Toxicity evaluation of poly(anhydride) nanoparticles as carriers for oral administration of drugs
Patricia Ojer Ojer
TESIS DOCTORAL
Pamplona, octubre 2013
TESIS DOCTORAL
Facultad de Farmacia Departamento de Farmacia y Tecnología Farmacéutica Departamento de Farmacología y Toxicología
Toxicity evaluation of poly(anhydride) nanoparticles as carriers for oral administration of drugs
Trabajo presentado por Dña.Patricia Ojer Ojer para obtener el Grado de Doctor
Fdo. Patricia Ojer Ojer
Pamplona, octubre 2013
El presente trabajo, titulado “Toxicity evaluation of poly(anhydride) nanoparticles as carriers for oral administration of drugs”, presentado por DÑA. PATRICIA OJER OJER para optar al grado de Doctor por la Universidad de Navarra en el área de tecnología Farmacéutica y Toxicología, ha sido realizado bajo nuestra dirección en los Departamentos de Farmacia y Tecnología Farmacéutica y de Farmacología y Toxicología de la Universidad de Navarra. Estimamos que puede ser presentado al tribunal que lo ha de juzgar.
Y para que así conste, firman la presente:
Fdo.: Dr. Juan M. Irache Garreta Fdo.: Dra. Adela López de Cerain Salsamendi
Pamplona, octubre 2013
Esta tesis doctoral se ha llevado a cabo gracias a las ayudas predoctorales de Formación (convocatoria 2007) y de movilidad internacional (convocatoria 2010) dentro del plan de Formación y de I+D del departamento de educación del Gobierno de Navarra y a la ayuda para la formación del personal investigador de la Asociación de Amigos de la Universidad de Navarra.
Las investigaciones realizadas en el presente trabajo se han desarrollado dentro del proyecto CAN “Nanotecnología y medicamentos” (REF 10828).
A mis padres, Inma y Vicente, y a mi hermano, Daniel.
“Lo esencial es invisible para los ojos”
Antoine de Saint-Exupéry
AGRADECIMIENTOS Deseo expresar mi agradecimiento a todas las personas e instituciones que durante estos últimos años han contribuido a que la presente tesis doctoral sea una realidad.
En primer lugar, quisiera agradecer especialmente a mis directores, el Dr. Juan Manuel Irache y la
Dra. Adela López de Cerain, la oportunidad de realizar esta tesis doctoral. Gracias por su inestimable labor de dirección, su estímulo para seguir creciendo intelectualmente y sobre todo, por la confianza depositada en mí y en mi trabajo.
Gracias a la Universidad de Navarra y en especial a los investigadores, profesores y personal de los
Departamentos de Farmacia y Tecnología Farmacéutica y de Farmacología y Toxicología, gracias por la ayuda prestada cuando la he necesitado.
A la asociación de Amigos y el Departamento de Educación del Gobierno de Navarra, por la aportación económica recibida a través de sus programas de ayudas para la realización de tesis doctorales y estancias en el extranjero.
I am grateful to the Department of Pharmaceutical Technology and Biopharmaceutics of the
University of Vienna, in particular to Prof. Franz Gabor and Prof. Michael Wirth for giving me the opportunity to join their group. Thanks to those people who I met in Vienna, and especially to Lukas
Neutsch for your friendliness and your “incubation times”.
A mis compañeros y amigos de los laboratorios de Galénica y Toxicología, ha sido un placer poder compartir con todos vosotros estos años, no sólo el trabajo dentro del laboratorio sino también los grandes momentos vividos fuera.
A Hugo. Muchísimas gracias por toda tu ayuda en estos años, porque no sólo te has limitado a realizar tu trabajo sino que además lo has hecho mucho más divertido, aunque la radio ayudaba bastante. Gracias por quedarte a trabajar horas que no te correspondían y por venir los fines de semana.
A Irene Esparza. Gracias por tu apoyo incondicional, tu sabiduría, tu infinita paciencia pero sobre todo gracias por tu amistad, siempre dispuesta a ayudar y a escuchar aunque para ello tuvieses que sacar al día 25 horas.
A todas las personas que durante estos años han formado parte de mi vida, besonders Camillo, well er an mich mehr geglaubt hat als ich selbst.
Todo esto no hubiera sido posible sin el cariño de mi familia, y en especial de mis padres. Este es también vuestro premio.
TABLE OF CONTENTS
ABBREVIATIONS...... 17
CHAPTER 1...... 21 GENERAL INTRODUCTION
CHAPTER 2 ...... 79 AIM AND OBJECTIVES
CHAPTER 3 ...... 83 EXPERIMENTAL DESIGN
CHAPTER 4 ...... 87 Spray-drying of poly(anhydride) nanoparticles for drug/antigen delivery
CHAPTER 5 ...... 113 Cytotoxicity and cell interaction studies of bioadhesive poly(anhydride) nanoparticles for oral antigen/drug delivery
CHAPTER 6 ...... 143 Toxicity studies of poly(anhydride) nanoparticles as carriers for oral drug delivery
CHAPTER 7 ...... 169 Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration
CHAPTER 8 ...... 195 GENERAL DISCUSSION
CHAPTER 9 ...... 207 CONCLUSIONS/CONCLUSIONES
15
ABREVIATIONS
ABREVIATIONS
%ID/g: Percentage of injected dose per gram 99mTc: 99mTechnetium ABI-007: Abraxane® ADA: Adenosine Deaminase AFM: Atomic Force Microscopy ALT: Alanine Transaminase APCs: Antigen-Presenting Cells AST: Aspartate Transaminase ATO: Atovaquone ATP: Adenosine Thriphosphate BCS: Biopharmaceutics Classification System BSA: Bovine Serum Albumin BSA-FITC: Bovine Serum Albumin-Fluorescein Isothiocyanate Conjugate Bw: Body weight CdSe: Cadmium Selenide CdTe: Cadmium Telluride CLSM: Confocal Laser Scanning Microscopy CMC: Critical Micelle Concentration CNTs: Carbon Nanotubes CT: Computed Tomography CTB: Cholera Toxin B subunit CYP: Cytochrome P450 metabolizing enzyme system DCFA: 2′7′-dichlorofluorescein diacetate DISS: Digital Image System DMF: Dimethylformamide DOX: Doxorubicin DPBS: Dulbecco´s Phosphate Buffered Saline DTXL: Docetaxel DTNB: 5,5-dithio-bis-2-nitrobenzoic acid EASAC: European Academies Science Advisory Council ELSD: Evaporative Light Scattering Detection EMA: European Medicines Agency EPR: Enhanced Permeability and Retention ESR: Erythrocyte Sedimentation Rate FDA: Food and Drug Administration FP7: Seventh Framework Program GCSF: Granulocyte Colony-Stimulating Factor GIT: Gastrointestinal Tract HGB: Hemoglobin HCT: Hematocrit 17 ABREVIATIONS
HER2: Human Epidermal growth factor Receptor 2 HGF: Hepatocytes Growth Factor HIV: Human Immunodeficiency Virus HPCD: 2-hydroxipropyl-β-cyclodextrin HPLC: High Performance Liquid Chromatography HPMA: N-(2-Hydroxypropyl) methacrylamide HS: Hot Saline antigenic extract HSA: Human Serum Albumin i.m.: intramuscular i.r.: intravitreous i.t.: intrathecal i.v.: intravenous ILSI RF/RSI: International Life Sciences Institute Research Foundation/Risk Science Institute InAs: Indium Arsenide InP: Indium Phosphide IRIV: Immunopotentiating Reconstituted Influenza Virosome ISO: International Organization for Standardization ITLC: Instant Thin Layer Chromatography JRC: Joint Research Centre of the European Commission
LD50: Lethal Dose 50% LDH: Lactate dehydrogenase LPS: Lipopolysaccharide LUVs: Large Unilamellar Vesicles MCH: Mean corpuscular hemoglobin MCHC: Mean Corpuscular Hemoglobin Concentration MCV: Mean Corpuscular Volume MLVs: Mutilamellar Vesicles MPS: Mononuclear Phagocyte System MTS: 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt MTT: 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide MWCNT: Multi-Walled Carbon Nanotubes NCS: Nanotoxicological Classification System NCTR: National Center of Toxicological Research NOAEL: No Observed Adverse Effect Level NNI: National Nanotechnology Initiative NP: Conventional poly(anhydride) nanoparticles NP-HPCD: Nanoparticles containing 2-hydroxypropyl-β-cyclodextrin OECD: Organisation for Economic Co-operation and Development Omp: Outer membrane proteins OVA: Ovalbumin PACA: Poly(akyl cyanocrylates)
18 ABREVIATIONS
PAMAM: Poly(amidoamine) PBS: Phosphate buffered saline PCL: Poly(Ɛ-caprolactone) PCS: Photon Correlation Spectroscopy PDI: Polydispersity Index PEG: Polyethylene glycol PEG-NP: Pegylated poly(anhydride) nanoparticles P-gp: P-glycoprotein PLA: Poly(lactic acid) PLDsis: Phospholipidosis PLGA: Poly(lactide-co-glycolide) PLT: Platelet count QDs: Quantum Dots RBC: Red Blood Corpuscles count RES: Reticular Endothelial System s.c.: subcutaneous SD: Spray-Drying SEM: Scanning Electron Microscopy SLN: Solid Lipid Nanoparticles SPECT-CT: Single-Photon Emission Computed Tomography STM: Scanning Tunneling Microscope SUVs: Small Unilamellar Vesicles or oligolamellar SWCNT: Single-Walled Carbon Nanotubes TBA: Tiobarbituric acid Tg: Glass transition temperature TNP: Targeted polymeric nanoparticles TUNEL: Terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling UPLC: Ultra Performance Liquid Chromatography VEGF: Vascular Endothelial Growth Factor WBC: White Blood Corpuscles count WPN: Working Party on Nanotechnology WPNM: Working Party on Manufactured Nanomaterials WST-1: 4-[3- (4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate XTT: 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide
19
CHAPTER 1 GENERAL INTRODUCTION
CHAPTER 1: GENERAL INTRODUCTION
1. Definitions
1.1. What Is Nanotechnology?
Nanotechnology has emerged as one of the central technologies in the XXI (1). Much more than in any other scientific field, the development and progress have been made possible through the convergence of chemistry, biology, physics, mathematics, engineering and others (2-4). However, although it seems that nanotechnology has recently appeared, this technology exits since long time before. Already in the V century B.C., colloidal gold was known in Egypt or China and, for instance, applied in medicine (5). The conceptual origins of nanotechnology were introduced in 1959 by physicist Richard Feymann (Nobel Prize winner in Physics in 1965) who described a process in which scientists would be able to manipulate and control individual atoms and molecules in his famous lecture “There is plenty of room at the bottom” (6). Over a decade later, in 1974, Professor Norio Taniguchi was the first who coined the term “nanotechnology” (7). However, at this time it was a more philosophical or theoretical problem. It was not until 1981, when nanotechnology emerged in the sense as we understand it today. This fact was made possible in part by the development of tools that enabled the understanding of materials far down to the nanoscale as the scanning tunneling microscope (STM) or the atomic force microscopy (AFM) (8, 9). On the definition of nanotechnology there is confusion and disagreement among experts. Thus, the National Nanotechnology Initiative (NNI) defines nanotechnology as the science of materials and phenomena in the range of 1 to 100 nm in diameter and agencies, including the Food and Drug Administration (FDA), continue to use this definition. However, this definition not encompasses three important requirements to be taken into account defining nanotechnology. First, the size limitation to the 1-100 nm range would exclude numerous materials and devices, especially in the pharmaceutical area. Moreover, it is important to bear in mind the requirement that the nano- structure is man-made. Otherwise you would have to include every naturally formed biomolecules and material particle, in effect redefining much of chemistry and molecular biology as “nanotechnology”. Finally, the most important requirement to consider in the definition of nanotechnology is that the nano-structure has special properties that are exclusively due to its nanoscale proportions. Due to this fact, alternative definitions that are practical and unconstrained by any arbitrary size limitations have been presented by different authors and agencies. In this context, Bawa and collaborators proposed a definition of nanotechnology that deal with the three requirements aforementioned and that it may considers most adequate. They defined nanotechnology as “the design, characterization, production, and application of structures, devices, and systems by controlled manipulation of size and shape at the nanometer scale (atomic, molecular, and macromolecular scale) that produces structures, devices, and systems with at least one novel/superior characteristic or property” (10). However, it should be kept in mind that there is still no comprehensive definition that would be generally and internationally accepted and legally binding
23 CHAPTER 1: GENERAL INTRODUCTION
(11). Figure 1 shows a scale, within the size range of nanotechnology, which compares visually the size of various biological components. Cells DNA Red Blood Cells Virus Atom Glucose
1 Å 1 nm 10 nm 100 nm 1000 nm 10000 nm
Figure 1. Scale of size. How small is small?
1.2. What Is Nanomedicine?
The convergence of nanotechnology and medicine has resulted in the interdisciplinary field of nanomedicine. The neologism “nanomedicine” as the exploitation of nanotechnology for human health, is a relatively new field of science and technology that appeared in the scientific literature at the end of the last century. The promotion of interdisciplinary research and the discovery of colloidal mechanisms of drug delivery in the 1960´s and 1970´s led to the development of the earlier nanomedicines. In this regard, one of the pioneers was Professor Peter Speiser, whose strategy for the controlled release was the development of miniaturized delivery systems (12, 13). As occurs with nanotechnology, there is no unique globally accepted definition for nanomedicine. Thus, The Medical Standing Committee of the European Science Foundation (ESF) defined nanomedicine as “the science and technology of diagnosing, treating, and preventing disease and traumatic injury, of relieving pain, and of preserving and improving human health, using molecular tools and molecular knowledge of the human body”. At the same time, the National Institutes of Health (NIH) considers the nanomedicine as “an offshoot of nanotechnology, which refers to highly specific medical interventions at the molecular scale for curing disease or repairing damaged tissues, such as bone, muscle, or nerve” (14). Nanomedicine is one of the main and most fascinating fields of nanotechnology and is heavily supported by public policy and investments due to the potential beneficial properties that it provides compared with conventional medicine. Applications of nanomedicine include nanostructure scaffolds for tissue replacement, nanostructures that allow transport across biological barriers, remote control of nanoprobes, integrated implantable sensory nanoelectronic systems and multifunctional chemical structures for drug delivery and targeting of disease. However, the majority of current commercial applications of nanomedicine are geared towards drug delivery to enable modes of 24 CHAPTER 1: GENERAL INTRODUCTION
action, as well as better targeting and bioavailability of existing medical substances. In this regard, the use of nanoparticles has been postulated as one of the more promising approaches for medical purposes, mostly for drug delivery applications. However, despite the wide and ever growing exploitation of these new nanosystems, toxicity studies are very sporadic and limited, and toxicity data concerning the release and the accumulation of potentially noxious by-products, are as scarce as fragmentary. Moreover, there is a lack of predictive methods and approved protocols to study the toxicity of these nanodevices, in both for in vitro and in vivo models. So, to provide guidelines and protocols there is a need to deepen understanding of the nanomedicine in order to establish the critical points that may affect toxicity.
1.3. Nanoparticles in medicine
From the medical point of view, nanoparticles are defined as submicron sized colloidal particles, with sizes ranging from 1 to 1000 nm in diameter (15, 16). The increasing interest in nanomedicine is driven in large part by fast pace of innovation and emerging successes of nanoparticles which have resulted in the clinical approval of a number of nanomedicines (Table I.). Nanoparticles can be formulated from diverse materials with unique architectures to serve, among other applications, as diagnostic tools, imaging or as drug-delivery vehicles. Drug delivery is one of the most important applications due to the great benefits they provide in the pharmaceutical field. In this regard, nanoparticles may (i) protect the drug from degradation, (ii) enhance drug absorption by facilitating diffusion through biological barriers, (iii) modify pharmacokinetic and drug tissue distribution profile, and/or (iv) improve intracellular penetration and distribution (17). However, although the safety of the carrier and of its eventual by-products seems to be as important as that of the active drug, studies to assess nanoparticle toxicity are scarce.
25 CHAPTER 1: GENERAL INTRODUCTION
Table I. Clinically approved nanoparticle-based therapeutics. From reference (18).
Composition Trade name Company Indication Administration Liposomal platforms Liposomal amphotericin B Abelcet Enzon Fungal infections i.v. Fungal and protozoal Liposomal amphotericin B AmBisome Gilead Sciences i.v. infections Malignant lymphomatous Liposomal cytarabine DepoCyt SkyePharma i.t. meningitis HIV-related Kaposi`s Liposomal daunorubicin DaunoXome Gilead Sciences i.v. sarcoma Combination therapy with Liposomal doxorubicin Myocet Zeneus cyclophosphamide in i.v. metastatic breast cancer Liposomal IRIV vaccine Epaxal Berna Biotech Hepatitis A i.m. Liposomal IRIV vaccine Inflexal V Berna Biotech Influenza i.m. SkyePharma, Liposomal morphine DepoDur Postsurgical analgesia Epidural Endo Age-related macular Liposomal veterporfin Visudyne QLT, Novartis degeneration, pathologic i.v. myopia, ocular histoplasmosis HIV-related Kaposi`s Ortho Biotech, sarcoma, metastatic breast Liposome-PEG doxorubicin Doxil/Caelyx i.m. Shering-Plough cancer, metastatic ovarian cancer Micellular estradiol Estrasorb Novavax Menopausal therapy Topical Polymeric platforms L-Glutamic acid, L-alanine, TEVA L-lysine and L-tyrosine Copaxone Multiple sclerosis s.c. Pharmaceuticals copolymer Methoxy-PEG-poly(D,L- Genexol-PM Samyang Metastatic breast cancer i.v. lactide) Taxol Severe combined immunodeficiency disease PEG-adenosine deaminase Adagen Enzon i.m. associated with ADA deficiency OSI Age-related macular PEG-anti-VEGF aptamer Macugen i.r. Pharmaceuticals degeneration Nektar, PEG-α-interferon 2a Pegasys Hepatitis B, hepatitis C s.c. Hoffmann-La Neutropenia associated with PEG-GCSF Neulasta Amgen s.c. cancer chemotherapy PEG-HGF Somavert Nektar, Pfizer Acromegaly s.c. PEG-L-asparaginase Oncaspar Enzon Acute lymphoblastic leukemia i.v., i.m. Poly(allylamine Renagel Genzyme End-stage renal disease Oral hydrochloride) Other platforms Abraxis Albumin-bound paclitaxel Abraxane BioScience, Metastatic breast cancer i.v. AstraZeneca Nanocrystalline aprepitant Emend Elan, Merck Antiemetic Oral Nanocrystalline fenofibrate Tricor Elan, Abbott Anti-hyperlipidemic Oral Elan, Wyeth Nanocrystalline sirolimus Rapamune Immunosuppressant Oral Pharmaceuticals ADA: Adenosine Deaminase; GCSF: Granulocyte Colony-Stimulating Factor; HGF: Hepatocytes Growth Factor; HIV: Human Immunodeficiency Virus; i.m.: intramuscular; i.r.: intravitreous; IRIV: Immunopotentiating Reconstituted Influenza Virosome; i.t.: intrathecal; i.v.: intravenous; PEG: Polyethylene Glycol; s.c.: subcutaneous; VEGF: Vascular Endothelial Growth Factor.
26 CHAPTER 1: GENERAL INTRODUCTION
2. Overview of the different classes of nanomaterials/nanoparticles
A wide range of nanoparticles formulated from diverse materials has been designed to be applied in medicine. These materials, called nanomaterials can be chemically “simple” molecules such as metallic, ceramic or carbon nanoparticles (pure elements) or they can be more complex if composed from different types of materials, for example, core-shell nanoparticles. Other nanomaterials have organized structures, including systems such as nanoliposomes and nanoemulsions. Based on the materials of which nanocarriers are mainly comprised, these can be broadly classified as: inorganic and organic nanomaterials.
2.1. Inorganic nanomaterials
Inorganic nanomaterials form a large and very disperse group that include ceramic, metallic and magnetic nanoparticles, carbon nanomaterials and quantum dots (QDs) among others. In early studies, inorganic nanoparticles demonstrated great potential as nanocarriers of therapeutic agents, vaccines and imaging agents. However, their clinical application is limited by concerns over toxicity, lack of biodegradability and persistent tissue accumulation. Therefore, relatively few inorganic nanoparticles have progressed to clinical application. The most used inorganic nanomaterials in nanomedicine, particularly as drug delivery systems, are explained in detail below.
2.1.1. Ceramic nanoparticles Ceramic nanoparticles are inorganic systems with porous characteristics that have emerged as drug vehicles more than ten years ago (19). These vehicles are biocompatible ceramic nanoparticles such as silica, titania and alumina. Ceramic nanoparticles can be used for drug delivery, especially biomacromolecular therapeutics because of their ultra-low size (less than 50 nm) and porous nature. Additionally, they protect the adsorbed particles against denaturation induced by extreme pH and temperature (20). However, one of the main concerns is that these particles are non-biodegradable, as they can accumulate in the body, thus causing undesirable effects (21, 22).
2.1.2. Carbon nanomaterials Carbon nanomaterials are composed of a distinct molecular form of carbon atoms that give them unusual thermal, mechanical and electronical properties (23). The two most important materials include fullerenes and carbon nanotubes (CNTs). Both materials can be used for tissue- or cell- specific delivery (24). Fullerenes are novel carbon allotrope with polygonal structure made up exclusively by 60 carbon atoms. These nanoparticles are characterized by having numerous point of attachment whose surfaces also can be functionalized for tissue binding (25). CNTs have been one of the most extensively used types of nanoparticles because of their high electrical conductivity and excellent strength. There are two classes of CNTs: single-walled carbon nanotubes (SWCNT) and
27 CHAPTER 1: GENERAL INTRODUCTION
multi-walled carbon nanotubes (MWCNT). MWCNT are larger and consist of many single-walled tubes stacked one inside the other. The nanoscale dimensions of SWCNT and MWCNT, along with their high surface area have compelled researchers to utilize them for the delivery of imaging and therapeutic agents and in the transport of DNA molecules into cells. They also have potential for vaccine delivery as they amplify the immunological response (26). However, the potential toxicity of fullerenes and CNTs has been postulated by several authors (27, 28). CNTs and fullerenes toxicity has been attributed to various factors, the ability to penetrate membranes due to the apolar character of fullerenes, the dispersant used to solubilize the CNTs and their potent reactions with different biochemical compounds.
2.1.3. Metallic nanoparticles Metallic nanoparticles are versatile agents with a variety of biomedical applications including their use in biosensing/imaging and cancer thermotherapy as well as for targeted drug and gene delivery (29-31). Gold, silver and copper are most commonly used (32, 33). Metal nanoparticles can easily be synthesized with a range of sizes (1–150 nm), are stable and can be modified by conjugation with various functional groups (29). Gold nanoparticles are spherical metallic nanoparticles consisting of a dielectric core covered by a thin metallic shell which is typically gold. These nanoparticles are the most extensively studied because their properties lend themselves to multiple applications, such as labeling, delivery, heating and sensing (34).
2.1.4. Magnetic nanoparticles Magnetic nanoparticles are increasingly being realized as important nanomaterials in the industrial sector and they are being widely used for biotechnological and biomedical applications. Iron oxide nanoparticles are the most commonly explored members of the broader class of magnetic nanoparticles. Magnetite (Fe3O4, ferromagnetic, superparamagnetic when the size is smaller than 15 nm) and maghemite (γ-Fe2O3, ferromagnetic) have proven particularly popular for biomedical applications because of their great biocompatibility. They have been investigated for use as biosensors, for imaging and for drug delivery (35, 36). However, it is interesting to note that, with a few exceptions, metallic and magnetic nanoparticles used in medicine includes almost exclusively transition elements and their toxicity can be high if they are not adequately detoxified, which combined with their prolonged retention in tissues, is of significant concern regarding its safety (36).
2.1.5. QDs QDs are semi-conductor nanoparticles that measure approximately 2–10 nm, usually consist of 10 to 50 atoms and fluoresce when stimulated by light. They are comprised of an inorganic core, an inorganic shell and an aqueous coating to which biomolecules can be conjugated. The size of the core determines the color of light emitted. Although QDs have been used in electronics and optics for 20 years, they have only recently been applied to nanomedical research. The most commonly used
28 CHAPTER 1: GENERAL INTRODUCTION
QDs for biomedical applications contain Cadmium Selenide (CdSe) or Cadmium Telluride (CdTe) (38). QDs containing Indium Phosphide (InP) and Indium Arsenide (InAs) are also frequently used (39, 40). Biomedical applications of QDs are primarily focused on their use as imaging agents (41). Moreover, QDs also have sufficient surface area to attach agents or biomarkers for simultaneous targeted drug delivery and in vivo imaging or for tissue engineering and diagnostic (42). However, clinical application of QDs is limited by the high toxicity of cadmium and selenium and their instability to photolysis and oxidative conditions, which could result in dissolution of QDs core and their slow elimination. Thus, crucial for biological applications, QDs must be covered with other materials allowing dispersion and preventing leaking of the toxic heavy metals (43).
2.2. Organic Nanomaterials
Organic nanomaterials are carbon based (with the striking exception of fullerenes and nanotubes that are considered inorganic) which are generally characterized by their biocompatibility and low toxicity. Organic nanomaterials have advantages such as high versatility to carry diverse drugs and conjugate to targeting agents which offers the potential for combining treatment and diagnosis, the so-called “theranostic” purpose. Organic nanomaterials can be classified into three main groups: lipid-based nanodevices, polymer therapeutics and polymeric nanoparticles. Figure 2 shows the structure of the most representative nanodevices from the lipid-based nanocarriers and polymer therapeutics groups.
a. Polymer-drug conjugate b. Polymeric micelle c. Dendrimers
Hydrophilic block Drug (shell)
Hydrophobic block (core)
5-20 nm 60-100 nm 60-200 nm
d. Liposomes e. Solid-lipid nanoparticles
Inner aqueous phase SUV
LUV
MLV Lipidic solid core Lipid bilayer
50 nm-1 μm 50 nm-10 μm
Figure 2. Structure of different nanodevices used for drug delivery purposes. Adapted from reference (44).
29 CHAPTER 1: GENERAL INTRODUCTION
2.2.1. Lipid-based nanocarriers encompass four main types of carriers: liposomes, nanoemulsions, lipid nanocapsules and solid lipid nanoparticles (SLN). a. Liposomes or (phospho)lipid vesicles, are self-assembled microscopic vesicles which posses a central aqueous cavity surrounded by a lipid membrane formed by concentric bilayer(s) (lamellas) (45). Liposomes are highly flexible delivery systems capable to incorporate hydrophilic substances (in the aqueous interior) or hydrophobic substances (in the lipid membrane). The production of liposomes, as membrane mimicking drug carriers, started long before any nanotechnology (46-48), with their first market appearance in 1986 in a cosmetic formulation by Dior, and is considered by some to be the archetypal nanoparticle (49). Liposomes are classified into three categories on the basis of their size and lamellarity (i.e. number of bilayers): (i) small unilamellar vesicles or oligolamellar (SUVs), (ii) large unilamellar vesicles (LUVs) and mutilamellar vesicles (MLVs) (Figure 2d). The surface of liposomes can be modified by covalent binding of natural (e.g. proteins, polysaccharides, glycolipids, antibodies, enzymes) or synthetic molecules (e.g. PEGs) (50-52). Moreover, by using lipids of different fatty-acid-chain lengths, scientists can construct liposomes to be temperature-sensitive or pH-sensitive, thereby permitting the controlled release of their contents only when they are exposed to specific environmental conditions (23). Liposomes are suitable for topical, i.v. and other parenteral administrations, but because they are susceptible to degradation in the gastrointestinal tract (GIT) they are rarely suitable for oral use. The components of liposomes are similar to natural human cell membranes; thus, they confer liposomal drug delivery with several intrinsic benefits for improving efficacy and safety of different drugs. Therefore, liposomes have been widely developed as drug delivery systems for the administration of chemotherapeutic agents for cancer and vaccines for immunological protection (53). Liposomes have been also proposed as carriers of radiopharmaceuticals, for diagnostic and imaging purposes as well as vehicles for nucleic acid-based medicines for gene therapy (54-57). However, their application has been slowed somewhat by stability issues, difficult manufacturing techniques, cost and because complement-mediated hypersensitivity reactions may occur in response to i.v. liposome administration (58). b. Lipid nanocapsules are biomimetic carriers consisting of an oil-filled core with a surrounding polymer shell. (59). They are characterized by a hybrid structure between polymer nanocapsules and liposomes. In general, they have an oil core of medium-chain triglycerides, which confers them special use for the loading of hydrophobic drugs, surrounded by a membrane made from a mixture of a wide variety of polymers/surfactants with hydrophilic segments such as PEGs. These types of polymers also enhance the intrinsic colloidal stability of the system minimizing their recognition by the mononuclear phagocyte system (MPS), a major drawback that often arises after i.v. injection of drug carriers. Moreover, the targeting capacities of these systems can be implemented by surface
30 CHAPTER 1: GENERAL INTRODUCTION
modification with specific biomolecules (e.g. antibody fragments, folic acid) enhancing the cell- targeting through molecular recognition processes (60-64). Their formulation is based on the phase-inversion temperature phenomenon of an emulsion leading to lipid nanocapsules formation with good monodispersion (65, 66). c. Nanoemulsions are oil-in-water dispersions of about 20-200 nm oil where the dispersed droplets are stabilized by an exterior film composed of surfactants and co-surfactants. Drugs are usually loaded into the dispersed oily. The advantages of nanoemulsions include simplicity, inexpensive preparation, high stability, capability to solubilize lipophilic substances and to protect them from degradation. Nanoemulsions are versatile and can be prepared from different aqueous solutions, surfactant mixtures and oily phases (67, 68). d. SLN were developed at the beginning of the 1990s as an alternative carrier system to emulsions, liposomes and polymeric nanoparticles. SLN are composed of solid lipids (i.e. lipids solid at room temperature and also at body temperature) stabilized by surfactant(s) and dispersed in an aqueous solution (Figure 2e). Most commonly, the pharmaceutical agent, mainly hydrophobic drugs, is dissolved or dispersed within the lipid. The production of SLN is carried out by two main manufacture methods: the high pressure homogenization technique (69) and the microemulsion technique (70). SLN exhibit several advantages such as protection of incorporated active compounds against degradation, excellent physical stability, high flexibility to modulate the release of the loaded compound and administration through multiple routes (orally, topically and intravenously) (71, 72). However, the drug loading capacity of conventional SLN is limited by drug solubility in the lipid melt, lipid matrix structure and the polymorphic state of the lipid matrix. Thus, drug expulsion after polymorphic transition during storage and relatively high water content of the dispersions have been observed (73-76). To minimize these drawbacks, lipid drug conjugate nanoparticles and the so-called nanostructure lipid carriers have been proposed (71, 77).
2.2.2. Polymer therapeutics Polymeric therapeutics is an “umbrella” term that emerged through the interdisciplinary research at the interface of polymer chemistry and biomedical sciences (78). Polymer therapeutics comprise a variety of complex macromolecular systems, their common feature being the presence of a rationally designed covalent chemical bond between a water-soluble polymeric carrier and the bioactive molecule(s). Although polymer therapeutics are nano-sized conjugates with sizes between 2- 200 nm, it should be noted that such constructs are quite distinct from polymeric nanoparticles. These water-soluble hybrid constructs encompasses at least three distinct classes including: polymer-drug conjugates, polymer-protein conjugates and supramolecular drug-delivery systems (e.g. polymer micelles, polyplexes and dendrimers).
31 CHAPTER 1: GENERAL INTRODUCTION
a. Polymer-drug/protein conjugates were firstly described by Ringsdorf in 1975 (79). Later Duncan and co-workers exploited the biological rationale and the mechanism of polymer-drug designs to pioneer a field that has remained of great scientific and therapeutic interest (80, 81). Polymer-drug conjugates are drug delivery technologies in which a drug is covalently bound to a water-soluble polymer carrier, normally via a biodegradable linker (often a peptidyl or ester linkage) (Figure 2a). Polymer-drug conjugates can improve drug solubility, circulation time (through the properties of the polymer carrier), drug targeting (by using appropriate linkers which respond to changes in physiological conditions such as temperature, pH, and the presence of enzymes) and decreased drug toxicity. Among others, the most popular polymers or macromolecular carriers are the followings: N-(2-hydroxypropyl)methacrylamide copolymers, PEGs, poly(glutamates) and albumin (82). Protein-polymer conjugates are similar to drug conjugates with the difference that in this case the biologically active molecule is a protein or macromolecule. PEG has mainly been the polymer of choice for preparing these polymer-protein conjugates. Abuchowski et al. in 1977 first introduced the conjugation of PEG to protein drugs (83). In this “pegylation” technology, linear or branched PEG derivates are coupled to the surface of the protein (84, 85). Binding polymers to therapeutically relevant proteins imparts several potential advantages including (i) its protection against degradation, (ii) longer circulation in blood stream and (iii) a decreased immune response. Thus, all of these properties result in a less-frequent administration to the patient (86, 87). b. Dendrimers are monodisperse macromolecules combining a number of unique characteristics including (i) a well defined, regularly hyperbranched and three dimensional architecture, (ii) a relatively low polydispersity and (iii) a high and tunable functionality (Figure 2c). This molecular structure confers the molecule properties substantially different than the linear counterparts (88). The first family of dendrimers was the poly(amidoamine)s (PAMAM) (89). Other families are the so-called polyamidoamine-organosilicon derivatives, poly(propyleneimine) dendrimers, multilingual, chiral, amphiphilic, micellar and Frechet-type dendrimers (90). Dendrimers possess three distinguished architectural components, namely (i) an initiator core; (ii) interior layers (generations) composed of repeating units, radically attached to the interior core, and (iii) exterior (terminal functionality) attached to the outermost interior generations (91). The chain-ends can be functionalized and, thus, modify the solubility, the miscibility and the reactivity of the resulting macromolecule (92). These highly functionalized materials enable drug incorporation into the core of the molecule and drug complexation and conjugation on the surface (93), being the ability of the molecules to enter the ramified tridimensional structure fully related to the size of the dendrimer. PAMAM dendrimers and other cationic dendrimers can also act as vectors in gene therapy (94). These macromolecules are terminated in amino groups which can interact with phosphate groups of nucleic acids ensuring the formation of transfection complexes, which are also known as dendriplexes (95).
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While dendrimer-based products are considered safe, a study published recently demonstrated that the marketed transfection reagent SuperFect®, consisting of partly hydrolyzed dendrimers interfered with cell signal transduction pathways via and oxidative stress-dependent mechanism even at doses that do not lead to over cytotoxicity (96). Thus, this study supports the importance of understand the genomic of biological effects of dendrimers prior to its clinical application. c. Polymeric micelles are nanostructures formed by the self-assembly of amphiphilic copolymers in aqueous media generally as a result of hydrophobic or ion pair interactions between polymer segments, above the so-called critical micelle concentration (CMC) (97, 98). Polymeric micelles are composed by internal and external zones named “core” and “shell”, respectively (Figure 2b). The different types of micelles used for drug delivery including regular micelles, reverse micelles and uni- molecular micelles. Regular micelles are self-assemblies of amphiphilic copolymers in an aqueous medium while reverse micelles are self assemblies of amphiphilic copolymers in a non-aqueous medium. Uni-molecular micelles are made from the block copolymers with several hydrophobic and hydrophilic regions in one molecule, which enables the self-assembly of one molecule into a micelle. The most broadly studied amphiphiles are derivatives of poly(ethylene oxide)-poly(propylene oxide) block copolymers (99). In the last years a profuse number of amphiphilic polymers based on styrene oxide and butylenes oxide and conjugates of hydrophilic PEG segments with hydrophobic blocks of phospholipids (100), poly(L-amino acids) (101) and polyesters (102) have been proposed. Reported advantages of micelles as delivery systems include simple preparation, increased drug solubility, reduced toxicity, increased circulation time, enhanced tissue penetration and targetability (103). However, the conventional micelles also have several disadvantages such as system instability over long periods of time, sustained release only for short periods of time, inadequate suitability for hydrophilic drugs and the need for system optimization with each drug (98). d. Polyplexes are synthetic gene carriers systems formulated from both nucleic acids (i.e. oligonucleotides, DNA or siRNA) and cationic polymers. Their production is regulated by ionic interactions with the negative charged phosphate groups of naked DNA or other gene material, resulting in the formation of positively charged complexes. As lipoplexes (complexes between lipids and DNA), these structures allowing the protection of DNA from degradation and facilitate the entry into the cell (104). Polyplexes used in gene therapy are based on the hypothesis that the complexes adsorb more effectively to the anionic plasma membrane of mammalian cells via electrostatic interactions then resulting in high transfection efficiency although the transport of the DNA into the nucleous is still not well understood. Different cationic polymers have been proposed as gene carriers for the transfecting foreign genes into mammalian cells in vitro including diethylaminoethyl-dextran, poly(2-dimethylaminoethyl methacrylamide), poly-L-lysine, poly(ethyleneimines) and various derivatives (105).
