Protein nanoparticles: Promising platforms for drug delivery applications

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Review

Protein nanoparticles: Promising platforms for drug delivery applications Annish Jain, Sumit K Singh, Shailendra K Arya, Subhas C Kundu, and Sonia Kapoor ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01098 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 2, 2018

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ACS Biomaterials Science & Engineering

Protein nanoparticles: Promising platforms for drug delivery applications Annish Jain1, Sumit K Singh1, Shailendra K Arya1, Subhas C Kundu2, Sonia Kapoor1,3* Department of Biotechnology, University Institute of Engineering and Technology, Panjab University, Chandigarh, 160014, India 1

3B’s Research Group, I3Bs – Biomaterials, Biodegradable and Biomimetics of University of Minho, AvePark, 4805-017 Barco, Guimarães, Portugal 2

3Amity

Institute of Molecular Medicine and Stem Cell Research, Amity University, Noida, 201313, Uttar Pradesh, India

*To

whom correspondence shall be addressed

Corresponding author: Dr Sonia Kapoor, Amity Institute of Molecular Medicine and Stem Cell Research, Amity University, Noida, 201313, Uttar Pradesh, India Email: [email protected] +91(0)-120-4392372 Fax: +91(0)-120-4392114

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Abstract Developing drug delivery systems using nanoparticles as carriers for small and large therapeutic molecules remains a rapidly growing area of research. The advantages of using proteins for preparing nanoparticles for drug-delivery applications include their abundance in natural sources, biocompatibility, biodegradability, an easy synthesis process and cost effectiveness. In contrast to several particulate systems like nanoparticles from metallic and inorganic/synthetic sources, the protein nanoparticles do not have limitations such as potential toxicity, large size and accumulation or rapid clearance from the body. In addition, protein-based nanoparticles offer opportunity for surface modification by conjugating other protein and carbohydrate ligands. This enables targeted delivery to the desired tissue and organ, which further reduces systemic toxicity. The use of protein nanoparticles for such applications could, therefore, prove to be a better alternative to maneuver and improve the pharmacokinetic and pharmacodynamic properties of the various types of drug molecules. In this review, while focusing on properties of few proteins such as silk protein fibroin, we attempt to provide an overview of the existing protein-based nanoparticles. We discuss various methods for synthesis of this class of nanoparticles. The review brings forth some of the factors that are important for the design of this class of nanoparticles and highlight the applications of the nanoparticles from these proteins. Keywords: Drug-Delivery; Proteins; Natural Polymers; Nanocarriers; Nanoparticles


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1. Introduction Nanotherapeutics have emerged as a platform which holds the potential to revolutionize the field of drug delivery. Initially, the drug delivery was based on administration of therapeutic molecules either by oral route or through any other injectable route. Administering therapeutic macromolecules such as proteins (often required during vaccination) and delivering many other essential pharmacologic agents including nucleic acids has been a big challenge.1 In the past few decades, there has been a paradigm shift towards the emphasis on enhancing the bioavailability of drugs in the body and improving the efficacy of therapeutics by designing nano-drug delivery systems. Nanotechnology refers to the techniques and methods for studying, designing and fabricating things such as particles, devices etc. at the nanometer scale where nano-platforms are of size between 1 nm to 1000 nm.2 Nanotechnology has led to the development of many novel carriers capable of providing a controlled release and targeted delivery of a wide range of therapeutic molecules such as proteins, peptides, genes, interleukins, growth factors and various chemical drugs. These nanocarriers are not only capable of offering a controlled drug release profile but also offer possibilities of loading multiple drugs simultaneously so that the advantage of drug synergism may truly be realized.3-5 To date many nanocarrier systems including quantum dots (QDs), liposome, dendrimers, micelles, fullerenes and nanoparticles have been designed in various forms. Each of them has unique structures and properties.6-9 Liposomes are the first nanosystems that got approved for the delivery of drugs and biomolecules in 1960s.10 The application of liposomes is, however, limited owing to the rapid release rate of hydrophilic drugs, very low encapsulation efficiency and poor stability.11 Nanoparticles have been prepared from a variety of materials such as metals, non-metals, polysaccharides and synthetic polymers. Nanoparticles from metallic and inorganic sources



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have been widely used to alter and improve the pharmacodynamic and pharmacokinetic properties of a variety of drug molecules, especially cancer chemotherapeutic agents. These drug carrying nanoparticles can increase the intracellular drug concentration and can be specifically targeted, thus, reducing toxicity to normal cells. However, their in vivo use is still limited due to certain factors like high particle toxicity, size, rapid clearance and their low scope of modification. In the synthetic polymers category, poly (lactic acid) (PLA) and poly(lactic-coglycolic acid) (PLGA) have gained an extensive popularity because of their long history of clinical use and widely explored characteristics such as drug release kinetics and degradation rate. Nano-systems based on these are a homogeneous product because of established processing methodologies and hence, their performance can be predicted and improved. However, the use of PLA and PLGA is limited in therapeutics due to their intrinsic properties and processing requirements. In particular, the acidic microenvironment inside these nanoparticles tends to create a dominant stress on the encapsulated drug or the therapeutic protein causing an aggregation of these within the nanoparticle, often resulting in incomplete release.12-15 Amongst the available options, nanoparticles that can be synthesized in a facile manner from a variety of naturally occurring or engineered proteins emerge as promising platform for drug delivery applications. Protein-based carriers have been reported in the form of prodrugs, drugconjugates, nanoparticles, microcapsules, hydrogels and 3-D scaffolds.16-18 Nanocarriers from proteins

are

non-toxic,

biodegradable,

easily

metabolizable,

and

possess

a

good

biocompatibility.19-23 Protein based nanocarriers can be degraded by the action of enzymes present inside the human body. In addition, protein nanoparticles have been found to elicit a weak or negligible immune response.24, 25 Moreover, the amphiphilic nature of proteins helps


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them interact with both hydrophilic as well as hydrophobic drugs and solvents. The abundance of hydroxyl, amino and carboxyl groups present in them makes them amenable for chemical modification. Therefore, protein nanoparticles can be covalently or non-covalently attached with one or different types of ligands and drug molecules. This offers excellent scope of surface modification.26,

27

For the past few decades, scientists have been utilizing the potential of

proteins including gelatin, silk-fibroin, albumin, gliadin and others derived from very diverse sources such as animals, plants, insects and recombinant protein expression systems (Table 1). In this review, we highlight potential of various proteins that can be fabricated as nanoparticles for effective targeted therapeutic delivery. We further discuss the synthesis techniques and applications of these protein nanoparticles in delivering therapeutic drugs, growth factors and hormones in various therapies. Table 1: Various proteins being used for fabricating nanoparticles: Merits and demerits as well as their applications are presented. Protein

Source

Size range of nanoparticles

Advantages/Applications

Disadvantages

Refs

Silk protein sericin

Silk worm, spiders and other arthropods

100-150 nm

Biodegradable, water soluble, good for hydrophobic drug delivery, easy synthesis, has moisture retention and anti-microbial properties Nanoparticles can be fabricated without the use of any solvents

Can cause in-vivo inflammation, low yield

28-33

Silk protein fibroin

Silk worm, spiders and other arthropods

100-200 nm

Biocompatible, non –toxic, recognized by Food and Drug Administration (FDA) for the development of a variety of nanotechnological tools Easy synthesis and modification High yield and entrapment efficiency, Can withstand heat sterilization process

Slow degradation rate

34-39

Albumin

Serum of cattle, humans etc. and eggs

50-200 nm

Biodegradable and biocompatible, non-toxic, highly soluble in physiological fluids, thermally stable Has high scope of surface

Anaphylaxis can occur

40-44



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modification. Can be prepared as nanospheres and nanocapsules. Protamine

Salmon fish

100-250 nm

Can be used to counter heparin activity, highly cell penetrating, efficient gene delivery, controllable size.

Low yield and complex synthesis process

45-47

Gliadin

Wheat

200-500 nm

Biodegradable , inexpensive, suitable for designing oral and topical drug carriers.

Rapidly degraded, large sized nanoparticles

48, 49

Legumin

Pea seed

250-400 nm

Biodegradable, bioadhesive in nature, inexpensive, easy to crosslink

Low yield.

50,51

Collagen

Found in connective tissues and flesh of mammals

120-150 nm

Biodegradable, biocompatible and non- antigenic, possess selfaggregation properties, thermally stable and easily sterilized

Religious constraints, chances of transmission of prions from animal source

52-56

Gelatin

Derived from collagen

100-300 nm

Inexpensive, water-soluble, easy to crosslink and sterilize, biodegradable, an FDA approved polymer recognized as “Generally Recognized as Safe” (GRAS). Used as a food supplement and in intravenous infusion. Widely used for encapsulating several biologically active molecules like proteins, growth factors and nucleic acids

Religious constraints, chances of transmission of prions from animal source.

