protection of the drug by Hydrophobic drugs can be dissolved in these sys- the lipids from chemical and enzymatic degrada- tems, enabling them to be administered as a unit tion delays the in vivo metabolism. Due to their dosage form. This leads to in situ solubilization of protective properties, SLNs and NLCs are of drug that subsequently can be absorbed by particular interest for peptide and protein deliv- lymphatic pathways, bypassing the hepatic first- ery by oral route [71]. pass effect [26,136]. The improved drug absorp- tion provided by S(M)EDDSs is contingent upon the maintenance of the drug in the solubilized 6. Mechanisms of interaction of lipid state until it can be absorbed from the GIT [70]. nanocarriers with cell membranes Since S(M)EDDSs provide ultra-low interfacial tensions and large o/w interfacial area, they The transport of nanocarriers across the cell have the advantages in possessing higher solubi- membrane is a complex biological process, often lization capacity than simple micellar solutions, difficult to understand because of its dynamic leading to the incorporation of poorly soluble nature [142]. The studies of biophysical interac- drugs inside the lipid phase [84]. The formation tions of nanocarriers with cell membranes eluci- of a variety of colloidal species on dispersion date the role of these systems in the cellular and subsequent digestion of S(M)EDDSs may uptake of different drugs and have also been also facilitate drug absorption [137].Namely, explored in recent years to predict toxicity these small droplets and the large surface area associated with these drug delivery systems. 6. Mechanisms of interaction of lipid nanocarriers with cell membranes 167

absorption of drug and lipids blood

gut wall mucus

adhesion gut drug drug release + mixed micelle lipid degradation products

SLN

+ bile salts degradation anchoring of lipase and co-lipase drug release micelle, drug solubilized diglyceride fatty acid

FIGURE 8.2 Mechanism of absorption enhancement by ultrafine dispersed lipids as either a nanoemulsion or SLNs in the gut [141].

Changes in the composition of the lipid nanocar- highly significant differences were observed riers may alter interactions with cells and tissues, between vehicles (microemulsion and dimethyl which could be explored to develop target- sulfoxide) on both studied cell lines. The intrinsic specific drug delivery systems. cytotoxicity of unloaded microemulsion was Sieniawska et al. [143] prepared stable, water- confirmed in this study, but differences in activ- dilutable microemulsions containing essential ity between essential oil and essential oil/micro- oils of citronella, mint, and eucalyptus and eval- emulsion were statistically significant only in uated their cytotoxic properties on Vero (normal case of mint oil. cell line established from the kidney of an adult Tiwari et al. [144] prepared microemulsion to African green monkey) and HeLa cell lines improve the solubility of ketoconazole and (human cancer cell line established from cervical enhance its antifungal activity compared to the adenocarcinoma). The cytotoxic properties of conventional dosage form. In vitro toxicity of microemulsions and essential oils in dimethyl the developed carriers was assessed on the sulfoxide were evaluated using MTT assay. The human lymphocytes cells using MTT assay. results of this study indicated that essential oils The human lymphocytes are treated with rose Ò in microemulsions had higher cytotoxicity than oil, surfactant (Tween 20), unloaded and essential oils in dimethyl sulfoxide, but the re- drug-loaded microemulsions. LD50 of both sults were statistically significant only in the unloaded and drug-loaded formulations case of mint oil on Vero cell line. Statistically, 168 8. Lipid nanocarriers for delivery of poorly soluble and poorly permeable drugs signifies lesser toxicity, compared to rose oil, Ban et al. [148] developed curcumin-loaded which attributes to its safer oral usage. SLNs and in the absorption study assessed gut This research of [145] focuses on the develop- permeation of the developed carriers using ment and characterization of lurasidone mucus-covered Caco-2 cell monolayers. Penetra- hydrochloride-SMEDDSs. The formulation was tion of the lipid particles across the gut epithe- further evaluated for in vitro cell uptake across lium was dependent on their size and surface Caco-2 cells. The results of confocal microscopy chemistry, due to the steric/interactive barrier and flow cytometry indicated that SMEDDSs of the mucus layer. were permeated deeply and thus increased intes- Du et al. [149] developed camptothecin (CPT)- tinal absorption and uptake across Caco-2 cell loaded SLNs for oral delivery. The cytotoxicity of monolayer as compared to the coumarin-6 CPT suspension, CPT SLN, and CPT-palmitic solution (lipophilic dye, which is used to mimic acid conjugate via a cleavable disulfide bond nature of the drug). The permeability of lurasi- linker (CPTeSSePA SLN) were evaluated done hydrochloride across cell monolayer was in vitro against Caco-2, HT-29, HepG2, and significantly increased with SMEDDSs compared MCF-7 cells. The cellular uptake behavior for to lurasidone hydrochloride suspension. The oral delivery was checked by confocal laser scan- enhanced permeation of lurasidone hydrochlo- ning microscopy using Caco-2 cells model. CPT ride could be due to nanosized droplets of SLN and CPTeSSePA SLN exhibited higher SMEDDSs, which provide close contact with cytotoxicities than CPT against all four cell lines apical membrane, solubility improvement, and after 24 h incubation. From the data, CPT SLN presence of excipients, such as Cremophor EL and CPTeSSePA SLN revealed much higher and Transcutol HP, which might have altered anticancer activity against all cell lines than CPT the fluidity of the membrane [146]. suspension. CPT SLN and CPTeSSePA SLN Pangeni et al. [147] designed o/w nanoemul- had enhanced cellular uptake compared to CPT, sions for oral delivery of quercetin to enhance its which may be ascribed to the improved lipid solubility and improve bioavailability and eval- solubility of the conjugate and nanoscale size of uated in vitro drug permeabilities through an the SLN compared with nonliposoluble and mm- artificial intestinal membrane and Caco-2 cell sized CPT particles. monolayer. The artificial intestinal membrane permeability assay showed significant increases in the permeability of a series of quercetin nano- 7. Conclusion and future perspectives emulsions when compared with aqueous disper- sion of quercetin used as a control. The results of Successful drug design with lipids depends in vitro permeability of quercetin in aqueous largely on understanding the physical-chemical dispersion, 0.3% NaCMC, and nanoemulsion and physiological factors that promote or inhibit across a Caco-2 cell monolayer revealed that the bioavailability. It requires a grasp of not only the permeability of quercetin from nanoemulsion drug candidate issues but also the role of the was greater than that of the aqueous dispersion drug delivery system or the potential for drug- and 0.3% NaCMC. The obtained results can be excipient interplay in vitro as well as in vivo explained by the presence of a surfactant conditions. Ò Ò (Labrasol ) and cosurfactant (Cremophor EL) With better understanding of the mechanisms combination in the nanoemulsion, which may in- of interactions with cell membranes, a rational crease membrane permeability by disrupting cell approach for drug discovery and development membranes and partitioning into the cell as well as for developing efficient drug delivery membrane. systems could be provided. References 169

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Nose-to-brain drug delivery: an alternative approach for effective brain drug targeting Amit Alexander1, Mukta Agrawal1, Mahavir Bhupal Chougule2,3,4, Shailendra Saraf 5, Swarnlata Saraf 5 1Rungta College of Pharmaceutical Sciences and Research, Bhilai, Chhattisgarh, India; 2Translational Bio-pharma Engineering Nanodelivery Research Laboratory, Department of Pharmaceutics and Drug Delivery, School of Pharmacy, University of Mississippi, University, MS, United States; 3Pii Center for Pharmaceutical Technology, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS, United States; 4National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS, United States; 5University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India

1. Introduction of body fluid and needs to cross the bloode brain barrier (BBB), which results in signifi- The oral and parenteral routes are most cantly lower drug concentration in the brain prevalent routes of drug administration. The [1,2]. Also, it is associated with hepatic first- benefits of these routes are well known, but pass metabolism, which causes degradation of apart from that, both the strategies have many drugs and bioactives. Along with this, many limitations, especially in the case of brain many other factors confine the central nervous disorder. The parenteral route is a painful system (CNS) acting drug’s concentration, method of drug administration and also needs reaching the brain from the systemic circulation, expert assistance for administration. On the and hinders the efficacy of the drug and in- other hand, the oral route is patient friendly. creases the peripheral side effect [1].Thus,to However, the drug administered via this route improve the therapeutic efficiency and mini- should follow the normal distribution pattern mize the side effects, an alternative and direct

Nanopharmaceuticals https://doi.org/10.1016/B978-0-12-817778-5.00009-9 175 © 2020 Elsevier Inc. All rights reserved. 176 9. Nose-to-brain drug delivery: an alternative approach for effective brain drug targeting delivery route to delivering drugs to the brain is proper clinical facilities, and better communica- highly desirable. tion between the researchers of the different The intranasal route offers a direct nose-to- regions [8]. Some commercially available intra- brain passage via olfactory and trigeminal nasal formulations for the treatment of various nerves and minimizes the exposure of the drug CNS disorders are listed in Table 9.1. to general circulation [3]. In the past 2 decades, The intranasal (i.n.) route is preferred over the nose-to-brain delivery was explored as a another route of drug administration for brain promising approach in the treatment of CNS dis- targeting due to its ability to directly deliver orders [4]. Previously, nasal drug delivery was the drug via neuronal pathways (olfactory and often used for local and systemic therapies due trigeminal nerve pathway). It bypasses the BBB to high vascularization [5]. Later, scientists and the hepatic first-pass metabolism, the two recognized that the direct connection between significant barriers of drug transport to the brain the brain and nasal cavity through olfactory from the periphery [9]. This route is suitable for and trigeminal neurons could be utilized for bet- various proteins, lipids, and drug moieties that ter brain targeting of drugs. The concept of nose- are susceptible to enzymatic degradation and to-brain drug delivery was discovered by W. H. harsh acidic environment of the gastrointestinal Frey II in 1989 [3]. Initially, the application of tract. The high vascularization and neuronal intranasal route was limited to brain targeting network of the nasal cavity facilitate the drug of insulin or insulin-like growth factor [6].How- absorption and improve the bioavailability in ever, studies have proven its suitability for many the brain as compared to the oral route. The other large-molecular-weight substances like drug is instilled in the deeper region of the nasal proteins, peptides, and other bioactives [4]. cavity, primarily absorbed via olfactory and Although some contradictions are also there, trigeminal neurons, and reaches to the olfactory among different group of scientists across the bulb via cellular transport [4]. The systemic globe [7], such paradoxes require a complete absorption is a secondary route in which a understanding of the technology, establishment limited amount of drug is entered and follows of uniform research protocols, availability of the regular path (crossing the BBB) to enter into

TABLE 9.1 List of commercially available conventional intranasal products for the treatment of brain disorders.

Product Active ingredient Dosage form Indication Company name References

Migranal DHE-45a Nasal spray Migraine Xcel Pharm [26,27] Stimate Desmopressin acetate Nasal spray Haemophillia A Rhone Poulenc Rorer [28,29] Syneral Nafarelin acetate Nasal spray Central precocious Roche lab [30,31] puberty Stadol Butorphanol tartrate Nasal spray Migraine ESi Lederle, Roxane labs [32,33] Zoming Zolmitriptan Nasal spray Migraine Astra Zeneca [34,35] DDAVP Desmopressin acetate Nasal spray Head trauma, polydipsia, Ferring Pharma, Aventis [35,36] and polyurea Pharma a Dihydroergotamine mesylate. Adapted from Selvaraj K, Gowthamarajan K, Karri V. Nose-to-brain transport pathways an overview: potential of nanostructured lipid carriers in nose-to- brain targeting. Artif Cell Nanomed Biotechnol 2018;46(8):2088e95. 2. Common brain disorders 177 the brain. Thus, owing to the minimum periph- 2. Common brain disorders eral exposure of drug, it also reduces the sys- temic side effects of CNS-acting agents. The use 2.1 Alzheimer disease of suitable permeation enhancer or amalgam- Age progression causes irreversible loss of ation with the novel carrier system enables the fi entry of large proteins and peptides and im- nerve cells in the brain. This may result in dif - proves brain targeting efficiency of i.n. route culty remembering things, with slight confusion, [10]. As cellular transport is the primary path thinking and responding slowly, etc. but severe memory loss, cognition and learning disability, of drug absorption, this route is suitable for fi both the lipophilic and hydrophilic molecules. dif culty in speech, abnormal behavior, and In comparison to the parenteral, surgical, and disturbing daily activity are the common signs other invasive strategies, the nasal route offers of Alzheimer disease (AD) [2,12]. Alzheimer is a noninvasive, patient-friendly technique, which a progressive, irreversible neurodegenerative is also convenient for the unconscious, pediatric, disorder that slowly destroys the brain cells and geriatric patients [2]. responsible for memory, learning, cognition, However, there are also some limitations that and other routine activities [4]. AD is mostly reduce the efficiency of i.n. route. First of all, the seen in old age but also sometimes observed in mucociliary clearance and low drug retention in young people, known as the younger stage AD. AD is the most common cause of dementia, the nasal cavity reduce the drug permeation e across the nasal mucosa. Also, nasomucosal around 60% 70% of all cases noticed world- enzymes cause degradation of various proteins, wide. As per the Alzheimer Association Report peptides, and bioactives. Secondly, due to the 2017, around 47 million in the world population small volume of the nasal cavity, only a small were suffering from dementia, of which 37 amount of drug can be instilled at once. More- million were reported to have AD. It is consid- over, the head position greatly affects drug ab- ered the sixth major cause of death in the United sorption through the neuronal system. So, States, and the statistics are supposed to reach to 131 million people by 2050 [13]. sometimes the patient needs to lie down or fi hold up their head during administration. AD was rst discovered by the German doc- Thirdly, the high vascularization facilitates sys- tor Alois Alzheimer in 1906, during the treat- temic absorption, which sometimes causes ment of his patent named August D. who was peripheral side effects and reduces the drug con- suffering from behavioral abnormalities and centration in the brain. Finally, the nasal epithe- memory loss. He observed shrinkage in the brain lium is less permeable for the polar drugs, which of the patient in the autopsy. Afterward, in 1910, can be overcome by the use of suitable perme- a psychiatrist Dr. Emil Kraepelin, a colleague of Dr. Alzheimer, named the disease as “Alz- ation enhancer. However, the excess of perme- ’ ” ation enhancer causes irritation to nasal heimer s Disease to honor his discovery. Then, mucosa [9]. in 1994, after a very long time, the U.S. Food Due to such disadvantages, novel carrier and Drug Administration (FDA) approved “Cognex” as the first AD drug. From then, until systems are used for nose-to-brain delivery, fi which circumvents the boundaries and increases now, we have had only have ve FDA-approved drug targeting to the brain. This chapter briefly drugs, including donepezil, galantamine, tacrine discusses some common CNS disorders, (Cognex), rivastigmine, and memantine. present therapies, and challenges to brain drug Although, there are various natural and syn- delivery [11]. thetic compounds available that are reported to have a promising anti-AD effect, others are still 178 9. Nose-to-brain drug delivery: an alternative approach for effective brain drug targeting under research such as curcumin, quercetin, In the year 1817, the disease was named PD after piperine, S14G-Humanin, nerve growth factor Dr. James Parkinson. Further, in 1912, a German (NGF), insulin, a-mangosteen, tarenflurbil, pathologist, Dr. Friedrich H. Lewy, reported the deferoxamine, risperidone, etc. Based on their existence of neuronal cytoplasmic inclusion in mechanism of action, the anti-AD drugs are of the brain region. Such bodies are named as two types: Lewy bodies in honor of this scientist. In 1919, Tretiakoff observed neurodegeneration in the (1) Symptomatic drug: The drug molecules that substantia nigra of the midbrain in PD patients. just reduce the symptoms of the disease and Afterward, the role of dopamine and its diminu- improve brain functions, including tion from the basal ganglia was reported in 1950. cognition, memory, and learning, fall into In the present scenario, PD is considered as the this category. These are mainly the second major neurodegenerative disorder asso- acetylcholine esterase (AChE) inhibitors, ciated with aging after AD [16]. The statistics which facilitate neurotransmission, e.g., indicate around 7 to 10 million people suffering donepezil, galantamine, rivastigmine, from PD worldwide. The disease prevalence is risperidone, piperine, etc. 41 per 100,000 after a person is in their forties, (2) Targeted drug: The drug molecules that which increases to approximately 1900 per mainly act on the pathological factors 100,000 after one is in their eighties. The global responsible for AD or directly act on the burden of PD has more than doubled in the target sites of the disease are considered as a last 26 years, from 2.5 million in 1990 to 6.1 targeted drug substances, e.g., million in 2016. deferoxamine, tarenflurbil, curcumin, NGF, Currently there is no complete cure for the H102 peptide, etc. [4]. PD; the available treatments only improve the symptoms of the disease. The primary and basic treatment of PD includes the combined therapy 2.2 Parkinson disease of levodopa with a dopamine agonist or other synergistic drugs like MAO-inhibitor, dopa Parkinson disease (PD) is a chronic neurode- decarboxylase inhibitor, COMT inhibitor, etc. generative disorder that primarily affects the But with the disease progression, these medi- motor system of the CNS. PD is mainly charac- cines become ineffective or sometimes produce terized by loss of various motor functions of other complications [17]. In extreme cases, sur- the brain, including movement difficulty, rigid- gery (deep brain stimulation) is also preferred ity, shaking, difficulty in walking, etc. Similar to reduce the severity of the disease. Rehabilita- to AD, the patient with PD also experiences tion and diet may also help as alternative behavioral abnormalities and dementia at the approaches. To find out a proper therapy for advanced stage of the disease. Along with this, the treatment of PD, various experiments are one may also suffer from anxiety, depression, being conducted. The research data suggest sleep, and sensory disbalance and emotional gene therapy, cell-based therapy as promising anomalies [14,15]. approaches to restore the brain function. At the PD has been well known to human beings same time, various novel drug delivery since ancient times. In ancient medical system approaches can also be a promising way to it is known as “Shaking palsy” (Western medi- improve the efficacy of existing drugs [18]. cine system) and “Kampavata” (in Ayurveda). 2. Common brain disorders 179

