Development of Novel Hollow Zein Nanoparticles for Nanoencapsulation of Curcumin

Home , Solid lipid nanoparticle

Siqi Hu, B.S.

University of Connecticut 2014

A Thesis

Submitted in Partial Fulfillment of the

Requirements for the Degree of

Master of Science

at the

University of Connecticut

2016 Approval Page

Master of Science Thesis

Development of Novel Hollow Zein Nanoparticles for Nanoencapsulation of

Curcumin

Presented by

Siqi Hu, B.S.

Major Advisor ______Maria-Luz Fernandez, Ph.D. Co-Major Advisor ______Yangchao Luo, Ph.D. Associate Advisor ______Christopher Blesso, Ph.D.

University of Connecticut

2016

Acknowledgements

Dr. Fernandez:

Thank you for accepting me into your laboratory and instructing me for three years. Without you, I would not have a chance to get involved in this research project. I have learned quite a few knowledges about basic nutrition and lipid metabolism from you. Getting involved in your guinea pig studies provided me a precious experience about working with animals. I found myself really interested in nutritional science and I will continue doing research in this area. I would also like to thank you and your graduate students for making me feel less lonely in a foreign country. I will never forget those happy days in your research team.

Dr. Luo:

I really appreciate the efforts you have made to help me completing this research project.

Thank you for providing me advises about how to design this study and revising my thesis. I also learned a lot from your graduate students. Moreover, I want to thank you for teaching me basic knowledge of food science and nanotechnology, those new techniques are impressive. I hope I will have a chance to work with your research group again in the future.

Dr. Blesso:

Thank you for providing me instructions about the animal study, although we did not have enough time to finish that part. Your seminar class taught me many useful presentation skills, which helped me preparing for my oral defense.

Lastly, I also want to recognize my family for supporting me in my academic career.

iii

Table of Contents

Approval Page ...... ii

Acknowledgements ...... iii

Table of Contents ...... iv

List of Abbreviations...... viii

List of Tables ...... x

List of Figures ...... xi

Chapter 1 Introduction ...... 1

Chapter 2 Litrature Review ...... 5

Nanoencapsulation and nutrients ...... 5

Polysaccharide-based NESs ...... 6

Preparations of polysaccharide-based NESs ...... 7

Applications of polysaccharide-based NESs for nutrients ...... 9

Lipid-based NESs ...... 11

Preparations of lipid-based NESs ...... 12

Applications of lipid-based NESs for nutrients ...... 13

Protein-based NESs ...... 15

Preparations of protein-based NESs ...... 16

Applications of protein-based NESs for nutrients ...... 18

Zein nanoparticles ...... 19

Introduction ...... 19

Self-assembly of zein nanoparticles ...... 21

Preparation of solid zein nanoparticles ...... 22

Preparation of hollow zein nanoparticles ...... 24

Strategies to improve encapsulation and delivery potentials of zein nanoparticles ...... 25

Coating of zein nanoaprticles ...... 25

Crosslinking of zein nanoaprticles ...... 25

Zein nanoparticles for nutrient delivery applications ...... 26

Curcumin...... 29

Introduction ...... 29

Problems in curcumin stability and bioavailability ...... 31

Chapter 3 Materials and methods ...... 36

Materials ...... 36

Preparation of nanoparticles ...... 36

Box-Behnken design ...... 36

Encapsulation of curcumin ...... 39 v

Nanoparticle characteristics ...... 39

Particle size, PDI and zeta potential ...... 39

Scanning electron microscopy (SEM) ...... 39

Lyophilization and redispersion of curcumin-loaded zein nanoparticles ...... 40

Fourier transform infrared (FT-IR) spectroscopy ...... 40

Fluorescence spectroscopy ...... 40

Encapsulation efficiency ...... 41

Stability ...... 41

Controlled release profile ...... 41

Statistics ...... 42

Chapter 4 Results and discussion ...... 43

Optimization of HZN/T characteristics using Box-Behnken design ...... 43

Particle size (Y1)...... 43

PDI (Y2)...... 48

Zeta potential (Y3) ...... 48

Verification of optimized formulation ...... 49

Effect of tannic acid on HZN ...... 50

Encapsulation and delivery potential of HZN for curcumin ...... 51 vi

Characterization of curcumin-loaded zein nanoparticles ...... 51

Interactions between curcumin and zein ...... 54

Re-dispersibility of freeze-dried powders ...... 58

Stability in simulated gastrointestinal conditions ...... 60

Kinetic release of curcumin ...... 63

Chapter 5 Conclusions ...... 65

References ...... 66

vii

List of Abbreviations

Hollow zein nanoparticles HZN

Solid zein nanoparticles SZN

Polydispersity index PDI

Generally regarded as safe GRAS

Tannic acid TA

Nanoencapsulation systems NESs

Ultraviolet UV

Solid lipid nanoparticle SLN

(−)-Epigallocatechin-3-gallate EGCG

Transmission electron microscopy TEM

Gastrointestinal tract GI tract

Low density lipoprotein LDL

Area under the curve AUC

Lactic-co-glycolic acid PLGA

Mean residence time MRT

Polyethyleneglycol-polylactide PEG-PLA

Dynamic light scattering DLS

Scanning electron microscopy SEM

Fourier transform infrared FT-IR

viii

Encapsulation efficiency EE

Simulated gastric fluid SGF

Simulated intestinal fluid SIF

Hollow zein nanoparticles with tannic acid HZN/T

Hollow zein nanoparticles without tannic acid HZN/NT

Solid zein nanoparticles with tannic acid SZN/T

List of Tables

Table 1. Experiment factors and levels ...... 37

Table 2. Experiment design (Box-Behnken design) ...... 38

Table 3. Statistical analysis results of particle size, PDI, and zeta potential ...... 47

Table 4. Predicted and actual results of surface response method ...... 50

Table 5. Particle size, PDI, and surface charge of different samples based on the optimized

formulation ...... 51

Table 6. Particle size, PDI, surface charge, and encapsulation efficiency of samples loaded with

different amount of curcumin ...... 53

Table 7. Particle size, PDI, surface charge, and encapsulation efficiency of different samples

loaded with equivalent amount of curcumin ...... 59

Table 8. Characteristics of redispersed lyophilized powder loaded with equivalent amount of curcumin ...... 59

Table 9. Particle size, PDI, and surface charge of samples after digestion ...... 61

List of Figures

Figure 1. Correlations between predicted and actual results of surface response method ...... 45

Figure 2. Selected surface response plots showing effects of fabrication conditions on particle size, PDI, and zeta potential ...... 46

Figure 3. SEM images of cast-dried zein nanoparticles ...... 53

Figure 4. Fourier transform infrared spectroscopy spectra of different samples ...... 55

Figure 5. Fluorescence emission spectra of free curcumin and curcumin-loaded samples ..... 57

Figure 6. The DLS curves of curcumin-loaded samples after digestion under different simulated gastric and intestinal conditions ...... 62

Figurre 7. The kinetic release profile of free curcumin and curcumin-loaded zein nanoparticles with different composition under simulated gastric and intestinal conditions...... 64

Abstract

Zein is the major storage protein from corn with strong hydrophobicity and unique solubility and has been studied as an excellent natural biomaterial for encapsulation and delivery of nutraceuticals. Although zein nanoparticles can be easily prepared by anti-solvent liquid-liquid dispersion method, the nanoparticles usually have a dimension greater than 150 nm with a solid core, which greatly limits their loading capacity and cellular uptake for encapsulation

applications. In this study, sodium carbonate was proposed as a sacrifice template and tannic acid

was used as a natural cross-linker to prepare hollow zein nanoparticles (HZN/T). The

formulation and fabrication process, including the amount of water, zein and sodium carbonate,

were optimized by surface response methodology (Box-Behnken design). The optimal HZN/T was then comprehensively characterized and compared with solid zein nanoparticles with tannic

acid (SZN/T). Our results indicated that both the amount of zein and sodium carbonate significantly affected the particle size, polydispersity index (PDI) and zeta potential, while the amount of water only had a significant effect on zeta potential. The optimal HZN/T exhibited a small dimension of 87.93 nm with a PDI of only 0.105 and a zeta potential (surface charge) of

-39.70 mV, indicating the nanoparticles were very homogenously distributed with excellent colloidal stability. Then, a lipophilic bioactive compound, curcumin, was adopted as a model lipophilic nutrient to explore the encapsulation and delivery potentials of HZN/T, in comparison with SZN/T and hollow zein nanoparticles without tannic acid (HZN/NT) prepared under the same conditions. The molecular interaction between curcumin and zein nanoparticles were

xii

investigated by fourier transform infrared spectroscopy and fluorescent spectrophotometer.

Associating with zein nanoparticles via hydrophobic interactions provided a hydrophobic and

neutral microenvironment for curcumin. Coincidently, when loaded at the same percentage

(10%), the encapsulation efficiency (95.82%) of HZN/T was higher than that of SZN/T (93.49%).

The physical characteristics of curcumin-encapsulated HZN/T almost remained the same having a size of 99.55 nm and PDI of 0.101, while the SZN/T showed a significantly larger particle size

(282.47 nm) and PDI (0.141). The stability and kinetic release profile of nanoparticles were tested in simulated gastrointestinal conditions. Tannic cross-linked nanoparticles were more resistant against the digestion under the simulated intestinal condition, while HZN/T showed a

better retained release profile compared to free curcumin. In summary, compared with traditional

SZN/T, the HZN/T developed in this study has promising features as potential oral delivery system for curcumin and other lipophilic nutrients/drugs.

xiii

Chapter 1

Introduction

Curcumin, a member of the polyphenol family, is a hydrophobic compound found in the herb

Curcuma longa. Previous studies supported that curcumin possesses anti-oxidant [1],

anti-inflammatory [2], anti-microbial [3], and anti-carcinogenic properties [4]. Nevertheless, the

compromised bioavailability, including poor absorption and rapid clearance from human body,

obstructed curcumin to be applied in therapeutics and functional foods [5]. In order to increase

its bioavailability, nanotechnology has been employed to develop delivery systems for curcumin.

Thus far, a variety of nanoparticles have been studied as potential vehicles to deliver curcumin

through different routes, either injection [6] or oral administration [7]. Although oral

administration, such as colloidal polymeric nanoparticles and nanoemulsions, has been a

preferred route over injection, many oral delivery systems of curcumin are prepared from either

synthetic polymers and/or surfactants, which are often associated with potential risk of toxicity.

Alternatively, food biopolymers, including polysaccharides and proteins, have received

increasing attention for their encapsulation and delivery potential for nutrients [8-10], due to

their naturally-occurring status with biodegradability and biocompatibility.

Zein nanoparticles are one of the recently studied biodegradable polymeric nanoparticles with

applications in drug delivery [11], biological macromolecule delivery [12], nutrient delivery [13] and tissue engineering [14,15]. Zein is a prolamine protein found as storage protein of maize and is generally regarded as safe (GRAS) as a food additive [16]. Compositional analysis indicated

that zein contains two thirds of non-polar amino acids [17], and one third of polar amino acids

[18,19]. Zein nanoparticles are commonly prepared by a liquid-liquid anti-solvent precipitation

method and such-prepared nanoparticles are often reported to have a dimension of 200 – 300 nm

with a solid internal core. Recently, Xu and colleagues reported a novel method to prepare hollow zein nanoparticles (HZN) by introducing sodium carbonate as a sacrifice template [20].

The HZN had a particle size around 60 nm and exhibited a significantly higher loading capacity than SZN. This is considered a novel methodology to prepare protein-based hollow nanoparticles,

as the core template sodium carbonate is removed simultaneously during the formation of zein

nanoparticles by anti-solvent process, while thermal or chemical treatment is usually required in the preparation of many other hollow nanoparticles [21,22]. Nevertheless, for a new

methodology, the comprehensive optimization, including fabrication procedures and nanoparticle

formulations, is needed to prepare HZN for delivery of nutrients.

As all other protein nanoparticles, a major challenge for zein nanoparticles as an oral delivery vehicle for nutrients is the stability under gastrointestinal conditions, where high concentration of salts, extreme pHs, and digestive enzymes are present. Although zein is relatively resistant

against the enzymatic digestion, it has been shown that zein nanoparticles without any

modification were rapidly hydrolyzed or aggregated [13,23], resulting in a burst effect of

encapsulated drugs/nutrients. Many strategies are available to possibly address this concern. For

instance, surface coating with another polymer [13,23,24] has demonstrated promising effects to

slower release rate of nutrients from zein nanoparticles. Crosslinking is another approach that

2 may be helpful to stabilize zein nanoparticles and improve their delivery potentials. Particularly, citric acid has been recently reported as a non-toxic chemical crosslinker to significantly prolong in vivo residence time of HZN [25]. However, heating at 50 ºC for 10 h is required in this process to create amide bonds between carboxylic groups of citric acid and amine groups of zein, which may negate its applications in encapsulating temperature-sensitive labile nutrients, such as curcumin. Previous studies have shown that binding proteins with tannins increase their stability and thus decreased digestibility [26]. Tannic acid belongs to the tannin family and contains abundant hydroxyl groups, allowing it to form hydrogen bonds with other compounds, including protein. The formation of tannic acid-protein complex was suggested to be based on non-covalent interactions between carbonyl groups of the protein and hydroxyl groups of the tannic acid [27]. Hydrophobic amino acids in zein, such as proline and phenylalanine, are also potential binding sites for tannic acid [28]. Therefore in order to increase the stability of zein nanoparticles in the gastrointestinal (GI) tract, tannic acid could be a possible option for the modification.

The first objective of this study was to explore the main and interactive effects of nanoparticle fabrication conditions and to optimize the formulation of HZN/T using surface response methodology with Box-Behnken design. Tannic acid was included in the formulation as a non-covalent crosslinker to stabilize HZN. Our hypothesis was that all fabrication conditions would have significant influence on all nanoparticle characteristics (i.e. particle size, PDI and zeta potential). The second objective was to evaluate the potential of prepared HZN/T as an oral

delivery vehicle for curcumin, with solid SZN/T as well as HZN without tannic acid studied as controls. The physicochemical properties, including morphology, intermolecular interactions, encapsulation efficiency, as well as stability and controlled release under simulated gastrointestinal conditions were comprehensively characterized. Our hypothesis was that the hollow structure would have promising effects on the encapsulation and delivery of curcumin over solid structure while tannic acid cross-linking would increase the stability of zein nanoparticles.

Chapter 2

Literature review

Nanoencapsulation and nutrients

Nowadays, the encapsulation technique has been considered as a hot spot in scientific research.

Encapsulation technique is the packaging of small compounds within a secondary material, which is called the matrix or shell, to form a small capsule [29]. After being encapsulated, the contents are isolated from the surrounding environment and are able to be released by certain triggers, such as pH change, mechanical stress, temperature, enzymatic activity, etc., thus enabling their controlled and targeted delivery. Thus far, encapsulation technology has been widely applied in different areas, such as pharmaceutics, cosmetics, chemistry, and agriculture.

In recent years, the encapsulation technique has been applied in food industry to improve the effectiveness of food additives, broaden the application range of food ingredients, and prolong the shelf life of foods [30].

Encapsulation can be divided into microencapsulation and nanoencapsulation, according to the size of the encapsulation capsule. Microencapsulation capsules generally have a size between 3 and 800 μm, while nanoencapsulation capsules range from 10 to 1000 nm [31]. The delivery

efficiency of bioactive compound to various sites within the body is negatively related with the

vehicle size [32]. Thus, nanoencapsulation has greater potential to enhance bioavailability and

cellular uptake of nutrients than microencapsulation. Bioactive nutrients such as vitamins,

polyphenols, carotenoids and minerals in foods are well known for their health benefits.

However, factors such as food matrix, harsh environment (e.g. stomach acid, digestive enzymes, etc.) in the gastrointestinal tract and interactions between different bioactive compounds may adversely affect their stability and absorption rate, leading to compromised bioavailability [33].

Therefore, nutrient delivery systems using nanoencapsulation are needed to improve bioavailability [34], achieve sustained release [35] and delay degradation [36] of nutrients.