57-60

Low mechanical strength and fast degradation

Elastin like protein (ELP)

Escherichia coli is the most used system for the production of ELPs

40-600 nm

Biodegradable and biocompatible, self-assembling features

Presence of endotoxin in case of bacterial expression system, degrades rapidly in presence of elastase and collagenase

61-66

Virus-like particles (VLPs)

Animal virus, Bacteriophages and Plant viruses

20-60 nm

Easy synthesis and inexpensive, improves antigenicity of poor immunogens, high drug-loading efficiency and excellent cellular uptake. Natural tropism and good targeting ability, easily modified, Used in vaccine, drug and DNA delivery, some vaccines based on VLPs, (Gardasil (Merck) and Cervarix (GlaxoSmithKline) are FDA-approved

Can cause potential immunogenicity when used for nonvaccine delivery

67-69


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2. Designing a nanoparticle The usage of nanoparticles for biomedical applications is aimed at a successful targeted drug delivery in a predictable manner with minimum toxicity to the host body. The nanocarriers must exhibit certain characteristic features which include: •

Non-toxic, effective and safe to use in-vivo.

•

Should have acceptable shelf life.

•

A high zeta potential to prevent particle aggregation.

•

Degradation must be slow to prevent sudden drug release.

•

The bi-products from degradation should be non-toxic, and easily metabolized and cleared from the body.

•

Easy and cost effective production and reproducibility.

Controlled tailoring of size, morphology, surface- and internal properties is necessary to fabricate protein nanoparticles with suitable properties. The small size of the nanoparticles is the most important characteristic that makes them special compared to other drug delivery systems. The interaction of nanoparticles with the cells and lipid bilayers plays a major role in its actual functioning as a drug or a therapeutic macromolecule carrier. A good nanoparticle should have an acceptable size of less than 200 nm with a spherical morphology. A study by Desai et al showed that 100 nm sized nanoparticles have 2.5 times greater cellular uptake as compared to 1 μm-sized particles and it is up to six times higher when compared with 10 μm sized microparticles.70 It is also reported that spherical nanoparticles have five times greater cellular uptake than the rod shaped nanoparticles71 which suggests that the cellular uptake of a nanocarrier is highly dependent on its size and shape. In addition to influencing the cellular uptake, the nanoparticle size is also known to manipulate drug loading, release and stability of



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nanoparticles.72-74 The size of protein nanoparticles can be easily modified by modulating the physico-chemical environmental parameters such as pH, ionic strength, and temperature. Sufficiently high internalization of virus like particles (VLPs) has inspired the researchers to develop protein particles that could mimic these viral structures so as to achieve a significantly higher cellular uptake.75 The protein nanoparticles often need to be chemically modified during/after synthesis to improve their stability and decrease their rate of degradation. This is usually achieved by inclusion of a synthetic crosslinker such as formaldehyde, glutaraldehyde or a natural crosslinker such as transglutaminase76or genipin which are much less toxic as compared to the synthetic ones. Surface properties such as surface charge and hydrophilicity/hydrophobicity are the major factors that determine the level of adsorption of various proteins such as opsonins on nanoparticles. Generally it is important for a nanocarrier to have a neutral and hydrophilic surface so as to resist the adsorption of plasma proteins in order to escape the uptake by macrophages.1,

77

It is reported that as much as 95% of foreign particles may undergo

opsonization and clearance by the reticuloendothelial system. PEGylation can be used as an approach to reduce adsorption of opsonins to protein nanoparticles.2,78 The protein nanoparticles can either be coated with PEG or nanoparticles may be synthesized from PEG-conjugated proteins. For instance, nanoparticles fabricated from gelatin conjugated with PEG show an improved plasma half-life of the encapsulated hydrophobic molecules and anti-inflammatory drugs.79 In addition to incorporating the design features such as shape, size and stability for improved performance, the protein nanoparticles have also been functionalized with tissuespecific molecular ligands for targeted drug delivery. Natural ligands such as protein receptors and antibodies and synthetic ligands such as smaller peptides and aptamers are being used for


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this purpose. These ligands target the nanoparticles to the desired site, thus, limiting the systemic exposure to the enclosed therapeutic molecule. In addition, owing to an affinity to tissue-specific cell surface molecules, these ligands assist in improving the retention of nanoparticles and slow down their loss from the tissues. Conjugation of desired ligand molecule with the protein nanoparticles’ surface is usually achieved by covalent bond formation between the functional groups on protein surface and the ligands. Surface coating techniques and electrostatic adsorption are also utilized for surface modification. Choice of ligand used for modifying the surface of protein nanoparticle depends on the cells/tissue/organ-site/ to be targeted such as the use of transferrin or anti-human epidermal growth factor receptor 2 (antiHER2) antibodies to target protein nanoparticles to brain80 and breast cancer cells81,

82,

respectively (Table 2). Table 2: Examples of ligands used for modification of various protein nanoparticles for targeted delivery are provided. S. No. 1 2 3

Ligand Biotinylated-EGF* Anti-CD3 antibody Cyclic pentapeptidecRGDfk) iRGD-EGFR antibody Folate Arginylglycylaspartic acid (RGD) peptide Cyclic RGD peptides CpGoligodeoxynucleotide

Protein Nanoparticle Gelatin Gelatin Silk fibroin

9

Elastin like protein (ELP)

NGR tripeptide

10

Trastuzumab-DM1

Virus-like particles (VLPs)

4 5 6 7 8

Silk fibroin Silk sericin BSA HSA Protamine

Target/Remarks Lung cancer cells CD3-positive T cell (leukemic) Human gastric carcinoma cell line MGC-803 HeLa tumor-bearing nude mice Tumour targeting Tumour vasculature Targeting Human melanoma cells (M21 cells) Countering the Th2-dominated immune response to prevent allergy Targets the CD13 receptor which is highly expressed in tumor vasculature and perivascular cells Targets HER2-positive breast cancer

*EGF; Epidermal Growth Factor


Ref. 83 84 85 86 87 88 89 90 91 92


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3. Protein nanoparticle synthesis strategies Extensive research and development in the field of nanotechnology has given birth to diverse strategies for fabrication of colloidal nanoparticles. Numerous distinct strategies which include salt precipitation (salting out), emulsion solvent extraction, coacervation or desolvation, polyelectrolyte complexation and electrospraying have been successfully used for the fabrication of protein-based nanoparticles. The size of protein nanoparticles fabricated using these methods commonly ranges from 100 to 1000 nm whereas methods yielding particle sizes of sub-100 nm are considerably less.93 Controlling and optimizing the final nanoparticle size i.e. the diameter of the nanoparticle is a key requirement for process success. In addition, these methods often need to be tailored for high-efficiency drug entrapment and retention of the desired pharmacologic activities of the loaded molecules. 3.1 Emulsion/solvent extraction Emulsion/solvent extraction method is conventionally adopted for polymeric nanoparticles but it could also be employed for yielding protein nanoparticles. In simplest terms, a polymer solution or an aqueous protein solution in organic solvent (W) is made to dissipate in a pertinent non-solvent such as castor oil, sesame oil or cotton seed oil (O) to form an emulsion system (O/W or W/O) under the conditions of mechanical stirring or sonication and the solvent/nonsolvent is removed subsequently to yield nanoparticles93-95 (Figure 1). Protein nanoparticles formed by this method can be chemically stabilized by adding crosslinkers or thermally stabilized by adding the W/O emulsion to pre-heated oil having temperature nearly or over 100 °C followed by purification. This technique yields particle of size generally larger than those obtained by other techniques such as coacervation/desolvation and others.96 Certain surface active agents like polysorbate-80, poly(vinyl alcohol) and organic solvents such as chloroform


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and ethyl-acetate are avoided as they have a tendency to alter bioactivity of proteins.97,

98

Moreover, they may result in unsolicited reactions against nanoparticles and thus, are required to be reduced significantly. The particle sizes are optimized by the use of surfactants that functions for the formation of smaller fluid particles in the emulsion. This facilitates the fabrication of smaller solid particles by surfactant-nanoparticle matrix interaction.99 Protein concentration and relative volume of water and oil phases (W:O) during emulsification are the critical parameters in the fabrication of the protein nanoparticles.96 For instance, the sizes of bovine serum albumin (BSA) nanoparticles ranging between 100–800 nm was found to be dependent on the relative W:O volume ratio and BSA concentration.100 3.2 Desolvation The desolvation or coacervation process is the method of choice for fabrication of protein based nanoparticles because of its simplicity and advantage of obtaining smaller size nanoparticles. The principle of this method is to reduce the solubility of an aqueous protein solution by using a desolvating agent such as ethanol, acetone and others resulting in a phase separation. Addition of desolvating agent tends to change the conformation of protein structure and decrease the solubility of protein leading to its precipitation in the form of protein nanoprecipitates101,