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3. Polymer-based nanoparticulate drug delivery systems
Although polymer-based nanoparticles are a group within organic nanomaterials, their classification in a different section is due to the special interest that this class of organic nanoparticulate systems represents in this work. Polymeric nanoparticles have been extensively investigated due to its potential as drug delivery systems. These potential have been recognized since the 1960´s (106). One of the pioneers in the development and evaluation of polymeric nanoparticles for drug delivery and for vaccines was Professor Peter Speiser, whose strategy for the controlled release was the development of miniaturized delivery systems (12, 13). His research group was focused at first in the development of polyacrylic microcapsules and in the late 1960s they developed the first nanoparticles. In parallel, albumin nanoparticles were developed for the first time in the Johns Hopkins Medical Institution in Baltimore (107). Another pioneer was Professor Patrick Couvreur, who described the production of biodegradable nanoparticles made of poly(methyl cyanoacrylate) and poly(ethyl cyanoacrylate) (108). At that time, other types of nanoparticles were also proposed as poly(acrylamide) (109) or poly(lactic acid) (PLA) nanoparticles (110). Polymeric nanoparticles are solid colloidal carriers ranging in size from about 10 to 1000 nm (111), and depending on the preparation method, nanospheres or nanocapsules can be obtained, with the therapeutic agent dissolved, entrapped, encapsulated or attached to the polymeric matrix (Figure 3). Nanocapsules are vesicular systems, in which the drug is confined to a cavity surrounded by a polymer layer, while nanospheres are matrix-type systems in which the drug is physically and uniformly dispersed (112). Besides, the active substance can also be adsorbed in the surface of both nanoparticles types. Nowadays, the role of polymeric nanoparticles in drug delivery systems covers multiple aspects, from the enhancement of the physical-chemical stability of the drug to the regulation of the drug release profile and targeting (113). Moreover, the modification of the physico-chemical properties and/or composition of their surface make possible the development of multifunctional nanoparticles capable of targeting and controlled release of therapeutic and diagnostic agents. Thus, by using polymeric nanoparticles a wide number of pharmaceutical and medical applications, impossible to reach with traditional medicines, can be achieved.
Polymeric core
Nanosphere
Nanocapsule
Water/oil liquid core Figure 3. Schematic representation of the structure of nanospheres and nanocapsules. Adapted from reference (65). 34 CHAPTER 1: GENERAL INTRODUCTION
3.1. Polymers used for the preparation of nanoparticles
Different polymers, both synthetic and natural, have been employed to prepare polymeric nanoparticles. Ideally, all the polymers applied in the biomedical field must be biocompatible, non- toxic, free of leachable impurities and readily processable (114). Compared to natural polymers which have a relatively short duration of drug release, synthetic polymers have a sustained release of the therapeutic agent over a period of days to several weeks. On the contrary, synthetic polymers are usually limited by the need of organic solvents and harsh formulation conditions (115, 116). As natural polymers, the most popular are chitosan and albumin. In a similar way, synthetic polymers such as poly(esters), poly(alkyl cyanocrylates) and poly(anhydrides) are also used.
3.1.1. Natural Polymers a. Chitosan is a modified natural carbohydrate polymer prepared by the partial N-deacetylation of chitin, the second most abundant natural polysaccharide in nature, which is derived from the cuticles of insect species or crustaceans such as crabs and shrimp (117). It is selected for the preparation of nanoparticles as it possesses some ideal properties such as biocompatibility, biodegradability and non- toxicity. Chitosan is soluble in weakly acid solutions, resulting in the formation of a cationic polymer with high charge density, and can therefore form polyelectrolyte complexes with a large variety of anionic polymers (118). Moreover, due to the presence of highly reactive amino groups along its structure, chitosan is susceptible to chemical or biological functionalization (119). Besides, it has excellent bioadhesive properties and the ability to enhance the penetration of large molecules across the mucosal surfaces, make it a suitable candidate for the design of mucosal drug delivery systems and vaccine formulations (120-123). b. Systems based on proteins including gelatin, collagen, casein, albumin and whey protein have been studied for delivering drugs, nutrients, bioactive peptides and probiotic organisms (124, 125). Protein-based nanoparticles are particularly interesting as they hold certain advantages such as greater stability during storage and in vivo, being non-toxic and non-antigenic and their ease to scale up during manufacture over other drug delivery systems (126-128). Among the available protein-based drug carrier systems, albumin nanoparticles have demonstrated to be a versatile macromolecular carrier. Albumin nanoparticles have been shown to be biodegradable and metabolized in vivo to produce innocuous degradation products, non-toxic and non-immunogenic (129). Besides, they can incorporate a great variety of drugs (e.g. hydrophilic, lipophilic, peptides, oligonucleotides) in a relatively non-specific fashion (130, 131). Commercially, albumins are obtained with significant quantities from egg white (ovalbumin (OVA)), bovine serum albumin (BSA), and human serum albumin (HSA). It is also possible to find albumins from soybeans, milk, and grains (132).
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3.1.2. Synthetic Polymers a. Poly(esters) are thermoplastic polymers with hydrolytically labile aliphatic ester linkages in their structure. Aliphatic polyesters with short aliphatic chains between ester bonds are the most used biodegradable polymers as they can degrade in the time required for most of the biomedical applications (118). Among them, copolymers between lactic and glycolic acids (poly(lactide-co- glycolide) (PLGA)), which are approved by the main regulatory agencies (e.g. FDA, European Medicines Agency (EMA)), are widely employed for the preparation of nanoparticles due to their biocompatibility and biodegradability (133, 134). PLGA is a poly(ester) composed by one or more of three different hydroxy acid monomers, glycolic acid and/or d-/l-lactic (135). These copolymers undergo hydrolysis of its ester linkages in the presence of water and the time required for the degradation is related to the ratio of monomers used in the preparation, since higher the content of glycolide units, the lower is the time required for its degradation (136). Moreover, these parameters also determine the encapsulation efficiency and release rate of drugs from the nanoparticles (137). However, PLGA degrades in the body producing its original monomers of lactic acid and glycolic acid which are the by-products of various metabolic pathways creating an acidic microenvironment (138). Thus, despite its obvious interest as nanoparticulate delivery systems, the use of these polymers as peptide or protein delivery systems may negatively affect the stability of the loaded compound due to the bulk degradation mechanism of the polymer and the acidic environment generated during this process (139-141). b. Poly(Ɛ-caprolactone) (PCL) is a FDA-approved semicrystalline, bioerodible, biodegradable and biocompatible polymer that can be used for the formulation of nanoparticles (44). Due to its semicrystallinity and hydrophobicity, the in vivo degradation of PCL is very slow making it more suitable for long-term delivery systems, extending over a period of more than one year (115, 142). In addition, PCL particles, unlike polylactides, do not generate an acidic environment that could negatively affect the loaded drug and it is also compatible with a lot of other polymers (143). c. Poly(akyl cyanocrylates) (PACA) are biodegradable, bioerodible and biocompatible polymers that have been used since at least 1966 due to their excellent adhesive properties (144, 145). However the employment of PACA polymers for the preparation of nanoparticles is more recent. From a pharmaceutical point of view, PACA nanoparticles have demonstrated good mucoadhesive properties and they are rapidly degraded by esterases in biological fluids. d. Poly(anhydrides) Since the present work has been focused on poly(anhydride) nanoparticles, the main characteristics of both polymer and nanoparticles have been explained in more detail.
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Poly (methyl vinyl ether -co-maleic anhydride) copolymers
The copolymers between methyl vinyl ether and maleic anhydride (commercialized as Gantrez® AN from ISP, Corp.) are a good example of the group of poly(anhydrides) (Figure 4).
OCH3
CH3 CH CH CH
O COC O n
Gantrez® AN Figure 4. Chemical structure of poly (methyl vinyl ether -co- maleic anhydride) or Gantrez® AN.
Those copolymers are widely employed for pharmaceutical and cosmetic applications as denture adhesives, thickening and suspending agents and as adjuvants for the preparation of transdermal patches. In addition, the ester derivatives are also employed as film-coating agents. From a general point of view, Gantrez® AN copolymers are highly soluble in acetone and display a low solubility in ethanol and isopropanol. On the other hand, these copolymers display a very low aqueous solubility; however, in contact with water the anhydride group can be hydrolyzed and dispersed to give uniform slurry. Table II summarizes the main physico-chemical properties of the four available Gantrez® AN copolymers.
Table II. Physico-chemical characteristics of the main Gantrez AN copolymers.
Commercial (a) Brookfield viscosity at 25ºC Molecular weight (b) Tg (ºC) name (mPa·s; 5-10%)
Gantrez® AN 119 213,000 15-35 152 Gantrez® AN 139 1,080,000 40-145 151 Gantrez® AN 149 1,250,000 45-136 153 Gantrez® AN 169 1,980,000 85-1400 154 (a) Brookfield RVT viscometer, spindle 3, 20 rpm. (b) Glass-transition temperature.
The hydratation process of the anhydride form of theses copolymers is a complex phenomenon. Thus, the process starts with an initial phase in which the polymer hydration occurs in contact with water. After this hydratation process (about 3 h), the copolymer begins to swell because of the hydrolysis phenomenon of the anhydride groups. Finally, in the third phase, the copolymer dissolves rapidly (146).
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The process of dissolution is influenced by the pH, temperature, molecular weight and the degree of substitution of the anhydride groups (147). Actually, Gantrez copolymers have a pKa of about 5.33 (weak acids), thus, the rate of dissolution is highly dependent on the pH of the medium. In neutral and basic conditions, this rate is significantly higher than in acidic conditions. Similarly, the temperature can highly affect the dissolution of the copolymer. Therefore, the hydrolysis of 10% copolymer dispersion in water takes more than 8 h, when the temperature is 40ºC, and only 15 min when the temperature is 90ºC (International Speciality Product ISP™). In an aqueous medium, Gantrez® AN can spontaneously react with molecules containing either hydroxyl (-OH) or primary amine (-NH2) residues to form stable bonds. These reactions can be useful to modify the surface of a co-polymer matrix and, thus, modify both their physico-chemical properties (i.e. degradability, stability and mechanical strength) and pharmaceutical characteristics (i.e. drug distribution, adhesivity and release rate). Nevertheless, in the same aqueous medium, the anhydride groups can also be hydrolyzed yielding two free carboxyl groups (Gantrez S). The resulting product shows a high aqueous solubility and cannot react with nucleophilic groups.
Poly(anhydride) nanoparticles
Nanoparticles from these copolymers (Gantrez ® AN 119) have been developed recently. Poly(anhydride) nanoparticles can be prepared under mild conditions using a solvent displacement method (148). Poly(anhydride) nanoparticles can incorporate a wide variety of drugs, including protein macromolecules, peptides and oligonucleotides. Another interesting feature is that these nanoparticles can be treated with crosslinking agents to modify the release profiles of the drug and the rate of degradation thereof. This crosslinking process is performed by direct incubation of preformed nanoparticles and the crosslinking agent without using traditional agents questionable from a toxicological point of view (glutaraldehyde derivatives carbodi-imide, etc.). One of the most important properties of poly(anhydride) nanoparticles is their ability to develop strong bioadhesive interactions with components of the gut mucosa. In addition, their surface can be easily modified by simple incubation with different excipients or ligands in order to modify their in vivo distribution and even to increase its affinity for the intestinal mucosa. Thus, for non-specific bioadhesion, poly(anhydride) nanoparticles can be coated with proteins (albumins) (148, 149), polysaccharides (e.g. chitosan and cyclodextrins) (150) and with PEGs or derivatives (151, 152). To develop specific bioadhesive interactions, proposed ligands include lectins (148, 153), flagellin of Salmonella enteritidis (154), lypopolysaccharides (155), simple molecules such as thiamine, mannosamine or derivatives of vitamin B12 (156-158). This versatility of poly(anhydride) nanoparticles makes them suitable carriers for easy administration of drugs that exhibit limited oral bioavailability as well as good candidates for oral immunotherapy.
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3.2. Preparation of polymeric nanoparticles (Bottom-up & Top-down techniques)
Several methods have been developed to prepare polymeric nanoparticles and selection of a particular one depends on the nature of the polymeric material forming the resulting nanoparticles and the properties of the employed drug molecules to load into nanoparticles. They can be classified into two main categories: bottom-up and top-down techniques. The bottom-up techniques are base on the use of monomers which are polymerized in situ (i.e. emulsion, microemulsion and interfacial polymerization). These methods based on polymerization reactions present some drawbacks such as inadequate biodegradability of the product and the presence of toxic residues. Top-down techniques involves the use of preformed polymers or purified natural macromolecules. These techniques are more commonly used to prepare biodegradable nanoparticles since they avoid the problems associated with bottom-up techniques. Among the different top-down techniques developed, two approaches are conventionally used. The former is based on spontaneous formation of nanoparticles whereas the latter is related with emulsification-based methods. Depending on the solubility or gelling properties of a dissolved polymer, nanoparticles can be formed spontaneously. The general principle of this method consists in the preparation of a solution of the polymer and then induce a phase separation by adding a non-solvent of the polymer (159, 160). Besides, nanoparticles from a hydrophilic polymer, such as alginate or chitosan, can be prepared based on its gelation properties by ionic gelation (161). Methods based on nano-emulsion templates derive from microparticle preparation techniques (162). The first step requires the formation of an emulsion and its homogenization, which can be achieve by high pressure homogenizers, microfluidizers and sonicators. Emulsification-based methods differ depending on the properties of the polymer and on the miscibility between the organic solvent and the aqueous phase. Thus, when hydrophobic polymers are used, some methods involve volatile and water-immiscible solvents that have to be eliminated by simple evaporation, leading to polymer precipitation (i.e. emulsification-solvent evaporation and double emulsion methods). In other methods, polymer precipitation occurs as a result of controlled diffusion processes using partially or fully water miscible organic solvents (salting out and emulsification- diffusion methods). Table III summarizes some of the main advantages and drawbacks of the methods used to prepare nanoparticles.
39 CHAPTER 1: GENERAL INTRODUCTION
Table III. Overview of the main advantages and drawbacks of some of the most used methods for the preparation of nanoparticles. Adapted from reference (44).
Preparation Advantages Disadvantages Examples of method polymers
Nanoprecipitation Simple, fast and Presence of surfactants Poly(esters) reproducible
Easy to scale up Use of organic solvents
Coacervation Easy to scale up Coacervate phase needs Proteins consolidation (i.e. cross- linkage)
Ionic Gelation Organic solvent free Low stability due to the Alginates, Chitosan method weakness of ionic interactions
Emulsification- Encapsulation of Organic solvents PLA, PLGA, PCL solvent evaporation hydrophobic and
Lipophilic drugs Possible coalescence of Amphiphilic nanodroplets during copolymers: PEG- evaporation PLGA, PEG-PCL, PEG-PACA
Salting out Minimization of the Purification step is PLA, PLGA stress to labile drugs critical to remove electrolytes
High loading efficiency
Easy to scale up PLA: Poly(lactic acid); PLGA: Poly(lactide-co-glycolide); PCL: poly(ε-caprolactone); PEG: Polyethylene glycol; PACA: Poly(akyl cyanocrylates).
Before obtaining nanoparticles ready to be applied in drug delivery or for other purposes, supplementary steps in the preparative process may be necessary. Normally, these steps may involve purification process in order to remove residues of organic solvents used during nanoparticle preparation as well as traces of unreacted polymers or/and molecules that have not been loaded into nanoparticles. Among purification methods, the most commonly used are tangential flow filtration (163), centrifugal filtration (164) and ultracentrifugation (159, 165). Furthermore, when nanoparticles are developed for parenteral or ophthalmic applications they have to be sterile. Normally, nanoparticles sterilization is carried out either by filtration before the filling process or by gamma-
40 CHAPTER 1: GENERAL INTRODUCTION
irradiation when filled in the definitive container (164). Finally, nanoparticles are usually presented as a dry powder in order to improve their stability as well as facilitate their manipulation, shipping and storage. Usually, the drying process of nanoparticles suspensions are achieved by lyophilization or spray-drying (165).
3.3. Behavior of polymeric nanoparticles after oral administration
Oral administration is the preferred route for drug delivery because is patient-friendly, painless and easy for self-medication. Oral bioavailability of drugs is strongly influence by their properties; solubility and permeability are two important parameters for their absorption via passive diffusion. Thus, the Biopharmaceutics Classification System (BCS) defines four categories of drugs based on their solubility and their permeability (166). The main mechanisms involved in the enhanced drug absorption by polymeric nanoparticles in oral drug delivery are: (i) protection of the drug from the harsh environment of the GIT, (ii) prolongation of the residence time in the gut by mucoadhesion, (iii) endocytosis of the particles and/or (iv) permeabilizing effect of the polymer. Moreover, specific delivery can be achieved by targeted nanoparticles. When nanoparticles are administered by the oral route, they diffuse into the liquid medium and reach the mucosal surface during their transit in the . Then, nanoparticles can be immobilized at the intestinal surface by an adhesion mechanism called “bioadhesion”; although if adhesion occurs on the mucus layer lining the mucosal surface, the term “mucoadhesion” is employed. These interactions are driven by hydrogen bonding, Van der Waals interactions, polymer chain interpenetration, hydrophobic forces and electrostatic/ionic interactions (167). Despite the duration of adhesion is limited by mucus clearance mechanisms (168), this phenomenon results in (i) a prolonged residence time and (ii) the establishment of a drug concentration gradient from the adhered nanoparticle till the surface of the mucosa. To increase residence time, diffusion in the mucus is critical. In this regard, PEG coating of nanoparticles surfaces has demonstrated to be an effective strategy to ensure rapid nanoparticle transport in mucus. Although PEG was first used to increase particles stability due to its chemical properties, PEG makes particle more hydrophilic and hence modulates their bioadhesive properties (167, 169). In fact, dense coating with PEG effectively minimizes adhesive interactions between nanoparticles and mucins, allowing penetration of nanoparticles (168, 170). The particle mobility also seems to be strongly dependent on surface charges. Transport rates were inversely related to particle surface potentials, with negatively charged particles displaying significantly higher transport rates than near neutral, or positively charged particles whose transport was severely limited, likely by particle aggregation and electrostatic adhesive interactions with mucin fibers (171). Therefore, nanoparticles interaction with the mucus layer is influenced by their surface characteristics. Once nanoparticles diffuse through the mucus layer they reach the intestinal epithelium, which is mainly composed of absorptive enterocytes for a large part sprinkled by mucus-producing Globet cells and M cells. Particle traslocation/absorption through the intestinal epithelium involves
41 CHAPTER 1: GENERAL INTRODUCTION
both paracellular and transcellular routes (Figure 5). However, paracellular route is limited because it utilizes less than 1% of the mucosal surface area. Moreover, juctional complexes (tight or adherens junctions) restrict or completely block the passage of macromolecules larger than approximately 1 nm. Hence, it is generally admitted that polymeric nanoparticles do not diffuse through the intestinal barrier by paracellular route. However, new potential modulators of the junctional proteins are developed to reversibly open membranous barriers and improve drug delivery by the paracellular way (172). Regarding transcellular route, two main endocytic mechanisms have been described: phagocytosis, which is restricted to M cells and phagocytic immune cells, and pinocytosis. The endocytic pathways differ with the size of the endocytic vesicle, the nature of the cargo and the mechanisms of vesicle formation (173). It is well established that uptake by enterocytes and M cells is size-dependent (174). Thus, small particles (50–100 nm) can be translocated by endocytosis through enterocytes while larger particles are more likely taken up and translocated by M cells. In this context, molecules transport through M cells have been exploited to orally deliver vaccines as these cells are specialized for antigens sampling in mucosal immunity.
Figure 5. Schematic representation of the different mechanisms of the uptake of nanoparticles in the intestinal epithelium. From reference (175).
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The distribution, bioadhesion and absorption of nanoparticles within the GIT can be modulated by associating different excipients and/or ligands to polymeric nanoparticles. Thus, coating nanoparticles with lectins has been demonstrated to increase the intensity of the bioadhesion phenomenon (176). On the other hand, the “decoration” of nanoparticles with vitamins (i.e. vitamin B12 derivatives or thiamine) (158), sugars or bacterial adhesins permits to target specific receptors or areas within the gut (177). Indeed, grafting or coating nanocarriers with ligands binding specific receptors can enhance their internalization and transport.
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4. Specific Applications of Polymeric nanoparticles as delivery systems
4.1. Tumor Targeting
The use of pharmacological agents developed using classical strategies is frequently limited by pharmacodynamics and pharmacokinetics problems such as low efficacy or lack of selectivity. Moreover, drug resistance at the target level due to physiological barriers or cellular mechanisms is also encountered. In addition, many drugs have a poor solubility, low bioavailability and they can be quickly cleared in the body by the reticular endothelial system (RES). Furthermore, the efficacy of different drugs such as chemotherapeutical agents is often limited by dose-dependent side effects as anticancer drugs, which usually have large volume of distribution and are toxic to both normal and cancer cells. Therefore, precise drug release into highly specified target involves miniaturizing the delivery systems to become much smaller than their targets. With the use of nanoparticles, targeting drug molecules to the site of action is becoming a reality resulting in a personalized medicine. In this regard, the rationale of using nanoparticles for tumor targeting is based in two main approaches. First, nanoparticles provide the ability to deliver a concentrate dose of drug in the vicinity of the tumor due to the enhanced permeability and retention (EPR) effect or active targeting by ligands on the surface of nanoparticles. Secondly, nanoparticles would reduce the drug exposure to health tissues by limiting drug distribution to the target organ minimizing the drug dose-dependent side effects.
4.1.1. Passive targeting with polymeric nanoparticles In general, nanoparticle delivery of anticancer drugs to tumor tissues can be achieved by passive or active targeting. Passive targeting refers to the preferential accumulation of nanoparticles (bearing no affinity ligands) at active sites. Passive targeting takes the advantage of the inherent physicochemical properties of nanoparticles (size, shape, flexibility...) and exploits the unique anatomical and pathophysiological abnormalities of tumor vasculature, such as EPR effect (178). Characteristics of the EPR effect include extensive angiogenesis, defective vascular architecture and impaired lymphatic drainage/recovery system of the tumors. Moreover, most solid tumors have elevated levels of vascular permeability factors which augment the permeability of tumor tissues in comparison to normal tissue, thereby also contributing to the EPR effect (179). Thus, the targeting effect of nanocarriers is achieved as they remain in the bloodstream, where the vessel structure is normal, but would extravasate through the leaky vasculature at the tumor site. Upon arrival at the target sites, the nanocarriers release the drug in the vicinity of the tumor cells. The absence of lymphatic drainage from the tumors contributes to retention of the nanocarriers, ultimately leading to the accumulation of high concentrations of drug at the tumor site (Figure 6) (180).
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Figure 6. Schematic representation of passive targeting of nanoparticles by EPR effect. From reference (181).
In this context, Maeda and collaborators were the first group, which in the 1980`s demonstrated the principle of passive targeting of colloidal particles to tumors (182, 183). In their initial studies, significantly higher concentrations of the cytotoxic drug neocarzinostatin was discovered in tumor tissue post administration of the drug conjugated with poly(styrene-co-maleic acid)-neocarzinostatin, in comparison to control experiments where the drug was administered in its free form. This led Maeda and colleagues to postulate that the enhanced accumulation of the colloidal particles in the tumor was attributed to the structural features of the tumor vasculature, an observation which was termed the EPR effect (184). Following the revolutionary work of Maeda, many other groups have greatly exploited this mechanism of passive targeting for the delivery of chemotherapeutics into polymeric nanoparticles. For example, Bhardwaj et al. synthesized paclitaxel- loaded PLGA nanoparticles stabilized with cationic surfactants, and applied this formulation to increase the oral bioavailability of the drug to treat chemical-induced breast cancer in rats (185). Their studies showed that paclitaxel-loaded nanoparticles improved efficacy in comparison with i.v. free paclitaxel since nanoparticles exhibited preferential accumulation in tumors; the EPR effect might be the key prospective explanation. Other report, demonstrated the superiority of doxorubicin (DOX) loaded in PACA nanoparticles in a murine hepatic metastases model (186). This work showed that the incorporation of DOX in PACA nanoparticles resulted in a much greater reduction of the number of metastases compared to free DOX. Recently, in an elegant experiment, Jäger and co- workers formulated core-shell polymeric nanoparticles to deliver two different anticancer drugs by passive tumor targeting (187). They prepared polymeric nanoparticles based on the self assembly of N-(2-Hydroxypropyl) methacrylamide (HPMA) copolymers and aliphatic biodegradable co-polyester as a vehicle for docetaxel (DTXL) and DOX. In vivo studies carried out in mice bearing EL-4 T cell lymphoma demonstrated that the use of nanoparticles simultaneously loaded with DTXL and DOX provided a more efficient suppression of tumor-cell growth when compared to the treatment with free drugs or with single-loaded nanoparticles (DTXL or DOX separately). Their experiments
45 CHAPTER 1: GENERAL INTRODUCTION
showed not only the selectivity of nanoparticles to be accumulated in tumors due to the EPR effect but also that by means of co-encapsulation the efficacy was increased for both drugs. The extensive efforts in developing nanoparticles for tumor targeting have ultimately resulted in the clinical translation of a number of passively targeted polymeric nanoparticles. An interesting example is Abraxane® (ABI-007) which was approved in 2005 by the FDA for the treatment of metastatic breast cancer (188, 189). ABI-007 is a formulation of albumin nanoparticles containing paclitaxel obtained by high-pressure homogenization (190). In a phase III clinical trial with metastatic breast cancer patients, ABI-007 demonstrated significantly higher tumor response rates (33% vs 19%) and longer times to tumor progression (23.0 vs 16.9 weeks) than the control formulation (Taxol®) (188). Besides metastatic breast cancer, ABI-007 has also been evaluated in clinical trials involving many other cancers including non-small-cell lung cancer (phase II trial) and advanced nonhematologic malignancies (phase I and pharmacokinetics trials) (191, 192).
4.1.2. Passive targeting with pegylated nanoparticles In order to achieve effective EPR mediated targeting, polymeric nanoparticles must have long-circulating half-lives that facilitate more opportunities for the passage of nanoparticles from systemic circulation into the disordered and permeable regions of tumor vasculature. Therefore, their usefulness is limited by their rapid blood clearance and recognition by the macrophages of the MPS. In order to overcome this drawback, pegylation emerged over a decade ago as one possible strategy. In 1994, based on previous work carried out by Illum and collaborators (193, 194), Gref and colleagues demonstrated that coating of PLA nanoparticles with PEG chains significantly increased the circulation half-lives of nanoparticles (195). Since then, there have been a myriad of pegylated polymeric nanoparticles reported in the literature with the benefits of pegylation demonstrated across a broad range of polymer molecular architectures and macromolecular assemblies (196, 197). In this context, pegylation has demonstrated to considerably reduce the opsonization of the nanoparticles and the rate of the complement system activation. Hence, nanoparticles are less recognized by the macrophages of the MPS and can remain in blood stream and distribute in tumors. The value that pegylation provides in drug delivery of chemotherapeutics have resulted in the clinical translation of a number of passively targeted pegylated nanoparticles. In general, these nanoparticles are pegylated polymeric micelle formulations that form from the self assembly of amphiphilic polymers at concentrations above the CMC, yielding nanoparticles which can encapsulate poorly water soluble drugs (198). Among the different formulations currently in clinical trials, Genexol-PM® is a novel Cremophor EL-free paclitaxel-encapsulated PEG-PLA micelle formulation developed for various cancers (199-201). Phase II studies have demonstrated the safety and efficacy of Genexol-PM® with high response rates in patients with metastatic breast cancer (202). Moreover, Genexol-PM® plus cisplatin combination chemotherapy has shown significant antitumor activity and allowed the administration of higher doses of paclitaxel compared to the Cremophor EL-based formulation in patients with advanced non-small-cell lung cancer (203).
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However, the success of PEG in transforming polymeric nanoparticles drug delivery has not been without its challenges. For example, the highly hydrophilic surface of pegylated nanoparticles might not result in optimal endocytic uptake by cancer cells within the tumor hence efficient drug delivery into tumors cells rather than in the tumor milieu could be hampered. Then, bearing in mind all previously mentioned, passive targeting strategies are not without limitations and therefore considerable efforts are now underway to improve the therapeutic effect by more precise control of the delivery of the drugs to the active sites.
4.1.3. Active targeting with polymeric nanoparticles Active targeting of nanoparticles is based on molecular recognition processes such as ligand- receptor or antigen-antibody interaction. Typically, these receptors/antigens are differentially over expressed on the cells membrane or in the extra-cellular matrix of the disease tissue so targeted nanoparticles can be utilized in applications where drug release is either intracellular or extracellular. In active tumor targeting nanoparticles are covalently coupled with the ligands. Then, the ligand coupled nanoparticles are received by the tumor cells expressing a homologous receptor on their surfaces. Thus, the specific ligand-receptor binding ensures that the nanoparticles carrying drugs will get attached specifically to the tumor cells facilitating delivery of drugs only to the tumor cells expressing receptor and not the normal healthy cells hence avoiding undesirable side effects. Several ligand-receptor systems have been already studied, such as folic acid receptor, epidermal growth factor, or metal receptors among others. As an approach, considering that folate receptors are over expressed on the surface of some human malignant cells and that cell adhesion molecules (i.e. selectins and integrins) are involved in metastatic events, nanoparticles bearing specific ligands such as folate may be used to target ovarian carcinoma while specific peptides or carbohydrates may be used to target integrins and selectins (204, 205). Another example is the use of galactolipids that bind to the asialoglycoprotein receptor of the human hepatoma HepG2 cells (206). In some cases, active targeting needs a receptor mediated cell internalization to occur. Thus it was demonstrated that the benefits of folate ligand coating were to facilitate tumor cell internalization and retention of gadolinium labelled-nanoparticles in the tumor tissue (207). Targeting with small ligands appears more likely to succeed since they are easy to handle and manufacture. Furthermore, it could be advantageous when the active targeting ligands are used in combination with the long circulating nanoparticles to maximize the likelihood of the success in active targeting of nanoparticles. One example of this approach is the design of PEG-coated PACA nanoparticles coupled to folic acid (208). These conjugated nanoparticles displayed a multivalent and hence stronger interaction with the surface of the malignant cells expressing high amounts of the folate receptor at their surface. In another study, nanoparticles based on gelatine and HSA were decorated by covalent attachment with the biotin binding protein NeutrAvidin®, enabling the binding of biotinylated drug targeting ligands by avidin–biotin complex formation (209). Using the human epidermal growth
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factor receptor 2 (HER2) receptor-specific antibody trastuzumab (Herceptin®) conjugated to the surface of these nanoparticles, a specific targeting to HER2-overexpressing cells, followed by effective internalization of these nanoparticles, can be obtained. While the potential benefit of ligand- mediated nanoparticle targeting is clear, there are few clinical validated studies so far. Hrkach and collaborators demonstrated the clinical benefits of targeted polymeric nanoparticles (TNP) containing the chemotherapeutic DTXL for the treatment of patients with solid tumors (210). DTXL-TNP were targeted to prostate-specific membrane tumor antigen which is expressed on prostate cancer cells and on the neovasculature of most non prostate solid tumors. In previous preclinical studies carried out in tumor-bearing mice, DTXL-TNP exhibited markedly enhanced tumor accumulation at 12 h and prolonged tumor growth suppression compared to a solvent-based DTXL formulation (sb-DTXL). Moreover, initial clinical data in patients with advanced solid tumors indicated that DTXL-TNP displayed a pharmacological profile differentiated from sb-DTXL, including pharmacokinetics characteristics consistent with preclinical data and cases of tumor shrinkage at doses below the sb- DTXL dose typically used in the clinic (210).
4.2. Oral drug delivery
In recent years, significant research has been done using polymeric nanoparticles as oral drug delivery vehicles for different purposes such as diabetes treatment or chemotherapy. Compared with other administration routes, oral administration of drugs represents the most comfortable and convenient route of administration.
4.2.1. Diabetes treatment Insulin, as a protein drug used to treat diabetes, has been conventionally administered via subcutaneous injection (211). Many researchers have attempted to administer insulin orally since its discovery in 1922 (212). However, the oral bioavailability of insulin is severely hampered by its inherent instability in the GIT and its low permeability across biological membranes in the intestine. Thus, to overcome these drawbacks, a possible strategy may be the use of polymeric nanoparticles which among other benefits, allow encapsulation of bioactive molecules, protect them against enzymatic and hydrolytic degradation and increase residence time in contact with the absorptive epithelium (213). Hydrophobic and hydrophilic polymers, including PLGA, PLA, PCL and chitosan have been used to fabricate nanoparticles for oral insulin delivery (175). In a recent study, Jain and collaborators developed insulin loaded folate coupled pegylated PLGA nanoparticles (214). In diabetic rats, the formulations with a mean diameter of 260 nm, exhibited a two-fold increase in the oral bioavailability (double hypoglycemia) without any hypoglycemic shock as compared to subcutaneously administered standard insulin solution.
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4.2.2. Oral chemotherapy Oral chemotherapy is especially appropriate since many anti-cancer therapies are optimally effective when given chronically for a continuous tumor exposure (215). Moreover, from the economic standpoint, oral administration is more attractive as it reduces the cost for hospitalization and infusion equipment supplies (216). However, despite the numerous advantages described above, there are also some inherent problems associated with the use of oral anticancer drugs. Thus, for some compounds, the risk of local irritation of the GIT may limit continued oral administration or result in poor compliance (217). Nevertheless the main limitation of oral administration is the low and highly variable oral bioavailability which is limited by factors such as rapid degradation, low solubility, poor membrane permeability or extensive pre-systemic metabolism. It has been widely demonstrated that the major determinants of oral bioavailability is the efflux by P-glycoprotein (P-gp) and/or intestinal and hepatic metabolism by cytochrome P450 metabolizing enzyme system (CYP). In this context, the encapsulation of anticancer drugs in bioadhesive nanoparticles may be an interesting approach to promote their oral absorption. Among the different bioadhesive polymeric nanoparticles currently under investigation, nanoparticles made from the poly(anhydride) poly(methylvinylether-co-maleic anhydride), termed poly(anhydride) nanoparticles have demonstrated to be a promising strategy for oral drug delivery (148). Thus, using fluorouridine, the potential of serum albumin-coated poly(anhydride) nanoparticles were evaluated. These nanoparticles were capable to specifically target the stomach mucosa hence minimizing their arrival to the small intestine where P-gp and CYP are located (152). From urine data, the FURD bioavailability was found to be 4-times higher when loaded in albumin-coated nanoparticles than in conventional ones (79% vs 21%, respectively). For the control oral solution this parameter was calculated to be only 11% (149). More recently, the combination between poly(anhydride) nanoparticles and cyclodextrins has been demonstrated effective to significantly improve the oral bioavailability of paclitaxel (218, 219). Apart to their well reputed activity as solubilizing agents, cyclodextrins have the capability to disturb and inhibit the activity of the intestinal P-gp (220). After oral administration of these nanoparticles to animals, paclitaxel plasma levels were found to be high and prolonged for at least 24 h post-administration. These sustained levels of the anticancer drug corresponded with a relative oral bioavailability of about 80% (218). This fact would be due to the combination of both bioadhesive and inhibitory properties of these cyclodextrin- poly(anhydride) nanoparticles. In fact, the nanoparticles would be capable to approach the paclitaxel– cyclodextrin complex till the surface of the mucosa where these carriers would remain immobilized in intimate contact with the absorptive membrane. Then, nanoparticles would progressively release their content to the gut environment. At this moment, the paclitaxel–cyclodextrin complex would dissociate yielding the free drug and the oligosaccharide molecules. Under these conditions, the cyclodextrins would interact with lipidic components of the membrane disturbing the activity of the efflux pump and CYP whereas the anticancer drug, simultaneously, would be absorbed. The same
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approach has been found by the pegylation of paclitaxel loaded poly(anhydride) nanoparticles (221). Pegylated nanoparticles have shown a high ability to cross the mucus layer and reach the surface of the enterocytes (152). In addition, PEG has shown a moderate inhibitory effect of both P-gp and CYP3A4 (222). Therefore, after oral administration of pegylated nanoparticles to animals, paclitaxel plasma concentration was maintained in a high and constant level for at least 48 h post- administration. More particularly, the relative oral bioavailability of nanoparticles pegylated with either PEG 2000 or PEG 6000 was calculated to be about 70% and 40% respectively (221).
4.3. Polymeric nanoparticles as adjuvants and delivery systems for oral vaccination
In the past decades, several new approaches in vaccine development have shown to offer significant advantages over traditional ones (i.e. attenuated vaccines and bacterines). The new generation of vaccines contains other defined antigens, called “subunit vaccines”. They include the use of vaccines based in proteins, synthetic peptides, and/or plasmid DNA (223-225). Although they offer advantages such as reduced toxicity compared to traditional ones, they are poorly immunogenic when administered alone. Therefore, the rationale for vaccine design against a specific pathogen requires the use of the right antigens and the appropriate adjuvant to elicit the corresponding immune and protective response. In this context, polymeric nanosystems have been proposed as new adjuvants more convenient to use, safer and easy to administer than the classical ones (such as alum) (226). Moreover, the new adjuvants based on nanoparticles may be susceptible to be used even through mucosal routes. In this regard, by using polymeric nanoparticles for mucosal vaccination via oral route, these systems not only may protect the antigens from its stomach degradation and/or act as adjuvants, but also can enhance the delivery of the loaded antigen to the gut lymphoid cells due to their ability to be captured and internalized by cells of the gut-associated lymphoid tissue. In fact, mucosal vaccination using antigens loaded or encapsulated in particles appears to have a sound scientific rationale based on the protection of an antigen from exposure to extreme pH conditions, bile and pancreatic secretions (153) and provide a depot-effect (227). In addition, the use of nanoparticles offers another advantage such as the possibility to load an immune-enhancer (i.e., cholera toxin B subunit (CTB) or lipopolysaccharide (LPS)) with the antigen in order to increase and induce the more appropriate immune response (228, 229). In this context, poly(anhydride) nanoparticles have demonstrated to be good candidates for oral vaccination and immunotherapy treatments since these nanoparticles have shown excellent properties as adjuvants and appropriate vehicles for the controlled release of antigens (230-233). Thus, Gomez and co-workers found enhancements in both Th1 and Th2 markers (IgG2a and IgG1, respectively) after oral administration of these nanoparticles loaded with OVA as allergen model (234). Moreover, these carriers were able to protect a model of sensitized mice to OVA from anaphylactic shock. In other elegant study, Salman and collaborators designed poly(anhydride) nanoparticles coated with mannose (recognized by lectine CLR type) or Salmonella Enteritidis derived flagellin (Toll like receptor 5 agonist) (177). Both
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coated nanoparticles demonstrated strong bioadhesive capacity to the gut oral mucosal, including Peyer´s patches post-oral administration (154, 157). Moreover, using OVA as antigen model, systemic and mucosal immune responses reported after oral administration, indicated that a single dose of OVA-mannose and OVA-flagellin nanoparticles, elicited higher and balanced systemic specific antibody responses (IgG1 (Th2-response) and IgG2a (Th1-response)) compared to non-coated ones. In addition, coated nanoparticles were able to elicit higher levels of intestinal secretory IgA after oral immunization compared to subcutaneous immunization.