Many nanoencapsulation systems (NESs) are prepared from either synthetic polymers and/or surfactants, which are often associated with potential risk of toxicity. Alternatively, many food ingredients, including polysaccharides, lipids and proteins, have received increasing attention for their encapsulation and delivery potential for nutrients [8-10]. In the following section, different types (e.g. nanoparticles, nanofibrils, nanomicelles, etc.) of NESs fabricated from food ingredients are discussed in detail, including their preparation methods, characterizations, and applications.

Polysaccharide-based NESs

Polysaccharides are polymeric carbohydrates consisting of glycoside linkage bonded monosaccharide molecules. Due to the following advantages, polysaccharides are widely applied in constructing NESs: (1) they are abundant from natural resources with low cost, including plants, algae, and animal. (2) their structure offers multiple functional groups for chemical modification to obtain novel physicochemical properties; (3) they are safe, biocompatible and biodegradable, as they can be degraded by enzymes and microorganisms in the human body [37];

(4) many polysaccharides exhibit mucoadhesive property to the mucosa of gastrointestinal tract

and thus promote absorption of the encapsulated nutrients [38]. The most commonly studied polysaccharides are: chitosan, pectin, alginate, and modified starch [39]. So far, numerous nutrients have been successfully encapsulated into polysaccharide-based NESs, including both micronutrients (vitamins and polyphenols) and macromolecular nutrients/therapeutics, such as peptides and proteins (insulin). In addition to forming NESs, polysaccharides are also often used as coating material for protein- and lipid-based NESs to bring new functionalities and multifunctions. To take a closer look at polysaccharide-based NESs, the preparation methods of three polysaccharide-based NESs, i.e. nanoparticles, nanofibrils and nanogels are introduced briefly in the following part.

Preparations of polysaccharide-based NESs

Nanoparticles are the most common form of NESs. In the following paragraphs, the two most widely used mechanisms for polysaccharide-based nanoparticles preparation, i.e. covalent crosslinking and ionic crosslinking, are introduced briefly. Covalent crosslinking has been employed in the early preparation of polysaccharide nanoparticles, such as chitosan nanoparticles

[39]. In this process, polysaccharide molecules are covalently bonded together with the covalent bonds formed between polysaccharide molecules and the cross-linking agent and thus nanoparticles are formed and stabilized. At the beginning, chitosan-based nanoparticles were fabricated by covalent crosslinker, glutaraldehyde, which reacts rapidly with amine groups in a chitosan backbone.[39]. However, glutaraldehyde was found to have cytotoxicity and resulted in lung/liver tissue lesions [40], therefore biocompatible crosslinkers, such as malic acid, tartaric

acid, and succinic acid [41,42], were utilized in chitosan nanoparticle fabrication. In the

cross-linking process, covalent bonds are formed between the carboxylic groups of natural acids

and the amino groups of chitosan. On the other hand, ionic crosslinking is another technique to

obtain polysaccharide-based nanoparticles, by which nanoparticles are formed via the electrostatic interactions between charged polysaccharide molecules and the counter-charged polyanions or polycations. For example, Calvo and colleagues fabricated chitosan nanoparticles with tripolyphosphate cross-linking for bovine serum albumin encapsulation in 1997 [43].

Nanofibrils are fibrils with length in the micrometer and width in the nanometric range, which can further form a network structure for compound encapsulation [44]. Cellulose is the most common polysaccharide for preparing nanofibrils. To obtain nanoscale cellulose fibers, raw materials (i.e. potato pulp, soybean stock, wheat straw, etc.) need to undergo intensive mechanical treatments, such as mechanical disintegration[45], high pressure homogenization [46] and refinement [47]. The mechanical force and pressure are able to break raw cellulose into the nano-scale directly. Recently, researchers discovered chemical or enzymatic pre-treatment could help individualizing cellulose fibrils by preventing inter-fibril hydrogen bond formation [48].

After chemical oxidation pre-treatment, cellulose nanofibrils with 3−5 nm in width were obtained [49]. Till now, cellulose nanofibrils have been utilized as a rheology modifier in foods, paints, cosmetics and pharmaceutical products[50]. Recently, nanofibrils were employed as a barrier coating to protect functional food substances, such as anthocyanins, from UV damage

[51].

Besides nanoparticles and nanofibrils, polysaccharide-based nanogels are also considered as

potential NESs for nutrient encapsulation. Nanogels are crosslinked polymeric particles with

high water content [52]. The water content in nanogels provide a suitable environment for

hydrophilic nutrient encapsulation, such as albumin [53]. Conventionally, polysaccharide

hydrogels are prepared by ionic gelation, which means formation of the nanogels is induced by

electrostatic interactions between polysaccharides and counter-charged ions. Pectin, a structural

hetero-polysaccharide extracted from primary cell walls of terrestrial plants, has been employed

in nanogel fabrication. Previous studies used heat treatment (at 60-80 ) and pH adjustment to

produce pectin nanogels [54,55]. Heat treatment and low pH value facilitates℃ the formation of

hydrogen bond induced cross-links and repulsion of methyl groups [56], by which a pectin

network is formed. Heating time and pH value were found to have effects on the morphology of

nanogels [54]. In addition, gelation of pectin can also be induced by calcium ions. In this process,

intermolecular cross-links were formed between calcium ions and the negatively charged

carboxyl groups of the pectin molecules, forming an ‘egg-box’ structure with interstices in which

the calcium ions may be packed and coordinated [53].

Applications of polysaccharide-based NESs for nutrients

As previously discussed, polysaccharides, having many unique physicochemical properties,

are ideal candidates for water soluble bioactive compounds due to their high hydrophilicity.

Furthermore, they are well known for their excellent thermal stability, compared to lipids and proteins, and thus they are extensively studied to encapsulate nutrients and protect them from

thermal degradation. For instance, ascorbic acid, also known as vitamin C, is related to collagen

biosynthesis, elimination of free radicals and immunity. Nevertheless, vitamin C turns into

inactive compounds after heat treatment [57]. Vitamin C was successfully incorporated into

chitosan nanoparticles by Jang et al. The result showed free vitamin C degraded rapidly with heat

treatment, while the Vitamin loaded in the nanoparticles remained over 40% stable [58].

After chemical modification, polysaccharide-based NESs are also capable for encapsulation of lipophilic nutrients, such as vitamin A, to improve their water solubility and stability. Vitamin A is a fat-soluble vitamin, which is indispensable for eye health and cell differentiation. Nowadays, vitamin A products have been applied in wrinkled skin [59] and eye syndrome therapy [60].

However, as a photo-sensitive substance, vitamin A is susceptible to ultraviolet (UV) radiation.

In order to overcome this unfavorable feature, Kim and colleagues encapsulated retinol into

chemically modified chitosan nanoparticles [61]. The chitosan shell might function as a barrier to

the UV light, and therefore the retinol was protected. The result showed the encapsulated retinol

was barely degraded [61]. Most natural polysaccharides contain abundant hydrophilic groups,

including hydroxyl, amino and carboxyl groups, indicating polysaccharide-based NESs can

increase water solubility of lipophilic nutrients. In the above study, the solubility of retinol

increased more than 1600-fold after chitosan encapsulation [61].

In addition to the abovementioned applications, polysaccharides have been shown to possess

strong mucoadhesive property that may also significantly contribute to a higher absorption rate

and bioavailability of encapsulated drugs/nutrients. Particularly, chitosan with positively charged

amino groups can electrostatically interact with negatively charged glycoproteins on the lining of mucosal tissues. For example, Dudhani and colleagues fabricated chitosan nanoparticles for catechin encapsulation [62]. They reported that catechin loaded chitosan nanopaticles and native

nanoparticles exhibited 40% and 32% mucoadhesivity, respectively. Meanwhile, other

polysaccharides, such as pectin and alginate, although negatively charged, they can interact with

mucosal tissues via hydrogen bonds as well as physical binding/adsorption. As previously

reported, pectin nanoparticles loaded with 5-fluorouracil, an anti-cancer drug, demonstrated great

adhesive property within the intestinal tract as the drug recovery rate in the intestine was

significantly raised by nanoencapsulation [63]. However, the mucoadhesive properties of

nutrients-loaded pectin-based nanoparticles have not been explored so far.

Lipid-based NESs

Lipids are naturally occurring hydrophobic or amphiphilic small molecules, including fatty

acids, sterols, monoglycerides, diglycerides, triglycerides, phospholipids, etc. Currently,

nanoemulsions, nanoliposomes, solid lipid nanoparticles and nanostructure lipid carriers are the

most extensively studied lipid-based NESs [64]. Saturated fatty acids (stearic acid [65], palmitic

acid), triglycerides [66], poloxamers [67] and amphiphilic lipids (lecithin [68],

phosphatidylcholine [69]) are ordinarily involved in the preparation of lipid-based NESs. All

four types of NESs mentioned above are available for large scale production via high pressure

homogenization [64], and thus they may be more suitable for industrial applications than

proteins- and polysaccharides-based NESs . Lipid contents in the NESs allow them to

11 encapsulate hydrophobic compounds with high encapsulation efficiency (>99% as previously reported) [70]. Nevertheless, high energy mechanical treatments required in the fabrication of lipid-based NESs, such as high pressure homogenization, microfludization and sonication [71], may increase production costs with potential risk of metal contamination. In the following section, the two most commonly studied lipid-based NESs, i.e. nanoemulsion and solid lipid nanoparticles, are introduced.

Preparations of lipid-based NESs

Nanoemulsion refers to the dispersion of one liquid phase in another immiscible liquid phase with nanoscale droplets [72]. Preparation of a nanoemulsion basically consists of two steps: First, preparation of the coarse pre-emulsion, during which the lipid phase carrying lipophilic nutrients is blended in the aqueous phase with emulsifiers. The lipid phase could be fish oil, medium chain fatty acids, vegetable oil, etc; Second, the mechanical emulsification, during which the coarse emulsion is further emulsified by intensive mechanical treatment, such as sonification, microfludization [73], and pressure homogenization [74]. Input of mechanical forces break the lipid phase into small droplets and then emulsifiers stabilize the newly formed droplets.

Depending on the formulation, droplet sizes of nanoemulsion could range from 50 nm to 500 nm

[71,75]. It should be noted that the mechanical treatment may result in inactivation of thermal/pressure sensitive nutrients [64].

Solid lipid nanoparticle (SLN), a novel lipid NES for drug [76] and nutrient [77] delivery, has attracted the attention of many researchers. SLN consists of emulsifiers and solid cores enfolding

12 hydrophobic contents. Currently, only two techniques are commonly exploited in large-scale

SLN production: hot homogenization technique and cold homogenization technique [78]. In the hot homogenization, melted lipids containing lipophilic contents are homogenized with hot aqueous emulsifier solution. To avoid thermal impact, the emulsifier solution should be pre-heated to the same temperature of the melted lipids. Then the homogenized emulsion is cooled down to allow re-crystallization of the lipid. For the cold homogenization technique, the content-containing lipid melt is cooled down to form lipid microparticles (with average size >50

μm) and these lipid microparticles are dispersed in a cold surfactant solution to yield a coarse suspension. Then this coarse suspension is homogenized at or below room temperature, the mechanical forces are strong enough to break the microparticles directly into nano-scale particles.

Compared to the hot homogenization, the cold homogenization is superior for encapsulating thermal sensitive nutrients. However, one should be aware of temperature increases in the homogenization process [64]. Moreover, cold homogenization can also increase the potential of

SLN for hydrophilic nutrient encapsulation, because this technique avoids, or minimizes, the melting of the lipid and thus minimizing loss of hydrophilic nutrients to the water phase [78].

Applications of lipid-based NESs for nutrients

Lipid-based NESs are mostly studied for encapsulation and delivery of lipophilic nutrients due to their strong association with the hydrophobic core of the NESs. Unsaturated fatty acids are considered as cardiovascular healthy nutrients. Unfortunately, those fatty acids, e.g. linolenic acid, are vulnerable to lipid oxidation during storage, shortening their shelf-life. Imai et al.

discovered oxidation rate of methyl linoleate (linoleic acid methyl ester) was inversely related

with the emulsion droplet size [79]. This phenomenon may be attributed to the presence of

emulsifiers at the oil/water interface preventing fatty acid molecules at the oil phase surface from

interacting with oxidative compounds [80]. All combined, lipid-based nanoencapsulation could

be a suitable technique to alleviate nutrient oxidation. Besides protecting sensitive bioactive

nutrients from oxidation, lipid-based NESs could also enhance antioxidant activity of

antioxidants. An in vitro study showed antioxidant capacity of umbelliferone, a member of the

coumarin family, was enhanced by SLN encapsulation. Umbelliferone loaded in SLN removed

about 75% of the total free radicals while free umbelliferone only removed about 51% [81]. It

was hypothesized that lipids in the SLN may function as intermediates for electron transfer,

increasing the quenching efficiency of umbelliferone [81].

Lipid-based NESs can also be utilized to achieve controlled-release of lipophilic compounds.

Some vitamins, e.g. vitamin A, exhibit toxicity at a high concentration. On the other hand, as

previously reported intake of excess amount of vitamin A impaired the absorption and utilization

of β-carotene, by down-regulating the activity of intestinal β-carotene cleavage enzyme. [82].

Therefore in order to ingest nutrients safely and efficiently, encapsulation and controlled release

of these nutrients may be exploited to minimize undesirable interactions. Jenning et al. reported that compared to the control group with retinol emulsion, retinol loaded in SLN achieved better sustained release pattern during a 24-hour period [83]. The solid lipid matrix might be

responsible for delaying retinol diffusion. In addition, slow and limited release of vitamin K from

SLN was previously reported by Liu et al [84]. In this study, SLN retained most entrapped

nutrient (>85%) for 56 hours in simulated gastrointestinal fluids, indicating the sustained release

of vitamin K in the digestion system. In conclusion, SLN may be an appropriate carrier to

achieve controlled-release of nutrients.

Protein-based NESs

Proteins are macromolecules consisting of one or more long chains of amino acid residues.

Proteins applied in nanoencapsulation are roughly divided into two categories: plant derived

protein (soy protein, gliadin, zein, etc.) and animal derived protein (albumin, casein, collagen,

whey protein, etc.). In the light of following features, protein is regarded as a competitive

candidate for the development of NESs for nutrient. First, unlike polysaccharides which are

highly hydrophilic molecules and not suitable for hydrophobic compound encapsulation,

protein-based NESs are available for both hydrophilic [85] and hydrophobic [86] molecule

encapsulation without additional modification. Secondly, compared to the energy-costing

treatments in the lipid-based NESs fabrication, preparation of protein-based NESs are relatively energy saving. Soy protein nanoparticles with an average size of 94.8 nm were fabricated without any high energy input[87]. Thirdly, functional groups such as carbonyl groups, amide groups, and polar/non-polar amino acid side chains enable surface modification of protein nanoparticles, thus making target delivery possible [88,89]. Instability of protein in the GI tract is

an obstacle to further application of protein-based NESs. To overcome this unfavorable property,

coatings [90] and cross-linkers [91] were involved in previous studies.

Preparations of protein-based NESs

The preparation methods of protein-based NESs could be categorized according to the solubility of proteins. Amphiphilic proteins are soluble in water and in non-polar solvents to some extent, which are commonly used to develop nanomicelles via a self-assemble process.

Briefly, proteins are dispersed in water solely or with the presence of polyions to allow self-assembly of micelles, and then these micelles may be further treated with high pressure homogenizer to achieve smaller size. Intramolecular electrostatic attractions and hydrophobic interactions are generally considered as the driving forces for the micelle formation. A protein derived from milk or casein, could be a good example. Casein molecules contain both hydrophobic and hydrophilic amino acid residues, allowing casein to self-assemble into spherical micelles by hydrophobic interactions. Nevertheless, the size and association degree of such-prepared micelles are limited by intramolecular electrostatic repulsion [92]. Then Holt et al. discovered interaction between serine-phosphate residue of casein and calcium phosphate, which provided a novel mechanism for micelle preparation [93]. Based on this mechanism, Semo et al. successfully produced casein micelles with a diameter round 150 nm for vitamin D2 delivery

[94]. Interestingly, Bachar and colleagues fabricated β-casein micelles without calcium ions [95].