2.3 Migraine analgesics like paracetamol, ibuprofen for the headache, and common medicines for nausea. Migraine is a headache disorder in which the If these are ineffective, the triptans and ergota- patient is suffering from recurrent, pulsating, mines are prescribed. Sometimes, caffeine may moderate to severe headache starting from either also be used for severe pain. Current research half of the brain, which may migrate to the other on migraine therapy focusses on the use of calci- half or the whole brain and lasts from 2 h to tonin gene related peptides (CGRPs), such as 3 days. Sometimes it may be associated with telcagepant and olcegepant, which claim to act vomiting, nausea, and light, sound, or smell on the pathophysiology of the pain. Unfortu- sensitivity [19]. Moreover, about one-third of nately, the phase III clinical trial conducted by migraine patients experience auras, which are Merck on telcagepant failed in 2011. Now there visual disturbances that signal the headache. is also study of CGRP monoclonal antibodies Genetic and environmental factors are believed for effective migraine therapy [22]. to be the two major cause of migraine. It is reported that about two-thirds of migraine cases are genetically based. The occurrence of 2.4 Schizophrenia migraine also varies with gender; females are more prone to the disease in comparison to Schizophrenia is a polygenic psychiatric dis- males, and this ratio increases after puberty. order characterized by behavioral disturbance, The study shows that around 75% of migraine cognitive, and perceptual abnormalities. It is patients are females [20]. generally observed beginning in young adult- Migraines have been well known from since hood and lasts for a long time. The person early human civilization. In the ancient world suffering from schizophrenia mainly experience (7000 BCE) trepanation (drilling a hole in the false belief, unusual visions and thinking, skull) was the preferable treatment for migraine. confused responses, emotional insecurity, lack The people at that time believed this procedure of motivation, hallucinations, and reduced social let the evil spirits escape from the mind. In the activities. Sometimes the patient hears imagi- 17th century, William Harvey also recommen- nary voices and has visions that do not exist in ded trepanation as effective migraine therapy. reality. Along with this, depression, anxiety, It was 1868 when a fungus “ergot” was first and poor mental health are the secondary symp- used in the treatment of migraine. Afterward, toms of schizophrenia [23,24]. in 1918, ergotamine was successfully isolated Various environmental (including modern from ergot and used in the treatment of lifestyle, cannabis addiction, nutritional defi- migraine. Then, in 1959, methysergide was syn- ciency during pregnancy, and parental age) thesized, and sumatriptan (the first triptan) and genetic factors are the prime causes of was developed in 1988 [21]. schizophrenia. The diagnosis is primarily based Migraine is generally divided into two types: on the behavior of the person and observation (1) migraine with aura, and (2) migraine without of the family members. The antipsychotic medi- aura. The pathophysiology of migraine disease is cines at their lowest possible dose are preferred not well known; some researchers believe that for the treatment of schizophrenia. Depending the CNS is primarily responsible for the pain, upon the symptoms and response of the patient whereas others believe that the peripheral to the medications, antidepressants and antianx- system including sensory neurons and blood iety drugs are prescribed in combination. Along vessels play an important role in disease initia- with this, proper counseling, rehabilitation, tion. The primary treatment utilizes regular emotional support, and job training assist in 180 9. Nose-to-brain drug delivery: an alternative approach for effective brain drug targeting fast recovery. In severe conditions, either volun- schizophrenia. In 1960, the autism term was tary or involuntary hospitalization is also established as a separate syndrome [29]. observed [25]. Statistics show that worldwide Genetic and environmental factors are the pri- around 0.3%e0.7% of the population experience mary cause of autism. Along with this, any kind schizophrenia during their lifespan. It is of drug, cocaine or alcohol addiction, and some observed that men are more prone to the disease infections (like Rubella) during pregnancy may as compared to women. Around 20% of people also be responsible for autism. It is believed respond to therapy and recover completely; that these factors may cause abnormal synaptic however, in 50% of the cases, the patient fails and neural connections, which cause misinter- to recover and has to live with schizophrenia pretations of the signals and are responsible for for the rest of their life. An average lifespan repetitive behaviors [30]. There are controversies with the disease is about 10e25 years [26]. among various scientists about the mechanism behind the disease, and the exact mechanism is still unknown. The diagnosis of the disease is 2.5 Autism mainly based on behavioral observation and interaction with the family and the affected Autism is a complex, heterogeneous develop- child. The two commonly used diagnostic mental and psychological disorder caused by the techniques are: anomalous connection between different brain (1) ADI-R (Autism Diagnostic Interview- regions. The common symptoms of autism are Revised) abnormal social behavior, difficulty in social (2) ADOS (Autism Diagnostic Observation interaction, inability in general communication, Schedule) stereotypical behavior, poor social skills, and cognitive impairment. The disease symptoms The primary treatment of the child with are mostly observed in childhood and gradually autism is mainly psychological and emotional increase with age [31]. The term “autism” origi- support. The education system, family, and nates from the Greek word autos, which means healthy surroundings ensure better results. isolated behavior, so autism is a neurodevelop- Improving the confidence and IQ level by giving mental syndrome in which an individual isolates some responsibility to the child will be beneficial themself from the surrounding culture. Reports most of the time. If the behavioral therapy fails, from the U.S. Centers for Disease Control and sometimes along with the behavioral therapies Prevention (CDC) claim the rate of disease prev- some medicines, including antipsychotic, anti- alence was approximately 1.5% in developed convulsant, antianxiety, and antidepressant countries in 2017. It was observed that boys are drugs may be given for treatment. Proper more prone to the disease than girls. Also, it is nutrition, quality lifestyle, personality develop- more frequently observed in white children ment classes, and music therapy may also be than children of color [27]. helpful [31]. Autism was first described in 1943, by Leo Kanner, as the inability of an individual to create normal social, emotional interaction with the 2.6 Cerebral palsy family and society. Kanner reported behavioral abnormalities of 11 children in his report [28]. Cerebral palsy (CP) is a group of neurological The first case of autism was reported by Kanner disorders that limits body activity and causes in 1938. At that time, the disease was confused permanent movement disability. CP can be with other similar syndromes like infantile defined as “set of permanent neurological 2. Common brain disorders 181 disorder resulted in distort posture, immobility as meningitis. It is characterized by severe head- of some or whole body and limited physical ache, pain in back and neck muscle, muscular movement and activities which are attributed stiffness [36]. The common symptoms of the to a non-progressive disturbance that occurs in disease are fever, shivering, cold, lethargy, infant’s brain or developing fetus” [32].The nausea, vomiting, blotchy or red rashes, loss of symptoms of cerebral palsy may vary from appetite, etc. The young population experiences very mild to severe spasms or movement very casual, nonspecific symptoms like drowsi- disability. The person/child with mild CP may ness, irritation, etc. The presence of rashes experience slight to severe difficulty in the move- indicates the bacterial, viral, or any other ment of one arm or leg or one side of the body, infection-generated meningitis [37]. Meningitis with the complete or partial sensory loss at that may directly affect the brain and spinal cord; side, sometimes associated with focal epilepsy. hence is considered as a life-threatening disor- In some cases, the spasm and dyskinesia are der. It is diagnosed by cerebrospinal fluid exam- experienced in all four limbs of the patient. ination (CSF). The primary treatment of disease Moreover, visual impairment and learning utilizes various antibiotics or sometimes anti- difficulties can also be observed occasionally. viral drugs, followed by corticosteroids in severe The individual with CP mostly becomes depen- conditions. The pneumococcal, meningococcal, dent on a wheelchair and needs assistance for mumps, and Hib vaccines are also used mobility [33]. sometimes. If it is not treated immediately, it CP is a neurological syndrome in which some may cause severe complications like epilepsy, lesions are formed in the brain during the devel- deafness, cognitive deficits, etc. [38,39]. opmental stage due to motor impairment. The disease was first reported in 1862, by William Little (an orthopedic surgeon). From then various attempts have been made to define and 2.8 Myasthenia gravis classify the disease, and recently, a proper defini- Myasthenia gravis is a chronic neuromuscular tion was proposed by the International Execu- disorder that weakens the skeletal muscle, more tive Committee for the definition of cerebral specifically affects the facial muscle and eye. palsy; cerebral palsy is described as a group of Histologically it is an autoimmune disorder permanent disorders of the development of that forms antibodies to destroy or block the movement and postures, causing activity limita- nicotinic cholinergic receptors present at the tion, that are attributed to a nonprogressive neuromuscular junction [40,41]. This interferes disturbance that occurred in the fetal or infant’s with the signal transmission from nerve to mus- brain. The motor disorder of cerebral palsy is cles and causes muscular contraction. It affects often accompanied by disturbance of sensation, approximately 200 persons per million popula- perception, cognition, communication, and tion, of which women under 40 years and men behavior, by epilepsy and by secondary muscu- over 60 years are more susceptible to the disease. loskeletal problems [34,35]. In most of the cases, acetylcholine esterase inhib- itors (like pyridostigmine, neostigmine) and 2.7 Meningitis various immunosuppressants (like prednisone, azathioprine) are useful for treatment. In some Acute inflammation in the protective covering severe conditions, the surgical removal of of the brain and spinal cord, meninges is known thymus gland can also be effective [42,43]. 182 9. Nose-to-brain drug delivery: an alternative approach for effective brain drug targeting

2.9 Stroke particles, microbes, parasites, and harmful toxins into the body [9]. The respiratory region is situ- Stroke is a physiological condition in which fl ated in the middle of the nose. It covers the brain cell death occurs due to poor blood ow largest area (approximately 130 cm2) of the nasal or excessive bleeding in the brain. In other cavity and is composed of goblet cells, basal words, it is a kind of brain attack in which the fi cells, and columnar epithelial cells (ciliated and de ciency of oxygen in brain cells results in nonciliated). The higher surface and high vascu- nerve cell death and thus affect memory and larization make it a prime region for drug other body functions like body movement, absorption in the systemic circulation [4].Itis muscle control, senses, etc. regulated by that also supplied with trigeminal sensory neurons, particular part of the brain [44]. Stroke may be which are responsible for signal transmission to divided into two types: the brain [48]. Next to this, the olfactory region - Ischemic stroke: Blockage in the brain blood is situated on the roof of the nose beneath the vessels that causes lack of blood flow cribriform plate of the skull. It is comprised of - Hemorrhagic stroke: Bleeding into the brain olfactory nerve cells, basal cells, supporting cells, due to blood vessel damage or rupture, microvillar cells, and trigeminal neurons in small resulting in bleeding in the interstitial or proportion. The olfactory region is responsible cerebrovascular space [45]. for direct connection with the brain via olfactory and trigeminal neurons, which facilitate the drug Tobacco smoking, high blood pressure, high transport to the brain [8]. blood cholesterol, diabetes, and arterial fibrilla- tion are the common risk factors responsible for stroke. The common symptoms are partial 4. Nose-to-brain drug transport pathways or whole-body paralysis or numbness of specific fi body parts like legs, arms, or face, dif culty in Due to the direct connection with the brain, speaking, walking, vision loss or blurred vision, the nasal cavity holds great potential for brain loss of the sensation, headache, vertigo, fatigue, targeting without the interference of the BBB etc. The disease can be diagnosed by MRI or and other peripheral factors. The drug transport CT scanning, sometimes associated with ECG. mostly takes place via a neural pathway (olfac- Stroke is considered as an emergency medical tory and trigeminal nerves) and through condition and is treated with statins, aspirin, sur- vascular route. In addition, CSF and lymphatic gery to remove a blockage in case of ischemic system are also involved in the passage of drug stroke, and warfarin for hemorrhagic stroke from nose-to-brain to a lesser extent [9]. Different [46,47]. brain transport mechanisms are discussed next.

