Protein-based nanoparticles: From preparation to encapsulation of active molecules

Accepted Manuscript Title: Protein-based nanoparticles: From preparation to encapsulation of active molecules Author: Mohamad Tarhini H´el`ene Greige-Gerges Abdelhamid Elaissari PII: DOI: Reference:

S0378-5173(17)30076-5 http://dx.doi.org/doi:10.1016/j.ijpharm.2017.01.067 IJP 16399

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

31-10-2016 26-1-2017 29-1-2017

Please cite this article as: Tarhini, M., Greige-Gerges, H., Elaissari, A.,Protein-based nanoparticles: From preparation to encapsulation of active molecules, International Journal of Pharmaceutics (2017), http://dx.doi.org/10.1016/j.ijpharm.2017.01.067 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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*Manuscript Click here to download Manuscript: Revised and modified version of Protein based nanoparticles Click here (AE).docx to view linked References

Protein-based nanoparticles: Form preparation to encapsulation of active molecules Mohamad Tarhini1,2, Hélène Greige-Gerges2, Abdelhamid Elaissari1* 1)

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Univ Lyon, University Claude Bernard Lyon-1, CNRS, LAGEP UMR 5007, 43 boulevard du 11 November 1918, F-69100, VILLEURBANNE, France 2) Faculty of Sciences, Lebanese University, B.P. 90656, Jdaidet El-Matn, Lebanon

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Corresponding author:[email protected] [email protected]

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Abstract

Nowadays, nanotechnology has become very integrated in the domain of pharmaceutical

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sciences since nanoparticle dispersions show various advantages as drug carriers. Among nanoparticles, the protein-based ones are of paramount importance. In fact, protein nanoparticles show many advantages over other types of nanoparticles, they are often non-toxic and

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biodegradable. In this review, the most common preparation methods of protein nanoparticles

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were targeted. In addition, the factors affecting their dispersions and the concepts of drug loading and drug release are also highlighted. It was obvious that each method can be optimized for a

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given protein. This issue was discussed in depth in the light of the current state of art, and supported by evidences for each method from the literature. In addition, it was concluded that the processing parameters strongly affect the properties of nanoparticles dispersion. Keywords: nanoparticles, protein, encapsulation, active molecules Graphical Abstract: Preparation methods of nanoparticles and proteins used with them. Red cases: animal protein, Green cases: plant protein

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1. Introduction The plant kingdom around us is full of active molecules that play a crucial role in the prevention or treatment of diseases. However, these molecules cannot achieve their nutritional or therapeutic values because of their chemical and/or biological instability and their low bioavailability. (Chen

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et al., 2011) (Esmaili et al., 2011). Therefore, new methods were needed to increase the stability of the active molecule to improve its absorption in the small intestines and control its release.

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During the past few decades, there has been an increasing interest for the development of micro and nanoparticles for effective drug, peptide, protein and DNA delivery. The difference between

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these systems is not limited to size; they also differ in their therapeutic applications and biopharmaceutical values. In general, the therapeutic active molecule is immobilized, adsorbed,

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attached, dissolved or encapsulated in the particle, allowing well control over drug release profiles (Orive et al., 2004). However, nanoparticles have a set of advantages far above than microparticles (Panyam and Labhasetwar, 2003)(Pinto Reis et al., 2006). Moreover, targeting to

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specific tissues such as inflamed and cancerous tissues may be limited only to nanoparticles (Avnir et al., 2011).

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The concept of controlled drug delivery is defined by the association of a drug with a carrier

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system. This association can modulate the pharmacokinetic of the drug. Different carriers or “nano-scale systems” were developed. Among them, we can cite nanoparticles (Leroux et al.,

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1995), liposomes (Bochot et al., 2002), surface-modified nanoparticles (Araujo et al., 1999) and solid-lipid nanoparticles (Müller et al., 2000). As for nanoparticles, they can control drug release, they affect the distribution and clearance of the drug and achieve an increase in drug efficacy and decrease in the side effects. These carriers show some advantages such as: 1) the drug loading in nanoparticles is relatively high, and the incorporation of the drug in the system can be obtained without any chemical reactions, which preserve the drug activity, 2) the possibility to have a site-specific targeting system by attaching specific ligands to the surface of the nanoparticle or using magnetic or specific guidance, and 3) the ability to use nanoparticles for various routes of administration (oral, nasal, parenteral, intraocular etc.) (Mohanraj and Chen, 2006a).

Fig. 1. Proteins used to build nanoparticles Page 2 / 39 Page 3 of 66

Among nanoparticles, protein-based nanoparticles (PBNs) have special interests because they are biodegradable, metabolizable, can be easily manipulated and there are various possibilities for surface alteration and/or modification for covalent drug attachment (C Weber et al., 2000). From

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PBNs, we can cite different types of nanoparticles elaborated from different proteins (Fig. 1). Proteins used in the nanoparticles domain can be classified as animal proteins and plant proteins and both have advantages and disadvantages. For animal proteins, the low toxicity of the end

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product (metabolite or product of degradation), gives them an advantage over synthetic polymers (Leo et al., 1997). The major drawback of animal proteins is the risk of infection from pathogen

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contamination. However, in some cases it is not actually a real problem since animal proteins can be sterilized (Pathak and Thassu, 2009).

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Regarding plant proteins, their hydrophobic character is the main advantage compared to animal proteins. This could lead to the avoidance of toxic chemical crosslinkers (Ezpeleta et al., 1996).

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In addition, plant proteins are also less expensive than animal proteins. This review introduces a comprehensive survey of literature for preparation of protein

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nanoparticles and their use for encapsulation of bioactive molecules for drug delivery

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applications. Firstly, the factors affecting the preparation of protein-based nanoparticles are presented. Then, the commonly used preparation methods for PBNs, using various types of

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proteins and their colloidal properties, are highlighted. After that, the drug loading rate and drug release concepts are explained. Finally, based on the state of the art, a guiding approach is suggested that can be used for the preparation of protein-based nanoparticles dispersions. 2. Factors affecting protein nanoparticles preparation The physico-chemical properties of the protein and the drug properties, which affect on the preparation and characteristics of nanoparticles, are presented in Fig. 2. Fig. 2. Factors influencing the preparation and performance of protein nanoparticles. 2.1 Protein composition The preparation of nanoparticles is influenced by the composition of protein, which in turn, depends on the source from which the protein derived. Usually, proteins are composed of different molecular weight fractions. Therefore, any variation in these fractions can have a significant influence on the nanoparticle characteristics (Pathak and Thassu, 2009). Page 3 / 39 Page 4 of 66

Human serum albumin (HSA) possesses a free thiol group that makes it capable to build dimers and higher aggregates. Langer et al. studied the influence of dimers and higher aggregates of HSA on the preparation of albumin nanoparticles (Langer et al., 2008). Results showed that the starting batch of HSA could influence the size of the prepared nanoparticles. In fact, the

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preparation is affected by the amount of high molecular HSA components; the higher amounts led to an increase of size and size distribution (polydispersity) of the obtained dispersions.

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Therefore, HSA-based nanoparticles could be prepared with almost predictable size (Fig. 3) (Langer et al., 2008).

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Fig. 3. Influence of the HSA batch on a) Particle diameter of HSA nanoparticles, and b) Polydispersity of HSA nanoparticles at various pH values. . Reproduced with permission from ref. (Langer et al., 2008) Copyright 2016 International Journal of Pharmaceutics As for plant proteins, they also have various molecular weight fractions. Moreover, the preparation of nanoparticles from plant proteins can be influenced by the presence of pigments.

2.2 Protein solubility

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nanoparticles (Pathak and Thassu, 2009).

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For this reason, the purification of the protein is important before the preparation process of

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The solubility of protein in aqueous or organic solvent is a major concern that regulates the choice of the preparation method and the characteristics of nanoparticles. The concept of the

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PBNs preparation is based on the differential solubility of the protein in aqueous and nonaqueous solvents, as depending on the polarity of the solvent, proteins can fold or unfold (Wang and Uludag, 2008). Duclairoir et al. (2014) prepared gliadin nanoparticles and used the solubility parameters to optimize the characteristics of these particles. The study showed that the size of gliadin nanoparticles could be controlled by its solubility parameters in both solvents used. The smallest size could be achieved when gliadin was solubilized in the solvent that has the same solubility of the protein (Duclairoir et al., 2014). Based on the isoelectric point (pI), protein exhibits pH-dependent water solubility. It was found that the size of HSA nanoparticles is significantly affected by the pH value of the aqueous solution. When the pH increased above the pI of albumin (pI = 5.05), the particle size decreased (Fig. 4) (Langer et al., 2003). At a pH away from the pI, the hydrophobic interactions in the protein are reduced, and consequently, resulting in less aggregation.

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Fig. 4. Influence of the pH value on the diameter (□) and yield (ᴏ) of HSA nanoparticles prepared in pure water and in 10 mM NaCl solution (∆). Reproduced with permission from ref. (Langer et al., 2003). Copyright 2016 International Journal of Pharmaceutics. 2.3 Surface properties

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The surface chemistry of PBN can be modified due to the presence of many surface functional groups in the protein. These modifications can alter many important features in the resulting nanoparticles, such as biodistribution, biocompatibility, drug loading and stability. The surface

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functional groups of the protein (amine, carboxyl, and thiol groups) are exposed by the

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conformational changes of the protein macromolecules(Pathak and Thassu, 2009).

The surface amino groups in the protein can be crosslinked using bifunctional crosslinkers such

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as glutaraldehyde. The crosslinking density depends on the protonation and deprotonation of the surface amino groups (Vandervoort and Ludwig, 2004). Normally, when the concentration of the crosslinker increases, denser particles can be formed due to the decrease of particle size. In

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addition, the surface amino groups in protein can be exploited for attachment with hydrophilic polymers such as polyethylene glycol (PEG). This addition can increase the circulation half-life

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of the functionalized nanoparticle by avoiding the phagocytic uptake (Kaul and Amiji, 2004).

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Furthermore, surface functional groups can be used for drug loading via electrostatic interaction, also, they can interact directly with the biological membranes. For instance, gliadin nanoparticles

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have bioadhesive ability through electrostatic interactions and hydrogen bonding to the intestinal membrane (Pathak and Thassu, 2009). For characterization of nanoparticles, elemental and spectroscopic analyses are usually used. Xray photoelectron spectroscopy has been utilized for studying the chemical compositions of particle surface. Moreover, Raman spectroscopy has been used for the identification of certain component in the particles. Infrared spectroscopy was also used to study the biopolymer interaction (Davidov-Pardo et al., 2015). 2.4 Drug properties In general, a drug can be loaded either by encapsulation inside the nanoparticle, or by interaction with the protein through covalent or noncovalent interactions. It is well known that the physicochemical properties of the drug (solubility, logP, and molecular weight) can influence the loading rate of the drug in nanoparticles. Page 5 / 39 Page 6 of 66

In the case of gelatin nanoparticles, higher encapsulation efficiency was reported for hydrophilic drugs than for hydrophobic ones (Vandervoort and Ludwig, 2004). For instance, doxorubicin was adsorbed onto gelatin-coated iron oxide nanoparticles for drug targeting using magnetic field. It was found that the adsorption of cationic doxorubicin onto gelatin nanoparticles

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increased with increasing pH due to the negative charge of gelatin at high pH values. On the other hand, the encapsulation of doxorubicin within gelatin-coated iron oxide nanoparticles

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showed a slower drug release than surface adsorbed nanoparticles (Gaihre et al., 2009).

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3. Preparation Methods

There are many methods for preparation of protein-based nanoparticles. Choice of the method depends on various factors such as the physico-chemical properties and the amino acid

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constituent of the protein used as a nanocarrier, and the properties of the drug to be encapsulated. In fact, the formation of nanoparticles depends on the increase of protein unfolding and the

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decrease of hydrophobic intramolecular interactions in the chosen protein. During nanoparticle formation, the protein undergoes conformational changes depending on its composition,

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methods (Pathak and Thassu, 2009).