102

(Figure 2). The formation of particles initially advances with an increase in size until a steady size is attained, which is succeeded by the gradual increase in the number of particles with almost same size.101 Lin et al. describe a pH-controlled desolvation process for fabricating 100 nm human serum albumin (HSA) nanoparticles.103 The particles are fabricated by using acetone as a desolvating agent for an aqueous solution of HSA at pH 7–9, followed by stabilization of particles by a cross-linker such as glutaraldehyde. In another study, for preparing HSA nanoparticles by desolvation with ethanol, the parameters such as rate of ethanol addition, pH of



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desolvating phase, HSA concentration as well as the purification conditions were optimized to obtain particles of size between 100 and 300 nm.104 It is found that the pH prior to desolvation procedure is the main factor affecting the size of nanoparticles, with high pH values yielding smaller nanoparticles.104 The pH and osmolarity are also found to be important factors in maintaining the stability of the nanoparticles with conditions leading to net-zero surface charge inducing more particle aggregation and hence an increase in size.104 When extended for heat treated β-lactoglobulin, a protein having low molecular weight (nearly 19 kDa) but isoelectric point (pI) almost similar to that of BSA (whose molecular weight is nearly 66 kDa), it led to the formation of 60 nm particles, indicating that size of nanoparticles correlated well with the size of the protein used.105 Chemically modified proteins such as polyethylene glycol modified HSA [HSA-mPEG]) can also be used as nanoparticle matrix. The properties of modified proteins may be different from the parent protein from which it is derived. Therefore, the modified protein may require different type of solvents and non-solvents for making nanoparticles than those required in case of unmodified protein.106 The protein nanoparticles obtained using desolvation are reported to have high encapsulation efficiencies. For instance, the BSA nanoparticles prepared using this method are shown to achieve very high encapsulation efficiencies for protein therapeutics viz. 95% and 90% for IFN-γ and for BMP-2, respectively.107,108 3.3 Salt precipitation The salting out approach, a method quite similar to the desolvation technique, is based on preparing protein coacervates by inducing hydrophobic-hydrophobic protein interactions in presence of high salt concentration. In contrast to desolvation in which the protein solution is added drop wise to a pool of desolvating agent, salt precipitation utilizes a slow addition of salting agent to the protein solution (Figure 3). The simplicity of this approach and relatively


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high packing efficiency makes it attractive, provided that the procedure does not adversely affect the conformational structure or bioactivity of the protein. In addition to high drug encapsulation efficiency and controlled drug release kinetics from the obtained nanoparticles, this method allows modulation of physicochemical characteristics and morphology of the nanoparticles. Insulin particles are designed using this approach by adding 0.5 M and higher concentrations of NaCl solution to the protein solution in acidic medium.109 The particles ranging from 100 to 1000 nm are obtained. The size of the nanoparticles is found to be strongly influenced by the pH of the solution.109Another example is the synthesis of silk fibroin nanoparticles prepared using potassium phosphate as salting out agent in aqueous solution.101, 110

Once the nanoparticles are formed, it is often required that they are further stabilized.

Stabilization of nanoparticles can be achieved by coating these pre-formed nanoparticles with either a cross-linker or by polyelectrolyte coating (considering the pI of protein and net polyelectrolyte charge). Both the approaches are quite easy and help to facilitate the stabilization of nanoparticles and prevent them from aggregation.109 The protein/polymer diffusion from emulsion into the solvent is the key step that differentiates the emulsion extraction approach from the salting out approach. This step is not required in the latter because of the presence of salts. Distilled water may be required to be added to decrease the ionic strength of the solution. Finally, the salting out agent in the aqueous solution can be eliminated by centrifugation and the sample may hence be purified.111, 112 This approach, however, suffers from a drawback of a high heterogeneity of the nanoparticle sizes obtained during fabrication process. 3.4 Polyelectrolyte complexation



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Proteins bearing active surface charge can function as emulsifying agents. The surface charge may exert a phenomenal colloidal stability by means of electrostatic interaction and steric hindrance. Since electrostatic interactions, which are highly dependent on pH, are the major contributing factors in the stabilization mechanism, the stabilized protein solution is quiet sensitive to pH. This phenomenon of pH dependent electrostatic interaction between proteins and other macromolecules can be exploited to design stable biocompatible nanoparticles and coacervates in order to facilitate controlled delivery of bioactive therapeutics. Coacervation complexes are formed as a result of macromolecular interaction due to electrostatic forces under aqueous conditions (Figure 4). As mentioned earlier, parameters like pH of the medium profoundly affect the formation of insoluble complexes between proteins and oppositely charged polymers. The use of polycations for nanoparticles fabrication requires a pH higher than the isoelectric point of the protein whereas, in case of polyanions, a pH lower than the isoelectric point of the protein is required.113 The cationic protein polymers are commonly employed to curtail long string-like DNA/RNA therapeutic molecules into nanoparticles by forming complexes with anionic oxygen atoms in the phosphodiester backbone of oligonucleotide.114 This type of observed interaction is rather entropic than enthalpic as small counter-ions are found to be released in the process.115 Since hydrophilic protein segments facilitate aqueous solubility of formed nanoparticles (which is a highly desirable feature for their systemic application), the combination-type polyelectrolytes are proposed to be particularly suited for the preparation of water-soluble complexes.113,

115

Serefoglou et al.

described the formation of water-soluble nanoparticles by means of columbic interaction between BSA and anionic polymers at a pH lower than the isoelectric point of BSA (~4.5).113 These complexes are found to contain 13-14 BSA molecules held intact by two polymeric


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molecules. The approach yields the particles of hydrodynamic radii of 65-83 nm. In addition to electrostatic interactions, hydrophobic interaction or hydrogen bonding can also facilitate protein/polymer complexation. In this case, the balance between the polymer-protein and polymer-polymer interactions govern the number of proteins and polymer in a complex.116 3.5 Electrospraying Electrodynamic spraying or electrospraying is an emerging and rather popular technique for fabricating sub-micron to nano-sized particles of protein molecules.117,

118

It is a process of

liquid atomization by concurrent generation and charging of droplets with the aid of electric force to overcome surface tension. In this approach, a stream of protein solution is emitted out of a capillary nozzle that is maintained at a relatively high potential. It is subjected to an electric field, which results in an elongated meniscus to form a jet. This is later deformed into semispherical aerosolized droplets, mainly due to electrostatic force (Figure 5). No additional mechanical energy but electric field alone is responsible for liquid atomization. Being highly charged, the nucleotides and other charged therapeutic molecules can easily be incorporated to these nanostructures with high loading efficacy. Nanoparticles fabricated with this approach have relatively low hydrodynamic diameters as the surface tension of the droplets is effectively countered with electric force. Gliadin nanoparticles fabricated with this process are shown to have the average particle size of 220 nm with a very high nanoparticle yield (>75%), and a drug loading efficiency of more than 72%.119 This process is scalable at industrial levels and is already being used in pharmaceutical industries to obtain powdered drug formulations. 4. Protein based nanoparticles Protein nanoparticles are being used as a successful candidates to deliver a variety of therapeutics molecules like nucleic acids, growth factors, vaccines and therapeutic drugs,



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chiefly to avoid the side effects of free therapeutic molecule and at the same time to increase its efficiency inside the host body. Loading of these therapeutics onto nanoparticles is usually done either by means of diffusion or by simply modifying the surface of protein nanoparticles for covalent or non-covalent binding.35, 120 In a study by Wang et al., gelatin nanoparticles loaded with two therapeutic proteins, namely bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF) showed simultaneous release of both the therapeutics resulting into an inhibitory effect on osteogenesis120. Similarly a signaling protein, vascular endothelial growth factor (VEGF) that stimulates the formation of blood vessels was loaded on silk fibroin nanoparticles via electrostatic binding and its in vitro release was evaluated.35 The anionic surface of protein nanoparticles can be modified by surface coating, for instance, by poly Llysine (PLL) in order to make it cationic so as to bind and deliver negatively charged therapeutic nucleic acid molecules. This approach has been used for modulating the surface properties of silk sericin nanoparticles to make it suitable for delivery of pEGFP-C1 plasmid DNA in mouse fibroblast cells .121 Nanoparticles derived from proteins have been extensively explored to deliver various anti-cancer drugs. The hydrophobic core present inside these protein nanoparticles effectively encapsulates these anti-cancer drugs, which are often hydrophobic in nature. These anti-cancer drugs can also be adsorbed to the surface of nanoparticles via electrostatic interactions. However, the surface bound drugs are much rapidly released compared to their counterpart that is entrapped inside the nanoparticles.122,123 The primary aim of using protein nanoparticles for cancer treatment is to improve therapeutic efficacy with low to minimal side effects for which protein nanoparticles are surface modified in order to achieve a site specific targeted drug delivery (as discussed in section 2).