4.1 Neuronal pathway 3. Anatomy of the nasal cavity The nasal cavity is supplied with two types of The human nasal cavity consists of three neurons: (1) olfactory receptor neurons, and subsequent regions with different functions: the (2) trigeminal sensory neurons. The olfactory vestibular, respiratory, and olfactory regions. nerve is primarily responsible for smell senses The vestibular region is the frontal-most part of and the trigeminal nerve transfers other senses the nose, containing hairy structures and mucus from the nasal mucosa to the brain. It offers a linings. It actively participates in the first line of direct way to access the brain and, hence, is body defense that prevents the entry of foreign considered a major pathway of nose-to-brain 5. Strategies to enhance nasal absorption 183 transport of drug [48]. The drug transfer via respiratory and olfactory region of the nose is these two nerves is explained below. diffused from the nasal mucosa to the trigeminal nerve endings and translocated to the different 4.1.1 Olfactory nerve pathway part of the brain such as the brain stem, medulla, The olfactory mucosa is enriched with olfac- pons, olfactory bulb, and also the forebrain [53]. tory receptor neurons. When the drug reaches the olfactory region, it interacts with the nerve endings of olfactory receptor neurons and trans- 4.2 Vascular pathway locates to CNS along the axonal length of olfac- The large absorption surface area and high tory neurons. The nerve bundle crosses the vascularization of the nasal cavity, particularly cribriform plate of ethmoid bone then enters the respiratory region, facilitate the drug absorp- the olfactory bulb [8]. The passage of drug cargo tion to the systemic circulation. Once a drug to the olfactory receptor neurons takes place by enters into general circulation, it reaches the transcellular or paracellular transport. Olfactory CNS/brain via a conventional path, i.e., by nerves deliver the bioactives to the olfactory bulb crossing the BBB as per the blood distribution and CSF. Further, the drug is distributed to a ratio [54]. The respiratory epithelium allows different region of the brain after mixing with the entry of both small and large, polar and interstitial fluid [49]. The axonal pathway is nonpolar moieties to the blood through paracel- also known as the intraneural route of drug lular and transcellular transport. Subsequently, transport to the brain, which is a slow process, the small, lipophilic molecules can cross the taking hours to days for drug transfer. In BBB and enter into the brain [8]. contrast, the paracellular or perineural channel, i.e., transfer between the epithelial or neural cells, is a faster way of drug passage to the brain, 5. Strategies to enhance nasal absorption which only takes a few minutes [50e52]. This route innervates the deeper brain regions like 5.1 Permeation enhancers cerebrum, cerebellum, and cortex. Apart from the physicochemical properties of 4.1.2 Trigeminal sensory nerve pathway drug and carrier systems, the nasal absorption Trigeminal nerve pathway connects the nose and bioavailability of the drug in CNS depend to the posterior region of the brain, including on the permeability of nasal mucosa. The para- medulla, pons, and spinal cord. It also has access cellular transport only allows the passage of to the olfactory bulb to a minor extent. The drug small hydrophilic molecules, whereas the trans- transport through the trigeminal nerve pathway cellular route permits the way for low- takes place either by the axonal route (intracel- molecular-weight lipophilic substances. In such lular transport) or via endocytosis. The trigemi- condition, owing to larger size, solubility, and nal nerve is the fifth cranial nerve and is polarity, most of the hydrophilic drugs, proteins, considered as one of the largest neurons of and peptides get stuck in the nasal cavity. CNS, comprised of maxillary, mandibular, and Improvement in nasal permeability could ophthalmic neurons. Among these, the maxillary resolve such an issue and facilitate the transfer and ophthalmic neurons are primarily respon- of bioactives via extracellular transport through sible for nose-to-brain connection [8]. Trigeminal olfactory and trigeminal neurons [8]. nerves are chiefly found in the respiratory region The permeation enhancers, including tight of the nasal cavity and in the dorsal olfactory junction modifiers, polymers, lipids, cyclodex- region to a lesser extent. The drug in the trin, bile salts, surfactants, etc. possess the ability 184 9. Nose-to-brain drug delivery: an alternative approach for effective brain drug targeting to improve the permeability of nasal epithelium. drugs/bioactives. Commonly, the enzyme inhib- Such compounds dissolve the membrane lipid itors belong to the class of proteases or pepti- and thereby reversibly modify the permeability dases [1]. In addition, some absorption [55,56]. Other common mechanisms by which enhancers, such as different salts and fusidic the permeation enhancer exerts their activity acid derivatives, also act as enzyme inhibitors are given below: [60]. Along with this, aprotinin, boroleucin, amastatin, borovaline, trypsin, and bestatin - By improving the contact between nasal inhibitors are other important compounds used epithelium and drug-carrier system to protect drugs from nasal enzymatic degrada- - By reducing the mucociliary clearance tion [57]. - By solubilizing the drug - By minimizing elasticity of the mucus layer - And by inhibiting the metabolizing enzymes 6. Novel drug delivery approaches Examples of some permeation enhancers used in intranasal formulations are saponins, Laureth- 6.1 Polymeric nanoparticles 9 (surfactant); fusidic acid derivatives, trihy- droxy salts (bile salts); oleic acid, caprylate, Polymeric nanoparticles primarily consist of laurate (fatty acid); EDTA, salicylic acid (chela- polymeric matrix in which the therapeutically tors); phospholipids, etc. [57,58]. active agents are either entrapped, encapsulated, Sometimes, permeation enhancers may or chemically conjugated [61e63]. The size of irritate the nasal mucosa and produce toxicity, polymeric nanoparticle is usually larger than which can be minimized by using suitable the micelle, i.e., 100e200 nm with higher Poly permeation enhancers at the appropriate concen- Dispersity Index (PDI) [64]. It offers a biocom- tration. In addition, this improves the drug patible, biodegradable, stable, and cost-effective permeation in the vascular region, which often carrier system with controlled or sustained leads to severe peripheral side effects [56]. release of the drug [65]. The polymer used may be of natural (like collagen, albumin, gelatin, 5.2 Enzyme inhibitor alginate chitosan, etc.) or synthetic origin (like polycaprolactones or polyacrylates), which Most of the protein, peptide, and some drug affect the biocompatibility, drug loading, release, moieties are highly susceptible to enzymatic toxicity, and stability profile of the nanoparticle degradation during transport through an epithe- [66]. Owing to the flexible characteristics, the lial barrier or in the nasal cavity, which reduces synthetic polymers sometimes are more benefi- their bioavailability in the brain region. The cial than the natural polymers [67]. Chitosan im- nasal mucosa is enriched with both exopeptidase parts mucoadhesiveness to the nanoparticle and and endopeptidase enzymes. The exopeptidases hence increases the retention time in nasal mu- are commonly used, belonging to mono- and cosa as well as reduces the mucociliary clear- diaminopeptidase classes, which cleave the ance. In addition, it loosens the tight junction peptides at C- and N-terminals. In contrast, between endothelial cells, hence, facilitates the endopeptidase enzymes like cysteine, serine, drug transfer via a paracellular route from the etc. break the internal peptide bonds [59]. Thus, nasal cavity to the brain [68]. Various studies various enzyme inhibitors are coadministered done using polymeric nanoparticles as carrier or added into the nanocarrier system to prevent for direct nose-to-brain delivery of drug are dis- the nasal metabolism or degradation of the cussed in Table 9.2. 6. Novel drug delivery approaches 185

TABLE 9.2 Polymeric nanoparticles for nose-to-brain delivery of therapeutics.

Drug Polymer Particle size Objective References

Tarenflurbil PLGA 133.13 nm Improve bioavailability of tarenflurbil in the brain [78] Huperzine A Chitosan- 153.2 nm Targeting to the brain via lactoferrin conjugation [79] PLGA bFGF PEG-PLGA 104.8e118.7 nm Effective targeting of proteins and peptides to the brain [80] Rivastigmine Chitosan 143.1 nm Improve bioavailability and nasal uptake of rivastigmine [81] Galantamine Chitosan 190 nm Improve galantamine entrapment into chitosan nanoparticle [69]

Galantamine Chitosan 48.3e68.3 nm Enhance therapeutic efficiency of galantamine and alter its [69] pharmacological and toxicological profile

Piperine Chitosan 248.5 nm Brain targeting of bioactive via nasal route [82] Neuroprotective PEG-PCL 70e90 nm To determine the brain targeting potential of [83] peptide (NAP) lactoferrin-modified nanoparticle

Hanafy et al. developed i.n. galantamine- drug. The ex vivo study shows higher drug chitosan complex nanoparticle for the treatment permeation and in vivo investigation demon- of AD and investigated the effect of drug- strated considerable improvement in bioavail- polymer complexation on the pharmacological ability and therapeutic efficiency of the drug behavior of the drug. The in vivo study on [75]. Similarly, Seju et al. utilized PLGA- male Wistar rats demonstrated no negative effect nanoparticle for nose-to-brain delivery of an anti- on the pharmacological profile of galantamine. It psychotic drug, olanzapine. The PLGA improved significantly increases the therapeutic potency of contact with nasal mucosa and facilitated drug the drug by reducing the AChE level. At the absorption and enhanced drug permeation. It same time, no toxicity or adverse effect was also increased the therapeutic efficiency of the observed [69]. Further, various other works drug [76]. have investigated the application of i.n. chitosan Tong et al. explored the ability of mucoadhe- nanoparticle for the treatment of cerebral sive PLGA-chitosan-based nanoparticle for brain ischemia [70],PD[71], schizophrenia and bipolar delivery of an antidepressant drug, desvenlafax- disorders [72], neural pain [73], and other brain ine. The drug was loaded into the i.n. mucoadhe- disorders. sive nanoparticle, to improve its concentration Another important synthetic polymer is p- in the brain and its therapeutic efficiency. The oly(lactic-co-glycolic acid) (PLGA), widely used combination of PLGA and chitosan imparts and approved by the FDA for preparation of excellent bioadhesion to the formulation and, nanoparticles. It is suitable for both the thus, facilitates drug absorption. They found hydrophilic and lipophilic moieties and offers a that the formulation considerably improved the biodegradable, controlled-release drug carrier drug pharmacokinetic and pharmacodynamic system [74]. Sharma et al. developed behavior and relieved the symptoms of depres- midazolam-loaded PLGA-nanoparticle for sion [77]. direct nose-to-brain delivery of the antianxiety 186 9. Nose-to-brain drug delivery: an alternative approach for effective brain drug targeting

6.2 Lipidic nanoparticles the drug permeation and provides controlled and prolonged release of the drug [87]. Further, Unlike the polymeric nanoparticles, lipid Youssef et al. developed almotriptan-loaded nanoparticles are colloidal, nanosized materials SLN and incorporated it into mucoadhesive composed of natural or synthetic lipid or combi- in situ gelling system to treat migraine. The nation of lipid, stabilized with an appropriate in situ gel imparts mucoadhesiveness to the surface acting agent [84,85]. Solid lipid nanopar- formulation and facilitates constant and higher ticles (SLNs) and nano lipid carriers (NLCs) are drug absorption for a prolonged duration. The the most promising and modern lipid nanopar- SLN successfully delivered the drug to the brain ticles, extensively used for brain targeting of with higher drug concentration. The formulation pharmacologically active substances [86]. Lipid didn’t impart any toxicity or irritability to the nanoparticles offer advantages over other nano- nasal mucosa [88]. Yasir et al. worked on i.n. carriers like biocompatibility, biodegradability, delivery of haloperidol- (antipsychotic agent) higher drug permeation, compatibility with a loaded SLN. The intranasal SLN demonstrated wide range of both hydrophilic and hydrophobic higher drug targeting efficiency than the oral molecules, less toxicity, controlled and formulation and a significant improvement in prolonged release, etc. However, low drug the local drug concentration in the brain [89]. encapsulation and susceptibility to degradation NLCs are considered as second-generation upon storage are the limitations of lipid nanocar- SLNs, as they overcome the limitation of poor rier. But, the next-generation carrier system like drug loading and stability issues [13]. A general SLNs and NLCs resolve these issues and hence structure of NLC is shown in Fig. 9.2. Various have become popular among researchers for research has focused on the utilization of NLC brain targeting [13]. A typical structure of SLN as a carrier system for treatment of a variety of is shown in Fig. 9.1. Rassu et al. utilized SLN CNS disorders like AD, PD, epilepsy, schizo- for intranasal administration of BACE1-siRNA phrenia, migraine, depression, anxiety, bipolar to the brain for the treatment of AD. They devel- disorder, etc. Some of the examples are discussed oped chitosan-coated, positively charged SLN in this section. Wavikar et al. developed for prolonged delivery of the drug to the brain. rivastigmine-loaded NLCs using in situ gelling The chitosan-modified nanoparticle increases

FIGURE 9.1 A general structure of solid lipid nanopar- ticle (SLN). FIGURE 9.2 General structure of NLC. 6. Novel drug delivery approaches 187 systems for the treatment of the neurodegenera- 6.4 Liposomes tive disorder. The NLC-based intranasal in situ gel demonstrated two-fold higher nasal perme- Liposomes are referred to as spherical lipidic ation and three-fold higher enzyme inhibition vesicles made of a concentric bilayer of phospho- activity [90]. Further, Khan and team investi- lipid with aqueous core material. The lipid con- gated the brain targeting efficiency of tent of liposome mimics the physiological temozolomide-loaded NLCs and found a membrane and thus offers a biocompatible and considerable improvement in the drug concen- biodegradable carrier system [12]. The lipid tration in the brain [91]. Apart from these, gets self-assembled as a nano-sized vesicle various other works focusing on the use of lipid enclosing an aqueous core when immersed into nanocarrier for intranasal drug delivery to the the aqueous solution. It is suitable for both the brain are discussed in Table 9.3. hydrophilic and hydrophobic moieties; the hy- drophilic drugs are encapsulated into the aqueous core while the lipophilic molecules get intercalated between the lipid bilayer (Fig. 9.3) 6.3 Inorganic nanoparticles [128]. It offers a nontoxic, biocompatible, and highly lipophilic carrier system for brain target- Inorganic nanoparticles are mainly composed ing, which has the ability to protect the bioactive of various metals or metal oxides and hold great substances from enzymatic degradation and the potential as diagnostic and therapeutic agents. surrounding environment [129]. Application of Studies demonstrated the application of liposome as carrier for nose-to-brain delivery of different inorganic nanoparticles like gold nano- therapeutics is explained in Table 9.5. particle, mesoporous silica nanoparticle, carbon Nageeb El-Helaly et al. developed an electro- nanotubes, quantum dots, magnetic nanopar- statically stable stealth liposome for brain target- ticles, etc. as radio or fluorescent-labeled agent ing of rivastigmine via the intranasal route to for biochemical analysis and as a carrier system treat neurodegenerative disorders like AD. for effective brain targeting. Depending on the Research shows that stability is the challenge nature of the material used, the individual nano- for liposomal preparation. Hence, the author particle holds unique novel properties like tar- used DDAB1 for electrostatic stability and PEG- geting to a specific site, controlled and DSPE to provide stealth nature and improve prolonged release of the active molecule, the stability of the formulation. The resultant car- external control, and regulation of in vivo rier system shows significantly higher drug behavior, high drug loading, etc. [114]. Although permeation, and pharmacokinetic behavior for brain targeting (Table 9.4), the inorganic thus increases the bioavailability of the drug in nanoparticles have not been much explored for the brain [130]. Another interesting approach is direct nose-to-brain delivery of therapeutics. the development of ghrelin-loaded surface- This may be because of some complexities, modified intranasal liposome with chitosan to such as irritant behavior to the nasal mucosa treat cachexia. The liposome was coated with and toxicity profiles of inorganic nanoparticles N-(2-hydroxy) propyl-3-trimethyl ammonium [115].

1 Didecyldimethyl ammonium bromide. 188 9. Nose-to-brain drug delivery: an alternative approach for effective brain drug targeting

TABLE 9.3 Lipidic nanoparticles for nose-to-brain delivery of therapeutics.