They indicated that nanoscale micelles can be obtained when the concentration of β-casein

exceeds the critical micellization concentration [95,96].

For proteins with low solubility in water or organic solvents, an anti-solvent method is

commonly used to create protein-based NESs. For water insoluble proteins, the concentration of

the protein-containing solvent is lowered by treatment such as evaporation or adding water.

When the attrition of the solvent reaches a critical level, the protein is no longer soluble, resulting in protein precipitation and formation of nanoparticles. For instance, nanoparticles based on zein, a prolamin-rich protein in corn, can be prepared by such method. Till now, liquid-liquid anti-solvent is the most common and simple techniques to prepare zein nanoparticles, in which nanoparticles are formed by pouring or shearing zein solution into water

[18]. As an example, Zou et al. fabricated zein nanoparticles with a particle size around 400 nm by liquid–liquid anti-solvent method for cranberry procyanidins encapsulation [97]. Recently, novel techniques have been employed to produce zein nanoparticles, such as supercritical anti-solvent method and flash nanoprecipitation, but those techniques are still based on the same basic mechanism as the conventional ones, which is inducing zein precipitation by solvent attrition. On the other hand, for proteins that are soluble in water but barely dissolved in organic solvents, the protein precipitation and nanoparticle formation are induced by adding organic solvents to protein aqueous solution. For example, Coester et al. developed a liquid-liquid anti-solvent technique to produce gelatin-based nanoparticles, which are widely used in other studies [98-100]. First, in order to obtain stable nanoparticles, gelatin is dissolved in water, followed by precipitation with acetone and centrifugation to remove low molecular weight molecules in the supernatant. Second, the sediment is re-dissolved with water and pH value adjusted to acidic condition. Third, gelatin particles are precipitated with acetone again and purified by centrifugation three times. Fourth, the particles are re-dispersed in acetone aqueous

solution (30% v/v) followed by acetone evaporation in water bath [100,101].

Applications of protein-based NESs for nutrients

Just like lipid-based NESs, protein-based NESs are also available to protect bioactive nutrients

from oxidative degradation. However, the protective mechanism of lipid-based NESs is only

isolating nutrients from oxidative agents, whereas some protein-based NESs possess an

anti-oxidative nature, which can further protect nutrients from oxidative stress. For instance,

Shpigelman and colleagues observed β-lactoglobulin nanoparticles encapsulated

(−)-Epigallocatechin-3-gallate (EGCG) were significantly protected from oxidative degradation

[102]. Compared with free EGCG, the encapsulated EGCG had a 33-fold lower degradation rate

for the first 48 h at room temperature, and a 3.2-fold lower average degradation rate for over 8 days. Free thiol groups in β-lactoglobulin molecules are anti-oxidant, which might protect EGCG from oxidation. Thus protein-based nanoencapsulation is alternative technique to protect

nutrients from degradation.

As mentioned in the previous section, protein-based NESs can achieve nutrient target delivery.

Therefore, it is possible to develop small intestine targeted protein-based NESs, which may enhance intestinal absorption of nutrient. Lectins are proteins or glycoproteins with high specificity for sugar molecules and therefore may be capable of binding to glycosylated mucus coated intestinal epithelial cells. Ezpeleta et al. modified gliadin nanoparticles with lectins and tested their affinity with mucus. As for the result, after a 90 minute-incubation, about 18% of the total mucin was bound to lectin-gliadin nanoparticles while the control group only achieved

about 7% [103]. What’s more, an in vivo absorption study suggested lectin conjugation

facilitated intestinal absorption of nanoparticles by almost 50 fold [104]. In such a context,

protein-based NESs with lectin conjugation provide a possible solution for poorly absorbed

nutrients. Besides lectin, antibodies have also been involved in protein nanoparitcle

modifications, resulting into accurate targeting delivery vehicles. Wagner et al. developed tumor

targeting anti-integrin antibody-albumin nanoparticles, which achieved specific cellular uptake

by melanoma cells. Moreover, antibody-modified protein nanoparticles loaded with anti-cancer drug exhibited higher cytotoxicity to tumor cells than the free drug, which means that those particles are favorable for developing cancer therapies [105]. Some natural nutrients, including astaxanthin, lycopene, and curcumin were found to have anti-tumor activities [106,107].

Although those neutraceuticals have lower side effect than synthetic drugs, their poor bioavailability should be of concern. Protein-based NESs modified with anti-tumor antibodies may address this problem.

Zein nanoparticles

Introduction

Zein is a storage protein found in protein bodies of maize, with average molecular weight about 40 kDa[108]. Zein is formed by a combination of prolamin- rich proteins in corn, which could be classified into four main types, α, β, γ, and δ zein, according to their solubility[109].

Among those types, α-zein is the most abundant component, accounting for about 75–85% of the zein in corn[18]. Previously reported SDS-PAGE analysis indicated -zein α containing two

sulfide bridge linked fractions with molecular masses of 22 kDa and 24 kDa, while β-zein appeared to have three different fractions of 14, 22, and 24 kDa[110]. Unfortunately, frequent precipitation and coagulation was found in solubilized-zein β and thus it is considered

unstable[111], therefore - αzein is the main constituent in commercial zein products instead of

β-zein. Further, primary sequence analysis of zein revealed that more than 50% of amino acid

residues in zein are hydrophobic, including leucine, proline, alanine, valine, isoleucine and

methionine; the other 40% consists of hydrophilic residues, i.e. glutamine, tyrosine, serine,

glycine, asparagine and threonine[19].

The primary structure indicates zein has an amphiphilic nature, resulting in its unique

solubility, which makes it soluble in aqueous solution of ethanol, methanol, and isopropanol,

instead of pure water or organic solvents[112]. By altering total charges on the zein molecules,

water with high concentration of urea, anionic surfactants or pH at 11 or above is also capable to

dissolve zein[113]. Depending on the solvent type, zein exhibits different solubility, for instance, diethylene glycol is able to dissolve zein at a concentration of 200 mg/ml, forming clear solution, while precipitation occurs in 70% v/v ethanol solution with equivalent zein concentration. To reach the same zein solubility, organic solvents with longer alkane chain require higher water content in the solution[114]. Considering the poor solubility in water and lack of certain indispensable amino acids (i.e. lysine and tryptophan)[18], zein is not a good source of dietary protein. Nevertheless, those physiochemical properties of zein make it suitable for applications in nutrient, drug, and gene encapsulation and delivery.

As a potential biopolymer vehicle for delivery of bioactive compounds and

biomacromolecules, zein has attracted considerable attention in the past decades [15,18,115].

Basically, the research interests of those previous studies were evoked by the following

characteristics of zein: First, zein is generally regarded as safe (GRAS) according to the Food

and Drug Administration, and has been used as food additive since 1937 [116], thus zein-based

NESs are commonly regarded as biocompatible and biodegradable and suitable for food related

applications. Second, zein molecules are able to provide a hydrophobic environment for

lipophilic bioactive compounds encapsulation in aqueous phase. Third, zein molecules are capable to self-assemble into nanoparticles easily via liquid-liquid anti-solvent precipitation

method. Fourth, zein is relatively resistant against the enzymatic digestion in the GI tract, which

is a desirable functionality for oral delivery [18]. Till now, zein has been developed into a variety

of different NESs, such as nanoparticles [117], nanofiber [118], and nanoemulsions [119]. This

review only focused on zein nanoparticles and their applications for encapsulation and delivery

of nutrients.

Self-assembly of zein nanoparticles

As an amphiphilic protein, zein is capable to form an organized structure without the guidance

of additional agents, which is called self-assembly process [120]. By using transmission electron

microscopy (TEM), fast fourier transform image, and circular dichroism, Wang and Padua

observed the process of cast-drying induced self-assembly of zein [120]. This process could be

divided into four steps: First, the hydrophobicity of zein solution decreased with ethanol

21 evaporation, providing a driving force for zein molecules to transform their secondary structure from α-helix to β-sheet. Second, due to strong hydrophobic interactions between β-sheets, zein molecules were packed side by side in an antiparallel form, forming a long ribbon. Third, the ribbon curled into a ring or toroid with a low density hole in the center. Fourth, with the evaporation continued, the - βsheet toroid shrank, resulting in close of the center hole. The proposed mechanism revealed conformation changes and interactions that occurred during zein self-assembly, which could significantly influence binding and encapsulating behavior of zein nanoparticles.

Preparation of solid zein nanoparticles

According to the structure, zein nanoparticles can be categorized into two different types, solid zein nanoparticles and hollow zein nanoparticles. Thus far, the anti-solvent precipitation method is the most frequently used technique for solid zein nanoparticles preparation. This technique only contains two simple steps: first, zein powder is dissolved in ethanol aqueous solution (55-90% v/v). Second, this solution is poured or sheared into water [18]. The dramatic decrease in ethanol concentration changes hydrophobicity of the solution and thus induces formation of zein nanoparticles. Zhong and colleagues investigated effects of several conditions, i.e. zein concentration, shearing speed, and ethanol concentration, on sizes of zein particle prepared by anti-solvent techniques [121]. The result indicated both shearing speed and ethanol concentration were negatively related with the particle size while zein concentration was positively related.

Increase in shearing speed possibly reduced droplet size of zein solution and thus forming

22 smaller particles; similarly, higher ethanol concentration postponed zein precipitation, allowing it to have more time for being broken into smaller droplets by shearing force. In contrast, with zein concentration increasing, viscosity of the stock solution also increased, obstructing the deformation of droplets and thus resulting in larger particles. Moreover, Podaralla et al. found zein nanoparticles formed at pH 6.8, which is around the isoelectric point of zein, had the smallest size [122], indicating that pH value also plays an important role in determining particle size of zein. The anti-solvent method is quite simple and relatively energy-saving. However, it requires a large amount of water, causing difficulties in the drying process during large-scale production.

Recently, several novel techniques were applied in preparation of solid zein nanoparticles, such as electrohydrodynamic atomization [123] and supercritical anti-solvent precipitation

[124,125]. These fabrication methods overcome the demerit of conventional anti-solvent method, while some limitations still need to be considered before applying those techniques to industrial production. For example, although the electrohydrodynamic atomization method is able to produce moisture-free nanoparticles without drying process, it requires high voltage (>14 kV), which may lead to potential hazard and high energy cost. The concentration of zein is also of concern, in the light of that high concentration of zein (10-15%) resulted in formation of irregular particles, which may be attributed to the increased zein solution viscosity [123].

Therefore more organic solvent is needed for dilution, which increases the production cost. The supercritical carbon dioxide anti-solvent technique is considered to have following advantages:

having a mild operating temperature, being environmentally friendly, single and easily controllable process [124]. Nevertheless, in this method, acetone and dimethyl sulfoxide were

used as the solvent and a high pressure was applied during the precipitating process. This may result in increase in the production cost and potential toxicity to operators caused by the presence of organic solvent.

Preparation of hollow zein nanoparticles

As discussed above, solid zein nanoparticles are commonly prepared by anti-solvent

precipitation method and such-prepared nanoparticles are often reported to have a dimension of

200-300 nm with a solid internal core [97,126]. Recently, Xu et al. employed sodium carbonate

as a sacrifice template to produce hollow zein nanoparticles and obtained particle size around 60

nm [20]. Moreover, the hollow nanoaprticles showed higher loading capacity for metformin than

the solid ones, in the light of more storage capacity provided by the cavity in the hollow core

[127]. Compared to the solid type, hollow nanoparticles are less commonly applied in

developing NESs due to the difficulties in fabrication. For instance, sacrifice templates

commonly used in hollow nanoparticle formation, require thermal or chemical treatment for

removal, which could result in protein-based nanoparticle denaturation [127]. Hollow

nanoparticles in this study were fabricated using sodium carbonate as the templates, which were

removed simultaneously during the formation of zein nanoparticles by anti-solvent process the

formation. Nevertheless, being a new methodology, a systematic optimization, which involves

fabrication procedures and nanoparticle formulations, is needed to develop hollow zein

nanoparticles into a delivery system for nutrients.

Strategies to improve encapsulation and delivery potentials of zein nanoparticles

Coating of zein nanoaprticles

Similar to other protein nanoparticles, an important characteristic of hollow zein nanoparticles

is the instability under gastrointestinal conditions, due to the presence of high concentration of

salts, extreme pHs, and digestive enzymes. Although zein is relatively hard to be digested, zein

nanoparticles without any modification were shown to rapidly hydrolyze the encapsulated

compounds leading to a burst effect of encapsulated drugs/nutrients [13,23]. Modifications such

as coating with another polymer were utilized to stabilize nanoparticles in digestive fluids

[23,24]. For instance, Luo and colleagues coated zein nanoparticles with chitosan for vitamin E

encapsulation [13]. Compared with the control, zein nanoparticles, chitosan coated zein nanoparticles at a zein/chitosan weight ratio of 5/1 decreased the cumulative release of vitamin E by almost 50% under a simulated gastrointestinal condition. This could be due to the chitosan coating delaying the hydrolysis of zein nanoparticles and thus obstructing the release of vitamin

Crosslinking of zein nanoaprticles

Besides coating, crosslinking is another technique that may stabilize zein nanoparticles and

improve their delivery potentials. As an example, Xu and colleagues recently reported

crosslinking with a non-toxic chemical crosslinker, citric acid, which significantly prolonged in

vivo residence time of zein nanoparticles [25]. Nevertheless, heating at 50 ºC for 10 h is 25

necessary in this crosslinking process to induce chemical bonding between carboxylic groups of

citric acid and amine groups of zein, which may result in instability of temperature-sensitive

labile nutrients, such as ascorbic acid. Previous studies have shown that binding proteins with

tannins increase their stability and thus decrease digestibility [26]. Tannic acid belongs to the

tannin family and contains abundant hydroxyl groups, allowing it to form hydrogen bond with

other compounds, including protein. The formation of tannic acid-protein complex was suggested to be based on the non-covalent interaction between carbonyl groups of the protein

and hydroxyl groups of the tannic acid [27]. Hydrophobic amino acids in zein, such as proline

and phenylalanine, are potential binding sites for tannic acid [128]. Therefore in order to increase

the stability of zein nanoparticles in the GI tract, tannic acid could be a possible option for the

modification.

Zein nanoparticles for nutrient delivery applications

Being a biocompatible and biodegradable biopolymer, zein nanoparticles have been intensively studied as an encapsulation system for nutrients, in order to enhance the oral bioavailability of lipophilic nutrients [129], improve the stability of sensitive bioactive

compounds [23], achieve sustained release of antioxidants [24], maintain the function of natural pigments [123], etc. According to the previous section, zein nanoparticles possess a hydrophobic

internal core, making them suitable for encapsulating lipophilic nutrients. Currently, studies on

nano-encapsulation of hydrophobic vitamins with zein nanoparticles have attracted interests of

researchers [13,23]. Experimental results showed vitamins were encapsulated in the

nanoparticles by molecularly dispersing them in the polymeric matrix, with a desirable

encapsulation efficiency (higher than 80%). After encapsulated in zein nanoparticles, more than

70% of the total vitamin D was preserved after 9.5 h of UV light exposure, while free vitamin D

only remained about 30%. Further, zein particles delayed the release of vitamin E in phosphate buffer saline with the help of hydrogen bonding and hydrophobic attractions. Both vitamin A and vitamin D were found to form hydrogen bonding with zein nanoparticles, indicating the potential of zein nanoparticles to achieve controlled release of other lipophilic vitamins [130]. Adding chitosan coating improved control-release profile and UV-protective function of zein

nanoparticles.