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concentration, preparation conditions (such as pH, ionic strength, solvent), and crosslinking

Despite the differences in the preparation methods of protein nanoparticles, all encapsulation

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methods are based on precipitation process of the used macromolecules. Precipitation of polymer and biomolecules occurs when the solubility of the macromolecules in a given solvent decreases. This can be done either by adding a non-solvent, or by changing the physico-chemical parameters such as pH, salinity or temperature of the protein solution (Miladi et al., 2014). From the thermodynamic point of view, it is known that Flory χ-parameter is the key factor governing the thermodynamic behavior of a given macromolecule in a given solution. The definition of this parameter is the free energy change (∆G) per solvent by shifting from solventsolvent contact to solvent-macromolecule contact. Flory χ-parameter is expressed by the following mathematical equation: (Eq.1)

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Where kB is Boltzmann constant, T is temperature, and A and θ parameters are expressed as follows: (Eq. 2)

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(Eq. 3)

From these equations, A is directly related to entropy variation (∆S), while θ temperature is both

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entropy (∆S) and enthalpy (∆H) related. When θ temperature equal T, then

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notice that,

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equal to zero, then, according the Eq. 1, χ parameter will equal to 1/2. Here, it is interesting to can be determined by light scattering measurement of a diluted

and a bad solvent if χ > 1/2 (Miladi et al., 2014).

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macromolecule solution and the solvent will be a good solvent for the macromolecule if χ < 1/2

Disulfides and thiol groups can be exposed during the preparation process by the unfolding of

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the protein. Then, the crosslinking process (thermally or chemically) will lead to the formation of crosslinked nanoparticles with entrapped drug molecules. Coacervation/desolvation and

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(Pathak and Thassu, 2009).

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emulsion based methods are most commonly used for the preparation of protein nanoparticles

details.

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In this section, the most used preparation methods for protein nanoparticles are discussed in

3.1 Desolvation

Briefly, desolvation process is the addition of a desolvating agent like alcohol or acetone to an aqueous solution of protein under stirring, this will lead to the dehydration of the protein and the change in its conformation from stretched to coil conformation (Elzoghby et al., 2015). The amino functional groups of the protein can be crosslinked in order to make the nanoparticles denser and guard the coacervates (Fig. 5). Albumin, gelatin, silk, whey and gliadin nanoparticles were prepared using desolvation process. In some cases, in order to reach a smaller and uniform size of nanoparticles, a second desolvation step is required. Despite the extensive use of desolvation technique in nanoparticles preparation, it has two major disadvantages, the use of organic solvent and the use of toxic crosslinkers (Elzoghby et al., 2015). Page 7 / 39 Page 8 of 66

Fig. 5. Preparation of protein nanoparticles by desolvation. Desolvation process of human serum albumin (HSA) was optimized by Weber et al. (C. Weber et al., 2000). Samples were prepared as follow: Aliquots of either 0.25 or 0.1 ml ethanol (total 8.0 ml) were added dropwise to 2.0 ml of 10% aqueous HSA solution under constant stirring.

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After each desolvation step, aliquots of 0.05 ml were taken from the samples, and crosslinked with glutaraldehyde. Glutaraldehyde is the most commonly used crosslinker that induces poly- or

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bifunctional crosslinks into the network structure of proteins by bridging the free amino groups of lysine or hydroxylysine residues. The particle size as well as the cumulative light intensity

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counts of the samples were determined by photon correlation spectroscopy (PCS) (Fig. 6) (C. Weber et al., 2000).

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Fig. 6. HSA nanoparticles prepared by desolvation method: A) Particle size and light intensity counts in the PCS measurement in correlation to the amount of ethanol added during the desolvation procedure. B) Percentage of dissolved HSA in the supernatant of nanoparticles in correlation to the amount of ethanol added during the desolvation procedure. C) Amino group content and percentage of dissolved HSA in the supernatant of nanoparticles in correlation to the amount of glutaraldehyde added. D) Particle size of the purified and unpurified nanoparticles in correlation to the amount of glutaraldehyde added. Reproduced with permission from ref. (C. Weber et al., 2000). Copyright 2016 International Journal of Pharmaceutics.

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Results showed that the particle size increased significantly by addition of up to 1.5-fold volume of ethanol relative to the volume of the initial HSA solution. Further addition of ethanol led to

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increasing the number of particles but did not affect the size (Fig. 6A/B) (C. Weber et al., 2000). The influence of crosslinker was also investigated. The increase of glutaraldehyde concentration did not affect the particle size; however, it significantly decreased the number of available amino groups on the surface of HSA nanoparticles (Fig. 6C/D). In order to produce a stable HSA nanoparticles, it was found that a crosslinker concentration about 40% glutaraldehyde was necessary(C. Weber et al., 2000).

Fig. 7. SEM of gelatin nanoparticles. Magnification=×30000. Reproduced with permission from ref. (Jain et al., 2008). Copyright 2016 Nanomedicine Human serum albumin (HSA) nanoparticles were also used as a doxorubicin carriers by Dreis et al. (Dreis et al., 2007). They were prepared using desolvation technique and doxorubicin was loaded either by adsorption to the nanoparticles surface or by incorporation into the particle Page 8 / 39 Page 9 of 66

matrix (Dreis et al., 2007). The characteristics of nanoparticles differ from one technique to another. In the adsorption method, the loading efficiency of nanoparticles was above 95% as long as the initial concentration of doxorubicin does not exceed 0.25 mg/ml. In this method, no significant change in size between empty and loaded nanoparticles was noticed. However, in the

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incorporation method, it was found that the characteristics of loaded nanoparticles depend on the concentration of doxorubicin, amount of the glutaraldehyde crosslinker, and the pH value during

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the desolvation process. The best nanoparticles dispersions were obtained at doxorubicin concentrations ≤1.0 mg/ml with 20.0 mg/ml HSA, with 100% crosslinking and without pH

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adjustment ( original pH 6.5) (Dreis et al., 2007).

Gelatin nanoparticles are commonly used as carriers for the didanosine. Nanoparticles coupled

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with mannose were prepared by a two-step desolvation technique. The size of nanoparticles was found to be in range of 248–325 nm (Fig. 7), and maximum drug loading was found to be 40.2%

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to 48.5%.

The zeta potential value of the obtained nanoparticles dispersions was 6.2 ± 0.12 mV. By grafting nanoparticles with mannose, the size of the particles increased while drug loading and

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zeta potential value decreased. However, by coupling with mannose, the drug uptake in the lung,

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liver and lymph node was significantly enhanced (Jain et al., 2008).

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Kundu et al. (2010) have prepared silk fibroin nanoparticles form two silk variants derived from domesticated Bombyxmori and tropical tasar silkworm Antheraeamylitta loaded with vascular endothelial growth factor (VEGF)(Kundu et al., 2010). Nanoparticles were prepared using desolvation method. The desolvating agent was dimethyl sulfoxide (DMSO). The TEM observation of the silk fibroin nanoparticles derived from liquid silk fibroin showed spherical granules without apparent aggregation or adhesion. The fibroin nanoparticles prepared from A. mylitta were smoother and morphologically more spherical compared to the B. mori particles which were coarser (Fig. 8) (Kundu et al., 2010). Fig. 8. TEM images of silk fibroin nanoparticles prepared from A. mylitta (a), silk fibroin nanoparticles prepared from B. mori (b), a single silk fibroin nanoparticles prepared from A. mylitta (c), a single silk fibroin nanoparticles prepared from B. mori (d). Reproduced with permission from ref. (Kundu et al., 2010). Copyright 2016 International Journal of Pharmaceutics.

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The size of nanoparticles was measured by dynamic light scattering (DLS) technique. The size and size distribution (polydispersity index) differ from one silk variant to another. A. mylitta nanoparticles possess a diameter around 157 ± 4 nm with a polydispersity index of 0.02. While B. mori nanoparticles have a diameter of 177 ± 3 nm with a polydispersity index of 0.027. By

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immersing the nanoparticles with fetal bovine serum containing medium, an increase of diameter by 20 nm was observed. This is possibly due to the adsorption of the serum proteins on to the

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surface of the silk nanoparticles (Kundu et al., 2010).

Moreover, desolvation method was used to entrap carbazol in gliadin nanoparticles (Arangoa et

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al., 2001). The size of the non-crosslinked particles was 460 ± 19 nm and zeta potential +27.5 ± 0.8mV. While the crosslinked nanoparticles decreased in size to become 453 ± 24 and the zeta

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potential 24.5 ± 0.5mV (Table 1). The oral bioavailability of carbazol increased dramatically over 49% when entrapped with gliadin nanoparticles and provided sustained release properties

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related to the bioadhesive capacity of gliadin nanoparticles with the stomach mucosa after oral administration (Arangoa et al., 2001). In Table 1, the variation in size values, yield and loaded carbazol are not significant; however, the value of zeta potential differs significantly. This

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variation is to be discussed.

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Table 1. Physicochemical properties of carbazol-loaded gliadin nanoparticles prepared by desolvation method. Reproduced with permission from ref. (Arangoa et al., 2001). Copyright 2016 Pharmaceutical Research. Gliadin nanoparticles were used to entrap amoxicillin for eradication of Helicobacter pylori in stomach. The eradication of Helicobacter pylori was more effective when amoxicillin was entrapped in gliadin nanoparticles because of the prolonged residence time that attributed to mucoadhesion (Umamaheshwari et al., 2004). Nanoparticles were also prepared by desolvation method, the surface morphology as investigated by SEM revealed a spherical shape with a smooth surface (Fig. 9). Gliadin nanoparticles have a size range of 285 ± 44 nm to 392 ± 20 nm with positive zeta potential values at maximum and minimum gliadin concentration, respectively (50-200 mg). The average zeta potential of drug loaded gliadin nanoparticles was 26.6 ± 0.8 mV (Umamaheshwari et al., 2004). Gliadin nanoparticles were prepared by desolvation method. In brief, gliadin was dissolved in ethanol/water phase (v/v: 7:3) then poured into a physiological saline (0.9% NaCl) containing

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pluronic as a stabilizer (0.5%) under constant magnetic stirring. Then, ethanol was eliminated by evaporation under reduced pressure and gliadin nanoparticles were purified by centrifugation (Arangoa et al., 2000).

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Fig. 9. SEM microphotograph of amoxicillin-loaded gliadin nanoparticles: gliadin 50 mg and amoxicillin 80 mg. Reproduced with permission from ref. (Umamaheshwari et al., 2004). Copyright 2016 AAPS PharmSciTech.

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3.2 Coacervation

Coacervation is based on the differential solubility of proteins in solvents depending on the

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solvent polarity, pH, ionic strength, or presence of electrolytes. The coacervation process reduces the solubility of the protein leading to phase separation. In coacervation technique, the liquid-

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liquid phase separation occurs when a salt is added to the protein solution giving rise to a polymer rich dense phase at the bottom and a transparent solution at the top, which results in the formation of desired nanoparticles (Elzoghby et al., 2015)(Fig. 10). Coacervation was used for

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preparation of various proteins including gelatin, silk, zein and legumin nanoparticles. Moreover, coacervation was implemented for preparation of BSA nanoparticles. Anhydrous

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ethyl alcohol was added to 150 ml BSA (5 mg/l in 10 mM Tris/HCl contained 0.02% sodium

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azide, pH 7.5) until the solution became turbid, then 150 µl of 25% glutaraldehyde was added as a crosslinker and the reaction was continued at room temperature (24°C) (Sailaja et al., 2011).

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Fig. 10. Preparation of protein nanoparticles by Coacervation. Noscapine-loaded HSA nanoparticles crosslinked with glutaraldehyde were prepared by Sebak et al. using a pH-coacervation method (Sebak et al., 2010). 100 mg of HSA was dissolved in 2 mL of water. Noscapine at a concentration ranging from 5–30 mg/mL was incubated with HSA solution for 4–8 h at room temperature. The pH was adjusted to 8 by the addition of 1 M NaOH. Nanoparticles were formed by adding 8 mL of ethanol drop wisely at a constant rate of 1 mL/min under constant magnetic stirring. After preparation, the particles were stabilized with crosslinking by adding 100 µL of 8% glutaraldehyde solution. The crosslinking was performed for at least 24 h under constant magnetic stirring at room temperature (Sebak et al., 2010). Results showed that size of the noscapine-loaded HSA nanoparticles was affected by pH of the solution. At pH 5–7, particles with large diameter and high size distribution were obtained. The particle size was highest at pH 7 and found to be approximately 2500 nm. At pH 8, the particles Page 11 / 39 Page 12 of 66

had optimal size of 175-200 nm with narrow size distribution. When viewed under SEM, the nanoparticles prepared at pH of 8–8.2 revealed uniform and spherical morphology (Sebak et al., 2010).

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The electrical behavior of the noscapine-loaded HSA nanoparticles was studied at different pH values. The surface charge was approximately null at pH 5, and it was increased by increasing pH to be maximal at pH 7. Above pH 7, the surface charge was slightly reduced and zeta

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potential of the prepared nanoparticles was -47 mV at pH 8 (Sebak et al., 2010).