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5. Nanotoxicology: Towards a safe and successful nanotechnology
5.1. Nano-specific toxic effects?
It is well known that nanotechnology is one of the most innovative areas of research and development which can find application in almost all essential aspects of our life. Thus, nanomaterials are steadily increasing and many products furnished with nanotechnology are released to the market. However, the successes of nanotechnology have not been associated with a parallel development of the adequate methodology to asses and manage the potential risks for humans. In this regard, in the “late lessons from early warnings for nanotechnology” it was pointed out that: “a new technology will only be successful if those promoting it can show is safe, but history is littered with examples of promising technologies that never fulfilled the true potential and/or caused untold damage because early warnings were ignored” (235). It is clearly demonstrated that the application of nanotechnology in health care defined as nanomedicine, offers new opportunities to improve medical therapies and diagnosis in all relevant pathological conditions including cancer. However, the same properties (small size, chemical composition, structure, large surface area and shape), which make nanoparticles so attractive in medicine, may contribute to the toxicological profile of nanoparticles in biological systems. Hence, to use the potential of nanoparticles in nanomedicine, full attention must be given to safety and toxicological issues (236). In respond to this concern, nanotoxicology has arisen as a multi-interdisciplinary field in order to properly assess the safety of nanoparticles before exploiting their advantages. So far, most of the nanotoxicological research has involved a limited group of engineered nanoparticles, mainly inorganic nanomaterials. The toxicity of liposomes, dendrimers, polymers and emulsions has been studied to a much more limited degree, although they are the most employed for medical therapies and diagnosis. This situation is not surprising as there is a considerable database on the safety of its bulk materials, which are already frequently used in healthcare products. Therefore, these organic nanoparticles have been considered safe and not much attention has been paid to its toxicity based on the safety of its bulk material. However, the properties and behavior at the nano level are completely different from the bulk materials thus the toxicological approach is substantially different from the classical way to address the adverse health effects. Thus, to properly assess nanoparticle toxicity two concepts should be highlighted: (i) Nanoparticles are developed for their unique properties in comparison to bulk materials. Since surface is the contact layer with the body tissue and crucial determinant of particle response, these unique properties need to be investigated from a toxicological standpoint. When nanoparticles are used for their unique reactive characteristics it may be expected that these same characteristics also have an impact on the toxicity of such particles.
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(ii) Nanoparticles are attributed qualitatively different physico-chemical characteristics from micron-sized particles, which may result in changed body distribution, passage of the blood barrier, and triggering of blood coagulation pathways. In view of these characteristics specific emphasis should be on investigations in pharmacokinetics and distribution studies of nanoparticles. What is currently lacking is a basic understanding of the biological behavior of nanoparticles in terms of distribution in vivo both at the organ and cellular level (2). Although current tests and procedures in drug and device evaluation may be appropriate to detect many risks associated with the use of these nanoparticles, development of new protocols and guidelines to properly detect all potential “nano-risks” are extremely urgent (237). Specific importance should be devoted to the toxicity of the empty non-drug-loaded nanoparticles. For nanodevices used in medicine the focus in most papers is mainly on obtained reduction of toxicity of the incorporated drug, whereas the possible toxicity of the carrier used is not considered. Particular attention must be taken in the case of slowly degradable or non-degradable nanoparticles used for drug delivery. After the delivery of the drug/antigen, nanoparticles residual components may accumulate in the body causing possible adverse effects. Moreover, nanoparticle size and persistency are two of the main aspects to consider as toxicity risk factors. In this regard, Müller and collaborators have proposed a nanotoxicological classification system (NCS) based on the structure of the BCS displayed in Figure 7 (238). The NCS would be useful as a first approach, since it allows a clear and straightforward justification of possible toxicological risks of nanoparticles used in medicine. However, as it was mentioned before, other physicochemical properties such as shape or surface may exert an influence on the overall adversity in cells and tissues (239).
Figure 7. NCS for a precise differentiation of nanotoxicological risks derived from nanoparticulate carriers in the future. Suggested by Müller and collaborators (238).
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Therefore, it is necessary to better understand the influence of these parameters on the potential nanoparticle toxicity. Recent studies by Warheit (240) and Murdock et al. (241), emphasize the urgent need for a thorough physical and chemical characterization of nanoparticles before conducting any toxicological study. Moreover, this characterization should be carried out in the media to be performed subsequent in vitro studies because sometimes, the protein components of the culture media may cause aggregation of the nanoparticles as well as its surface modification. In this context, table IV summarizes the more significant nanoparticle physico-chemical properties that may be controlled in order to improve biocompatibility hence reducing toxic effects. Consequently, prior to evaluate the toxicity of nanoparticles by in vitro or in vivo studies, it should be considered in early stages, designing nanoparticles with appropriate physico-chemical characteristics that take into account not only its potential uses but also its risks.
Table IV. Physico-Chemical Nanoparticle Properties of relevance for Nanotoxicology.
Characteristic Biological Behavior
‐ The smaller nanoparticles are, the more the surface area they have per unit mass; thus more chemical molecules may attach to this surface, which would enhance
SIZE its reactivity and result in an increase of toxic effects ‐ Key factor with respect to translocation across cell barriers or along neuronal pathways
‐ Spherical nanoparticles with identical surface functionalization are generally SHAPE more toxic and more efficiently ingested than rod-shape particles
‐ Charge: Positively charged nanoparticles have a greater tendency of interacting with biological components compared with neutral and anionic. In consequence, cationic nanoparticles are generally defined as more toxic
SURFACE ‐ Chemistry: Reactive groups on particle surface influence their interaction with PROPERTIES biological material. Thus they would certainly modify the biological effects, affecting its safety mainly by causing oxidative stress
‐ Area: Due to their larger surface area, nanoparticles have greater tendency to agglomeration/aggregation which may result in undesirable effects
‐ Non-biodegradable nanoparticle may be accumulated unintentionally in different STABILITY tissues like the brain , gut, spleen and lymph nodes leading toxicity
‐ Once nanoparticles interact with the biological environments in the human body CORONA they are immediately covered by matrix molecules surrounding the particle thereby determining the impact and potential toxicity
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5.2. In vitro assessment of nanoparticle toxicity
In vitro toxicological assessment is a vitally important tool for nanotoxicology and is the next step that should be conduct when addressing the evaluation of nanoparticles toxicity after its physico- chemical characterization. The interaction, influence and possible toxicity of nanoparticles at cellular level are an essential focus in understanding nanoparticle compatibility versus toxicity. In addition, compared to in vivo studies, in vitro studies benefit from being faster, lower cost, allowing greater control, and minimizing ethical concerns. Consequently, in vitro models provide the possibility to investigate toxic effects on human cells extensively, which cannot be conducted in vivo. However, the extension of results from in vitro experiments for the prediction of in vivo toxicity is problematic due to the in vitro exposure conditions which usually feature much higher concentrations and exposure times than found in the cellular environment in vivo. Commonly, in vitro toxicity studies have used monocultures of cells that are specific to organs of the body. For example, macrophage cells are used as a model to study the effects of nanoparticles on the human immune system as well as dendritic cells. Additionally, specific cells such as the human hepatoma cell line HepG2 cells are also used for example for the liver and the human colon carcinoma cell line Caco-2 for the intestine. However, although monoculture systems provide the basis for high-throughput analysis for nanotoxicology, they do not represent a realistic model of how nanoparticles will interact with a specific organ of the body. Thus, it is essential that co-culture systems mimicking the in vivo system are established and used for the specific organs that are in danger or interacting with nanoparticles. However, these systems will not be able to be completely definitive of the in vivo situation. For in vitro studies, there are a number of different toxicological endpoints that researchers have used to assess the potential adverse effects that nanoparticles may have on organs of the human body. These include numerous different biochemical- and molecular-based testing strategies, investigating the potential for nanoparticles to cause cytotoxicity, inflammation, oxidative stress, cell proliferation and genotoxicity. The most commonly used in vitro assessment techniques are summarized in table V.
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Table V. Overview of common in vitro toxicological techniques used in nanotoxicology. Adapted from reference (242).
Cellular property/process Assay type Category Assays probed
MTT, MTS, XTT, WST-1, ATP, Metabolic activity Proliferation Alamar Blue
DNA synthesis [3H]thymidine incorporation
LDH, Trypan Blue, Neutral Red, Viability Necrosis Membrane integrity Propidium iodide
Apoptosis Membrane structure Annexin-V
Esterase activity/membrane Calcein acetoxymethyl/ethidium Live-Dead integrity homodimer
DNA fragmentation Comet, Sister Chromatid Exchange DNA damage DNA double-strand breakage TUNEL
Presence of reactive oxygen DCFA, Rhodamine123 species
C11-BODIPY, TBA assay for Lipid peroxidation malondialdehyde Mechanistic Presence of lipid Oxidative stress Amplex Red hydroperoxides
Antioxidant depletion DTNB
Superoxide dismutase activity Nitro blue tetrazolium
Superoxide dismutase Immunobloting expression
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; MTS: 3-(4,5-dimethylthiazol-2- yl)-5-(3-crboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; WST-1: 4-[3- (4- iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate. XTT: 2, 3-bis (2-methoxy-4- nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide; LDH: Lactate dehydrogenase; TUNEL: Terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP- digoxigenin nick end labeling; DTNB: 5,5-dithio-bis-2-nitrobenzoic acid; DCFA: 2′7′- dichlorofluorescein diacetate; ATP: Adenosine thriphosphate; TBA: Thiobarbituric Acid.
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In particular, for cytotoxicity studies, it is important to know in advance the sensitivity of cultured cells to changes in their environment as temperature or pH changes, among others, since the nanoparticles may be responsible for these changes. It should be noted that nanoparticles frequently interfere with commonly used cytotoxicity assays that are based on the detection of fluorescence or absorbance of various probes. In this context, the enzyme LDH (lactate dehydrogenase) can absorb to the surface of nanoparticles, masking their toxicity and thus providing a false-negative toxic result (243). Moreover, Worle-Knirsch and collaborators reported that CNTs interact with the MTT formazan used and provided false-negative toxicity (244). Therefore, the control of the experimental conditions is crucial to ensure that the cytotoxicity results are due only to the addition of the nanoparticles rather than to the culture conditions. Another important aspect to consider when assessing the potential toxicity of any form of nanoparticles, is to investigate how the particles enter the cells and in which compartments they may be found within or, which is also possible, if the nanoparticle stay attached to the cell membrane. This is of great importance, as it has been shown that the uptake and behavior can influence the cellular response following nanoparticles exposure (245). To date, there is a lack of consensus in the published literature on in vitro nanoparticle toxicity studies due to variable methods, materials, and cell lines. Standardization in experimental set up such as choice of model (monoculture/co-culture systems) and exposure conditions (cell confluence, exposure duration, nanoparticle-concentration ranges and dosing increments) is necessary in order for comparisons between studies conducted by different groups to be effective and also to predict in vivo responses in order to reduce and avoid extensive testing using laboratory animals. Nevertheless, to evaluate nanoparticle toxicity, in vitro models are insufficient to predict possible hazards to humans and to carry on successful transition through the clinical trials to the markets (246). Taking this into account, data obtained for in vitro studies require verification from in vivo experiments to accurately assess nanotoxicity.
5.3. In vivo assessment of nanoparticle toxicity
In the past years, the majority of nanotoxicity research has focused on cell culture systems; however, the data from these studies could be misleading and will require verification from in vivo studies to evaluate the impact of nanoparticle exposure within intact living organisms. In vivo studies are typically undertaken in rodents, although recently, zebrafish (Danio rerio) has emerged as a promising in vivo model for nanotoxicity studies (247). Despite of the fact that in vivo studies bear ethical consideration, due to the use and sacrifice of animals, these studies allow examination of long-term effects, tissue localization, biodistribution and retention/excretion of nanoparticles. To carry out adequate in vivo studies is necessary to fully understand the overall behavior that nanoparticles could exhibit within the organism: (i) nanoparticles can be administered or can enter into the body via six principal routes: i.v., dermal, subcutaneous,
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inhalation, intraperitoneal and oral, (ii) adsorption and/or interaction can occur when the nanostructures first interact with biological components (proteins, cells), (iii) afterward they can be absorbed and therefore distribute to various organs in the body and may remain the same structurally, be modified, or metabolized, or excreted (iv) they enter the cells of the organ where nanoparticles can be degraded or reside in the cells for an unknown amount of time before leaving to move to other organs or to be excreted. Currently there are insufficient data to understand the direct relationship between the physicochemical properties of nanoparticles and their in vivo behavior, thus, understand this relationship would provide a basis for evaluating the response and toxicity of nanoparticles and could lead to predictive models for assessing the toxicity of these new derived-nanotechnology materials.
Beyond the simple LD50 (lethal dose for 50% of participating animals) study, in vivo nanoparticle toxicity studies typically focus on one or more of three major areas: changes in blood serum chemistry and cell population, changes in tissue morphology, examined using histology, or the overall nanoparticle biodistribution. Typically, in vivo experiments utilize several of these, and other, techniques to maximize the toxicity information gained. As with in vitro studies, one must consider many aspects prior to beginning studies, such as nanoparticle choice and characterization, prior to exposure, and choice of relevant model system. Additionally, one must also consider issues of exposure route, nanoparticle dosing and specific tissue/organ or protein/cell type of interest, to examine histological or serum/hematological changes, respectively. Last but not least, it is also important when evaluating in vivo nanotoxicity, to conduct systematic studies to investigate chronic and acute exposure doses of nanoparticles with different physical or chemical variables such as size, charge, and hydrophobicity. However, a major concern, not only in nanotoxicology but in general toxicology, is to conduct animal testing following the principles of the 3 Rs: Replacement, Reduction and Refinement. So, as for now, the use of animals is necessary to evaluate the safety of nanoparticles in vivo, and there are currently no validated guidelines, in vivo nanotoxicological studies should be performed, whenever possible, following the principles of the 3 Rs. In this context, the standard procedures and guidelines proposed by the Organization for Economic Cooperation and Development (OECD), which are currently used to test and assess chemicals, are being modified to encompass these principles. Thus, in assessing in vivo toxicity of nanoparticles, the use of the OECD guidelines may be considered adequate because these guidelines following the principles of the 3 Rs; besides, it is noteworthy that several studies have shown that OECD guidelines can be adapted to assess in vivo nanotoxicity leading to valuable and consistent results (248-250).
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5.4. Nanotoxicology regulation: Is the current regulation robust enough to handle risks of nanomaterials?
Usually it is hard to figure out whether the inconsistencies observed in nanotoxicological studies are due to improper characterization of material itself or due to an inadequate or flawed design of the study conducted (239). In fact, the vast number of applications and the enormous amount of variables influencing the characteristics of the nanomaterials make particularly difficult the elaboration and standardization of appropriate nanotoxicological protocols. Therefore, drugs and diagnostics based on nanoparticles present challenges for regulatory agencies such as the FDA and the EMA, which do not have specific guidance documents for nanoparticles per se. Indeed, there is still no final consensus on which validated toxicological assays are appropriate and meaningful enough in the case of nanoparticles, how they optionally have to be adapted to this material, or whether new assays have to be developed and validated. The existing medical regulations are expected to be applied to nano-enabled drugs and diagnostics, but additional regulations, which consider their applicability to nanoparticles, may be required. In United States, the FDA has formed in 2006 the Nanotechnology Task Force to facilitate the regulation of nanoproducts. In addition, within the FDA´s National Center of Toxicological Research (NCTR), there are nanotechnology programs which are focused on two main areas: (i) evaluating the potential of nanomaterials in FDA-regulated products and (ii) modernizing toxicology approaches to enhance product safety. In this context, The Nanotechnology Characterization Laboratory has published a set of protocols relevant for the safety assessment of nanomedicines (251). In EU, within the Seventh Framework Program (FP7) several projects, such as NanoTEST, have been addressing the standardization of testing strategies for the safety assessment of nanoparticles for medical applications (252). Furthermore, the European Academies Science Advisory Council (EASAC) and the Joint Research Centre of the European Commission (JRC) recently published a report entitled “Impact of Engineered Nanomaterials on Health: Considerations for Benefit–Risk Assessment” (253, 254). The report seeks to identify gaps in current knowledge and provides a set of recommendations for further research; the authors emphasize the importance of ‘safety-by-design’ for the successful implementation of the emerging nanotechnologies. The International Organization for Standardization (ISO) has published several standards that are relevant for the classification of engineered nanomaterials (255). In addition, the OECD has launched the programme work named “Safety of Manufactured Nanomaterials” and within it has established the OECD working Party on Nanotechnology (WPN) to advise upon emerging policy issues of science, technology, and innovation related to the responsible development of nanotechnology (256). WPN has published a list of physicochemical properties that should be fully characterized for the different nanomaterials. However, the discussion about which properties are essential to fully characterize and describe nanoparticles is not finished yet.
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Therefore, the implementation of the regulatory mechanisms and standardized protocols to address the risk associated to nanotechnology in United States and Europe as well as in the other countries around the world is in progress. However, although numerous efforts have been carried out in response to specific calls for proposal of nanosafety projects or by independent projects studies in the various fields of nanotoxicology, there is still a lack of consistent data that need to be bridged. As illustrated in Figure 8, this requires, as first main step, performing adequate characterization to identify the potential risks associated to nanomedicines and thereby carry out nanoparticles toxicological assessment through methods and protocols developed by the interdisciplinary work. Thus, only a combination of efforts by the subjects with responsibility, researchers and public authorities can adequately address the challenge of complexity proposed by nanotoxicology.
Nano-enabled products development (Safety by design)
• Chemical composition • Particle size and size distribution • Morphology (crystalline/amorphous, shape) Potential Hazards Physico-Chemical • Surface chemistry, coating, functionalization Identification Characterization • Degree of agglomeration/aggregation • Distribution under experimental conditions (e.g. media with/without proteins) •Surface reactivity (corona) and/or surface load (zeta potential).
Nanotoxicity evaluation (in vitro/in vivo studies)
Assess current tools Adapt/Design new and their limitations Protocols/Methods (NPs interference with assay systmes) (Academia, goverments, industry) Integrate various aproaches into testing strategies (Adapt existing OECD guideilnes)
Figure 8. Schematic representation of the evaluation process of toxicity of nanoparticles for medical purposes highlighting the importance of an adequate physic-chemical characterization to identify potential risks.
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6. References
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250. M. Ema, A. Matsuda, N. Kobayashi, M. Naya, and J. Nakanishi. Dermal and ocular irritation and skin sensitization studies of fullerene C60 nanoparticles. Cutan Ocul Toxicol. 32:128-134 (2013). 251. Nanotechnology Characterization Laboratory (NCL). National Cancer Institute. U.S. National Institutes of Health. www.ncl.cancer.gov. 252. NanoTEST. Alternative testing strategies for the assessment of the toxicological profile of nanoparticles used in medical diagnosis. (7th Framework Programme). www.nanotest-pf7.eu. 253. European Academies Science Advisory Council (EASAC). Impact of Engineered Nanomaterials on Heath: Considerations for Benefit-Risk Assessment. EASAC Policy Report - JRC Reference Report. http://www.easac.eu/home/reports-and-statements/detail- view/article/first-joint.html (2011). 254. Joint Research Centre (JRC). Impact of Engineered Nanomaterials on Health: Considerations for Benefit-Risk Assessment - EASAC Policy Report - JRC Reference Report. http://publications.jrc.ec.europa.eu/repository/handle/111111111/22610 (2011). 255. International Organization for Standardization (ISO). ISO/TC 229 – Nanotechnologies. http://www.iso.org/iso/home/store/catalogue_tc/catalogue_tc_browse.htm?commid=381 983&published=on&includesc=true. (2005). 256. Organization for Economic Cooperation and Development (OECD). Safety of Manufactured Nanomaterials. http://www.oecd.org/env/ehs/nanosafety/. (2006).
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CHAPTER 2 AIM AND OBJECTIVES
CHAPTER 2: AIM AND OBJECTIVES
AIM & OBJECTIVES
Until today, many polymeric nanoparticles have demonstrated great potential as oral drug delivery systems. However, its clinical development sometimes is hindered by the limited knowledge of their possible harmful effects. Therefore, understanding of the interactions of polymeric nanoparticles with biological systems according to their physico-chemical properties and to assess their toxicity is clearly required in order to develop effective and safe nanomedicines for humans. In this context, an extensive research carried out in the department of Pharmacy and Pharmaceutical Technology, have proven that poly(anhydride) nanoparticles designed from the copolymer of methyl vinyl ether and maleic anhydride (Gantrez® AN 119) are ideal candidates as vehicles for oral delivery of drugs and antigens. Thus, it was considered really interesting to study its toxicity in relation to their physico-chemical characteristics by using appropriate protocols and guidelines. The general aim of this research project was to evaluate the toxicity of different poly(anhydride) nanoparticles intended for oral delivery. For this purpose, this aim was divided in the following objectives:
1. To develop a scalable spray-drying procedure for the preparation of different types of poly(anhydride) nanoparticles in order to simplify the method used so far.
2. To evaluate the in vitro toxicity of the poly(anhydride) nanoparticles in HepG2 and Caco-2 cells as well as their capability to interact and be internalized by intestinal cells.
3. To investigate the toxicological profile of these carriers by the oral route through acute and sub-acute toxicity studies (according to OECD guidelines) and biodistribution studies.
4. To approach the acute toxicity and biodistribution of the poly(anhydride) nanoparticles by the i.v. route.
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CHAPTER 3 EXPERIMENTAL DESIGN
CHAPTER 3: EXPERIMENTAL DESIGN
Nanoparticle formulation design and method development
• Selection of saccharide and Nanoparticles toxicity saccharide/polymer ratio selection •Spray-drying vs Lyophilization assessment
• Cytotoxicity evaluation Applicability: In vitro • Hemocompatibility studies Encapsulation of model drugs • Nanoparticles-Cell interaction studies [Hot saline antigenic complex (HS) of Brucella ovis and Atovaquone (ATO)] • Acute toxicity: OECD guideline 425 (limit test) In vivo , • Sub-chronic toxicity: Oral OECD guideline 407 • Biodistribution: In vivo/ex vivo studies using 99mTc radiolabelled nanoparticles
• Acute toxicity:
In vivo , OECD guideline 425 • Biodistribution: Intravenous In vivo/ex vivo studies using 99mTc radiolabelled nanoparticles
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CHAPTER 4 Spray-drying of poly(anhydride) nanoparticles for drug/antigen delivery
Spray-drying of poly(anhydride) nanoparticles for drug/antigen delivery
Patricia Ojer1, Hesham Salman1, Raquel Da Costa Martins1, Javier Calvo2, Adela López de Cerain1,
Carlos Gamazo1, Jose Luis Labandeira2, and Juan M. Irache1
1 University of Navarra, C/Irunlarrea 1, 31080-Pamplona, Spain. 2 Tres Cantos Medicines Development Campus, GlaxoSmithKline, 28760, Tres Cantos, Spain.
ABSTRACT
Gantrez® AN nanoparticles can be obtained by desolvation of the poly(anhydride) with an aqueous medium, followed by a purification step and freeze-drying. In order to simplify this method, a spray- drying procedure was evaluated here. For this purpose, suspensions of nanoparticles were mixed with carbohydrates before their drying. Lactose, in a saccharide/polymer ratio of 2, was found to be the most suitable excipient regarding the physico-chemical characteristics and stability of the resulting nanoparticles. These conditions can be successfully applied to the preparation of conventional, combined cyclodextrin/polyanhydride or pegylated nanoparticles. The capabilities of the new procedure were also studied by the characterization of nanoparticles loaded with either an antigenic extract (HS from Brucella ovis) or atovaquone (ATO). In all cases, the new formulations displayed similar physico-chemical characteristics that those of nanoparticles obtained by lyophilization. In summary, the spray-drying procedure can be an adequate alternative to lyophilization in the preparative process of poly(anhydride) nanoparticles.
Published in Journal of Drug Delivery Science and Technology 20(5):353-359 (2010).
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CHAPTER 4: Spray-drying of poly(anhydride) nanoparticles for drug/antigen delivery
1. INTRODUCTION
Over the few past decades, the technology of particle size reduction to nanometers has significantly advanced. The use of nanoparticles for drug delivery purposes may permit to achieve a personalized health care, rational drug design and targeted drug delivery (1). In principle, pharmaceutical nanoparticles can be of interest to conduct the loaded drug to its site of action or absorption and, as a consequence, improve the efficacy of the therapy and/or increase the bioavailability of the active molecule. In recent years, poly(anhydride) nanoparticles obtained from the copolymer between methyl vinyl ether and maleic anhydride (Gantrez® AN) have been developed and proposed as carriers with bioadhesive properties for the mucosal delivery of drugs (2, 3) and antigens (4, 5). In addition, due to the interesting ability of this polymer to incorporate different compounds, functionalized nanodevices can be easily produced including nanoparticles containing cyclodextrins(6), mannose- coated nanoparticles (7) or pegylated nanoparticles (8). In all cases, these carriers have been obtained by desolvation of the copolymer in a solution of acetone with a mixture of ethanol and water, followed by a purification step and freeze-drying. Lyophilization is a well known technique which is usually employed for the preservation of biodegradable nanoparticles (9, 10). Nevertheless, one important drawback associated with this technique is related with the long duration of the process. Therefore, alternatives to a lyophilization step are always welcomed if they can simplify and shorten the preparative process. In this context, one possible solution can be the spray-drying (SD) technique. Spray-drying is a manufacturing process commonly used in the industry, which converts a liquid into a homogeneous dry powder in a one-step process (11). The process involves the atomization of a liquid feedstock into a spray of droplets and contacting the droplets with hot air in a drying chamber. It offers a quicker process and has advantages such as its capability to influence and modulate the physico-chemical properties of the resulting solids (12). In addition, another important advantage is that this process can be applied to the effective drying of proteins without any significant degradation (13). On the other hand, spray-drying has been successfully applied in the production of a number of microdevices for drug delivery purposes, including poly(+/-)lactide microparticles (14), polycaprolactone microspheres (15) or albumin microspheres (16). On the contrary, spray-drying has been scarcely used for drying polymer nanoparticles. In this context, the first attempt was to spray poly(Ɛ-caprolactone) (PCL) nanocapsules in the presence of silicon dioxide particles as drying adjuvant (17). Thus, the nanoparticle suspensions led to microparticles presenting different and homogeneous nanocoating after the drying process (18, 19). Similarly, other soluble supports (i.e. polyvinylpyrrolidone K30 and mannitol) have also been proposed to dry polyester nanocapsules (20). Nevertheless, large amounts of these excipients (excipient/nanocapsules ratio of 10 by weight) were needed to obtain adequate reconstitution in aqueous media (20). 91 CHAPTER 4: Spray-drying of poly(anhydride) nanoparticles for drug/antigen delivery
The aim of this work was to develop a spray-drying procedure for the preparation of different types of poly(anhydride) nanoparticles in order to simplify the method used so far for the production of these carriers. In addition, the capabilities of the new procedure were also studied by the incorporation and characterization of the nanoparticles loaded with either an antigenic extract (HS from Brucella ovis) or ATO, used as models of active compounds.
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2. MATERIALS AND METHODS
2.1. Chemicals, reagents and solutions
Poly (methyl vinyl ether-co-maleic anhydride) or poly(anhydride) (Gantrez® AN 119; Mw 200,000) was kindly gifted by ISP (Barcelona, Spain). Polyethylene glycol 6000 (PEG6000) was provided by Fluka (Switzerland). 2-hydroxypropyl-β-cyclodextrin (HPCD) was provided by Sigma– Aldrich (Steinheim, Germany). Acetone was obtained from VWR Prolabo (Fontenay-sous-Bois, France). Deionized water (18.2MΩ resistivity) was prepared by a water purification system (Wasserlab, Pamplona, Spain). ATO was obtained from ChemPacific Corp (Baltimore, United States). The hot saline antigenic extract (HS) from Brucella ovis was obtained following the procedures detailed later. Mannitol, lactose, glucose and maltose were of Ph. grade and were purchased from Roig Pharma S.A. (Barcelona, Spain). All other chemicals and solvents used were of analytical grade.
2.2 Extraction and characterization of the HS antigen
The HS was obtained from the strain B. ovis REO 198 by the hot saline method previously described (21). The total protein was determined by the BCA™ Protein Assay method (22) using bovine serum albumin (BSA) as standard. The 2-keto-3-deoxyoctonate (KDO, exclusive marker of lipopolysaccharide (LPS)) analysis, corrected for 2-deoxyaldoses, was performed by the method of Warren modified by Osborn (23). The batch of antigen used to prepare the vaccine formulation contained 66.4±10.6% protein and 39.5±3.8% rough lipopolysaccharide (R-LPS).
2.3. Preparation of poly(anhydride) nanoparticles
All types of nanoparticles were prepared by solvent displacement followed by incorporation of an excipient and, finally, dried by spray-drying. In order to study the influence of the nature and amount of saccharide on the physico-chemical properties of the nanoparticles, the following saccharides were studied: lactose, mannitol, glucose and maltose.
2.3.1. Conventional poly(anhydride) nanoparticles (NP-SD) Briefly, 500 mg of poly(anhydride) were dissolved and stirred in 30 mL acetone. Then, the desolvation of the polymer was induced by the addition of 15 mL deionized water under magnetic stirring to the organic phase. In parallel, 1 g of saccharide was dissolved in 10 mL deionized water and added to the nanoparticle suspension under agitation for 5 min at room temperature. Finally, the suspension was dried in a Mini Spray-dryer Büchi B191 (Büchi Labortechnik AG, Switzerland). For this purpose, the following parameters were selected: inlet temperature: 90°C; outlet temperature 60°C; spray-flow 600 L/h, and aspirator at 90% of the maximum capacity. During the process the nanoparticle suspension was maintained under moderate agitation. The recovered powder was stored in closed vials at room temperature.
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For comparisons, nanoparticles prepared in the same way but in absence of any type of saccharide were used as control. Similarly, nanoparticles obtained by desolvation and dried by lyophilization (NP-L) were prepared as described previously (24).
2.3.2. Poly(anhydride) nanoparticles containing 2-hydroxypropyl-β-cyclodextrin (NP-HPCD- SD) Briefly, 500 mg of poly(anhydride) were dissolved and stirred in 20 mL acetone. Then, 10 mL acetone containing 125 mg HPCD were added to the polymer solution under magnetic stirring and incubated for 30 min. Nanoparticles were obtained by the addition of 15 mL deionized water under magnetic stirring to the organic phase. In parallel, 1 g of lactose was dissolved in 10 mL deionized water and added to the nanoparticle suspension under agitation for 5 min at room temperature before drying of this suspension as described in 2.3.1. The recovered powder was stored in closed vials at room temperature. For comparisons, nanoparticles obtained by desolvation and dried by lyophilization (NP- HPCD-L) were prepared as described previously (6).
2.3.3. Pegylated poly(anhydride) nanoparticles (PEG-NP-SD) In this case, 62.5 mg PEG6000 were dissolved in 15 mL acetone and mixed with 15 mL acetone containing 500 mg poly(anhydride) and incubated under magnetic stirring for 1 h. Then, nanoparticles were obtained by the addition of 15 mL deionized water under magnetic stirring to the organic phase. The solvents were eliminated under reduced pressure (Buchi R-144, Switzerland) and nanoparticles were purified by double centrifugation in water at 17,000 rpm for 20 min (Sigma 3K30, Germany). Nanoparticles were then resuspended in 25 mL deionized water containing 1 g of lactose and, finally, dried as described in 2.3.1. The recovered powder was stored in closed vials at room temperature. For comparisons, pegylated nanoparticles obtained by desolvation and dried by lyophilization (PEG-NP- L) were prepared as described previously (8).
2.4. Preparation of HS-loaded nanoparticles
To obtain conventional HS-loaded nanoparticles (NP-HS-SD), 20 mg of the HS from Brucella ovis were dispersed in 2 mL deionized water under sonication. Then, this aqueous phase was added to 30 mL acetone containing 500 mg poly(anhydride). After incubation for 1 h under magnetic stirring at room temperature, nanoparticles were obtained by the addition of 15 mL deionized water and incubation for 5 min at room temperature. Then, 10 mL of water containing 1 g of lactose was added and the suspension was dried in a Mini Spray-dryer Büchi B191 (Büchi Labortechnik AG, Switzerland) under the experimental conditions described above (see 2.3.1). For the preparation of pegylated HS-loaded nanoparticles (PEG-NP-HS-SD), 20 mg of Brucella ovis HS were dispersed in 2 mL deionized water under sonication. Then, this aqueous phase
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was added to 15 mL acetone containing 500 mg poly(anhydride). The mixture was incubated for 30 min under magnetic stirring at room temperature. On the other hand, 62.5 mg PEG6000 were dissolved in 15 mL acetone and stirred for 20 min. Then, this PEG solution was added to the HS and the polymer mixture and maintained under magnetic agitation for 1 h at room temperature. After incubation, nanoparticles were formed by the addition of 15 mL deionized water and further incubation at room temperature for 5 min. The solvents were eliminated under reduced pressure (Buchi R-144, Switzerland) and nanoparticles were purified by double centrifugation at 17,000 rpm for 20 min (Sigma 3K30, Germany). Nanoparticles were resuspended in 25 mL deionized water containing 1 g of lactose. Finally, the suspension was dried in a Mini Spray-dryer Büchi B191 (Büchi Labortechnik AG, Switzerland), under the experimental conditions described above (see 2.3.1).
2.5. Preparation of atovaquone-loaded nanoparticles
This formulation was prepared by encapsulation of ATO complexed with HPCD in poly(anhydride) nanoparticles (ATO-HPCD-NP-SD). For this purpose, 20 mg of HPCD and 2.5 mg ATO were dissolved and incubated in 10 mL of ethanol overnight. Then, the ethanol was evaporated up to 5 mL under reduced pressure. On the other hand, 500 mg poly(anhydride) were dissolved in 25 mL of acetone. After dissolution, the ethanolic phase containing ATO-HPCD complexes was added and the mixture was incubated for 20 min under agitation at room temperature. The nanoparticles were formed after addition of 15 mL deionized water to the acetone phase under magnetic stirring. Then, 10 mL of water containing 1 g of lactose was added and the suspension was dried in a Mini Spray-dryer Büchi B191 (Büchi Labortechnik AG, Switzerland) under the experimental conditions described above (see 2.3.1).
2.6. Characterization of nanoparticles
2.6.1. Physico-chemical characterization The particle size and the zeta potential of nanoparticles were determined by photon correlation spectroscopy (PCS) and electrophoretic laser doppler anemometry, respectively, using a Zetamaster analyzer system (Malvern Instruments, United Kingdom). The samples were diluted in deionized water and measured at room temperature with a scattering angle of 90°. All measurements were performed in triplicate. The morphological examination of the spray-dried powders was obtained by scanning electron microscopy (SEM) in a Zeiss DSM 940A scanning electron microscope (Oberkochen, Germany) coupled with a digital image system (DISS) Point Electronic GmBh. Previously, a small amount of the spray-dried powders was diluted with deionized water and the resulting suspension was centrifuged at 17,000 rpm (Sigma 3K30, Germany) for 20 min in order to eliminate sugars. Finally, the pellet was shaded with a 12 nm gold layer in a Hemitech K 550 Sputter-Coater.
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The spray-drying process yield was determined as the difference between the total amount of solids contained in nanoparticle suspensions dried and the sum of the powder collected after the drying process.
2.6.2. Quantification of HPCD in nanoparticles The amount of HPCD associated to the nanoparticles was estimated by quantification of HPCD content in the supernatants collected from the purification step, using a high performance liquid chromatography - evaporative light scattering detection (HPLC-ELSD) (25). The supernatants recovered during the purification step were diluted in 10 mL in water. A 1 mL aliquot of the supernatants was transferred to auto sampler vials, capped and placed on the HPLC auto sampler. The apparatus used for the HPLC analysis was an Agilent model 1100 series LC (Waldbronn, Germany) coupled with an ELSD 2000 (Alltech, Illinois, United States). An ELSD nitrogen generator (Alltech) was used as the source of the nitrogen gas. Data acquisition and analysis were performed with a Hewlett-Packard computer using the ChemStation G2171 AA program. Separation was carried out at 50°C on a reversed-phase ZorbaxEclipse XDB-Phenyl column (2.1 mm x 150 mm; particle size 5 μm) obtained from Agilent Technologies (Waldbronn, Germany). This column was protected by a 0.45 μm filter (Teknokroma, Spain). ELSD conditions were optimized in order to achieve maximum sensitivity: the drift tube temperature was set at 115°C, the nitrogen flow was maintained at 3.2 L/min and the gain was set to 1. The mobile phase composition was a mixture of acetonitrile (A) and water (B) in a gradient elution at a flow-rate of 0.25 mL/min. The injection volume was 5 μL. Under these experimental conditions, retention times for HPCD and poly(anhydride) were 10.26 ± 0.06 min and 1.08 ± 0.05 min respectively. For quantification, 30 mg of dry powder were dispersed in 10 mL water. Then, the suspensions of nanoparticles were centrifuged twice at 17,000 rpm for 20 min at 4°C and the supernatants recovered to quantify the free HPCD and poly(anhydride). The amount of HPCD and poly(anhydride) forming the nanoparticles were calculated as the difference between the initial amount of cyclodextrin and poly(anhydride) added and the amount of both recovered in the supernatants.