In the last decades, the demand for health related functional foods has increased considerably

[131]. Thus far, quite a few studies on cytotoxicity and biological efficacy of nutrient-loaded zein nanoparticles have been conducted to support that zein nanoparticles are potential encapsulation vehicles for functional foods delivery. Resveratrol, a polyphenol naturally found in a variety of plant species, vegetables, and fruits, has antioxidant, antiaging, cardioprotective, and anti-tumor effects. Nevertheless, due to the poor water solubility, rapid blood clearance rate and chemical labile property, oral bioavailability of resveratrol is limited [129]. As reported by Penalva and colleagues, resveratrol was successfully encapsulated into zein nanoparticles, which were prepared by a desolvation method followed by ultrafiltration purification. They reported that with the encapsulation, resveratrol achieved high and prolonged plasma levels in a Wistar rat model up to 48 h. The enhanced bioavailability may be due to the nanoparticle matrix, which functioned

27 as a barrier to prevent rapid clearance and burst release of resveratrol. They also found oral administration of the resveratrol loaded nanoparticles protected lipopolysaccharide challenged mice from inflammatory symptoms and mediators of endotoxic shock. Those findings could support that nanoencapsulated resveratrol has health promoting effects and may be applied in dietary interventions to prevent inflammatory induced chronic diseases. In addition, zein nanoparticles have been recently reported to encapsulate cranberry procyanidins, an oligomer formed by monomeric units of flavans [97]. As a potent antioxidant, a previous in vitro study suggested procyanidins were able to protect against LDL oxidation, which may help lowering the risk of cardiovascular diseases [132]. Nevertheless, the intestinal metabolism could digest procyanidins into small aromatic compounds, resulting in compromised oral bioavailability [133].

On the other hand, procyanidins have cytotoxicity by mediating nitric oxide induced cell apoptosis [134]. Zou et al. reported zein nanoparticle encapsulated procyanidins appeared to have a significantly lower cytotoxicity in human promyelocytic leukemia HL-60 cells compared to the control procyanidins solution. The hydrogen bonding and hydrophobic attraction of zein nanoparticles may obstruct the release of procyanidins and thus apoptosis induced cytotoxicity was alleviated. With reduced toxicity, the safety of nanoencapsulated cranberry procyanidins as a dietary supplement is improved. In conclusion, those results advocate that zein nanoparticles are satisfactory nanoencapsulation system for nutrient delivery.

Curcumin

Introduction

Curcumin is a bioactive polyphenol found in rhizomes of turmeric (Curcuma longa), which is

a member of the ginger family. In 1815, Vogel and Pelletier firstly isolated this bright yellow

chemical from Curcuma longa and named it curcumin [135]. Then in 1910, Milobedzka and

Lampe identified the chemical structure of curcumin [136]. Curcumin is identified as

1,7-bis-(4-hydroxy-3-methoxyphenyl)-hepta-1,6-diene-3,5-dione, which can be presented in a

molecular formula of C21H20O6 [137]. At room temperature, curcumin appears as a powder of

bright yellow-orange color, with a molecular weight of 368.39 g/mol. It is a hydrophobic

compound that is insoluble in water but is able to dissolve in organic solvent, including methanol,

ethanol, acetone, and dimethyl sulfoxide. However, in alkaline aqueous solution, the curcumin molecule is deprotonated and its water solubility increases. As previously reported, curcumin has been found to have three different pKa values of 8.54, 9.30 and 10.69, corresponding to the dissociation of protons in the enolic group and the two phenolic groups, respectively [138].

Typically, UV/visible absorption spectra indicate curcumin has maximum absorption at 430 nm and 415–420 nm in methanol and acetone, respectively [139].

It was reported that curcumin molecules could quench excited superoxide radicals by donating protons to those superoxides [140]. Basically, two major sites in the curcumin molecules are responsible for the proton donation: The first one is the central methylene group, which is adjacent to the two highly activated carbonyl groups in the feruloylmethane skeleton. In the

redox reaction, massive delocalization of the unpaired electron on the adjacent oxygen atoms

will induce breakdown of C–H bonds in the methylene group, providing protons to free radicals.

The second one is the hydroxyl groups locate on phenolic rings of curcumin molecules. At the

beginning, one phenolic hydroxyl group is able to donate protons and thus turns the curcumin

molecule into a phenoxyl radical. The ability for this phenoxyl radical to scavenge the oxidizing

free radicals is dramatically higher than the original molecule. Then the phenolic radical can

further donate the second hydrogen atom to free radicals from the other phenolic hydroxyl group, thereby forming a diradical. This diradical may be further degraded into smaller phenols such as ferulic acid, vanillic acid and feruloylmethane or converted into stable compounds like quinones [137].

The natural source of curcumin, turmeric, has been used as herb medicine and food colorant in

Asian countries for centuries. Besides the anti-oxidant property, abundant research findings also support that curcumin may have favorable pharmacological properties such as anti-inflammatory, antimicrobial, and neuroprotective activities [2,141,142]. What’s more, curcumin may possess anti-carcinogenic activities by modulating tumor suppressor genes and inducing cell apoptosis in multiple human cancer cell lines [143-145]. Nevertheless, curcumin is poorly absorbed and rapidly metabolized in human body, leading to compromised bioavailability. Therefore applications of curcumin in therapeutics and functional foods are limited. In order to improve its bioavailability, nanotechnology was applied in curcumin delivery system development. Thus far, quite a few NESs have been investigated as potential vehicles to deliver curcumin, such as

nanoparticles [146] and nanoemulsions [147]. The following paragraphs will provide a closer look to the stability and bioavailability problems of curcumin and the improvements in those aspects brought by nanoencapsulation.

Problems in curcumin stability and bioavailability

Although possessing a variety of promising properties, curcumin is considered as physicochemically unstable, which could have unfavorable effects on its shelf life and quality.

Previous in vitro studies on kinetics of curcumin degradation indicated curcumin is labile under basic conditions [148]. Curcumin molecules in the pH range of 1-7 are in the neutral form with the conjugated diene structure, thus they are relatively stable and have a half-life of more than 15

hours. Nevertheless, the dissociation of protons takes place under pH values above neutral, leading to a rapid hydrolytic degradation, and resulting in a half-life of curcumin of less than 10 minutes. Feuric acid and feruloylmethane were found to be the hydrolysates of curcumin, while the latter could be further degraded into smaller molecules such as vanillin and acetone [149].

Wang and colleagues reported curcumin decomposed rapidly in 0.1 M buffer and solution,

especially under neutral-basic pH conditions [150]. After the incubation in 0.1 M phosphate

buffer and serum-free medium for 30 minutes at 37, about 90% of the total curcumin were

degraded. Trans-6-(4'-hydroxy-3’-methoxyphenyl)-2, 4-dioxo-5-hexenal has been identified as

the major decomposition product of this process. Moreover, curcumin is also unstable under

exposure to UV/visible radiation, producing several photolysis products. Further investigation

identified singlet oxygen as a possible mediator for the photo fading of curcumin [151]. Recently,

Suwannateep and colleagues reported at a similar concentration of 0.1 mg/mL, free curcumin

degraded completely after 4 hour exposure in sunlight, while 80.8 ± 0.8% of the

nanoencapsulated curcumin in ethyl cellulose was preserved. The increase in photo stability was

probably due to accumulation of curcumin inside the polymeric spheres, resulting in less

exposure of curcumin molecules to sunlight and reactive oxygen species. Later on, Zhou et al.

found low density lipoprotein (LDL) nanomicelles extracted from egg yolks protected curcumin

from hydrolysis at alkaline pH [152]. They reported that free curcumin degraded about 15%

within 60 min under pH 12, whereas LDL nanomicelle encapsulation reduced the degradation

rate to only 5.8%. This difference might be caused by the hydrophobic environment inside LDL

micelles, which protected curcumin molecules from hydrolysis. According to these findings, it is

obvious that nanoencapsulation has improved physiochemical stability of curcumin.

Besides physiochemical instabilities, previous in vivo studies have revealed extremely low bioavailability of curcumin in both murine and human subjects, especially through oral routes. In vivo studies on curcumin bioavailability could be tracked back to 1978, Bo Wahlström and G.

Blennow reported only trace amount of curcumin was detected in plasma after oral administration of curcumin at a high dose of 1 g/kg in a Sprague-Dawley rat model, while most curcumin were lost in the feces, indicating that curcumin was barely absorbed in the intestine

[153]. Then in a later study, with curcumin oral administration at a dose of 2 g/kg, serum curcumin level in Albina Wistar rats reached only 1.35 ± 0.23 µg/mL after about 1 hour, whereas in human subjects with the equivalent dose, serum curcumin levels were barely detectable, and

only reached 0.006 ± 0.005 µg/mL after 1 hour [154]. Therefore, to further apply curcumin in therapeutics and functional foods, its bioavailability should be improved. In recent years, NESs

have been employed to improve the gastrointestinal absorption of curcumin, thereby resulting in

a higher blood level and lower kinetic elimination and thus improve the bioavailability of

curcumin. A pharmacokinetic study conducted by Onoue and colleagues could be a good

example [155]. In this study, curcumin (100 mg/kg) and curcumin nanoemulsion (20 mg/kg)

were orally administered to Sprague–Dawley rats. The nano-formulated curcumin showed a

maximum plasma curcumin level of 451 ± 166 ng/mL, whereas free curcumin had a maximum

plasma concentration of 35 ± 8 ng/mL after oral dosing. About a two-fold increase in area under

the curve (AUC) was found in this study for the curcumin nanoemulsion over the free curcumin.

Overall, the nanoencapsulation increased oral bioavailability of curcumin by nine-fold. These

results indicate that nanoencapsulation can significantly increase bioavailability of curcumin

even with a lower dose.

Rapid clearance in the circulation system is responsible to the poor bioavailability of curcumin.

As shown in a pharmacokinetic study, the plasma half-life of unencapsulated curcumin in rats

appeared to be only 74.2 ± 5.9 minutes [156]. On the other hand, after incorporated into a

polymeric nanoparticle, the half-life of curcumin increased to 135 ± 45 minutes, suggesting that

nanoencapsulation delayed the blood clearance of curcumin. A more recent study showed oral

consumption of curcumin loaded poly lactic-co-glycolic acid (PLGA) nanoparticles at 50 mg/kg

gave a mean residence time (MRT) of 259 ± 11 minutes in rat models, whereas free curcumin

only reached 176 ± 18 minutes with a 20-fold higher dose [157]. The nanoencapsulation may protect curcumin from enzymatic metabolism in the liver [158] and thus formulated curcumin

appeared to have longer half-life and retention time in the circulation system.

A previous study has reported that the antioxidant property of curcumin allows it to inhibit amyloid β oligomer and fibril formation in an Alzheimer’s disease model, implying that curcumin may be applied in prevention or treatment of Alzheimer’s disease [159]. Unfortunately, due to the presence of the blood-brain barrier, curcumin can barely enter the brain tissue.

According to Pan et al., after intraperitoneal injection for 1 hour, only a trace amount of curcumin was detected in the brain tissue of a mouse model [160]. Nevertheless, several studies have suggested nanoencapsulation may help curcumin penetrating the blood-brain barrier. For example, Cheng et al. examined effects of nanoencapsulated curcumin on Tg2576 mice, which are models for Alzheimer's disease [161]. It was reported polyethyleneglycol-polylactide

(PEG-PLA) encapsulated curcumin achieved six-fold higher AUC and MRT in the brain of

Tg2576 transgenic mice than the control curcumin solution. Blood-brain barrier only allows low molecular weight drugs and highly lipophilic small molecules to pass through it and enter the brain [162]. Therefore PEG-PLA nanoencapsulation may help curcumin entering the brain by further increasing its hydrophobicity. Surprisingly, they also reported that nanoencapsulated curcumin also showed significant improvements in working and cue memory of the transgenic mice. It is obvious that nanoencapsulation could help increasing the bioavailability and the neuroprotective activity of curcumin in the brain.

Nanoencapsulation of curcumin has been reported to increase its biological efficacy through

intravenous administration. Compared to oral route, this route usually increases the biological efficacy as it avoids the first pass metabolism in the digestive system and hepatic portal system. .A pharmacokinetic study showed orally administered curcumin at a dose of 500 mg/kg gave 0.06 ± 0.01 µg/mL maximum blood level in rats, while higher maximum blood level was achieved (0.36 µg/mL) with only 10 mg/kg of intravenously administrated curcumin.

Nanoecapsulation further improved the bioavailability of intravenously administrated curcumin.

For instance, Sun and colleagues injected curcumin loaded SLN to rats through tail vain at a dose of 2 mg/kg and obtained lower clearance rate and higher AUC compared with the free curcumin

group, showing curcumin in the SLN treated group had significantly better bioavailability. With

SLN encapsulation, the bioavailability of curcumin increased by 25% [6]. However, from the nutrition aspect, intravenous administration is not recommended. When compared with oral administration, intravenous administration is invasive and may potentially cause infections and blood vessel damages.

Chapter 3

Materials and methods

Materials

Zein, sodium carbonate (Na2CO3, purity≥99.0%,), and tannic acid (ACS reagent) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Curcumin (purity≥98%) was obtained from ACROS Organics (Geel, Belgium). Other chemicals were of analytical grade and obtained from Thermo Fisher Scientific (Pittsburgh, PA, USA).

Preparation of nanoparticles

Hollow zein nanoparticles with tannic acid (HZN/T) were prepared by using anti-solvent precipitation method with Na2CO3 used as sacrifice templates [20]. A certain amount of zein powder (100-500 mg) was dissolved in 10 mL 70% v/v aqueous ethanol to form a stock solution.

The sacrifice templates were formed by pouring 0.7 mL of pure ethanol into 0.3 mL deionized water containing Na2CO3 (2.5-7.5 mg). Then, 1 mL of zein stock solution was mixed with the template solution. Pre-established amount of deionized water (5-15 mL) with 2 mg tannic acid was pipetted gradually to the mixture under constant magnetic stirring. Particles were stirred for another 30 min for stabilization. HZN without tannic acid and SZN/T were prepared in parallel

as controls for comparison purpose. In the SZN/T preparation, Na2CO3 was pre-dissolved in 1

mL water (no formation of sacrifice templates), rather than 70% v/v aqueous ethanol solution.

Box-Behnken design

A Box-Behnken design with 15 runs, 3 factors and 3 levels, was utilized to optimize

formulations of HZN/T. Design-Expert software (Trial Version 8.0.6, Stat-Ease Inc., MN) was

used to develop a quadratic response surface, which was designed for testing main effects,

quadratic effects of independent variables, and interactions between them. The software

generated a quadratic model based on the following equation:

= + + + + + + + + + 2 2 2 푌 퐴0 퐴1푥1 퐴2푥2 퐴3푥3 퐴4푥1푥2 퐴5푥1푥3 퐴6푥2푥3 퐴7푥1 퐴8푥2 퐴9푥3 Y represents the response variable needs to be optimized; A1-A9 are regression coefficients of

independent factors, showing their interactions and quadratic terms; X1, X2 and X3 are the coded

levels of explanatory variables. Selection of independent variables, i.e. amount of additional

water (X1), Na2CO3 (X2) and zein (X3), was based on previous study [20], and the detail information was shown Table 1. Independent factors were divided into three levels: -1, 0 and 1, representing low, medium and high values, respectively. Table 2 presented the study design matrix established by the software. Dependent variables of this study were: particle size (Y1), polydispersity index (PDI) (Y2) and zeta-potential (Y3).

Table 1. Experiment factors and levels

Factors Level (-1) Level (0) Level (+1)

Water (mL), X1 5 10 15

Sodium (mg), X2 2.5 5 7.5

Zein (mg), X3 10 30 50

Table 2. Experiment design (Box-Behnken design)

Run X1 X2 X3

1 0 0 0

2 0 1 -1

3 1 0 1

4 0 -1 1

5 1 1 0

6 1 0 -1

7 -1 -1 0

8 -1 0 1

9 0 0 0

10 0 -1 -1

11 0 0 0

12 1 -1 0

13 -1 1 0

14 -1 0 -1

15 0 1 1

Encapsulation of curcumin

Formulations of curcumin loaded zein nanoparticles were based on the optimal formulation

obtained from Box-Behnken design. The preparation followed the same procedures for

abovementioned zein nanoparticles in Section 2.2, with curcumin being dissolved in zein stock

solution. Different zein/curcumin mass ratio (5:1-100:1) was investigated to optimize the

encapsulation efficiency.

Nanoparticle characteristics

Particle size, PDI and zeta potential

Particle size, PDI, and zeta potential of samples were measured using a dynamic light

scattering analyzer (DLS analyzer, Nano Zetasizer ZS, Malvern Instruments, Ltd.,

Worcestershire, UK), which was equipped with alternative 50 mW laser beam at a scattering

angel of 173° and wavelength of 532 nm. To avoid multiple scattering, all samples were diluted

by 20 times prior to particle size and PDI measurements. All measurements were performed in

three replicates at 25 °C.