The stability of the nanoparticles was also investigated. Noscapine-loaded HSA nanoparticles

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prepared at pH 8.2 were stored for 5 days in water at 4°C and at predefined times the samples were analyzed with regard to size. The particle size increased slightly to 190 nm, but the

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dispersion was found to be stable after 5 days (Sebak et al., 2010)

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Irache et al. (1995) studied the effect of pH, surfactant and ionic strength on the size of legumincontaining nanoparticles prepared by pH coacervation method followed by crosslinking using glutaraldehyde under the following experimental conditions (pH 6.8; ionic strength 0.103 M;

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surfactant content 0.33% w/v). Then, coacervates have been crosslinked by adding different

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volumes of 25% w/v aqueous glutaraldehyde solution under constant stirring at room temperature for 0-6 h (Irache et al., 1995).

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Results showed the decease of coacervates size by increasing pH, and the optimal pH was found to be in the neutral pH range (6.5-7). At this pH domain, the smallest size of the coacervtes was obtained with the same coacervates yield (Fig. 11A). It was also reported that, nanoparticle size and yield were decreased by increasing the ionic strength (Fig. 11B)(Irache et al., 1995). Fig. 11. Influence of experimental conditions on the size and yield of legumin nanoparticles: A) pH; B) Ionic strength; C) Surfactant concentration and D) Concentration of crosslinker glutaraldehyde. Reproduced with permission from ref. (Irache et al., 1995). Copyright 2016 International Journal of Pharmaceutics.

Moreover, the influence of surfactant content on the size and yield of legumin coacervates was also studied. Coacervates yield was increased by increasing the surfactant concentration, however, particles size was not significantly affected (Fig. 11C). Finally, the effect of crosslinking on the particles size was investigated. Results revealed that in order to stabilize the Page 12 / 39 Page 13 of 66

colloidal suspension, at least 0.05 mg glutaraldehyde/mg legumin should be used. Under this condition (0.05 mg glutaraldehyde/mg legumin), nanoparticles were found to be stable after 2 h of incubation. When a high amount of glutaraldehyde (0.1 & 0.3 mg/mg) was used in the formulation, instantaneous stable dispersions were obtained. However, such conditions did not

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prevent the aggregation of legumin coacervates (Fig. 11D)(Irache et al., 1995).

Casein nanoparticles were also prepared by simple coacervation in the presence of calcium

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(Penalva et al., 2015) and the influence of presence of either lysine or arginine on the physicochemical properties and stability of nanoparticles was also studied. Photon correlation

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spectroscopy and electrophoretic laser doppler anemometry were used to measure the particle size distribution and zeta potential of the formulated dispersions, respectively. The morphology

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and shape of nanoparticles were examined using scanning electron microscopy (Penalva et al., 2015).

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The results showed that when nanoparticles were prepared in the presence of a basic amino acid, the mean hydrodynamic diameter of the resulting particles decreased significantly. The polydispersity index reflecting the size distribution was reported to be slightly lower than that

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in Table 2.

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obtained for dispersions prepared in the absence of the amino acid. The results are summarized

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Table 2. Physico-chemical properties of casein nanoparticles. Reproduced with permission from ref. (Penalva et al., 2015). Copyright 2016 Food Hydrocolloids. 3.3 Emulsification

There are two main strategies in literature concerning emulsification by solvent evaporation method. The first method depends on the preparation of single emulsion (oil in water (o/w)), and the second depends on the preparation of double emulsions (water-in-oil-in-water (w/o/w)). These methods utilize high-speed homogenization or ultrasonication, followed by evaporation of the solvent during the continuous magnetic stirring at room temperature or under reduced pressure. Afterwards, the solidified nanoparticles can be collected by ultracentrifugation (Sailaja et al., 2011) (Fig. 12). Fig. 12. Preparation of protein nanoparticles by 1) single emulsion and 2) double emulsion.

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In an emulsion based method to prepare HSA nanoparticles (Mishra et al., 2006), olive oil was used as an oil phase and was slowly added to the aqueous protein solution under mechanical stirring, followed by ultrasonication step. Phosphatidyl choline was used as a stabilizer surfactant and was added in the oil phase. The crosslinker glutaraldehyde was added to the emulsion in

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order to obtain particles with size ranging from 100 to 800 nm. Protein concentration and the aqueous phase volume (w/o: v/v) ratio were influential factors on the hydrodynamic particle size.

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It was found that the increase in both w/o phase volume ratio and protein concentration led to the increase in the size of particles. (Mishra et al., 2006).

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Gupta et al. studied the effect of gelatin particles encapsulating the fluorescent molecule fluorescein isothiocyanate-dextran. In this study, crosslinked gelatin nanoparticles were prepared

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via water-in-oil (w/o) microemulsion system. Dispersions were prepared inside the aqueous cores of sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/n-hexane reverse micelles. The prepared

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particles have spherical morphology with average size 37 ± 0.84 nm as pointed out by transmission electron microscopy (TEM) (Fig. 13A). In phosphate buffer, pH 7.4, it was found that 80% of the encapsulated fluorescent molecules were released of from the prepared particles

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through 6 hours. More interestingly, it was found that even at high concentration (500 µg/ml),

(Gupta et al., 2004).

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gelatine particles endocytosed by the fibroblast cells had no cytotoxicity (i.e. 100% cell viability)

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Gelatin particles were also prepared via a single w/o emulsion technique (Bajpai and Choubey, 2006). The prepared particles were loaded with chloroquine phosphate (CP), anti-malarial drug. Particles were smooth and spherical in shape with an average diameter ranging from 100 to 300 nm according to scanning electron microscopy analysis (Fig. 13B). The zeta potential measurements showed an increase in positive potential of the particles surface after loading chloroquine phosphate molecules onto particles (Bajpai and Choubey, 2006). This could be attributed to the adsorption of chloroquine phosphate on the particles surface rather than real encapsulation. Fig. 13. Gelatin nanoparticles images by A) TEM (Gupta et al., 2004), and B) SEM (×30000). Reproduced with permission from ref. (Bajpai and Choubey, 2006). Copyright 2016 Journal of Materials Science: Materials in Medicine.

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In another study, Rössler et al. (1995) prepared collagen microparticles via emulsification technique. Their work resulted in the formation of spherical particles with a diameter ranging from 3 to 40 µm (Rössler et al., 1995).

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Moreover, they stated that the most efficient way to control the particle size is denaturation at a relatively low temperature, which should be higher than 39 °C, the approximate melting point of collagen. Furthermore, the small differences in the denaturation time at higher temperatures

cr

resulted in high differences in particle size. The collagen particles were completely degraded

minutes of heating at 100 °C (Rössler et al., 1995).

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after 30 minutes of incubation in pepsin media, however they were completely stable after 30

In a recent study, casein particles were prepared in order to carry the hydrophobic anti-cancer

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drug, flutamide. Particles were fabricated via oil-in-water emulsification then stabilized by ionic crosslinking with the polyanionic crosslinker sodium tripolyphosphate. The prepared

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nanoparticles had a spherical shape with a size below 100 nm and possessed positive zeta potential (+7.54 to +17.3 mV). Flutamide was dispersed inside the nanoparticle protein matrix. Flutamide-loaded nanoparticles showed a longer circulation time, a delayed blood clearance of

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flutamide, and an extended half-life of flutamid from 0.88 hours to 14.64 hours after intravenous

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administration. Moreover, it was shown that by varying sodium tripolyphosphate concentration (i.e. crosslinking density), the biodegradability of casein in trypsin solution could be controlled

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(Elzoghby et al., 2013). 3.4 Nanoprecipitation

Nanoprecipitation or solvent displacement method requires two miscible solvents, where both protein and drug must be soluble in one solvent and insoluble in the other one. Nanoprecipitation occurs by a rapid desolvation of the protein when the protein solution is added to the nonsolvent. Indeed, as soon as the protein containing solvent diffuses into the dispersing medium, the polymer precipitates involving immediate drug entrapment (Fig. 14) (Fessi et al., 1989). Fig. 14. Preparation of protein nanoparticles by nanoprecipitation. By using another strategy, protein solution in 70–80% aqueous alcohol containing the drug could be added to water containing a stabilizer (e.g., sodium caseinate, Tween 80, or poloxamer) under vigorous stirring. When the alcohol concentration decreases below a critical level required for Page 15 / 39 Page 16 of 66

dissolving protein, protein becomes insoluble and precipitates to form nanoparticles. Thus, nanoprecipitation is suitable for preparation of drug-loaded nanoparticles from hydrophobic proteins (e.g. zein, gliadin, etc...). In addition, this technique is rapid and easy that enables the fabrication of small size particles with a unimodal distribution. This process is advantageous

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compared to conventional emulsification methods that require strong shear forces to reduce droplet sizes to submicrometers (Elzoghby et al., 2015).

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In this method, the physico-chemical properties of the resulted nanoparticles are affected by the criteria of addition of the organic phase to the aqueous phase such as injection rate, agitation

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speed, the method of addition of organic phase to the aqueous one and the organic/aqueous phase volume ratio. In addition, nanoparticles characteristics are determined by the nature and

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concentration of their components. Although, the use of surfactant is not required to ensure the formation of nanoparticles via nanoprecipitation process, but the particle size is affected by the

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surfactant nature and concentration (Rao and Geckeler, 2011).

In 2012, Lee et al. prepared gelatin nanoparticles using nanoprecipitation method. Water and ethanol were used as solvent non-solvent, respectively. Particles shape and size were discussed

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for both crosslinked and non-crosslinked particles. The crosslinked nanoparticles have a

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unimodal size of 251 nm, low polydispersity index of 0.096 and uniformly round shape. However, the non-crosslinked nanoparticles showed an irregular shaped morphology due to the

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particles aggregation (Fig. 15) (Lee et al., 2012). Fig. 15. SEM images of gelatin nanoparticles prepared by nanoprecipitation; a) crosslinked and b) non-crosslinked. Reproduced with permission from ref. (Lee et al., 2012). Copyright 2016 Bioprocess and Biosystems Engineering. Zein nanoparticles were also prepared by pH controlled nanoprecipitation method with 6,7dihydroxycoumarin as a model of hydrophobic compounds (Podaralla and Perumal, 2010). Both blank and loaded zein nanoparticles shared a similarity in particle size distribution. However, the mean particle size of loaded nanoparticles (365 ± 63 nm) was smaller than the size of blank nanoparticles (460 ± 54 nm). As for zeta potential, blank nanoparticles (-16 mV) were more negatively charged than the particles containing loaded active molecule (-11.34 mV). Finally, the yield of nanoparticles was greater than 95% and the encapsulation efficiency of Coumarin was found to be around 62% (Podaralla and Perumal, 2010).

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3.5 Nano Spray Drying Spray drying, as shown by its name, is a technique used to dry liquids into particles under a continuous process. It is very suitable when treating heat sensitive molecules such as protein preventing them from degradation. In this technique, a simple tuning of the method parameters

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can easily control particles characteristics such as particle size, bulk density, and flow properties. For these reasons, spray drying is widely and commercially applied for the production of protein

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drugs. Usually, the nanoparticles formulation introduced to spray drying consists of an aqueous suspension containing a drying enhancer in the form of a soluble compound. The process of

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spray drying can be divided into four steps: (1) atomization of the nanoparticles suspension into a spray, (2) spray-air contact, (3) drying of the spray and (4) separation of the dried end product

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from the drying gas (Lee et al., 2011)(Vauthier and Bouchemal, 2009).

Nano spray drying approach was used effectively to prepare bovine serum albumin (BSA)

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nanoparticles of 460 nm in size. Moreover, it was also proved the successful use of this approach for preparation of proteins for pulmonary, nasal, and controlled oral delivery (Lee et al., 2011).

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Fig.16. The Nano spray dryer B-90.

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Nowadays, the progress in nanotechnology of spray drying led to the develpoment of a novel technology at the spray head, heating system and particle collector of the Nano Spray Dryer B-90

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(Fig. 16). The new nano spray dryer B-90 uses a vibrating mesh technology to generate fine droplets. In brief, the spray head contains piezoelectric crystals responsible for the vibration motion. This head is incorporated with a spray cap containing a thin perforated membrane with precise size holes. When an ultrasonic frequency is introduced, the crystals will vibrate and generate a piezoelectric effect, which in turn leads to the vibration of the mesh upwards and downwards in synchronization with the current generated. This consequently leads to injecting of fine and precisely sized droplets from the holes and generating the aerosols (Fig. 17). Fig. 17. The functional principle of mesh vibration occurring at the piezoelectric driven spray head of the Nano Spray Dryer B-90. Reproduced with permission from ref. (Lee et al., 2011). Copyright 2016 International Journal of Pharmaceutics. The feasibility of nano spray dryer B-90 and the effect of experimental conditions on the production of BSA nanoparticles were investigated (Lee et al., 2011). Results showed that the factors that predominantly affect the particle size and morphology of the particles were the spray Page 17 / 39 Page 18 of 66

mesh size and the surfactant concentration, respectively (Fig. 18). Other factors like the flow rate of the drying gas and the inlet temperatures did not influence particles size and morphology. Optimized smooth spherical nanoparticles (median size: 460 ± 10 nm, yield: 72 ± 4%) were produced using the 4 m spray mesh at BSA concentration of 0.1% (w/v), surfactant concentration

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of 0.05% (w/v), drying gas flow rate of 150 L/min and inlet temperature of 120◦C (Lee et al., 2011).