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The engineering and use of virus-like particles (VLPs) have opened new avenues in the applications of protein nanoparticles in drug delivery, gene delivery and especially in vaccine delivery. Display of epitopes on virus-like Particles (VLPs) is a very popular technique for increasing the immunogenicity of antigens which are poorly immunogenic in nature. The VLPbased vaccines can be used to draw out long-lasting and high-titer antibody responses against several target antigens and in some cases even self-antigens. VLP-based vaccines are presently licensed and commercialized, and many other vaccine candidates are under preclinical and clinical studies.69, 124 Apart from their use in therapeutic delivery, protein nanoparticles have shown a tremendous scope in bio-imaging. This has been enabled by means of entrapping fluorescent dyes such as Rhodamine B into the tightly packed inner compartments of protein nanoparticles.125 Luminiscent protein nanoparticles with appropriate quantum yield have also been prepared as in case of silk protein fibroin with the help of microwave assisted hydrothermal treatment of protein.126 These carbonaceous nanoparticles show better biocompatibility as compared to other systems such as quantum dots along with excellent luminescence suggesting their applications in bioimaging. 4.1 Silk proteins Silk fiber is obtained from arthropods including silkworms and spiders which belong to the Lepidoptera class.127,128,129 The most extensively characterized and used silk, often known as mulberry silk, is derived from the silkworm Bombyx mori (Bombycidae family). Silk isolated from the cocoons of B. mori have been historically used for industrial, domestic, and medicinal purposes. Silk fiber from silkworm consists of two different proteins: sericin and fibroin. Silk sericin is the glue protein that binds and strengthens the silk fiber (fibroin). Silk fibroin



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represents a structural fibrous protein. Silk proteins have been extensively explored for preparing nano-drug carrier. 4.1.1 Sericin Sericin, a globular protein rich in polar amino acids including serine and aspartic acid, is one of the main components of the silk proteins. It is derived from the cocoons of silkworms and represents 20-30% of the total cocoon weight of mulberry species. It acts as a glue in order to bind fibroin protein present in the silk fibers together and, thus, enhances the strength of silk fibers. Sericin is also a by-product of the silk textile industries and is obtained as a water soluble fraction by several degumming processes. These include boiling of the cocoons in sodium carbonate solution, also known as alkaline extraction or by the use of enzymes such as alkaline protease and alkylase.130-132 For a long time, sericin has been known to cause in-vivo inflammation, which makes its removal necessary from silk fibers.28,

29

However, in recent

studies, sericin is reported to be non-immunogenic and biocompatible.30,31,133,134 Sericin blended with other polymers like poloxamer and PEG has been used as 3-dimensional scaffold, biofilms, hydrogels and nanoparticles.32,87,121,135-141 It is shown that silk sericin, in combination with activated polyethylene glycol (PEG) or poloxamer F-127 and F-87, can self-assemble to form nanoparticles of size ranging 200-400 nm, or 100 nm, respectively.32,138 Several studies have demonstrated the potential of sericin nanoparticles to be used as a nano-drug carrier for the delivery of genes and drugs for anti-cancer and anti-hypercholesterolemia therapy. Huang et al. have investigated the use of folate conjugated sericin nanoparticles having size of around 120 nm for efficient tumor targeting by covalently linking doxorubicin drug to sericin nanoparticles through pH-sensitive hydrazone bonds in order to obtain a sustained release when taken up into acidic intracellular endo-lysosomal compartments.87 Another study has reported the successful


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use of 100-150 nm sized sericin nanoparticles fabricated with one step desolvation technique for transfecting mouse fibroblast cells with pEGFP-C1 plasmid DNA bound to sericin nanoparticles, suggesting its potential applications for gene delivery.121 Recently, it has been demonstrated that sericin nanoparticles could be fabricated without the use of any solvents.33 Sericin has been shown to have moisture retention and anti-microbial properties. Therefore, a considerable effort has been made towards integrating sericin with silver nanoparticles, as a component of nanoparticles or as a capping agent.142, 143 4.1.2 Fibroin Fibroin, which constitutes 65-85% of total silk proteins, is the major protein present in silk fibers. Silk fibroin is the protein that gives silk its exceptional physical and chemical properties such as flexibility, mechanical strength, ability to self-assemble, low immunogenicity, biodegradability and biocompatibility.34 Fibroin from B. mori silk is a glycoprotein macromolecule composed of more than 5000 amino acids with a repeated sequence of six amino acid residues (Gly-Ala-Gly-Ala-Gly-Ser)n along with few other bulky chain amino acids.144 Fibroin is composed of a heavy chain (approximately 360 kDa) and a light chain (approximately 26 kDa), which are held together by a single disulphide bond.145 Fibroin is usually obtained from cocoons by degumming the silk fibers. This is a process that removes sericin (the glue like protein which holds fibroin together) by boiling in water. The degummed fibers are solubilized in chaotropic salts such as LiBr (Figure 6). Silk protein fibroin exits in two forms, the first one is a water-soluble state known as Silk I while the other is water insoluble form known as Silk II. Silk I, predominantly present in regenerated silk fibroin, is a mixture of α –helix & random-coils along with few β-sheets.121,

122

Silk I is unstable to

mechanical or shear stress and gets converted to Silk II. The native silk fibroin form, is water



Page 20 of 74

insoluble because of the extensive network of tightly packed anti-parallel β-sheets formed by the formation of hydrogen bonds among the hydrophobic amino acids of silk fibroin.123The regenerated silk fibroin is amenable to various processing conditions and retains excellent stability and mechanical properties as well as biocompatibility, which makes it highly sought after protein for developing nanocarriers. Silk fibroin is potentially used as a natural nanocarrier to deliver various therapeutic agents like anti-cancer drugs, proteins, growth factors and peptide molecules. These are either entrapped or simply adsorbed onto the surface of nanoparticles via electrostatic force of attraction. Initially, silk has been used as a coating material to coat nanoparticles and Quantum dots to improve their stability and prevent aggregation.149,

150

Lately, there have been several studies that explored silk fibroin

nanoparticles as a novel drug carrier with controllable size and negligible cytotoxicity.83,

35

Studies have shown that silk nanoparticles are remarkably stable owing to a highly negative zeta potential and are able to carry drugs that are of hydrophobic nature. Silk nanoparticles have also been found to be biocompatible to human blood under flow as well as quasi-static conditions, with negligible inflammation and coagulation.151 This suggests that silk fibroin nanoparticles would be as suitable for systemic drug delivery as they are for local drug delivery. Silk fibroin nanoparticles loaded with anti-cancer agents such as curcumin, doxorubicin, docetaxel, paclitaxel, gemcitabine and others have been evaluated for drug delivery in breast, colon, pancreatic, bone and lung cancer therapies36,123,152,153 (Table 3). Recently, a carrier-in-carrier system has been developed in which curcumin-encapsulating fibroin nanoparticles are further uptaken by mesenchymal stem cells (MSCs). Curcumin is encapsulated in silk nanoparticles with a high drug loading content of up to 32% with nanoparticles accumulated in the cytosol of mesenchymal stem cells. Subsequently, these


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MSCs efficiently release the extracellular vesicles with nanoparticles. The system has a potential for combining regenerative cell therapy and nanomedicine.154 Zhao et al., have prepared silk fibroin nanoparticles with an average diameter of 50 nm by solution-enhanced dispersion by supercritical fluid CO2 .155 The silk protein fibroin nanoparticles have been used for delivering an anti-inflammatory drug, Indomethacin (IDMC). Kundu et al., used a simple desolvation technique for synthesizing silk nanoparticles in DMSO. The fibroin particles synthesized using fibroin from two different sources, Antheraea mylitta and B. mori, have an average size of 157 nm and 177 nm, respectively, in deionized water. The study has evaluated the in-vitro release of vascular endothelial growth factor (VEGF) from the nanoparticles and found that the maximum release was 35% on the fifth day.35 Lozano-P´erez et al., have shown the ability of silk fibroin nanoparticles to encapsulate and release the natural antioxidant quercetin, used as a free radical scavenger.156 Nanoparticles exhibited significant encapsulation efficiency of 70% with the quercetin loaded particle exhibiting a size of 171 nm. This was slightly higher than the size of unloaded nanoparticles (139 nm). We have synthesized protein nanoparticles (having size between 124 and 150 nm) by desolvation method using regenerated B. mori fibroin (Figure 7i). We have previously shown that deposition of nanoparticles fabricated from fibroin of A. mylitta using a similar desolvation method could suitably alter the topology of titanium implant surfaces so as to promote initial osteoblast adhesion and the sustained release of gentamicin (an anti-bacterial) from the nanoparticles could prevent bacterial infection and, hence, failure of implants, suggesting that multiple functionalities can be incorporated in the protein nanoparticles.157An exciting area of investigation is the fabrication of silk fibroin- magnetic nanoparticles by the methods such as suspension-enhanced dispersion by supercritical CO2. Such magnetic fibroin nanoparticles have been evaluated for



transdermal delivery and for systemic drug delivery.158,

159

Page 22 of 74

Drug delivery to a particular

organ/tissue is highly desirable to reduce the toxicity and non-specific effects associated with systemic exposure to drug. Therefore, silk fibroin nanoparticles have been conjugated with specific moieties for targeted drug delivery. For example, cyclic pentapeptide cRGDfk, iRGDEGFR antibody, SP5-52 and folic acid have been conjugated with silk nanoparticles to specifically target the tumor cells which overexpress the αvβ3 integrin receptor, EGFR receptor, and folate receptor, respectively.85,86,152,153 Table 3: Silk protein fibroin based nanoparticles: sizes, various drugs being loaded and specific target with brief description are outlined. S. No.