Scanning electron microscopy (SEM)

The morphology of nanoparticles were observed by scanning electron microscopy (SEM).

Freshly prepared samples were first cast-dried on an aluminum pan using vacuum desiccator

(VDC-11, Jeio Tech, Korea) overnight. Then the samples were mounted onto specimen stub,

coated with a thin layer of gold by a sputter coater and observed using SEM at 11.5 kV

(JSM-6330F, JEOL Ltd., Tokyo, Japan).

Lyophilization and redispersion of curcumin-loaded zein nanoparticles

Freshly produced curcumin-loaded nanoparticles were first frozen at -80 °C, and then freeze-dried by Labconco Freezone (Labconco Corp., Kansas City, MO) under -80 °C and

vacuum overnight. The freeze-dried samples were re-dispersed in deionized water at 1 mg/mL, followed by particle size, PDI and zeta potential measurements.

Fourier transform infrared (FT-IR) spectroscopy

FT-IR spectrum analysis was performed to determine the chemical structures of curcumin-loaded nanoparticles and their individual ingredients (i.e., zein, curcumin and tannic

acid). The freeze-dried samples were then mounted onto ATR crystal for analysis utilizing a

Nicolet iS5 FT-IR spectrometer (Thermo Scientific, Waltham, MA, USA). The spectra were

obtained at 500-4000 cm-1 wavenumbers with a resolution of 4 cm-1.

Fluorescence spectroscopy

The binding between curcumin with zein nanoparticles was determined using fluorescence

spectrophotometry [163]. Fluorescence measurements were carried out in a PerkinElmer LS 55 fluorescence spectrometer (PerkinElmer Instruments, UK). Curcumin-loaded zein nanoparticles

(loaded with 10% curcumin) were selected for fluorescence measurement. The excitation wavelength was 420 nm, and the emission spectra were recorded from 440 to 610 nm. The slit

widths were both 10 nm for excitation and emission. Two free curcumin controls were prepared.

One was dissolved and diluted with pure ethanol to the same curcumin concentration as in nanoparticles, whereas the other was prepared by pre-dissolving curcumin in ethanol followed by

appropriate dilution with Na2CO3 to achieve equivalent pH and ionic strength, as in the

nanoparticles.

Encapsulation efficiency

Briefly, freshly prepared curcumin-loaded zein nanoparticles were centrifuged at 17,217 g for

30 min to remove unstable particles and excess curcumin. Curcumin in the precipitate was extracted by 1 mL pure ethanol with vortexing for 1 min. Curcumin concentrations were

determined by a UV-Vis spectrophotometer (Evolution 201, Thermo Scientific, Waltham, MA,

USA) at 428 nm with a standard curve (R2=0.999). The encapsulation efficiency (EE) was

calculated according to the following equation:

( ) ( ) (%) = × 100 ( ) 푇푇푇푇푇 푐푐푐푐푐푐푐푐 푐푚 − 푃푐푒푐푐푐푐푇푇푇푒푐 푐푐푐푐푐푐푐푐 푐푚 퐸퐸 Stability 푇푇푇푇푇 푐푐푐푐푐푐푐푐 푐푚

The stability of curcumin-loaded nanoparticles was evaluated in simulated gastric and

intestinal conditions, according to our previous study [152]. Brifely, 1 mL of freshly prepared

nanoparticles was added to 9 mL of simulated gastric fluid (SGF, pH 2 or pH 4, with 1 mg/mL

pepsin) or simulated intestinal fluid (SIF, pH 7, with 10 mg/mL pancreatin), followed by incubation at 37 °C for 2 or 4 h, respectively. Particle size, PDI and zeta potential were measured

after each incubation.

Controlled release profile

The pH dependent controlled release study was performed by dialysis membrane method [7].

SGF (pH 4), and SIF (pH 7) premixed with equal volume of ethanol were prepared as release

medium to create sink conditions for curcumin. Briefly, 3.7 mg of freeze-dried curcumin loaded

nanoparticle powders were dispersed in 3 mL SGF release medium was loaded into dialysis bag

with a molecular weight cutoff of 12-14 kDa (Fisherbrand®, Fisher Scientific, Pittsburgh. PA).

The release was carried out at 37 °C consecutively in SGF for 2 h and then SIF for another 4 h

under moderately shaking. The 1 mL of release medium was extracted from the reservoir at

predetermined time point, and fresh release medium was replenished. Curcumin concentration

was determined as previously described using UV-vis spectrophotometer.

Statistics

All measurements were performed in triplicate and data were expressed as mean ± standard

deviation. All data were first tested for homogeneity of variances using Levene’s test, and then

One-way ANOVA with Turkey’s Honest Significant Difference test (if Levene’s test was

negative, P>0.05) or Games Howell test (if Levene’s test was positive, P<0.05) was employed to

determine differences among experimental results. P<0.05 was considered as the significance level. The analyses were carried out using SPSS statistical software package (SPSS, Version 22.0,

Chicago, USA).

Chapter 4

Results and discussions

Optimization of HZN/T characteristics using Box-Behnken design

Among all different types of response surface designs, Box-Behnken design has been found to be more efficient than many other designs and widely applied in analytical system optimization

[164]. In this study, each experimental run was measured in triplicate with amount of tannic acid fixed at 2 mg, and the average value was recorded. After collecting all experimental data, the design-expert software suggested the quadratic model to be the best fit model. The software generated three-dimensional graphs to depict relationships between amount of ingredients and each characteristics of HZN/T (Figure 2). In the 3-D plot, one factor was fixed at a constant value when main effects and interactions of the other two factors were being analyzed.

Comparing to statistical analysis with only numbers, these graphs exhibited changes in the response surface in a more vivid form.

Particle size (Y1)

Particle size of HZN/T ranged from 88.44 to 218.70 nm, significantly dependent upon the amount of sodium carbonate (X2) and the amount of zein (X3), as indicated in Table 3. The following quadratic equation depicts the relationships with a R2=0.9757 (Figure 1), indicating a good fit:

= 127.03 0.46 + 16.23 + 50.69 + 2.2 + 4.56 14.47 + 14.04 2 1 1 2 3 1 2 1 3 2 3 1 푌 −+ 13.푋03 + 17.푋67 푋 푋 푋 푋 푋 − 푋 푋 푋 2 2 푋2 푋3 43

As indicated in the above equation and Figure 2A, both the amounts of sodium carbonate and

zein were positively related to particle size, with the amount of zein exhibiting a much greater

impact, however, water did not have a significant affect. Generally, when the zein concentration

was low, the particle size remained stable with a fixed zein concentration, due to the relatively

small size of carbonate crystals being formed during the precipitation process. Nevertheless, the particle size of HZN/T rapidly increased thereafter as the amount of sodium carbonate further increased, especially when the zein concentration was low. This may be explained by the excessive amount of sodium carbonate in the dispersion causing the aggressive growth of crystal size due to supersaturation. Similar observations were also reported in the case of hollow silica nanoparticles, showing that the shape and size of hollow nanoparticles depended on the sizes of the internal sacrifice templates [165]. In contrast, when zein concentration was high, such effect

of templates was weakened. This was probably owing to the insufficient of templates compared

to zein molecules and thus the formation of larger solid particles becoming possible. On the other

hand, the particle size of HZN/T increased in a much more rapid behavior with the increase of

zein concentration, regardless of the amount of templates. It is notable that increasing zein

concentration in the dispersion induced formation of thicker coating around the templates,

resulting in the rapid growth of particle size of HZN/T.

Table 3. Statistical analysis results of particle size (Y1), PDI (Y2), and zeta potential (Y3)

Particle size (Y1) PDI (Y2) Zeta potential (Y3) Parameters* Coefficient P-value Coefficient P-value Coefficient P-value

Intercept 127.03 N/A 0.081 N/A -32.13 N/A

X1 -0.46 0.9141 0.010 0.1420 -8.41 <0.0001

X2 16.23 0.0099 -0.023 0.0092 2.94 0.0013

X3 50.69 <0.0001 0.025 0.0070 2.70 0.0019

X1*X2 2.20 0.7141 -0.013 0.1594 0.17 0.7948

X1*X3 4.56 0.4580 -0.016 0.1025 -0.40 0.5582

X2*X3 -14.47 0.0513 0.003 0.7236 -0.95 0.1966

X1*X1 14.04 0.0634 0.016 0.1141 2.93 0.0069

X2*X2 13.03 0.0784 -0.008 0.3795 -0.42 0.5541

X3*X3 17.67 0.0303 0.036 0.0079 1.25 0.1175

* X1, amount of water; X2, amount of sodium carbonate; X3, amount of zein.

PDI (Y2)

PDI of HZN/T (Y2) varied from 0.054 to 0.177, and the correlation between PDI and independent factors can be well explained by this equation with a R2=0.9326 (Figure 1):

= 0.081 + 0.009875 0.023 + 0.025 0.013 0.016 + 0.003

2 1 2 3 1 2 1 3 2 3 푌 + 0.016 푋 −0.008042푋 + 0.036푋 − 푋 푋 − 푋 푋 푋 푋 2 2 2 푋1 − 푋2 푋3 As shown in above equation, the coefficient of X1 was extremely low, and thus it is clear that the amount of water was not an important variable for determining the PDI. On the other hand, both X2 and X3 were significant affecting factors because their coefficients were relatively high.

Generally, the PDI decreased with increasing of sodium carbonate, while increased with increasing of zein concentration, as reflected by the negative and positive value of their respective coefficients. A higher PDI indicated a less homogeneous distribution of particle size when zein concentration was high but the template amount was low. Nevertheless, it is worth mentioning that all the PDI values were smaller than 0.3, indicating that all samples were considered as homogeneous nanoparticles with mono-distributed dimensions.

Zeta potential (Y3)

As illustrated in Figure 2C and D, zeta potential of HZN/T ranged from -16.9 to -41.5 mV and the statistical analysis indicated that it was significantly affected by all three factors (Table 3)

The relationships between zeta potential and independent factors is dictated by the following equation with a R2=0.9891 (Figure 1) showing a good fit:

= 32.13 8.14 + 2.94 + 2.70 + 0.17 0.40 0.95 + 2.93 2 2 1 2 3 1 2 1 3 2 3 1 푌 − − 0.42푋 + 1.25푋 푋 푋 푋 − 푋 푋 − 푋 푋 푋 2 2 − 푋2 푋3 Compared with the amount of zein and sodium carbonate, the amount of water exhibited much more dramatic effects on the zeta potential. The effect of sodium carbonate was primarily explained by the ionic screening effect on the electrical double layer of zein molecules [166].

Although serving as a sacrifice template for the formation of hollow structure, the addition of sodium carbonate also introduced a lot of salts as sodium ions were released from solubilized crystals upon pouring the mixture into water during liquid-liquid dispersion process. The increase of ionic strength caused screening effects, thus reducing the surface potential of proteins.

This may also dictate the drastic effect of water amount on zeta potential. By increasing the volume of water during liquid-liquid dispersion process, both concentrations of zein and sodium carbonate were greatly reduced, and therefore the extent of screening effect was consequently attenuated, resulting in a significant increase of magnitude of zeta potential.

Validation of optimized formulation

Optimization of HZN/T properties was conducted by the Design-expert software with goals of achieving minimum particle size, PDI and zeta potential. The optimized formulation provided by the software was 11.45 mL of additional water, 11.2 mg of zein and 2.5 mg of sodium carbonate.

To verify the validity of the optimized formulation, three batches of HZN/T were fabricated based on the optimized formulation, and the particle size, PDI and zeta potential were measured and compared with theoretical values. As shown in Table 4, the actual particle size, PDI and zeta 49 potential was 87.93 ± 1.73 nm, 0.105 ± 0.023 and -39.70 ± 1.51 mV, respectively, all of which were in a good agreement with predicted results.

Table 4. Predicted and actual results of surface response method

Sets Size (nm) PDI Zeta potential (mV)

Predicted 77.48 0.120 -39.94

Actual 87.93±1.73 0.105±0.023 -39.70±1.51

Effect of tannic acid on HZN

To evaluate the function of tannic acid cross-linking on zein nanoparticles, the basic particulate characteristics were compared among HZN/T, HZN without tannic acid (HZN/NT), as well as SZN with tannic acid (SZN/T) as a control (Table 5). The HZN/NT and HZN/T had much smaller particle size compared with the SZN/T. Meanwhile, it is worth mentioning that although not statistically significant, the PDI values of both HZN/NT and HZN/T were lower than that of SZN/T as well. Similar observation was reported in a previous study, which attributed the smaller size of HZN to the effect of pre-formed sodium carbonate crystals acting as heterogeneous nucleation sites, thus preventing the formation of large particles during dispersion process [20]. On the other side, tannic acid did not exhibit apparent effect on particle size and

PDI. The SZN/T had a size of 230.40 nm with a negative zeta potential of -31.03 mV, which were both in a good agreement with the literature [13,24,121]. The zeta potential of HZN/NT and

HZN/T was -36.93 and -39.70 mV, respectively, being significantly greater than that of SZN/T.

This may be in part explained by the smaller particle size of hollow structured particles, thus

providing a larger surface area which increased electric charges per surface area unit [167].

Table 5. Particle size, PDI, and surface charge of different samples based on the optimized

formulation

Sample* Size (nm) PDI Zeta potential (mV)

HZN/T 87.93±1.73a 0.105±0.023a -39.70±1.51a

HZN/NT 92.44±2.24a 0.09±0.010a -36.93±1.55a

SZN/T 230.40±1.85b 0.173±0.054a -31.03±1.33b

* HZN/T, hollow zein nanoparticles with tannic acid; HZN/NT, hollow zein nanoparticles without tannic acid; SZN/T, solid zein nanoparticles with tannic acid. Data in the same column with different superscript letter were significantly different (p < 0.05).

Encapsulation and delivery potential of HZN for curcumin

Characterization of curcumin-loaded zein nanoparticles

To optimize the encapsulation of curcumin in HZN/T, five loading percentages were studied and the results are shown in Table 6. Compared with blank HZN/T particles, the particle size remained unchanged (86.69 – 99.55 nm) until curcumin loading reached 20%, when it significantly increased to 114.20 nm. This indicated that entrapment of curcumin caused a significant expansion of the hollow core when the loading was too high, which might also explain the saturation of curcumin in the core at 20% loading. This hypothesis was further confirmed by the EE results that a significant reduction in EE at 20% loading. The high zeta potential together with small PDI values evidenced that the stable nanoparticles with narrow size

distribution were obtained. Considering all characteristics as a whole, the HZN/T with 10%

curcumin loading was selected as the optimal one for further experiments. Consequently,

curcumin was also encapsulated into the HZN/NT and SZN/T at 10% loading to compare the

effects of tannic acid and hollow/solid structure on the particle characteristics. As shown in Table

6, while the addition of tannic acid had a negligible impact, the SZN/T with a solid core

exhibited a significantly greater particle size and a lower EE, suggesting the advantage of hollow

structure on the encapsulation of nutrients over solid structure.