Particle Drug Lipid or oil phase size Objective References

Microemulsion Rivastigmine Capmul MCM 53e55 nm Improve pharmacokinetic profile and [92] brain bioavailability of rivastigmine Paliperidone Oleic acid, Polyoxyl 40 27.31 nm To develop paliperidone-loaded [93] hydrogenated intranasal microemulsion for treatment castor oil and Caprylocaproyl of schizophrenia polyoxylglycerides Olanzapine Oleic cis-9-octadecenoic acid 23.87 nm Effective treatment of schizophrenia [94] Buspirone Isopropyl myristate 35.7e Improve drug concentration and [95] hydrochloride 36.2 nm bioavailability in the brain Tramadol Isopropyl myristate, Polyethylene 16.69 nm Improve therapeutic efficiency of drug [96] glycol-400, Propylene glycol

Curcumin Capmul MCM, DHA rich oil <20 nm To evaluate role of DHA in brain [97] delivery of curcumin via i.v. and i.n. route Quetiapine Capmul MCM 35.31 nm Effective brain targeting bioavailability [98] fumarate of drug Solid lipid nanoparticle (SLN) Quetiapine Solid lipid: Glycerol monostearate 117.8 nm Improve therapeutic efficiency of drug to [99] fumarate treat schizophrenia and antipsychotics

Vincristine Solid lipid: Cetyl palmitate 100e Enhance brain delivery of drug [100] sulfate 169 nm Ganciclovir Solid lipid: Glyceryl monostearate 113.7e Investigate tissue distribution of drug in [101] 142.5 nm the brain Rivastigmine Solid lipid: Tocopherol succinate 15.6 nm Improve brain targeting of drug in the [102] brain Rosmarinic Solid lipid: Glyceryl monostearate 149.2 nm Evaluate the brain targeting efficiency [103] acid and soy lecithin of SLN Ondansetron Solid lipid: Glyceryl monostearate 320e Investigate CNS targeting efficiency of [104] HCl 498 nm ondansetron-loaded SLN Haloperidol Solid lipid: Glyceryl monostearate 115e Study the stability and effect of SLN on [89] 156 nm brain targeting efficiency Tarenflurbil Solid lipid: Glyceryl monostearate 89.21 nm Improve concentration of drug in the [78] brain via intranasal administration Rivastigmine Solid lipid: Compritol 888 ATO 82.5 nm To develop rivastigmine-loaded SLN by [105] applying quality by design approach Nano lipid carrier (NLC) GDNF Solid lipid: Precirol ATO 205.9 nm To evaluate neuroprotective and [106] Liquid lipid: Mygliol regenerative behavior of Chitosan-TAT-based nasal NLC 6. Novel drug delivery approaches 189

TABLE 9.3 Lipidic nanoparticles for nose-to-brain delivery of therapeutics.dcont'd

Particle Drug Lipid or oil phase size Objective References

Clonazepam Solid lipid: Compritol 888 209.6e Improve brain targeting of drug via [107] Liquid lipid: Oleic acid and 288.8 nm nasal olfactory mucosa glyceryl monooleate Artemether Solid lipid: Trimyristin 123.4 nm Optimize drug-loaded NLC via central [108] Liquid lipid: Medium chain triglycerides composite design Protein Solid lipid: Precirol ATO5 114 nm Design and optimize chitosan-coated [109] Liquid lipid: Mygliol NLC for brain targeting Duloxetine Solid lipid: Glyceryl monostearate 137.2 nm Evaluate brain targeting efficiency of [110] Liquid lipid: Capryol PGMC NLC upon nasal administration Efavirenz Solid lipid: Precirol ATO 5 162 nm Develop and optimized [111] Liquid lipid: Captex P 500 efavirenz-loaded NLC for effective brain targeting via nasal route Rivastigmine Solid lipid: Glyceryl monostearate and 123.2 nm To explore the brain targeting [112] Capmul MCM C8 (3:2) efficiency of NLC Liquid lipid: Stearic acid Temozolomide Solid lipid: Gelucire 141.28e To optimize brain pharmacokinetic and [91] Liquid lipid: Vitamin E 220.11 nm drug efficiency via spinographic imaging after i.n. administration to brain

Embelin Solid lipid: Cetyl palmitate 152 nm Investigate brain targeting efficiency of [113] Liquid lipid: Octyldodecanol NLC for treatment of epilepsy

chitosan chloride to impart surface charge and improve the therapeutic efficiency of the (anionic) to the carrier system. The surface drug. The liposome significantly enhances the modification with negatively charged polymer nasomucosal permeability of drug and provide facilitates interaction with the nasal mucosa, a safer means of drug targeting [132]. In addi- thus improving the drug permeation. It was tion, Bender et al. developed GDNF2-loaded delivered as an aerosol preparation. The liposome for intranasal delivery to the brain to prepared formulation enhances the brain target- treat PD. The intranasal liposome successfully ing efficiency and shows higher bioavailability targets the bioactive to the brain and signifi- of the drug [131]. In the same sequence, Upad- cantly improves overall bioavailability hyay et al. tried to improve the bioavailability of GDNF [133]. Apart from these, a number of of quetiapine fumarate for the treatment of studies have been conducted by using liposome schizophrenia. The oral administration of quetia- as a carrier system for direct nose-to-brain target- pine fumarate is hindered by hepatic first-pass ing of the drug by using various surface-acting metabolism. Hence, the author developed an agents, targeting molecules and other modifica- intranasal to avoid gastrointestinal degradation tions to improve the efficiency of the system.

2 Glial cell lineederived neurotrophic factor. 190 9. Nose-to-brain drug delivery: an alternative approach for effective brain drug targeting

TABLE 9.4 Application of inorganic nanoparticle for nose-to-brain delivery of therapeutics.

Type of inorganic Metal/metal Particle Drug nanoparticle derivatives size Objective References e Quantum CdSe/ZnS 15e20 nm To explore suitability of quantum dots [116] dots nanocrystals for nose-to-brain delivery e Quantum CdSe/ZnS 95.3 nm Brain targeting of diagnostic agent for [117] dots in vivo imaging of brain e Quantum Cadmium 20e25 nm Study microglial activation by [118] dots selenide nanoparticulate stimulation, in vivo imaging hNgR-Fc Gold Gold 15 nm Improve brain targeting and therapeutic [119,120] protein nanoparticle efficiency of protein and peptide e Gold Gold 13 nm Study the biodistribution, safety, and [121] nanoparticle efficacy of gold nanoparticle after intranasal administration

Resveratrol Gold Gold 10.30 nm Effective brain targeting of resveratrol [122] nanoparticle and as biomarker agent e Mesoporous Silica 220 nm Investigate the application of [123] silica mesoporous silica nanoparticle as nanoparticle carrier for nose-to-brain drug delivery e Silica SiO2 115 nm Evaluate the adverse effect of silica [124] nanoparticle nanoparticle on brain e Carbon e 5e15 nm Study the effect of carbon nanotube on [125] nanotube function and viability of brain macrophages

Mesenchymal Magnetic Iron oxide 5.22 nm Improve homing of stem cell [126] stem cells nanoparticle Carmustine Magnetic Iron (II) chloride 30e50 nm Effective nose-to-brain drug delivery via [127] nanoparticle tetrahydrate and iron (III) olfactory nerve pathway chloride hexahydrate

Such extensive utilization suggested liposome as imparts transparent or milky white appearance a potential carrier system for nose-to-brain to the nanoemulsions [144,145]. It possesses a delivery of therapeutic agents. higher surface area than the other carrier system, and also the smaller size is responsible for the 6.5 Nanoemulsions stable formulation without any sign of sedimen- tation, creaming, coalescence, etc. It improves Nanoemulsions are nanosized w/o or o/w the solubility of poorly soluble drug substances. emulsion of two immiscible liquids stabilized In addition, it has the ability to protect the drug with suitable surfactant. The standard globule from the surrounding environment (pH and size of nanoemulsion was supposed to less physiological condition), oxidation, and enzy- than 100 nm. However, 300 nm is also reported matic degradation [145]. in some literature. The smaller droplet size 6. Novel drug delivery approaches 191

Various preclinical and clinical investigations demonstrated nanoemulsion as a promising carrier system that promotes the permeation of small lipophilic molecules across the nasal epithelium (Table 9.6) [86]. Columbo et al. stud- ied the efficiency of kaempferol in treatment of glioma when delivered intranasally by encapsu- lating in the mucoadhesive nanoemulsion. The formulation improves the nasal permeation and preserves the antioxidant potency of the drug. The in vivo study indicated 4.5e5-fold higher drug concentration in the brain. More- over, the higher cytotoxicity to the tumor cell makes it a suitable candidate for the treatment of glioma [150]. Further, the intranasal nanoe- mulsion was also investigated for schizophrenia FIGURE 9.3 A typical structure of liposome. treatment. Boche et al. developed quetiapine

TABLE 9.5 Application of liposome for nose-to-brain delivery of therapeutics.

Particle Drug Lipid size Objective References

Rivastigmine Egg phosphatidylcholine 178.9 nm Improve brain distribution of drug and [134] reduce systemic side effects via i.n. route

Galantamine Soy phosphatidylcholine 112 nm Evaluate the AChE inhibition activity of [135] hydrobromide drug

Rivastigmine Soy lecithin 10 mm For effective delivery of drug to the brain [136] via nasal route Cyclovirobuxine D Soy lecithin 72.03e To evaluate brain targeting efficiency of [137] 86.5 nm polysorbate 80-coated liposome and to increase bioavailability of drug in the brain

H102 Egg phosphatidylcholine, 112.2 nm Enhanced brain targeting of peptide [138] DSPE-PEG Ferric ammonium Soy lecithin 40 nm For effective delivery of iron to the brain [139] citrate bFGF Soy phosphatidylcholine 128 nm Prevent cerebral ischemia in stroke patients [140] Donepezil Distearyl-sn-glycerol-3- 102 nm Enhance brain bioavailability of drug for [141] phosphocholine (DSPC) effective treatment of AD Rivastigmine CPP, DSPE-PEG-NHS 178.9 nm Improve brain distribution and reduce [134] side effect of drug Risperidone Soy phosphatidylcholine 91e Effective brain targeting of risperidone to [142] 106 nm treat schizophrenia Ghrelin Soy lecithin 10 mm Improve brain delivery of ghrelin [143] 192 9. Nose-to-brain drug delivery: an alternative approach for effective brain drug targeting

TABLE 9.6 Application of nanoemulsion for nose-to-brain delivery of therapeutics.

Globule Drug Oil phase size Objective References

Riluzole Sefsol 23.92 nm Improve brain bioavailability of riluzole [146] Cyclosporine-A Flaxseed oil 272 nm Investigate biodistribution and [147] pharmacokinetics of drug in brain upon i.n. administration

Selegiline Grape seed oil and 61.43 nm Effective brain delivery and improved [148] Sefsol 218 (1:1) bioavailability to treat Parkinson disease

Tramadol Isopropyl myristate 136.3 nm Improve therapeutic efficiency of drug [96] Quetiapine Capmul MCM 144 nm Improve brain targeting via intranasal route [149] fumarate

Kaempferol Egg lecithin and 170.4 nm Develop mucoadhesive nasal formulation for effective [150] medium-chain triglycerides brain targeting fumarateeloaded nanoemulsion and investi- 6.6 Dendrimers gated its brain targeting efficiency. Significantly higher drug transport and targeting efficiency Dendrimers are nanosized, three-dimensional were observed by direct nose-to-brain delivery complex polymeric structures with higher of nanoemulsion [149]. Brain delivery of zolmi- surface functionalization and high drug- e loading efficiency (Fig. 9.4) [13]. Win-Shwe triptan, an antimigraine drug loaded fi bioadhesive nanoemulsion via intranasal route et al. evaluated the brain targeting ef ciency of was studied by Abdou et al. The nanoemulsion PAMAM dendrimer after administration of a successfully delivered the drug to the brain at single intranasal dose on mice model. The ani- high concentration and rapid onset of action mal organs (different parts of brain including was achieved, which is essential for migraine the hippocampus, olfactory bulb, cerebrum, therapy. Study shows significant improvement and cortex, and blood samples) were evaluated in pharmacokinetic behavior of the formulation for the presence of a biomarker. The study indi- in the brain [151]. Similarly, Parikh et al. utilized cated the transfer of carrier system takes place nanoemulsion to improve the bioavailability of via olfactory neuron path as well as through riluzole for the treatment of amyotrophic lateral systemic circulation upon intranasal administra- tion. The intranasal PAMAM dendrimer shows sclerosis, a progressive neurodegenerative disor- fi der. It is prone to P-glycoprotein efflux transpor- signi cant brain targeting ability (Table 9.7) tation and restricted by the BBB. The intranasal [152]. Earlier, Perez utilized PAMAM dendrimer for nanoemulsion improves brain bioavailability of 3 the drug and, hence, the therapeutic efficiency brain delivery of siRNA . They developed [146]. Along with these, numerous other studies siRNA-complexed dendrimer incorporated into fi in situ mucoadhesive chitosan-based nasal gel. con rm that the intranasal nanoemulsion fi presents a potential carrier system for the To check the brain targeting ef ciency, the pre- treatment of CNS disorders. pared drug carrier system was radiolabeled.

3 32P-small interference RNA. 6. Novel drug delivery approaches 193

FIGURE 9.4 Basic structure of dendrimer showing different generations and function surface groups with highly branched structure.

TABLE 9.7 Application of dendrimer for nose-to-brain delivery of therapeutics.

Drug Polymer Particle size Objective References e PAMAM-NH2 (G4) 5.7 nm To study the neurotoxicity of PAMAM [152] dendrimer after intranasal administration siRNA poly(amidoamine) (G7) e To study the radioactivity of brain after nasal [153] administration of si-RNA-loaded dendriplex

Paeonol PAMAM 72.4e96.5 nm Improved brain targeting of drug [154]

The thermoresponsive in situ gel increases the in situ gelling systems. They observed a signifi- nasal retention time of the system and facili- cant amount of dendrimer nanocomposite in tates drug absorption via nasal mucosa. At the brain, which further increases by using thesametime,thedendrimerimprovesbrain in situ gelling system upon intranasal adminis- targeting efficiency via an olfactory neural tration. The brain targeting was achieved by pathway, and hence, higher radioactive mate- neuronal pathway. All these studies show that rial was observed in the olfactory region the dendrimer serves as a potential carrier sys- [153]. Recently, another study done by Xie tem for brain targeting via intranasal route et al. also relies on the findings of the above [154]. However, systematic investigations are studies. They also synthesized PAMAM den- needed to establish an approach for commercial drimer and merged this nanocomposite with application. 194 9. Nose-to-brain drug delivery: an alternative approach for effective brain drug targeting

7. Recent advancements in the clinical surface-modified or targeted formulations are trials of nanoparticles via the nose-to-brain used for more effective intranasal delivery to delivery the brain. The novel carrier system made of mucoadhesive polymers or high-viscosity The delivery of drugs using conventional formulation increases the retention time and dosage form such as solution or suspension via reduces the mucociliary clearance of the drug. the nose-to-brain delivery has been studied in The use of permeation enhancer facilitates the clinical trials. There is a lack of clinical trials drug absorption. Similarly, nanocarriers also that are focused on the nose-to-brain delivery protect the drug from enzymatic degradation of drug-loaded nanoparticles. One of the prom- and assist in enhancing the absorption. In addi- ising delivery systems is nanoemulsions, which tion, the surface-modified nanocarrier system represent a promising strategy for nose-to-brain promotes drug targeting to a site of action. More- delivery for the treatment of CNS diseases. over, the proper nasal administration technique Technion developed an electronic nose-based is also essential to facilitate neural absorption nanomaterial for diagnosis of diseases such as from the dorsal olfactory region of the nasal cancer, kidney failure, etc. via breath samples. cavity to the brain. Currently, most of the This trial is completed (NCT01206023). Howev- nanocarrier-based intranasal strategies are under er, the results of this trial were not published at clinical or preclinical investigation stages. A sys- the time of writing. In addition to pharmacoki- tematic understanding of nasal drug targeting netic and pharmacodynamic studies under and amalgamation with a novel nanocarrier clinical trial, the toxicity of nanoparticles should system is hoped to offer better treatment of be tested to calculate the benefit-to-risk ratio. It is chronic CNS disorders. expected that with the advancement of formula- tion strategies for the nose-to-brain delivery, Acknowledgments these nanoparticle-based delivery systems will be tested under clinical trials in the near future. The authors would like to acknowledge the Department of Pharmaceutics and Drug Delivery, School of Pharmacy, Uni- versity of Mississippi, USA, for providing start-up support to Dr. Chougule’s lab. 8. Summary

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Trends in the intellectual property (IP) landscape of drug delivery systems: 30 years of growth and evolution Amanda Starling-Windhof, Asur Srinivasan, Tina Tomeo, Anne Marie Clark, Peter Mattei CAS, a Division of the American Chemical Society, Columbus, OH, United States

1. Introduction drugs, protecting their R&D investments. Generic drug makers or drug delivery system innovators One of the most critical drivers of innovation in also contribute to the drug delivery system IP the drug delivery field is commercialization landscape as they offer enhancements to build potential, which is facilitated by intellectual upon others’ research with new innovations. property (IP) protection, primarily in the form This chapter presents a detailed discussion on of patents. After the launch of a new drug, phar- the evolution of the global IP landscape around maceutical companies continue to research the drug delivery over the past 30 years. Using active pharmaceutical ingredient (API) seeking global patent records and value-added data to improve the drug’s chemical and physical from CAS’s curated content collection, we iden- forms as well as developing novel and specialized tify key trends in drug delivery over this period, delivery systems. The goal of these systems is to paying particular attention to the three most improve the performance and effectiveness of widely used drug delivery systems: oral, topical, the active ingredient and/or the patient experi- and parenteral. Based on this analysis, we high- ence by offering easier dosing schedules, lower light emerging opportunities within the drug de- dose formulations, or a change from injections livery landscape and anticipate the focus of to oral delivery, for example. In addition to future IP growth in the field. We will also discuss providing an enhanced drug product for patients, the importance and core requirements of a pro- these improvements to the initial API invention gram to proactively monitor the IP landscape also offer organizations the opportunity to to identify opportunities and defend existing IP acquire additional patent protection for their assets.