Nanoparticle size (nm)

Drug loaded

Target/remarks

Refs.

1

130

Paclitaxel

Human gastric cancer cell lines BGC-823 and SGC-7901

[160]

2

150-170

Endothelial growth factor

Murine squamous

[35]

cell carcinoma cells

3

100-200

Doxorubicin

MCF-7 human breast cancer cell line

[161]

4

150-160

Methotrexate

Feline fibroblast (AH927) cells

[162]

5

130

Doxorubicin

Multidrug resistant human breast cancer cells (MCF-7/ADR )

[163]

6

200-500

Paclitaxel

Prolonged drug release [164] for 2 weeks

7

200-500

Floxuridine

HeLa cells (Human cervical cancer cell line)


[165]

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8

40-120

Insulin

Enhanced resistance of [37] insulin to trypsin digestion

9

150–700

Curcumin

[166] Silk -curcumin nanoparticles showed better anti-aging properties compared to free curcumin drug

10

60-100

Arginylglycylaspartic acid (RGD) peptide

RGD loaded silk fibroin nanoparticles displayed intestinal anti-inflammatory properties

[167]

11

120

Salinomycin and Paclitaxel

Both the drug together showed superior inhibition of tumor growth compared to mono-therapy

[168]

12

209

Indocyanine- green dye

Image-guided photothermal therapy for glioblastoma

[169]

4.2 Albumin Being a major plasma protein, which biologically functions as a transporter of various proteins, albumin has an unambiguous edge over other biomaterials for fabrication of nanoparticles. Since it is a major transporter protein, albumin is able to bind to various drugs and peptide moieties through non-covalent interactions. In addition to non-covalent interactions, the presence of reactive groups like amino, thiol and carboxyl on the surface of albumin nanoparticles facilitate covalent ligand binding and surface modification.170 Furthermore, albumin has been found to be freely soluble in water and dilute saline with solubility as high as 40% (w/v) at pH 7.4. In addition, albumin is stable in the pH range 4.0 -9.0 and exhibits



Page 24 of 74

thermal stability at 60 °C up to 10 hours without any deleterious effects.44 Studies have shown that albumin accrues in solid tumors171 making it a potential macromolecule for site-specific drug delivery system for antitumor drugs. Reasons like high stability, high solubility in physiological fluids, biodegradability, non-immunogenicity, non-toxicity, availability in pure form and ease of preparation makes albumin an attractive macromolecular carrier for preparation of nanospheres and nanocapsules. Albumin can be isolated from a large number of sources including human, bovine, rat, and chicken. However, Bovine Serum Albumin (BSA) has been used most extensively for nanoparticle fabrication to facilitate controlled and prolonged release of drugs.172 Tacrolimus (TAC), clinically used as immunosuppressant to avoid organ rejection during organ transplantation, has been embedded in BSA nanoparticles.173These TAC embedded nanoparticles have been prepared by emulsificationdispersion technique and are found to be of size around 189.5 nm (diameter). The drug encapsulation efficiency has been assessed to be around 85%. The in vitro drug release from TAC-BSA nanoparticles exhibits a biphasic pattern with an initial burst and subsequently sustained and prolonged release which could possibly be due to the substrate inhibition action in indigenous proteases. BSA has been found to be an even better carrier for this hydrophobic drug as compared to commercially used PROGRAF® as the kidney uptake and nephrotoxicity is lesser in case of BSA-TAC nanoparticles. Albumin bound nanocarrier systems of size ~ 130 nm have been formulated to target tumors in various cases171 (Table 4). One such formulation, the albumin-bound paclitaxel (Abraxane®, ABI-008) has been approved by FDA for the clinical superiority of this approach in metastatic breast cancer.174 Table 4: Albumin based nanoparticles used in drug delivery: Their sizes in nm, the drug loaded and target cells are mentioned.


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S. No.

Size (nm)

Drug loaded

Target/remarks

Refs.

1

565-750

10-Hydroxycamptothecin (HCPT)

Stability and solubility of the loaded drug improved, efficient liver targeting

[68]

2

50-150

Doxorubicin

MOLT-4 Lymphoblastic leukemia cell line, MESSA/DX-5 Multidrug resistant uterine sarcoma cells

[122]

3

200-220

5-Flurouracil

Controlled and prolonged drug release (in-vitro) over a time period of 1600 min is achieved

[175]

4

70-80

Tamoxifen

Human breast adenocarcinoma MCF-7 cells

[176]

5

50-60

Curcumin

Human breast adenocarcinoma MCF-7 cells

[177]

6

145-200

Paclitaxel

Ovarian adenocarcinoma [178] SKOV-3 cells and Cervical adenocarcinoma HeLa cells

7

160-165

Doxorubicin

DOX loaded aptamerfunctionalized BSA nanoparticles show higher cellular uptake and strong cell inhibitory efficacy against cancerous MCF-7 cells

[179]

8

120

Beta-carotene

Chlorin e6 (Ce6) modified BSA nanoparticles caused tumor suppression effect when irradiated by 660-nm

[180]



Page 26 of 74

light. 9

200

Capsaicin (CAP)

CAP-loaded [181] albumin nanoparticles showed biphasic release with significant antioxidant activity in an in vitro assay

10

216

Etoposide and Berberine for dual drug delivery

A549 lung cancer cells

[182]

11

120-180

Gemcitabine

Gemcitabine loaded albumin nanoparticles were more effective than gemcitabine alone for inhibiting tumor growth

[183]

12

110

Entecavir and Glycyrrhetinic Acid co delivery

Glycyrrhetinic Acid enhanced accumulation of Entecavir in liver

[184]

13

100-278

Oxytocin

Brain delivery

[185]

4.3 Protamines Protamines have their origin in late haploid phase of spermatogenesis. These small arginine rich nuclear proteins (~4 kDa) replace histones and promote condensation and stabilization of DNA in sperm head as they allow a denser packaging of DNA in spermatozoon than histones. Protamines assist in carrying sperm DNA to the nuclei of the eggs after fertilization, where subsequently DNA is decompressed and used for protein synthesis.186 These proteins can easily be extracted from the mature salmon fish testicles or can be synthetically prepared. Protamines are exploited for decades for its use in therapeutics to counter heparin activity during surgery and as an excipient for slow release of insulin.45, 46 Taking inspiration from the natural biological function of protamines, Junghans et al, explored the idea of cellular delivery


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of oligonucleotides (ODNs) using protamine nanoparticles.187 They found that optimized concentration of these protamines/ODN mixtures self-assemble into particles (protamines being cationic molecules spontaneously assemble with negatively charged ODNs). These DNA/RNA oligonucleotide carrying protamine particles have come to be known as ‘proticles’. Many therapeutic molecules such as oligonucleotides for gene therapy and peptide based drugs for immunotherapy require a cytosolic delivery as either they have their receptors located in the cytosol or their active site is an intracellular organelle. This makes it necessary for them to voyage across the cytosolic compartment. However, short half-life of these drugs owing to their susceptibility to enzymatic degradation impedes their delivery by conventional methods. The cell penetrating properties of protamine make them a promising vector for cytosolic delivery of such therapeutic molecules resulting in minimum degradation.47 Since protamines possess certain sequences which mimic the nuclear localization signal, protamine-based nanoparticles can be exceptionally useful for nuclear delivery of therapeutic molecules, especially the oligonucleotides.188 Investigation in this field is a need of hour as oligonucleotide based therapeutic molecules as well as protein and peptide based drugs represents a fast growing and promising therapeutic class. Vasoactive intestinal peptide (VIP), a potent system pulmonary vasodilator, is a promising peptide molecule for treatment of idiopathic pulmonary arterial hypertension (IPAH).189,

190

The patients suffering from IPAH have an increased

number of VIP-receptors, namely, VPAC-1 as well as VPAC-2 in pulmonary blood vessels despite of low VIP serum concentration.189, 190 Encapsulation of vasoactive intestinal peptide into protamine-oligonucleotide nanoparticles (proticles) prevents its rapid enzymatic degradation in addition to maintaining a sustained drug release.189 VIP-loaded proticles (prepared by mixing oligonucleotide, VIP and protamine in aqueous phase in the mass ratio