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3.6 Nanoparticle albumin bound technology

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Fig. 18. FE-SEM images of BSA particles (A) run 1 (0%, w/v Tween 80) and (B) run 11 (0.05%, w/v Tween 80), showing the effect of surfactant (Tween 80) on the shape of particles. (C) run 2 (4.0 m spray mesh), (D) run 5 (5.5 m spray mesh) and (E) run 8 (7.0 m spray mesh), showing the effect of spray mesh size on the size of particles. Reproduced with permission from ref. (Lee et al., 2011). Copyright 2016 International Journal of Pharmaceutics.

Nanoparticle albumin bound technology (NAB) is usually applied for the encapsulation of poorly

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water-soluble drugs; it is considered as a safe and suitable technique when it comes to intravenously administrated nanoparticles. In this method, the selected drug and protein (e.g. albumin) have to be mixed in an aqueous solvent, and then the drug-albumin nanoparticles were

d

formed after the solution passes through a jet under high pressure. The size of obtained

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nanoparticles was in the range 100–200 nm (Desai, 2016). The NAB technology was used to encapsulate drugs such as paclitaxel (130 nm) and curcumin

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(130-150 nm). HSA (1000 mg) was dissolved in 50 ml of water saturated with chloroform, and curcumin (150 mg) was dissolved in 3 ml chloroform saturated with water. These two solutions were mixed and homogenized at 20,000 psi for nine cycles. Then, chloroform was removed from the colloidal suspension via evaporation under reduced pressure at 25°C for 15 min. Afterward, the formed dispersion was lyophilized. The obtained nanoparticles showed markedly high water solubility than free drug (Elzoghby et al., 2015)(Kim et al., 2011)(Fig. 19). Fig. 19. Preparation of albumin bound curcumin nanoparticles using NAB technology The use of NAB technology was successfully extended for preparation of protein nanparticles other than albumin. Lactoferrin nanoparticles were prepared to encapsulate gambogic acid using this NAB-based method. In brief, gambogic acid (22.2 mg) was dissolved in 1.0 mL methylene dichloride. Another solution of bovine lactoferrin (5% w/v) was prepared. The first solution was mixed with 4 ml of the second one, and then followed by homogenization for 5 minutes at low Page 18 / 39 Page 19 of 66

agitation speed in order to obtain a crude emulsion. This emulsion was then transferred into 40 kHz sonicator cell under sonication power of 400 W for 2 min in ice-water bath. Next, methylene dichloride was rapidly removed at 40°C by rotary evaporation at reduced pressure for 30 min. The filtration of the dispersion was done through 0.45 mm pore sized membranes. Finally, the

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dispersion was lyophilized for 48 hours under cryoprotectant agent-free conditions (Zhang et al., 2013).

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NAB technology is known by its association with disulfide bounding via homogenization. This feature could be an advantage over other traditional solvent-based methods where it avoids many

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potential drawbacks (Elzoghby et al., 2015). In addition, NAB technology leads to different final states of albumin than the other methods (Kim et al., 2011).

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Curcumin-loaded HSA nanoparticles were prepared using NAB technology (Kim et al., 2011). It was observed that the increase of mean particle size and curcumin loading were induced by the

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increase of curcumin concentration. While, the decrease of particle size and curcumin loading was caused by the decrease of the organic/water volume ratio. In that research, the conditions used were: HAS concentration 2% (w/w), curcumin concentration 50 mg/ml, and organic

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phase/water to water ratio 1:19. These conditions led to the formation of particles with a mean

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size less than 150 nm with a satisfactory curcumin loading. The resulting nanoparticles had a mean hydrodynamic particle size of 139.6± 2.9 nm as determined by DLS technique, zeta

Ac ce p

potential was -23.4 ± 1.2 mV, and the curcumin loading was 7.2 ± 2.5%. In addition, the prepared particles showed spherical morphology with particle size 130–150 nm, as examined by field emission scanning electron microscope (FE-SEM) imaging (Fig. 20) (Kim et al., 2011). Fig. 20. SEM imagery of curcumin loaded HSA nanoparticles (original magnification: × 30,000). Reproduced with permission from ref. (Kim et al., 2011). Copyright 2016 International Journal of Pharmaceutics. 3.7 Self Assembly Protein micelles can be created spontaneously when the individual protein chains are dissolved in an aqueous solution beyond the threshold concentration (Critical micelle concentration or CMC) and critical solution temperature (CMT) to form nano-sized aggregates (Batrakova et al., 2006).

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Micellar structures could be hardened and stabilized by crosslinking between the polymer chains. The obtained crosslinked micelles of nano-sized scale are stable upon dilution and resistant to variations in experimental conditions (e.g. pH, ionic strength, solvents, etc.) and shear forces

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(Batrakova et al., 2006). Hydrophilic proteins e.g. albumin, can be modified hydrophobically to obtain amphiphilicity. The hydrophobically modified protein can then be assembled in micellar shaped nanospheres

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when added to an aqueous solution. In addition, the hydrophobic core can act as a container of

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poorly soluble active molecules.

For example, paclitaxel was successfully entrapped (loaded) by octyl-modified BSA protein particles (more lipophilic than normal BSA). On the other hand, self-assembled micelles

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synthesized by conjugating hydrophilic methoxypoly(ethylene glycol) to the hydrophobic plant protein, zein, had an enhancing effect on the solubility and stability of curcumin and augmented

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its cellular uptake, as compared with the free curcumin in drug resistant ovarian cancer cells (Elzoghby et al., 2015).

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In nature, casein took the form of sub micellar structures that encapsulate calcium phosphate (a very essential molecule for the growth of infants). Casein micelles are formed with different

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casein variants. The core is formed by αs1-, αs2-, and β-caseins, and the outer layer is formed by

Ac ce p

κ-casein, which generates a strong steric repulsion that helps in stabilizing the structure (Fig. 21) (Davidov-Pardo et al., 2015)

Fig. 21. Schematic representation of a casein micelle. Casein micelles could be produced in vitro by self-assembly of the casein units around a hydrophobic compound. To encapsulate hydrophobic compounds, it is important to create a proper pH and ionic strength conditions. For this reason, different salts can be used such as potassium phosphate, calcium chloride, and potassium citrate (Davidov-Pardo et al., 2015). Pan et al. (2014) used disintegration/reassembly method as an alternative to produce casein micelles to encapsulate curcumin. This method was explained as follow: casein micelles were disintegrated by introducing it in aqueous alkaline conditions at pH 12. The pH was then shifted to pH 7 where the casein units are self-assembled leading to active molecule encapsulation (Fig.

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22). This method significantly increases stability and bioavailability of the encapsulated hydrophobic curcumin (Pan et al., 2014).

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Fig. 22. The proposed principle of the pH driven encapsulation of curcumin in self-assembled casein nanoparticles. Reproduced with permission from ref. (Pan et al., 2014). Copyright 2016 Soft Matter. In another study, silk sericin protein, from non-mulberry Antheraeamylitta tropical tasar silk

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cocoons coupled with poloxamer, was successfully self-assembled into micellar nanostructures (SS-P) and have the ability to carry both the hydrophilic inulin and the hydrophobic paclitaxel

us

(Mandal and Kundu, 2009).

Poloxamer is known with its ability to micellar formation. By adding it to the protein, it can help

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in decreasing the size of the resulted micelles. Poloxamer has an amphiphilic property, which allows it to form micelles above the CMC. This could help in efficient drug encapsulation and extend the circulation time in the body. The size of nanoparticles ranged between 100 and 110

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nm and the low polydispersity index showed that the particles were homogeneously dispersed in solvent without aggregation (Mandal and Kundu, 2009).

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Moreover, it was found that the encapsulation of inulin inside the hydrophilic core of the particle

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led to an increase in size by 7 nm (the average diameter size is 123 ± 5 nm and the polydispersity index is 0.27). After 10 days of incubation, the particles were stable with an increase of 11 nm in

Ac ce p

size. These results suggested that the drug-encapsulated particles could be used effectively as carrier molecules for targeted drug delivery applications. In addition, hydrophobic paclitaxel was encapsulated efficiently inside the hydrophobic core of the micelles, which in turn increased the size by 15 nm. This study revealed the importance of silk protein as a drug carrier (Mandal and Kundu, 2009).

Other self-assembled proteins could be applicable in drug delivery. Ferritin, naturally existing in almost all living organisms, is a good candidate. Its assembly and disassembly are pH dependent. Nanoparticles resulted from self-assembly of ferritin sub-units can maintain an intact structure at pH 9 and temperature up to 85 °C. However, at pH value around 2 to 3, ferritin nanoparticles can be fragmented (disassembled) into sub-units. Moreover, several bio active molecules have been conjugated onto the surface of ferritin nanoparticles for drug delivery purposes such as doxorubicin, 5-fluorouracil, and gadoteridol (Lee et al., 2015). Page 21 / 39 Page 22 of 66

In addition, other types of proteins, called small heat shock proteins (sHsp), could be used as self-assembled carrier nanoparticles. The sHsp derived from Methanococcus Jannaschii was experienced for this reason. It has 24 sub-units and differ from ferritin by the presence of large 3 nm pores which allow ease diffusion of small molecules inside and outside the pores (Schoonen

ip t

and van Hest, 2014). It was used as efficient carrier for different anti-cancer agents such as doxorubicin (Lee et al., 2015).

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3.8 Electrospraying

Protein-based particles (fibers or capsules) can be produced using electrospinning methods,

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where electric field is applied to pull a protein solution through a small nozzle. In brief, protein and drug are dispersed in a common suitable solvent and placed within a capillary tube. Then,

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protein solution is pulled out of the capillary tube by applying a high voltage electric field, forming a thin jet. On the collection plate, the formation of either fibers or capsules depends on

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the strength of the electric field and the nature of the protein solution (Fig. 23). The evaporation of solvent takes place when the electrosprayed jet travels from the capillary to the collector, so the distance must be long enough in order to completely evaporate the solvent, otherwise, protein

d

particles will fuse together (Davidov-Pardo et al., 2015).

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Fig. 23. Schematic representation of electrospraying technique.

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Giner et al. (2010) used electrospraying technique in order to prepare docosahexaenoic acid (DHA)-loaded zein nanoparticles (Torres-Giner et al., 2010). Optical microscope was used to show bright field and fluorescence images of the ultrathin matrices of blank zein and zein-DHA nanoparticles. From the obtained results, it was found that the encapsulation of DHA inside zein nanoparticles did not affect size and round shaped morphology. The size of encapsulated nanoparticles was either the same as the blank or in the shortest dimension was approximately 530 nm for pure zein and 490 nm for DHA-zein (Torres-Giner et al., 2010). Moreover, Rubia et al. (2012) prepared whey protein (WP) nanoparticles for encapsulating the antioxidant β-carotene using electrospinning method. Active molecule was encapsulated successfully with high encapsulation efficiency (90%). The obtained dispersions were relatively stable and electrospraying technique demonstrated its feasibility in the field of drug delivery (López-Rubio and Lagaron, 2012).

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However, a major drawback of electrospraying technique is that many proteins cannot be used alone to create electrospun fibers due to their complex macromolecular and 3D structures in combination with their strong inter- and intramolecular forces. To overcome this problem, surfactants, plasticizers, or reducing agents can be incorporated in the protein solutions

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(Elzoghby et al., 2015).