2.6.3. Quantification of PEG The amount of PEG associated to the nanoparticles, as well as the amount of poly(anhydride) in pegylated nanoparticles were estimated by HPLC coupled to and ELSD detector (26). Separation was carried out at 40°C on a PL aquagel-OH 30 column (300 mm×7.5 mm; particle size 8 μm) obtained from Agilent Technologies (GB, United Kingdom). ELSD conditions were optimized in order to achieve maximum sensitivity: the drift tube temperature was set at 110 °C, the nitrogen flow was maintained at 3 L/min and the gain was set to 1. The mobile phase composition was a mixture of methanol (A) and water (B) in a gradient elution at a flow rate of 1 mL/min. Under
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these experimental conditions, retention times for PEG 6000 and the polymer were 6.92 ± 0.02 and 4.48 ± 0.06 min, respectively. The amount of PEG associated to the nanoparticles was estimated by quantification of PEG content in the supernatants collected from either the nanoparticles purification step (for freeze-dried nanoparticles) or the dry powder (for the spray-drying nanoparticles), using HPLC-ELSD. Supernatants recovered during the purification step were diluted with water until a final volume of 10 mL was attained. For spray-dried nanoparticles, 30 mg of powder was dispersed in a total volume of 10 mL of deionized water before centrifugation at 17,000 rpm for 20 min at 4°C. In all cases, 20μl of these supernatants were injected onto the HPLC column. The amount of PEG associated to nanoparticles was calculated as the difference between the initial PEG added and the amount of PEG recovered in the supernatants during the purification step. Similarly, the amount of poly(anhydride) was estimated by the difference in the same way.
2.6.4. HS loading in the nanoparticles For the HS quantification, 10 mg of dry powdered nanoparticles, both NP-HS-SD and PEG- NP-HS-SD, were resuspended in 1 mL of deionized water and centrifuged (17,000 rpm, 20 min, 4°C). Then, the pellet was dissolved in acetone/dimethylformamide (DMF) (3:1) and kept for 1 h at - 80°C. Samples were centrifuged at 17,000 rpm for 20 min at 4°C and the pellets were washed with 1 mL acetone and kept 30 min at -80°C. After centrifugation under the same conditions, pellets were resuspended in 500 μL of ultrapure water in order to quantify antigenic proteins by the BCA Protein™ Assay. In any case, each type of nanoparticle was assayed in triplicate. The calculations were carried out via the application of a calibration curve of free HS obtained after proteins precipitation with acetone/DMF in ultrapure water (r2 > 0.999). HS loading was expressed as the amount of HS (in μg) per mg nanoparticles.
2.6.5. Study of the Structural Integrity of the Entrapped HS To evaluate the effect of the manufacturing process on the HS protein profile and antigenicity, proteins from spray-dried nanoparticles were extracted with acetone/DMF (3:1) as described above, and assayed by SDS-PAGE (21).
2.6.6. Quantification of ATO content The amount of ATO loaded into nanoparticles was calculated by UPLC (Ultra performance liquid chromatography). Briefly, the apparatus was an Agilent model 1100 series LC and a diode-array detector set at 254 nm. Data were collected and processed by chromatographic software MassLynx 4.1 (Waters). The chromatographic system was equipped with a reversed – phase 50 x 2.1 mm UPLC Acquity BEH C18 column (50 mm x 2.1 mm, 1.7 μm; Waters). The gradient elution buffers were A (Methanol and 0.1 % formic acid) and B (water and 0.1 % formic acid). The column was eluted with a linear gradient consisted of 90% A over 0.5 min, 90 to 10 % over 0.5 to 4 min, 10% over 4 to 4.5 min, returned to 90 for 0.5 min and kept for a further 1 min before the next injection. Total run was 97 CHAPTER 4: Spray-drying of poly(anhydride) nanoparticles for drug/antigen delivery
5 min, volume injection was 5 μL and the flow rate of 500 μL/min. The limit of quantification was calculated to be 0.5 μg/mL with a relative standard deviation lower than 2.5%. For analysis, 5 mg of dry powder were digested with 1 mL acetonitrile. After filtering through 0.2 μm PTFE filter (Millipore, MA, United States), the samples were analyzed and quantified by UPLC. All samples were assayed in triplicate and results were expressed as the amount of ATO (in μg) per mg nanoparticles.
2.7. Statistical analysis
Values of P<0.05 were considered as a statistically significant difference. All calculations were performed using SPSS® statistical software program (SPSS® 15.0, Microsoft, United States).
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3. RESULTS
3.1. Effect of saccharides on the characteristics of poly(anhydride) nanoparticles (NP-SD)
When nanoparticle suspensions (obtained by desolvation of an acetone solution of the polymer with water) were directly dried in the spray-drying apparatus, the subsequent dispersion of the dry powder in an aqueous medium gave always heterogeneous dispersions characterized by the presence of large aggregates of polymer as well as some microparticles in the aqueous phase with a size higher than 2.0 μm. In order to solve this drawback our strategy was to incorporate in the nanoparticle dispersions, pharmaceutical excipients capable to protect nanoparticles against the drying stress induced in the spray-drying apparatus. Figure 1 shows the influence of the saccharide/polymer ratio, when either lactose or mannitol were used, on the mean size of the resulting poly(anhydride) nanoparticles. Interestingly, similar results were found for both types of excipients. Thus, for a saccharide/polymer ratio of 0.5, a similar phenomenon to that observed without any type of excipient was found, large and particles bigger than 2 μm were detected. On the contrary, when the ratio was 1.0 or higher, the dispersion of the dry powder in water gave homogeneous suspensions of nanoparticles with a mean size lower than 200 nm. Furthermore, by increasing the saccharide/polymer ratio a slightly decrease of the size of the resulting nanoparticles was observed (Figure 1.). Table I summarizes the influence of the type of saccharide on the physico-chemical characteristics of the resulting nanoparticles for a saccharide/polymer ratio of 2. This ratio was the most suitable in order to obtain the optimal nanoparticle size using the lowest amount of saccharide.
Lactose Mannitol Figure 1. Influence of the saccharide/poly(anhydride) ratio on the diameter of the 2000 nanoparticles obtained by spray- drying and after resuspension in
Size (nm) 150 purified water. Data expressed as 100 mean ± SD (n=3). Saccharide: 50 lactose or mannitol. 0 01234 Saccharide/Polymer ratio (w/w)
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Table I. Influence of the type of saccharide on the physico-chemical characteristics of nanoparticles obtained by spray-drying. Experimental conditions; saccharide/polymer ratio of 2. Data expressed as the mean ± SD (n=6). PDI: polydispersity index. Adhesivity: adhesion of the resulting powder to the cyclone and receptacle glass walls of the spray-drying apparatus: (-) no adhesion, (+) very slight adhesion, (+++) intensive adhesion, (++++) very intensive adhesion.
Size Zeta potential Yields Excipient Adhesivity (nm) PDI (mV) (%) Lactose 149 ± 1 0.09 ± 0.02 -57.90 ± 0.95 67.5 ± 0.4 + Mannitol 135 ± 1 0.09 ± 0.03 -59.40 ± 1.99 62.3 ± 0.7 - Glucose 129 ± 1 0.13 ± 0.05 -60.80 ± 0.33 54.7 ± 1.7 ++++ Maltose 128 ± 1 0.13 ± 0.06 -60.83 ± 0.37 59.1 ± 1.3 +++
In all cases, the mean size and zeta potential of nanoparticles was similar and independent of the type of saccharide used. Thus, the diameter of nanoparticles was about 130-150 nm and the surface charge of the resulting carriers was around –60 mV. Similarly, in all cases, the particles displayed and spherical shape and a smooth surface. Figure 2 shows SEM photograph corresponding to nanoparticles prepared in the presence of lactose.
2 μm
Figure 2. SEM photograph of poly(anhydride) nanoparticles obtained by spray-drying in the presence of lactose.
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Finally, another important aspect was the amount of poly(anhydride) transformed into nanoparticles after desolvation and the yield of the spray-drying process. In the former, more than 90% of poly(anhydride) was transformed into nanoparticles. In the latter, the yield of the spray-drying process was calculated to be between 55 and 68%, depending on the type of saccharide used. Nevertheless, both lactose and mannitol appeared to be more efficient than glucose or maltose to induce the production of more homogeneous populations of nanoparticles. In fact, the polydispersity of nanoparticles was lower with lactose or mannitol than when either glucose or maltose was used. In addition, the use of glucose or maltose resulted in the production of a sticky and hygroscopic powder, which hampered their recovery and a significantly lower yield. According to these results, the stability of the nanoparticles formulated with either mannitol or lactose was evaluated after their storage in close vials at 25°C one year after their preparation. Thereafter, nanoparticles formulated with lactose maintained all of their original physicochemical characteristics (size: 145 nm; PDI: 0.1; zeta potential: -60 mV). However, for nanoparticles prepared in the presence of mannitol, both the size (about 430 nm) and polydispersity (PDI of 0.5) were dramatically increased (Figure 3).
1) 20 Lactose 15 Mannitol
10
Intensity (%) (%) Intensity 5 Intensity (%)
0 0.1 1 10 100 1000 10000
Size (d.nm) (d.nm) 2) 20 Lactose
15 Mannitol
10
Intensity (%) (%) Intensity (%) Intensity 5
0 0.1 1 10 100 1000 10000 SizeSize (d.nm)(d.nm)
Figure 3. Particle size distribution of nanoparticles obtained by spray-drying. 1) Size immediately after preparation, 2) Size one year after reparation of nanoparticle batch.
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3.2. Preparation and characterization of NP-HPCD-SD and PEG-NP-SD
From these results it was clear that the most appropriate saccharide for the preservation of the physico-chemical characteristics of nanoparticles appeared to be lactose at an excipient/polymer ratio of at least 2. Under these experimental conditions, cyclodextrin-poly(anhydride) nanoparticles (NP-HPCD) and pegylated nanoparticles (PEG-NP) were prepared by spray-drying and compared with nanoparticles obtained by lyophilization. Table II summarizes these results.
Table II. Influence of the preparative method on the physico-chemical characteristics of nanoparticles obtained by either spray-drying or lyophilization. NP-HPCD: 2-hydroxypropyl-β- cyclodextrin-poly(anhydride) nanoparticles, PEG-NP: poly(anhydride) nanoparticles pegylated with PEG 6000. Data expressed as mean values ± standard deviations (n=6).
HPCD PEG Size Zeta potential Formulations PDI Yields (%) associated associated (nm) (mV) (µg/mg NP) (µg/mg NP) NP-HPCD 168 ± 1.0 0.10 ± 0.03 -52.8 ± 0.5 74.2 ± 0.8 71.9 ± 3.2 - Spray-Drying
NP-HPCD 140 ± 1.2 0.08 ± 0.02 -58.4 ± 1.5 81.8 ± 2.4 68.4 ± 4.3 - Lyophilization
PEG-NP 126 ± 0.6 0.17 ± 0.01 -54.1 ± 3.5 76.4 ± 0.5 - 55.6 ± 2.3 Spray-Drying
PEG-NP 158 ± 0.9 0.11 ± 0.02 -62.9 ± 1.0 80.9 ± 1.7 - 53.5 ± 0.9 Lyophilization
In all cases, little differences in the physico-chemical characteristics of nanoparticles obtained by either spray-drying of lyophilization were observed. Interestingly the amount of poly(anhydride) transformed into nanoparticles was also higher than 90% and the amount of PEG6000 or HPCD associated to nanoparticles was found to be statistically similar when the carriers were produced by spray-drying or lyophilization (p<0.05). On the contrary, when nanoparticles were produced by spray-drying, the resulting nanoparticles displayed a slightly higher polydispersity than when obtained after lyophilization; although this parameter was always above 0.3. Similarly, batches produced by spray-drying showed always a lower yield than when produced by lyophilization (about 68% vs 90%). The morphological analysis by scanning electron microscopy showed that the incorporation of HPCD in or pegylation of poly(anhydride) nanoparticles clearly modified the aspect of the resulting nanoparticles. Thus, the presence of the cyclodextrin or PEG6000 produced rough and
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irregular shaped nanoparticles, whereas a smooth surface is typical for conventional nanoparticles (Figure 4).
(A) (A.1) (B) (B.1)
500 nm 1 μm
(C) (C.1)
1 μm
Figure 4. SEM photographs of nanoparticles obtained by spray-drying. (A) NP-SD. (A.1) Magnification of a section of photograph (A). (B) NP-HPCD-SD. (B.1) Magnification of a section of photograph (B). (C) PEG-NP-SD. (C.1) Magnification of a section of photograph (C).
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3.3. Preparation and characterization of nanoparticles encapsulating model drugs
In order to study the capability of the new method to produce drug- or antigen-loaded nanoparticles, the HS of Brucella ovis and ATO were selected as model of active molecule to incorporate into poly(anhydride) nanoparticles prepared by spray-drying. Table III summarizes the main physicochemical characteristics of these formulations. For HS loaded nanoparticles, all formulations were characterized by a homogeneous size of about 150 nm, with a low PDI (≈0.2) and a negative zeta potential. On the other hand, the HS loading was found to be significantly higher for conventional nanoparticles (NP-HS-SD) than for pegylated ones (PEG- NP-HS-SD) (p<0.05).
Table III. Physico-chemical characteristics of HS-loaded nanoparticles and ATO-loaded nanoparticles obtained by spray-drying.Data expressed as the mean ± standard deviation (n=6). NP- HS-SD: Hs-loaded poly(anhydride) nanoparticles; PEG-NP-HS-SD: HS-loaded in pegylated nanoparticles; ATO-HPCD-NP-SD: atovaquone loaded in hydroxypropyl cyclodextrin- poly(anhydride) nanoparticles. PDI: polydispersity index.
Size Zeta HS loading ATO loading Formulations PDI potential Yields (%) (nm) (mV) (µg/mg NP) (µg/mg NP)
NP-HS-SD 141 ± 2 0.18 ± 0.02 - 49.03 ± 0.4 72.3 ± 0.7 34.9 ± 0.4 -
PEG-NP-HS-SD 155 ± 2 0.21 ± 0.01 - 42.3 ± 1.4 74.4 ± 1.2 27.9 ± 2.0 -
ATO-HPCD-NP-SD 305 ± 7 0.12 ± 0.03 - 34.12 ± 3.6 71.8 ± 1.5 - 27.2 ± 0.5
Figure 5 shows the SDS-PAGE of the HS extracted from the two different nanoparticle formulations. By comparing the apparent molecular weight of the antigenic proteins, it could be seen that the protein profile and the integrity of the HS extracted from the nanoparticles was similar to that of free HS and, furthermore, did not reveal the presence of additional protein bands as consequence of the entrapment and/or spray-drying procedures. Finally, ATO-loaded nanoparticles displayed a size of about 305 nm with a negative surface charge. The antiprotozoan was successfully incorporated with values of encapsulation efficiency higher than 87%.
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Figure 5. SDS-PAGE and Coomasie blue stain profiles of free and entrapped HS from B. ovis REO 198. Lanes: 1: Molecular Mass Marker Precision Plus ProteinTM KaleidoscopeTM Standards (BioRad); 2: free HS from B. ovis REO 198; 3: HS extracted from conventional HS-loaded nanoparticles (NP-HS-SD); 4: HS extracted from pegylated HS-loaded nanoparticles (PEG-NP-HS- SD).
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4. DISCUSSION In the present study we have developed a new strategy to facilitate and simplify the production of poly(anhydride) nanoparticles. These carriers have been currently produced after the desolvation of the copolymer (dissolved in acetone) with a mixture of ethanol and water and, subsequent, lyophilization of the resulting nanoparticles (6, 8, 24). Overall a typical production takes about 50 h. So, in order to simplify and shorten this process we decided to exchange the lyophilization step by a drying process in a spray-drier apparatus. This strategy is not new and, in the last years, different authors have been suggested and described it as a real alternative for lyophilization of microparticles and nanoparticles (11, 20, 27). In fact, the spray-drying method offers some important advantages such as its rapidity and, in some cases, the possibility to modulate the physicochemical characteristics of the resulting powder (12). During the optimization of the spray-drying process of poly(anhydride) nanoparticles, we noticed two main problems. The former was that after the spray-drying of poly(anhydride) suspensions, the yield of the process was low and the resulting powders were extremely difficult to redisperse in water. The latter was that this irreversible process was amplified when ethanol was present in the nanoparticle suspensions. These observations are in accordance with a previous work published by Muller and collaborators, in which they observed a similar phenomenon with PCL nanocapsules (17). They argued the need of an adjuvant (colloidal silicon dioxide 50% w/w) in order to induce a dilution effect which would reduce the possibilities for nanoparticle interaction and fusion due to the temperature of the process. In our case, we decided to incorporate saccharides or polyols in the poly(anhydride) nanoparticle suspensions. Polyols, mono and disaccharides, are generally considered adequate and GRAS excipients for a number of pharmaceutical operations, including spray-drying. Moreover, these excipients may also be interesting in terms of producing dry powders with adequate flow properties, which can be of use when facilitating the filling of capsules, vials or sachets. In this context, lactose and maltose (disaccharides), glucose and mannitol (monosaccharides) were chosen for the spray- drying of poly(anhydride) nanoparticles. In a first approach, it was demonstrated that a saccharide/poly(anhydride) ratio of at least 2 was necessary to produce a homogeneous reconstitution of nanoparticles in an aqueous environment. On the other hand, it was also clear that both lactose and mannitol displayed a higher capacity to protect the spray-dried nanoparticles than glucose or maltose (Table I). This fact could be related with a combination of the two following factors: hygroscopicity and glass transition temperature (Tg). In fact, glucose and maltose are very hygroscopic in their amorphous states and interact strongly with water molecules (12, 28, 29) whereas lactose and mannitol are considered poorly hygroscopic (28, 30, 31). Therefore, this high ability to interact with water molecules would be responsible for the production of a more sticky powder when glucose or maltose rather than mannitol or lactose were used.
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On the other hand, when either mannitol or lactose were used the resulting powders demonstrated adequate flow properties. Moreover, nanoparticles produced in the presence of either lactose or mannitol displayed similar physico-chemical characteristics to those produced by lyophilization (6, 8, 24). However, when both types of poly(anhydride) nanoparticles were stored for a long time at room temperature, their behavior after resuspension in water was completely difference (Figure 3). In fact, nanoparticles produced in the presence of mannitol appeared to aggregate and, after resuspension in water, displayed a significantly higher size and PDI. This fact may be explained by the lower Tg of mannitol (of about 13ºC (32)) compared to that of lactose (114ºC (33)). Thus, a low Tg would promote crystallization during storage and further creation of crystalline bridges (34), which consequently negatively affected the stability of nanoparticles. In this context, Yoshinari and collaborators described that the polymorphic transition from δ- to β - form of mannitol is triggered by moisture with a consequent morphological change in which water molecules would act as a molecular loosener to facilitate this conversion as a result of multi-nucleation (30). In contrast, the high Tg of lactose may provide an advantage in the stabilization of dried nanoparticles due to a better preservation of the amorphous state. Furthermore, taking into account that the poly(anhydride) nanoparticles are mainly intended for the encapsulation of biologically active molecules, several studies have suggested that the election of an amorphous carbohydrate as drying excipients may be more suitable for the preservation of both the structure and activity of therapeutic proteins (35). Based on these results, lactose was chosen for the preparation of nanoparticle formulations (Table II). In all cases, nanoparticles prepared by spray-drying can be considered similar to those produced by lyophilization. Nevertheless, a slight increment on the PDI of nanoparticle batches produced by spray-drying was noticed. The applicability of the spray-drying technique was also evaluated with either drug or antigen-loaded formulations by the incorporation of the HS from Brucella ovis or ATO into nanoparticles. For HS-loaded nanoparticles, the amount of antigen entrapped was considered high in spite of pegylation of nanoparticles decreased the antigen loading of the resulting carriers. More important, the preparative process did affect neither the integrity nor the composition of proteins included in the HS. In this point, it is interesting to highlight the maintenance of the profile and integrity of the outer membrane proteins (Omp 31, Omp 25, Omp 22 and L-Omp 19), which are of particular importance to induce protection against a Brucella infection (21, 36). On the other hand, ATO is a highly lipophilic drug. For this reason it was necessary to prepare a drug-cyclodextrin complex in order to increase the drug encapsulation in nanoparticles. Under these experimental conditions, the drug loading was calculated to be about 27 µg/mg with encapsulation efficiency close to 90%. These values are similar to those previously reported by Cauchetier and collaborators related with the encapsulation of this antiprotozoan in PCL nanocapsules (37).
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5. CONCLUSIONS For the preparation of poly(anhydride) nanoparticles by spray-drying procedure, both the solvent in which these carriers were sprayed as well as the presence of carbohydrates appear to be key parameters influencing the characteristics of the final product. Thus, for the preparation of nanoparticles by this procedure the presence of ethanol has to be avoided. On the other hand, lactose at a saccharide/polymer ratio of 2 appears to provide the best conditions to produce a dry powder that can be easily reconstituted in a nanoparticle suspension by simple addition of water. More importantly, the characteristics of nanoparticles produced by spray-drying were similar to those previously reported and obtained by lyophilization. Finally, the spray-drying procedure can be applied to the preparation of different types of poly(anhydride) nanoparticles containing sensitive proteins, antigenic extracts or drugs.
Acknowledgements This work was supported by grants from the “Instituto de Salud Carlos III” (number: PI070326; Res. 15/10/2007) and Department of Health of the Government of Navarra (Res. 2118/2007) in Spain. Patricia Ojer was also financially supported by grant from the Education Department of the Government of Navarra in Spain. We also thanks to Dr. Jon Etxeberria Uranga, (Materials Department, CEIT, 20018 San Sebastian, Spain) and Prof. Dr. Rafael Jordana (Zoology and Ecology Department, University of Navarra, 31080 Pamplona, Spain) for SEM photographs.
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6. References
1. G.A. Hughes. Nanostructure-mediated drug delivery. Nanomedicine. 1:22-30 (2005). 2. P. Arbos, M.A. Campanero, M.A. Arangoa, and J.M. Irache. Nanoparticles with specific bioadhesive properties to circumvent the pre-systemic degradation of fluorinated pyrimidines. J Control Release. 96:55-65 (2004). 3. K. Yoncheva, S. Gomez, M.A. Campanero, C. Gamazo, and J.M. Irache. Bioadhesive properties of pegylated nanoparticles. Expert Opin Drug Geliv. 2:205-218 (2005). 4. H.H. Salman, C. Gamazo, M.A. Campanero, and J.M. Irache. Salmonella-like bioadhesive nanoparticles. J Control Release. 106:1-13 (2005). 5. S. Gomez, C. Gamazo, B. San Roman, M. Ferrer, M.L. Sanz, S. Espuelas, and J.M. Irache. Allergen immunotherapy with nanoparticles containing lipopolysaccharide from Brucella ovis. Eur J Pharm Biopharm. 70:711-717 (2008). 6. M. Agueros, P. Areses, M.A. Campanero, H. Salman, G. Quincoces, I. Penuelas, and J.M. Irache. Bioadhesive properties and biodistribution of cyclodextrin-poly(anhydride) nanoparticles. Eur J Pharm Sci. 37:231-240 (2009). 7. H.H. Salman, C. Gamazo, M.A. Campanero, and J.M. Irache. Bioadhesive mannosylated nanoparticles for oral drug delivery. J Nanosci Nanotechnol. 6:3203-3209 (2006). 8. K. Yoncheva, E. Lizarraga, and J.M. Irache. Pegylated nanoparticles based on poly(methyl vinyl ether-co-maleic anhydride): preparation and evaluation of their bioadhesive properties. Eur J Pharm Sci. 24:411-419 (2005). 9. B. Magenheim and S. Benita. Nanoparticle characterization: a comprehensive physicochemical approach. STP Pharma. 1:221-241 (1991). 10. E. Allemann, R. Gurny, and E. Doelker. Drug-loaded nanoparticles - preparation and drug targeting issues. Eur J Pharm Biopharm. 39:173-191 (1993). 11. C. Freitas and R.H. Mullera. Spray-drying of solid lipid nanoparticles (SLN TM). Eur J Pharm Biopharm. 46:145-151 (1998). 12. J. Broadhead, S.K.E. Rouan, and C.T. Rhodes. The spray drying of pharmaceuticals. Drug Dev Ind Pharm. 18:1169-1206 (1992). 13. M. Adler, M. Unger, and G. Lee. Surface composition of spray-dried particles of bovine serum albumin/trehalose/surfactant. Pharm Res. 17:863-870 (2000). 14. R. Bodmeier and H.G. Chen. Preparation of biodegradable poly(+/-)lactide microparticles using a spray-drying technique. J Pharm Pharmacol. 40:754-757 (1988). 15. P. Giunchedi, B. Conti, L. Maggi, and U. Conte. Cellulose acetate butyrate and polycaprolactone for ketoprofen spray-dried microsphere preparation. J Microencapsul. 11:381-393 (1994).
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16. F. Pavanetto, I. Genta, P. Giunchedi, B. Conti, and U. Conte. Spray-dried albumin microspheres for the intra-articular delivery of dexamethasone. J Microencapsul. 11:445-454 (1994). 17. C.R. Muller, V.L. Bassani, A.R. Pohlmann, C.B. Michalowski, P.R. Petrovick, and S.S. Guterres. Preparation and characterization of spray-dried polymeric nanocapsules. Drug Dev Ind Pharm. 26:343-347 (2000). 18. A. Raffin Pohlmann, V. Weiss, O. Mertins, N. Pesce da Silveira, and S. Staniscuaski Guterres. Spray-dried indomethacin-loaded polyester nanocapsules and nanospheres: development, stability evaluation and nanostructure models. Eur J Pharm Sci. 16:305-312 (2002). 19. P. Tewa-Tagne, S. Briancon, and H. Fessi. Spray-dried microparticles containing polymeric nanocapsules: formulation aspects, liquid phase interactions and particles characteristics. Int J Pharm. 325:63-74 (2006). 20. P. Tewa-Tagne, S. Briancon, and H. Fessi. Preparation of redispersible dry nanocapsules by means of spray-drying: development and characterisation. Eur J Pharm Sci. 30:124-135 (2007). 21. R. Da Costa Martins, C. Gamazo, and J.M. Irache. Design and influence of gamma- irradiation on the biopharmaceutical properties of nanoparticles containing an antigenic complex from Brucella ovis. Eur J Pharm Sci. 37:563-572 (2009). 22. P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, and D.C. Klenk. Measurement of protein using bicinchoninic acid. Anal Biochem. 150:76-85 (1985). 23. M.J. Osborn. Studies on the gram-negative cell wall. I. Evidence for the role of 2-Keto-3- deoxyoctonate in the lipopolysaccharide of Salmonella Thypimurium. Proc Natl Acad Sci USA. 50:499-506 (1963). 24. P. Arbos, M. Wirth, M.A. Arangoa, F. Gabor, and J.M. Irache. Gantrez AN as a new polymer for the preparation of ligand-nanoparticle conjugates. J Control Release. 83:321-330 (2002). 25. M. Agueros, M.A. Campanero, and J.M. lrache. Simultaneous quantification of different cyclodextrins and Gantrez by HPLC with evaporative light scattering detection. J Pharm Biomed Anal. 39:495-502 (2005). 26. V. Zabaleta, M.A. Campanero, and J.M. Irache. An HPLC with evaporative light scattering detection method for the quantification of PEGs and Gantrez in PEGylated nanoparticles. J Pharm Biomed Anal. 44:1072-1078 (2007). 27. M.V. Chaubal and C. Popescu. Conversion of nanosuspensions into dry powders by spray drying: a case study. Pharm Res. 25:2302-2308 (2008). 28. A. Sokolovsky. Effect of humidity on hygroscopic properties of sugars and caramel. J Ind Eng Chem. 29:1422-1423 (1937).
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29. S. Jaya and H. Das. Effect of maltodextrin, glycerol monostearate and tricalcium phosphate on vacuum dried mango powder properties. J Food Eng. 63:125-134 (2004). 30. T. Yoshinari, R.T. Forbes, P. York, and Y. Kawashima. Moisture induced polymorphic transition of mannitol and its morphological transformation. Int J Pharm. 247:69-77 (2002). 31. P.F. Fox and P.L.H. McSweeney. Advanced Dairy Chemistry. Volume 3: Lactose, water, salts and minor constituents. Springer Sciences, New York, 2009. 32. L. Yu, D.S. Mishra, and D.R. Rigsbee. Determination of the glass properties of D-mannitol using sorbitol as an impurity. J Pharm Sci. 87:774-777 (1998). 33. L.S. Taylor and G. Zografi. Sugar-polymer hydrogen bond interactions in lyophilized amorphous mixtures. J Pharm Sci. 87:1615-1621 (1998). 34. H.K. Chan, A.R. Clark, J.C. Feeley, M.C. Kuo, S.R. Lehrman, K. Pikal-Cleland, D.P. Miller, R. Vehring, and D. Lechuga-Ballesteros. Physical stability of salmon calcitonin spray-dried powders for inhalation. J Pharm Sci. 93:792-804 (2004). 35. A. Millqvist-Fureby, M. Malmsten, and B. Bergenstahl. Spray-drying of trypsin - surface characterisation and activity preservation. Int J Pharm. 188:243-253 (1999). 36. R. Kittelberger, F. Hilbink, M.F. Hansen, G.P. Ross, G.W. de Lisle, A. Cloeckaert, and J. de Bruyn. Identification and characterization of immunodominant antigens during the course of infection with Brucella ovis. J Vet Diagn Invest. 7:210-218 (1995). 37. E. Cauchetier, M. Deniau, H. Fessi, A. Astier, and M. Paul. Atovaquone-loaded nanocapsules: influence of the nature of the polymer on their in vitro characteristics. Int J Pharm. 250:273-281 (2003).
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CHAPTER 5 Cytotoxicity and cell interaction studies of bioadhesive poly(anhydride) nanoparticles for oral antigen/drug delivery
Cytotoxicity and cell interaction studies of bioadhesive poly(anhydride) nanoparticles for oral antigen/drug delivery
Patricia Ojer 1, Lukas Neutsch 2, Franz Gabor 2, Juan Manuel Irache 1, and Adela López de Cerain 3
1 Department of Pharmacy and Pharmaceutical Technology. University of Navarra, Irunlarrea 1, E-31008 Pamplona, Spain. 2 Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Pharmacy Center, Althanstrasse 14, A-1090 Vienna, Austria. 3. Department of Pharmacology and Toxicology, University of Navarra, Irunlarrea 1, E-31008 Pamplona, Spain.
ABSTRACT
The use of bioadhesive polymers as nanodevices has emerged as a promising strategy for oral delivery of therapeutics. In this regard, poly(anhydride) nanoparticles have shown great potential for oral drug delivery and vaccine purposes. However, despite extensive research into the biomedical and pharmaceutical applications of poly(anhydride) nanoparticles, there are no studies to evaluate the interaction of these nanoparticles at a cellular level. Therefore, the main objectives of this study were to evaluate the cytotoxicity as well as the cell interaction of different poly(anhydride) nanoparticles: conventional (NP), nanoparticles containing 2-hydroxypropyl-β-cyclodextrin (NP-HPCD) and nanoparticles coated with polyethylene glycol 6000 (PEG-NP). For this purpose, nanoparticles were prepared by solvent displacement method and labelled with BSA-FITC (Bovine Serum Albumin- Fluorescein Isothiocyanate conjugate). Nanoparticles displayed a size about 175 nm with negative surface charge. Cytotoxicity studies were developed by MTS and LDH assays in HepG2 and Caco-2 cells. Results showed that only in HepG2 cells, NP and NP-HPCD induced significant cytotoxicity at the highest concentrations (1 and 2 mg/mL) and incubation times (48 and 72 h) tested. Studies to discriminate between cytoadhesion and cytoinvasion were performed at 4ºC and 37ºC in Caco-2 cell line as intestinal cell model. Nanoparticles showed cytoadhesion to the cell surface but not internalization; PEG-NP was the most bioadhesive followed by NP-HPCD and NP as demonstrated by flow cytometry. Finally, cellular localization of particles by fluorescence confocal microscopy confirmed the association of these nanoparticles with cells. Thus, this study demonstrated the safety of NP, NP-HPCD and PEG-NP at cellular level and its bioadhesive properties within cells.
Published in Journal of Biomedical Nanotechnology 9(11):1891-1903 (2013).
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CHAPTER 5: Cytotoxicity and cell interaction studies of bioadhesive poly(anhydride) nanoparticles for oral antigen/drug delivery
1. INTRODUCTION
Nanotechnology is one of the fastest growing sectors of the high-tech economy. In the last two decades it has been widely used in the pharmaceutical industry, medicine and engineering technology (1, 2). However, while the number of nanotechnological products continues to increase, studies to characterize their effects after exposure and address their potential toxicity are scarce. Due to the nanometer size of these products, their properties differ substantially from those bulk materials of the same composition (3). These properties which make materials on a nanometer scale so attractive, such as small size, chemical composition, structure, large surface area and shape may contribute to the toxicological profile in biological systems (4). In this regard, the toxicity of metal- based nanoparticles, ultrafine particles, asbestos fibers and dust particles has been largely studied, particularly in lung injury (3, 5, 6). The harmful effects observed for these nanoparticles were correlated with common biological mechanisms such as inflammation and oxidative stress. Thus, Donalson and collaborators proved that nanoparticulate forms (< 50 nm) of titanium oxide, aluminum oxide and carbon black increased the pulmonary toxicity inflammation parameters 10 times more than administration of fine particles of the same products (7). On the other hand, Pan and co-workers demonstrated in four different cell lines that one of the smallest gold nanoparticles tested has greater toxicity than other gold nanoparticles with higher sizes (8). Furthermore, Hohr and co-workers observed an increase in pulmonary inflammatory reaction in rats after inhalation of the nanoparticulate form of TiO2 in comparison with the microparticulate form (9). Taking into account the above-mentioned, it has been further shown that the toxicological profile and biological response to the different nanoparticles are closely related with their physicochemical properties. Regarding to nanomedicine - the application of nanotechnology to healthcare -, exposure to nanoparticles for medical purposes involves intentional contact or administration; therefore, understanding the properties of nanoparticles, evaluate their cytotoxicity as well as their behavior in adequate cell models and perform in vivo toxicity and biodistribution studies are crucial before clinical use. However, the potential adverse effects of the nanoparticles have not been extensively studied, possibly because in nanomedicine, mainly biodegradable, biocompatible and/or pharmaceutical approved materials are used (10, 11). In this context, Poly(methyl vinyl ether-co-maleic anhydride) [Gantrez® AN] nanoparticles have been considered promising platforms for drug delivery and other biomedical applications. In the last years, poly(anhydride) nanoparticles have successfully been developed as oral drug delivery systems (12), immunization (13) or allergy treatment (14). In addition, surface modification of these carriers can be easily carried out by simple incubation between the polymer (or the plain nanoparticles) and molecules showing hydroxyl or amino groups. For instance, coating poly(anhydride) nanoparticles with hydrophilics compounds such as PEGs is an adequate strategy to obtain bioadhesive carriers for mucosal delivery and to target the small intestine (15). Furthermore, cyclodextrin- poly(anhydride) nanoparticles have been proposed as effective carriers to both develop 117 CHAPTER 5: Cytotoxicity and cell interaction studies of bioadhesive poly(anhydride) nanoparticles for oral antigen/drug delivery intense bioadhesive interactions between the gut and increase loading capacity of lipophilic drugs such as paclitaxel (16). However, although there are plenty of studies supporting the efficacy of these polymeric nanoparticles, the role of nanoparticle physicochemical properties on their in vitro toxicity and their interaction with cells needed to be investigated thoroughly. Thus, the aim of this work was to evaluate in vitro toxicity by means of MTS and LDH assays of plain, pegylated and cyclodextrin poly(anhydride) nanoparticles using two different cell lines: the human hepatoma cell line HepG2 and the human colon carcinoma cell line Caco-2. In addition, interaction studies between nanoparticles and Caco-2 cells (single cells and monolayers) were carried out using a combination of flow cytometry and fluorescence microscopy.
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2. MATERIALS AND METHODS
2.1. Chemicals, reagents and solutions
Poly(methyl vinyl ether-co-maleic anhydride) or poly(anhydride) (Gantrez® AN 119; Mw 200,000) was provided by ISPcorp. (Waarwijk, The Netherlands). Polyethylene glycol 6000 (PEG6000) was provided by Fluka (Switzerland). 2-hydroxypropyl-β-cyclodextrin (HPCD) was provided by Sigma–Aldrich (Steinheim, Germany). Acetone was obtained from VWR Prolabo (Fontenay-sous-Bois, France). Deionized water (18.2MΩ resistivity) was prepared by a water purification system (Wasserlab, Pamplona, Spain). BSA-FITC and lactose were purchased from Sigma (St. Louis, MO. United States). All other chemicals and solvents used were of analytical grade.
2.2. Preparation of poly(anhydride) nanoparticles
All types of nanoparticles were prepared by desolvation of the polymer in acetone and dried in a Mini Spray-dryer Büchi B290 (Büchi Labortechnik AG, Switzerland) as described previously (17).