The morphology of curcumin-loaded nanoparticles was observed under SEM as shown in

Figure 3. Generally, the hollow structured nanoparticles (HZN/NT and HZN/T, Figure 3A and B,

respectively) were smaller than the one with solid core (SZN/T, Figure 3C). However, compared

with the dimensions determined by DLS (Table 6), it is also apparent that nanoparticles with

tannic acid, i.e. HZN/T and SZN/T, exhibited a significantly larger size in the SEM images. This

might be in part owing to the fact that tannic acid, as a crosslinker, induced aggregation and growth of particles, because its local concentration increased dramatically as evaporation of water during the cast-drying process before the SEM observation. Our hypothesis is in line with a previous study reporting that the significant aggregation of zein nanoparticles occurred with a high concentration of tannic acid in the formulation [168]. In addition, the hollow nanoparticles were very homogenous and well separated (Figure 3A and 3B), while the solid nanoparticles tended to clump together without clear partition (Figure 3C). Similar observations have also been reported in previous studies zein nanoparticles prepared with low-energy input techniques

Interactions between curcumin and zein

FT-IR was used to investigate the intermolecular interactions among different ingredients in

the nanoparticles, and the spectra are shown in Figure 4. Apparently, no significant shift was

found in the vibration peaks ranging from 3200 to 3400 cm–1, indicating no dramatic change of hydrogen bonding in zein [23]. However, in the spectra of the HZN/T and SZN/T samples

(Figure 4B, 4D), the vibration peaks at 758/759 cm-1 could be assigned to C=C in benzene rings of tannic acid [169], indicating its presence in the particles. The peaks at 832/833 and 880 cm-1 were found in all nanoparticles, attributing to out-of-plane CCH group of aromatic ring

connected with “enolic” and “keto” part of curcumin molecule [170,171] and implying the

presence of curcumin. Interestingly, peaks at 1113 cm-1 and 1272 cm-1 in the curcumin spectra

(data not shown), representing aromatic rings of curcumin [171], were shifted to 1121-1123 cm-1

and 1281-1284 cm-1, respectively. This indicated the hydrophobic interactions between zein and

curcumin. With the presence of tannic acid, new peaks at 1199 cm-1 and 1317 cm-1 were

observed (Figure 4B and 4D), which were shifted from peaks at 1165 cm-1 and 1304 cm-1 in the

tannic acid spectra (data not shown), probably resulted from hydrophobic interactions between

pentagalloyl glucose of tannic acid and proline residues of zein [172]. Zou et al. [168] also

reported that tannic acid was non-covalently associated with zein nanoparticles. Additionally,

another noticeable shift was the amide I bond at 1645 cm-1 of zein molecules (Figure 4A) when

tannic acid was added in the nanoparticle formulation (Figure 4B and 4D), which may be owing

to the conformational change in protein structure induced by the interactions between zein and

The molecular binding between curcumin and zein in different samples was further

investigated by fluorescence spectroscopy, as presented in Figure 5. The fluorescence of

curcumin that was dispersed in pure ethanol had a peak at 529 nm, whereas curcumin that was pre-dissolved in ethanol and then diluted with water to the equivalent final ethanol concentration and pH value in nanoparticles (about 10% and 9.5, respectively) did not show any fluorescence

intensity. As previously reported, the photochemical properties and fluorescence spectra of curcumin significantly depend on the environmental polarity and pH [173,174]. Therefore this

discrepancy was caused by dramatic decrease in the environmental hydrophobicity and increase

in the pH value. Nevertheless, compared with the curcumin controls, curcumin in all three

nanoparticles exhibited significant fluorescence intensity with the peaks being blue shifted to 522

nm, indicating a more hydrophobic and neutral environment provided by the encapsulation.

These findings are in accordance with the literature [175,176]. Moreover, the fluorescence

intensity of the SZN/T was slightly higher than that of the HZN/NT and HZN/T. This may be

explained by the fact that the solid internal core provides more hydrophobic microenvironment

with additional binding sites for curcumin and thus gave rise to the fluorescence intensity.

Re-dispersibility of freeze-dried powders

Lyophilization has been considered as an effective method to improve long-term storage stability of nanoparticles, however, many nanoparticles made from hydrophobic polymers (such as zein) are no longer able to re-constitute in water after freeze-drying. Zein nanoparticles prepared by traditional liquid-liquid dispersion method cannot be re-constituted in water, unless aqueous/ethanol solutions or aqueous solutions of other organic solvents are used. In this study, all three types of curcumin-encapsulated zein nanoparticles were freeze-dried and then re-dispersed in deionized water at a concentration of 1 mg/mL to evaluate physiochemical properties of re-constituted products. Compared to original characteristics (Table 7), the particle size was reduced by 140 and 10 nm for solid and hollow structured nanoparticles, respectively

(Table 8). The PDI value in re-constituted SZN/T was significantly greater than the both hollow nanoparticles. Particle size and PDI of the reconstituted nanoparticles have been recognized as significant parameters for examining quality loss after the freeze-drying process [177]. Therefore, the reduced particle size with elevated PDI suggested quality loss and less stability of solid nanoparticles during freeze-drying process.

Table 7. Particle size, PDI, surface charge, and encapsulation efficiency of different samples

loaded with equivalent amount of curcumin

Sample* Size (nm) PDI Zeta potential EE (%)

(mV)

HZN/T 99.55±5.28a 0.101±0.027a -35.0±0.90a 95.82±0.56a

HZN/NT 94.66±4.63a 0.097±0.010a -37.10±1.74a 97.23±0.60b

SZN/T 282.47±29.92b 0.141±0.069a -34.30±1.51a 93.49±0.22c

Table 8. Characteristics of redispersed lyophilized powder loaded with equivalent amount of curcumin (1 mg/mL)

Sample* Size (nm) PDI Zeta potential (mV)

HZN/T 89.99±4.75a 0.111±0.014a -40.10±1.71a

HZN/NT 86.06±2.06a 0.094±0.017a -37.90±2.65a

SZN/T 143.0±4.65b 0.248±0.027b -39.80±1.10a

Stability in simulated gastrointestinal conditions

To explore the potential of HZN as oral delivery vehicles, the stability was determined in

simulated gastric and intestinal conditions, respectively. Three conditions were tested, i.e.

fast-status (pH 2) and fed-status (pH 4) gastric conditions with pepsin as digestive enzyme, and intestinal condition (pH 7) with pancreatin as digestive enzyme. As shown in Table 9, incubation

at fast-status gastric condition for 2 h induced severe aggregation in all three samples, ending up with precipitates and microparticles larger than 1 µm with PDI>0.3. It is proposed that the destabilization of zein nanoparticles was mainly due to the protein aggregation/denaturation at extremely acidic pH, rather than the enzymatic digestion by pepsin, because the disintegration of zein nanoparticles by pepsin was not observed. This can be deduced from the size distribution curve (Figure 6B) that only one peak of larger size was observed. Nevertheless, when incubated at fed-status gastric conditions, zein nanoparticles exhibited better stability. Although the particle sizes of all samples were similar to the original particles, the size distribution of hollow nanoparticles exhibited a better profile than solid ones (Figure 6C). The increased ratio of small particles in SZN/T indicated that the partial digestion took place causing the disintegration of larger particles to smaller ones [178]. On the other hand, under intestinal condition, the nanoparticles with tannic acid (HZN/T and SZN/T) showed a better resistance to digestion by

pancreatin, which was able to degrade α-zein dimers while pepsin was not [179]. The HZN particles without tannic acid were disintegrated into multiple smaller fractions (Figure 6D), explaining its smaller mean particle size and larger PDI value (Table 9).

Table 9. Particle size, PDI, and surface charge of samples after digestion under pH 2, 4 and

pH Sample* Size (nm) PDI Zeta potential (mV)

HZN/T 1791.25±372.08a 0.325±0.058a 6.05±0.49a

2 HZN/NT 1275.5±137.94a 0.613±0.033b 0.22±0.81b

SZN/T 1346.25±250.40a 0.386±0.110a 4.86±0.07c

HZN/T 99.78±2.48a 0.092±0.009a -25.50±7.26a

4 HZN/NT 90.54±6.65a 0.146±0.086ab -25.00±7.87a

SZN/T 300.67±2.42b 0.245±0.005b -32.23±7.45a

HZN/T 879.90±60.34a 0.337±0.040a -13.67±0.503a

7 HZN/NT 419.90±24.29b 0.563±0.055b -13.57±0.569a

SZN/T 1099.33±30.89c 0.331±0.019a -13.10±0.436a

Kinetic release of curcumin

Based on the stability data, the zein nanoparticles were not stable at pH 2 and therefore kinetic release of curcumin was only studied at pH 4 and 7 (Figure 7). The diffusion of free curcumin

across the dialysis membrane was found to be linear, with 70% detected at pH 4 followed by another 15% at pH 7. Curcumin in all three nanoparticles shared similar release profile that only about 30% of the total curcumin was released at pH 4 followed by another 10-15% release at pH

7. Burst release was not observed, indicating they all had good controlled release properties.

Interestingly, no dramatic difference was found between the hollow and the solid samples. We

hypothesized that although hollow nanoparticles were more stable than solid ones, their smaller

size and greater surface area as well as thinner zein layer may facilitate the release of curcumin

from the core. Similar results were also reported in a previous study on the release profile of

metformin from HZN and SZN [20].

Chapter 5

Conclusions

In conclusion, HZN/T with diameters less than 100 nm were developed by exploring sodium carbonate as a sacrifice template and the formulation was comprehensively optimized by surface response methodology. Particle size, PDI and zeta potential of HZN/T were significantly

influenced by the amount of sodium carbonate and zein, while the amount of water only had a

significant effect on zeta potential. Compared with SZN/T, HZN/T had smaller particle size,

higher curcumin encapsulation efficiency and better stability upon reconstitution. Encapsulation

in zein nanoparticles provided curcumin a strong hydrophobic microenvironment in aqueous

solution and hydrophobic interaction were considered to be the major driving force for the

encapsulation. In terms of their stabilities under simulated gastrointestinal conditions, tannic acid

cross-link zein nanoparticles were more resistant against the digestion by pancreatin than

non-crosslinked ones. All zein nanoparticles exhibited good controlled release properties but no dramatic difference was found between solid and hollow nanoparticles. In summary, tannic acid cross-linked HZN would be a suitable vehicle for encapsulation and oral delivery of curcumin

and other lipophilic nutrients/drugs.

References 1. Weber WM, Hunsaker LA, Abcouwer SF, Deck LM, Vander Jagt DL. Anti-oxidant activities of curcumin and related enones. Bioorganic & medicinal chemistry, 13(11), 3811-3820 (2005). 2. Satoskar RR, Shah SJ, Shenoy SG. Evaluation of anti-inflammatory property of curcumin (diferuloyl methane) in patients with postoperative inflammation. International journal of clinical pharmacology, therapy, and toxicology, 24(12), 651-654 (1986). 3. De R, Kundu P, Swarnakar S et al. Antimicrobial activity of curcumin against Helicobacter pylori isolates from India and during infections in mice. Antimicrobial agents and chemotherapy, 53(4), 1592-1597 (2009). 4. Limtrakul P, Lipigorngoson S, Namwong O, Apisariyakul A, Dunn FW. Inhibitory effect of dietary curcumin on skin carcinogenesis in mice. Cancer letters, 116(2), 197-203 (1997). 5. Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB. Bioavailability of curcumin: problems and promises. Molecular pharmaceutics, 4(6), 807-818 (2007). 6. Sun J, Bi C, Chan HM, Sun S, Zhang Q, Zheng Y. Curcumin-loaded solid lipid nanoparticles have prolonged in vitro antitumour activity, cellular uptake and improved in vivo bioavailability. Colloids and Surfaces B: Biointerfaces, 111, 367-375 (2013). 7. Shaikh J, Ankola DD, Beniwal V, Singh D, Kumar MNVR. Nanoparticle encapsulation improves oral bioavailability of curcumin by at least 9-fold when compared to curcumin administered with piperine as absorption enhancer. European Journal of Pharmaceutical Sciences, 37(3–4), 223-230 (2009). 8. Abd El-Salam MH, El-Shibiny S. Formation and potential uses of milk proteins as nano delivery vehicles for nutraceuticals: A review. International Journal of Dairy Technology, 65(1), 13-21 (2012). 9. Hu B, Huang Q-r. Biopolymer based nano-delivery systems for enhancing bioavailability of nutraceuticals. Chinese Journal of Polymer Science, 31(9), 1190-1203 (2013). 10. McClements DJ. Nanoscale Nutrient Delivery Systems for Food Applications: Improving Bioactive Dispersibility, Stability, and Bioavailability. Journal of Food Science, 80(7), N1602-N1611 (2015). 11. Lai LF, Guo HX. Preparation of new 5-fluorouracil-loaded zein nanoparticles for liver targeting. Int J Pharm, 404(1-2), 317-323 (2011). 12. Regier MC, Taylor JD, Borcyk T, Yang Y, Pannier AK. Fabrication and characterization of DNA-loaded zein nanospheres. Journal of nanobiotechnology, 10, 44 (2012). 13. Luo Y, Zhang B, Whent M, Yu LL, Wang Q. Preparation and characterization of zein/chitosan complex for encapsulation of alpha-tocopherol, and its in vitro controlled release study. Colloids and surfaces. B, Biointerfaces, 85(2), 145-152 (2011). 14. Dong J, Sun Q, Wang J-Y. Basic study of corn protein, zein, as a biomaterial in tissue engineering, surface morphology and biocompatibility. Biomaterials, 25(19), 4691-4697 (2004). 66

15. Paliwal R, Palakurthi S. Zein in controlled drug delivery and tissue engineering. Journal of controlled release : official journal of the Controlled Release Society, 189, 108-122 (2014). 16. Janes ME, Kooshesh S, Johnson MG. Control of Listeria monocytogenes on the Surface of Refrigerated, Ready-to-eat Chicken Coated with Edible Zein Film Coatings Containing Nisin and/or Calcium Propionate. Journal of Food Science, 67(7), 2754-2757 (2002). 17. Righetti PG, Gianazza E, Viotti A, Soave C. Heterogeneity of storage proteins in maize. Planta, 136(2), 115-123). 18. Luo Y, Wang Q. Zein-based micro- and nano-particles for drug and nutrient delivery: A review. Journal of Applied Polymer Science, 131(16), n/a-n/a (2014). 19. Geraghty D, Peifer MA, Rubenstein I, Messing J. The primary structure of a plant storage protein: zein. Nucleic acids research, 9(19), 5163-5174 (1981). 20. Xu H, Jiang Q, Reddy N, Yang Y. Hollow nanoparticles from zein for potential medical applications. Journal of Materials Chemistry, 21(45), 18227-18235 (2011). 21. Peng S, Sun S. Synthesis and Characterization of Monodisperse Hollow Fe3O4 Nanoparticles. Angewandte Chemie, 119(22), 4233-4236 (2007). 22. Zeng H, Cai W, Liu P et al. ZnO-Based Hollow Nanoparticles by Selective Etching: Elimination and Reconstruction of Metal−Semiconductor Interface, Improvement of Blue Emission and Photocatalysis. ACS Nano, 2(8), 1661-1670 (2008). 23. Luo Y, Teng Z, Wang Q. Development of Zein nanoparticles Coated with Carboxymethyl Chitosan for Encapsulation and Controlled Release of Vitamin D3. Journal of agricultural and food chemistry, 60(3), 836-843 (2012). 24. Luo Y, Wang TTY, Teng Z, Chen P, Sun J, Wang Q. Encapsulation of indole-3-carbinol and 3,3′-diindolylmethane in zein/carboxymethyl chitosan nanoparticles with controlled release property and improved stability. Food Chemistry, 139(1–4), 224-230 (2013). 25. Xu H, Shen L, Xu L, Yang Y. Controlled delivery of hollow corn protein nanoparticles via non-toxic crosslinking: in vivo and drug loading study. Biomedical microdevices, 17(1), 8 (2015). 26. Taylor J, Bean SR, Ioerger BP, Taylor JR. Preferential binding of sorghum tannins with γ-kafirin and the influence of tannin binding on kafirin digestibility and biodegradation. Journal of Cereal Science, 46(1), 22-31 (2007). 27. Van Buren JP, Robinson WB. Formation of complexes between protein and tannic acid. Journal of agricultural and food chemistry, 17(4), 772-777 (1969). 28. Jobstl E, O'Connell J, Fairclough JP, Williamson MP. Molecular model for astringency produced by polyphenol/protein interactions. Biomacromolecules, 5(3), 942-949 (2004). 29. Augustin MA, Hemar Y. Nano-and micro-structured assemblies for encapsulation of food ingredients. Chemical society reviews, 38(4), 902-912 (2009). 30. Gouin S. Microencapsulation: industrial appraisal of existing technologies and trends. Trends in Food Science & Technology, 15(7–8), 330-347 (2004). 31. Ezhilarasi PN, Karthik P, Chhanwal N, Anandharamakrishnan C. Nanoencapsulation 67