Electrospraying technique could have an advantage over other conventional methods. The

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nanoparticles created by this technique do not require any template or surfactant. Furthermore,

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this method leads to high drug loading efficiency and self-dispersion (Elzoghby et al., 2015). 3.9 Salting Out

The concept of salting out is based on the separation of water-miscible solvent from aqueous

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solution via salting out effect. Briefly, protein and drug must be dissolved in the same solvent (i.e. acetone), which is subsequently emulsified in an aqueous gel containing the salting out

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agent (electrolytes such as magnesium chloride, calcium chloride, magnesium acetate or non electrolytes such as sucrose) and a colloidal stabilizer such as polyvinylpyrrolidone or hydroxyehtylcellulose. Later, a dilution step with water or aqueous solution to enhance the

d

diffusion of acetone into the aqueous phase is necessary, which leads to the formation of

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nanospheres. Finally, the elimination of solvent and salting out agent are done via cross flow

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filtration (Fig. 24) (Nagavarma et al., 2012). Fig. 24. Preparation of nanoparticles by salting out technique. Salting out technique has certain advantages such as it minimizes stress to protein carriers. The process does not include a temperature modification, which is suitable for heat sensitive substances. On the other hand, the disadvantages of this technique is related to its restriction to lipophilic drugs as well as the costly nanoparticles washing step (Nagavarma et al., 2012). Salting out was used by Lammel et al. (2010) in order to prepare silk nanoparticles from an aqueous silk fibroin solution (5 mg/mL) by adding potassium phosphate solution (1.25 M, pH 8) (Lammel et al., 2010). Briefly, the silk solution was mixed with potassium phosphate in a volumetric ratio of 1:5. Particles were stored for 2 hours at 4°C then centrifuged at 2000 g for 15 min. Subsequently, particles were redispersed in purified water and washed three times (Lammel et al., 2010). Page 23 / 39 Page 24 of 66

In this study, the influence of different parameters (ionic strength, pH and protein concentration) on particle formation was investigated. Results showed that the increase of ionic strength of potassium phosphate buffer and changes in pH had drastic effect on salting out efficiency (Fig. 25A/B). Moreover, particle size could be controlled by modifying the concentration of the

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protein (Fig. 25C) as well as the value of zeta potential could be controlled by modifying the pH of the preparation (Fig. 25D) (Lammel et al., 2010).

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Fig. 25. Effect of experimental conditions on the characteristics of silk fibroin nanoparticles prepared by salting out technique. A) Salting out efficiency as a function of ionic strength of potassium phosphate; B) Salting out efficiency as a function of pH; C) Average size of silk particles as a function of protein concentration; D) Zeta potential of particles produced by salting out with 1.25 M potassium phosphate at different pH conditions. Reproduced with permission from ref. (Lammel et al., 2010). Copyright 2016 Biomaterials.

3.10 Crosslinking

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In order to achieve a sustained drug delivery and increase the stability of the protein, crosslinking methods are followed. In this regard, there are different types of crosslinkers (e.g. chemical,

d

ionic, thermal and enzymatic) that are usually used in protein-based nanoparticles preparation

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(Elzoghby et al., 2015).

HSA nanoparticles were crosslinked using 8% glutaraldehyde aqueous solution by Langer et al.

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(2003). Their goal was to study the effect of crosslinking on the size and isoelectric point (pI) of nanoparticles. For this reason, they prepared nanoparticles by using different glutaraldehyde concentrations. The concentrations were chosen according to the calculation of the amount of crosslinkers necessary for quantitative crosslinking of the 59 ε-amino groups of lysine in the HSA molecules in the particle matrix. From these calculations, the concentrations chosen were 40, 100 and 200% of the calculated amount (Langer et al., 2003). The obtained results showed that glutaraldehyde concentration has no effect on the particle size. However, pI of the nanoparticles decreased by increasing glutaraldehyde concentration (Fig. 26). The decrease in pI could be induced by the chemical grafting reaction of the lysine side chains of the protein (Langer et al., 2003). Fig. 26. Influence of the crosslinking process, at different glutaraldehyde concentrations, on the diameter (□) and pI (O) of the resulting HSA nanoparticles. Reproduced with permission from ref. (Langer et al., 2003). Copyright 2016 International Journal of Pharmaceutics. Page 24 / 39 Page 25 of 66

Crosslinkers are effective and can affect the particle stability in principle. However, crosslinkers cannot be removed properly and can induce toxicity to biological systems. In addition, the washing step, which requires dialysis process, is time consuming (Varca et al., 2016). For this reason, Varcaet al. (2016) investigated another method of crosslinking based on

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irradiation. BSA nanoparticles were exposed to γ-irradiation in phosphate buffer (pH 7.2) in the absence and/or presence of ethanol and methanol at 30% and 40% (v/v), respectively. Results

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showed that irradiation has a significant effect on BSA particle size, so by controlling the irradiation dose, crosslinking density and subsequently the particle size could be controlled. It

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was found a correlation between particle size and crosslinking density. This gives the possibility to produce nanoparticles in a one-step procedure and may allow simultaneous crosslinking and

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sterilization (Varca et al., 2016).

At the end part of this review, Tables 3&4 resume the data cited in this review. Table 3 stated

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the characteristics of each protein mentioned in this state of the art, the preparation methods used to obtain nanoparticles from each protein, and the bioactive molecules that carried by these nanoparticles. While Table 4 resumes the colloidal properties (Size, polydispersity index, zeta

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potential, and morphology) of each protein as a function of its preparation method. These data

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4. Drug Loading

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will be analyzed later in this review in the discussion part.

The drug loading of the nanoparticles is generally defined as the amount of drug bounded per mass of polymer. It is usually expressed in moles or mg of drug per mg polymer, and it could also be given on a percentage basis based on the polymer (Jahanshahi and Babaei, 2008). A high drug loading capacity is a criterion of a successful nanoparticle system; such system has a reduced quantity of matrix material for administration. The loading of the drug in a nanoparticle system can be done by two methods, either by incorporating the drug during nanoparticles preparation (incorporation method), or after the preparation of nanoparticles by mixing the carrier with a concentrated drug solution (adsorption/absorption technique) (Mohanraj and Chen, 2006a). The solid state drug solubility in the polymer matrix reflected by the polymer composition, the molecular weight, the drug polymer interaction, and the presence of end functional groups are

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major factors in drug loading and entrapment efficiency (Mohanraj and Chen, 2006b). Moreover, greater loading efficiency were shown when the protein is loaded at or close to its isoelectric point, and has minimum solubility and maximum adsorption (Jahanshahi and Babaei, 2008).

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In order to measure the drug binding to the protein nanoparticles, the total amount of drug used (W) must be well known after centrifugation of a part of the particles suspension, the nonentrapped drugs (w) must be present in the clear supernatant. The amount of the free drug can be

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calculated by UV spectrophotometry, fluorescence spectrophotometry or high performance liquid chromatography (HPLC), by plotting a standard calibration curve of drug concentration

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versus absorbance. The amount of free drug present in the supernatant (w) must then be subtracted from the total amount of drug (W), and (W-w) will become the amount of drug

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entrapped within the particles. Then drug entrapment percentage can be determined as follow: (Jahanshahi and Babaei, 2008).

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The encapsulation efficiency refers to the ratio of the amount of drug encapsulated/absorbed to the total amount of drug used, with regard to the final drug delivery system of the dispersion of

d

nanoparticles (Jahanshahi and Babaei, 2008).

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Kim et al.(2011) studied the loading efficiency on curcumin-loaded HSA nanoparticles prepared by NAB technology (Kim et al., 2011). Loading efficiency was determined as follows: 1 mg of

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loaded nanoparticles was dissolved in 10 ml of ethyl acetate/propanol (9:1, v/v) and sonicated for 30 min to guarantee the complete extraction of curcumin from nanoparticles. Curcumin concentration in the solution was determined by HPLC, and curcumin loading efficiency was defined by the equation mentioned above. It was found that the curcumin loading increased in parallel with curcumin concentration, while it decreased by decreasing the organic solvent to water ratio. The optimum curcumin loading (7.2 ± 2.5%) was obtained when HSA concentration of 2% (w/w), a curcumin concentration of 50 mg/ml, and an organic phase to water ratio of 1:19 were used (Kim et al., 2011). 5. Drug Release Drug release and protein biodegradation are very important features for a sophisticated nanoparticle system. Therefore, drug release is affected by the solubility of the drug, drug diffusion through the nanoparticle matrix, nanoparticle degradation, and the processes of erosion Page 26 / 39 Page 27 of 66

and diffusion. In other words, the release mechanism is governed by solubility, diffusion and nanoparticle biodegradation (Mohanraj and Chen, 2006a). In the case of nanospheres, the drug is uniformly distributed in the nanoparticle matrix, and the

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release of the drug depends on its diffusion and on the matrix degradation. Thus, if the diffusion rate is faster than the degradation of the matrix, the release will depend mainly on diffusion, otherwise, it depends on degradation of encapsulating matrix. The release of drug from protein-

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based nanoparticles (PBNs) can also depend on many circumstances, like the protein erosion or degradation, diffusion of the drug through pores, release from the surface of polymer, and pulsed

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delivery initiated by applied oscillating magnetic or sonic field (Couvreur and Puisieux, 1993). The release profile has been studied in vitro by different methods such as artificial membranes,

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dialysis bag, agitation followed by centrifugation, and centrifugal ultrafiltration. In most cases, the agitation followed by centrifugation was used. However, due to the time consuming nature

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and technical difficulties encountered in the separation of nanoparticles from release media, the dialysis technique was generally preferred (Mohanraj and Chen, 2006a).

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The release of hydrophilic drugs from gelatin nanoparticles was governed by zero order kinetics,

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however, hydrophobic drugs were released by pseudo zero order kinetics (Vandervoort and Ludwig, 2004). Duclairoir et al. studied the release of drugs of varying polarity from gliadin

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nanoparticles (Duclairoir et al., 2003). It was found that hydrophobic drugs have a slower release as compared to hydrophilic drugs. This was attributed to the high affinity between hydrophobic drugs and hydrophobic gliadin protein. In addition, hydrophilic drugs showed a burst release followed by slower drug diffusion from the nanoparticles matrix. 6. Applications and clinical usage

Protein-based nanoparticles (PBNs), recently reported, are of great interest due to their various advantages. In fact, they prove their competence in the therapeutic and clinical fields. Several formulations have been developed and suggested as potential future therapeutic products (Babapoor et al., 2011; Pimentel et al., 2009; Wahome et al., 2012). In addition, some PBNs have been officially approved by the US food and drug administration such as Abraxane ®(Food and Drug Administration, 2015).

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Abraxane® is based on formulation of paclitaxel-loaded albumin nanoparticles prepared by nanoparticle albumin bound (NAB) technology, also known as NAB-paclitaxel. Paclitaxel is an antineoplastic agent with anticancer activity against different types of cancer such as breast, ovarian, and non-small cell lung cancer (Montero et al., 2005). Practically, different paclitaxel

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formulations already exist for cancer treatment such as Cremophor® and Taxol®. However, these formulations can contribute in serious toxicities. Therefore, the development of a safer (non-

successfully utilized as a paclitaxel vehicle.

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toxic) alternative was necessary (Gradishar, 2006). For this reason, human albumin was In fact, the clinical trials showed that NAB-

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paclitaxel has a low toxicity, high antitumor activity and leads to increasing intra-tumor paclitaxel concentrations. These results suggested that albumin was not only more safe

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alternative but also more efficient. This suggestion is based on the hypothesis that paclitaxel delivery using human serum albumin (HAS) induces endogenous albumin-mediated pathways,

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which enhances the drug delivery into the tumor (Gradishar, 2006). Moreover, the presence of functional groups on the surface of protein-based nanoparticles (e.g. carboxylic and amino groups) facilitates the surface modification of these particles, which make

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them suitable for tumor targeting strategies. Lee et al. (2006) developed a passive targeting

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system composed of gelatin-doxorubicin nanoparticles coupled with poly(ethyl glycol) (PEG). The PEGylation of particles was found to prolong the circulation half-life, reduce the

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immunogenicity, lower the cytotoxicity effect and significantly inhibited tumor growth up to 82% compared to non PEG containing particles (Lee et al., 2006). On the other hand, protein-based nanoparticles (PBNs) surface can be modified by addition of targeting ligands such as peptides, antibodies, vitamins, hormones, enzymes, etc… These surface modifications allow for specific targeting and accumulation of the particles on desired tumor. Different successful attempts of active targeting of cancer cells were done; some of them are summarized in Table 3

Table 3. Protein nanoparticles designed for active targeting of cancer cells. HSA nanoparticles were also used as a drug carrier across the blood-brain barrier (BBB). They were used to carry oximes such as HI 6 dichloride monohydrate and HI 6 dimethane sulfonate into a blood-brain barrier in vitro model as a counteract against organophosphorus compounds. The in vitro study showed that both HI 6 salt formulations were well transported over the Page 28 / 39 Page 29 of 66

endothelial cell model. Both formulations showed significant advantage over free oxime and showed two times higher reactivation of acetylcholinesterase than that was inhibited by the organophosphorus compounds.