2.2.1. Conventional poly(anhydride) nanoparticles (NP) Briefly, 500 mg of poly(anhydride) were dissolved and stirred in 30 mL acetone. Then, the desolvation of the polymer was induced by the addition of 15 mL purified water under magnetic stirring to the organic phase. In parallel, 1 g of lactose was dissolved in 10 mL purified water and added to the nanoparticle suspension under agitation for 5 min at room temperature. Finally, the suspension was dried in the Mini Spray-dryer Büchi B290. The recovered powder was stored in closed vials at room temperature.
2.2.2. Poly(anhydride) nanoparticles containing 2-hydroxypropyl-β-cyclodextrin (NP-HPCD) Briefly, 500 mg of poly(anhydride) were dissolved and stirred in 20 mL acetone. Then, 10 mL acetone containing 125 mg HPCD were added to the polymer solution under magnetic stirring and incubated for 30 min. Nanoparticles were obtained by the addition of 15 mL purified water under magnetic stirring to the organic phase. In parallel, 1 g of lactose was dissolved in 10 mL purified water and added to the nanoparticle suspension under agitation for 5 min at room temperature. Finally, the suspension was dried in the Mini Spray-dryer Büchi B290. The recovered powder was stored in closed vials at room temperature.
2.2.3. Pegylated poly(anhydride) nanoparticles (PEG-NP) In this case, 62.5 mg PEG 6000 were dissolved in 15 mL acetone and mixed with 15 mL acetone containing 500 mg poly(anhydride) and incubated under magnetic stirring for 1 h. Then, nanoparticles were obtained by the addition of 15 mL purified water under magnetic stirring to the organic phase. The solvents were eliminated under reduced pressure (Büchi R-144, Switzerland) and nanoparticles were purified by double centrifugation at 17,000 rpm for 20 min (Sigma 3K30,
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Germany). The pellet was resuspended in 25 mL purified water containing 1 g of lactose. Finally, the suspension was dried in the Mini Spray-dryer Büchi B290. The recovered powder was stored in closed vials at room temperature.
2.3. Preparation of BSA-FITC poly(anhydride) nanoparticle conjugates
To assess the association of poly(anhydride) nanoparticles with Caco-2 cells, fluorescently labelled nanoparticles were prepared by encapsulation of BSA-FITC in the nanoparticles. For this purpose, 2.5 mg BSA-FITC was incubated with the polymer (and eventually with HPCD or PEG 6000) in acetone for 30 min. at room temperature. Then nanoparticles were obtained and purified as described above (see section 2.2.).
2.4. Characterization of the nanoparticles
2.4.1. Size and zeta potential The particle size and the zeta potential of nanoparticles were determined by photon correlation spectroscopy (PCS) and electrophoretic laser Doppler anemometry, respectively, using a Zetamaster analyzer system (Malvern Instruments Ltd., Worces-tershire, United Kingdom) and a ZetaPlus zeta potential analyzer (Brookhaven Instruments Corp., Holtsville, NY). The diameter of the nanoparticles was determined after dispersion in purified water (1/10) and measured at 25 ºC by dynamic light scattering angle of 90 ºC. The zeta potential was determined by diluting the samples in a 0.1 mM KCl solution adjusted to pH 7.4. All measurements were performed in triplicate. The morphological examination of the nanoparticles was obtained by scanning electron microscopy (SEM) in a Zeiss DSM 940A scanning electron microscope (Oberkochen, Germany) coupled with a digital image system (DISS) Point Electronic GmBh. Previously, a small amount of the spray-dried powders was diluted with purified water and the resulting suspension was centrifuged at 17,000 rpm (Sigma 3K30, Germany) for 20 min in order to eliminate sugars. Finally, the pellet was shaded with a 12 nm gold layer in a Hemitech K 550 Sputter-Coater.
2.4.2. Quantification of HPCD and PEG6000 The amount of HPCD and PEG6000 associated to the nanoparticles as well as the amount of poly(anhydride) transformed into nanoparticles were estimated using two different high performance liquid chromatography (HPLC) methods previously described (18, 19). Specifically the apparatus used for the HPLC analysis was a model 1100 series Liquid Chromatography, Agilent (Waldbronn, Germany) coupled with an evaporative light scattering detector (ELSD) 2000 Alltech (Illinois, United States). An ELSD nitrogen generator Alltech was used as the source for the nitrogen gas. Data acquisition and analysis were performed with a Hewlett-Packard computer using the ChemStation G2171 AA program.
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The amount of HPCD and PEG associated to nanoparticles was calculated as the difference between the initial HPCD and PEG and the amount of those recovered in the supernatants. Similarly, the amount of poly(anhydride) was estimated by the difference in the same way.
2.5. Cell Culture
Human hepatoma cell line HepG2 and the colon carcinoma cell line Caco-2 were obtained from the ATCC collection (Maryland, United States). HepG2 cells were grown in DMEM (Dulbecco´s modified Eagle´s medium) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Caco-2 cells were grown in RPMI-1640 with 10% fetal bovine serum, 4 mM L-glutamine and 150 µg/mL gentamycine. Tissue culture reagents were from Sigma (St. Louis, MO, United States) and Gibco Life Technologies Ltd. (United Kingdom). Cells were maintained under standard cell culture conditions (5% CO2, 95% humidity and 37ºC) and subcultured by trypsinization.
2.5.1. Cytotoxicity assays To test the cytotoxicity induced by the different poly(anhydride) nanoparticles, MTS and LDH assay were carried out in both HepG2 and Caco-2 cell lines. Cells were cultured in 96-well plates (Falcon®, United States). The optimum cell concentration as determined by the growth profile of the cell lines was 2.5 x 104 cells/mL (100 µL of cell solution/well). All tests were performed using sub-confluent cells in the logarithmic growth phase. In conducting the experiments it was used culture media without serum to prevent interactions between serum proteins and nanoparticles. MTS and LDH assays were performed in samples containing nanoparticles and culture medium only (without cells). The results demonstrated no interaction between nanoparticles and the different assays components. The stock solution of nanoparticles was performed in Dulbecco´s Phosphate Buffered Saline (DPBS) w/o calcium and magnesium. Before the nanoparticles addition, cell monolayers were washed with DPBS w/o calcium and magnesium. The following concentrations were used: 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL for 4, 24, 48 and 72 h. The concentration range for the nanoparticle formulations and the times of exposure were selected based on literature information (20, 21) as well as on preliminary studies performed in our laboratory. Assays results were confirmed by microscopic observation. Cell morphology was assessed with a Nikon Eclipse TS-100 phase-contrast microscope.
2.5.1.1. Mitochondrial function Cell viability was evaluated by measuring mitochondrial function with the MTS assay
® (CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay) purchased from Promega (Madison, United States) that is based on the protocol described for the first time by Mossman (22). In brief, cells were treated with different nanoparticle formulations for the indicated concentrations and incubation times. The control received culture medium only. After the treatment, the cultured media containing the nanoparticles were removed, cells were washed with DPBS and 100 µL of fresh media
121 CHAPTER 5: Cytotoxicity and cell interaction studies of bioadhesive poly(anhydride) nanoparticles for oral antigen/drug delivery were added to each well. MTS was added to the cultures and the assay was conducted according to
® the manufacturer`s instructions (Cell Titer 96 AQueous Non-Radioactive Cell Proliferation Assay Technical Bulletin #TB169, Promega Corporation). Finally, absorbance readings were carried out at a wavelength of 490 nm using a microplate spectrophotometer system (Spectra MR™ microplate, DYNEX). The relative cell viability of each nanoparticle formulation (%), corrected by the background absorbance of the culture medium, was calculated as follows: [A]sample/[A]control X 100. Where [A]sample is the absorbance of the test sample and [A]control is the absorbance of control sample. Cell survival values above 70% were considered as non-cytotoxic.
2.5.1.2. LDH leakage Cytotoxicity of nanoparticles was assessed by measuring membrane integrity with the LDH assay (Cytotox 96® Non-Radioactive Cytotoxicity Assay) provided by Promega (Madison, United States). Briefly, after the cells were exposed to nanoparticles (NP, NP-HPCD and PEG-NP) at the concentrations and incubations times mentioned, the cultured media were collected and the LDH activity was determined using the LDH assay. In order to calculate LDH max, 10 µL of lysis solution (10X) was added to the control wells. The absorbance of each sample was spectrophotometrically measured at 490 nm with a microplate spectrophotometer system (Spectra MR™ microplate, DYNEX). The relative cytotoxicity of each formulations (% LDH leakage), corrected by the background absorbance of the culture medium, was calculated as follows: [A]sample/[A]LDH max X 100. Where [A]sample is the absorbance of the test sample and [A]LDH max is the absorbance of the LDH max.
2.6. Nanoparticle interaction studies
2.6.1. Association assay to Caco-2 monolayers The interaction of nanoparticle formulations with Caco-2 monolayers was used as a tool to study the association of the drug delivery systems with an established cell culture model for the gastrointestinal epithelium. Cells were cultured for 10-14 days on 96-well plates at a density of 1.7 x 104/well. The monolayers were washed three times with PBS (Phosphate Buffered Saline) and incubated with the different fluorescently labelled formulations at a nanoparticle concentration of 0.5 mg/mL (BSA- FITC labelled NP, NP-HPCD and PEG-NP). After removing the supernatants, non-stably cell- associated nanoparticles were removed by washing three times with 100 µL PBS each. Then the cell- associated fluorescence was determined using a microplate fluorescence reader (Spectrafluor
Fluorometer, Tecan, Austria) set to λex= 485 nm and λem= 520 nm. In order to discriminate between particle association (cytoadhesion and cytoinvasion) and particle binding to the cell surface (cytoadhesion), experiments were performed at either 37 or 4ºC.
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2.6.2. Flow cytometry To evaluate the amount of nanoparticles associated with single cells, 50 µL cell suspension (5 x 106 cells/mL) were mixed with 50 µL nanoparticle suspension and incubated for 1 h at 4ºC. Unbound and loosely associated particles were removed by centrifugation and washing with 400 µL PBS (5 min, 1000 rpm, 4ºC). The cell pellet was resuspended in 1000 µL PBS and analyzed on an EPICS XL-MLC analytical flow cytometer (488/525 nm; Beckman Coulter, Brea, United States) using a forward versus side scatter gate for inclusion of the single-cell population and exclusion of debris and cell aggregates. Intact single cells are located in gate A and gate E, whereas cell fragments are detected in gate B. To verify that cell viability was not compromise by nanoparticles addition, cells without any addition of nanoparticles (PBS-treated cells) were carried along as a negative control in every measurement. A minimum of 2000 cells was acquired per run, and triplicate samples were analyzed for each experiment. The mean channel number of the logarithmic fluorescence intensity of the individual peaks was used for further calculations.
2.6.3. Fluorescence microscopy To visualize the binding characteristics of the fluorescent labelled nanoparticles to Caco-2, images of both, single cells and monolayers were obtained using a Zeiss Axio Observer.Z1 microscopy system using LD Plan-Neofluoar objectives equipped with phase contrast and differential interference contrast (DIC) equipped with LED illumination system “Colibri”.
2.6.4. Inmunofluorescence Caco-2 monolayers were grown on glass cover slides placed in 24-well plates and washed twice with 500 µL PBS prior to the assay. After 1 h incubation with the different fluorescently labelled nanoparticles at 4ºC in PBS the monolayers were fixed with methanol at -20ºC, cell layers were washed and rehydrated. The tight junction associated protein ZO-1 was stained for 1 h at room temperature with a primary antibody (610966; BD Biosciences, San Jose, United States) diluted 1:100 in a 1% solution of BSA in PBS. Upon washing thrice with PBS-BSA (1% v/v) solution, the monolayers were incubated with a 1:100 dilution of a secondary goat anti-mouse immunoglobulin- RPE antibody (Dako Denmark A/S, Glostrup, Denmark) and with a dilution 1:20 of Hoechst 33342 nucleic acid stain (Invitrogen), respectively for 30 min at room temperature. Finally cell layers were washed three times and mounted for microscopy using a drop of FluorSave (FluorsaveTM Reagent, 345789, Calbiochem®, Merck Biosciences) on a slide. Corresponding negative controls were performed in the same way applying PBS-BSA solution without the primary antibody. Inmunofluorescence images of labelled nanoparticles and cells were obtained by fluorescence microscopy.
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2.7. Statistical analysis
All experiments were performed in triplicate, and the results were presented as mean ± standard deviation. The experimental data obtained in cytotoxicity studies were analyzed using non- parametric test "Kruskal Wallis" and "U Mann-Whitney" with Bonferroni adjustment. p-values less than 0.05 were considered as a statistically significant difference. All calculations were performed using SPSS® statistical software program (SPSS® 15, Microsoft, United States).
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3. RESULTS
3.1. Nanoparticles characterization
The main physicochemical characteristics of poly(anhydride) nanoparticles formulations are summarized in Table I. All formulations exhibited a homogeneous size of about 175 nm, with a polydispersity index (PDI) less than 0.3 and a negative zeta potential. Nanoparticles size was also determined in PBS and cell culture medium and no significant change in mean size was observed (data not shown). This is in agreement with previously obtained results (23).
Table I. Physico-chemical characteristics of nanoparticles. Data expressed as the mean ± standard deviation (n=6). NP: poly(anhydride) nanoparticles; NP-HPCD: hydroxypropyl cyclodextrin- poly(anhydride) nanoparticles; PEG-NP: pegylated nanoparticles. PDI: polydispersity index.
Size Zeta Potential Np formation yield PDI µg ligand/mg Np (nm) (mV) (%) NP 167 ± 1 0.11 ± 0.04 -44.6 ± 2.5 97 ± 1.1
NP-HPCD 172 ± 3 0.23 ± 0.02 -36.1 ± 1.7 98 ± 1.2 71.9 ± 3.1
PEG-NP 185 ± 2 0.19 ± 0.06 -32.1 ± 2.5 96 ± 2.2 55.6 ± 2.2
The morphological analysis by scanning microscopy (Figure 1.) showed that NP, HPCD-NP and PEG-NP consisted of a homogeneous population of spherical particles with a smooth surface in case of NP and in contrast with rough surface in case of HPCD-NP and PEG-NP. For NP-HPCD, the amount of associated cyclodextrin was calculated to be about 72 µg/mg whereas for PEG-NP the amount of PEG associated to nanoparticles was about 56 µg/mg. In addition, more than 95% of poly(anhydride) was transformed into nanoparticles for all the formulations.
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Figure 1. SEM photographs of nanoparticles. (A) NP. (A.1) Magnification of a section of photograph (A). (B) NP-HPCD. (B.1) Magnification of a section of photograph (B). (C) PEG-NP. (C.1) Magnification of a section of photograph (C).
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3.2. Mitochondrial Function (MTS assay)
The effect of poly(anhydride) nanoparticle formulations in the mitochondrial function and by extension the relative cell viability of HepG2 and Caco-2 cells is shown in Figure 2. All nanoparticle formulations showed no significant cytotoxic effect on Caco-2 viability while NP and NP-HPCD exhibit significant reductions in relative HepG2 viability (<70%). Thus, cell survival in Caco-2 was over 70% for all the concentration range and incubation times for both, NP, NP-HPCD and PEG- NP, although at the highest concentrations tested and long incubation times (48 and 72h) there is a decrease in cell survival. On the contrary, in HepG2, NP and NP-HPCD showed dependent significant viability decrease at longer incubations times. In fact, cell viability was below 30% for NP and NP-HPCD following 72 h exposure at the highest concentration tested (2 mg/mL). Moreover NP-HPCD showed a significant increase in cytotoxicity at 1 mg/mL after 48 h. The results of the assay also showed a slight viability decrease in cells treated with PEG-NP following 72 h exposure at 2 mg/mL. After 48 h and 72 h, the cytotoxic effect appeared to be concentration dependent for all the nanoparticles tested in both cell lines.
3.3. LDH leakage
Figure 3 shows the effect of poly(anhydride) nanoparticles on relative LDH toxicity. Similarly to the results obtained in the MTS assay not significant increase in LDH release was observed in Caco-2 cells compared to controls, where the relative LDH release was always around 40%. With regard to results obtained in HepG2 cells, these also corresponded with results obtained in MTS assay. Following 48 and 72 h exposure, the LDH leakage gradually increased for NP and NP-HPCD formulations at the highest concentrations (1-2 mg/mL), reaching approximately 80% of relative LDH release. Furthermore, a slight increase in LDH leakage was observed with PEG-NP but it was not significant compared to controls.
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4 h 24 h 48 h 72 h 4 h 24 h 48 h 72 h
A 140 D 140
120 120
100 100
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40 40 Relative cell viability (%) viability cell Relative Relative cell viability(%) 20 * * 20
0 0 5 .5 1 2 5 0 5 1 2 25 50 0 0. 1 .2 .25 .062 0. 0 0625 0.12 0 0 0. mg/mL mg/mL B 140 E140
120 120
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80 80 Caco2 HepG2 60 * 60 *
Relative viability (%) viability Relative 40 40 Relative cell viability (%) viability cell Relative 20 * * 20 0 0 5 1 2 1 2 2 25 0.5 0.5 .1 .250 .250 .06 0 0 .0625 0.125 0 0 0 mg/mL mg/mL C 140 F 140
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40 40 Relative cell viability (%) Relative cell viability (%) viability cell Relative 20 20
0 0 5 0 .5 1 2 .5 1 2 0 25 0 6 .125 .250 0.12 0.25 0 0 0.0625 0.0 mg/mL mg/mL
Figure 2. Effect of nanoparticles on the viability of HepG2 and Caco-2 cells measured by MTS assay following 4, 24, 48 and 72 h incubation at various nanoparticle concentrations. (A)(D) NP. (B)(E) NP-HPCD. (C)(F) PEG-NP. Results presented as % viability relative to controls and expressed as the mean ± SD (n=6). * Statistically significant difference compared with controls (p<0.05).
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4 h 24 h 48 h 72 h 4 h 24 h 48 h 72 h 100 A D 100 * 80 80 * 60 * 60
40 40 Relative release LDH (%) 20 Relative release LDH (%) 20
0 0
L .5 1 2 L .5 1 2 625 0 25 0 RO 0 .1 T . 0.125 0.250 0 0.250 0 0.0625 ONTRO CON C mg/mL mg/mL
B 100 E 100 * 80 * 80
60 60 * *
40 Caco2 40 HepG2
Relative release LDH (%) 20 Relative release LDH (%) 20
0 0
0 1 2 L 5 1 2 L 2 50 O 625 125 0.5 2 0.5 R 0 . . T 0 0.25 TRO 0.1 0 N 0. 0.0625 ON CO C mg/mL mg/mL C 100 F100
80 80
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40 40 Relative releaseLDH (%) Relative release LDH (%) 20 20
0 0 5 .5 1 2 0 .5 1 2 25 50 0 5 0 62 1 .2 .0 0. 0 TROL 0.125 0.2 0 N 0.0625 CONTROL CO mg/mL mg/mL
Figure 3. Influence of nanoparticles on the membrane integrity of Caco-2 cells, measured by LDH assay after 4, 24, 48 and 72 h incubation at various nanoparticle concentrations. (D) NP. (E) NP- HPCD. (F) PEG-NP. * Statistically significant difference compared with controls (p<0.05). Results presented as % LDH release relative to controls and expressed as the mean ± SD (n=6).
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3.4. Nanoparticle/cell interaction on Caco-2 monolayers
To evaluate the interaction of the nanoparticles with the gastrointestinal epithelium, fluorescence-labelled nanoparticles were incubated on Caco-2 monolayers to determine their behavior in an in vitro intestinal epithelium model. In preliminary tests, BSA-FITC was identified as a suitable marker molecule and the different fluorescence-labelled formulations were prepared. To ensure that the fluorescence quantified and visualized in cells was due to BSA-FITC that is stably associated/entrapped with/in nanoparticles, the release of BSA-FITC from nanoparticles was studied over 4 h in PBS. Since the release was found to lie well below 10%, it can be assumed that the fluorescence measured was mainly due to the cell-associated nanoparticles. For the cytoadhesion studies in Caco-2, nanoparticles concentration of 0.5 mg/mL was used. The selection was based on the absence of toxicity at this nanoparticle concentration, as determined by the MTS and LDH assays. Figure 4 illustrates the association of BSA-FITC labelled NP, NP-HPCD and PEG-NP with Caco-2 monolayers at either 4ºC or 37ºC following 4 h incubation. According to the results at both temperatures the cell-associated fluorescence intensity decreased with time for all nanoparticle formulations. In fact, the fluorescence intensity decreased more markedly after 3 h incubation at 37ºC, especially for NP and NP-HPCD. In case of NP, a decrease of 40% of the starting values was observed at 37ºC within 3 h incubation while at 4ºC the decrease was about 13%. Thereafter, the rate of decline was reduced resulting in a cell-associated fluorescence intensity of 21% at 4ºC and 45% at 37ºC after 4 h. For NP- HPCD, at 37ºC and 4ºC the decrease in cell associated fluorescence was about 15% for both temperatures following 3 h incubation and remained constant after 4 h. Similar results were obtained for PEG-NP but at 37ºC the remarkable decrease in cell associated fluorescence intensity took place within 2 h incubation (about 20%) whereas at 4ºC the decline occurred during the first 3 h. To visualize the interaction of nanoparticles in Caco-2 monolayers fluorescence microscopy images were obtained at the end of the association assay. Figure 4 shows the association of BSA- FITC labelled NP-HPCD with Caco-2 monolayers at both temperatures, following 4 h incubation and extensive cells washing. Green fluorescence, arising from BSA-FITC labelled nanoparticles was found distributed throughout the imaged area of the cell monolayer. The fluorescence appears to follow the contour of cells on the apical surface.
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900
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300 NP 37ºC NP 4ºC (a) 0
Cell-associated mean fluorescence intensity 0 60 120 180 240 Time (min)
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Cell-associated mean fluorescence intensity 0 60 120 180 240 Time (min)
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300 PEG-NP 37ºC PEG-NP 4ºC 0
Cell-associated mean fluorescenceintensity 0 60 120 180 240 Time (min)
Figure 4. Influence of the temperature on the association of nanoparticles to Caco-2 monolayers loaded with 0.5 mg/mL of BSA-FITC labelled nanoparticles. (a) NP at 37ºC and (b) at 4ºC following 4 h incubation. Data expressed as the mean ± standard deviation (n=3).
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3.5. Interaction between BSA-FITC nanoparticles and Caco-2 single cells
To clarify whether the surface modification of nanoparticles modify the interaction with Caco-2 cells, the different formulations of BSA-FITC labelled nanoparticles were examined with respect to their association to the cells, the viability and the agglomeration of Caco-2 single cells after binding. For this purpose 0.5 mg/mL of nanoparticles were incubated with single cells for 1 h at 4ºC followed by flow cytometric analysis. These results are summarized in Figure 5.
A: 72.7% A: 70.3% A B: 4.4% B B: 5.4% E: 20.2% E: 27.8%
A: 65.2% D A: 63.6% C B: 3.9% B: 4.2% E: 30.3% E: 29.1%
Figure 5. Forward versus side scatter histograms of Caco-2 single cells. Control cells (A); cells treated with BSA-FITC labelled NP (B); NP-HPCD (C) and PEG-NP (D) after 1 h incubation at 4ºC.
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Regarding cell viability, without nanoparticles addition, 73% of the cells were detected in gate A (single cells), 20 % in gate E (agglomerated cells) and 4.4% in gate B (debris, Figure 5). These results were similar to those obtained with nanoparticles. Thus the influence of the different BSA- FITC labelled nanoparticles on viability of Caco-2 single cells after 1 h exposure, is suggested to be negligible. Taking into account the cell-bound fluorescent intensity (refers to gate A), the values obtained were 4.32 ± 2.14, 7.12 ± 1.85 and 9.28 ± 2.65 for BSA-FITC labelled NP, NP-HPCD and PEG-NP respectively. Therefore, although no differences were observed between nanoparticles formulations in Caco-2 monolayer experiments according to quantitative single cell experiments PEG-NP and NP-HPCD showed higher cell binding than NP.
3.6. Fluorescence Microscopy of Caco-2 single cells
Complementary information on the association of nanoparticles by Caco-2 cells was collected via fluorescence microscopy. Single cells were incubated with the nanoparticles under the same experimental conditions mentioned above. As illustrated in Figure 6, only low amounts of NP (Fig. 6A) were associated with the cell membrane. In contrast, for NP-HPCD and PEG-NP, high amount of nanoparticles were bound to the cell surface (Figure 6B and C).
3.7. Confocal Laser Scanning Microscopy of Caco-2 Monolayers
To corroborate the results from the cellular association assays and to visualize nanoparticle binding as well as tight junctions integrity in Caco-2 monolayers, confocal laser scanning microscopy (CLSM) was used (Figure 7). Cells were double-stained with anti-ZO1 (tight junctions, red) and Hoechst 33342 (nuclei, blue). Related to tight junctions integrity, a continuous ring of red fluorescence, arising from ZO-1 staining, at cell-cell contacts is clearly visible in the control cell layer (Figure 7A). Fluorescence at the point of contact between the cells can also be seen in cell layers incubated with nanoparticles (Figure 7B-D), therefore, nanoparticles addition to cells did not affect tight junctions integrity. Regarding nanoparticle-cell interactions, homogenous distribution of green fluorescence, arising from BSA-FITC labelled nanoparticles, at cell membrane is visible for the three nanoparticle formulations. However, slight differences are observed. Thus, for NP-HPCD and PEG- NP the amount of membrane-bound nanoparticles seems to be higher than for NP.
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A.1 A.2
B.1 B.2
C.1 C.2
Figure 6. Confocal images of Caco-2 single cells after incubation with BSA-FITC labelled NP (A), NP-HPCD (B), and PEG-NP (B). The left column shows transmission images (A.1, B.1 and C.1) and the right one represents the fluorescence images (A.2, B.2, and C.2). A 50 µl cell suspension (5 x 106 cells/mL) was incubated for 1 h at 4ºC.
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A C
B D
Figure 7. Association of BSA-FITC nanoparticles with Caco-2 monolayers. Green: BSA-FITC- labelled nanoparticles, blue: Hoechst 33342-labelled nuclei and red: tight junction associated protein ZO-1. (A) Control cell layers not subjected to nanoparticle incubation. (B) NP, (C) NP-HPCD and (D) PEG-NP.
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4. DISCUSSION
The employment of bioadhesive polymers as nanoparticulate systems has been mostly used towards application to oral drug delivery. These nanosystems provide some advantages compared to oral delivery of no encapsulated drugs. They can protect the therapeutic agent from the harsh environment of the GIT (Gastrointestinal tract) and increase in situ residence time and intimate contact with intestinal mucosa (24). In this context, poly (anhydride) nanoparticles have demonstrated great potential to develop interactions within the GIT. The presence of anhydride residues confers the ability to modify its surface with different molecules or ligands which allows to vary their biodistribution within the gut or/and their bioadhesive potential. As a result these nanoparticles may improve the oral bioavailability of different drugs such as paclitaxel (25), atovaquone (ATO) (26) or fluorouridine (27) and/or facilitate the interaction and presentation of the loaded antigen or allergen with the antigen- presenting cells (APCs) (28). The promising results obtained with these nanoparticles encouraged us to evaluate their cytotoxicity and cell-interaction. The following types of poly(anhydride)nanoparticles have been studied: conventional nanoparticles (NP), nanoparticles containing 2-hydroxypropyl-β-cyclodextrin (NP-HPCD-) and pegylated nanoparticles (PEG-NP). The present work was performed with nanoparticles of well-defined sizes of about 160 nm and narrow size distribution (PDI<0.3) with negative surface charge. Nanoparticles morphology was found to be affected by the association of either HPCD or PEG as shown in Figure 1. This could mean that formulations containing either HPCD or PEG have larger surface area than conventional nanoparticles to establish bioadhesive interactions. Hence rougher nanoparticles should interact in a larger extent than smooth ones. Assessing nanoparticles toxicity at cellular level is an important issue to consider for evaluating their potential bioactivity and a first step before testing toxicity in vivo. Furthermore, select adequate cell models to perform in vitro studies are critical to obtain valuable results that complement in vivo studies. In this context, human hepatocarcinoma-derived HepG2 cells and human colonic adenocarcinoma-derived Caco-2 cells were chosen as models of two important targets for nanoparticles toxicity upon oral exposure like the liver and the gastrointestinal epithelium. The human-derived Caco-2 cell model has been widely used in in vitro studies of epithelial transport/interaction processes, due to its homology to human intestinal epithelial absorptive enterocytes (29, 30). Furthermore, the International Life Sciences Institute Research Foundation/Risk Science Institute (ILSI RF/RSI) Nanomaterial Toxicity Screening Working Group has recommended the use of the immortal cell line, Caco-2 (31). This cell line has been extensively characterized and has been demonstrated to exhibit a faithful representation of both the in vivo structural characteristics and the molecular level to mirror the differentiation of human intestinal cells (32). 136 CHAPTER 5: Cytotoxicity and cell interaction studies of bioadhesive poly(anhydride) nanoparticles for oral antigen/drug delivery
For HepG2 cells, this cell line derived from human liver carcinoma has widely been used in nanotoxicological studies (33). Moreover, this cell line retains some of the functions of fully differentiated primary hepatocytes as some metabolic competence and it has been widely used as a model system for hepatotoxicity studies (34). The results for the MTS and LDH assays showed a concentration-dependent cytotoxic effect. However, by comparing the cytotoxicity of nanoparticles to Caco-2 versus HepG2 cells, a difference in the respective cellular viabilities was noted at equal tested concentrations with both cytotoxicity assays. HepG2 cells were more sensitive than Caco-2 cells to poly(anhydride) nanoparticles, in particular at the highest concentrations (1 and 2 mg/mL). In addition, PEG-NP were less cytotoxic than NP and NP-HPCD in both cell types and assays. It is not possible to understand the mechanism underlying these observations but some differences in membrane composition or structure might be the cause. On the other hand, poly(anhydride) is a biocompatible and biodegradable (bioerodible) polymer and pegylation has been widely used to increase nanoparticles biocompatibility (35) and this is may account for the lower cytotoxicity of PEG-NP compared with the conventional and NP-HPCD. In any case, the in vitro safety of these poly(anhydride) nanoparticles could be assumed, since all the formulations were well tolerated by both cell lines showing cytotoxicity only at very high concentrations and long incubation times in HepG2 cells. In fact, thinking on in vivo studies, these concentrations would be too high to be used in clinical trials. The association studies carried out in Caco-2 monolayers showed that although the cell- associated fluorescence intensity obtained at 37ºC was higher than at 4ºC, results were not different enough to ensure that nanoparticles were taken up by cells. Indeed, it is known that at 4ºC, the fluidity of the cell membrane and the metabolism of the cell are reduced to a minimum, so cell- associated fluorescence intensity primarily reflects the binding of nanoparticles to the surface (cytoadhesion). In contrast, upon incubation at 37ºC cells are metabolically active and energy- dependent transport processes are initiated. Thus, the detectable amount of cell-associated nanoparticles refers to a combined effect of binding (cytoadhesion) and uptake (cytoinvasion) (36, 37). Thus, at 37ºC, BSA-FITC that is taken up by cells, would be quenched in the acidic environment of the lysosome. So, if there was uptake, the curve at 37ºC should show a steeper decrease than the curve at 4ºC. As this is not the case, because both curves show the same decrease, there was no substantial energy-dependent uptake of nanoparticles. These outcomes were in agreement with previous studies developed by Arbos and co-workers where they observed that while Sambucus nigra agglutinin coated poly(anhydride) nanoparticle were taken up by cells, plain poly(anhydride) nanoparticles showed only cytoadhesion (23). Hence, considering all above, the different poly(anhydride) nanoparticles tested were capable to establish bioadhesive interactions but not to be taken up by cells.
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These findings were corroborated via flow cytometric analysis using Caco-2 single cells. The slight increased observed in the ratio of cells in gate A (healthy single cells) may be due to the fact that binding of nanoparticles to the cell surface changes granularity of the cells and also because the high bioadhesive properties of the nanoparticles could induce cell aggregation. Furthermore, the different cell fluorescence intensity observed between NP, NP-HPCD and PEG-NP confirmed that surface modification confers larger surface area and subsequently leading to higher cell binding. The above outcomes were confirmed by CLSM observation of the interaction between the fluorescent nanoparticles and cell monolayers. The images corroborated the high affinity of poly(anhydride) nanoparticles to Caco-2 cell surface as well as the heterogeneity of the particle localization over the monolayer. Moreover, comparing to control group, after incubation with poly(anhydride) nanoparticles, the staining for ZO-1 proteins showed a continuous ring appearance between contiguous cells. This fact correlated well with cytotoxicity results.
5. CONCLUSIONS
This study is the first to evaluate the in vitro toxicity and cell interaction of NP, NP-HPCD and PEG-NP. The results for the cytotoxicity studies in HepG2 and Caco-2 cells after exposure to the different poly(anhydride) nanoparticles revealed that viability starts to decrease only at very high concentrations and incubations times, highlighting the safety of nanoparticles, independently of their chemical composition and surface properties. In addition, association studies in Caco-2 cells showed cytoadhesion to the cell surface but not internalization. Regarding flow cytometry experiments, due to the rough surface, the highest cytoadhesion was observed for PEG-NP followed by NP-HPCD and the lowest correspond to NP. Finally, cellular localization of particles by fluorescence confocal microscopy demonstrated the strong association of the poly(anhydride) nanoparticles with cells membranes due to their bioadhesive properties.
Acknowledgments This work was supported by the Ministry of Science and Innovation in Spain (Project SAF2008-02538) by Caja Navarra Foundation (Grant number 10828), and by the Department of Education, Government of Navarra, Spain.
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6. References
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16. M. Agueros, L. Ruiz-Gaton, C. Vauthier, K. Bouchemal, S. Espuelas, G. Ponchel, and J.M. Irache. Combined hydroxypropyl-beta-cyclodextrin and poly(anhydride) nanoparticles improve the oral permeability of paclitaxel. Eur J Pharm Sci. 38:405-413 (2009). 17. P. Ojer, H. Salman, R. Da Costa Martins, J. Calvo, A. López de Cerain, C. Gamazo, J.L. Lavandera, and J.M. Irache. Spray-drying of poly(anhydride) nanoparticles for drug/antigen delivery. J Drug Del Sci Tech. 20:353-359 (2010). 18. M. Agueros, M.A. Campanero, and J.M. lrache. Simultaneous quantification of different cyclodextrins and Gantrez by HPLC with evaporative light scattering detection. J Pharm Biomed Anal. 39:495-502 (2005). 19. V. Zabaleta, M.A. Campanero, and J.M. Irache. An HPLC with evaporative light scattering detection method for the quantification of PEGs and Gantrez in PEGylated nanoparticles. J Pharm Biomed Anal. 44:1072-1078 (2007). 20. N. Lewinski, V. Colvin, and R. Drezek. Cytotoxicity of nanoparticles. Small. 4:26-49 (2008). 21. C.I. Vamanu, M.R. Cimpan, P.J. Hol, S. Sornes, S.A. Lie, and N.R. Gjerdet. Induction of cell death by TiO2 nanoparticles: studies on a human monoblastoid cell line. Toxicol In Vitro. 22:1689-1696 (2008). 22. T. Mosmann. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 65:55-63 (1983). 23. P. Arbos, M. Wirth, M.A. Arangoa, F. Gabor, and J.M. Irache. Gantrez AN as a new polymer for the preparation of ligand-nanoparticle conjugates. J Control Release. 83:321-330 (2002). 24. J. Haas and C.M. Lehr. Developments in the area of bioadhesive drug delivery systems. Expert Opin Biol Ther. 2:287-298 (2002). 25. V. Zabaleta, G. Ponchel, H. Salman, M. Agueros, C. Vauthier, and J.M. Irache. Oral administration of paclitaxel with pegylated poly(anhydride) nanoparticles: permeability and pharmacokinetic study. Eur J Pharm Biopharm. 81:514-523 (2012). 26. J. Calvo, J.L. Lavandera, M. Agueros, and J.M. Irache. Cyclodextrin/poly(anhydride) nanoparticles as drug carriers for the oral delivery of atovaquone. Biomed Microdevices. 81:1015-1025 (2011). 27. P. Arbos, M.A. Campanero, M.A. Arangoa, and J.M. Irache. Nanoparticles with specific bioadhesive properties to circumvent the pre-systemic degradation of fluorinated pyrimidines. J Control Release. 96:55-65 (2004). 28. A.I. Camacho, R. Da Costa Martins, I. Tamayo, J. de Souza, J.J. Lasarte, C. Mansilla, I. Esparza, J.M. Irache, and C. Gamazo. Poly(methyl vinyl ether-co-maleic anhydride) nanoparticles as innate immune system activators. Vaccine. 29:7130-7135 (2011). 29. P. Artursson and C. Magnusson. Epithelial transport of drugs in cell culture. II: Effect of extracellular calcium concentration on the paracellular transport of drugs of different
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lipophilicities across monolayers of intestinal epithelial (Caco-2) cells. J Pharm Sci. 79:595- 600 (1990). 30. A.R. Hilgers, R.A. Conradi, and P.S. Burton. Caco-2 cell monolayers as a model for drug transport across the intestinal mucosa. Pharm Res. 7:902-910 (1990). 31. G. Oberdorster, A. Maynard, K. Donaldson, V. Castranova, J. Fitzpatrick, K. Ausman, J. Carter, B. Karn, W. Kreyling, D. Lai, S. Olin, N. Monteiro-Riviere, D. Warheit, and H. Yang. Principles for characterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy. Part Fibre Toxicol. 2:8 (2005). 32. M.D. Peterson, W.M. Bement, and M.S. Mooseker. An in vitro model for the analysis of intestinal brush border assembly. II. Changes in expression and localization of brush border proteins during cell contact-induced brush border assembly in Caco-2BBe cells. J Cell Sci. 105 ( Pt 2):461-472 (1993). 33. Y. Yuan, C. Liu, J. Qian, J. Wang, and Y. Zhang. Size-mediated cytotoxicity and apoptosis of hydroxyapatite nanoparticles in human hepatoma HepG2 cells. Biomaterials. 31:730-740 (2010). 34. J. Zou, Q. Chen, X. Jin, S. Tang, K. Chen, T. Zhang, and X. Xiao. Olaquindox induces apoptosis through the mitochondrial pathway in HepG2 cells. Toxicology. 285:104-113 (2011). 35. K. Knop, R. Hoogenboom, D. Fischer, and U.S. Schubert. Poly(ethylene glycol) in drug delivery: pros and cons as well as potential alternatives. Angew Chem Int Ed Engl. 49:6288- 6308 (2010). 36. M. Wirth, C. Kneuer, C.M. Lehr, and F. Gabor. Lectin-mediated drug delivery: discrimination between cytoadhesion and cytoinvasion and evidence for lysosomal accumulation of wheat germ agglutinin in the Caco-2 model. J Drug Target. 10:439-448 (2002). 37. A. Weissenboeck, E. Bogner, M. Wirth, and F. Gabor. Binding and uptake of wheat germ agglutinin-grafted PLGA-nanospheres by caco-2 monolayers. Pharm Res. 21:1917-1923 (2004).