techniques for food bioactive components: a review. Food and Bioprocess Technology, 6(3), 628-647 (2013). 32. Hughes GA. Nanostructure-mediated drug delivery. Nanomedicine: nanotechnology, biology and medicine, 1(1), 22-30 (2005). 33. Rein MJ, Renouf M, Cruz-Hernandez C, Actis-Goretta L, Thakkar SK, da Silva Pinto M. Bioavailability of bioactive food compounds: a challenging journey to bioefficacy. Br J Clin Pharmacol, 75(3), 588-602 (2013). 34. Qi K, Ma Q, Remsen EE, Clark CG, Wooley KL. Determination of the Bioavailability of Biotin Conjugated onto Shell Cross-Linked (SCK) Nanoparticles. Journal of the American Chemical Society, 126(21), 6599-6607 (2004). 35. Mukherjee B, Santra K, Pattnaik G, Ghosh S. Preparation, characterization and in-vitro evaluation of sustained release protein-loaded nanoparticles based on biodegradable polymers. International journal of nanomedicine, 3(4), 487-496 (2008). 36. Liu F, Wang D, Sun C, Gao Y. Influence of polysaccharides on the physicochemical properties of lactoferrin–polyphenol conjugates coated β-carotene emulsions. Food Hydrocolloids, 52, 661-669 (2016). 37. Jain A, Gupta Y, Jain SK. Perspectives of biodegradable natural polysaccharides for site-specific drug delivery to the colon. Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques, 10(1), 86-128 (2007). 38. Pan Y, Li YJ, Zhao HY et al. Bioadhesive polysaccharide in protein delivery system: chitosan nanoparticles improve the intestinal absorption of insulin in vivo. Int J Pharm, 249(1-2), 139-147 (2002). 39. Liu Z, Jiao Y, Wang Y, Zhou C, Zhang Z. Polysaccharides-based nanoparticles as drug delivery systems. Advanced drug delivery reviews, 60(15), 1650-1662 (2008). 40. Fürst W, Banerjee A. Release of Glutaraldehyde From an Albumin-Glutaraldehyde Tissue Adhesive Causes Significant In Vitro and In Vivo Toxicity. The Annals of Thoracic Surgery, 79(5), 1522-1528 (2005). 41. Bodnár M, Hartmann JF, Borbély J. Nanoparticles from Chitosan. Macromolecular Symposia, 227(1), 321-326 (2005). 42. Bodnar M, Hartmann JF, Borbely J. Preparation and characterization of chitosan-based nanoparticles. Biomacromolecules, 6(5), 2521-2527 (2005). 43. Calvo P, Remunan‐Lopez C, Vila‐Jato JL, Alonso MJ. Novel hydrophilic chitosan‐ polyethylene oxide nanoparticles as protein carriers. Journal of Applied Polymer Science, 63(1), 125-132 (1997). 44. Zhang Y, Nypelö T, Salas C, Arboleda J, Hoeger IC, Rojas OJ. Cellulose Nanofibrils. Journal of Renewable Materials, 1(3), 195-211 (2013). 45. Malainine ME, Mahrouz M, Dufresne A. Thermoplastic nanocomposites based on cellulose microfibrils from Opuntia ficus-indica parenchyma cell. Composites Science and Technology, 65(10), 1520-1526 (2005). 46. Leitner J, Hinterstoisser B, Wastyn M, Keckes J, Gindl W. Sugar beet cellulose 68

nanofibril-reinforced composites. Cellulose, 14(5), 419-425 (2007). 47. Nakagaito AN, Yano H. The effect of morphological changes from pulp fiber towards nano-scale fibrillated cellulose on the mechanical properties of high-strength plant fiber based composites. Applied Physics A, 78(4), 547-552 (2003). 48. Saito T, Kimura S, Nishiyama Y, Isogai A. Cellulose Nanofibers Prepared by TEMPO-Mediated Oxidation of Native Cellulose. Biomacromolecules, 8(8), 2485-2491 (2007). 49. Saito T, Nishiyama Y, Putaux J-L, Vignon M, Isogai A. Homogeneous Suspensions of Individualized Microfibrils from TEMPO-Catalyzed Oxidation of Native Cellulose. Biomacromolecules, 7(6), 1687-1691 (2006). 50. Siró I, Plackett D. Microfibrillated cellulose and new nanocomposite materials: a review. Cellulose, 17(3), 459-494 (2010). 51. Zhao Y, Simonsen J, Cavender G, Jung J, Fuchigami LH. Nano-cellulose coatings to prevent damage in foodstuffs. (Ed.^(Eds) (Google Patents, 2014) 52. Oh JK, Drumright R, Siegwart DJ, Matyjaszewski K. The development of microgels/nanogels for drug delivery applications. Progress in Polymer Science, 33(4), 448-477 (2008). 53. Kim TH, Park YH, Kim KJ, Cho CS. Release of albumin from chitosan-coated pectin beads in vitro. International Journal of Pharmaceutics, 250(2), 371-383 (2003). 54. Lin L, Xu W, Liang H et al. Construction of pH-sensitive lysozyme/pectin nanogel for tumor methotrexate delivery. Colloids and Surfaces B: Biointerfaces, 126, 459-466 (2015). 55. Chang C, Wang ZC, Quan CY et al. Fabrication of a novel pH-sensitive glutaraldehyde cross-linked pectin nanogel for drug delivery. Journal of biomaterials science. Polymer edition, 18(12), 1591-1599 (2007). 56. Sharma B, Naresh L, Dhuldhoya N, Merchant S, Merchant U. An overview on pectins. Times Food Processing Journal, 23(2), 44-51 (2006). 57. Yuan J-P, Chen F. Degradation of Ascorbic Acid in Aqueous Solution. Journal of agricultural and food chemistry, 46(12), 5078-5082 (1998). 58. Jang K-I, Lee HG. Stability of Chitosan Nanoparticles for l-Ascorbic Acid during Heat Treatment in Aqueous Solution. Journal of agricultural and food chemistry, 56(6), 1936-1941 (2008). 59. Varani J, Warner RL, Gharaee-Kermani M et al. Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin. The Journal of investigative dermatology, 114(3), 480-486 (2000). 60. Kim EC, Choi J-S, Joo C-K. A Comparison of Vitamin A and Cyclosporine A 0.05% Eye Drops for Treatment of Dry Eye Syndrome. American Journal of Ophthalmology, 147(2), 206-213.e203 (2009). 61. Kim D-G, Jeong Y-I, Choi C et al. Retinol-encapsulated low molecular water-soluble chitosan nanoparticles. International Journal of Pharmaceutics, 319(1–2), 130-138 69

(2006). 62. Dudhani AR, Kosaraju SL. Bioadhesive chitosan nanoparticles: Preparation and characterization. Carbohydrate Polymers, 81(2), 243-251 (2010). 63. Subudhi BM, Jain A, Jain A et al. Eudragit S100 Coated Citrus Pectin Nanoparticles for Colon Targeting of 5-Fluorouracil. Materials, 8(3) (2015). 64. Fathi M, Mozafari MR, Mohebbi M. Nanoencapsulation of food ingredients using lipid based delivery systems. Trends in Food Science & Technology, 23(1), 13-27 (2012). 65. Bocca C, Caputo O, Cavalli R, Gabriel L, Miglietta A, Gasco MR. Phagocytic uptake of fluorescent stealth and non-stealth solid lipid nanoparticles. International Journal of Pharmaceutics, 175(2), 185-193 (1998). 66. Bandyopadhyay S, Katare OP, Singh B. Optimized self nano-emulsifying systems of ezetimibe with enhanced bioavailability potential using long chain and medium chain triglycerides. Colloids and Surfaces B: Biointerfaces, 100, 50-61 (2012). 67. Muller RH, Maassen S, Weyhers H, Mehnert W. Phagocytic uptake and cytotoxicity of solid lipid nanoparticles (SLN) sterically stabilized with poloxamine 908 and poloxamer 407. Journal of drug targeting, 4(3), 161-170 (1996). 68. Zhou H, Yue Y, Liu G et al. Preparation and Characterization of a Lecithin Nanoemulsion as a Topical Delivery System. Nanoscale Research Letters, 5(1), 224-230 (2009). 69. Yilmaz E, Borchert H-H. Design of a phytosphingosine-containing, positively-charged nanoemulsion as a colloidal carrier system for dermal application of ceramides. European Journal of Pharmaceutics and Biopharmaceutics, 60(1), 91-98 (2005). 70. Yang S, Zhu J, Lu Y, Liang B, Yang C. Body distribution of camptothecin solid lipid nanoparticles after oral administration. Pharmaceutical research, 16(5), 751-757 (1999). 71. Anton N, Benoit J-P, Saulnier P. Design and production of nanoparticles formulated from nano-emulsion templates—A review. Journal of Controlled Release, 128(3), 185-199 (2008). 72. Mason TG, Wilking JN, Meleson K, Chang CB, Graves SM. Nanoemulsions: formation, structure, and physical properties. Journal of Physics: Condensed Matter, 18(41), R635 (2006). 73. Jafari SM, Assadpoor E, Bhandari B, He Y. Nano-particle encapsulation of fish oil by spray drying. Food Research International, 41(2), 172-183 (2008). 74. Yuan Y, Gao Y, Zhao J, Mao L. Characterization and stability evaluation of β-carotene nanoemulsions prepared by high pressure homogenization under various emulsifying conditions. Food Research International, 41(1), 61-68 (2008). 75. Talegaonkar S, Mustafa G, Akhter S, Iqbal ZI. Design and Development of Oral Oil-in-Water Nanoemulsion Formulation Bearing Atorvastatin: In Vitro Assessment. Journal of Dispersion Science and Technology, 31(5), 690-701 (2010). 76. Varshosaz J, Ghaffari S, Khoshayand MR, Atyabi F, Azarmi S, Kobarfard F. Development and optimization of solid lipid nanoparticles of amikacin by central composite design. Journal of liposome research, 20(2), 97-104 (2010). 77. Trapani A, Mandracchia D, Di Franco C et al. In vitro characterization of 6-Coumarin 70

loaded solid lipid nanoparticles and their uptake by immunocompetent fish cells. Colloids and Surfaces B: Biointerfaces, 127, 79-88 (2015). 78. Müller RH, Mäder K, Gohla S. Solid lipid nanoparticles (SLN) for controlled drug delivery – a review of the state of the art. European Journal of Pharmaceutics and Biopharmaceutics, 50(1), 161-177 (2000). 79. Imai H, Maeda T, Shima M, Adachi S. Oxidation of Methyl Linoleate in Oil-in-Water Micro- and Nanoemulsion Systems. Journal of the American Oil Chemists' Society, 85(9), 809-815 (2008). 80. Nakaya K, Ushio H, Matsukawa S, Shimizu M, Ohshima T. Effects of droplet size on the oxidative stability of oil-in-water emulsions. Lipids, 40(5), 501-507). 81. Lacatusu I, Badea N, Murariu A, Oprea O, Bojin D, Meghea A. Antioxidant Activity of Solid Lipid Nanoparticles Loaded with Umbelliferone. Soft Materials, 11(1), 75-84 (2013). 82. van Vliet T, van Vlissingen MF, van Schaik F, van den Berg H. β-Carotene Absorption and Cleavage in Rats Is Affected by the Vitamin A Concentration of the Diet. The Journal of Nutrition, 126(2), 499-508 (1996). 83. Jenning V, Schäfer-Korting M, Gohla S. Vitamin A-loaded solid lipid nanoparticles for topical use: drug release properties. Journal of Controlled Release, 66(2–3), 115-126 (2000). 84. Liu C-H, Wu C-T, Fang J-Y. Characterization and formulation optimization of solid lipid nanoparticles in vitamin K1 delivery. Drug Development and Industrial Pharmacy, 36(7), 751-761 (2010). 85. Dubey RR, Parikh JR, Parikh RR. Effect of heating temperature and time on pharmaceutical characteristics of albumin microspheres containing 5-fluorouracil. AAPS PharmSciTech, 4(1), E4 (2003). 86. Zhao D, Zhao X, Zu Y et al. Preparation, characterization, and in vitro targeted delivery of folate-decorated paclitaxel-loaded bovine serum albumin nanoparticles. International journal of nanomedicine, 5, 669-677 (2010). 87. Teng Z, Luo Y, Wang Q. Carboxymethyl chitosan–soy protein complex nanoparticles for the encapsulation and controlled release of vitamin D3. Food Chemistry, 141(1), 524-532 (2013). 88. Teng Z, Luo Y, Wang T, Zhang B, Wang Q. Development and application of nanoparticles synthesized with folic acid conjugated soy protein. Journal of agricultural and food chemistry, 61(10), 2556-2564 (2013). 89. Wartlick H, Michaelis K, Balthasar S, Strebhardt K, Kreuter J, Langer K. Highly Specific HER2-mediated Cellular Uptake of Antibody-modified Nanoparticles in Tumour Cells. Journal of drug targeting, 12(7), 461-471 (2004). 90. Luo Y, Zhang B, Cheng W-H, Wang Q. Preparation, characterization and evaluation of selenite-loaded chitosan/TPP nanoparticles with or without zein coating. Carbohydrate Polymers, 82(3), 942-951 (2010). 91. Perera G, Greindl M, Palmberger TF, Bernkop-Schnürch A. Insulin-loaded poly(acrylic 71

acid)-cysteine nanoparticles: Stability studies towards digestive enzymes of the intestine. Drug Delivery, 16(5), 254-260 (2009). 92. Horne DS. Casein structure, self-assembly and gelation. Current Opinion in Colloid & Interface Science, 7(5–6), 456-461 (2002). 93. Kruif CG, Holt C. Casein Micelle Structure, Functions and Interactions. In: Advanced Dairy Chemistry—1 Proteins: Part A / Part B. Fox, PF, McSweeney, PLH (Eds.) (Springer US, Boston, MA, 2003) 233-276. 94. Semo E, Kesselman E, Danino D, Livney YD. Casein micelle as a natural nano-capsular vehicle for nutraceuticals. Food Hydrocolloids, 21(5–6), 936-942 (2007). 95. Bachar M, Mandelbaum A, Portnaya I et al. Development and characterization of a novel drug nanocarrier for oral delivery, based on self-assembled β-casein micelles. Journal of Controlled Release, 160(2), 164-171 (2012). 96. Portnaya I, Cogan U, Livney YD et al. Micellization of Bovine β-Casein Studied by Isothermal Titration Microcalorimetry and Cryogenic Transmission Electron Microscopy. Journal of agricultural and food chemistry, 54(15), 5555-5561 (2006). 97. Zou T, Li Z, Percival SS, Bonard S, Gu L. Fabrication, characterization, and cytotoxicity evaluation of cranberry procyanidins-zein nanoparticles. Food Hydrocolloids, 27(2), 293-300 (2012). 98. Balthasar S, Michaelis K, Dinauer N, von Briesen H, Kreuter J, Langer K. Preparation and characterisation of antibody modified gelatin nanoparticles as drug carrier system for uptake in lymphocytes. Biomaterials, 26(15), 2723-2732 (2005). 99. Tseng C-L, Su W-Y, Yen K-C, Yang K-C, Lin F-H. The use of biotinylated-EGF-modified gelatin nanoparticle carrier to enhance cisplatin accumulation in cancerous lungs via inhalation. Biomaterials, 30(20), 3476-3485 (2009). 100. Zwiorek K, Kloeckner J, Wagner E, Coester C. Gelatin nanoparticles as a new and simple gene delivery system. Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques, 7(4), 22-28 (2005). 101. Coester CJ, Langer K, van Briesen H, Kreuter J. Gelatin nanoparticles by two step desolvation--a new preparation method, surface modifications and cell uptake. Journal of microencapsulation, 17(2), 187-193 (2000). 102. Shpigelman A, Israeli G, Livney YD. Thermally-induced protein–polyphenol co-assemblies: beta lactoglobulin-based nanocomplexes as protective nanovehicles for EGCG. Food Hydrocolloids, 24(8), 735-743 (2010). 103. Ezpeleta I, Arangoa MA, Irache JM et al. Preparation of Ulex europaeus lectin-gliadin nanoparticle conjugates and their interaction with gastrointestinal mucus. International Journal of Pharmaceutics, 191(1), 25-32 (1999). 104. Hussain N, Jani PU, Florence AT. Enhanced Oral Uptake of Tomato Lectin-Conjugated Nanoparticles in the Rat. Pharmaceutical research, 14(5), 613-618). 105. Wagner S, Rothweiler F, Anhorn MG et al. Enhanced drug targeting by attachment of an anti αv integrin antibody to doxorubicin loaded human serum albumin nanoparticles. 72