Page 28 of 74

1:1:0.6) formed nanostructures by self-assembly under the influence of strong ionic interaction between biomolecules.191,

192

The formed protamine-stabilized nanoparticles possess a size

between 177 and 251 nm with an encapsulation efficiency of about 80% and an onsite VIP release of 77-87%.45 In another study, Pali-Schöll et al. report the use of protamine with CpGoligodeoxynucleotide as a tool in immunotherapy to prevent allergen-induced TH-2 response. The conventional alum adjuvants are reported to evoke an initial IgE-boost among several other side effects.192-195 On the other hand, the CpG-oligodeoxynucleotides (ODNs) direct the immune response towards TH-1 pathway. The CpG-ODNs are DNA sequences that possess CpG motives and consist of repetitive phosphorylated guanine and cytosine in the phosphodiester backbone. These CpG motives are readily recognized by TLR-9, which is expressed on plasmacytoiddentritic cells and B-cells in humans. Hence, CpG-ODNs induce a biased TH-1 immune response.90,

196

The study determines the immunological properties of

these CpG-ODN complexedproticles against allergen Ara H2 in mice model. The nanoparticles are prepared by self-assembly so as to make up the final concentration to 100 µg/ml CpGODNs and 300 µg/ml protamines in water. This approach yields nanoparticles of hydrodynamic diameter of ~215 nm and provides promising results in terms of immune modulatory effects of CpG-ODNs by directing the immune response of Ara H2 towards TH-1 pathway rather than inducing TH-2 response. This suggests that proticles could be promising biodegradable drug vector for immunotherapy to treat the allergic ailments. Some similar studies related to protamine nanoparticles are outlined in Table 5. Table 5: Protamine based nanoparticles in therapeutic delivery S. No.

Size (nm) Drug loaded

Target/remarks

Refs.

1

150-225

SCC7 cells

[197]

Heparin Derivatives


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2

275-365

Doxorubicin

MCF-7 cells

[198]

3

250

Tripterine

Increased the accessibility of the drug [199] molecules by overcoming mucosal barriers in rabbit model

4

120-135

Paclitaxel

C6 glioma cells

[200]

5

120-130

Secretoneurin

Good loading efficiency of up to 65% in proticle matrix

[201]

6

110-130

5-Fluorouracil

A547 cells and HeLa cells

[202]

4.4 Gliadin Nanoparticles of vegetal origin are prepared from proteins like wheat gluten derived gliadin and pea seed derived vicilin or legumin for biological applications.48 Gliadin, a major protein present in wheat gluten is a water-insoluble protein and is often extracted from gluten using ethanol. Gliadin has peculiar mucoadhesive properties, which make it a suitable polymer for designing oral and topical drug carriers capable of anchoring to the mucus layer.49 The use of Gliadin as a nanoparticle material is attributed to its biocompatibility, biodegradability, natural origin and being a plant protein, a high certainty to be free of prions. Gliadin nanoparticles (GNP) show an affinity towards the upper gastrointestinal regions, while their presence is found to be low in other gastrointestinal regions making them a suitable polymer for targeted drug delivery.20 3This protein is rich in lipophilic and neutral residues, which can promote interaction within biological tissue by hydrophilic – hydrophilic interaction. At the same time, gliadin forms strong hydrogen bonding with the mucosa because of neutral amino acids. Gliadin’s hydrophobicity and low solubility in aqueous phase permits fabrication of nanoparticles that have ability to protect the loaded drug and support controlled release.204 Ezpeleta et al. demonstrated preparation of a gliadin-based nanocarrier system for the administration of all-



trans-retinoic acid (RA).

Page 30 of 74

All- trans-retinoic acid leads to increase in proliferation and

differentiation of epithelial tissues, and is used for the treatment of psoriasis, acne, ichthyosis, hyperkeratosis, and epithelial tumors by reduction of the size of sebaceous glands as well as sebum secretion.48 Retinoic acid-loaded gliadin nanoparticles, prepared by desolvation method, are found to be of size ~500 nm (diameter). This method results in fabrication of gliadin nanoparticles with a yield of ~90% of the initial protein. These nanoparticles are stable for over 4 days in PBS, pH 7.4. However, they are observed to be degraded rapidly over 3 hours in PBS solution containing trypsin, which can be countered by chemical cross-linking of nanoparticles with glutaraldehyde. The nanoparticles show an entrapment efficiency of 75-97% RA subjected to RA to initial protein ratio. The in vitro drug release profile of the RA-gliadin nanocarrier system is found to be biphasic in nature with an initial burst, which releases about 20% of the loaded drug, followed by zero-order diffusion with a drug release rate of 0.065 mg/h. In another study, gliadin nanoparticles are used to administer cyclophosphamide, a cyclic phosphamide ester of mechlorethamine known to cross link DNA and prevent cellular division, to induce apoptosis in breast cancer MCF-7 cells.90 Desolvation followed by electrospraying technique, yields cyclophosphamide-loaded gliadin nanoparticles of size ~220 nm and a drug loading efficiency of ~72%. The drug release pattern of this nanocarrier system is assessed to be biphasic in nature with an initial burst followed by steady release of cyclophosphamide from the nanocarrier over a period of 48 hours. The bi-phasic release occurs most likely due to the initial quick release of drug present on the surface of nanoparticles in the aqueous medium which is followed by the diffusion of the drug from the inside of the nanoparticles.90 Owing to its mucoadhesive properties, gliadin is particularly explored for drug delivery against gastric pathogens, including Helicobacter pylori, which cause gastric ulcers as well as gastric


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adenocarcinoma. Such gastric pathogens often penetrate the gastric mucus, resulting in the evasion from conventional therapeutic drugs during treatment regimen. Gliadin nanoparticlesmediated delivery of amoxicillin, clarithromycin and omeprazole as single therapy or triple therapy is shown to be highly effective in controlled and targeted release of these antimicrobial drugs and in eradication of gastric pathogens.205-208 4.5 Legumin Legumin is one of the main storage proteins in pea seeds (Pisumsativum L.) which belongs to 11s globulin group of proteins. It has a molecular mass between 300 and 400 kDa.50 Legumin is an aluminous protein and is rich in acidic amino-acids. It is composed of six subunits held together forming an oligomeric structure. The solubility of this protein is substantially reduced during coacervation process. This leads to phase separation and subsequently the formation of nanoparticles. Nanoparticles derived from legumin are bioadhesive in nature and have large surface area which is an indicative of high interactive potential with biological surfaces.50 Mirshahi et al. have attempted to fabricate a colloidal carrier system from legumin in the form of micro- and nanoparticles in order to achieve sustained release and targeted delivery of drugs. It is shown that legumin after aggregation and chemical crosslinking with glutaraldehyde, has a tendency to bind together to form nanoparticles.51 In an initial attempt to fabricate nanoparticles from legumin, pH-coacervation method followed by glutaraldehyde cross-linking has been tried which enables avoiding the organic solvents and yields nanoparticles of size nearly 250 nm in diameter as assessed by scanning electron microscope.51 However this method yields as low as 27% of the initial protein as nanoparticles. These nanoparticles are observed to be quite stable in PBS (pH 7.4) and follow zero-order degradation.51 Glutaraldehyde concentration is reported to have no significant role in the size, percentage



Page 32 of 74

yield and surface charge of the nanoparticles, however, longer half-life of nanoparticles can be obtained by an increase in glutaraldehyde concentration. Initial drug release studies with methylene blue, used as model hydrophilic drug, show a biphasic drug release pattern with rapid initial release followed by slower release rates. The second phase of drug release is found to be inversely proportional to the degree of cross-linking. Importantly, cross-linking with glutaraldehyde reduces the antigenic epitopes of legumin resulting in reduced immunogenicity of the protein.209Even though legumin based nanoparticles have small size, good stability and low antigenicity, further studies need to be carried out for optimizing legumin nanoparticle yield and establishing its usefulness in biomedical applications. 4.6 Collagen Collagen is considered as one of the most abundant proteins present in the animal body representing almost 30% of the total protein mass. It is fibrous in nature and occurs in the form of elongated fibrils. Collagen is especially found in the connective tissues and flesh of mammals including bones, blood vessels, skin, cartilage, cornea, ligament, and tendons. Structurally, collagen possesses a triple helix structure, which is generally made up of two homologous chains (α-1) and one supplementary chain (α-2) that varies slightly in its chemical composition.210, 211These chains are polypeptide in nature and are coiled around one another in a cable form. Each has a distinct turn in the reverse direction. They are connected together mainly by hydrogen bonds between CO- and NH- groups. The molecular weight of collagen is 300 kDa and its structure is rope shaped having a length of 300 nm and a width of 1.5 nm.210,211 Collagen plays a significant role in the formation of organs and tissues and is therefore, widely used in medical field for regeneration and drug delivery applications. Collagen is involved in various functions of cells. It is a material of choice in biomedical applications due to its