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CHAPTER 6 Toxicity studies of poly(anhydride) nanoparticles as carriers for oral drug delivery
Toxicity studies of poly(anhydride) nanoparticles as carriers for oral drug delivery
Patricia Ojer1,2, Adela López de Cerain2, Paloma Areses3, Ivan Peñuelas3, and Juan M. Irache1
1 Department of Pharmacy and Pharmaceutical Technology. University of Navarra, Pamplona, 31008, Spain. 2 Department of Nutrition and Food Sciences, Physiology and Toxicology. University of Navarra, Pamplona, 31008, Spain. 3 Radiopharmacy Unit. Department of Nuclear Medicine, University Clinic of Navarra, Pamplona, 31008, Spain.
ABSTRACT
In order to evaluate the acute and subacute toxicity of poly(anhydride) nanoparticles as carriers for oral drug/antigen delivery three types of poly(anhydride) nanoparticles were assayed: conventional (NP), nanoparticles containing 2-hydroxypropyl-β-cyclodextrin (NP-HPCD) and nanoparticles coated with polyethylene glycol 6000 (PEG-NP). Nanoparticles were prepared by a desolvation method and characterized in terms of size, zeta potential and morphology. For in vivo oral studies, acute and sub-acute toxicity studies were performed in rats in accordance to the OECD 425 and 407 guidelines respectively. Finally, biodistribution studies were carried out after radiolabelling nanoparticles with 99mTechnetium (99mTc). The results showed that nanoparticle formulations displayed a homogeneous size of about 180 nm and a negative zeta potential. The lethal dose 50%
(LD50) for all the nanoparticles tested was established to be higher than 2000 mg/kg body weight (bw). In the sub-chronic oral toxicity studies at two different doses (30 and 300 mg/kg bw), no evident signs of toxicity were found. Lastly, biodistribution studies demonstrated that these carriers remained in the gut with no evidences of particle translocation or distribution to other organs. Thus, poly(anhydride) nanoparticles (either conventional or modified with HPCD or PEG6000) showed no toxic effects, indicating that these carriers might be a safe strategy for oral delivery of therapeutics.
Published in Pharmaceutical Research 29(9):2615-2627 (2012).
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1. INTRODUCTION
In the last decades, the development of nanoparticles for medical and pharmaceutical applications has received a great interest (1-3). Among other, polymeric nanoparticles offer interesting advantages for drug delivery purposes (4-6). As pharmaceutical dosage forms, polymeric nanoparticles may protect the loaded therapeutic agent against extreme pH conditions and/or enzymatic degradation. Their surface modification with ligands permits to drive their distribution in vivo and, thus, improve their targeting properties for a specific tissue or groups of cells within the body (5, 7). This functionalization of nanoparticles would be useful to promote the arrival of the encapsulated drug to its ideal site for action or absorption and, thus, reach a particular target inside the cell or improve its bioavailability (8). Last but not least, it is also important to remember the capability of polymeric nanoparticles to control the release of the loaded drug (5). Although numerous efforts have been carried out to exploit desirable properties of polymeric nanoparticles, attempts to evaluate potentially undesirable effects are limited in comparison (9). The same properties as its small size and large surface area, which make nanoparticles so attractive for medical intentions, may provoke undesirable tissue accumulation and subsequent long-term toxicity (10). Therefore there is a pressing need for careful consideration of nanoparticles toxicity. Hence, nanotoxicology as a new science is becoming a trending topic. Nevertheless, to date, the first challenge that nanotoxicology has to face is the lack of special regulation to deal with potential risks of nanoparticles. In order to solve this problem, different organizations are developing new regulatory initiatives, such as thus from both the European Committee for Standardization (CEN/TC 352) and the International Organization for Standardization (ISO/TC 229), in order to ensure that products derived from nanomedicine are safe without hindering innovation (11) and, thus, support commercialisation and market development (12). In parallel, since 2006, activities related to the development or revisions of test guidelines to assess nanotoxicology have been performing by the Working Party on Manufactured Nanomaterials (WPNM) within the Organization for Economic Cooperation and Development (OECD) (13). In this way, WPMN has recommended the guidelines 425 and 407 for the evaluation of oral acute and sub-acute toxicity of nanomaterials, respectively (14). In the last years, promising poly(anhydride) based nanoparticles have been developed from the copolymer of methyl vinyl ether and maleic anhydride. This polymer shows a great potential for oral drug/antigen delivery due to its well-studied bioadhesive properties when formulated as nanoparticles (15-17). This capability to establish bioadhesive interactions can be modulated by the modification of poly(anhydride) nanoparticles with different compounds such as PEGs or cyclodextrins (17, 18). In this regard, a study conducted by Yoncheva and collaborators demonstrated that pegylated nanoparticles possessed high affinity to adhere to the small intestine compared to conventional nanoparticles (19) and these nanoparticles may be suitable carriers for DNA (20). In another study it was corroborated the synergistic effect of the combination between bioadhesive
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nanoparticles and cyclodextrins on the oral bioavailability of paclitaxel and other drugs ascribed to the groups II and IV of the biopharmaceutics classification system (BCS) (21, 22). The aim of this study was to evaluate the toxicological profile of different types of poly(anhydride) nanoparticles: conventional (NP), nanoparticles containing 2-hydroxypropyl-β- cyclodextrin (NP-HPCD) and nanoparticles coated with PEG 6000 (PEG-NP). More particularly, in this work we report their safety through acute and sub-acute toxicity studies. Additionally, in vivo biodistribution of these nanoparticles after oral administration was also investigated.
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2. MATERIALS AND METHODS
2.1. Chemicals, reagents and solutions
Poly(methyl vinyl ether-co-maleic anhydride) or poly(anhydride) (Gantrez® AN 119; Mw 200,000) was provided by ISPcorp. (Waarwijk, The Netherlands). Polyethylene glycol 6000 (PEG6000) was provided by Fluka (Switzerland). 2-hydroxypropyl-β-cyclodextrin (HPCD) was provided by Sigma–Aldrich (Steinheim, Germany). Acetone was obtained from VWR Prolabo (Fontenay-sous-Bois, France). Deionized water (18.2MΩ resistivity) was prepared by a water purification system (Wasserlab, Pamplona, Spain). 99Mo-99mTc generator (Drytec; GE Healthcare Bio- science, United Kingdom) was eluted with 0.9% NaCl following the manufacturer’s instructions.
SnCl2·2H2O and HCl were from Panreac (Barcelona, Spain); 0.9% NaCl was purchased from Braun (Barcelona, Spain) and NaOH from Fluka (Switzerland). The anaesthetic isoflurane (Isoflo™) was from Esteve, (Barcelona, Spain) and the euthanasic T-61 from Intervet (Madrid, Spain). All other chemicals and solvents used were of analytical grade.
2.2. Preparation of poly(anhydride) nanoparticles
Poly(anhydride) nanoparticles were prepared from a dissolution of Gantrez® AN 119 in acetone by a simple desolvation method previously described (15). The resulting suspensions were always dried in a Mini Spray-dryer Büchi B290 (Büchi Labortechnik AG, Switzerland) as described previously (23). Three different types of nanoparticles were evaluated: conventional nanoparticles (NP), nanoparticles containing HPCD (NP-HPCD) and pegylated nanoparticles with PEG 6000 (PEG-NP).
2.2.1. Conventional poly(anhydride) nanoparticles (NP) Briefly, 500 mg of the copolymer of methyl vinyl ether and maleic anhydride (Gantrez® AN 119) were dissolved and stirred in 30 mL acetone. Then, the desolvation of the polymer was induced by the addition of 15 mL purified water under magnetic stirring to the organic phase. In parallel, 1 g of lactose was dissolved in 10 mL purified water and added to the nanoparticle suspension under agitation for 5 min at room temperature. Finally, the suspension was dried in the Mini Spray-dryer Büchi B290. The recovered powder was stored in closed vials at room temperature.
2.2.2. Poly(anhydride) nanoparticles containing 2-hydroxypropyl-β-cyclodextrin (NP-HPCD) Briefly, 500 mg of the copolymer of methyl vinyl ether and maleic anhydride were dissolved and stirred in 20 mL acetone. Then, 10 mL acetone containing 125 mg HPCD were added to the polymer solution under magnetic stirring and incubated for 30 min. Nanoparticles were obtained by the addition of 15 mL purified water under magnetic stirring to the organic phase. In parallel, 1 g of lactose was dissolved in 10 mL purified water and added to the nanoparticle suspension under
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agitation for 5 min at room temperature. Finally, the suspension was dried in the Mini Spray-dryer Büchi B290. The recovered powder was stored in closed vials at room temperature.
2.2.3. Pegylated poly(anhydride) nanoparticles (PEG-NP) In this case, 62.5 mg PEG were dissolved in 15 mL acetone and mixed with 15 mL acetone containing 500 mg of the copolymer of methyl vinyl ether and maleic anhydride and incubated under magnetic stirring for 1 h. Then, nanoparticles were obtained by the addition of 15 mL purified water under magnetic stirring to the organic phase. The solvents were eliminated under reduced pressure (Büchi R-144, Switzerland) and nanoparticles were purified by double centrifugation at 17,000 rpm for 20 min (Sigma 3K30, Germany). The pellet was resuspended in 25 mL purified water containing 1 g of lactose. Finally, the suspension was dried in the Mini Spray-dryer Büchi B290. The recovered powder was stored in closed vials at room temperature.
2.3. Characterization of the nanoparticles
2.3.1. Size and zeta potential The mean hydrodynamic diameter and the zeta potential of nanoparticles were determined by photon correlation spectroscopy (PCS) and electrophoretic laser Doppler anemometry, respectively, using a Zetamaster analyzer system (Malvern Instruments, United Kingdom) and a ZetaPlus zeta potential analyzer (Brookhaven Instruments Corp., Holtsville, NY). The diameter of the nanoparticles was determined after dispersion in purified water (1/10) and measured at 25 ◦C by dynamic light scattering angle of 90 ºC. The zeta potential was determined by diluting the samples in a 0.1 mM KCl solution adjusted to pH 7.4. All measurements were performed in triplicate. The morphological examination of the nanoparticles was obtained by scanning electron microscopy (SEM) in a Zeiss DSM 940A scanning electron microscope (Oberkochen, Germany) coupled with a digital image system (DISS) Point Electronic GmBh. Previously, a small amount of the spray-dried powders was diluted with purified water and the resulting suspension was centrifuged at 17,000 rpm (Sigma 3K30, Germany) for 20 min in order to eliminate sugars. Finally, the pellet was shaded with a 12 nm gold layer in a Hemitech K 550 Sputter-Coater.
2.3.2. Quantification of HPCD and PEG The amount of HPCD and PEG associated to the nanoparticles as well as the amount of the polymer transformed into nanoparticles were estimated using two different high performance liquid chromatography (HPLC) methods previously described (24, 25). Specifically the apparatus used for the HPLC analysis was a model 1100 series Liquid Chromatography, Agilent (Waldbronn, Germany) coupled with an evaporative light scattering detector (ELSD) 2000 Alltech (Illinois, United States). An ELSD nitrogen generator Alltech was used as the source for the nitrogen gas. Data acquisition and analysis were performed with a Hewlett-Packard computer using the ChemStation G2171 AA program.
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On one hand, PEG separation was carried out at 40ºC on a PL aquagel-OH 30 column (300mm × 7.5 mm; particle size 8 μm) obtained from Agilent Technologies (GB, United Kingdom). The mobile phase composition was a mixture of methanol (A) and water (B) in a gradient elution at a flow rate of 1 mL/min. On the other hand, HPCD separation was carried out at 50ºC on a reversed- phase Zorbax Eclipse XDB-Phenyl column (2.1mm × 150 mm; particle size 5 μm) obtained from Agilent Technologies (Waldbronn, Germany). This column was protected by a 0.45 μm filter (Teknokroma, Spain). The mobile phase composition was a mixture of acetonitrile (A) and water (B) in a gradient elution at a flow-rate of 0.25 mL/min. In all cases, for ELSD quantifications, the drift tube temperature was set at 115◦ C, the nitrogen flow was maintained at 3.2 l/min and the gain was fixed to 1. The amount of HPCD and PEG associated to nanoparticles was calculated as the difference between the initial HPCD and PEG and the amount of those recovered in the supernatants. Similarly, the amount of the poly(anhydride) copolymer was estimated by difference in the same way.
2.4. Labelling of nanoparticles with 99mTc
Poly(anhydride) nanoparticles were labeled with 99mTc by reduction with tin chloride (26). Briefly, 1 mCi of freshly eluted 99mTc pertechnetate was reduced with 0.03 mg/mL stannous chloride and the pH was adjusted to 4 with 0.1N HCl. Then, nanoparticles (NP, NP-HPCD or PEG-NP) in water were added to the pre-reduced 99mTc mixture. The suspension was vortexed for 30 s and incubated at room temperature for 10 min. The overall procedure was carried out in helium-purged vials using helium-purged solutions to minimize oxygen content and avoid oxidation of tin chloride. The radiochemical purity was examined by a double-solvent instant thin layer chromatography (ITLC) system using silica gel coated fiber sheets (Polygram® sil N-RH, Macherey-Nagel, Düren, Germany) with methyl ethyl ketone (first solvent) and 18% sodium acetate (second solvent) as mobile phases. After labeling, nanoparticles were mixed with non-labelled ones to adjust the required dose (either 30 mg/kg or 300 mg/kg) and the volume was adjusted to 1 mL.
2.5. Animals
The experimental protocols involving animals were carefully reviewed and approved by the Ethical Committee for Animal Experimentation of the University of Navarra (Spain). Eight week old male and female Wistar rats were purchased from Harlan (Horst, The Netherlands) and employed for acute and sub-acute toxicity studies as well as biodistribution studies. On the day of arrival, the animals were weighed in order to assure that bw variation did not exceed ± 20% (27, 28). They were randomly housed in groups in polycarbonate cages with stainless steel covers to allow acclimatization to the environmental conditions (12 h day/night cycle, temperature 22 ± 2ºC, relative humidity 55 ± 10%, standard diet ‘‘2014 Teklad Global 14% Protein Rodent Maintenance Diet” from Harlan Iberica Spain and water ad libitum).
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2.6. Acute-toxicity study
This study was designed according to the OECD guideline 425 for the Testing of Chemicals (27). The procedure of the limit test was applied because no toxicity was expected after a single oral dose: a limit dose of 2000 mg/kg bw was selected. Twenty female Wistar rats were divided into four groups (n=5): Group I (Control), Group II (NP), Group III (NP-HPCD) and Group IV (PEG-NP). A single dose of 2000 mg/kg bw was administered by gavage to each animal in 2 mL/100 g bw of purified water, except for the control group, that received purified water only (vehicle). After the administration each animal was observed for a period of 48 h for signs of toxicity or mortality. If no such signs were observed after 48 h, then the same dose was administered to animal number 2. This procedure was sequentially followed until a total of five animals were administered. The animals were fasted 4 h prior to dosing and 3 h after the nanoparticles were administered. Individual animal weights were registered on day 0 and weekly during 14 days. Animals were observed individually for any clinical signs or any symptoms of toxicity at different time intervals after administration (10 min, 30 min, 1 h, 3 h, and 6 h) and daily during 14 days. On completion of the study, on day 14, blood samples were extracted from the retro-orbital sinus under isoflurane anesthesia. The following hematological parameters were analyzed: hemoglobin (HGB; g/L), hematocrit (HCT, %), red blood corpuscles count (RBC; 1012/L), white blood corpuscles count (WBC; 103/µL), absolute erythrocyte indices and differential WBC. Biochemical analyses of plasma samples were performed with a Hitachi 911™ (Roche Diagnostics) analyzer using the protocols obtained from Roche for the determination of the standard parameters: total protein (g/dL), albumin (g/dL), glucose (mg/dL), aspartate transaminase (AST; U/l), alanine transaminase (ALT; U/l), cholesterol (mg/dL), creatinine (mg/dL), urea (mg/dL) and total bilirrubin (mg/dL).
Thereafter, the animals were sacrificed in CO2 chamber, subjected to necropsy and various organs were collected and fixed for further histopathological examination.
2.7. Sub-acute toxicity study
This study was designed according to the OECD guideline 407 for the Testing of Chemicals (28). The dose selected of each nanoparticle type was based on the expected therapeutic dose according to previous studies (22), 30 mg/kg bw, and ten times the reported value, 300 mg/kg bw. Animals were randomly divided into seven groups, containing five male and five female rats per group. Group I served as the control. Groups II and III received NP, 30 mg/kg bw and 300 mg/kg bw, respectively. Groups IV and V were treated with NP-HPCD, 30 mg/kg bw and 300 mg/kg bw, respectively. Finally, Groups VI and VII received PEG-NP, 30 mg/kg bw and 300 mg/kg bw, respectively. The animals were administered the respective doses for a period of 28 days, once daily, by oral gavage. During this period they were observed for any clinical signs of toxicity, mortality or changes in bw. Blood samples were collected before the first administration, on day 15, and at the 152 CHAPTER 6: Toxicity studies of poly(anhydride) nanoparticles as carriers for oral drug delivery
end of the study from the retro-orbital sinus under anesthesia and hematological and biochemical parameters were analyzed (see Acute toxicity study). On day 28, animals were sacrificed in CO2 chamber, subjected to necropsy and various organs were collected, weighed and fixed for further histopathological examination.
2.8. Histopathological studies
Tissue samples from different body organs (including thymus, kidney, liver, pulmonary, spleen, stomach, liver and intestines) were taken during necropsy, fixed in 4% formaldehyde solution, dehydrated and embedded in paraffin. Paraffin sections (3 µm) were cut, mounted onto glass slides, and dewaxed and stained with haematoxylin and eosin (H&E) for the subsequent histopathological examination.
2.9. In vivo and ex vivo biodistribution studies with radiolabelled nanoparticles
For this study we only used female rats which were divided in groups of three animals each. The groups were the same as described in the “Sub-acute toxicity study”. The radiolabelled dose (1 mCi) was administered on days 1, 15 and 28 of the study. After the administration of nanoparticles, animals were anesthetized with 2% isoflurane and placed in prone position on the gammacamera. The gammagraphic studies were performed in a single-photon emission computed tomography (SPECT–CT) (Symbia; Siemens Medical System, United States). A high-resolution low-energy parallel-hole collimator was used. The scan parameters for computed tomography (CT) were 130 kV, 30 mA s, 1 mm slices and Flash 3D iterative reconstruction with a Gaussian filter with a full-width at half maximum of 8.4.. For image acquisition the gammacamera was programmed to reach 500.000 events with a static program. The images were acquired 8 h after the administration of the radiolabelled nanoparticles. For ex vivo studies, 24 h after the administration of nanoparticles, animals were euthanized with T-61 (after anesthesia with 2% isoflurane gas). Then blood, urine and different organs (lungs, heart, spleen, pancreas, liver, kidneys, bone, gut and muscle) were collected and the radioactivity of each organ measured in a gamma counter (Compugamma CS, RIA; LKB Pharmacia, Finland) calibrated for 99mTc energy. For practical reasons, the GIT (Gastrointestinal tract) of animals was divided as follows: stomach, small intestine, caecum and rectum. All of these sections were first washed by careful gentle injection of 0.9% NaCl through the lumen. The washing liquids were recovered, weighed and also measured in the gamma counter. Finally, results were expressed as the percentage of injected dose per gram (%ID/g).
2.10. Statistical analysis
Data were expressed as the mean ± SD of at least three experiments. For the nanoparticles characterization the Student t test was used. Other statistical significance analysis were carried out
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using the non-parametric Mann-Whitney U test. P values of < 0.05 were considered as statistically significant. All calculations were performed using SPSS® statistical software program (SPSS® 15.0, Microsoft, United States).
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3. RESULTS
3.1. Nanoparticles characterization
The main physicochemical characteristics of poly(anhydride) nanoparticles formulations are summarized in Table I. Overall the different nanoparticle formulations exhibited a homogeneous mean size of about 170-190 nm, with a low polydispersity index (PDI). Conventional nanoparticles (NP) displayed a mean size slightly smaller than that observed for NP-HPCD and PEG-NP. These observations were confirmed by scanning electron microscopy (Figure 1). The morphological analysis of the three types of nanoparticles revealed that NP and PEG-NP displayed a round shape while NP- HPCD were characterized by an irregular aspect. In addition, NP-HPCD showed rough surface in comparison with the smooth shape observed for NP.
Table I. Physico-chemical characteristics of nanoparticles. NP: poly(anhydride) nanoparticles; NP-HPCD: hydroxypropyl cyclodextrin-poly(anhydride) nanoparticles; PEG-NP: pegylated nanoparticles. PDI: polydispersity index. Data expressed as the mean ± standard deviation (n=6).
Formulation Size (nm) PDI Zeta Potential Np formation yield µg ligand/mg Np (mV) (%) NP 170 ± 2 0.15 ± 0.03 -45.3 ± 2.5 98 ± 3.1
NP-HPCD 189 ± 1.6 0.14 ± 0.01 -35.9 ± 3.5 96 ± 4.2 87.7 ± 2.2
PEG-NP 182 ± 2.1 0.25 ± 0.02 -34.6 ± 3.7 97 ± 2.3 45.7 ± 1.9
On the other hand, the incorporation of either HPCD or PEG in the nanoparticles decreased the negative zeta potential of the resulting carriers. Concerning the yield of the process, in all cases, this parameter was calculated to be very high and close to 100 %. Finally, the amount of HPCD associated to nanoparticles was calculated to be about 87 µg/mg. For pegylated nanoparticles, the amount of PEG linked to nanoparticles was 46 µg/mg.
3.2. Acute-toxicity study
Throughout all the observation period, the animals dosed with the nanoparticles displayed neither any sign of toxicity nor any abnormal behavior. Similarly, in all cases, the hematological and biochemical parameters analyzed showed normal values that did not differ from the control group (data not shown). Finally, gross and histological pathological examination of the vital organs did not exhibit any evidence of toxicity during the animal necropsies (data not shown). Thus, the different poly(anhydride) nanoparticles were found to be orally safe at the single limit dose of 2000 mg/kg bw.
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(A) (B) (C)
Figure 1. SEM photographs of nanoparticles. (A) NP. (B) NP-HPCD. (C) PEG-NP.
3.3. Sub-acute toxicity
During the sub-acute toxicity study no mortality was observed in the different treatment groups. Detailed physical examinations conducted weekly did not demonstrate any unusual change in behavior and no signs of toxicity were observed throughout the study. Thus, long-term administration of the poly(anhydride) nanoparticles had no adverse effects on the general health of animals. No significant differences were observed in bw of the animals of treatment groups compared with control ones (Figure 2). All the hematological and biochemical values were found to be within the normal range with no difference between the control and the treatment groups for both male and female animals (Figures 3 and 4). The relative organ weight of heart, liver, kidneys, adrenals, thymus, spleen, ovaries and testis were also unaffected by the treatments (data not shown). Regarding histology, the gross and histopathology of various organs revealed that the natural architecture remained normal. No dose related toxicity lesions were observed. Histological findings of a female dosed with the highest dose, 300 mg/kg bw, are presented in Figure 5.
Group I Group I 150 Group II Group II (A) Group III 150 Group III Group IV (B) Group IV Group V Group V Group VI Group VI Group VII Group VII 100 100
50 50 Body Weight(% initial) of Body Weight(% ofinitial)
0 0 y k y k a e a ek ek e th d th d we week d wee h we week w st nd r t st week th week 0 0 nd rd 1 2 3 4 1 2 3 4 Time (weeks) Time (weeks) Figure 2. Body weight (bw) registered of female (A) and male rats (B) through sub-acute toxicity study.
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1200 (A) Group I 1000 Group II 800 Group III Group IV 600 Group V 400 Group VI 100 Group VII Units 80
60
40
20
0 l) l) 3 ) ) ) /L) 12 / µ g L %) %) (%) 3 / (p /d (%) 3 /µl) T s s ( H (g e e 10 B (g C t t hile ( G H C y (x H MC cy p MCV (µm CH o LT P RBC (x10 WBC (x10 M ph sino Monoc o ym E L 1200 (B) 1000 Group I Group II 800 Group III 600 Group IV Group V 400 Group VI 100
Units Group VII 80
60
40
20
0 ) 3 ) ) ) 12 /l) µl) L) % (% 3 / /d (%) ( (%) 3 /µl 0 µm (pg) s e 10 ( H (g il C tes h HCT (x1 (x C yte p HGB (g/L) C M c o MCV o ocy n LT (x10 h n i P RB WBC MCH p s Mo o ym E L Figure 3. Hematological parameters in female (A) and male rats (B) evaluated for sub-acute toxicity.
10 (A.I) 10 (B.I) Group I Group I 8 Group II 8 Group II Group III 6 6 Group III Group IV 4 4 Group IV Group V 0.5 Group V Units Units Group VI 0.5 0.4 Group VI Group VII 0.4 0.3 0.3 Group VII 0.2 0.2 0.1 0.1 0.0 0.0 ) ) ) ) L L) L L L) L /d /d /d /d g/d g g g ( mg ( in (g/dL) in ( in (m e e e um n b ni ubin (mg/dL) rote in i r P in Al at Albumin ( al eat re t Bilirubin (mg/d Total Prot C o Cr l T ta o Total Bili T 250 Group I 250 (B.II) Group I (A.II) Group II Group II 200 Group III 200 Group III Group IV Group IV 150 Group V 150 Group V Group VI Group VI Units Units 100 100 Group VII Group VII
50 50
0 0 ) ) ) ) ) ) /L L L L L /d /d /d /dL IU g g mg l ( l (m (mg ALT ( AST (IU/L) o ALT (IU/L) AST (IU/L) o se ose (m r er Urea (mg/d e Urea (mg/dL) luc lest luco olest G G h ho C C Figure 4. Biochemical parameters in female (A.I and A.II) and male rats (B.I and B.II).
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Figure 5. Light photomicrographs of organ tissues after 28 days administration of poly(anhydride) nanoparticles: (I) NP; (II) NP-HPCD and (III) PEG-NP. The images presented here are from the females highest dose group, 300 mg/kg bw. The images are from thymus (a-c), stomach (d-f), intestine (g-i), Peyer patches (j-l), spleen (m-o) and liver (p-r).
3.4. In vivo biodistribution studies
In order to evaluate the biodistribution of orally administered nanoparticles, the different types of poly(anhydride) nanoparticles were labelled with 99mTc. The labelling yield was always over 90%. In this study, the animals (as in the sub-acute toxicity study) received orally every day a dose of nanoparticles (either 30 mg/kg or 300 mg/kg bw) during 28 days. On days 1, 15 and 28, the dose of nanoparticles included 1 mCi of 99mTc labelled carriers. Figure 6 shows the localization of radiolabelled nanoparticles 8 h post administration of the dose corresponding to day 15. The images revealed that nanoparticles were located in the stomach and distal parts of the GIT with no evidences 158 CHAPTER 6: Toxicity studies of poly(anhydride) nanoparticles as carriers for oral drug delivery
of distribution in other organs or nanoparticle translocation. This distribution was found to be similar for all the formulations and doses tested. Similarly, the distribution patterns of nanoparticles (NP, NP-HPCD and PEG-NP) within the animals were found to be similar on the images captured on days 1, 15 or 28 (data not shown).
Figure 6. Localization of 99mTc labeled A nanoparticles (NP) 8 h post administration of the dose (300 mg/kg) corresponding to day 28th. Coronal (left) and sagittal (right) SPECT-CT fused 6 mm-thick images. Sagittal cut was made following the A-B line. Color bar indicates relative radioactivity concentration. Arrow: stomach; arrowhead: small intestine and caecum.
B
3.5. Ex vivo biodistribution studies
According to the previously obtained images, the highest accumulation of radioactivity was found in the stomach, small intestine, caecum and large intestine. The %ID/g of radiolabelled nanoparticles (NP, NP-HPCD and PEG-NP) in each one of these organs 24 h after oral administration of the dose corresponding to day 28 is represented in Figure 7. A relative low concentration of radioactivity was also found in the liver and the kidneys, but this represented less than 0.05% ID/g in all the cases (data not shown). Comparing the different types of formulations, the radioactivity in the stomach and the large intestine was significantly higher for PEG-NP than for NP and NP-HPCD (p<0.05). Similarly, the
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radioactivity found in the washing liquids of the GIT of animals was found to be significantly higher for animals treated with PEG-NP than with NP or NP-HPCD (data not shown). Despite NP and NP-HPCD showed similar behavior, NP-HPCD seemed to pass slower through the stomach and small intestine. In fact, slightly higher radioactivity concentration was found in these organs for NP-HPCD but this difference was not statistically significant.
NP NP-HPCD PEG-NP 2.0 *
1.5
1.0
% ID/g % *
0.5
0.0 e ch ne a in um i st c st e e e Stom Int Ca Int ll rge Sma La Organs
Figure 7. Ex vivo biodistribution studies of 99mTc-labelled poly(anhydride) nanoparticles. Animals were sacrificed 24 h post-administration, organs extracted and radioactivity of each organ measured. * Statistically significant difference (p<0.05). Results expressed as the mean ± SD (n=3).
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4. DISCUSSION
From a general point of view, in vivo toxicity studies are more desirable since direct verification of the effect of nanoparticles toward the human body is achieved. Nevertheless, to date, the reported toxicity studies have mainly focused on in vitro examinations rather than in in vivo experiments. This fact can be due to the easy in execution as well as in the control and interpretation of the experiments compared with in vivo tests. The copolymers of methyl vinyl ether and maleic anhydride and their ether and salt derivatives (Gantrez® series) are widely employed in a diverse range of topical and oral pharmaceutical formulations as thickening and suspending agents, denture adhesives, film-coating agents and adjuvants for transdermal patches (29, 30). In the last years, these copolymers have also been employed to prepare nanoparticles with bioadhesive properties as vehicles for oral drug/antigen delivery (15-17, 21-25). These carriers offer a high versatility derived from the presence of anhydride residues which can easily react (under mild conditions) with different ligands and excipients and, thus, yielding “decorated” nanoparticles with improved targeting or controlled release properties. As a consequence these nanoparticles may facilitate the interaction and presentation of the loaded antigen or allergen with the antigen-presenting cells (APCs) (16) and/or improve the oral bioavailability of different drugs such as fluorouridine (15), paclitaxel (21) or atovaquone (ATO) (22). In spite of these interesting results, no information about the toxicological profile of these nanoparticles was still known. Although Gantrez® polymers alone are generally regarded as non-toxic and non-irritant for oral administration (31), their properties may change completely upon transformation in a nanoparticulate system. This is because the size, charge and surface modifications of the nanoparticles often decide their fate in vivo (32, 33). In this context, the main objective of this work was to evaluate the toxicity of the following types of poly(anhydride) nanoparticles when orally administered: conventional nanoparticles (NP), nanoparticles containing 2-hydroxypropyl-β- cyclodextrin (NP-HPCD) and pegylated nanoparticles (PEG-NP). For this purpose we decided to follow the standard procedures and guidelines proposed by the OECD which are currently used to test and assess chemicals. For the acute toxicity study, a limit test based on the oral administration to laboratory animals of a single oral dose of 2 g nanoparticles per kg bw in water was applied. This dose was around 200-times higher than the usual dose employed in previous studies with these nanoparticles (16, 18, 21). In all cases, the administered dose had no toxic effect. In fact, neither any death occurred nor abnormal or toxic responses were observed in the rats during the 14 day observation period. In addition no macroscopic pathological alterations attributed to nanoparticles were found in the necropsies. These findings were consistent with previous results with nanoparticles containing chitosan. Thus, Sonaje and collaborators (34) reported an apparent absence of toxicity in mice treated with 100 mg/kg chitosan nanoparticles with a mean size of about 220 nm. In the same way, the DL50 of gold nanoparticles coated with chitosan (10-50 161 CHAPTER 6: Toxicity studies of poly(anhydride) nanoparticles as carriers for oral drug delivery
nm) was found to be greater than 2000 mg/kg (35). On the contrary other types of nanoparticles, such as zinc or titanium oxide particles, appear to induce some toxicological problems. Thus, Wang and collaborators reported that the oral administration to mice of a large dose of 5 g/kg bw of titanium oxide nanoparticles (25, 80 or 155 nm) showed no obvious acute toxicity. However, animals treated with very fine nanoparticles (25 or 80 nm) displayed important changes of serum biochemical parameters associated to liver injury and nephrotoxicity (36). In another interesting report, it was demonstrated that the oral administration of 5 g/kg bw of zinc microparticles (1080 nm) induced more severe liver damage than zinc nanoparticles (60 nm), while these small ones could induce heavier renal damage and anemia (37). This different toxicological profile between polymeric and metallic particles may be ascribed, at least in part, to the capabilities of chitosan and poly(anhydride) to yield carriers able to develop sustained adhesive interactions with components of the mucus layer and/or the membrane epithelia (19, 38, 39). This fact would minimize the “translocation” and entry into the circulation for these polymeric nanoparticles compared with “non-adhesive” ones such as metallic nanoparticles. Interestingly, an absence of toxicological effects were also observed during the sub-acute toxicity studies with the three types of poly(anhydride) nanoparticles. All the animals survived the duration of the study, with no significant changes in clinical signs or bw. In these experiments, the hematological parameters of all the animals were found to be within the normal ranges with no differences between the control and experimental groups (p>0.05). These results suggested that poly(anhydride) nanoparticles are nontoxic when administered orally as they did not affect the circulating red cells, hematopoiesis or leucopoiesis that could otherwise have caused hematological disorders (40). With regard to biochemical parameters analyzed, which may reflect alterations in blood enzymes and are used to diagnose organ diseases (i.e. from heart, liver or kidney), no significant differences (p>0.05) between control and treated groups of both male and female animals were also observed. Again, this fact confirmed that poly(anhydride) nanoparticles did not generate cumulative and latent biochemical changes following multiple administrations even using doses 30- times higher than those reported in other studies (15-21). Moreover, all the outcomes aforementioned were corroborated with the macroscopic and histological analysis of the target organs. Indeed, no toxic lesions were evident in any of the organs evaluated and all the organs tested showed unaffected natural architecture. In spite of the number of sub-acute oral toxicity studies with nanoparticles is very scarce, the presented results appears to be in line with other previously reported involving gold nanoparticles or hydrogel nanoparticles. In the former, Dhar and co-workers reported the absence of any hematological or biochemical abnormalities observed with gellam gum-reduced gold nanoparticles (14 nm) orally administered daily at a dose of 300 mg/kg during 28 days (41). In the latter, nanoparticles obtained by the combination of hydroxyl propyl methyl cellulose and polyvinyl pyrrolidone (100 nm) were 28-day daily administered to rats and none of the treated animals evidenced toxic effects on vital organs or metabolic abnormalities (42).
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In order to gain insight about the fate of poly(anhydride) nanoparticles in the body, the carriers were radiolabelled with 99mTc and orally administered to rats. Neither imaging nor gamma counter data showed radioactivity concentration in liver, kidneys or spleen, confirming that nanoparticles were in no way absorbed through the intestine and transported to the blood circulation. These findings are different to that observed for other types of nanoparticles such as zinc (36) or silver nanoparticles (43). In this last report, describing a sub-acute study in rats, it was demonstrated that the toxicity of silver nanoparticles (60 nm) is related with their accumulation in the kidneys after absorption through the gut (43). Therefore, the safety of poly(anhydride) nanoparticles would be directly related to the absence of a “translocation” phenomenon. Comparing the three types of poly(anhydride) nanoparticles, it was observed that the radioactivity values quantified in the GIT of animals treated with PEG-NP were three times higher than for animals dosed with NP-HPCD or NP values (Figure 7). This slow transit through the gut for PEG-NP is consistent with previous observations reported by Yoncheva and collaborators, who demonstrated that pegylation of nanoparticles increased the bioadhesive capability of the resulting carriers (18, 19). In these studies it was postulated that pegylated poly(anhydride) nanoparticles would display a PEG-surface layer in a “brush” conformation. Due to this morphology, pegylated nanoparticles would be capable of diffuse across the mucus protective layer and reach the surface of the enterocytes. As a consequence, the residence time of these nanoparticles in the gut would be longer than for other types of carriers (i.e. NP or NP-HPCD).