Therefore, this

formulation could

lead to

a better

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organophosphorus-poisoning antidote therapy (Dadparvar et al., 2011). 7. Discussion

Table 4 summarizes the characteristics of each protein mentioned in this review, the preparation

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methods used to obtain nanoparticles from each protein, and the bioactive molecules that carried

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by these nanoparticles. From these information, we can conclude that each protein has a tendency to encapsulate either hydrophobic or hydrophilic molecules. Gelatin, silk, gliadin, and legumin have higher encapsulation efficiency for hydrophilic drugs. While collagen, casein, and

an

zein proteins have higher encapsulation efficiency for hydrophobic drugs. Albumin, however, has the ability to bioconjugate with hydrophilic drugs and interact with highly hydrophobic drugs

M

(Table 4). Accordingly, we can conclude that the choice of the protein for the nanoparticles preparation depends on the drug water solubility.

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Table 4. Animal and plant proteins: characteristics, nanoparticles preparation techniques, and the encapsulated active molecules

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Moreover, each protein has a certain set of characteristics that increase its specificity to be a better carrier for a certain drug. Albumin, for example, is the most abundant plasma protein,

Ac ce p

which makes it nontoxic, biodegradable, and nonimmunogenic. It also has a binding ability to a large spectrum of drugs and it is extremely robust to various conditions. Gelatin and collagen possess many carboxyl groups with possible crosslinking function (Table 4). These characteristics are highly important when choosing the method of preparation of nanoparticles. From the literature, we can observe that nanoparticles derived from a certain protein have their proper preparation methods that are widely used. The reported methods are illustrated in Fig. 27. Fig. 27. The commonly used preparation methods for various types of proteins: Red circles: Animal proteins, Green circles: Plant proteins Table 5 resumes the colloidal properties (Size, polydispersity index, zeta potential, and morphology) for each protein as a function of its preparation method. This table obviously indicates that the preparation method is not the only criterion that affects the resulting colloidal properties of the nanoparticles. Despite that, in some cases, different studies on the same protein Page 29 / 39 Page 30 of 66

using the same nanoparticles preparation method can result in similar colloidal properties. For example, albumin nanoparticles prepared by desolvation technique, in two different studies, have approximately the same size (150 and 157 nm), same polydispersity index (0.1 for both), same morphology (spherical), and approximately the same zeta potential (-31.9 and -30) (Table 5).

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However, in other cases, colloidal properties were completely different between various studies despite the use of the same protein and the same preparation method. This differentiation could

cr

be the result of the difference in the experimental conditions within the preparation method.

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Table 5. Colloidal properties of PBNs for each protein and its preparation methods. This claim is supported by the work of Sadeghi et al. (2014) (Sadeghi et al., 2014). They studied the effect of different desolvating agents on BSA nanoparticles properties prepared by

an

desolvation technique using ethanol, and acetone, and a mixture of them (ethanol:acetone 70:30 and 50:50). Spherical nanoparticles were obtained by using ethanol and a mixture of ethanol and

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acetone as desolvating agents. While spherical and rod-shaped nanoparticles were obtained by using acetone only. The polydispersity index values of BSA NPs in this study were 0.045 ± 0.007, 0.065± 0.013, 0.091 ± 0.012, and 0.120 ± 0.016 for ethanol (100), Et:Ac (70:30), Et:Ac

te

d

(50:50), and acetone (100), respectively (Fig. 28) (Sadeghi et al., 2014)

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Fig. 28. The effect of different desolvating agents and solvent ratios on particle size and polydispersity of BSA nanoparticles. Particle size (solid line), and polydispersity index (dashed line). Reproduced with perimission from ref. (Sadeghi et al., 2014). Copyright 2016 Journal of Nanoparticle Research. This example justifies how the variation in the experimental conditions can affect the resulting colloidal properties of nanoparticles. Therefore, the choice of the preparation method must be followed by testing all variations in the experimental conditions to obtain the perfect nanoparticles for a certain study. This step is important because the colloidal properties can affect the encapsulation efficiency and the administration of the nanoparticles. Thus, size, polydispersity index, zeta potential and morphology of the prepared nanoparticles must be decided depending on the type of drug to be encapsulated and the route of administration for these nanoparticles. Form this analysis we can conclude that in order to prepare a drug carrier, one must follow a specific approach. This methodology is illustrated in Fig. 29 and detailed in this paragraph.

Page 30 / 39 Page 31 of 66

First, the researcher must know the drug that he wants to encapsulate. Then, the identification of physico-chemical property of the drug must be done. The solubility profile and stability spectrum of the drug allow the researcher to go to the next step which is the choice of the administration

ip t

route (Fig. 29). Fig. 29. Methodology of preparation of PBNs.

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When selecting a route for nanoparticles administration, it is important to consider the stability of the drug in the biological fluids. In addition, anatomical and physiological characteristics of

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the administration route and the target site are greatly important. Then, the choice of administration route is very crucial in the preparation, and it does not depend on the drug only,

an

but also on the protein used as a carrier (Fig. 29).

The choice of protein is based on the properties of drug, and on the future of the nanoparticles to be prepared. The properties of the chosen protein e.g. molecular weight, surface functionality,

M

hydrophobicity, etc., can affect particle size, drug loading and loading efficiency, and dissolution or release profile of the drug to the surrounding environment of nanoparticles (Fig. 29),

d

Finally comes the choice of the proper method of preparation. It mainly depends on the

te

properties of drug and protein. Moreover, it depends on the stability of both in the preparation conditions of a certain method. Virtually, different methods could be appropriate for one system,

Ac ce p

however, these methods must be tested to identify the most suitable one for the desired system. 8. Conclusion

Nanoparticles are considered a breakthrough in the field of pharmaceuticals. They have the ability to target a specific site in vivo and protect the encapsulated active molecules from biodegradation and undesirable metabolism. Protein nanoparticles, however, have unique properties when compared to other nanoparticles since they are derived from natural sources that are widely present in nature, easy to manipulate, and most importantly are often nontoxic and do not leave undesirable biodegradation products. Protein nanoparticles can be prepared through a variety of methods. Each method has its advantages, disadvantages and steps that can be controlled to tune the nanoparticles end product. Therefore, the choice of the preparation method is crucial, which depends on the characteristics of the protein and its final use for drug encapsulation, delivery and therapy. Page 31 / 39 Page 32 of 66

Moreover, in the same preparation method, the alteration of experimental conditions can also lead to different properties. The control of protein concentration, solvents, temperature, pH, and crosslinker, etc., allow the researcher to tune the properties of the final nanoparticles to be

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suitable for the final application . Finally, as it shown in this review, protein nanoparticles can encapsulate a large spectrum of active molecules and have the ability to penetrate into cancerous cells and blood brain barrier,

cr

since they are at the nanoscale dimension and can be modified to target a specific area. This is considered a great advantage of protein nanoparticles over other drug carriers since it allows the

us

use of active molecules that are not stable in their free form to treat a wide range of diseases. Therefore, the research in the field of protein nanoparticles is of great importance for

an

development of new methodologies in drug delivery and therapy, which possibly can lead to a

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te

d

M

significant breakthrough in the treatment of major and chronic diseases.

Page 32 / 39 Page 33 of 66

This research did not receive any specific grant from funding agencies in the public, commercial,

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te

d

M

an

us

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or not-for-profit sectors.

Page 33 / 39 Page 34 of 66

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Duclairoir, C., Nakache, E., Marchais, H., Orecchioni, a.-M., 2014. Formation of gliadin nanoparticles: Influence of the solubility parameter of the protein solvent. Colloid Polym. Sci. 276, 321–327.

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Duclairoir, C., Orecchioni, a. M., Depraetere, P., Osterstock, F., Nakache, E., 2003. Evaluation of gliadins nanoparticles as drug delivery systems: A study of three different drugs. Int. J. Pharm. 253, 133–144.

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Elzoghby, A.O., Elgohary, M.M., Kamel, N.M., 2015. Implications of Protein- and PeptideBased Nanoparticles as Potential Vehicles for Anticancer Drugs, 1st ed, Protein and Peptide Nanoparticles for Drug Delivery. Elsevier Inc.

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Elzoghby, A.O., Helmy, M.W., Samy, W.M., Elgindy, N.A., 2013. Novel ionically crosslinked casein nanoparticles for flutamide delivery: Formulation, characterization, and in vivo pharmacokinetics. Int. J. Nanomedicine 8, 1721–1732.

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Esmaili, M., Ghaffari, S.M., Moosavi-Movahedi, Z., Atri, M.S., Sharifizadeh, A., Farhadi, M., Yousefi, R., Chobert, J.M., Haertlé, T., Moosavi-Movahedi, A.A., 2011. Beta caseinmicelle as a nano vehicle for solubility enhancement of curcumin; food industry application. LWT - Food Sci. Technol. 44, 2166–2172.

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Ezpeleta, I., Irache, J.M., Stainmesse, S., Chabenat, C., Popineau, Y., Orecchionp, A., 1996. Gliadin nanoparticles for the controlled release of all-trans- retinoic acid. Int. J. Pharm. 131, 191–200.

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Fessi, H., Puisieux, F., Devissaguet, J.P., Ammoury, N., Benita, S., 1989. Nanocapsule formation by interfacial polymer deposition following solvent displacement. Int. J. Pharm. 55, 1–4.

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Gaihre, B., Khil, M.S., Lee, D.R., Kim, H.Y., 2009. Gelatin-coated magnetic iron oxide nanoparticles as carrier system: Drug loading and in vitro drug release study. Int. J. Pharm. 365, 180–189. Gradishar, W.J., 2006. Albumin-bound paclitaxel: a next-generation taxane. Expert Opin. Pharmacother. 7, 1041–1053. Gupta, A.K., Gupta, M., Yarwood, S.J., Curtis, A.S.G., 2004. Effect of cellular uptake of gelatin nanoparticles on adhesion, morphology and cytoskeleton organisation of human fibroblasts. J. Control. Release 95, 197–207. Irache, J.M., Bergougnoux, L., Ezpeleta, I., Gueguen, J., Orecchioni, A.M., 1995. Optimization and in vitro stability of legumin nanoparticles obtained by a coacervation method. Int. J. Pharm. 126, 103–109. Jahanshahi, M., Babaei, Z., 2008. Protein nanoparticle : A unique system as drug delivery vehicles. J. Biotechnol. 7, 4926–4934. Jain, S.K., Gupta, Y., Jain, A., Saxena, A.R., Khare, P., Jain, A., 2008. Mannosylated gelatin nanoparticles bearing an anti-HIV drug didanosine for site-specific delivery. Nanomedicine 4, 41–8.

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Kaul, G., Amiji, M., 2004. Biodistribution and Targeting Potential of Poly(ethylene glycol)modified Gelatin Nanoparticles in Subcutaneous Murine Tumor Model. J. Drug Target. 12, 585–591.

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Kim, T.H., Jiang, H.H., Youn, Y.S., Park, C.W., Tak, K.K., Lee, S., Kim, H., Jon, S., Chen, X., Lee, K.C., 2011. Preparation and characterization of water-soluble albumin-bound curcumin nanoparticles with improved antitumor activity. Int. J. Pharm. 403, 285–291. Kundu, J., Chung, Y. Il, Kim, Y.H., Tae, G., Kundu, S.C., 2010. Silk fibroin nanoparticles for cellular uptake and control release. Int. J. Pharm. 388, 242–250.

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Lammel, A.S., Hu, X., Park, S.H., Kaplan, D.L., Scheibel, T.R., 2010. Controlling silk fibroin particle features for drug delivery. Biomaterials 31, 4583–4591.

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Langer, K., Anhorn, M.G., Steinhauser, I., Dreis, S., Celebi, D., Schrickel, N., Faust, S., Vogel, V., 2008. Human serum albumin (HSA) nanoparticles: Reproducibility of preparation process and kinetics of enzymatic degradation. Int. J. Pharm. 347, 109–117.

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Lee, E.J., Khan, S.A., Park, J.K., Lim, K.H., 2012. Studies on the characteristics of drug-loaded gelatin nanoparticles prepared by nanoprecipitation. Bioprocess Biosyst. Eng. 35, 297–307.

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Lee, E.J., Lee, N.K., Kim, I.S., 2015. Bioengineered protein-based nanocage for drug delivery. Adv. Drug Deliv. Rev. 106, 157–171.

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Lee, G.Y., Park, K., Nam, J.H., Kim, S.Y., Byun, Y., 2006. Anti-tumor and anti-metastatic effects of gelatin-doxorubicin and PEGylated gelatin-doxorubicin nanoparticles in SCC7 bearing mice. J. Drug Target. 14, 707–716.

