Protein-based nanocarriers as promising drug and gene delivery systems

Journal of Controlled Release 161 (2012) 38–49

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Journal of Controlled Release

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Review

Protein-based nanocarriers as promising drug and gene delivery systems

Ahmed O. Elzoghby a, Wael M. Samy b, Nazik A. Elgindy a,⁎a Department of Industrial Pharmacy, Faculty of Pharmacy, Alexandria University, El-Khartoum Square, Azarita, Alexandria 21521, Egyptb Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Beirut Arab University, Beirut, Lebanon

⁎ Corresponding author. Tel.: +20 3 4878248; fax: +E-mail address: [email protected] (N.A. Elgindy)

0168-3659/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.jconrel.2012.04.036

a b s t r a c t
a r t i c l e i n f o
Article history:Received 30 January 2012Accepted 23 April 2012Available online 28 April 2012

Keywords:Protein nanocarriersAnimal proteinsPlant proteinsDrug deliveryGene delivery

Among the available potential colloidal drug carrier systems, protein-based nanocarriers are particularly in-teresting. Meeting requirements such as low cytotoxicity, abundant renewable sources, high drug binding ca-pacity and significant uptake into the targeted cells, protein-based nanocarriers represent promisingcandidates for efficient drug and gene delivery. Moreover, the unique protein structure offers the possibilityof site-specific drug conjugation and targeting using various ligands modifying the surface of proteinnanocarriers. The current review highlights the main advances achieved in utilizing protein nanocarriers asnatural vehicles for drug and gene delivery tasks with respect to types, advantages, limitations, formulationaspects as well as the major outcomes of the in vitro and in vivo investigations. The recently emerged tech-nologies in the formulation of protein nanocarriers including using recombinant proteins as alternatives tonative ones and new non-toxic crosslinkers as alternatives to the toxic chemical crosslinkers are alsodiscussed.

© 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382. Animal proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

2.1. Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392.2. Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402.3. Albumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402.4. Milk proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

2.4.1. Casein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412.4.2. Whey proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

2.5. Silk proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422.6. Elastin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

3. Plant proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433.1. Zein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433.2. Gliadin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443.3. Soy proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443.4. Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

4. Advantages and pharmaceutical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465. Drawbacks and challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

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rights reserved.

1. Introduction

In recent years, there has been a considerable interest in the de-velopment of novel drug delivery systems using nanotechnology[1,2]. Polymeric materials used for preparing nanoparticles for drugdelivery must be at least biocompatible and best biodegradable. The

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Fig. 1. Scanning electron micrographs of small interfering RNA (siRNA)-encapsulatedtype B gelatin nanoparticles and siRNA containing NiMOS [32].

39A.O. Elzoghby et al. / Journal of Controlled Release 161 (2012) 38–49

use of natural polymers has been very well described in the literaturefor fabrication of nanoparticles [3,4]. Obviously, there is growing in-terest in developing protein nanocarriers as GRAS (generally reg-arded as safe) drug delivery devices due to their exceptionalcharacteristics, namely biodegradability, nonantigenicity, high nu-tritional value, abundant renewable sources and extraordinary bind-ing capacity of various drugs. Proteins have the possibility of lessopsonization by the reticuloendothelial system (RES) through anaqueous steric barrier in addition to their excellent functional prop-erties including emulsification, gelation, foaming and water bindingcapacity [5–7]. Moreover, protein nanoparticles can be easily pre-pared and scaled up during manufacture [6,7].

Owing to multiple functional groups in the primary sequences ofpolypeptides, protein nanoparticles can be exploited to create differ-ent interactions with therapeutic compounds and subsequently formthree-dimensional networks offering a variety of possibilities for re-versible binding of active molecules, protecting them in a matrix aswell as specific targeting to the site of action [6,7]. Furthermore, pro-tein nanoparticles possess acceptability as metabolizable naturallyoccurring components. Hydrolysis of proteins by digestive enzymesgenerates bioactive peptides that may exert a number of physiologi-cal effects in vivo [5]. This review embodies an in-depth discussionof the nanoparticulate drug delivery systems that make use of pro-teins as drug carriers.