Nanopharmaceuticals https://doi.org/10.1016/B978-0-12-817778-5.00010-5 201 © 2020 Elsevier Inc. All rights reserved. 202 10. Trends in the intellectual property (IP) landscape of drug delivery systems

2. Overview of the IP landscape for drug covered, as indicated by the increase in the delivery systems average density of the filed patents (shown as the blue [light gray in print version] to orange Patents describing drug delivery systems in [dark gray in print version] color change in the the form of tablets and capsules can be found figure). This evolution may be due to more as early as the 19th century [1]. However, the aggressive IP protection strategies, with the goal number of patents for drug delivery systems of minimizing competitors’ freedom to operate did not experience much growth until the with specific active ingredients, for example. 1980s. Even then, it wasn’t until the turn of Fig. 10.1 also reveals a significant rise in the the 21st century that the IP landscape for drug number of countries from which these patents delivery systems truly took off. originate, as reflected by the increasing thickness The past 30 years have witnessed consider- of the line shown on the graph. Analyzing this able growth in both the volume and complexity trend in more detail highlights considerable of patents for drug delivery technologies. change in the geographical origin of drug deliv- Fig. 10.1 shows the number of patents published ery patents over the past 30 years. Fig. 10.2 annually between 1988 and 2018, as well as their shows the top contributors to the drug delivery complexity and number of source countries. patent literature by country. Starting in the late 1990s, the IP landscape for While the United States has dominated the IP drug delivery systems has experienced remark- landscape for drug delivery technologies over able growth. Despite a dip in the number of the past 30 years, China is playing an increas- new patents filed between 2009 and 2010, and ingly large role. The last 2 decades have seen again between 2015 and 2016, the field has China rapidly and substantially expand its IP benefited from year-on-year growth in the vol- footprint. In contrast, Japan has seen its share ume of patent filings. This expansion coincides of patents for drug delivery systems decrease with increasing complexity of the technologies relative to the overall growth in global patent fil- ings. Two decades ago, Japan was second to the

FIGURE 10.1 Increase in the volume, complexity, and number of country assignees of drug delivery system patents pub- lished between 1988 and 2018. Source: CAS content collection. 2. Overview of the IP landscape for drug delivery systems 203

FIGURE 10.2 Top countries of origin of drug delivery system patents for the 3 decades spanning 1989 to 2018. Source: CAS content collection.

United States in terms of drug delivery patent across the pharmaceutical field, the dominant output. Today, its share of IP in this field is com- categories of drug delivery technologies have parable with leading European countries such as remained unchanged over the past 30 years. Germany and the United Kingdom. Oral drug delivery systems, perhaps unsurpris- ingly, have been the mainstay of patent output, fl 2.1 Industry trends re ecting the historical importance of oral routes of administration within the pharmaceutical in- While the aforementioned analysis highlights dustry. These routes of administration include macroscopic changes in global drug delivery IP, traditional tablets and capsules, as well as oral it is perhaps more useful to evaluate the trends suspensions and alternative user-friendly oral that have occurred at the industry level. Our dosage forms such as instant drinks and gran- analysis of the patent literature highlights five ules. Controlled-release drug delivery systems, key commercial sectors responsible for drug another important subset of oral drug delivery delivery IP: pharmaceutical, biotechnology, cos- systems, are consistently the third most widely metics and personal care, food and nutraceuti- patented delivery technology by pharmaceutical cals, and polymers. Fig. 10.3 shows the top stakeholders. However, while oral dosage forms three patented drug delivery technologies over are among the oldest types of delivery system, the 3 decades between 1989 and 2018 for each formulations are becoming increasingly sophisti- of these key industries. cated, with tamper-proof opioids and sequen- Drug delivery systems continue to drive inno- tially released multi-API delivery systems vation and ensure the delivery of a healthy developed in recent years. return on R&D investment for conventional Despite these advances, some drugs cannot small molecule drugs. The findings in Fig. 10.3 tolerate passage through the gastrointestinal highlight that, while patent output has grown tract without degradation. Topical systems are 204 10. Trends in the intellectual property (IP) landscape of drug delivery systems

FIGURE 10.3 Top three drug delivery systems by industry for the 3 decades between 1989 and 2018. Source: CAS content collection. the second most important family of drug deliv- therapeutics and viral gene therapies has put ery technologies within this field for each of the considerable focus on novel parenteral drug de- decades assessed in this study, while injectable livery systems. Likewise, new hormone thera- forms are becoming increasingly important, pies, such as those used to fight inflammatory especially for biotechnology-derived pharma- diseases using targeted steroids or bone diseases ceutical products. using parathyroid hormone analogues, require Considerable advances in biotechnology- their own delivery mechanisms. Furthermore, derived therapeutics or “biologics” over the the use of radiolabeling techniques to track past 30 years have driven research into innova- delivery methods and application of signal mol- tive drug delivery systems for these novel treat- ecules as cell penetration agents have resulted in ment strategies. As with the pharmaceutical a wealth of patents in this area. sector, oral and topical drug delivery systems The cosmetics and personal care industry is have dominated biotechnology IP over the full another market sector where drug delivery tech- period studied. However, while an analysis of nologies play a key role. Topical, oral, and trans- patents filed between 1989 and 1998 finds dermal drug delivery systems represent the controlled-release drug delivery systems to be three most common patent categories within the third most important family of drug delivery this industry, with little change in the impor- technologies, the last 2 decades have seen paren- tance of each of these technologies over the teral drug delivery systems become more impor- past 30 years. Transdermal drug delivery sys- tant. This is likely due to the substantial clinical tems refer to approaches that involve systemic and commercial success of biologics and their delivery of the drug to the bloodstream through transition to an established therapeutic strategy. the skin, and differ from topical systems, which As the use of peptide- and protein-based offer slower rates of absorption and are designed drugs continues to mature, expansion in this for localized treatment. That topical and trans- field has necessitated the development of dermal feature prominently is perhaps unsur- delivery technologies specifically designed for prising, given the dominance of products such these types of therapeutics. Similarly, the emer- as lotions and moisturizers designed to slow gence of novel treatments based on nucleotide the signs of aging or treat acne, dermatitis, and 2. Overview of the IP landscape for drug delivery systems 205 other skin conditions within the cosmetics indus- for this purpose include nanocomposites and try. Oral drug delivery technologies are equally disposable infusion products, both of which important for oral hygiene products such as have seen growing attention in recent years. mouthwash formulations, some of which are Polymers play an essential role in the manufac- now designed to treat gingivitis or periodontitis. ture of these technologies in the pharmaceutical The food industry is a further source of drug sector as well as in related fields of science and delivery patents, reflecting the size of the nutra- are increasingly the focus of patent applications. ceuticals market, as well as the fact that some API and formulation excipients are derived from foodstuffs. Garlic, for example, is a 2.2 Technology trends medicinally active food that is reported to have antimicrobial, antithrombotic, and anticancer Analyzing the patents for drug delivery sys- properties and is widely taken as a supplement tems across all industries over the past 3 decades [2], while phospholipids derived from food sour- also provides useful insight into the types of ces such as soybeans and egg yolk are used in technologies that are being developed. Fig. 10.4 pharmaceutical formulations [3]. Beyond IP highlights the eight most significant classifica- associated with food extracts, patents in this tions for drug delivery systems patents by category also include designs such as beverage decade. It should be noted that patents are not caps that are used for the delivery of food sup- exclusive to the type of drug delivery system plements or health drinks. they disclose. For example, many patents that The data in Fig. 10.3 shows that the food disclose an oral drug delivery technology will industry has witnessed a more marked change also specify the specific form of the delivery sys- in drug delivery trends than most other market tem, such as a tablet and/or a capsule. While segments. While the data shows that oral drug typically if a patent discloses a tablet dosage delivery systems have dominated patents over form, an oral drug delivery system will also be the period 1989e2018, growth in the number of disclosed, this is not necessarily reciprocal. The patents in liquid oral and effervescent drug data present discrete concerns on the patents delivery systems has come at the expense of that disclose drug delivery systems that occur controlled-release, topical, and sugar-coated in many areas of application and are therefore solid oral drug delivery systems. indicative of prevailing trends in the disclosed Finally, the polymer and plastic industry is a information of drug delivery system patents. rapidly evolving field for drug delivery IP. The As highlighted by the industry data shown most commonly patented drug delivery technolo- earlier, oral drug delivery systems have played gies in this field are those relating to controlled- a key role in a wide range of industries, release systems. Specific polymer chemistries including the pharmaceutical and biotechnology have long played an important role in the devel- sectors, for the past 30 years. The share of patents opment of these types of formulations and have associated with oral drug delivery systems, as been the main subject of patents in this market well as for tablets and capsules specifically, has sector for the past 3 decades. Interestingly, the increased significantly over this period. Growth last decade has seen a shift away from patented in oral drug delivery patents has been fueled topical systems toward targeted or “smart” by novel and more user-friendly dosage forms drug delivery technologies. These systems are such as granules and gels, as well as the powders designed to reduce the potential for adverse used for instant drink formulations, and these side effects in other parts of the body by deliv- trends will be discussed further in the following ering the drug locally. Technologies designed section. 206 10. Trends in the intellectual property (IP) landscape of drug delivery systems

FIGURE 10.4 Proportion of patents (gray bars) and patent volume growth (blue line; dark gray in print version) for the eight most common classifications for drug delivery system patents for the 3 decades spanning 1989 to 2018. Source: CAS content collection.

Interestingly, growth in the percentage share autoimmune conditions remain important thera- of oral drug delivery systems comes at the peutic fields for drug delivery IP, volumes of expense of IP based on controlled-release, patents in these areas have all decreased as a topical, and transdermal drug delivery systems. proportion of the total output over the past 3 de- Notably, while transdermal drug delivery sys- cades. This is due to both a reduction in the rate of tems accounted for 13% of filings in the top eight growth in these areas, as well as the impact from classifications of drug delivery system patents expansion in other fields. registered between 1989 and 1998, in the last The data presented in this study highlights decade this has fallen to 7%. While the overall that the three major classes of drug delivery sys- picture is that patent registrations have grown tems are oral, topical, and parenteral approaches. across the board, it is interesting to note that Fig. 10.6 shows that the past 3 decades have seen technologies such as these have received less considerable growth in the proportion of patents R&D focus relative to other fields. for oral drug delivery systems relative to topical As one might expect, patents for drug delivery and parenteral technologies. The following sec- systems cover a broad range of therapeutic fields, tions discuss the changes in the patent landscape reflecting the diverse range of diseases and condi- for these three key drug delivery systems to tions that are the focus of modern medicine. understand the nature of these changes in detail Fig. 10.5 shows the top disease areas for drug and explore the reasons for this evolution. delivery patents for the 3 decades up to 2018. Most notably, the data highlights cancer as a 3. Trends in oral drug delivery IP growing focus of drug delivery IP, with mam- mary gland, prostate gland, and lung neoplasms all increasing their share of patents. Interestingly, Oral drug delivery is generally considered the while glaucoma, acne, AIDS, psoriasis, and preferred route of administration, as it offers 3. Trends in oral drug delivery IP 207

FIGURE 10.5 Proportion of patents (gray bars) and patent volume growth (blue line; dark gray in print version) in the leading disease areas for drug delivery IP for the 3 decades spanning 1989 to 2018. Source: CAS content collection.

FIGURE 10.6 Change in the proportion of patents for oral, topical, and parenteral drug delivery systems published during the 3 decades spanning 1989 to 2018. Source: CAS content collection. convenience for patients, presents a large degree solubility of many APIs means that various of flexibility in terms of dosage form design, and enabling technologies can be used to improve formulations can be more cost-effective to manu- oral bioavailability [6], including those based facture [4]. These delivery systems draw upon a on particle size reduction and solid dispersion broad range of technologies to achieve safe, [7e9], as well as lipid-based approaches such effective, and predictable results. Controlled- as microemulsions and self-emulsifying drug release formulations, for example, which deliver delivery strategies [10]. Moreover, with alterna- the API orally to body or by localized adminis- tive oral formulations such as granules, gels, tration to suitable body part over an extended and powders gaining traction for use in over- period [5], rely on the careful application of the-counter medications, technologies to excipient and copolymer technologies to achieve improve taste, mouthfeel, and appearance are the required release profile. Issues with the becoming increasingly important. 208 10. Trends in the intellectual property (IP) landscape of drug delivery systems

The breadth of technologies employed in the from the leading regions for the 3 decades up to development of oral drug delivery systems high- 2018. lights the enormous importance of IP protection Although the United States continues to be a for the successful commercialization of safe and major player in the oral drug delivery space, effective pharmaceutical products. Much about China’s impact in the field has grown substan- current trends and potential opportunities in tially over the past 30 years, rising from just 2% oral drug delivery can be gleaned by analyzing of global output in the decade spanning the patent literature. 1989e2008 to 34% in the last decade. Another notable entrant is India, taking a 4% share of IP in the last decade. While other countries have 3.1 Overall trends in oral drug delivery also expanded their patent output over this IP same time frame, this growth has not kept pace with the landscape as a whole. Consequently, The rapid growth in the number of patents for the overall proportion of drug delivery IP from oral drug delivery systems over the past 3 de- countries such as Japan, the United Kingdom, cades mirrors that of the wider drug delivery France, and Italy has declined over the past IP landscape, as shown in Fig. 10.7, which 3 decades. highlights the growth in patents for these tech- nologies between 1988 and 2018. Following 3.2 Trends in oral drug delivery moderate but steady growth in patenting activ- technologies ity during the late 1980s and early 1990s, the number of patents filed annually for oral drug Analysis of the patent literature also provides delivery technologies has rapidly increased since insight into the types of oral drug delivery the turn of the 21st century. This growth is in systems being developed. Fig. 10.9 shows patent large part due to the rapid expansion in patent growth of the leading categories of oral drug output from China, as further explored in delivery systems for the 3 decades up to 2018 Fig. 10.8, which highlights the volume of patents (blue lines; dark gray in print version), while

FIGURE 10.7 Volume of oral drug delivery system patents filed annually between 1988 and 2018. Source: CAS content collection. 3. Trends in oral drug delivery IP 209

FIGURE 10.8 Country origin of oral drug delivery patents for the 3 decades up to 2018. Source: CAS content collection.