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biodegradability, biocompatibility, non-antigenicity, an ability to easily penetrate a lipid-free interface as well as self-aggregation and cross-linking properties.52 Collagen-based nanoparticles are thermally stable and can be easily sterilized.53 Collagen nanoparticles have been prepared for systemic drug delivery of theophylline, used as a model drug.54 Collagen nanoparticles, prepared by emulsification method have been explored for sustained and slow release of lipophilic steroids such as glucocorticoids55. 17β-estradiol-hemihydrate loaded collagen nanospheres of mean diameter of 123 nm are reported to exhibit a prolonged estradiol release in hormone replacement therapy.212 It is apparent that collagen nanoparticles with a controllable size can be synthesized with its large surface area and good drug binding properties. 4.7 Gelatin Gelatin is a natural water-soluble polymer that can be obtained by hydrolysis of collagen in either alkaline or acidic medium or by thermal or enzymatic degradation of collagen.213 The primary and most abundant source of gelatin is porcine skin, followed by bovine skin.57 Gelatin contains both cationic and anionic groups and has a triple helical structure because of the presence of repeating triplets of glycine, alanine and proline residues.213 It is widely recognized as a biodegradable, biocompatible and multifunctional biopolymer and hence, finds various applications in medical and pharmaceutical industries.58,

59

First commercially used

gelatin dates back to 1685214 and later in World War I for intravenous infusion.215 Gelatin is an FDA approved polymer that has been classified as “Generally Recognized as Safe” (GRAS). Owing to its established evidence of safety, it is used as a food supplement and in intravenous infusion as plasma expanders, commercially available as Gelafundin™, Gelafusal™. To use gelatin, it must be crosslinked physically, biologically or chemically by various cross-linking



agents such as glutaraldehyde, glyceraldehyde60,

216

Page 34 of 74

etc. so as to increase its mechanical

strength and lower its degradation rate and solubility in aqueous solutions.217 Gelatin-based nanoparticles, prepared by desolvation method, require subsequent cross-linking to enhance their stability.218 The gelatin nanoparticles are widely used for encapsulating several biologically active molecules like bovine serum albumin (BSA), BMP-2 and bFGF growth factors as well as DNA and RNA oligonucleotides.120, 219-221 Li et al. encapsulated BSA as a model therapeutic protein molecule, the release of which is shown to be diffusion-controlled.220 Similarly, the studies on the release of various growth factors such as BMP-2 and bFGF in vitro (used to inhibit osteogenesis) show that the release rate is highly dependent on degree of gelatin cross-linking rather than gelatin type.120 Depending on the preparation methods viz. alkaline hydrolysis or acid hydrolysis of collagen, gelatin obtained from collagen degradation exhibits variable isoelectric points. The release profile of a particular molecule in a specific medium may be modulated by the isoelectric point of the gelatin used for encapsulating that molecule. Gelatin nanoparticles can be surface-modified to carry DNA and RNA oligonucleotides via ionic interactions.221 The loading capacity of gelatin nanoparticle in these cases is observed to be dependent on incubation medium and nanoparticle zeta potential. Lu et al., prepared Paclitaxel loaded gelatin nanoparticles sized from 600 nm to 1000 nm which are found to be capable of targeting and killing human RT4 bladder transitional cancer cells.222 Gelatin can be easily conjugated with synthetic polymers such as PEG. The PEGylated gelatin nanoparticles improve the plasma half-life of the encapsulated hydrophobic molecules and anti-inflammatory drugs.79 These nanoparticles are also used to encapsulate chloroquine phosphate, an anti-malarial drug, thereby, reducing the side effects caused by its systemic exposure.223 Chloroquine phosphate release from gelatin nanoparticles is shown to occur via


Page 35 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


diffusion in a manner that increases with an increase in temperature. The diffusion decreases in physiological fluids and is found to have an optimal release rate near the physiological pH (7.4). Addition of cross-linker glutaraldehyde to the nanoparticles decreases the drug release rate.223A major property of gelatin nanoparticles is their accumulation in macrophages and macrophages rich organs and the ease with which gelatin nanoparticles can crossover the conventional blood-brain barrier.224 Gelatin, therefore, is extensively used as a vector for delivering various anti-cancer drugs, herbal extracts and therapeutic bio-macromolecules (Table 6). Table 6: Gelatin based nanoparticles in drug delivery S. No.

Size (nm) Drug loaded

Target/remarks

Refs.

1

600-1000 Paclitaxel

Human RT4 bladder cancer cells

[222]

2

300-9000 Paclitaxel

Accumulation in organs especially in

[225]

liver, small intestine, and kidney 3

100-200

Paclitaxel

Maximum drug release in alkaline environment and lower drug release at acidic conditions

[226]

4

110

Cisplatin

Higher drug retention in tumor compared to free drug

[227,228]

5

40–200

Cardamom extract

Human glioblastoma U87MG cell lines

[229]

6

150–250

Gemcitabine

Panc-1 human pancreatic ductal adenocarcinoma cells

[230]

7

150-250

wt-p53 tumour suppressor gene

Panc-1 human pancreatic adenocarcinoma cells

[231]

8.

150

Tramadol HCl

Study shows gelatin as a suitable carrier for sustained release of water-

[232]



Page 36 of 74

soluble drugs 9

182-188

Sesamol

MCF-7 breast cancer cells

[233]

10

40–200

Cardamom extract

Human glioblastoma cancer U87MG cells

[234]

4.8 Recombinant proteins Advancements in the field of recombinant technology have opened a whole new platform to exploit proteins of different functionalities and properties for various applications.235-237 We can now exploit microorganisms and various other cell types as biofactories to produce proteins of interest in order to save time and minimize immunological response. This approach is a boon when the availability of native protein is limited in nature, extraction process is expensive or the protein has undesired immunogenic response in the host. Few of these proteins like Elastin like polypeptides (ELPs) and virus shell proteins (VSPs) have grown popular over few recent years.69,238-243 4.8.1 Elastin Like Polypetides (ELPs) Elastin is a naturally occurring protein that is an essential component of extracellular matrix of tissues. Primary sequence of elastin is composed of repetitive conserved motifs VGVAPG, VPGG, APGVG and VPGVG. Elastin possess elasticity and it can also inflect cellular behavior to promote tissue repair.244 The cost factor associated with large scale isolation of elastin from its natural source switched the focus to recombinant Elastin like polypeptides (ELPs) that is both

structurally

biocompatibility.66,

and 245

functionally

similar

to

elastin

and

possess

exceptional

ELPs are thermosensitive biopolymers that remains hydrated below a

temperature known as the transition temperature (Tt).246,247 On introduction to temperature


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above Tt, their hydrophobic domains interact and self-assemble to form highly ordered structures in the form of nanoparticles.248 Besides temperature, the protein concentration, pH of the solution and ionic strength determines the designing of ELP nanoparticles.249 E. coli is the most commonly used system for large scale production of ELP while other biosystems such as yeast and plants are also used as ELP biofactories.246,250,251 ELPs are used to coat highly hydrophobic materials to support cellular growth and to improve their functionality.244 ELPs have been used as vehicles for drug delivery.252-256 ELP-based drug delivery systems have been developed for controlled release of peptide drugs to treat type 2 diabetes.257 Engineered nanoformulations of hydrophobic drugs conjugated to the C-terminus of ELPs have also been developed by self-assembly in order to promote tumor regression in mouse models.258 Wu et al. described the synthesis of stimuli-responsive nanoformulations of ELPs by electro spraying to construct ELP nanoparticles for encapsulation and activated release of doxorubicin.259. Herrero-Vanrell et al. investigated Poly (VPAVG) micro-and nanoparticles as a system for the controlled release of Dexamethasone phosphate (DMP). Poly (VPAVG) was prepared as stable particles by self-assembly with a size < 3 μm above its transition temperature (∼30 °C). These self-assembled particles were able to encapsulate appreciable amount of the drug when selfassembling was carried out in a co-solution of polymer-DMP.260, 261 Recombinant technology approach allows the production of fusion proteins with ELPs to integrate multiple features in the fabricated nanoparticles. For instance, engineered fusion proteins comprising of Stromal cell-derived factor-1 (SDF1) and ELP were produced which self-assembled to form nanoparticles of size 600 nm262. When evaluated for wound healing in diabetic mice, the biological activity of SDF1-ELP nanoparticle in vivo was found to be significantly superior to that of free SDF1 or free ELP.262 ELP peptide unit expressed as a fusion protein with



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hydrophilic aspartic acid-rich peptide unit has been shown to form nanoparticles of diameter 40 nm at temperature above transition phase temperature.