2. Animal proteins

Animal proteins represent good raw materials since they have theadvantages of synthetic polymers and the advantages of absorbabilityand low toxicity of the degradation end products [8].

2.1. Gelatin

Gelatin is a denatured protein that is obtained from collagen byacid and alkaline hydrolysis. It has a long history of safe use in phar-maceuticals, cosmetics, as well as food products and it is consideredas GRAS material by the FDA [9]. It has a relatively low antigenicitybecause of being denatured. Its functional groups are accessible forvarious chemical modifications, which may be especially useful indeveloping targeted drug delivery vehicles [10]. Gelatin is a poly-ampholyte having both cationic and anionic groups along withhydrophobic groups. The repeating sequences of glycine, prolineand alanine amino acid triplets are responsible for its triple helicalstructure [11]. Gelatin nanoparticles have been reported for suc-cessful delivery of various drugs including anticancer [8,12], anti-HIV [13], antimalarial [14], antimicrobial [15,16], skeletal musclerelaxant [16], analgesic [17], anti-inflammatory [18], anti-diabetic[19,20], topical ophthalmic [21] drugs, protein synthesis inhibitors[22], tissue-type plasminogen activator [23] as well as gene delivery[10,24]. Various methods including desolvation [25], coacervation-phase separation [12], emulsification-solvent evaporation [14], re-verse phase evaporation [26] and nanoprecipitation [16] havebeen used to prepare gelatin nanoparticles.

Desolvation of native gelatin by a desolvating agent (e.g., alcoholor acetone) produces large nanoparticles with a wide size range.Therefore, a two-step desolvation process was adopted by Coesteret al. [25] to discard low MW gelatin molecules and then precipitatesmaller nanoparticles with a narrow size distribution. Although theuse of glutaraldehyde (GA) as a crosslinker leads to improvementof the mechanical properties and stability of nanoparticles, its hightoxicity may limit the applications of the final product. Therefore,the use of non-toxic crosslinking agents was emerged as an alterna-tive [17]. Genipin is a naturally occurring crosslinking agent with anegligible cytotoxicity (~10,000 times less than GA) [27]. Recombi-nant human gelatin (rHG) nanoparticles were prepared using genipinas a crosslinker and showed a great potential for protein drug delivery

in terms of sustained release, less initial burst and safety. About 50% ofthe model protein (FITC-BSA) was released from the nanoparticlesover 10 days [28]. In another study, a mixture of a water solublecarbodiimide (CDI) and N-hydroxysuccinimide (NHS) was usedby Qazvini and Zinatloo [17] as a non-toxic crosslinker of gelatinnanoparticles. Using paracetamol as a model drug, both drug en-trapment and loading efficiency were higher in the CDI/NHScrosslinked nanoparticles (27 and 7.3%); than that of nanoparticlescrosslinked with GA (10 and 4%), respectively [17]. The recombinantenzyme microbial transglutaminase, an acyltransferase that formsintra- and intermolecular isopeptide bonds in and between many pro-teins by crosslinking the ε-amino groups of the amino acid lysine tothe side chain amide group of glutamine, was used by Fuchs et al. [29]to crosslink gelatin nanoparticles producing particles of defined sizebelow 250 nm and narrow size distribution.

A coacervation-phase separation method was used to preparepaclitaxel-loaded gelatin nanoparticles where an aqueous solutionof sodium sulfate was added slowly to gelatin solution containingTween 20 followed by addition of isopropanol containing paclitaxel[12]. Compared to the conventional paclitaxel formulation whichuses cremophor/ethanol as solubilizers, the nanoparticles showedlonger retention and higher accumulation in organs and tissues (av-erage of 3.2-fold). Gelatin nanoparticles were also prepared via a singlewater-in-oil emulsification/solvent evaporation method by mixing anaqueous phase of both gelatin and drug with an oil phase of polymeth-ylmethacrylate then crosslinked with glutaraldehyde saturated toluene[14]. A reverse phase evaporationmethod was used by Gupta et al. [26]to prepare gelatin nanoparticles (~37 nm) inside the inner aqueouscore of reverse micellar droplets formed by dissolving the surfactantsodium bis(2-ethylhexyl) sulfosuccinate (AOT) in n-hexane [30]. In an-other study, Lee et al. [16] prepared drug-loaded gelatin nanoparticlesby nanoprecipitationmethod usingwater as a solvent, ethanol as a non-solvent and poloxamer as a stabilizer. About 80% of loaded tizanidinehydrochloride was released around 15 h after start-up of the releaseexperiment. Recently, a novel water-in-water emulsion technique wassuccessfully used by Zhao et al. [20] to prepare D,L-glyceraldehyde-crosslinked gelatin-poloxamer 188 nanoparticles as insulin pulmonaryadministration system. The nanoparticles promoted insulin pulmonaryabsorption effectively and showed good relative pharmacological bio-availability [20].