FIGURE 10.9 Proportion of patents (gray bars) and patent volume growth (blue line; dark gray in print version) of leading categories of oral drug delivery system IP for the 3 decades up to 2018. Source: CAS content collection. the gray bars in Fig. 10.9 highlight how each of IP landscape over the last 3 decades. Together, these categories compares over this time period. these dosage forms consistently account for Analysis of this data shows that pharmaceu- around half of all oral drug delivery patents in tical tablets and capsules have dominated the each decade. While the share of patents obtained 210 10. Trends in the intellectual property (IP) landscape of drug delivery systems for these technologies has varied slightly from between 2009 and 2018, down from 5% for the decade to decade, the overall picture is that decade between 1989 and 1998, while the share they remain an important field of research. of patents for lozenges has fallen from 4% for Interestingly, patents for pharmaceutical the decade between 1989 and 1998 to 2% for granules and powders have both steadily the decade between 2009 and 2018. increased over the past 30 years. In the decade e spanning 1989 98, granules and powders 3.3 Trends in disease areas and accounted for 6% of total oral drug delivery IP pharmaceutical substances each. The last decade has seen the share of IP for granules and powders increase to 10% and Given the widespread use of drugs designed for 9%, respectively. Additionally, the share of pat- oral administration, patents for drug delivery sys- ents for controlled-release dosage forms almost tems span a wide range of therapeutic fields. e doubled from 5% for the decade 1989 98 to 9% Fig. 10.10 highlights the top disease areas associ- e for the decade 1999 2008. However, the last ated with patents filed in the 3 decades up to 2018. decade has seen the share of total IP for As with the overall picture of therapeutic controlled-release formulations fall slightly to applications for drug delivery technologies, can- 7% of the total IP landscape. cer fields have experienced considerable growth The proportion of IP associated with oral sus- in the share of oral drug delivery IP over the past pensions, gels, solutions and syrups has largely 3 decades. Inflammation, diabetes, and rheuma- remained the same over the 3 decades studied toid arthritis fields have also seen a rise in their in this analysis, indicating that the volume of share of patents. Therapeutic fields that have patents for these technologies has increased at lost some of their share of the IP landscape for a similar rate to the growth in oral drug delivery oral drug delivery technologies include Alz- IP overall. However, the share of total patents for heimer disease, autoimmune diseases, multiple both pharmaceutical lozenges and liposomes has sclerosis, psoriasis, and stroke. decreased over the period studied. Liposome Fig. 10.11 highlights the top 25 pharmaceu- fi systems now account for just 3% of patents led tical substances mentioned in patents for oral

FIGURE 10.10 Proportion of patents (gray bars) and patent volume growth (blue line; dark gray in print version) for major disease areas associated with oral drug delivery IP for the 3 decades up to 2018. Source: CAS content collection. 3. Trends in oral drug delivery IP 211

FIGURE 10.11 Top 25 pharmaceutical substances, primarily anticancer drugs, associated with oral drug delivery patents between 2009 and 2018 (blue; dark gray in print version). Data for the decade between 1999 and 2008 (gray) is also presented overlapping to demonstrate the growth. Source: CAS content collection. drug delivery systems filed between 2009 and drug used to treat many types of cancer, 2018, and the percentage increase from the previ- including those of the bladder, breast, lung, ous decade. ovaries, and pancreas [15]. The majority of the substances on this list An analysis of the IP landscape for oral drug are chemotherapy drugs, with the top three delivery systems highlights an evolving land- compoundsdpaclitaxel, cisplatin, and scape. The most notable change over the past doxorubicindall used as anticancer agents. 3 decades has been the rate at which China has Thelargestdifferenceintheshareofpatent made such an impact to IP in this field. Around output is for metformin, a diabetes medication half of all patents still relate to tablets and cap- [11]. Other large increases in patent output sules, with their share of the IP landscape include those associated with oxaliplatin, an remaining relatively constant over the last 30 anticancer agent used to treat advanced years. However, patents for technologies colorectal cancer [12]; rapamycin, a macrolide relating to pharmaceutical powders and gran- drug with immunosuppressant properties that ules have experienced growth above that of the is used to prevent organ transplant rejection field as a whole. Patents for chemotherapy drugs and inhibit restenosis following angioplasty continue to dominate the oral drug delivery [13,14]; and gemcitabine, a chemotherapy field, with the percentage of new patents 212 10. Trends in the intellectual property (IP) landscape of drug delivery systems designed for this therapeutic area growing the trends in these delivery systems over the decade on decade. past 3 decades can be inferred from an analysis of the patent literature. 4. Trends in topical drug delivery IP 4.1 Overall trends in topical drug Topical drug delivery systems encompass a delivery IP broad range of technologies designed to support The annual number of patents for topical drug the delivery of therapeutic agents to superficial delivery systems has steadily increased since the tissues such as the skin, eyes, nose, and vagina late 1980s (Fig. 10.12), with topical systems for localized treatment. Two distinct but and currently the second most patented drug deliv- related groups of drug delivery technologies ery technologies after oral dosage forms. As that will also be discussed in this section are with the growth in patents for oral drug delivery transdermal drug delivery systems, which systems, much of this increase is due to the rapid enable therapeutic agents to penetrate through expansion in IP output from China. However, the skin and into the bloodstream [16], and inha- despite year-on-year growth for much of the lation drug delivery systems, which enable both past 3 decades, the number of patents filed in local and systemic exposure via the lungs [17]. the past two consecutive years has fallen after Topical drug administration offers several ad- reaching an all-time high in 2015. vantages over other delivery routes, as it is Fig. 10.13 highlights the contribution to noninvasive and convenient for patients, can topical drug delivery IP from the five most active result in an improved physiological and phar- countries in this field over the past 3 decades. macological response, and in some cases reduce Three decades ago, Japan and the United States systemic toxicity [18]. In the case of transdermal dominated the topical drug delivery IP land- drug delivery, these systems circumvent the scape. However, while US patent output in this gastrointestinal tract and potential challenges area increased over the following 2 decades associated with hepatic first-pass metabolism, and remains the leading source of new patents differences in pH conditions, and changes in in the last decade, the number of patents from plasma levels, potentially offering improved Japan has actually fallen in volume. Combined drug bioavailability [18]. with a substantial increase in the number of pat- Each of the organs mentioned above has ents from China, this has resulted in a significant evolved to be highly adept at controlling the decrease in the percentage share of topical drug movement of molecules both into and out of delivery IP from Japan. Interestingly, the propor- the body, and each poses a specific set of chal- tion of new patents from France and Germany lenges for drug administration. As a result, has remained relatively constant over the past topical, transdermal, and inhalation drug deliv- 3 decades, indicating that IP output has kept ery systems depend on a range of effective pace with the overall growth in global patent enabling technologies to bypass the barriers to output. effective treatment [18,19]. Key technologies associated with topical drug delivery include creams, ointments, gels, lotions, sprays, solu- 4.2 Trends in topical drug delivery tions, and suspensions. The extensive range of technologies technologies used to develop topical, vaginal, ophthalmic, nasal, inhalation, and transdermal A more in-depth analysis of the patent litera- drug delivery systems emphasizes the commer- ture over the past 30 years reveals subtle changes cial importance of IP protection. Much about in the types of topical drug delivery systems 4. Trends in topical drug delivery IP 213

FIGURE 10.12 Volume of topical drug delivery system patents filed annually between 1988 and 2018. Source: CAS content collection.

FIGURE 10.13 Country origin of topical drug delivery patents for the 3 decades up to 2018. Source: CAS content collection. being developed. Fig. 10.14 highlights the lead- Notably, while the number of patents for ing topical drug delivery systems and technolo- pharmaceutical ointments, creams, lotions, and gies by volume for the 3 decades up to 2018 as liposomes has increased in volume over the indicated by the blue lines (dark gray in print past 3 decades, their percentage share has fallen version), while gray bars highlight changes in slightly as other types of systems have experi- the share of each category over the same time enced greater growth. Indeed, concerted efforts period. 214 10. Trends in the intellectual property (IP) landscape of drug delivery systems

FIGURE 10.14 Proportion of patents (gray bars) and patent volume growth (blue line; dark gray in print version) of leading categories of topical drug delivery system for the 3 decades up to 2018. Source: CAS content collection. in the fields of vaginal, ophthalmic, nasal, inhala- increased share of patents for gels, sprays, and so- tion, and transdermal drug delivery have seen lutions in the latter 2 decades assessed in this these systems maintain or increase their share study. of IP. While other reviews highlight advances in these fields in more detail [17e19],itisworth 4.3 Trends in disease areas and noting the breadth of technologies that are being pharmaceutical substances applied for these types of drug delivery system. Patents for vaginal drug delivery systems based As one might expect, topical drug delivery on pressure sensitive adhesives and hydrogels systems are dominated by ointments and creams have been filed in the last few years [20,21],while that are most commonly used to treat skin com- novel ophthalmic delivery systems based on sus- plaints. However, an analysis of the patent liter- pensions and colloidal carriers have also been ature over the past 3 decades highlights rapid recently published [22,23]. The number of inhaler growth in a number of key areas. Fig. 10.15 devices available on the market has steadily reveals the most commonly indexed disease increased over the past 2 decades, with novel areas associated with topical drug delivery pat- breath-enhanced, breath-actuated, and vibrating ents for the past 3 decades. mesh nebulizer designs supporting more efficient This analysis highlights significant growth in drug delivery [19]. Furthermore, improvements patents associated with inflammation and neo- in structure, velocity, and electrically based trans- plasms. While these disease categories dermal drug delivery systems have also accounted for a combined 6% of patents for the enhanced the dermal permeation of technologies leading disease areas 2 decades ago, the last associated with this route of administration [18]. decade saw these patents increase their share to Many of these changes are reflected in the 27%. This increase has come at the expense of 5. Trends in parenteral drug delivery IP 215

FIGURE 10.15 Proportion of patents (gray bars) and patent volume growth (blue line; dark gray in print version) of major disease areas associated with topical drug delivery IP for the 3 decades up to 2018. Source: CAS content collection.

IP for conditions such as skin disease, acne, and of key changes over the past 3 decades. As psoriasis, although each of these fields has expe- with oral drug delivery systems, China has rienced some growth in the absolute number of rapidly expanded its patent output. Patents for patents filed. vaginal, ophthalmic, and transdermal drug Fig. 10.16 highlights the top 25 substances delivery systems are sharing a greater propor- with pharmacological activity indexed in topical tion of topical drug delivery IP. Notably, patents drug delivery patents for the 2 decades between describing drug delivery systems for inflamma- 1999 and 2018. tion and neoplasms have rapidly increased. The list reflects the diverse range of pharma- ceutical substances that are used for topical treat- ments, and includes chemotherapy agents, 5. Trends in parenteral drug delivery IP corticosteroids, vitamins, analgesics, and antiin- flammatories. By far the largest increase in citing Many therapeutic agents are unsuitable for patents was for endo-borneol, a monoterpene oral administration, and will either degrade un- alcohol that has been shown to assist in the trans- der the harsh pH conditions of the stomach, port of drugs through the skin [24] and to pass through the gastrointestinal tract without enhance bloodebrain barrier permeation [25]. being absorbed, or undergo metabolism in the Patents associated with the antibiotics genta- liver. Parenteral drug products, defined by the micin, ciprofloxacin, and erythromycin also United States Pharmacopoeia as “injections and experienced a large increase in the decades implanted drug products that are injected 1999e2008 and 2009e18, indicating growth in through the skin or other external boundary topical antibacterial treatments. tissue, or implanted within the body to allow Our analysis of the patent landscape for the direct administration of the active drug sub- topical drug delivery systems shows a number stance(s) into blood vessels, organs, tissues or 216 10. Trends in the intellectual property (IP) landscape of drug delivery systems

FIGURE 10.16 Top 25 pharmaceutical substances associated with topical drug delivery patents between 2009 and 2018 (blue; dark gray in print version). Data for the decade between 1999 and 2008 (gray) is also presented for comparison. Source: CAS content collection. lesions” [26], offer an alternative means of granted market approval over this period. The achieving appropriate bioavailability of trends in these delivery systems over the past therapeutics. 3 decades can be better understood by analyzing Over the past 30 years the range of parenteral the patent literature. delivery systems has expanded to include inno- vative technologies such as biodegradable im- 5.1 Overall trends in parenteral drug plants, intramuscular depot injections, and delivery IP colloidal drug carriers such as nanoparticles and liposomes [27]. Advanced parenteral formu- The rate of growth in IP for parenteral drug lations have emerged that can deliver targeted, delivery systems over the past 30 years can be sustained, and controlled drug delivery, over- largely divided into three periods, as shown in coming some of the complexities associated Fig. 10.17. Following modest expansion in the with administering drugs by injection or infu- late 1980s and early 1990s, the period sion [27]. Furthermore, advances in biotech- 1998e2008 saw a rapid increase in patent output. nology over the past 30 years have resulted in Since 2008, however, the rate of growth has pla- a wide range of novel biopharmaceuticals that teaued and even decreased, with the 3 years necessitate parenteral drug delivery, with following 2014 seeing falling patent output. several first-in-class biotechnology products 5. Trends in parenteral drug delivery IP 217

FIGURE 10.17 Volume of parenteral drug delivery system patents filed annually between 1988 and 2018. Source: CAS con- tent collection.

Fig. 10.18 shows the contribution to paren- delivery areas, the United States has dominated teral drug delivery IP from the most active coun- the field over the past 3 decades, maintaining a tries in this field over the past 3 decades. Our steady share of patents over this time frame. analysis shows that as seen in other drug Interestingly, US impact on parenteral drug

FIGURE 10.18 Country origin of parenteral drug delivery patents for the 3 decades up to 2018. Source: CAS content collection. 218 10. Trends in the intellectual property (IP) landscape of drug delivery systems delivery IP is greater than for oral and topical (dark gray in print version) in Fig. 10.19 present drug delivery technologies, highlighting the the most patented parenteral drug delivery tech- country’s strong R&D footprint in this space. nologies by volume for the 3 decades up to Notably, China does not appear to be having 2018, while the gray bars in Fig. 10.19 highlight as much impact on parenteral drug delivery sys- changes in the share of each category over the tems as for other routes of administration, with same time period. the country only entering the top five players This analysis reveals that the largest share of in the last decade. India, on the other hand, has parenteral drug delivery patents over this time rapidly emerged as a major source of parenteral period have been for intravenous injection tech- drug delivery patents. Between 1989 and 1998, nologies. The volume of patents for these technol- patents from India made up an extremely small ogies have increased largely in line with share of global IP in this area. In the last decade, expansion in the overall field over the past 3 - the country’s output was second only to the decades. The next largest categories of delivery United States, highlighting the country’s fast system, intramuscular and subcutaneous pace of growth within this space over the past injections, have both increased their share of the 20 years. injectables IP space since the decade 1989e98, As with other types of drug delivery systems, while intraperitoneal injections have experienced Japan’s impact on the IP landscape has waned, a substantial increase in IP. Intrathecal drug with the country contributing an increasingly delivery systems, which include injections to the small share of patents. The proportion of new pat- spinal canal, have also entered the list of leading ents from the United Kingdom and Italy has also parenteral technologies in the past 2 decades. fallen slightly, while Germany and France have It is worth noting that the share of patents kept pace with expansion in global patent output. described with the general term “pharmaceutical injections” has decreased over this time period. Given the growth in the share of intravenous, 5.2 Trends in parenteral drug delivery subcutaneous, intramuscular, intraperitoneal, technologies and intrathecal injections, this change perhaps reflects the more precise indexing of patented Deeper analysis of the patent literature high- technologies, rather than a decrease in the share lights several interesting findings. The blue lines of injectable technologies as a whole.

FIGURE 10.19 Proportion of patents (gray bars) and patent volume growth (blue line; dark gray in print version) of leading categories of parenteral drug delivery system by patent volume for the 3 decades up to 2018. Source: CAS content collection. 5. Trends in parenteral drug delivery IP 219

Controlled-release formulations have main- Technologies designed for anticancer applica- tained a steady share of the evolving parenteral tions have experienced significant growth, in IP landscape. Parenteral suspensions are tradi- line with trends observed in the oral and topical tionally favored for this role [27], however, in drug delivery space. Breast, prostate, and lung recent years various colloidal systems have cancer applications make up the largest propor- been engineered for the controlled release of tion of these. parenterally administered drugs, with these sys- One of the most notable trends identified in tems capable of supporting both water-soluble these plots is the rapid fall in new patents and insoluble drugs. describing parenteral drug delivery systems for AIDS since the decade 1989e98. Similarly, pat- ents coindexed with terms for infections, multi- 5.3 Trends in disease areas and ple sclerosis, and autoimmune diseases also pharmaceutical substances take a smaller share of the IP landscape in the last decade than 3 decades ago. Rheumatoid Our analysis of parenteral drug delivery arthritis and inflammation, on the other hand, space suggests the leading therapeutic applica- have both seen their share of IP increase. tions for infusion and injectable technologies Fig. 10.21 lists the top 25 pharmaceutically have evolved over the past 30 years. Such change active substances indexed in parenteral drug de- is perhaps not unexpected, given the rapid livery patents for the 2 decades between 1999 advances in medical science, particularly in the and 2018. As with the leading substances fi biotechnology eld. Fig. 10.20 highlights described in oral drug delivery system patents, the most commonly coindexed disease terms the majority of the substances featured in this associated with parenteral drug delivery patents list are chemotherapy drugs. Indeed, the top for the past 3 decades.