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Lee, S.H., Heng, D., Ng, W.K., Chan, H.K., Tan, R.B.H., 2011. Nano spray drying: A novel method for preparing protein nanoparticles for protein therapy. Int. J. Pharm. 403, 192–200. Leo, E., Angela Vandelli, M., Cameroni, R., Forni, F., 1997. Doxorubicin-loaded gelatin nanoparticles stabilized by glutaraldehyde: Involvement of the drug in the cross-linking process. Int. J. Pharm. 155, 75–82. Leroux, J.C., Cozens, R., Roesel, J.L., Galli, B., Kubel, F., Doelker, E., Gurny, R., 1995. Pharmacokinetics of a novel HIV-1 protease inhibitor incorporated into biodegradable or enteric nanoparticles following intravenous and oral administration to mice. J. Pharm. Sci. 84, 1387–91. López-Rubio, A., Lagaron, J.M., 2012. Whey protein capsules obtained through electrospraying for the encapsulation of bioactives. Innov. Food Sci. Emerg. Technol. 13, 200–206. Mandal, B.B., Kundu, S.C., 2009. Self-assembled silk sericin/poloxamer nanoparticles as nanocarriers of hydrophobic and hydrophilic drugs for targeted delivery. Nanotechnology 20, 355101. Miladi, K., Ibraheem, D., Iqbal, M., Sfar, S., Fessi, H., Elaissari, A., Avicenne, R., 2014. Page 36 / 39 Page 37 of 66

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Rössler, B., Kreuter, J., Scherer, D., 1995. Collagen microparticles: preparation and properties. J. Microencapsul. 12, 49–57. Sadeghi, R., Moosavi-Movahedi, A.A., Emam-jomeh, Z., Kalbasi, A., Razavi, S.H., Karimi, M., Kokini, J., 2014. The effect of different desolvating agents on BSA nanoparticle properties and encapsulation of curcumin. J. Nanoparticle Res. 16.

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Vauthier, C., Bouchemal, K., 2009. Methods for the Preparation and Manufacture of Polymeric Nanoparticles. Pharm. Res. 26, 1025–1058. Wahome, N., Pfeiffer, T., Ambiel, I., Yang, Y., Keppler, O.T., Bosch, V., Burkhard, P., 2012. Conformation-specific Display of 4E10 and 2F5 Epitopes on Self-assembling Protein Nanoparticles as a Potential HIV Vaccine. Chem. Biol. Drug Des. 80, 349–357. Wang, G., Uludag, H., 2008. Recent developments in nanoparticle-based drug delivery and targeting systems with emphasis on protein-based nanoparticles. Expert Opin. Drug Deliv. 5, 499–515. Weber, C., Coester, C., Kreuter, J., Langer, K., 2000. Desolvation process and surface characterisation of protein nanoparticles. Int. J. Pharm. 194, 91–102. Weber, C., Kreuter, J., Langer, K., 2000. Desolvation process and surface characteristics of HSA-nanoparticles. Int. J. Pharm. 196, 197–200. Zhang, Z.-H.. b, Wang, X.-P.., Ayman, W.Y.., Munyendo, W.L.L.., Lv, H.-X.., Zhou, J.-P.., 2013. Studies on lactoferrin nanoparticles of gambogic acid for oral delivery. Drug Deliv. 20, 86–93.

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List of Abbreviations BLG: β-lactoglobulin BSA: Bovine Serum Albumin

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CMC: Critical Micelle Concentration CMT: Critical Micelle Temperature

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DLS: Dynamic Light Scattering ELS: Electrophoretic Light Scattering

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HSA: Human Serum Albumin LC: Loading Capacity

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LE: Loading Efficiency OVA: Ovalbumin

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PBNs: Protein Based Nanoparticles PCS: Photon Correlation Spectroscopy

pI: Isoelectric Point

SLS: Static Light Scattering

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SEM: Scanning Electron Microscopy

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PDI: Poly Dispersity Index

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TEM: Transmission Electron Microscopy WGE: Wheat Germ Agglutinin WPI: Whey Protein Isolates ZP: Zein Nanoparticle

Page 39 / 39 Page 40 of 66

Table(s)

Table 1: Physicochemical properties of carbazol loaded gliadin nanoparticles prepared by desolvation method. Reproduced with permission from ref. (Arangoa et al., 2001). Copyright 2016 Pharmaceutical Research.

Size (nm) NP CL-NP

Zeta potential (mV) 27.5 ± 0.8 24.5 ± 0.5

460 ± 19 453 ± 24

Yield (%)

Loaded carbazole (µg/mg) 12.57 ± 1.23 12.23 ± 0.78

89.6 ± 4.5 86.8 ± 5.7

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a

NP, non-hardened gliadin nanoparticles; CL-NP, cross-linked gliadin nanoparticles. Experiment performed in a 10−4 M HCl solution. Values represent the mean ± standard deviation (n = 6).

cr

b

NP-1

None

305 ± 56

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Table 2: Physicochemical Properties of casein nanoparticles. Reproduced with permission from ref. (Penalva et al., 2015). Copyright 2016 Food Hydrocolloids.

0.45 ± 0.02

-9.8 ± 0.2

NP-2

Lysine

170 ± 4

0.25 ± 0.02

-11.9 ± 0.9

NP-3

arginine

184 ± 2

0.25 ± 0.01

-9.4 ± 0.2

Size (nm)

PDI

Zeta potential (mV)

NP-1: Casein nanoparticles prepared without lysine

M

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Stabilizing agent

NP-2: Casein nanoparticles prepared with a lysine/casein ration of 0.12 (by weight g/g)

te

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NP-3: Casein nanoparticles prepared with an arginine/casein ration of 0.12 (by weight g/g)

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Table 3: Protein nanoparticles designed for active targeting of cancer cells

Protein HSA

BSA

Gelatin

Ligand

Active molecule

Reference



Monoclonal antibody (DI17E6)



Doxorubicin



(Wagner et al., 2010)



Biotin (Vitamin B)



Methotrexate



(Taheri et al., 2011)



Folate (Vitamin B)



Paclitaxel



(Zhao et al., 2010)



Folate



Vinblastine sulfate



(Zu et al., 2009)



Epidermal growth factor (EGF)



Ciplatin



(Tseng et al., 2009)



EGF



EGFR2R-lytic



(Gaowa et al., 2014)

hybrid peptide

Page 41 of 66

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15-250 kDa

Isoelectric pH 7-9 (Type A) 4-5 (Type B)

Characteristics

 

low toxicity, biodregradability of end products and biocompatility higher encapsulation efficiency for hydrophilic drugs [18] denatured form of collagen used as ingredient in drug formulations possess many carboxyl functional groups for cross-linking

Collagen

300 kDa

ce pt

ed

  

4.7-4.8

 

Albumin

Ac



66.5 kDa (Elzoghby et al., 2015)

4.7

   

 

Preparation Techniques

us

Gelatin

Molecular Weight

M an

Protein

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Table 4: Animal and plant proteins: characteristics, nanoparticle preparation techniques, and the encapsulated active molecules

low toxicity, biodegradability, and immunogenicity structral protein with high content of glycine, hydroxyproline and hydroxylysine contain carboxyl group and secondary amino groups with possible cross-linking function bioconjugate hydrophilic drug interact with highly hydrophobic drugs [18] extremely robust to various conditions bind to many drugs such as indole compound, penicillin and benzodiazepins non-toxicity, biodegradability and immunogenicity the most abundant plasma protein









 

 







Bioactive Molecules

w/o emulsion (Gupta et al., 2004)(Bajpai and Choubey, 2006)(Zhao et al., 2012) Coacervation-phase separation (Teng et al., 2005) Two-step desolvation (Jain et al., 2008)(Nahar et al., 2008)(Qazvini and Zinatloo, 2011)(Kumar et al., 2011) Nanoprecipitation (Lee et al., 2012)

   

Alkaline hydrolysis (Nicklas et al., 2009) phase separation method (Niu et al., 2009) Emulsification (Rössler et al., 1995) Coacervation (Arnedo et al., 2002)(Sebak et al., 2010) Desolvation (Dreis et al., 2007)(Kufleitner et al., 2010)(Jiang et al., 2015) Nab technology (Kim et al., 2011)(Gradishar, 2006) W/O Emulsion (Mishra



     



       

FITC-Dex (Gupta et al., 2004) Paclitaxel (Teng et al., 2005) Didanosine (Jain et al., 2008) Chloroquine phosphate (Bajpai and Choubey, 2006) Amphotericin B (Nahar et al., 2008) Gatifloxacin (Lee et al., 2012) Tizanidine hydrochloride (Lee et al., 2012) Paracetamol (Qazvini and Zinatloo, 2011) Indomethacin (Kumar et al., 2011) Insulin (Zhao et al., 2012) Estradiol-hemihydrate (Nicklas et al., 2009) Tetracaine (Rössler et al., 1995)

Phosphodiester oligonucleotide (Arnedo et al., 2002) Noscapine (Sebak et al., 2010) Doxorubicine (Dreis et al., 2007) Obidoxime (Kufleitner et al., 2010) Docetaxel (Jiang et al., 2015) Curcumin (Kim et al., 2011) Paclitaxel (Gradishar, 2006) Azidothymidine (Mishra et al., 2006)

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ip t

et al., 2006) Nano-spray drying (Lee et al., 2011)

cr α-zein: 22–27 kDa β-zein: 44 kDa γ-zein: 14 kDa (Elzoghby et al., 2015)

   

Produced by certain insects andspiders; as a support to enzyme immobilization; easy processing to different morphologies

M an

4.6 (Elzoghby et al., 2015)



easy to incorporate hydrophobic drug very tolerable to robust conditions nontoxic of the degradable end product no disulfide bridge

6.228 (Elzoghby et al., 2015)

   

used to entrap hydrophobic drug has antibacterial activity protects ivermecin from photodegradation low toxicity of degradable end products, biocompatible,

Ac

Zein

αs1: 23 kDa αs2: 25 kDa β: 24kDa κ: 19 kDa (Elzoghby et al., 2015)

3–5

ed

Casein

Silk fibroin 60–150 kDa

ce pt

Silk

us



  

 

  



 



Gliadin

25-100 kDa (Elzoghby et

6.8

 

plant protein derived from wheat non-toxicity, biocompatible and biodegradable



Desolvation (Kundu et al., 2010) Salting out (Lammel et al., 2010) Capillary microdot technique (Gupta et al., 2009) Self assembly (Mandal and Kundu, 2009) Self assembly (Esmaili et al., 2011)(Shapira et al., 2010)(Zimet et al., 2011)(Pan et al., 2014) o/w emulsification (Elzoghby et al., 2013) Coacervation (Penalva et al., 2015) Nanoprecipitation (da Rosa et al., 2015)(Podaralla and Perumal, 2010) Phase separation (Xu et al., 2011)(Lai and Guo, 2011) Liquid-liquid Dispersion (Zou et al., 2012)(Zhong et al., 2009) Electrospraying (TorresGiner et al., 2010) Desolvation (Arangoa et al., 2001)(Duclairoir et al., 2002)(Duclairoir et

     

VEGF (Kundu et al., 2010) Alcian blue (Lammel et al., 2010) Rhodamine B (Lammel et al., 2010) Curcumin (Gupta et al., 2009) FITC-inulin (Mandal and Kundu, 2009) Paclitaxel (Mandal and Kundu, 2009)



Curcumin (Esmaili et al., 2011)(Pan et al., 2014) Paclitaxel (Shapira et al., 2010) ω-3 fatty acid (Zimet et al., 2011) Flutamid (Elzoghby et al., 2013) Folic acid (Penalva et al., 2015)

      

 

Thymol (da Rosa et al., 2015) Carvacrol (da Rosa et al., 2015) 6,7-dihydroxycoumarin (Podaralla and Perumal, 2010) Metformin (Xu et al., 2011) Cranberry procyanidins (Zou et al., 2012) ω-3 Fatty acid (Torres-Giner et al., 2010) Fish oil (Zhong et al., 2009) 5-fluorouracil (Lai and Guo, 2011)

  

Carbazol (Arangoa et al., 2001) Vitamin E (Duclairoir et al., 2002) Linalool and linalyl acetate (Duclairoir

  

Page 44 of 66

Soy Proteins

121 kDa

 

4.8



ip t

al., 2003)(Ezpeleta et al., 1996)(Umamaheshwari et al., 2004) Nanoprecipitation (Duclairoir et al., 2014)

cr

abundant and inexpensive plant protein highly soluble in water and easy to incorporate hydrophilic drug non-toxicity and reusable end products