40 A.O. Elzoghby et al. / Journal of Controlled Release 161 (2012) 38–49

Bhavsar and Amiji [31] developed a uniquemulticompartmental oralDNA delivery system termed “nanoparticles-in-microsphere oral sys-tem” or NiMOS based on encapsulation of type B gelatin nanoparticlesin poly(ε-caprolactone) (PCL)microspheres. A schematic representationof NiMOS is illustrated in Fig. 1 [32]. NiMOs were able to protect orallyadministered DNA during transit from the stomach and release thenanoparticles in the small and large intestine [31,32].

Recently, gelatin nanocomplexes with polysaccharides or synthet-ic polymers were successfully utilized for drug delivery. Zorzi et al.[33] developed new hybrid nanoparticles via ionic gelation betweencationized gelatin and the anionic polysaccharides, dextran sulfateand chondroitin sulfate for delivery of plasmid DNA (pEGFP) to theocular surface. In another study, cisplatin-loaded gelatin/poly(acrylicacid) nanoparticles were successfully prepared where a complex wasformed between platinum of cisplatin and carboxylic groups in thenanoparticles [34]. Cisplatin-loaded nanoparticle formulation was su-perior to free cisplatin in anticancer effect on murine hepatic H22tumor-bearing mice model. In another study, composite nanoparticlesbased on glycidyl methacrylated dextran and gelatin were fabricatedby a facile synthesis method assisted by self-assembly without usingany organic solvents where the release of bone morphogenetic protein(BMP)wasmaintained formore than 12 days under degradation condi-tions by dextranase [35]. Recently, nanoparticles were fabricated viaself-assembly between partially purified ellagitannins (PPE) and gelatin[36]. The factors including PPE-to-gelatin mass ratio, pH, temperatureand reaction time strongly influenced the fabrication of nanoparticles.

2.2. Collagen

Collagen is the primary structural material of vertebrates and is themost abundant mammalian protein accounting for about 20–30% oftotal body proteins [37]. It has beenwidely used inmedical applicationsdue to its biodegradability, weak antigenecity and superior biocompat-ibility [37]. However, some disadvantages of collagen-based systemsarose from the difficulty of assuring adequate supplies and their poormechanical strength. Improvement of the physical and chemical prop-erties can overcome some of the drawbacks in collagen-based applica-tions [38].

Formation of collagen nanospheres was driven by electrostaticforces with sodium sulfate employed as a desolvating agent to facilitategreater charge–charge interactions between plasmid DNA and collagen[39]. The biodegradable collagen-based nanospheres were thermallystable, readily achieving their sterilization. Moreover, nanoparticlescould be taken up by the RES and enabled an enhanced uptake of exog-enous compounds, such as anti-HIV drugs, into a number of cells, espe-cially macrophages, whichmay be an additional advantage of collagen-based nanoparticles as a systemic delivery carrier [40]. Due to theirsmall size with a large surface area, high adsorptive capacity and abilityto disperse in water to form a clear colloidal solution, collagen-basednanoparticles have been used as a sustained release formulation for an-timicrobial agents or steroids [41]. Delivery of hydrocortisone from col-lagen nanoparticles was not affected by the pH of the receptor mediumor its binding affinity to the particles [37].