FIGURE 10.20 Proportion of patents (gray bars) and patent volume growth (blue line; dark gray in print version) for major disease areas associated with parenteral drug delivery IP for the 3 decades up to 2018. Source: CAS content collection. 220 10. Trends in the intellectual property (IP) landscape of drug delivery systems

FIGURE 10.21 Top 25 pharmaceutical substances associated with parenteral drug delivery patents between 2009 and 2018 (blue; dark gray in print version). Data for the decade between 1999 and 2008 (gray) is also presented for comparison. Source: CAS content collection. three indexed substances, paclitaxel, cisplatin, The patent landscape for parenteral drug and doxorubicin, are the same anticancer agents delivery systems shows a number of key changes that topped the equivalent list for oral drug over the past 3 decades. Despite a rapid increase delivery IP. in the rate of patent growth between 1998 and The pharmaceutical substances that have 2008, growth in this field has plateaued and experienced the largest increase in citations has begun to slow in recent years. While China between the decades spanning 1999e2008 and has not yet expanded its patent output in this 2009e18 are oxaliplatin and gemcitabine, anti- field to the same extent as for oral and topical cancer agents described previously in Chapter drug delivery, patent output from India has 3 as receiving additional attention as orally grown significantly since the turn of the 21st administered drugs, as well as trastuzumab, a century. chemotherapy drug used to treat breast cancer and adenocarcinomas of the stomach and gastro- esophageal junction [28]. Tamoxifen, another 6. Emerging trends in drug delivery IP anticancer drug that is used to treat breast can- cer, has also seen a notable increase in patent ref- Our overview of patents for drug delivery erences [29]. systems highlights an evolving landscape, in 6. Emerging trends in drug delivery IP 221 which technological advances can rapidly open systems has accelerated rapidly, as shown in up new opportunities for innovation. Many of Fig. 10.22, with the most significant growth the trends observed in the previous sections seen in their application for topical and oral have taken place over relatively short time drug delivery. spans; significant changes in this market can Fig. 10.23 highlights the specific nanotech- occur within the space of a decade or less. With nology areas that are most prominent in the IP IP protection essential for successful commer- landscape over the last 20 years, with micelles, cialization of therapeutic products, maintaining nanoemulsions, and nanocapsules being most awareness of emerging trends in the landscape prominent. Solid lipid nanoparticles containing is critical to guiding development and strategy a lipophilic core are now well established for decisions. In this section, we consider four spe- the controlled release of pharmaceutical agents cific areas of drug delivery that have demon- [33]. However, recent years have seen expansion strated rapid growth in recent years and are in nanoscale delivery systems that release drug expected to be key thrusts of innovation over in response to stimuli, including pH, tempera- the next decade: targeted drug delivery systems, ture, enzymes, and light [34e37]. nanoscale drug delivery systems, immunoconju- Immunoconjugates, which consist of anti- gates, and formulations and dosage forms. bodies connected by a chemical linker to a Targeted drug delivery systems first emerged molecular “payload,” such as a cytotoxic drug during the late 1990s; however, it has only been compound or radioisotope, are another emerging in the last few years that IP for these technologies delivery technology that has experienced consid- has really begun to gain traction. These systems, erable IP growth in the last 5 years. Antibody- which are designed to focus the effects of thera- drug conjugates (ADCs) are one of the most peutic agents in a localized area, are commonly important families of immunoconjugates and used for oncology applications where mini- mizing systemic exposure to highly potent or toxic drugs that may cause severe adverse side effects is particularly important. A wide variety of liposome, nanoparticle, and polymeric micelle-based strategies involving covalent and noncovalent attachment of the drug to the carrier have been developed [30]. Polymer-drug sys- tems based on polyethylene glycol and N-(2- hydroxypropyl) methacrylamide, as well as superparamagnetic iron oxide nanoparticles and condensed magnetic clusters, have been the focus of intense research interest [31]. Nanoscale drug delivery systems are also a key innovation focus in drug delivery research currently. An increasingly important subset of nanoscale technologies are controlled-release drug delivery systems [32]. Patents in this area can be found as early as the mid-1990s, yet FIGURE 10.22 Growth rate of nanotechnology in each growth in this space remained relatively slow type of drug delivery system 1999e2018. Percentage within until the middle of the 2010s. Since then, IP for each bar indicates growth compared to the previous 5 years. controlled-release nanoscale drug delivery Source: CAS content collection. FIGURE 10.23 Detail of specific nanotechnology areas cited in drug delivery IP 1999e2018. Block size proportional to relative patent volume. Source: CAS content collection. 6. Emerging trends in drug delivery IP 223 have shown particular promise as anticancer considerable expansion. This growth may reflect agents. As of 2018, four ADCs have been the growing importance of injectables and infu- approved by the US Food and Drug Administra- sion technologies, where these technologies play tion, with around 60 in clinical trials [38]. a valuable role. However, other categories of Recent years have seen growth in patents for drug delivery system also have made significant all three components of ADC systems [39,40]. advances over this time. For example, intrave- In terms of antibodies, the development of nous injections, a subset of parenteral drug deliv- masked proproteins, conditionally active anti- ery systems, have come to occupy an increasingly bodies, and antibodies that target noninternaliz- important position in the last decade, while intra- ing antigens have generated particular interest. peritoneal injections have also emerged as one of Novel linker technologies employing various the leading categories of drug delivery IP in the conjugation and cleavage strategies, including last 5 years. those based on MTGase- and sortase-mediated Fig. 10.24 takes a more detailed look at the linkage have recently been reported, while pat- changes in delivery route and dosage form for ents for cytotoxic drug payloads such as benzo- the pharmaceutical, biotechnology, cosmetics diazepines, duocarmycins, and tubulysins have and personal care, and food and nutraceuticals also been published in the last 5 years. sectors over the past 15 years. Notably, some sec- A final area of increasing focus for innovation tors have experienced more change than others; in drug delivery over recent years is formulations the biotechnology sector in particular shows a and dosage forms. Novel formulations of existing good deal of variation in the leading drug deliv- drugs are continuously being developed to ery technologies. Indeed, a number of new improve drug stability, increase patient accep- dosage form trends have emerged within the tance and convenience, comply with regulatory biotechnology industry in recent years. Notable requirements, and/or enable more personalized entrants to the leading dosage form categories medicine approaches. In this way, the same active within the last decade include pastes and pow- ingredient is often now being made available in ders, while intraperitoneal injections have again multiple dosage forms or drug delivery systems emerged as key delivery route within the past simultaneously to serve different needs. We see 5 years. the impact of these strategies increasingly in the Innovations in dosage forms for pharmaceuti- drug delivery IP landscape with patents specif- cals are often linked to desired routes of delivery. ically focused on formulations and dosage forms. Many of the delivery routes that are currently As noted above, oral, topical, and parenteral preferred in the pharmaceutical field are those systems account for the majority of drug delivery that have dominated the IP space for the past IP over the past 3 decades. Oral administration 2 decades. However, as with the overall picture, routes are likely to continue to be preferred intravenous injections and intraperitoneal injec- because they are usually relatively less compli- tions have more recently emerged as important cated and widely accepted by patients. For this drug delivery routes. From our analysis, the reason, tablets and capsules continue to be the traditional dosage forms such as tablets, dominant dosage form of across the whole drug capsules, powders, solutions, and suspensions delivery IP landscape, while pharmaceutical are still the most widely patented drug delivery powders have been among the leading categories technologies. of formulation IP for many years. However, The cosmetics and food/nutraceuticals sec- within the last decade, two categories of formula- tors reveal little change in drug delivery IP in tions, suspensions and emulsions, have shown recent years, with topical drug delivery systems 224 10. Trends in the intellectual property (IP) landscape of drug delivery systems

FIGURE 10.24 Leading drug delivery routes and dosage forms by industry, between 2004 and 2018. Source: CAS content collection. continuing to dominate cosmetics IP, and oral interesting to note that oil-in-water emulsions drug delivery systems still leading within the have recently gained ground from powders as food/nutraceuticals sector. That said, it is one of the five leading dosage formulations. 6. Emerging trends in drug delivery IP 225

Changes in the IP landscape for formulations the leading focus of new patents. Recent growth and dosage forms are also driven by trends in in IP for analgesics has also been observed. desired pharmaceutical activity. Fig. 10.25 high- In the cosmetics and personal care field, the lights emerging trends in pharmaceutical activ- last few years have seen a number of new cate- ity by oral, parenteral, and topical drug gories emerge. Antibacterial agents have delivery systems. emerged as a key focus of oral drug delivery sys- Within the pharmaceutical sector, the leading tem patents, while hair growth stimulants have applications for each of these drug delivery sys- become a focus of parenteral systems and anti- tems has largely remained the same over the past aging cosmetics have emerged as a focus of decade, with antiinflammatory, antitumor, and topical systems. Skin moisturizers have also antiviral agents, as well as vaccines, currently experienced rapid growth, entering the top five

FIGURE 10.25 Most common pharmaceutical activity by drug delivery system and sector, between 2004 and 2018. Source: CAS content collection. 226 10. Trends in the intellectual property (IP) landscape of drug delivery systems most patented cosmetics drug delivery technolo- grow rapidly over the coming years, it is impor- gies in the last 5 years. tant that researchers, business decision makers, Similarly, within the food and nutraceuticals and other IP stakeholders have a strategy to sector, the current leading drug delivery systems maintain awareness of the landscape on an on- have evolved. Antidiabetic agents have ree- going basis. Maintaining current awareness of merged as a key focus of oral drug delivery IP, the IP landscape is critical as it ensures that while antiobesity agents and nutrients have on-going research is fully informed by the become a dominant focus of parenteral systems work of others in the field and that those hoping within the past 5 years. A large proportion of to commercialize their discoveries invest topical drug delivery patents for antiaging cos- resources and time focused on novel work that metics have also been received in recent years. can be patented and does not infringe on others’ In addition to the new types of drugs being already disclosed innovations. There is nothing developed, two other key drivers for emerging worse than finally achieving a desired research innovation in drug delivery are new develop- outcome after extensive investment, only to ments in related technology fields and advance- find out that someone else had accomplished it ments in our underlying understanding of previously and thus it cannot be patented, specific biomolecular processes. For example, in commercialized, or marketed. In addition to recent years advances in polymer science and keeping one up to date on the state of the art in nanomaterials have had direct impact on IP in the field and avoiding unnecessary investments, the transdermal drug delivery space. Similarly, IP monitoring also helps researchers maintain changes in our understanding of metabolism awareness of their competitors’ strategy and and degradation of certain drug classes, identifi- can help them identify emerging opportunity cation of new drug targets, and new insights areas to support their forward-looking plans. regarding bioavailability have spurred new IP As all published works can be considered dis- in the areas of target and controlled-release closures of novel ideas from the standpoint of drug delivery. In addition, recent advances are intellectual property rights, it is important that also blurring the lines between traditional sys- any IP monitoring strategy not only consider tem definitions in drug delivery. One extreme newly published patents and patent applications example is a recently reported dosage form the but also include sources such as journal articles, size of a pea that once in the stomach, mechani- conference presentations, Internet content, etc. cally injects insulin into the stomach lining tis- How comprehensive one’s monitoring strategy sues. So here we have a chemically sensitive needs to be is highly dependent on many factors, peptide hormone that needs to be administered including the importance of commercializing the in an injectable form, loaded into an oral dosage work, the amount of investment being made in form that subsequently injects the drug [41].We the research, and the current stage of develop- envision that it is these breakthroughs as much ment. The general rule is that the more time as advances in the active ingredients themselves and money that is on the line, and the closer that are likely to influence future IP blooms in one is to filing a patent, the more diligent one drug delivery systems. must be in ensuring awareness of new develop- ments in the technology space of interest. All researchers should strive to maintain 7. IP monitoring strategies awareness of the work being done in their field, and there is a wide variety of open-source and As the volume of published research in the commercial tools that can be helpful in that drug delivery space is expected to continue to effort. Most global government patent offices 8. Conclusion 227 offer free search tools on their websites that pro- is complex and often not found by basic search vide basic capabilities such as a lookup by inven- engines built primarily for common words. For tor, keyword, patent number, or publication those in academic settings, the university library date. Common Internet search engines, such as can be a wonderful resource to help you find the Google, also allow you to search or set up alerts best solutions. However, those working on com- on topics, authors, or organizations of interest. mercial research will usually need to license These resources are free and convenient to ac- solutions directly from the producers. cess, making them a helpful starting point for At key decision points in the research process, monitoring the published literature. However, when significant investment decisions must be be aware that these resources have significant made, and when preparing a patent application, limitations in their coverage and search capabil- it is important to seek guidance from profes- ities. Thus, it is likely that in the drug delivery sionals to ensure you have a full view of the land- space, they will provide only a small portion of scape and maximize the value of the intellectual the publications relevant to your interests, property associated with your discovery. Two creating a significant awareness gap that could key professionals that are critical are a patent at- be a major risk for anyone hoping to commer- torney and a professional IP searcher. In aca- cialize their research work. demic settings, a technology transfer office may When significant monetary and/or legal con- provide these resources, while commercial orga- sequences are attached to the possibility of nizations either employ or contract with these missing one or more publications, a comprehen- professionals. Regardless of the path to get there, sive strategy is required to ensure you have a it is critical that all inventors make sure a thor- complete picture of the landscape to advise ough prior art search has been completed by a decision-making. There are a wide range of com- knowledgeable patent searcher before a patent mercial tools and services available to support is drafted and filed. This key step expedites pat- these needs. Key criteria to consider when select- ent prosecution, ensures application fees are not ing an information solution are the breadth of wasted, and allows an attorney to draft the broad- publications and patent offices covered, with est defensible claims related to your invention. particular focus on coverage in your field of in- Though monitoring the research landscape terest; the currency of the information provided may seem a peripheral or lower-priority task to (i.e., how long from the publication date does it a busy researcher, it is in reality a critical under- take for publications to be accessible?); and the taking that can save wasted time and resources. comprehensiveness and precision of the search Thus, it is highly encouraged for all those work- capabilities. In the drug delivery field, it is ing in the rapidly evolving field of drug delivery important to consider coverage of key journals to carefully consider key aspects of IP strategy and conferences, as well as patent offices and select high-quality resources to support you relevant to your commercialization interests. in staying up to date not just in the drug delivery Premium information can be obtained via infor- field itself but also in the related technology areas mation solutions such as SciFinder and STNext, and underlying biomolecular insights that will which feature CAS databases. These platforms impact drug delivery advances of the future. support precise searches using chemical struc- tures and names as well as biosequences, in addition to keywords. In drug discovery, intel- 8. Conclusion lectually indexed content can also be very impor- tant to achieving efficient, comprehensive Our review of drug delivery system patents searches, as chemical and scientific terminology published over the past 3 decades highlights an 228 10. Trends in the intellectual property (IP) landscape of drug delivery systems expanding and evolving landscape. The IP space will evolve. Scientific researchers, patent profes- for drug delivery systems has experienced sus- sionals, and business leaders around the world tained growth, particularly in the last 20 years, across commercial, academic, and government fueled in large part by rapid expansion from sectors rely on our solutions and services to China and India and continued output from US advise discovery and strategy. Leverage our and European markets. Patents for oral drug unparalleled content, specialized technology, delivery systems have come to dominate the and unmatched human expertise to customize field, due to the importance of these technologies solutions that will give your organization an within the pharmaceutical and biotechnology information advantage. With more than 110 sectors, while topical drug delivery systems years of experience, no one knows more about continue to be the focus of the cosmetics market. scientific information than CAS. Learn more at Drug delivery systems for chemotherapy appli- www.cas.org. cations are taking an increasing share of the IP CAS e Where Science and Strategy Converge. space, a trend further supported by the promi- nence of anticancer agents in lists of the most commonly cited pharmaceutical substances in References patents. Moreover, recent trends reveal the [1] Bishop TH. 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Overview of milling drug delivery with great excitement. techniques for improving the solubility of poorly water-soluble drugs. Asian J Pharm Sci 2015;10: 255e74. 8.1 About CAS [8] Davis MT, Egan DP, Kuhs M, Albadarin AB, Griffin CS, Collins JA, Walker GM. Amorphous solid CAS, a division of the American Chemical So- dispersions of BCS class II drugs: a rational approach ciety, partners with R&D organizations globally to solvent and polymer selection. Chem Eng Res Des fi 2016;110:192e9. to provide actionable scienti c insights that help [9] Sareen S, Mathew G, Joseph L. Improvement in solubi- them plan, innovate, protect their innovations, lity of poor water-soluble drugs by solid dispersion. Int and predict how new markets and opportunities J Pharm Investig 2012;2:12e7. References 229

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[37] Liu J, Detrembleur C, De Pauw-Gillet M-C, Mornet S, [39] Kennedy S. Antibody Drug Conjugates: The Patent Jerome^ C, Duguet E. Gold nanorods coated with Mes- Landscape for a New Class of Cancer Treatment. Dil- oporous Silica shell as drug delivery system for remote worth IP; 2018. https://www.dilworthip.com/ near infrared light-activated release and potential antibody-drug-conjugates-patent-landscape-new- phototherapy. Small 2015;11. https://doi.org/ class-cancer-treatment. 10.1002/smll.201402145. [40] Storz U. Antibody-drug conjugates: intellectual prop- [38] Chalouni C, Doll S. Fate of antibody-drug conjugates in erty considerations. mAbs 2015;7:989e1009. cancer cells. J Exp Clin Cancer Res 2018;37. https:// [41] Abramson A, et al. An ingestible self-orienting system doi.org/10.1186/s13046-017-0667-1. for oral delivery of macromolecules. Science 2018. https://doi.org/10.1126/science.aau2277. Index

Note: ‘Page numbers followed by “f” indicate figures and “t” indicates tables’.