Biomaterials, 31(8), 2388-2398 (2010). 106. Jyonouchi H, Sun S, Iijima K, Gross MD. Antitumor Activity of Astaxanthin and Its Mode of Action. Nutrition and Cancer, 36(1), 59-65 (2000). 107. Kuppusamy P, Yusoff MM, Maniam GP, Ichwan SJA, Soundharrajan I, Govindan N. Nutraceuticals as potential therapeutic agents for colon cancer: a review. Acta Pharmaceutica Sinica B, 4(3), 173-181 (2014). 108. Ravindran Girija A, Balasubramanian S, Dhandayudhapani B et al. Biocompatible fluorescent zein nanoparticles for simultaneous bioimaging and drug delivery application. Advances in Natural Sciences: Nanoscience and Nanotechnology, 3(2), 025006 (2012). 109. Momany FA, Sessa DJ, Lawton JW, Selling GW, Hamaker SAH, Willett JL. Structural Characterization of α-Zein. Journal of agricultural and food chemistry, 54(2), 543-547 (2006). 110. Sousa FFO, Luzardo-Álvarez A, Blanco-Méndez J, Otero-Espinar FJ, Martín-Pastor M, Sández Macho I. Use of 1H NMR STD, WaterLOGSY, and Langmuir monolayer techniques for characterization of drug–zein protein complexes. European Journal of Pharmaceutics and Biopharmaceutics, 85(3, Part A), 790-798 (2013). 111. Shukla R, Cheryan M. Zein: the industrial protein from corn. Industrial Crops and Products, 13(3), 171-192 (2001). 112. Osborne TB. THE AMOUNT AND PROPERTIES OF THE PROTEIDS OF THE MAIZE KERNEL. 2. Journal of the American Chemical Society, 19(7), 525-532 (1897). 113. Turner JE, Boundy JA, Dimler RJ. Zein: A heterogeneous protein containing disulfide-linked aggregates. Cereal Chem, 42, 452-461 (1965). 114. Wall JS, Paulis JW. Corn and sorghum grain proteins. Advances in cereal science and technology, 2, 135-219 (1978). 115. Elzoghby AO, Samy WM, Elgindy NA. Protein-based nanocarriers as promising drug and gene delivery systems. Journal of Controlled Release, 161(1), 38-49 (2012). 116. Hansen DW. Manufacture of laminated films. (Ed.^(Eds) (Google Patents, 1937) 117. Chen H, Zhong Q. A novel method of preparing stable zein nanoparticle dispersions for encapsulation of peppermint oil. Food Hydrocolloids, 43, 593-602 (2015). 118. Karthikeyan K, Guhathakarta S, Rajaram R, Korrapati PS. Electrospun zein/eudragit nanofibers based dual drug delivery system for the simultaneous delivery of aceclofenac and pantoprazole. International Journal of Pharmaceutics, 438(1–2), 117-122 (2012). 119. Wang Y-H, Wan Z-L, Yang X-Q, Wang J-M, Guo J, Lin Y. Colloidal complexation of zein hydrolysate with tannic acid: Constructing peptides-based nanoemulsions for alga oil delivery. Food Hydrocolloids, 54, Part A, 40-48 (2016). 120. Wang Y, Padua GW. Nanoscale Characterization of Zein Self-Assembly. Langmuir, 28(5), 2429-2435 (2012). 121. Zhong Q, Jin M. Zein nanoparticles produced by liquid–liquid dispersion. Food Hydrocolloids, 23(8), 2380-2387 (2009). 122. Podaralla S, Perumal O. Influence of Formulation Factors on the Preparation of Zein nanoparticles. AAPS PharmSciTech, 13(3), 919-927 (2012). 73

123. Gomez-Estaca J, Balaguer MP, Gavara R, Hernandez-Munoz P. Formation of zein nanoparticles by electrohydrodynamic atomization: Effect of the main processing variables and suitability for encapsulating the food coloring and active ingredient curcumin. Food Hydrocolloids, 28(1), 82-91 (2012). 124. Hu D, Lin C, Liu L, Li S, Zhao Y. Preparation, characterization, and in vitro release investigation of lutein/zein nanoparticles via solution enhanced dispersion by supercritical fluids. Journal of Food Engineering, 109(3), 545-552 (2012). 125. Zhong Q, Jin M, Xiao D, Tian H, Zhang W. Application of Supercritical Anti-Solvent Technologies for the Synthesis of Delivery Systems of Bioactive Food Components. Food Biophysics, 3(2), 186-190 (2008). 126. Wu Y, Luo Y, Wang Q. Antioxidant and antimicrobial properties of essential oils encapsulated in zein nanoparticles prepared by liquid–liquid dispersion method. LWT - Food Science and Technology, 48(2), 283-290 (2012). 127. El-Toni AM, Habila MA, Labis JP et al. Design, synthesis and applications of core-shell, hollow core, and nanorattle multifunctional nanostructures. Nanoscale, 8(5), 2510-2531 (2016). 128. Jöbstl E, O'Connell J, Fairclough JPA, Williamson MP. Molecular Model for Astringency Produced by Polyphenol/Protein Interactions. Biomacromolecules, 5(3), 942-949 (2004). 129. Penalva R, Esparza I, Larraneta E, González-Navarro CJ, Gamazo C, Irache JM. Zein-Based Nanoparticles Improve the Oral Bioavailability of Resveratrol and Its Anti-inflammatory Effects in a Mouse Model of Endotoxic Shock. Journal of agricultural and food chemistry, 63(23), 5603-5611 (2015). 130. Park C-E, Park D-J, Kim B-K. Effects of a chitosan coating on properties of retinol-encapsulated zein nanoparticles. Food Science and Biotechnology, 24(5), 1725-1733 (2015). 131. Siró I, Kápolna E, Kápolna B, Lugasi A. Functional food. Product development, marketing and consumer acceptance—A review. Appetite, 51(3), 456-467 (2008). 132. Kris-Etherton PM, Keen CL. Evidence that the antioxidant flavonoids in tea and cocoa are beneficial for cardiovascular health. Current opinion in lipidology, 13(1), 41-49 (2002). 133. Déprez S, Brezillon C, Rabot S et al. Polymeric proanthocyanidins are catabolized by human colonic microflora into low-molecular-weight phenolic acids. The Journal of nutrition, 130(11), 2733-2738 (2000). 134. Shao ZH, Hsu CW, Chang WT et al. Cytotoxicity induced by grape seed proanthocyanidins: Role of nitric oxide. Cell Biology and Toxicology, 22(3), 149-158). 135. Vogel H, Pelletier J. Curcumin-biological and medicinal properties. J. Pharma, 2, 50 (1815). 136. Miłobȩdzka J, Lampe V. Zur Kenntnis des curcumins. Berichte der deutschen chemischen Gesellschaft, 43(2), 2163-2170 (1910). 137. Ghosh S, Banerjee S, Sil PC. The beneficial role of curcumin on inflammation, diabetes and neurodegenerative disease: A recent update. Food and Chemical Toxicology, 83, 74

111-124 (2015). 138. Shen L, Ji H-F. Theoretical study on physicochemical properties of curcumin. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 67(3–4), 619-623 (2007). 139. Goel A, Kunnumakkara AB, Aggarwal BB. Curcumin as “Curecumin”: From kitchen to clinic. Biochemical Pharmacology, 75(4), 787-809 (2008). 140. Mishra B, Priyadarsini KI, Bhide MK, Kadam RM, Mohan H. Reactions of superoxide radicals with curcumin: probable mechanisms by optical spectroscopy and EPR. Free radical research, 38(4), 355-362 (2004). 141. Cole GM, Lim GP, Yang F et al. Prevention of Alzheimer's disease: Omega-3 fatty acid and phenolic anti-oxidant interventions. Neurobiology of Aging, 26(1, Supplement), 133-136 (2005). 142. De R, Kundu P, Swarnakar S et al. Antimicrobial activity of curcumin against Helicobacter pylori isolates from India and during infections in mice. Antimicrobial agents and chemotherapy, 53(4), 1592-1597 (2009). 143. Choudhuri T, Pal S, Agwarwal ML, Das T, Sa G. Curcumin induces apoptosis in human breast cancer cells through p53-dependent Bax induction. FEBS Letters, 512(1-3), 334-340 (2002). 144. Kuo M-L, Huang T-S, Lin J-K. Curcumin, an antioxidant and anti-tumor promoter, induces apoptosis in human leukemia cells. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1317(2), 95-100 (1996). 145. Chen H, Zhang ZS, Zhang YL, Zhou DY. Curcumin inhibits cell proliferation by interfering with the cell cycle and inducing apoptosis in colon carcinoma cells. Anticancer research, 19(5A), 3675-3680 (1999). 146. Mohanty C, Sahoo SK. The in vitro stability and in vivo pharmacokinetics of curcumin prepared as an aqueous nanoparticulate formulation. Biomaterials, 31(25), 6597-6611 (2010). 147. Yu H, Huang Q. Improving the Oral Bioavailability of Curcumin Using Novel Organogel-Based Nanoemulsions. Journal of agricultural and food chemistry, 60(21), 5373-5379 (2012). 148. Tønnesen HH, Karlsen J. Studies on curcumin and curcuminoids. Zeitschrift für Lebensmittel-Untersuchung und Forschung, 180(5), 402-404). 149. Tønnesen HH, Karlsen J. Studies on curcumin and curcuminoids. Zeitschrift für Lebensmittel-Untersuchung und Forschung, 180(2), 132-134). 150. Wang Y-J, Pan M-H, Cheng A-L et al. Stability of curcumin in buffer solutions and characterization of its degradation products. Journal of Pharmaceutical and Biomedical Analysis, 15(12), 1867-1876 (1997). 151. Tønnesen HH, Karlsen J, Henegouwen GB. Studies on curcumin and curcuminoids VIII. Photochemical stability of curcumin. Zeitschrift für Lebensmittel-Untersuchung und Forschung, 183(2), 116-122). 152. Zhou M, Wang T, Hu Q, Luo Y. Low density lipoprotein/pectin complex nanogels as 75

potential oral delivery vehicles for curcumin. Food Hydrocolloids, 57, 20-29 (2016). 153. Wahlström B, Blennow G. A study on the fate of curcumin in the rat. Acta pharmacologica et toxicologica, 43(2), 86-92 (1978). 154. Shoba G, Joy D, Joseph T, Majeed M, Rajendran R, Srinivas PS. Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers. Planta medica, 64(4), 353-356 (1998). 155. Onoue S, Takahashi H, Kawabata Y et al. Formulation Design and Photochemical Studies on Nanocrystal Solid Dispersion of Curcumin with Improved Oral Bioavailability. Journal of Pharmaceutical Sciences, 99(4), 1871-1881 (2010). 156. Xie X, Tao Q, Zou Y et al. PLGA Nanoparticles Improve the Oral Bioavailability of Curcumin in Rats: Characterizations and Mechanisms. Journal of agricultural and food chemistry, 59(17), 9280-9289 (2011). 157. Tsai Y-M, Jan W-C, Chien C-F, Lee W-C, Lin L-C, Tsai T-H. Optimised nano-formulation on the bioavailability of hydrophobic polyphenol, curcumin, in freely-moving rats. Food Chemistry, 127(3), 918-925 (2011). 158. Li S-D, Huang L. Pharmacokinetics and Biodistribution of Nanoparticles. Molecular pharmaceutics, 5(4), 496-504 (2008). 159. Yang F, Lim GP, Begum AN et al. Curcumin inhibits formation of amyloid β oligomers and fibrils, binds plaques, and reduces amyloid in vivo. Journal of Biological Chemistry, 280(7), 5892-5901 (2005). 160. Pan M-H, Huang T-M, Lin J-K. Biotransformation of curcumin through reduction and glucuronidation in mice. Drug metabolism and disposition, 27(4), 486-494 (1999). 161. Cheng KK, Yeung CF, Ho SW, Chow SF, Chow AHL, Baum L. Highly Stabilized Curcumin Nanoparticles Tested in an In Vitro Blood–Brain Barrier Model and in Alzheimer’s Disease Tg2576 Mice. The AAPS Journal, 15(2), 324-336 (2012). 162. Alam MI, Beg S, Samad A et al. Strategy for effective brain drug delivery. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 40(5), 385-403 (2010). 163. Sahu A, Kasoju N, Bora U. Fluorescence Study of the Curcumin−Casein Micelle Complexation and Its Application as a Drug Nanocarrier to Cancer Cells. Biomacromolecules, 9(10), 2905-2912 (2008). 164. Ferreira SL, Bruns RE, Ferreira HS et al. Box-Behnken design: an alternative for the optimization of analytical methods. Analytica chimica acta, 597(2), 179-186 (2007). 165. Chen JF, Ding HM, Wang JX, Shao L. Preparation and characterization of porous hollow silica nanoparticles for drug delivery application. Biomaterials, 25(4), 723-727 (2004). 166. Hunter RJ. Zeta potential in colloid science: principles and applications (Academic press, 2013). 167. Yin Win K, Feng S-S. Effects of particle size and surface coating on cellular uptake of polymeric nanoparticles for oral delivery of anticancer drugs. Biomaterials, 26(15), 2713-2722 (2005). 168. Zou Y, Guo J, Yin SW, Wang JM, Yang XQ. Pickering Emulsion Gels Prepared by 76

Hydrogen-Bonded Zein/Tannic Acid Complex Colloidal Particles. Journal of agricultural and food chemistry, 63(33), 7405-7414 (2015). 169. Socrates G. Infrared and Raman characteristic group frequencies: tables and charts (John Wiley & Sons, 2004). 170. Kolev TM, Velcheva EA, Stamboliyska BA, Spiteller M. DFT and experimental studies of the structure and vibrational spectra of curcumin. International Journal of Quantum Chemistry, 102(6), 1069-1079 (2005). 171. Hu K, Huang X, Gao Y, Huang X, Xiao H, McClements DJ. Core-shell biopolymer nanoparticle delivery systems: synthesis and characterization of curcumin fortified zein-pectin nanoparticles. Food Chem, 182, 275-281 (2015). 172. Baxter NJ, Lilley TH, Haslam E, Williamson MP. Multiple Interactions between Polyphenols and a Salivary Proline-Rich Protein Repeat Result in Complexation and Precipitation. Biochemistry, 36(18), 5566-5577 (1997). 173. Chignell CF, Bilskj P, Reszka KJ, Motten AG, Sik RH, Dahl TA. SPECTRAL AND PHOTOCHEMICAL PROPERTIES OF CURCUMIN. Photochemistry and Photobiology, 59(3), 295-302 (1994). 174. Bong P-H. Spectral and photophysical behaviors of curcumin and curcuminoids. Bulletin-Korean Chemical Society, 21(1), 81-86 (2000). 175. Pan K, Zhong Q, Baek SJ. Enhanced Dispersibility and Bioactivity of Curcumin by Encapsulation in Casein Nanocapsules. Journal of agricultural and food chemistry, 61(25), 6036-6043 (2013). 176. Sahu A, Kasoju N, Bora U. Fluorescence study of the curcumin− casein micelle complexation and its application as a drug nanocarrier to cancer cells. Biomacromolecules, 9(10), 2905-2912 (2008). 177. Abdelwahed W, Degobert G, Stainmesse S, Fessi H. Freeze-drying of nanoparticles: formulation, process and storage considerations. Advanced drug delivery reviews, 58(15), 1688-1713 (2006). 178. Teng Z, Li Y, Wang Q. Insight into Curcumin-Loaded β-Lactoglobulin Nanoparticles: Incorporation, Particle Disintegration, and Releasing Profiles. Journal of agricultural and food chemistry, 62(35), 8837-8847 (2014). 179. Hurtado-López P, Murdan S. Zein microspheres as drug/antigen carriers: A study of their degradation and erosion, in the presence and absence of enzymes. Journal of microencapsulation, 23(3), 303-314 (2006).