5. CONCLUSIONS
Acute and sub-acute toxicity studies of poly(anhydride) nanoparticles administered by the oral route to rats demonstrated the absence of adverse effects related to either the treatment or sex. The distribution of the three different nanoparticle formulations appeared to be restricted to the GIT of animals and no evidences of “traslocation” or absorption of particulates was found. However, the residence within the gut of PEG-NP was found to be significantly higher than for conventional nanoparticles or NP-HPCD. Overall, the current findings confirm that poly(anhydride) nanoparticles are safe for both oral short-term and prolonged administrations.
Acknowledgments This work was supported by the Ministry of Science and Innovation in Spain (projects SAF2008-02538) and Caja Navarra Foundation (Grant 10828). Patricia Ojer was also financially supported by a grant from the Department of Education of the Government of Navarra in Spain.
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6. References
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CHAPTER 7 Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration
Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration
Patricia Ojer1,2, Adela López de Cerain2, María Sánchez3, Ivan Peñuelas3, and Juan M. Irache1
1 Department of Pharmacy and Pharmaceutical Technology. University of Navarra, Pamplona, 31008, Spain. 2 Department of Pharmacology and Toxicology. University of Navarra, Pamplona, 31008, Spain. 3 Radiopharmacy Unit. Department of Nuclear Medicine, University Clinic of Navarra, Pamplona, 31008, Spain.
ABSTRACT
Prior to in vivo studies, the hemocompatibility of the different poly(anhydride) nanoparticles (conventional (NP), nanoparticles containing 2-hydroxypropyl-β-cyclodextrin (NP-HPCD) and nanoparticles coated with polyethylene glycol 6000 (PEG-NP)) were studied in vitro at three different concentrations (0.25, 0.5 and 1 mg/mL). To evaluate the acute toxicity of different intravenously administered poly(anhydride) nanoparticles rats were exposed to two different doses (50 and 150 mg/kg body weight (bw)). Toxic effects were assessed via general behavior, hematological and serum biochemical parameters and histopathological observation of the animals. Biodistribution were assessed at a dose of 50 mg/kg bw using radiolabelling nanoparticles with 99mTechnetium (99mTc). The results showed that the different poly(anhydride) exhibited a homogenous size of about 175 nm and a negative zeta potential. Nanoparticles did not induce any agglutination effect neither hemolytic activity in the hemocompatibility studies carried out. In the acute toxicity study animals dosed with 150 mg/kg bw showed acute episodes of respiratory distress. In fact, 2/6 animals dosed with NP and NP-HPCD died. The histopathological examinations revealed apparent pathological changes characterized by the presence of foamy cells in the liver, spleen and lungs (Phospholipidosis (PLDsis)). Finally, biodistribution studies showed that nanoparticles accumulation occurred predominantly in organs of the reticular endothelial system (RES). Thus, although all nanoparticle formulations demonstrated to be hemocompatible in vitro and in vivo, the intravenous (i.v.) toxicity was dose dependent, with the presence of foamy cells in spleen, liver and lungs as the most relevant finding observed.
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CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration
1. INTRODUCTION
Research conducted in nanomedicine has enabled the development and of new and/or improved diagnostic and therapeutic methods. In this context, the use of biodegradable polymers based nanoparticles has been identified as promising candidates in different pharmaceutical fields. Among the various routes of administration, polymeric nanoparticles administer by i.v. route have received much attention. Their use offers advantages such as carriers for otherwise insoluble or poorly soluble drugs, imaging agents and gene delivery purposes (1). Furthermore, as drug delivery devices, injectable polymeric nanoparticles can be seen to prolong the blood half life of drugs, increase their efficacy and represent very attractive properties for targeting applications to improve the selectivity in drug delivery. Their efficacy largely depends on the control of their biodistribution within the body. Such a distribution after i.v. administration is considerably influenced by their interactions with the biological environment and their physicochemical properties, including particle size, surface charge, morphology, and surface hydrophilicity (2). Indeed, following i.v. administration, nanoparticles can accumulate in different organs through systemic circulation and induce potential toxicity. This fact has been widely demonstrated in the last years by different authors in relation to engineered nanomaterials due to its no biodegradability (3, 4). On the contrary, for biodegradable PCL (5) and polyacrilamide nanoparticles (6) it has been reported absence of toxicity in an acute toxicity study after i.v. administration. Hence, using biocompatible and biodegradable polymers has been outlined as a good option to develop nanoparticles for i.v. administration. In this regard, the most common choices are polymers as polyesters (e.g. PCL and poly(lactide-co-glycolide) (PLGA)) (7, 8) and macromolecules (e.g. albumin and chitosan) (9, 10). However, although there are a number of studies regarding the evaluation of the efficacy of polymeric nanoparticles as drug delivery devices, knowledge on i.v. toxicity of these nanoparticles is scarce. Therefore, evaluation (parallel to the study of its effectiveness) of the potential toxic effects of these nanoparticles is required, given that specific mechanisms and pathways through which nanoparticles may exert their toxic effects remain largely unknown. In the recent years, poly(methyl vinyl ether-co-maleic anhydride) [Gantrez® AN] nanoparticles have been considered promising platforms for drug delivery and other biomedical applications. Poly(anhydride) nanoparticles have successfully been developed as oral drug delivery systems (11-13), immunization (14-16) or allergy-immunotherapy treatment (17, 18). In addition, the oral toxicity of plain, cyclodextrin and pegylated poly(anhydride) nanoparticles has been evaluated in vivo confirming its oral safety (19). Particularly, the investigations demonstrated that LD50 for all nanoparticles tested was higher than 2000 mg/kg bw and the sub-chronic oral toxicity studies at two different doses (30 and 300 mg/kg bw) showed no evident signs of toxicity. Given that the efficacy and safety of these nanoparticles has been widely demonstrated by the oral route, the assessment of their toxicity by the i.v. route has been the objective of this study, in order to evaluate the possibility of administering such formulations by the i.v. route as a new strategy 173 CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration that may be useful for some specific applications. To this aim, three different types of poly(anhydride) nanoparticles have been studied: conventional (NP), nanoparticles containing 2- hydroxypropyl-β-cyclodextrin (NP-HPCD) and nanoparticles coated with PEG6000 (PEG-NP). Specifically, the influence of these nanoparticles on erythrocyte sedimentation and agglutination as well as their hemolytic activity was tested in vitro. Then, single dose in vivo toxicity studies of the different poly(anhydride) nanoparticles were carried out. In addition, in vivo biodistribution after i.v. administration using radiolabelled nanoparticles to assess qualitative and quantitative tissue distribution in rat was also investigated.
174 CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration
2. MATERIALS AND METHODS
2.1. Chemicals
Poly(methyl vinyl ether-co-maleic anhydride) or poly(anhydride) (Gantrez® AN 119; Mw 200,000) was provided by ISP corp. (Waarwijk, The Netherlands). Polyethylene glycol 6000 (PEG6000) was provided by Fluka (Switzerland). 2-hydroxypropyl-β-cyclodextrin (HPCD) was provided by Sigma–Aldrich (Steinheim, Germany). Acetone was obtained from VWR Prolabo (Fontenay-sous-Bois, France). Ultrafiltrated water (18.2MΩ resistivity) was prepared by a water purification system (Wasserlab, Pamplona, Spain). 99Mo-99mTc generator (Drytec; GE Healthcare Bio- Science, United Kingdom) was eluted with 0.9% NaCl following the manufacturer’s instructions. SnCl2 and HCl were purchased from Panreac (Barcelona, Spain) and 0.9% NaCl from Braun (Barcelona, Spain). The anesthetic isoflurane (Isoflo™) was from Esteve (Barcelona, Spain). All other chemicals and solvents used were of analytical grade.
2.2. Preparation of poly(anhydride) nanoparticles
Poly(anhydride) nanoparticles were prepared from a dissolution of Gantrez® AN 119 in acetone by a simple desolvation method following by a dried step in a Mini Spray-dryer Büchi B290 (Büchi Labortechnik AG, Switzerland) as described previously (20). Three different types of nanoparticles were evaluated: conventional nanoparticles (NP), nanoparticles containing HPCD (NP- HPCD) and pegylated nanoparticles with PEG 6000 (PEG-NP).
2.2.1. Conventional poly(anhydride) nanoparticles (NP) Briefly, 500 mg of the copolymer of methyl vinyl ether and maleic anhydride were dissolved and stirred in 30 mL acetone. Then, the desolvation of the polymer was induced by the addition of 15 mL ultrafiltrated water under magnetic stirring to the organic phase. In parallel, 1 g of lactose was dissolved in 10 mL ultrafiltrated water and added to the nanoparticle suspension under agitation for 5 min at room temperature. Finally, the suspension was dried in the Mini Spray-dryer Büchi B290. The recovered powder was stored in closed vials at room temperature.
2.2.2. Poly(anhydride) nanoparticles containing 2-hydroxypropyl-β-cyclodextrin (NP-HPCD) Briefly, 500 mg of the copolymer of methyl vinyl ether and maleic anhydride were dissolved and stirred in 20 mL acetone. Then, 10 mL acetone containing 125 mg HPCD were added to the polymer solution under magnetic stirring and incubated for 30 min. Nanoparticles were obtained by the addition of 15 mL ultrafiltrated water under magnetic stirring to the organic phase. In parallel, 1 g of lactose was dissolved in 10 mL ultrafiltrated water and added to the nanoparticle suspension under agitation for 5 min at room temperature. Finally, the suspension was dried in the Mini Spray-dryer Büchi B290. The recovered powder was stored in closed vials at room temperature.
175 CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration
2.2.3. Pegylated poly(anhydride) nanoparticles (PEG-NP) In this case, 62.5 mg PEG were dissolved in 15 mL acetone and mixed with 15 mL acetone containing 500 mg of the copolymer of methyl vinyl ether and maleic anhydride and incubated under magnetic stirring for 1 h. Then, nanoparticles were obtained by the addition of 15 mL ultrafiltrated water under magnetic stirring to the organic phase. The solvents were eliminated under reduced pressure (Büchi R-144, Switzerland) and nanoparticles were ultrafiltrated by double centrifugation at 17,000 rpm for 20 min (Sigma 3K30, Germany). The pellet was resuspended in 25 mL ultrafiltrated water containing 1 g of lactose. Finally, the suspension was dried in the Mini Spray-dryer Büchi B191. The recovered powder was stored in closed vials at room temperature.
2.3. Characterization of the nanoparticles
2.3.1. Size and zeta potential The mean hydrodynamic diameter and the zeta potential of nanoparticles were determined by photon correlation spectroscopy (PCS) and electrophoretic laser Doppler anemometry, respectively, using a Zetamaster analyzer system (Malvern Instruments, United Kingdom) and a ZetaPlus zeta potential analyzer (Brookhaven Instruments Corp., Holtsville, NY). The diameter of the nanoparticles was determined after dispersion in purified water (1/10) and measured at 25ºC by dynamic light scattering angle of 90 ºC. The zeta potential was determined by diluting the samples in a 0.1 mM KCl solution adjusted to pH 7.4. All measurements were performed in triplicate. The morphology of the nanoparticles was examined using a scanning electron microscope (Zeiss DSM 940A SEM; Oberkochen, Germany) with a digital imaging capture system (DISS) (Point Electronic GmBh; Halle, Germany). For this purpose, a small amount of the spray dried powders was resuspended in ultrapure water and centrifuged at 27,000×g for 20 min at 4°C to eliminate the sugars. Then, supernatants were rejected and the pellets were resuspended in water and mounted on a plate adhered with a double-sided adhesive tape onto metal stubs and dried.
2.3.2. Quantification of HPCD and PEG The amount of HPCD and PEG associated to the nanoparticles as well as the amount of the polymer transformed into nanoparticles was estimated using two different high performance liquid chromatography (HPLC) methods previously described (21, 22). Specifically the apparatus used for the HPLC analysis was a model 1100 series Liquid Chromatography, Agilent (Waldbronn, Germany) coupled with an evaporative light scattering detector (ELSD) 2000 Alltech (Illinois, United States). An ELSD nitrogen generator Alltech was used as the source for the nitrogen gas. Data acquisition and analysis were performed with a Hewlett-Packard computer using the ChemStation G2171 AA program. The amount of HPCD and PEG associated to nanoparticles was calculated as the difference between the initial HPCD and PEG and the amount of those recovered in the supernatants. Similarly, the amount of the poly(anhydride) copolymer was estimated by difference in the same way.
176 CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration
2.4. Labelling of nanoparticles with 99mTc
Poly(anhydride) nanoparticles were labelled with 99mTc by reduction of 99mTc-Pertecnetate (99mTcO4-) with stannous chloride as described previously (23). Briefly, 20 µl of a stannous chloride solution adjusted to pH 1 with 0.1 N HCl with a final concentration of stannous of 0.03 mg/mL were added to 1 mg of the different nanoparticle formulation (NP, NP-HPCD and PEG-NP) followed by the addition of 56 MBq of freshly eluted 99mTcO4- in 0.5 mL of NaCl 0.9%. The final suspension was vortexed for 30 seconds and incubated at room temperature for 10 min and showed a pH of 4. The overall procedure was carried out in helium-purged vials using helium-purged solutions to minimize oxygen content and avoid oxidation of tin chloride. Just before administration to the animals, radiolabelled nanoparticles were mixed with non-labelled ones to adjust the required dose for administration to animals of 50 mg/kg bw.
2.5. Hemocompatibility studies
2.5.1. Preparation of erythrocytes Whole blood was collected from three healthy volunteers in heparinised tubes (lithium heparin). The study was approved by the Committee on Ethics of the Clinic University of Navarra, and the volunteers signed a written consent. The experiments on human blood were performed on the day of its collection. Blood from the three volunteers was centrifuged 10 min at 1500 x g and room temperature separately. The supernatant consisting on peripheral blood mononuclear cells and a plasma band was discarded with a Pasteur pipette. The pellet consisting on erythrocytes was washed three times with isosmotic PBS (Phosphate Buffered Saline) w/o calcium and magnesium (1:1 v/v). A 4% suspension of erythrocytes in PBS was used for experiments. All the experiments were performed in triplicate.
2.5.2. Erythrocyte sedimentation and agglutination Different concentrations of poly(anhydride) nanoparticles (0.25, 0.5 and 1 mg/mL) in 100 µL of erythrocyte suspension were incubated together in eppendorfs (1:1 v/v) for 1 h at 37ºC. A negative control of erythrocytes treated with PBS and a positive control of erythrocytes treated with Triton X-100 0.1% were included in all the experiments. Erythrocytes sedimentation and agglutination was recorded by using a camera.
2.5.3. Hemolysis assay Hemolysis induced by nanoparticle treatment was assessed photometrically. 100 µL of nanoparticle suspensions (0.25, 0.5 and 1 mg/mL) were incubated (1:1 v/v) with the erythrocyte suspension at 37ºC for 1 h. After incubation all samples were centrifuged for 10 min at 1500 x g and the supernatants were recovered. Free hemoglobin (HGB) was measured photometrically in the supernatant at 540 nm. The percentage of hemolysis of each nanoparticle formulation, corrected by
177 CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration the spontaneous absorbance or erythrocytes incubated with PBS suspension was calculated as follows:
[A] sample [Eq. 1] % Hemolysis = X 100 [A] 100% hemolysis
Where, [A]sample is the absorbance of the erythrocytes incubated with nanoparticle suspension and [A]100% hemolysis is the absorbance of the supernatant of the erythrocyte incubated with Triton X-100 (0.1%) solution in PBS suspension. All samples were analyzed in triplicate.
2.6. Animals
The experimental protocols involving animals were carefully reviewed and approved by the Ethical Committee for Animal Experimentation of the University of Navarra (Spain) (Protocol number 044-11). Eight week old female Wistar rats were purchased from Harlan (Horst, The Netherlands) and employed for both single dose toxicity studies and biodistribution studies. On the day of arrival, the animals were weighed in order to assure that bw variation did not exceed ± 20% (24). They were randomly housed in groups in polycarbonate cages with stainless steel covers to allow acclimatization to the environmental conditions (12 h day/night cycle, temperature 22 ± 2ºC, relative humidity 55 ± 10%, standard diet ‘‘2014 Teklad Global 14% Protein Rodent Maintenance Diet” from Harlan Iberica Spain and water ad libitum).
2.7. Single dose toxicity study
The design of the study was based on the Organisation for Economic Co-operation and Development (OECD) guideline 425 for the Testing of Chemicals (24) with some modifications. Animals were treated with either saline (control group) or a suspension of nanoparticles in saline. The dose selected of each nanoparticle formulation was based on the results of a preliminary study carried out in our laboratory in which there was not apparent toxicity: 50 mg/kg bw and three times the reported value, 150 mg/kg bw. Animals were injected via tail vein with saline or test suspensions at a volume of 0.5 mL/100 g bw. Animals were randomly divided into seven groups, containing six female Wistar rats per group. First group served as control and for each nanoparticle formulations (NP, NP-HPCD and PEG-NP) animals received a dose of either 50 mg/kg bw or 150 mg/kg bw. The groups were named as: NP50, NP150, NP-HPCD50, NP-HPCD150, PEG-NP50 and PEG-NP150 respectively. Individual animal weights were registered on day 0 and weekly during 14 days. Animals were observed individually for any clinical signs or any symptoms of toxicity at different time intervals after administration (10 min, 30 min, 1 h, 3 h and 6 h) and daily during 14 days. On completion of the study, on day 14, blood samples were extracted from the retro-orbital sinus under isoflurane 178 CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration anesthesia. The following hematological parameters were analyzed: hemoglobin (HGB; g/L), hematocrit (HCT, %), red blood corpuscles count (RBC), white blood corpuscles count (WBC), absolute erythrocyte indices and differential WBC. Biochemical analyses of plasma samples were performed with a Hitachi 911™ (Roche Diagnostics) analyzer using the protocols obtained from Roche for the determination of the standard parameters: total protein (g/dL), albumin (g/dL), glucose (mg/dL), aspartate transaminase (AST; U/l), alanine transaminase (ALT; U/l), cholesterol (mg/dL), creatinine (mg/dL), urea (mg/dL) and total bilirrubin (mg/dL).
Thereafter, the animals were sacrificed in CO2 chamber, subjected to necropsy and various organs (spleen, heart, liver, kidneys, adrenal glands, ovaries and thymus) were collected and fixed for further histopathological examination.
2.8. Histopathological studies
Tissue samples from different body organs were taken during necropsy, fixed in 4% formaldehyde solution, dehydrated and embedded in paraffin. Paraffin sections (3 micrometers) were cut, mounted onto glass slides, and dewaxed and stained with haematoxylin and eosin (H&E) for the subsequent histopathological examination.
2.9. In vivo and ex vivo biodistribution studies with radiolabelled nanoparticles
For in vivo studies, female rats were anesthetized with 2% isoflurane in 100% O2 and placed in prone position on a gammacamera (SYMBIA-T2 TruePoint®; Siemens, Munich, Germany). Either one of the radiolabelled nanoparticle suspensions (99mTc-NP, 99mTc-NP-HPCD and 99mTc-PEG-NP) or free 99mTcO4- were intravenously administered in the tail vein of the animals (50 mg/kg bw in 0.5 mL/100 g bw, 56 MBq). Five consecutive single-photon emission computed tomography (SPECT- CT) studies (90 images, 15 seconds/image, 2 detectors, matrix 128 x 128, zoom 1) where acquired. SPECT-CT data were exported to PMOD program and volumes of interest (VOIs) drawn on computed tomography (CT) images for quantification. For ex vivo studies, a second group of animals (n=3) was sacrificed 5 h after the i.v. administration of the radiolabelled nanoparticle formulations. Then, blood, urine, feces and different organs (lungs, heart, spleen, pancreas, liver, kidneys, stomach, small intestine, large intestine, caecum, rectum, brain, bone and muscle) were collected, weighted and the radioactivity of each organ measured in a gamma counter (1282 Compugamma CS, LKB Pharmacia, Finland) calibrated for 99mTc energy. Finally, results were expressed as the percentage of injected dose per gram (%ID/g).
2.10. Statistical analysis
Data were expressed as the mean ± SD of at least three experiments. For the nanoparticles characterization the Student t test was used. Other statistical significance analysis were carried out using the non-parametric Mann-Whitney U test. P values of < 0.05 were considered as statistically
179 CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration significant. All calculations were performed using SPSS® statistical software program (SPSS® 15.0, Microsoft, United States).
180 CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration
3. RESULTS
3.1. Nanoparticles characterization
The main physicochemical characteristics of poly(anhydride) nanoparticles formulations are summarized in Table I. All formulations exhibited a homogeneous size of about 175 nm, with a polydispersity index (PDI) lower than 0.3 and a negative zeta potential.
Table I. Physico-chemical characteristics of nanoparticles. NP: poly(anhydride) nanoparticles; NP- HPCD: hydroxypropyl cyclodextrin-poly(anhydride) nanoparticles; PEG-NP: pegylated nanoparticles. PDI: polydispersity index. Data expressed as the mean ± standard deviation (n=6).
µg ligand/mg Formulation Size (nm) PDI Zeta Potential Nanoparticle (mV) formation yield (%) Nanoparticle NP 159 ± 1.2 0.17 ± 0.05 -42.9 ± 1.5 97 ± 2.1
NP-HPCD 180 ± 2.3 0.21 ± 0.03 -38.2 ± 3.3 98 ± 3.3 74.6 ± 2.2
PEG-NP 183 ± 2 0.23 ± 0.04 -31.7 ± 2.3 97 ± 3.1 53.4 ± 2.8
The morphological analysis by scanning microscopy (Figure 1) revealed that NP, HPCD-NP and PEG-NP consisted of spherical particles with a smooth surface in case of NP. In contrast HPCD-NP and PEG-NP displayed a rough surface. For NP-HPCD, the amount of associated cyclodextrin was calculated to be about 75 µg/mg whereas for pegylated nanoparticles, the amount of PEG linked to nanoparticles was 53 µg/mg. Finally, concerning the yield of the process, in all cases, this parameter was calculated to be very high and close to 100 %.
Figure 1. SEM photographs of nanoparticles. (A) NP. (A.1) Magnification of a section of photograph (A). (B) NP-HPCD. (B.1) Magnification of a section of photograph (B). (C) PEG-NP. (C.1) Magnification of a section of photograph (C).
181 CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration
3.2. Erythrocyte sedimentation and agglutination
Erythrocyte sedimentation rate (ESR) was determined by the observation of the sediment height in a given time interval, usually 1 h. Compared to control, the different poly(anhydride) nanoparticles did not induce any agglutination effect, which suggests that the integrity of the erythrocytes membrane remained intact (data not shown).
3.3. Hemolysis study
The hemolytic activity of different poly(anhydride) nanoparticles was tested after incubation with erythrocyte suspension 1 h at 37ºC at different concentrations. The addition of the nanoparticles at different concentrations did not cause hemolysis (Figure 2). Indeed the percentage of hemolysis obtained for the treatments and by adding PBS (spontaneous HGB release) were all about 8%.
120
100
80
0.25 mg/mL 60 0.5 mg/mL 1 mg/mL
% Hemolysis 40
20
0 Triton X-100 (0.1%) PBS NP NP-HPCD PEG-NP
Figure 2. Hemolytic activity of poly(anhydride) nanoparticles at concentrations of 0.25, 0.5 and 1 mg/mL. Total cell lysis and spontaneous HGB release were achieved by application of Triton X-100 (1%) and PBS respectively. Data expressed as mean ± SD (n=3).
3.4. Single dose toxicity study
Throughout all the observation period, poly(anhydride) nanoparticles at a dose of 50 mg/kg bw did not produce the death of animals. Moreover, no abnormal clinical signs or behaviors were detected neither in treated nor in control groups. On the contrary, all animals dosed with 150 mg/kg bw showed signs of passivity, piloerection and respiratory distress (Table II). These symptoms were more severe in animals treated with NP and NP-HPCD and continued 3 h after dosing. Indeed, 2/6 animals died after dosing 150 mg/kg bw of NP and NP-HPCD respectively. In both cases the deaths occurred about 3 h after administration. However, in animals treated with PEG-NP these symptoms were milder than the observed in animals treated with NP and NP-HPCD and disappeared 1 h after administration. Indeed, PEG-NP at a dose of 150 mg/kg bw did not cause any deaths. In all cases, the reported toxic 182 CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration effects returned to normal gradually the next day after administration and mortality was not observed during the study.
Table II. Mortality rate and symptoms scored after single i.v. administration of vehicle and nanoparticle suspensions doses of 50 mg/kg bw or 150 mg/kg bw. Saline: Control, NP50/150: poly(anhydride) nanoparticles; NP-HPCD50/150: hydroxypropyl cyclodextrin-poly(anhydride) nanoparticles; PEG-NP50/150: pegylated nanoparticles.
Respiratory Treatment Piloerection Passivity Mortality distress Saline - - - 0/6
NP50 - - - 0/6
NP-HPCD50 - - - 0/6
PEG-NP50 - - - 0/6
NP150 ++ +++ +++ 2/6
NP-HPCD150 ++ +++ +++ 2/6
PEG-NP150 + ++ + 0/6 Severity of the symptoms: (-) no symptoms; (+) weak; (++) moderate and (+++) strong.
According to weight register, no significant differences were observed in bw of the animals of treatment groups compared with control ones. Furthermore, all the hematological and biochemical values obtained on day 14th were found to be within the normal range with no differences between the control and the treatment groups (data not shown). The relative organ weight of heart, kidneys, adrenals, thymus and ovaries were also unaffected by the nanoparticles. However, the i.v. administration of the different poly(anhydride) nanoparticles induced an increase in the relative weight of spleens (p<0.05) (Figure 3). Furthermore, PEG-NP induced a significant increase of the liver relative weight at both doses compared with the control group (p<0.05).
183 CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration
15 * * 10
CONTROL 5 NP50 1.0 * * * NP150 0.8 * * * NP-HPCD50 0.6 NP-HPCD150 0.4 PEG-NP50 Relative organs weight(%) 0.2 PEG-NP150 0.0 Spleen Liver
Figure 3. Spleen and liver relative organ weights. Data expressed as mean ± SD (n=6) * (p<0.05) statistically significant difference compared with control.
3.5. Histopathology
Regarding histology, the gross and histopathology of spleen, liver and lungs revealed apparent pathological changes for NP, NP-HPCD and PEG-NP at a dose of 150 mg/kg bw (Figure 4). Actually, an intense proliferation of foamy cells located in the red pulp and lymphoid follicles were observed in the spleen. Moreover, lungs showed normal areas with moderate inflammatory infiltration in the alveolar septa, highlighting the presence of small groups of foamy cells associated. In the liver, small periportal hemorrhages were found as well as massive and diffuse infiltration of foamy cells between hepatocytes.
3.6. In vivo biodistribution studies
For all the formulations tested, nanoparticles disappeared rapidly from the blood-stream during the first 10 min. after their i.v. administration. Then, during the following min, NP was the first to reach the liver and disappear from the lungs and NP-HPCD and PEG-NP remained longer in the lungs and reached the liver later. Moreover, the SPECT-CT images obtained 100 min after administration, clearly showed different biodistribution patterns for the different type of radiolabelled poly(anhydride) nanoparticles (Figure 5) although nanoparticles accumulation occurred predominantly in organs of the RES. Thus, while 99mTc-NP was mainly located in the liver, 99mTc- HPCD-NP and 99mTc-PEG-NP were mainly located in the lungs and also in the kidneys. On the contrary, free 99mTc-pertechnetate used as control was basically located in the stomach.
184 CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration
Figure 4. Histological sections of spleen, liver and lung samples collected two weeks after single i.v. administration of different poly(anhydride) nanoparticles at a dose of 150 mg/kg bw. Accumulation of foamy cells was detected (black arrows).
3.7. Ex vivo biodistribution studies
Additional experiments without imaging permitted more detailed quantification of the amount of radioactivity in the different organs at 5 h post-administration (Figure 6). According to the results obtained, the highest accumulation of radioactivity was found in the kidneys, following by the liver, lungs, spleen, urine and blood. A relative low concentration of radioactivity was also found in other organs as hearth, intestines, stomach and thymus, but in all cases represented less than 1% ID/g (data not shown). Comparing the different formulations, the radioactivity in the spleen, kidney, blood and urine were similar. However, the highest value of radioactivity in the liver corresponded with 99mTc-HPCD- NP (10.33%±0.92) following by 99mTc-PEG-NP (9.72%±0.95) and 99mTc-NP (7.9%±0.22). Moreover, the radioactivity measured in the lungs was found to be significantly lower (p<0.05) for animals treated with NP (1.97±0.54) than for HPCD-NP (8.29±0.65) and PEG-NP (8.66±0.48). Thus, although 5 h after administration all formulations seemed to be accumulated in the same organs, 99mTc-NP-HPCD and 99mTc-PEG-NP appeared to remain longer in the lungs.
185 CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration
Figure 5. 3D summed SPECT-CT images of the localization 100 min after administration of the radiolabelled nanoparticles. (A): 99mTc-NP, (B): 99mTc-NP-HPCD, (C): 99mTc-PEG-NP and (D): free 99mTc-pertechnetate.
15
NP NP-HPCD 10 PEG-NP %ID/g
5
*
0 r n s d e y o in lee o Live p Ur Lungs S Bl Kidne Organs
Figure 6. Ex vivo biodistribution studies of 99mTc-labelled poly(anhydride) nanoparticles. Animals were sacrificed 5 h post-administration, organs extracted and radioactivity of each organ measured. Results expressed as the mean ± SD (n=3) * (p<0.05) statistically significant difference.
186 CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration
4. DISCUSSION
In the last years, the copolymers of methyl vinyl ether and maleic anhydride have been employed to prepare nanoparticles with bioadhesive properties as vehicles for oral drug/antigen delivery (25, 26). Recently, a comprehensive oral biodistribution study has been carried out in conventional poly(anhydride) nanoparticles (NP), nanoparticles containing 2-hydroxypropyl-β- cyclodextrin (NP-HPCD) and pegylated nanoparticles (PEG-NP) (19). The results showed that these nanoparticles remained within the gut after their administration and no evidences of particulate absorption were found. Therefore, as the effects of these nanoparticles in the circulatory system are still unknown, evaluate their behavior and safety by i.v. route before testing its therapeutic effectiveness is mandatory since this route of administration represents the worst case scenario of toxicity among all the different administration routes. Taking this into account, the aim of the present study was to assess the in vitro hemocompatibility, potential acute toxicity and biodistribution of NP, NP-HPCD and PEG-NP following a single i.v. injection in rats. Our results demonstrated the hemocompatibility of the different poly(anhydride) nanoparticles independently of their concentration or surface coating. Indeed, none of the formulations caused any adverse effects on erythrocytes, neither agglutination nor hemolysis. This outcome was consistent with findings of Bender and co-workers who revealed that lipid-core nanocapsules with sizes about 130 nm did not induce hemolysis when nanoparticles were uncoated or coated with chitosan (27). At a dose of 50 mg/kg bw, rats exposed to the different poly(anhydride) formulations by i.v. route did not showed abnormal signs of behaviors throughout the acute study suggesting that NP, NP-HPCD and PEG-NP could be well tolerated at this dose level. On the contrary, when nanoparticles were evaluated at the higher dose (150 mg/kg bw) evident signs of toxicity were observed. Thus, 30% of the animals treated with the high dose of either NP or NP-HPCD died (see Table II). However, among animals treated with PEG-NP mortality was not observed. Nevertheless, in all groups, the toxicity observed were consistent with an acute episode which was characterized by passivity, piloerection and respiratory distress. However, the acute episode was resolved within 3 h for NP and NP-HPCD, or 1 h for PEG-NP. On the other hand, evident histopathological changes were observed in the spleen, lungs and liver of animals (see Figure 4). These changes were put in evidence by the presence of macrophages with foamy material in their cytoplasm in the observed organs. This disorder, called PLDsis, appears to be caused by cationic amphiphilic drugs, which stimulate uptake and/or accumulation of phospholipids by cells (28). However, PLDsis is usually reversible and after the end of the treatment, the phospholipids levels return to normal and the ultrastructural changes diminish or disappear (29). Due to this, there is a prevailing theory that PLDsis is primarily an adaptative response to drug exposure rather than a toxic response. In this regard, even though poly(anhydride) nanoparticle do not possess a cationic amphiphilic nature, it seems that following i.v. administration, macrophages 187 CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration located in the spleen, liver and lungs recognized the different types of poly(anhydride) nanoparticles. As macrophages are involved in uptake and metabolism of foreign molecules and particulates (30), they could phagocyte and metabolize the internalized nanoparticles adopting a foamy morphology. Then high dose administered could contribute to the accumulation of foamy cells in the different organs and this would induce pathological changes as the hepatosplegnomegaly observed. All of these observations would be directly related with the biodistribution of nanoparticles after i.v. administration. Thus, after administration, nanoparticles would interact with serum of plasma proteins in a phenomenon called “opsonization” (31). This fact would occur with the three types of nanoparticles; although the class of serum proteins adsorb on the surface of nanoparticles may be different and thus their fate might be different. There is growing evidence which suggest that opsonization of pegylated or sterically protected carriers occur efficiently. One example is complement activation by long-circulating liposomes, which result in surface opsonization with C3b and iC3b (32, 33). In this way, poly(anhydride) nanoparticles have been found to efficiently interact with proteins of the complement. Concretely, Gomez and Camacho evaluated nanoparticle-complement interactions by analyzing of C3a and the opsonin C3b, demonstrating that poly(anhydride) nanoparticles can be strong complement fixing surfaces (34, 35). In any case, after i.v. administration of nanoparticles, these would reach the lung capillaries, in which it would be facilitated, at least in part, the transient formation of nanoparticle aggregates. This fact has been observed previously by other authors. Thus, Yang and collaborators described a similar effect in mice after the administration of chitosan-coated PLGA nanoparticles of about 200 nm (36). Nanoparticles accumulation in lungs was also reported by Xie and co-workers for 80 nm silica nanoparticles after i.v. administration in mice (37). In our case, this effect would be more pronounced for NP and NP-HPCD than for PEG- NP, probably by a different protein adsorption pattern on the surface of nanoparticles. These aggregates may cause transient embolism in the lungs capillaries inducing respiratory distress as observed in animals. However, the dissociation of aggregates would soon proceed inducing a loss of accumulated nanoparticles in the lungs and then, the recovery observed in animals. Once the nanoparticles would leave the lungs they would be captured the monocyte-macrophage system located in the liver (i.e. Kuppfer cells) and spleen (i.e. red-pulp macrophages). This effect appeared to be more rapidly for NP than for NP-HPCD or PEG-NP. Thus, 2 h, post-administration, NP appeared to be located mainly in the liver (data not shown). On the contrary, for NP-HPCD and PEG-NP, the amount of radioactivity measured in lungs (5-h post administration) was about 4-time higher than that observed with NP (see Figure 6). In this regard, different studies have demonstrated that on exposure to blood, nanoparticles of different surface characteristics, size and morphology attract different arrays of opsonins as well as
188 CHAPTER 7: Hemocompatibility, acute toxic effects and biodistribution of poly(anhydride) nanoparticles following intravenous administration other plasma proteins which may account for the different pattern in the rate and site of nanoparticle clearance for the blood-stream (2, 38, 39). Finally, the fraction of nanoparticles that would evade their capture by macrophages would reach the kidneys in which they would accumulate or trap in the glomerule. This behavior observed for poly(anhydride) nanoparticles is similar to the biodistribution pattern found for chitosan (40) and PLGA nanoparticles (41). Thus, Zhang and co-workers reported that N-octyl-O-sulfate chitosan nanoparticles intravenously administered were accumulated with time in kidneys. In another study conducted by Giri and collaborators, surface modified PLGA nanoparticles for hepatitis B treatment were found to be mainly accumulated in blood, kidneys, liver, spleen and lungs suggesting that this biodistribution pattern could be due to the combined activity of the circulating blood passing through organs as well as the particle uptake by the monocyte-macrophage system. Until the end of the study, animals showed normal behavior and the results of biochemical and hematological analysis at day 14th were found within the normal values comparing to control. Therefore, as PLDsis has been described as a reversible process, it may occur that the histopathological changes finally disappear. However, this hypothesis should be confirmed by recovery studies. Furthermore, although pegylated nanoparticles did not result in increased residence time in blood, in relation to the single dose toxicity studies carried out, PEG-NP demonstrated to be safer than NP or NP-HPCD.
5. CONCLUSIONS
This study presents a comprehensive evaluation of hemocompatibility, biodistribution and acute i.v. toxicity of conventional (NP), nanoparticles containing 2-hydroxypropyl-β-cyclodextrin (NP-HPCD) and nanoparticles coated with PEG6000 (PEG-NP). All nanoparticle formulations demonstrated to be hemocompatible independent of their surface coating. After i.v. administration of a dose of 50 mg/kg bw, which revealed no evident toxicity, the different poly(anhydride) nanoparticles were distributed in the kidneys, liver, spleen and lungs. The single dose toxicity studies have revealed that the i.v. toxicity of the different poly(anhydride) nanoparticles was dose dependent. Only the highest dose of 150 mg/kg bw was toxic, with the presence of foamy cells in spleen, liver and lungs as the most relevant finding observed.
Acknowledgments This work was supported by the Ministry of Science and Innovation in Spain (projects SAF2008-02538) and Caja Navarra Foundation (Grant 10828). Patricia Ojer was also financially supported by a grant from the Department of Education of the Government of Navarra in Spain.