A migraine, 179 Fourth generation, 95e96, 96f Adsorption, distribution, myasthenia gravis, 181 Freeze drying, 106e107 metabolism, and elimination Parkinson disease (PD), 178 G (ADME) studies, 121e122, 128 schizophrenia, 179e180 Alzheimer disease, 177e178 stroke, 182 Generally Recognized as Safe status (GRAS), 2e3 Antigen-presenting cells (APCs), 136 symptomatic drug, 178 targeted drug, 178 Genotoxicity, 126 Autism, 180 e Brain toxicity, 127 Glass solution, 97 98 B Gnetum gnemon L. (Gg),19e20, 20f BCS. See Biopharmaceutics C Gold (Au) nanoparticles, 82e83 e classification system (BCS) Cellular uptake capability, 29 32 H Bioactive compounds Chemo-angiogenic therapy, 62 biochemical barriers, 21e22 Chemo-enzyme prodrug therapy, High pressure homogenizers (HPH), 2e3 biological barriers, 21e22 64e65 complexity, 20e21 Chemo-immunotherapy, 61e62 High-throughput screening e e Chemo-photothermal therapy, 63 (HTS), 54 nanocarriers, 22 29, 23t 25t e biokinetic profile, 33, 34te35t Chemo-radiotherapy, 64 Hot melt extrusion (HME), 112 114, 113f cellular uptake capability, 29e32 Copper (Cu) nanoparticles, 85 challenges, 33e37, 36t Coprecipitation (CP) technique, Hydrophilicity/hydrophobicity herbal nanoparticles for breast 108e109 properties, 55 cancer, 34e37 Critical micelle concentration Hydrophilic-lipophilic balance (HLB) values, 8 lipid-based nanocarrier systems, (CMC), 47 22e29 Cryogenic processing, 107 I e safe-by-design bioactive-loaded Curcuma,17 18 Immune responses after nanocarrier D vaccination, 136e137 system, 29, 31t Immunotoxicity, 126 fi Dasatinib (DAS), 55 surface modi cation, 32 Inorganic nanoparticles, 80e86, Dendrimers, 192e193, 193f physical barriers, 21, 21f e 187, 190t e Dermal toxicity, 126 127 therapeutic agents, 17 20 gold (Au) nanoparticles, 82e83 e Differential scanning calorimetry Curcuma,17 18 metal oxide nanoparticles, e (DSC), 7 Gnetum gnemon L. (Gg),19 20, 20f 83e84 Drug-loading methods, 49 Physalis angulata L., 20, 20f silver (Ag) nanoparticles, 80e82 Silybum marianum (L.), 19, 19f E Intellectual property (IP) landscape fi e Zingiber of cinale,18 19 Electrostatic spinning, 109e111, 110f emerging trends, 220e226, 221f fi e Biokinetic pro le, 33, 34t 35t Encapsulation parameters, 5 industry trends, 203e205, 204f fi Biopharmaceutics classi cation Enzyme inhibitor, 184 monitoring strategies, 226e227 e system (BCS), 152 153 Ethosomes, 76e78 oral drug delivery trends, Botulism, 142 Eutectic mixtures, 98, 98f 206e212 e Brain disorders, 176t, 177 182 disease areas, 210e212, 210f e F Alzheimer disease, 177 178 pharmaceutical substances, e autism, 180 Fifth generation, 96 97 210e212, 210f e e cerebral palsy, 180 181 First generation, 92 93 technologies, 208e210, 209f meningitis, 181 Fluid-bed coating, 111 overview, 202e206

231 232 Index

Intellectual property (IP) landscape Liver toxicity, 127 Nanoparticulate systems, wound (Continued) Lyophilization, 106e107 healing parenteral drug delivery trends, M copper (Cu) nanoparticles, 85 215e220 ethosomes, 76e78 e disease areas, 219e220 Melt agglomeration, 114 115, 115f inorganic nanoparticles, 80e86 e overall trends, 216e218 Melting method, 111 116 gold (Au) nanoparticles, 82e83 pharmaceutical substances, Melting-solvent method, 116 metal oxide nanoparticles, 83e84 219e220 Metal and nonmetal inorganic silver (Ag) nanoparticles, 80e82 technologies, 218e219, 218f NPs, 142 lipid-based nanoparticles, 74e79 e technology trends, 205e206, 206f Metallic NPs (MeNPs), 146 147 liposomes, 74, 75te76t e topical drug delivery trends, Metal oxide nanoparticles, 83 84 nanoemulsions, 78e79 212e215 cerium oxide nanoparticles, 84 nanostructured lipid carriers, 79 e disease areas, 214e215 iron oxide nanoparticles, 83 84 penetration enhancer vesicles overall trends, 212 titanium dioxide nanoparticles, 84 (PEVs), 76, 77t pharmaceutical substances, zinc oxide NPs (ZnONPs), 83 selenium nanoparticles, 86 214e215 Molecular mobility, 100 silicon nanoparticles, 85e86 e technologies, 212e214 Multidrug resistance (MDR), 49 54, solid lipid nanoparticles, 79 Intranasal route, 176e177 50f stages, 74e79, 75f In vitro assay interferences, 125e126 chemotherapeutics codelivery, transfersomes, 78 e In vitro toxicity, 124e125 49 54, 50f Nanotoxicity chemosensitizers, 51e53, 52t e K absorption, 122 124 downregulating gene agents, adsorption, distribution, e e KinetiSol technique, 115 116 53 54 metabolism, and elimination L N (ADME) studies, 121e122, 128 e e brain toxicity, 127 Lipid-based nanocarrier systems, Nanocarriers, 22 29, 23t 25t e 22e29 biokinetic profile, 33, 34te35t dermal toxicity, 126 127 e e drug delivery systems (DDSs), 121 liposomes, 24 26 cellular uptake capability, 29 32 e nanoemulsions, 26e27, 28t challenges, 33e37, 36t drug molecules, 122 123 solid lipid nanoparticles (SLNs), herbal nanoparticles for breast engineered nanomaterials, 123e124 28e29, 30t cancer, 34e37 Lipid nanocarriers, oral drug delivery lipid-based nanocarrier systems, genotoxicity, 126 biopharmaceutics classification 22e29 immunotoxicity, 126 system (BCS), 152e153 safe-by-design bioactive-loaded liver toxicity, 127 nanomedicines, 122e124 classification and composition, nanocarrier system, 29, 31t 153e155, 154t surface modification, 32 nanotechnology products regulation, future perspectives, 168 Nanocytotoxicity, 146e147 129 gastrointestinal tract (GIT) Nanoemulsions, 78e79, 190e192, nephrotoxicity, 127 organ-on-chip systems, 128 irritation, 155 192t mechanisms with cell membranes, Nanomedicines, 122e124 perspectives, 129 166e168 Nanoparticle-based vaccines pharmacokinetics of nanoparticles, 122f nonvesicular lipid nanocarriers, antigen-presenting cells (APCs), 136 e risk assessment process, 124 156 164 companies and clinical trials, e permeability enhancement 143e146, 146t theranostics, 122 124 e in vitro assay interferences, strategies, 164 166, 165f immune responses after vaccination, e e 125 126 safety, 155 136 137 e solubility enhancement strategies, liposomes, 137e141 in vitro toxicity, 124 125 e in vivo of human response, 164 166, 165f metal and nonmetal inorganic NPs, e types, 155e164 142 127 128 whole-animal models, 128e129 vesicular lipid nanocarriers, 155e156 metallic NPs (MeNPs), 146e147 Lipid nanoparticles, 1e2, 74e79, nanocytotoxicity, 146e147 Nasal and pulmonary delivery, 11 186e187, 186f polymeric NPs, 142e143 Nasal cavity anatomy, 182 Liposomes, 74, 75te76t, 137e141, vaccine types, 136t, 137, 138te139t Nephrotoxicity, 127 Neuronal pathway, 182e183 187e190, 191f, 191t virus-like particles (VLPs), 141e142 Index 233

Nonvesicular lipid nanocarriers, liposomes, 187e190, 191f, 191t pH-sensitive codelivery systems, 156e164 nanoemulsions, 190e192, 57e58 lipid nanoparticles, 162 192t redox-sensitive codelivery microemulsions, 156e157 polymeric nanoparticles, 184e185 systems, 58e59 nanoemulsions, 157 O targeted codelivery, 60 nanostructured lipid carriers transactivator of transcription (NLCs), 163 Ocular delivery, 9 (TAT), 60 self-emulsifying drug delivery Olfactory nerve pathway, 183 Polymeric nanoparticles, 142e143, systems (SEDDSs), 158 Organ-on-chip systems, 128 184e185 self-microemulsifying drug delivery P e R systems (SMEDDSs), 158 159 Parenteral administration, 10e11 self-nanoemulsifying drug-delivery Reconstructed human epidermis Parkinson disease (PD), 178 (RhE) technology, 8e9 systems (SNEDDSs), 159 PEGSS-C18 copolymer, 59 smartLipids particles, 164 Redox-sensitive codelivery systems, Penetration enhancer vesicles (PEVs), e solid lipid nanoparticles 58 59 76, 77t Reticuloendothelial system (RES), 47 (SLNs), 79, 162 e Permeation enhancer, 183 184 Risk assessment process, 124 solid self-emulsifying drug delivery Pharmacokinetics of nanoparticles, systems (S-SEDDSs), 160 47f S Nose-to-brain drug delivery PH-sensitive codelivery systems, Safe-by-design bioactive-loaded brain disorders, 176t, 177e182 e e 57 58 nanocarrier system, 29, 31t Alzheimer disease, 177 178 Physalis angulata L., 20, 20f Second generation, 93e95 autism, 180 Polymeric nanomicelles Selenium nanoparticles, 86 cerebral palsy, 180e181 application, 60e65 Silicon nanoparticles, 85e86 meningitis, 181 characterization, 46e49 Silver (Ag) nanoparticles, 80e82 migraine, 179 techniques, 48e49 Silybum marianum (L.), 19, 19f myasthenia gravis, 181 chemo-angiogenic therapy, 62 Solid dispersions (SD) Parkinson disease (PD), 178 e chemo-enzyme prodrug therapy, applications, 99 schizophrenia, 179 180 64e65 challenges, 99e101 stroke, 182 chemo-immunotherapy, 61e62 classification, 92e97, 92fe93f symptomatic drug, 178 chemo-photothermal therapy, 63 drug release mechanism, 98e99 targeted drug, 178 chemo-radiotherapy, 64 fifth generation, 96e97 central nervous system (CNS) acting, fi e e chemotherapeutics codelivery, rst generation, 92 93 175 177 49e56 fourth generation, 95e96, 96f clinical trials, 194 dasatinib (DAS), 55 melting method, 101, 111e116 intranasal route, 176e177 e e Dox, 54 55 hot melt extrusion (HME), nasal absorption strategies, 183 184 high-throughput screening (HTS), 112e114, 113f enzyme inhibitor, 184 54 KinetiSol technique, 115e116 permeation enhancer, 183e184 hydrophilicity/hydrophobicity melt agglomeration, 114e115, 115f nasal cavity anatomy, 182 properties, 55 melting-solvent method, 116 nose-to-brain drug transport mitigate side effects, 56 melt-solvent methods, 102 pathways, 182e183 multidrug resistance (MDR), molecular mobility, 100 neuronal pathway, 182e183 49e54, 50f second generation, 93e95 olfactory nerve pathway, 183 critical micelle concentration solvent evaporation method, trigeminal sensory nerve (CMC), 47 102e111 pathway, 183 drug-loading methods, 49 coprecipitation (CP) technique, vascular pathway, 183 micelles, 46e49 108e109 novel drug delivery approaches, e e preparation methods, 47 48 cryogenic processing, 107 184 193 principles, 46e49 electrostatic spinning, 109e111, dendrimers, 192e193, 193f reticuloendothelial system (RES), 47 110f inorganic nanoparticles, stimuli-responsive codelivery, fluid-bed coating, 111 187, 190t e e e 56 59, 58t freeze drying, 106 107 lipidic nanoparticles, 186 187, PEGSS-C18 copolymer, 59 lyophilization, 106e107 186f 234 Index

Solid dispersions (SD) (Continued) lipid nanoparticles, 1e2 Spray drying, 103e106, 104fe105f, spray drying, 103e106, 104fe105f, microemulsion, 2e3 105t 105t nanotoxicology, 3 Stability of formulations, 7e8 supercritical fluid (SCF) physicochemical properties, 5e8 Supercritical fluid (SCF) technology, technology, 107e108 differential scanning calorimetry 107e108 vacuum drying, 106 (DSC), 7 Surface modification, 32 solvent method, 101 encapsulation parameters, 5 T structure-based classification, particle morphology, 7 e 97e98 particle size, 6 Theranostics, 122 124 e eutectic mixtures, 98, 98f release profile, 7e8 Third generation, 95 96 fi e glass solution, 97e98 stability of formulations, 7e8 Toxicity pro ling, 3 5 solid solutions, 97, 97f zeta potential, 6 Transactivator of transcription techniques, 101e116 preclinical toxicological studies, 3 (TAT), 60 third generation, 95e96 solidified emulsion technologies, Transfersomes, 78 administration routes, 8e11 2e3 Trigeminal sensory nerve pathway, drug bioavailability, 8e11 toxicity profiling, 3e5 183 nasal and pulmonary Solid solutions, 97, 97f V e delivery, 11 Solvent evaporation method, 102 111 Vacuum drying, 106 ocular delivery, 9 coprecipitation (CP) technique, e e Vascular pathway, 183 oral administration, 9 10 108 109 Virus-like particles (VLPs), 141e142 parenteral administration, 10e11 cryogenic processing, 107 reconstructed human epidermis electrostatic spinning, 109e111, 110f W (RhE) technology, 8e9 fluid-bed coating, 111 Whole-animal models, 128e129 topical and dermal routes, 8e9 freeze drying, 106e107 Wound healing, 73e74, 75t, 76t, 77t, drug delivery systems, 1e2 lyophilization, 106e107 78e86 fi e e e emulsi ers and polymers, 1 2 spray drying, 103 106, 104f 105f, Z high pressure homogenizers (HPH), 105t 2e3 supercritical fluid (SCF) technology, Zeta potential, 6 fi e hydrophilic-lipophilic balance (HLB) 107e108 Zingiber of cinale,18 19 values, 8 vacuum drying, 106