Collagen nanoparticles were used to enhance dermal delivery ofretinol and facilitated a faster and higher transportation of the incor-porated drug through the skin than the freshly precipitated drug [37].Similarly, nanoparticles of Chondrosia reniformis sponge collagen en-hanced the transdermal delivery of 17β-estradiol hemihydrate inhormone replacement therapy [42]. The drug-loaded nanoparticlesincorporated in a hydrogel enabled a prolonged estradiol releaseand yielded considerably enhanced estradiol absorption comparedto a commercial gel free from nanoparticles. Recently, chitosanmicro-spheres loaded with BMP-2-derived synthetic peptide were incorpo-rated into a porous nano-hydroxyapatite/collagen/poly-L-lactic acid(PLLA) scaffold. The synthetic peptide was released in a temporally

controlled manner, depending on the degradation of both incorporat-ed chitosan microspheres and PLLA matrix [43].

2.3. Albumin

Over the past decades, albumin has emerged as a versatile macromo-lecular carrier for therapeutic and diagnostic agents. Albumin has beenshown to be non‐toxic, non-immunogenic, biocompatible, biodegradableand metabolizable in vivo into innocuous degradation products [44,45].In addition, it is likely that endogenous albumin, with the half-life of19 days in the blood circulation, may play an important role for improv-ing the pharmacokinetic profile as well as drug targeting properties ofmany novel drugs [45,46]. Commercially, albumins are obtained fromegg white (ovalbumin), bovine serum (bovine serum albumin, BSA)and human serum (human serum albumin, HSA). Albumin-basednanoparticles as drug delivery systems have been previously reviewed[7]. The techniques used to prepare albumin nanoparticles include deso-lvation, emulsification, thermal gelation, nano spray drying, nab (nano-particle albumin bound)-technology and self-assembly [7].

Albumin nanoparticles have gained considerable attention as drugdelivery vehicles because they offer several advantages: first, albuminnanoparticles exhibit high binding capacity of various drugs becauseof the multiple drug binding sites present in the albumin molecule[44]. Due to their high content of charged amino acids (e.g. lysine, argi-nine, aspartate and glutamate), albumin-based nanoparticles couldallow the electrostatic adsorption of positively (e.g. ganciclovir) or neg-atively charged (e.g. oligonucleotide) molecules [44]. Second, HSAnanoparticles represent a promising strategy for targeted delivery ofanticancer drugs to tumor cells [45,46]. Their enhanced uptake insolid tumors is mediated by binding of albumin to albumin-bindingproteins, such as membrane-associated gp60 (albondin) and secretedprotein, acidic and rich in cysteine (SPARC). Albondin receptor on theendothelial cells of tumor vessels allows transcytosis of albumin acrosscontinuous endothelium while overexpressed SPARC results in accu-mulation of albumin within the tumor interstitium [46]. Abraxane®(paclitaxel-albumin nanoparticles prepared by nab-technology) wassuccessfully developed for treating metastatic breast cancer [45]. In ad-dition to Abraxane®, the nab‐technology platformhas been successfullyused to encapsulate other anticancer lipophilic drugs such as docetaxel,the mTOR inhibitor rapamycin, the heat-shock protein inhibitor Hsp90,the tubulin polymerization/topoisomerase I dual inhibitor 5404 and anovel taxane CY196. The efficacy of these albumin drug nanoparticleshas been demonstrated in preclinical studies in multiple tumor xeno-graft models [45]. Third, HSA nanoparticles enable the delivery of a va-riety of drugs across the BBB into the brain [47]. Dadparvar et al. [47]found that the oximes (HI 6 dimethanesulfonate and HI 6 dichloridemonohydrate) adsorbed to HSA nanoparticles demonstrated twotimes higher reactivation of organophosphorous-inhibited AChE thanthe free oximes indicating successful brain delivery. Fourth, HSA nano-particle preparations appear to be a suitable agent for gene therapy be-causeHSAmight avoid undesired interactionswith serum that are oftenencountered after i.v. injection of transfection complexes, protect theoligonucleotides from nuclease digestion and also appear to befusogenic carrier able to target the nucleus [48]. Lastly, the presence offunctional groups on the albumin nanoparticle surfaces enables theirsurface modification with various ligands (e.g. folate) for site-specificdrug targeting [49].