 

  

Coacervation (Irache et al., 1995)(Mirshahi et al., 2002)(Gao et al., 2014) Desolvation (Teng et al., 2012)(Teng et al., 2013) Thermal Gelation (Zhang et al., 2012)(Zhang et al., 2015)

    

et al., 2003) Benzalkonium chloride (Duclairoir et al., 2003) trans-retinoic acid (Ezpeleta et al., 1996) Amoxicillin (Umamaheshwari et al., 2004) Curcumin (Teng et al., 2012)(Teng et al., 2013) Coumarin 6 (Zhang et al., 2012) Folic acid (Teng et al., 2013) Linoleic acid (Gao et al., 2014) Vitamin B12 (Zhang et al., 2015)

Ac

ce pt

ed

i.e Legumin

us



protect carrier from breaking down by stomach acid easy to entrap hydrophilic drugs due to its water solubility

M an



al., 2015)

Table 5: Colloidal properties of PBN for each protein and its preparation methods

Gelatin Desolvation

Size (nm)

248 (Jain et al., 2008) 204 (Nahar et

Collagen

Albumin

Silk

157 (Dreis et al., 2007) 173

157 (Kundu et al., 2010)

Casein

Zein

Gliadin

Legumin

464 (Ezpeleta et al., 1996) 460 (Arangoa et al.,

232 (Irache et al., 1995) 243 (Mirshahi

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Coacervation

Size

PDI

ip t 0.020(Kundu et al., 2010)

Low PDI(Umamaheshwari et al., 2004)

0.081 (Irache et al., 1995) 0.18 (Mirshahi et al., 2002)

Sp(Jiang et al., 2015)

Sp(Kundu et al., 2010)

Sp(Ezpeleta et al., 1996)(Umamaheshwari et al., 2004)(Duclairoir et al., 2003)

Sp (Irache et al., 1995)(Mirshahi et al., 2002)

10.5 (Jain et al., 2008) 20.6 (Nahar et al., 2008) 3.53 (Kumar et al., 2011)

-31.9 (Dreis et al., 2007) -56 (Kufleitner et al., 2010) -30 (Jiang et al., 2015)

-26.15 (Kundu et al., 2010)

-3.5 (Ezpeleta et al., 1996) 27.5 (Arangoa et al., 2001) 26.6 (Umamaheshwari et al., 2004) -1 (Duclairoir et al., 2003)

-18.7 (Mirshahi et al., 2002)

600 (Lu et al., 2004)

150 (Sebak et al., 2010) 258 (Arnedo et al., 2002) <0.4 (Sebak et al., 2010)

us

M an

0.1(Kufleitner et al., 2010) 0.170(Jiang et al., 2015)

cr

et al., 2002) 170 (Gao et al., 2014)

Ac

ZP (mV)

2001) 312 (Umamaheshwari et al., 2004) 450 (Duclairoir et al., 2003)

ed

Mrph.

(Kufleitner et al., 2010) 150 (Jiang et al., 2015)

ce pt

PDI

al., 2008) 280 (Qazvini and Zinatloo, 2011) 219 (Kumar et al., 2011) 0.439(Jain et al., 2008) 0.104(Nahar et al., 2008) 0.45(Qazvini and Zinatloo, 2011) 0.329(Kumar et al., 2011) Sp (Jain et al., 2008)(Nahar et al., 2008)(Qazvini and Zinatloo, 2011)(Kumar et al., 2011)

154 (Penalva et al., 2015) 0.24 (Penalva et al.,

232 (Irache et al., 1995) 243 (Mirshahi et al., 2002) 0.081 (Irache et al., 1995) 0.18 (Mirshahi

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ip t

Sp (Sebak et al., 2010)(Arnedo et al., 2002) -47 (Sebak et al., 2010) -24.8 (Arnedo et al., 2002) 172 (Mishra et al., 2006)

PDI

Ac

Mrph.

37 (Gupta et al., 2004) 250 (Zhao et al., 2012) 300 (Bajpai and Choubey, 2006) 0.19 (Zhao et al., 2012) 0.135 (Bajpai and Choubey, 2006) Sp (Gupta et al., 2004)(Zhao et al., 2012)(Bajpai and Choubey, 2006) -21.1 (Zhao et al., 2012) 201 (Bajpai and Choubey, 2006) 251 (Lee et al., 2012)

ZP

Nanoprecipitation

Size

ed

Size

ce pt

Emulsification

M an

ZP

2015) Sp (Penalva et al., 2015) -17.6 (Penalva et al., 2015) 100 (Elzoghby et al., 2013)

cr

Sp (Lu et al., 2004)

us

Mrph.

0.232 (Mishra et al., 2006)

0.59 (Elzoghby et al., 2013)

Sp (Mishra et al., 2006)

Sp (Elzoghby et al., 2013)

19.47 (Mishra et al., 2006)

17 (Elzoghby et al., 2013)

et al., 2002) Sp (Irache et al., 1995)

-18.7 (Mirshahi et al., 2002)

153 (da Rosa et al., 2015) 460 (Podaralla and Perumal,

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ip t Round (Lee et al., 2012)

ZP

Size

Ac

PDI

460 (Lee et al., 2011)

ce pt

Size PDI Mrph.

Nab technology

ed

ZP

Spray drying

Mrph. ZP

Self-assembly

Size

PDI

2010) 0.173 (da Rosa et al., 2015) 0.46 (Podaralla and Perumal, 2010) Sp (da Rosa et al., 2015)(Podaralla and Perumal, 2010) 31.73 (da Rosa et al., 2015) -16 (Podaralla and Perumal, 2010)

cr

Mrph.

us

0.096 (Lee et al., 2012)

M an

PDI

Sp (Lee et al., 2011) Negatively charged (Lee et al., 2011) 137.3 (Kim et al., 2011) 0.31 (Kim et al., 2011) Sp (Kim et al., 2011) -23.4 (Kim et al., 2011) 105 (Mandal and Kundu, 2009) 0.21 (Mandal and

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us

Mrph.

ZP Size

M an

Electrospraying

PDI Mrph.

PDI Mrph. ZP

530 (TorresGiner et al., 2010) Round (TorresGiner et al., 2010)

500 (Lammel et al., 2010) Sp (Lammel et al., 2010) `(Lammel et al., 2010)

Ac

PDI: Poly dispersity indexe Mrph: Morphology ZP: zeta potential SP: Spherical

ed

ZP Size

ce pt

Salting out

ip t

cr

Kundu, 2009) Round (Mandal and Kundu, 2009)

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M

Ac ce p

te

d

Figure 1: Proteins used to build nanoparticles

an

us

cr

ip t

Figure(s)

Figure 2: Factors that influence the preparation and performance of protein nanoparticles

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Figure 3: Influence of the HSA batch on A: Particle diameter of HSA nanoparticles; and B: Polydispersity of HSA nanoparticles at various pH. Reproduced with permission from ref. (Langer et al., 2008) Copyright 2016 International Journal of Pharmaceutics.

Figure 4: Influence of the pH value on the diameter (□) and yield (ᴏ) of HSA nanoparticles prepared in pure water and in 10 mM NaCl solution (∆). Reproduced with permission from ref. (Langer et al., 2003). Copyright 2016 International Journal of Pharmaceutics.

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Figure 5: preparation of protein nanoparticles by desolvation .

Figure 6: HSA nanoparticles prepared by desolvation method: A) particle size and light intensity counts in the PCS measurement in correlation to the amount of ethanol added during the desolvation procedure. B) Percentage of dissolved HSA in the supernatant of the nanoparticles in correlation to the amount of ethanol added during the desolvation procedure. C) amino group content and percentage of dissolved HSA in the supernatant of the nanoparticles in correlation to the amount of glutaraldehyde added. D) particle size of purified and unpurified nanoparticles in correlation to the amount of glutaraldehyde added. Reproduced with permission from ref. (C. Weber et al., 2000). Copyright 2016 International Journal of Pharmaceutics.

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Figure 7: Gelatine Nanoparticles, SEM imagery (x30000). Reproduced with permission from ref. (Jain et al., 2008). Copyright 2016 Nanomedicine.

Figure 8: TEM images of silk fibroin nanoparticles prepared from A. mylitta (a), silk fibroin nanoparticles prepared from B. mori (b), a single silk fibroin nanoparticles prepared from A. mylitta (c) , a single silk fibroin nanoparticles prepared from B. mori (d). Reproduced with permission from ref. (Kundu et al., 2010). Copyright 2016 International Journal of Pharmaceutics.

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Figure 9: Scanning electron microphotograph of amoxicilline loaded gliadin nanoparticles: gliadin 50 mg and amoxicillin 80 mg. Reproduced with permission from ref. (Umamaheshwari et al., 2004). Copyright 2016 AAPS PharmSciTech.

Figure 10: Preparation of protein nanoparticles by Coacervation.

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Figure 11: Influence of experimental conditions on the size and yield of legumin nanoparticles: A)pH; B)Ionic strength; C)Surfactant concentration and D)Concetration of cross-linker glutaraldehyde. Reproduced with permission from ref. (Irache et al., 1995). Copyright 2016 International Journal of Pharmaceutics.

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Ac ce p

Figure 12: Preparation of protein nanoparticles by 1) single emulsion and 2) double emulsion.

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an

Figure 13: Gelatin nanoparticles imagery by A) TEM (Gupta et al., 2004), and B) SEM (x30000). Reproduced with permission from ref. (Bajpai and Choubey, 2006). Copyright 2016 Journal of Materials Science: Materials in Medicine.

Figure 14: Preparation of protein nanoparticles by nanoprecipitation.

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Figure 15: SEM images of Gelatin nanoparticles prepared by nanoprecipitation a)cross-linked; b)noncross-linked. Reproduced with permission from ref. (Lee et al., 2012). Copyright 2016 Bioprocess and Biosystems Engineering.

Figure 16: The nano spray dryer B-90.

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Figure 17: The functional principle of mesh vibration occurring at the piezoelectric driven spray head of the Nano Spray Dryer B-90. Reproduced with permission from ref. (Lee et al., 2011). Copyright 2016 International Journal of Pharmaceutics.

Figure 18: FESEM images of BSA paritcles (A) run 1 (0%, w/v Tween 80) and (B) run 11 (0.05%, w/v Tween 80), showing the effect of surfactant (Tween 80) on the shape of particles. (C) run 2 (4.0 m spray mesh), (D) run 5 (5.5 m spray mesh) and (E) run 8 (7.0 m spray mesh), showing the effect of spray mesh size on the size of particles. Reproduced with permission from ref. (Lee et al., 2011). Copyright 2016 International Journal of Pharmaceutics.

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Figure 19: Preparation of albumin-bound curcumin nanoparticles using nab technology.

Figure 20: SEM imagery of curcumin loaded HSA nanopaticles (original magnification 30,000x). Reproduced with permission from ref. (Kim et al., 2011). Copyright 2016 International Journal of Pharmaceutics.

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Figure 21: Schematic representation of a casein micelle.

Ac ce p

Figure 22: The proposed principle of the pH-driven encapsulation of curcumin in self-assembled casein nanoparticles. Reproduced with permission from ref. (Pan et al., 2014). Copyright 2016 Soft Matter.

Figure 23: Schematic representation of electrospraying technique.

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Figure 24: Preparation of nanoparticles by salting out.

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Figure 25: Effect of experimental conditions on the characteristics of silk fibroin nanoparticles prepared by salting out: A) Salting out efficiency as a function of ionic strength of potassium phosphate; B) Salting out efficiency as a function of pH; C) Average size of silk particles as a function of protein concentration; D) Zeta potential of particles produced by salting out with 1.25 M potassium phosphate at different pH. Reproduced with permission from ref.

(Lammel et al., 2010). Copyright 2016 Biomaterials. Figure 26: Influence of the crosslinking process with different amounts of glutaraldehyde on the diameter (□) and pI (O) of the resulting HSA nanoparitlces. Reproduced with permission from ref. (Langer et al., 2003). Copyright 2016 International Journal of Pharmaceutics.

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Figure 27: Widely used preparation methods for each protein: Red circles:Animal proteins, Green circles: plant proteins.

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Figure 28: The effect of different desolvating agent/ solvent ratios on particle size and polydispersity of BSA NPs, particle size (solid line), and polydispersity index (dashed line). Reproduced with perimission from ref. (Sadeghi et al., 2014). Copyright 2016 Journal of Nanoparticle Research

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Figure 29: Methodology of preparation of PBN.

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