263

These nanoparticles displayed

antibody binding domains on their surfaces, which allowed immobilization of antibodies and later formation of large, visually detectable complexes in the presence of target molecule (antigen) suggesting the usefulness of ELP based nanoparticles in homogeneous turbidity immunoassays.263 4.8.2 Virus like Particles (VLPs) Engineering viral capsids as noninfectious protein-based nanoparticles is a nascent field of research and nanoparticles, thus, fabricated are termed as virus-like particles or VLPs. VLPs are coat proteins of viral capsids that that form homogeneous nanoparticles by self-assembly. Since VLPs consist only of the coat proteins and are devoid of genome of Virus particles, they are typically noninfectious. VLPs have been shown to be highly effective delivery system that have potential to overcome many of the challenges encountered by nanoparticles for delivery of therapeutic cargo. VLPs can encapsulate and deliver therapeutic molecules such as proteins, small peptide drugs, siRNA and chemotherapeutic drugs.69,264-270 PEGylated VLPs have also been formed so as to modify the surface of the particle and to add a variety of functional ligands which improves their affinity to targeted site and reduce phagocytosis.265,271 VLPs that are being actively developed originate from three main sources, namely: Animal virus, Bacteriophages and Plant viruses. Animal virus derived VLPs are mostly engineered from Hepatitis B virus. This virus can be engineered to fabricate two different types of VLPs, one which is derived from the assembly of core internal protein capsid and the other derived from using surface antigens which have a capability to interact with lipids to form nanoparticles. 272274

Bacteriophages like MS2, Qβ and P22 have also been used to derive VLPs. All of the three


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are composed of nucleic acid filled capsid and can be produced in hosts including E. coli and yeast cells or can be alternatively synthesized as cell free proteins. Most of these viral capsids can be enriched either by size-exclusion chromatography, differential centrifugation, or immobilized metal affinity chromatography and can be self-assembled into VLPs.273,275-281 Virus of plant origin including cowpea chlorotic mottle virus (CCMV) and cowpea mosaic virus (CPMV) have been investigated to engineer VLPs. CCMV VLPs can be produced in yeast or E. coli cells and purified using size exclusion chromatography or immobilized metal affinity chromatography.282,283 However CPMV VLPs are produced using insect and plant cells .284, 285 MS2 derived VLPs have been successfully employed to encapsulate chemotherapeutic drugs taxol and doxorubicin. Taxol encapsulated MS2 VLPs of size ~50nm in diameter were found to be thermally stable upto 64 °C.264,

286

Nucleic acid-loaded VLPs have been

synthesized for several uses including delivery of vaccines and vaccine adjuvants,287 gene288, micro RNA289-291, and gene knockdown systems264,292 For instance, the suitability of MS2 VLP for siRNA delivery was recently explored. MS2 VLPs were loaded with a cocktail of siRNAs that were able to silence expression of cyclin A2, B1, D1 and E1 in cells. It was found that siRNA-loaded VLPs were stable for more than 3 months in PBS at 4 °C and protect encapsidated siRNA from RNase mediated degradation. These gene silencing systems were able to induce apoptosis in > 90% of Hep3B cells within 36 hours at a siRNA concentration of 150 pM without substantially affecting the viability of normal hepatocytes.264

5. Conclusion and future prospects



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It is evident that proteins from a variety of sources can be fabricated as nanoparticles using easy, cost-effective and eco-friendly synthesis process with less use of toxic chemicals. Each protein has a well-established natural mechanism for their metabolization which leads to facile degradation of nanoparticles within the host with non-toxic by-products. They have the potential to be used as the natural counterpart to the synthetic and inorganic sources available for the fabrication of nanoparticles for targeted drug delivery applications. This review highlights that protein nanoparticles, as a vector, possess various advantages and applications in delivering anti-cancer drugs, peptide hormones, growth factors and even in the delivery of genetic material like DNA and RNA. Several approaches are applied to functionalize the surfaces of the protein nanoparticles with desired ligands. These include fabrication from modified biomaterials, coating nanoparticles with synthetic polymers post-fabrication and using non-covalent binding to place ligands on surface of nanoparticles. One of the primary goals of surface modification of nanoparticles is the site specific drug delivery. Some of the major concerns with the use of protein nanoparticles include (i) Mostly obtained in variable size range, (ii) structural changes may change the original property of native protein, (iii) Presence of endotoxin while obtaining recombinant protein from a bacterial expression system (iv) Low yield of some protein based nanoparticles, (v) can cause in-vivo inflammation or transmission of prions in some cases (vi) some proteins like protamine and ELPs are rapidly degraded(vii) nanoparticles fabricated from proteins mostly exhibit biphasic drug release pattern with initial burst release. A lot of effort is being devoted to overcome these challenges. Low yield in case of protein nanoparticle can be countered by using the recombinant protein approach along with further research on improving its downstream processing. Presence of endotoxin in recombinant proteins can be prevented by using yeast or plants as an expression


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system. Blending of protein with other biocompatible polymers can be used to tailor physicochemical properties of protein nanoparticles and address the problems of rapid degradation and initial burst drug release. Generally for any product to be commercially successful it needs to have an easy synthesis mechanism within a feasible cost. Approval by FDA of certain protein based products such as Abraxane®, ABI-008 (Paclitaxel albumin bound nanoparticles) for metastatic breast cancer and Gelafundin™, Gelafusal™ as plasma expanders for intravenous infusion has created a landmark in the history for the development of protein nanoparticles as drug carriers. From a technological perception, the extended pharmacokinetic studies and a better understanding of various key factors involved in controlling the release rate along with devising ways to prolong the drug release can be highly beneficial. These investigations will prove instrumental in turning the idea of protein nanoparticle technology into a practical application as the next generation of drug delivery system. Acknowledgement: This work is supported by the financial grant support by SERB-Department of Science and Technology (project number ECR/001173/2016), Department of Biotechnology (project number BT/PR18562/BIC/101/424/2016) and University Grants Commission (project: F.30301/2016 (BSR)), Govt. of India to SK. SCK presently holds an ERA Chair Full Professor position at 3B´s Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Portugal, supported by European Union Framework Programme for Research and Innovation HORIZON 2020 under grant agreement nº 668983 FoReCaST. Authors’ contributions SK and SCK designed the concept, AJ, and SKS wrote the paper along with SK and SKA. SCK reviewed the manuscript, SK and SCK revised the manuscript. All the authors discussed, commented, revised and approved the final manuscript. Conflict of interests



Authors declare no conflict of interest.


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FIGURE LEGENDS Figure 1: Emulsion/Solvent Extraction technique for preparing protein nanoparticles: aqueous protein phase and non-aqueous (oil) phase are combined and emulsified to yield protein nanoparticles. The thermal stabilization of the nanoparticles is also shown. T, Temperature Figure 2: Fabrication of protein nanoparticles by desolvation method. Organic solvents are most commonly used as desolvating agents. Figure 3: Preparation of protein nanoparticles by salt precipitation. Figure 4: Fabrication of protein nanoparticles by polyelectrolyte complexation method: The pH of the protein solution is varied to be well above or below its isoelectric point and is then complexed with oppositely charged poly-ion to form coacervates. Figure 5: Production of protein nanoparticles by electrospraying method. Also known as nanospraydrying, this method leads to drying and particle formation in one single-step. Figure 6: Schematic diagram of the process for isolation of silk fibroin protein from Bombyx mori silkworm cocoons by degumming (removal of silk protein sericin) them in Na2CO3 solution. Figure 7: (i) Synthesis of Fibroin nanoparticles by using desolvation method. Transmission electron micrographs of synthesized nanoparticles is shown, scale bar = 100 nm. (Kapoor S, Unpublished data) (ii) Fibroin nanoparticles were deposited on Titanium surface and the morphology, roughness and hydrophobicity were compared with bare Ti surface. (A) Scanning electron microscopic image of bare and fibroin deposited Ti implant surface. Magnified image is in inset. (B) Atomic force microscopy of specified area of bare and nanoparticle deposited Ti surface. (C) Water droplet contact angle measurement of bare and nanoparticle deposited Ti surface. (iii). The osteoblast adhesion promoting ability of fibroin nanoparticles (alone (Ti-Np) and antibacterial gentamicin loaded (Ti-GNp)) deposited on Ti surface is compared with bare Ti surface. (A) The adhesion of osteoblast cells was determined at indicated time points Hoechst staining of nuclei. The arrows denote nuclei. (B) Live/Dead analysis of osteoblast cells after 3 day of the seeding. TCP, tissue culture plate. (ii and iii, reproduced with permission from reference [157], Copyright, 2016 Elsevier Inc).


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Protein nanoparticles: Promising platforms for drug delivery applications Annish Jain, Sumit K Singh, Shailendra K Arya, Subhas C Kundu, Sonia Kapoor


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Protein nanoparticles: Promising platforms for drug delivery applications

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