HSA–protamine sulfate–DNA ternary proticles were successfullyutilized as a nonviral gene delivery vehicle where they combined ahigh transfection potential with a good biocompatibility, low cytotox-icity and efficient uptake by cells [50]. Li and Yao [51] prepared BSA–dextran conjugates by Maillard reaction and their nanoparticles weresimultaneously formed via self-assembly and successfully loadedwith ibuprofen. Heating was used to crosslink the core and strongercrosslinks were optionally made with glutaraldehyde [51].

Fig. 2. Degradation of βLG and BSA nanoparticles with orwithout pepsin at pH2.0 (A) andwith or without trypsin in pH 7.4 PBS (B) [60].


2.4. Milk proteins

Milk proteins are natural vehicles for bioactives [6]. They can beclassified into two categories according to their structure: flexibleproteins for caseins and globular proteins for whey proteins [6,52].

2.4.1. CaseinCasein, the major milk protein, is inexpensive, readily available, non-

toxic and highly stable. As a natural food product, this GRAS protein isbiocompatible and biodegradable. Many of the structural and physico-chemical properties of caseins facilitate their functionality in drug deliv-ery systems including binding of ions and small molecules, exceptionalsurface-active and stabilizing properties, excellent emulsification andself-assembly properties together with superb gelation and water bind-ing capacities [52]. Additionally, caseins are not sensitive to temperature,whereas whey proteins show important denaturation at temperaturesabove 70 °C [52]. Mainly four casein phosphoproteins, αS1-, αS2-, β-and κ-casein exist in cow milk with molecular weights between 19 and25 kDa and an average isoelectric point (pI) between 4.6 and 4.8 [64].Casein-based nanoparticles as drug delivery systems were previouslyreviewed [6].

Caseins are amphiphilic proteins that can be thought as block copol-ymers with high levels of hydrophobic or hydrophilic amino acid resi-dues. Therefore, caseins exhibit a strong tendency to self-assembleinto spherical caseinmicelles 50–500 nm in diameter [53]. Only recent-ly casein micelles were harnessed for delivering exogenous hydropho-bic bioactives. Casein micelles effectively protected vitamin D2 and theω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) againstUV-light-induced degradation and oxidation, respectively [54]. Modelhydrophobic chemotherapeutic drugs such as mitoxantrone, vinblas-tine, irinotecan, docetaxel and paclitaxel were successfully entrappedwithin β-casein (β-CN)-based nanomicelles [55,56]. The gastric digest-ibility of β-CN was suggested as a possible targeting mechanism forstomach cancer.

Following simulated digestion of paclitaxel-loadedβ-CNnanomicelleswith pepsin, paclitaxel retained its cytotoxic activity to human gastric car-cinoma cells [57]. Without prior simulated gastric digestion, thenanovehicles were non-cytotoxic, suggesting that β-CN may protect theupper GIT regions including buccal and esophageal epithelia from un-toward toxicity of paclitaxel. Recently, Bachar et al. [58] successfully de-veloped β-CN nanomicelles for oral delivery of celecoxib with highencapsulation loads. The drug-loaded micelles were lyophilized withoutany cryo-protectants and were fully reconstituted by rehydrationsuggesting the protein itself acts as a cryo-protectant. The complexationof the poorly soluble chemopreventive agent curcumin with β-CN mi-celles was investigated by Esmaili et al. [59] who found that presence ofβ-CN increased the solubility of curcumin at least 2500‐fold with the hy-drophobic interactions being predominant. The cytotoxicity of curcuminto human leukemia cell line was enhanced in the presence of β-CN mi-celles [59].

2.4.2. Whey proteinsBeta-lactoglobulin (βLG), the major whey protein in cow milk and

its principal gelling agent, is a small (18.3 kDa) globular protein withtwo disulfide bridges and a free thiol group which is inaccessible tosolvent at or below neutral pH. Because it can maintain a stable glob-ular conformation, βLG is known to be stable at low pH and highly re-sistant to proteolytic degradation in the stomach [60]. Alpha-lactalbumin (αLA), the second most prevalent whey protein in cowmilk, is a smaller globular metalloprotein with four disulfide bridges[61]. The physicochemical properties of the whey proteins suggestthat they may be suitable for drug delivery applications [61].