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6. References
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12. M. Agueros, S. Espuelas, I. Esparza, P. Calleja, I. Penuelas, G. Ponchel, and J.M. Irache. Cyclodextrin-poly(anhydride) nanoparticles as new vehicles for oral drug delivery. Expert Opin Drug Deliv. 8:721-734 (2011). 13. J. Calvo, J.L. Lavandera, M. Agueros, and J.M. Irache. Cyclodextrin/poly(anhydride) nanoparticles as drug carriers for the oral delivery of atovaquone. Biomed Microdevices. 13:1015-1025 (2011). 14. R. Da Costa Martins, J.M. Irache, J.M. Blasco, M.P. Munoz, C.M. Marin, M. Jesus Grillo, M. Jesus De Miguel, M. Barberan, and C. Gamazo. Evaluation of particulate acellular vaccines against Brucella ovis infection in rams. Vaccine. 28:3038-3046 (2010). 15. H.H. Salman, J.M. Irache, and C. Gamazo. Immunoadjuvant capacity of flagellin and mannosamine-coated poly(anhydride) nanoparticles in oral vaccination. Vaccine. 27:4784- 4790 (2009). 16. A.I. Camacho, R. Da Costa Martins, I. Tamayo, J. de Souza, J.J. Lasarte, C. Mansilla, I. Esparza, J.M. Irache, and C. Gamazo. Poly(methyl vinyl ether-co-maleic anhydride) nanoparticles as innate immune system activators. Vaccine. 29:7130-7135 (2011). 17. S. Gomez, C. Gamazo, B. San Roman, M. Ferrer, M.L. Sanz, S. Espuelas, and J.M. Irache. Allergen immunotherapy with nanoparticles containing lipopolysaccharide from Brucella ovis. Eur J Pharm Biopharm. 70:711-717 (2008). 18. S. Gomez, C. Gamazo, B. San Roman, A. Grau, S. Espuelas, M. Ferrer, M.L. Sanz, and J.M. Irache. A novel nanoparticulate adjuvant for immunotherapy with Lolium perenne. J Immunol Methods. 348:1-8 (2009). 19. P. Ojer, A.L. de Cerain, P. Areses, I. Penuelas, and J.M. Irache. Toxicity studies of poly(anhydride) nanoparticles as carriers for oral drug delivery. Pharm Res. 29:2615-2627 (2012). 20. P. Ojer, H.H. Salman, R. Da Costa Martins, J. Calvo, A. López de Cerain, C. Gamazo, J.L. Lavandera, and J.M. Irache. Spray-drying of poly(anhydride) nanoparticles for drug/antigen delivery. J Drug Del Sci Tech. 20:353-359 (2010). 21. M. Agueros, M.A. Campanero, and J.M. lrache. Simultaneous quantification of different cyclodextrins and Gantrez by HPLC with evaporative light scattering detection. J Pharm Biomed Anal. 39:495-502 (2005). 22. V. Zabaleta, M.A. Campanero, and J.M. Irache. An HPLC with evaporative light scattering detection method for the quantification of PEGs and Gantrez in PEGylated nanoparticles. J Pharm Biomed Anal. 44:1072-1078 (2007). 23. M. Agueros, P. Areses, M.A. Campanero, H. Salman, G. Quincoces, I. Penuelas, and J.M. Irache. Bioadhesive properties and biodistribution of cyclodextrin-poly(anhydride) nanoparticles. Eur J Pharm Sci. 37:231-240 (2009).
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24. Organization for Economic Cooperation and Development. Test No. 425: Acute Oral Toxicity: Up-and-Down Procedure. Guidelines for the Testing of Chemicals. Paris: OECD Publishing; 2006. 25. J.D. Reboucas, J.M. Irache, A.I. Camacho, I. Esparza, V. Del Pozo, M.L. Sanz, M. Ferrer, and C. Gamazo. Development of poly(anhydride) nanoparticles loaded with peanut proteins: the influence of preparation method on the immunogenic properties. Eur J Pharm Biopharm (2012). 26. V. Zabaleta, G. Ponchel, H. Salman, M. Agueros, C. Vauthier, and J.M. Irache. Oral administration of paclitaxel with pegylated poly(anhydride) nanoparticles: permeability and pharmacokinetic study. Eur J Pharm Biopharm. 81:514-523 (2012). 27. E.A. Bender, M.D. Adorne, L.M. Colome, D.S. Abdalla, S.S. Guterres, and A.R. Pohlmann. Hemocompatibility of poly(varepsilon-caprolactone) lipid-core nanocapsules stabilized with polysorbate 80-lecithin and uncoated or coated with chitosan. Int J Pharm. 426:271-279 (2012). 28. M.J. Reasor, K.L. Hastings, and R.G. Ulrich. Drug-induced phospholipidosis: issues and future directions. Expert Opin Drug Saf. 5:567-583 (2006). 29. L.A. Chatman, D. Morton, T.O. Johnson, and S.D. Anway. A strategy for risk management of drug-induced phospholipidosis. Toxicol Pathol. 37:997-1005 (2009). 30. T.M. Saba. Physiology and physiopathology of the reticuloendothelial system. Arch Intern Med. 126:1031-1052 (1970). 31. D.E. 3rd Owens, and N.A. Peppas. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. Int J Pharm. 307:93-102 (2006). 32. J. Szebeni, L. Baranyi, S. Savay, J. Milosevits, R. Bunger, P. Laverman, J.M. Metselaar, G. Storm, A. Chanan-Khan, L. Liebes, F.M. Muggia, R. Cohen, Y. Barenholz, and C.R. Alving. Role of complement activation in hypersensitivity reactions to doxil and hynic PEG liposomes: experimental and clinical studies. J Liposome Res. 12:165-172 (2002). 33. S.M. Moghimi and J. Szebeni. Stealth liposomes and long circulating nanoparticles: critical issues in pharmacokinetics, opsonization and protein-binding properties. Prog Lipid Res. 42:463-478 (2003). 34. S. Gomerz, C. Gamazo, B. San Roman, C. Vauthier, M. Ferrer and J.M. Irache. Development of a novel vaccine delivery system based on Gantrez nanoparticles. J. Nansci Nanotechnol. 6:3283-3289 (2006) 35. A.I. Camacho, R. Da Costa Martins, I. Tamayo, J. de Souza, J.J. Lasarte, C. Mansilla, I. Esparza, J.M. Irache, and C. Gamazo. Poly(methyl vinyl ether-co-maleic anhydride) nanoparticles as innate immune system activators. Vaccine. 29:7130-7135 (2011).
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36. R. Yang, S.G. Yang, W.S. Shim, F. Cui, G. Cheng, I.W. Kim, D.D. Kim, S.J. Chung, and C.K. Shim. Lung-specific delivery of paclitaxel by chitosan-modified PLGA nanoparticles via transient formation of microaggregates. J Pharm Sci. 98:970-984 (2009). 37. G. Xie, J. Sun, G. Zhong, L. Shi, and D. Zhang. Biodistribution and toxicity of intravenously administered silica nanoparticles in mice. Arch Toxicol. 84:183-190 (2010). 38. F. Alexis, E. Pridgen, L.K. Molnar, and O.C. Farokhzad. Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm. 5:505-515 (2008). 39. H.M. Patel and S.M. Moghimi. Serum-mediated recognition of liposomes by phagocytic cells of the reticuloendothelial system - the concept of tissue specificity. Adv Drug Deliv Rev. 32:45-60 (1998).39. 40. C. Zhang, G. Qu, Y. Sun, T. Yang, Z. Yao, W. Shen, Z. Shen, Q. Ding, H. Zhou, and Q. Ping. Biological evaluation of N-octyl-O-sulfate chitosan as a new nano-carrier of intravenous drugs. Eur J Pharm Sci. 33:415-423 (2008). 41. N. Giri, P. Tomar, V.S. Karwasara, R.S. Pandey, and V.K. Dixit. Targeted novel surface- modified nanoparticles for interferon delivery for the treatment of hepatitis B. Acta Biochim Biophys Sin (Shanghai). 43:877-883 (2011).
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CHAPTER 8 GENERAL DISCUSSION
CHAPTER 8: GENERAL DISCUSSION
GENERAL DISCUSSION
Nanomedicine is a burgeoning field of research with tremendous prospects for the improvement of the diagnosis and treatment of human diseases. However, the biomedical application of engineered nanoparticles may be hampered by two concerns. First, the methods for production and purification of nanoparticles have to be balanced against the actual benefits offered by the new technology in order to obtain nanomedicines capable of being produced on an industrial scale and thus get into de market. Secondly, and the most important, the biological behavior and toxicological properties of new nanomedicines must be carefully assessed. To date, our understanding of the interactions of nanomaterials with biological systems is limited and thus it is unclear how nanomedicine-based products might produce harmful biological responses. In this regard, nanotoxicology has emerged in the last years to recognize and avoid potential risks associated with nanoparticles. For this to be achieved, detailed physical and chemical characterization of nanoparticles is imperative as it is known that the physico-chemical properties of nanoparticles have a direct influence on its behavior and hence on its toxicity. Although certain relationships between these characteristics and their potential toxicity have been established in general terms, it is considered that the toxicity of each novel nanoparticulate system has to be evaluated on a case by case basis, and should not be applicable to other nanosystems. Therefore, efforts are needed to improve the standardization of assays used for in vitro and in vivo testing of nanoparticles. The copolymers of methyl vinyl ether and maleic anhydride (Gantrez®) are widely employed in a diverse range of topical and oral pharmaceutical formulations due to its adhesive properties and safety profile. Likewise, these copolymers have demonstrated to be biocompatibles and bioerodibles. Therefore, a decade ago, our research group employed for the first time these copolymers to prepare biocompatible and bioerodible nanoparticles with bioadhesive properties as vehicles for oral drug/antigen delivery. We designed different types of poly(anhydride) nanoparticles as carriers for oral delivery: conventional nanoparticles (NP), nanoparticles containing 2-hydroxypropyl-β- cyclodextrin (NP-HPCD) and pegylated nanoparticles (PEG-NP) which have demonstrated high versatility and efficacy by loading different drugs/antigens. Thus, these nanoparticles could be good candidates to make the leap to clinical studies. However, before proceeding to clinical studies, design a scalable manufacturing method and evaluate its safety is undoubtedly necessary. Bearing in mind that modify the manufacturing process may alter the physicochemical characteristics of nanoparticles, and hence its toxicity, the first step carried out in the present research project was to develop a new scalable manufacture method ensuring that the physicochemical properties of the different poly(anhydride) nanoparticles remain unaltered. When poly(anhydride) nanoparticles were firstly developed, they were produced by desolvation of the polymer with a mixture of ethanol and water and subsequent lyophilization. Although this method of preparation and preservation resulted in nanoparticle formulations with 197 CHAPTER 8: GENERAL DISCUSSION
physicochemical characteristics suitable for oral administration, this strategy also had some drawbacks that hindered its scale-up and subsequent use in clinical studies. Thus, the time necessary to product and preserve poly(anhydride) nanoparticles was about 50 h. Furthermore, it should be noted that lyophilization is highly expensive and reserved for products with high added value. On the other hand, the method of preparation involved the use of two organic solvents such as ethanol and acetone. Therefore, to shorten the process we decided to exchange the lyophilization step by a drying process in a spray-dryer apparatus. During the optimization of the spray-drying process of poly(anhydride) nanoparticles we noticed that the yield of the process was low and the resulting powders were extremely difficult to redisperse in water. Moreover, these problems were amplified when ethanol was present in the nanoparticle suspensions. Thus we decided to incorporate an adjuvant to induce a dilution effect which may reduce the possibilities for nanoparticle interaction and fusion due to the process temperature. Furthermore, ethanol was removed, whereby the preparation process involved acetone and water only. Carbohydrates, concretely lactose, maltose, glucose and mannitol, were the selected adjuvant agents that we decided to incorporate in the poly(anhydride) nanoparticle suspension because its well- establish use as excipients in the pharmaceutical industry. After assessing the ability of the different carbohydrates to protect the spray-dried nanoparticles, it was demonstrated that a saccharide/poly(anhydride) ratio of at least 2 was necessary to produce a homogeneous reconstitution of nanoparticles in an aqueous environment. In addition, among the different carbohydrates evaluated, lactose was the one that provided the best conditions to produce a dry powder from the poly(anhydride) nanoparticles suspension. Compared with nanoparticles produced by lyophilization, the main physico-chemical characteristics (size, zeta potential and PDI) of the different nanoparticles evaluated were similar to those prepared by spray-drying when lactose was used as adjuvant, demonstrating that the spray- drying technique is an adequate alternative to preserve poly(anhydride) nanoparticles as dry powders. Thus, throughout this research project, by using spray-drying technique, nanoparticles of well-defined sized about 160-170 nm, narrow size distribution (PDI<0.3) and with negative surface charge were obtained. Likewise, nanoparticles morphology was similar to those prepared by lyophilization. The applicability of the spray-drying technique was evaluated with either drug or antigen- loaded formulation by incorporation of the HS from Brucella ovis or ATO. The results demonstrated that the preparative process did not affect neither the integrity/composition of the proteins included in the HS nor the encapsulation efficiency of ATO. Indeed, it has been found that the preparation of poly(anhydride) nanoparticles by spray-drying not only allows the incorporation of drugs or sensitive molecules, but could also enhance the ability of these nanoparticles as stimulators of immune responses. In this context, a study published by our research group, which addresses the application of these nanoparticles in peanut allergy immunotherapy, demonstrated that spray-dried
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poly(anhydride) nanoparticles loaded with peanut proteins showed different immunomodulatory properties than nanoparticles produced by lyophilization (1). Particularly, these differences were due to the greater stability that spray-dried nanoparticles displayed, which resulted in a slower release rate of the allergen, and therefore an efficient stimulation of immune responses. After improving the manufacture method by using the spray-drying technique, we proceeded to assess their possible harmful effects. When addressing the evaluation of nanoparticles toxicity, the first step that should be conducted is to evaluate their cytotoxicity and cell interaction since these studies are the basis for understanding the biocompatibility of the nanoparticles prior to in vivo studies. Likewise, it is very important to note that if nanoparticles are highly toxic in vitro, the modification of physico-chemical particles characteristics should be considered before proceeding with in vivo toxicity studies as it would not be ethically appropriate to conduct studies involving animals if it is expected that they are going to suffer due to the high toxicity of the nanoparticles. In this research project, to evaluate the cytotoxicity of the different poly(anhydride) nanoparticles, two different cell lines were chosen as models of two important targets for nanoparticles toxicity upon oral exposure: the human hepatocarcinoma-derived HepG2 cells (as liver model) and the human colonic adenocarcinoma derived Caco-2 cells (as gastrointestinal epithelium model). Although the results from the MTS and LDH assays showed a concentration-dependent cytotoxic effect, this cytotoxicity appeared only at very high concentrations and long incubation times in HepG2 cells (1-2 mg/mL at 48-72 h). Thus, the in vitro safety of the different poly(anhydride) nanoparticles was demonstrated. This fact allowed establishing the basis to assess its safety in vivo because, although it is difficult to extrapolate the results from in vitro to in vivo studies, the absence of cytotoxicity makes it ethically acceptable to conduct in vivo studies. The results obtained corroborated the biocompatibility of the different poly(anhydride) nanoparticles and in turn, its bioadhesive character was clearly confirmed after conducting association studies in Caco-2 cells, which is directly related to their previously reported oral efficacy (2-4). Furthermore, this study enabled to establish a direct relation between the physico-chemical characteristics of the designed nanoparticles and its adhesive properties. In this regard, HPCD-NP and PEG-NP, which displayed a rough surface on the SEM images, showed higher cell adhesion than NP with smooth surface. Therefore, for the first time, the specific surface properties of the poly(anhydride) nanoparticles were demonstrated by in vitro cell interaction studies because their behavior in cultured cells was well correlated with the assumption that larger surface area of nanoparticles results in higher cell binding. Given the efficacy of poly(anhydride) nanoparticles as oral delivery systems and its in vitro safety, the in vivo toxicity evaluation by this route was considered the adequate next step. For this purpose, acute and sub-acute toxicity studies were conducted according to the standard procedures
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and guidelines proposed by the OECD which are recommended at the regulatory level to assess chemicals safety. Since so far there are no specific protocols validated for nanoparticles or nanomaterials, we decided to follow OECD guidelines because they make general recommendations regarding the study protocols without taking into account the kind of product to be tested. Therefore, these guidelines could be easily adapted to conduct nanotoxicological studies. The OECD guidelines have been established in order to obtain reliable and reproducible results and give recommendations regarding different aspects of the test procedure such as, experimental animal model (species, sex and number of animals, housing and feeding conditions), dose levels to be tested; observation period; clinical examination procedure; hematology; blood and urine analytical biochemistry; pathology (gross necropsy and histopathology). They also include recommendations on the treatment and evaluation of the results, and the items to be included in a test report. Moreover, OECD guidelines are in continuous revision and updating in order to incorporate scientific progress in the field and also to encompass the principles of the 3 Rs (Replacement, Reduction and Refinement) which is an essential aspect to consider when conducting animal studies. The results obtained in acute and sub-acute toxicity studies demonstrated the safety of the different poly(anhydride) nanoparticles by the oral route. The acute toxicity study was performed according to OECD Guideline 425 “Acute Oral Toxicity: Up-and-Down Procedure” that is one of the three guidelines that were validated and accepted at the regulatory level as alternatives to the OECD guideline 401 “Acute Oral Toxicity”, that was deleted on 2001 (5). The procedure of the limit test was applied and a single dose of 2000 mg/kg bw was administered by gavage. After the administration of this dose that was approximately 200-times higher than the usual therapeutic dose employed previously, no toxic effects were reported in the animals during the 14-day study and no alterations were observed in the necropsies. The sub-acute toxicity studies were carried out according to Guideline OECD 407 “Repeated dose 28-days Oral Toxicity Study in Rodents” (6). The results obtained confirmed that poly(anhydride) nanoparticles do not generate cumulative and latent changes following multiple oral administrations during 28 days, even using doses 30-times higher than those reported in previous studies. Thus, for the different poly(anhydride) nanoparticles, the NOAEL (No Observed Adverse Effect Level) after repeated dose 28-day toxicity study was 300 mg/kg bw. In addition, in the biodistribution studies, where the different nanoparticles were radiolabelled with 99mTc and orally administered to rats, the safety of poly(anhydride) nanoparticles was confirmed as no evidences of translocation or absorption of nanoparticles was found. Moreover, PEG-NP showed longer residence time within the GIT, followed by NP-HPCD and lastly NP. These differences are consistent with those found in the in vitro studies, and therefore may also be explained in line with their different surface characteristics. Despite of the fact that poly(anhydride) nanoparticles were designed for oral delivery, due to the promising results obtained in terms of efficacy and safety by this route (2-4, 7), it was consider of
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great interest to evaluate their behavior and safety by i.v. route before assessing their therapeutic effectiveness. Following the same strategy that was applied for oral safety assessment of the different poly(anhydride) nanoparticles, prior to carry out in vivo toxicity studies, nanoparticles hemocompatibility was tested in vitro. The results demonstrated that all the poly(anhydride) nanoparticles were hemocompatible as none of the formulations caused any harmful effect on erythrocytes at any of the concentrations tested (0.25-1 mg/mL). Once the hemocompatibility was demonstrated in vitro, the in vivo safety of nanoparticles was assessed. For this purpose, acute toxicity studies, according with OECD guidelines, were performed (5). The acute toxicity study was carried out at two different doses of 50 and 150 mg/kg bw, being the lower dose based on preliminary studies developed in our laboratory. Although at a dose of 50 mg/kg bw the animals exposed to nanoparticles intravenously did not show abnormal signs of behavior throughout the study, at the higher dose (150 mg/kg bw) evident signs of toxicity were observed in all groups, characterized by passivity and respiratory distress. In fact, 30% of the animals died because of the administration of NP and NP-HPCD. It is interesting to note that, in surviving animals, the acute episode was resolved in 1 h for PEG-NP group and in 3 h for NP and NP-HPCD groups suggesting that, although the polymer in form of nanoparticles seems to provoke toxicity when it is administered intravenously, adding PEG to the nanoparticle formulation appears to reduce its toxicity somehow. Thus, once again, it is clear the relationship between the physico-chemical characteristics of the nanoparticles and its toxicity, and according to this study, the surface characteristics of the different poly(anhydride) nanoparticles is a key aspect. Thus, the i.v. acute toxicity study allowed concluding that the poly(anhydride) nanoparticles were clearly toxic at the highest dose tested. Furthermore, it is noteworthy that evident histopathological changes were observed in the spleen, lungs and liver of animals for the three types of poly(anhydride) nanoparticles, characterized by the presence of macrophages with foamy material in their cytoplasm. This disorder, called PLDsis is usually dose-dependent and reversible, so there is a prevailing theory that PLDsis is primary an adaptative response to drug exposure rather than a toxic response. To date PLDsis has been mainly reported for cationic amphiphilic drugs although some studies have also reported this disorder for Pluronic F68, cerium and silica oxide nanoparticles, PAMAM dendrimers and PEG (8-12). In the present work, if PLDsis had been found in the animals that were treated with PEG- NP only, the disorder would have been associated with the presence of PEG, which is already reported to cause PLDsis (8). However, the disorder was observed for the three types of nanoparticles, so, in this case PLDsis is due not only to the PEG but also to the polymer. Therefore, this is the first time that PLDsis has been described for polymeric nanoparticles administered by the i.v. route.
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Moreover, as it was mention before, for the three nanoparticle formulations evaluated by the i.v. route, NP and NP-HPCD were found to be the most toxic and PEG-NP the less toxic. This fact could be due to the adsorption of different blood proteins onto the nanoparticles by a phenomenon called opsonization. Actually, it is well known that one of the key points to control when performing i.v. administration is the opsonization phenomenon, and this is directly related to the physico- chemical characteristics of the nanoparticles. Thus, as our three types of formulations have different physicochemical properties, the opsonization pattern may be different. The results obtained in the biodistribution studies support this hypothesis. Thus, it was observed that after the i.v. administration, although all nanoparticles accumulated in the lungs, NP disappeared more rapidly than NP-HPCD and PEG-NP, which remained in the lungs for a longer time. Nanoparticles accumulation in the lungs is consistent with the acute respiratory distress observed in the animals during the single dose toxicity studies; this symptom was particularly intensive for NP and NP-HPCD formulations. Therefore, these results clearly demonstrate once again, that the physico-chemical characteristics lead the biological/toxicological behavior of nanoparticles. Accordingly, it is not possible to extrapolate the outcomes obtained with one type of nanoparticles to other types. This study corroborates that the individual characteristics of each system will determine its toxicity, and as a consequence, any change in these characteristics makes necessary to re-evaluate their toxicity. Taking into account the aforementioned, the present work has established a clear relationship between the physico-chemical characteristics of the poly(anhydride) nanoparticles tested and their toxicity. In Table I there is an attempt to correlate some physicochemical characteristics with a nanoparticle biological behavior which has been in some cases demonstrated in this study and, in other cases it could be expected according to the available data of this and other studies. Moreover, it is important to note that the physico-chemical characteristics of spray-drying nanoparticles were similar to those obtained by lyophilization which means that the outcomes obtained in the present study apply also to the lyophilized nanoparticles, as long as their physico- chemical characteristics remain similar.
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Table I. Correlation of poly(anhydride) nanoparticles physicochemical characteristics with their biological behavior/toxicity.
Characteristic Biological behavior
• Nanoparticles are not uptaken by Caco-2 cells so they do not provoke intracellular toxicity. SIZE • After oral administration, poly(anhydride) nanoparticles 160- 170 nm do not translocate to systemic circulation. (no translocation) • After i.v. administration, poly(anhydride) nanoparticles do not translocate across neuronal pathways.
• Nanoparticles do not disturb cell membranes in HepG2 and Caco-2 cells. CHARGE • After oral administration, nanoparticles do not interact Negative surface charge with any biological component of the gut. (no membrane disruption) • After i.v. administration, there is an opsonization phenomenon STABILITY • Nanoparticles do not accumulate in tissues following Nanoparticles are bioerodible multiple administrations. (no accumulation)
• NP displaying a smooth surface have less bioadhesive SURFACE SHAPE capacity. (different bioadhesive • NP-HPCD and PEG-NP displaying a rough surface result properties) in large surface area and therefore greater bioadhesive potential.
• NP are less hydrophilic and its surface is mainly composed of carboxylic groups highly reactive, thus NP can react easily with molecules containing –OH, -NH or – SH among others. • NP-HPCD surface is composed of carboxylic groups belonging to the poly(anhydride) and to HPCD, as it was SURFACE CHEMISTRY demonstrated that they are located in & out the (different opsonization nanoparticle. Thus NP-HPCD may react with the same pattern) kind of molecules that NP, but more efficiently. • For PEG-NP, several studies have demonstrated that PEG chains form a brush layer surrounding nanoparticle surface. Ethylene groups present in the surface are less reactive, thus this strategy is employed to design more compatible nanoparticles, especially in case of i.v. administration.
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Therefore, according to the Nanotoxicological Classification System (NCS) (13), as the poly(anhydride) nanoparticles tested in this work have displayed a size above 100 nm, they are degradable and are not taken up by cells, NP, NP-HPCD and PEG-NP would be classified as Class I. This is in agreement with the results obtained for these nanoparticles by the oral route, where the formulations have demonstrated to be safe and thus good candidates to be used in medicine. Bearing in mind the design of oral and i.v. study, one may conclude that in order to carry out an appropriate nanotoxicological study, once the physico-chemical characterization of the nanoparticles is known, a preliminary in vitro study should be conducted as a first approach, since it provides a clear and straightforward estimation of possible toxicological risks prior to in vivo studies and even allows us to give answers to the results obtained from in vivo studies. Related to in vivo studies, the guidelines followed in this study have been proven to be suitable to evaluate the safety/toxicity of the poly(anhydride) nanoparticles since it has been demonstrated that these nanoparticle formulations are safe when they are administered orally but not intravenously. Thereby, considering all the above, this research project has permitted to establish the basis for assessing the toxicity of polymeric nanoparticles intended for oral delivery of drugs and/or antigens.
204 CHAPTER 8: GENERAL DISCUSSION
References
1. J. De Souza Reboucas, J.M. Irache, A.I. Camacho, I. Esparza, V. Del Pozo, M.L. Sanz, M. Ferrer, and C. Gamazo. Development of poly(anhydride) nanoparticles loaded with peanut proteins: the influence of preparation method on the immunogenic properties. Eur J Pharm Biopharm. 82:241-249 (2012). 2. P. Arbos, M.A. Campanero, M.A. Arangoa, and J.M. Irache. Nanoparticles with specific bioadhesive properties to circumvent the pre-systemic degradation of fluorinated pyrimidines. J Control Release. 96:55-65 (2004). 3. H.H. Salman, J.M. Irache, and C. Gamazo. Immunoadjuvant capacity of flagellin and mannosamine-coated poly(anhydride) nanoparticles in oral vaccination. Vaccine. 27:4784- 4790 (2009). 4. V. Zabaleta, G. Ponchel, H. Salman, M. Agueros, C. Vauthier, and J.M. Irache. Oral administration of paclitaxel with pegylated poly(anhydride) nanoparticles: permeability and pharmacokinetic study. Eur J Pharm Biopharm. 81:514-523 (2012). 5. Organization for Economic Cooperation and Development. Test No. 425: Acute Oral Toxicity: Up-and-Down Procedure. Guidelines for the Testing of Chemicals. Paris: OECD Publishing; 2006. 6. Organization for Economic Cooperation and Development. Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents. Guidelines for the Testing of Chemicals. Paris: OECD Publishing; 2001. 7. P. Ojer, A.L. de Cerain, P. Areses, I. Peñuelas, and J.M. Irache. Toxicity studies of poly(anhydride) nanoparticles as carriers for oral drug delivery. Pharm Res. 29:2615-2627 (2012). 8. A. Bendele, J. Seely, C. Richey, G. Sennello, and G. Shopp. Short communication: renal tubular vacuolation in animals treated with polyethylene-glycol-conjugated proteins. Toxicol Sci. 42:152-157 (1998). 9. J.Y. Ma, R.R. Mercer, M. Barger, D. Schwegler-Berry, J. Scabilloni, J.K. Ma, and V. Castranova. Induction of pulmonary fibrosis by cerium oxide nanoparticles. Toxicol Appl Pharmacol. 262:255-264 (2012). 10. G. Magnusson, T. Olsson, and J.A. Nyberg. Toxicity of Pluronic F-68. Toxicol Lett. 30:203- 207 (1986). 11. S.T. Stern, P.P. Adiseshaiah, and R.M. Crist. Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Part Fibre Toxicol. 9:20 (2012). 12. M.J. Reasor, K.L. Hastings, and R.G. Ulrich. Drug-induced phospholipidosis: issues and future directions. Expert Opin Drug Saf. 5:567-583 (2006).
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13. R.H. Muller, S. Gohla, and C.M. Keck. State of the art of nanocrystals-special features, production, nanotoxicology aspects and intracellular delivery. Eur J Pharm Biopharm. 78:1-9 (2011)
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CHAPTER 9 CONCLUSIONS/CONCLUSIONES
CHAPTER 9: CONCLUSIONS/CONCLUSIONES
CONCLUSIONS
The experimental work compiled in this research project has been focused on the evaluation of the oral and i.v. toxicity of poly(anhydride) nanoparticles. All the results obtained for this work have led us to conclude the following:
1. Preparation of conventional poly(anhydride) nanoparticles, nanoparticles containing 2- hydroxypropyl-β-cyclodextrin and pegylated nanoparticles by the spray-drying procedure.
1.1. The different poly(anhydride) nanoparticles were easily prepared by the solvent displacement method using only water and acetone.
1.2. The addition of lactose at a saccharide/polymer ratio of 2 provided the best conditions to produce a dry powder that were easily reconstituted in a nanoparticle suspension by simple addition of water and kept their physico-chemical characteristics one year after its preparation.
1.3. By employing the new industrial scale-up method, the time required to prepare the nanoparticles was shorten and the physico-chemical characteristics of the nanoparticles produced were similar to those obtained by lyophilization.
2. Cytotoxicity assessment and in vitro interactions studies in human cell lines.
2.1. Poly (anhydride) nanoparticles showed concentration and incubation time dependent cytotoxic effects in HepG2 and Caco-2 cells. Thus, only at the highest concentrations and incubation times (1-2 mg/mL; 48-72 h) tested, conventional and cyclodextrin nanoparticles proved to be cytotoxic, so poly(anhydride) nanoparticle safety were clearly demonstrated in vitro.
2.2. The bioadhesive character of poly(anhydride) nanoparticles was confirmed in the interaction studies carried out in the Caco-2 cell line, and its adhesive properties were directly related with their surface characteristics. Thus, the highest cytoadhesion was observed for PEG-NP followed by NP-HPCD and the lowest corresponded to NP
3. Evaluation of acute and sub-chronic oral toxicity according to OECD guidelines and biodistribution assessment by Technetium radiolabelling.
3.1. Acute and sub-chronic oral toxicity studies, conducted according to the OECD guidelines 425 and 407, confirmed the oral safety of the different poly(anhydride) nanoparticles.
3.2. At single dose, the LD50 was greater than 2,000 mg/kg bw in rat by the oral route.
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3.3. At repeated doses, during 28 days, the NOAEL was 300 mg/kg bw, a dose 30-times higher than the therapeutic dose.
3.4. Biodistribution studies with 99mTechnetium showed that, following oral administration, the different poly(anhydride) nanoparticles are located mainly in the intestine and excreted by feces.
3.5. Pegylated-poly(anhydride) nanoparticles shower slower transit through the gut than conventional or cyclodextrin nanoparticles. This fact confirmed that pegylation of nanoparticles increase their adhesive properties.
4. Evaluation of hemocompatibility, biodistribution and acute i.v. toxicity of different poly(anhydride) nanoparticles
4.1. In human erythrocytes, all nanoparticle formulations demonstrated to be hemocompatible independent of their concentration and physico-chemical characteristics.
4.2. At single dose by i.v. route, poly(anhydride) nanoparticles showed dose dependent toxicity. Thus, although at a dose of 50 mg/kg bw, the different formulations did not show evident signs of toxicity, at a dose of 150 mg/kg bw, all formulations revealed to be toxic and its toxicity was dependent on its physico-chemical characteristics.
4.3. The toxicity of poly(anhydride) nanoparticles were characterized by an respiratory distress acute episode, being more serious in the case of conventional and cyclodextrin nanoparticles, where 30% of the animals died.
4.4. In the histopathological examination, the most relevant finding was the presence of foamy cells in the spleen, liver and lungs. These findings demonstrated, for the first time, that intravenously administered polymeric nanoparticles could provoke phospholipidosis.
4.5. Intravenously, biodistribution studies showed that, the different poly(anhydride) nanoparticles are rapidly cleared from the circulation and accumulate in the liver, spleen, lungs and kidneys. Thus, at short times, the distribution pattern was dependent on the physico- chemical characteristics of the formulations, which in turn seem to drive the opsonization pattern of the nanoparticles and therefore its toxicity.
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CONCLUSIONES
El trabajo experimental recogido en esta memoria se ha enfocado hacia la evaluación de la toxicidad oral e intravenosa de las nanopartículas de poli(anhídrido). Los resultados obtenidos en esta tesis doctoral nos han permitido concluir lo siguiente:
1. Obtención de nanopartículas de poli(anhídrido) convencionales, con 2-hidroxipropil-β- ciclodextrina y poli-etilenglicol 6000 por la técnica del “spray-drying”:
1.1. Las diferentes nanopartículas de poli(anhídrido) se prepararon fácilmente mediante el método del desplazamiento del disolvente empleando únicamente agua y acetona.
1.2. La adición de lactosa con un ratio sacárido/polímero de 2 y posterior secado por “spray drying” permitió obtener formulaciones en forma de polvo seco que se reconstituyeron fácilmente en agua y mantuvieron sus características físico-químicas un año después de su fabricación.
1.3. Mediante la aplicación del nuevo método escalable a nivel industrial se disminuyó el tiempo requerido para preparar las nanopartículas obteniéndose formulaciones con características físico- químicas similares a las nanopartículas preparadas anteriormente por liofilización.
2. Evaluación de la citotoxicidad e interacción in vitro en células humanas:
2.1. Las nanopartículas de poli(anhídrido) mostraron efectos citotóxicos dependientes de la concentración y el tiempo de incubación en las líneas celulares HepG2 y Caco-2. Así, únicamente a las concentraciones más altas y mayores tiempos de incubación (1-2 mg/mL; 48- 72 horas) las nanopartículas convencionales y con ciclodextrina demostraron ser citotóxicas, por lo que su seguridad in vitro quedó claramente demostrada.
2.2. El carácter bioadhesivo de las nanopartículas de poli(anhídrido) quedó confirmado en los estudios de interacción llevados a cabo en la línea celular Caco-2 y sus propiedades adhesivas dependieron directamente de sus características de superficie. Así, las nanopartículas de poli(anhídrido) pegiladas y con ciclodextrina mostraron una mayor adhesión a la superficie de las células, debido probablemente a su mayor área de superficie en comparación con las nanopartículas convencionales.
3. Evaluación de la toxicidad aguda y sub-crónica por vía oral según las guías de la OCDE y evaluación de la biodistribución mediante radiomarcaje con Tecnecio:
3.1. Los estudios de toxicidad oral aguda y sub-crónica, llevados a cabo según las directrices de las guías de la OCDE 425 y 407, demostraron la seguridad por vía oral de las diferentes formulaciones de nanopartículas de poli(anhídrido). 211 CHAPTER 9: CONCLUSIONS/CONCLUSIONES
3.2. A Dosis única, la DL50 fue superior 2.000 mg/kg peso en rata por vía oral.
3.3. A dosis repetidas durante 28 días, se obtuvo una NOAEL de 300 mg/kg peso, que es una dosis 30 veces superior a la dosis terapéutica.
3.4. Los estudios de biodistribución con Tecnecio, mostraron que tras su administración oral, las distintas formulaciones se localizan principalmente en el intestino y son eliminadas por heces.
3.5. Las nanopartículas con poli-etilenglicol 6000 mostraron un tránsito más lento en comparación con las convencionales y con ciclodextrina. Este hecho confirmó que la pegilación de las nanopartículas de poli(anhídrido) aumenta sus propiedades adhesivas.
4. Evaluación de hemocompatibilidad, toxicidad aguda y biodistribución por vía intravenosa de las distintas nanopartículas de poli(anhídrido):
4.1. En eritrocitos humanos, todas las formulaciones demostraron ser hemocompatibles independientemente de la dosis y de sus características físico-químicas.
4.2. A dosis única por vía intravenosa, las nanopartículas de poli(anhídrido) mostraron toxicidad dependiente de la dosis. Así, si bien a una dosis de 50 mg/kg peso las distintas formulaciones no produjeron síntomas evidentes de toxicidad, a una dosis de 150 mg/kg peso todas las formulaciones resultaron ser tóxicas y su toxicidad dependió de sus características físico- químicas.
4.3. La toxicidad de las nanopartículas de poli(anhídrido) se caracterizó por provocar un episodio agudo de distrés respiratorio, siendo más grave en el caso de nanopartículas convencionales y con ciclodextrina donde provocó la muerte del 30% de los animales.
4.4. En el análisis histopatológico, el hallazgo más significativo fue la presencia de células con aspecto espumoso en el bazo, hígado y pulmón. Estos hallazgos demostraron, por primera vez, que nanopartículas poliméricas por vía intravenosa pueden producir fosfolipidosis.
4.5. Por vía intravenosa, los estudios de biodistribución demostraron que las diferentes nanopartículas de poli(anhídrido) desaparecen rápidamente de la circulación sanguínea y se acumulan principalmente en el hígado, bazo, pulmones y riñones. Así, a tiempos cortos, el patrón de distribución dependió de las diferentes características físico-químicas de las formulaciones que a su vez parecen dirigir el patrón de opsonización de las mismas y por lo tanto su toxicidad.
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