Small molecular weight, highly unfolding and less hydrophobicproteins such as βLG are preferred for preparing nanoparticles [60].Therefore, βLG nanoparticles (131 nm) were prepared by a deso-lvation method using acetone as a desolvating agent followed by

glutaraldehyde crosslinking [62]. It was found that pre-heating ofβLG solution to 60 °C and subsequent pH readjustment to 9.0 reducedthe size of nanoparticles to 59 nm with improved uniformity [60].Under similar conditions, βLG nanoparticles were smaller and moreuniform than BSA nanoparticles due to lower hydrophobic interac-tions of βLG than that of BSA. Degradation experiments showed thatβLG nanoparticles were more stable than BSA nanoparticles in acidicand neutral environments with and without added proteolytic en-zymes (Fig. 2). This could be attributed to the denser matrix structureand lesser amount of the basic amino acid composition of βLG com-pared to BSA [60].

Thermally induced βLG-epigallocatechin-3-gallate (EGCG) co-assemblies conferred significant protection to the potent antioxidantnutraceutical EGCG against oxidation and degradation [63]. Optimalnano-entrapment was obtained when EGCG was added to the pre‐heated βLG solution during cooling and vortexing. Nanoparticles ofβLG could interact with curcumin forming a 1:1 complex at 25 °Cwhere curcumin binds to the central calyx of βLG through hydropho-bic interaction [64]. The nanoparticles were found to encapsulate cur-cumin with >96% efficiency with the solubility of curcumin wassignificantly enhanced to 625 μM compared to its aqueous solubility(30 nM) [64].

Giroux et al. [65] successfully prepared whey protein nanoparticlesby pH-cycling treatment and thermal processing. In the pH-cycling

image of Fig.�2

Fig. 3. Resistance of native insulin and Ins–SFN bioconjugates against trypsin digestion(A), Scanning electron micrograph of silk particles produced by salting out with potas-sium phosphate (B) [71,72].


treatment, low temperature crosslinking of denatured whey proteincould be used to produce stable nanoparticles from solublewhey proteinpolymers [65]. Stable nanoparticles with a diameter ranging from 100 to300 nm were formed by acidification of diluted polymer dispersionsfollowed by pHneutralization [65]. On the other hand, after thermal pro-cessing, native whey proteins may form gels or aggregates. The heat sta-bility of native whey proteins was improved by incorporating proteinsolutions in nanoscale micelles of w/o microemulsions to form wheyprotein nanoparticles by thermal pretreatment at 90 °C for 20 min [66].

Stable electrostatic nanocomplexes were formed when an excessof negatively charged pectin was added to βLG below its isoelectricpoint (pI 5.18) where it is positively charged [67]. βLG–pectinnanocomplexes showed a very good colloidal stability and effectivelyprotected DHA against oxidation [67]. In another study, chitosan–whey protein Maillard conjugates formed nanoparticles crosslinkedby tripolyphosphate for oral delivery of catechin [68]. The particleswere positively charged below pH 7.2, indicating their stability toaggregation under GI conditions with 56.5% of the encapsulated cate-chin was released after 24 h [68].

2.5. Silk proteins

Silk is generally defined as protein polymers that are spun into fi-bers by some lepidoptera larvae such as silkworms, spiders, scorpions,

mites and flies [69]. Silk proteins are promising materials for drug de-livery and tissue engineering due to their biocompatibility, slow bio-degradability, self-assembly, excellent mechanical properties andcontrollable structure and morphology [69]. Additionally, silk is lessinflammatory than other common biodegradable polymers such aspoly(lactide) [70].

The silkworm Bombyx mori produces silk to weave its cocoon andits major components are fibroin and sericin. Fibroin is a fibrous pro-tein constituting the core of silk, while sericin is a glue-like proteinthat envelops fibroin fibers with successive sticky layers that help inthe formation of a cocoon [70]. Novel insulin-silk fibroin nanoparticle(Ins-SFN) bioconjugates were prepared by Yan et al. [71] where thecrystalline silk nanoparticles could be conjugated covalently with in-sulin via the crosslinking reagent glutaraldehyde through the ε-NH3

present on the surface of nanoparticles. The bioconjugation of insulinwith silk fibroin nanoparticles strikingly improved its in vitro stabilityin both human serum and trypsin solutions (Fig. 3A) [71]. In anotherstudy, silk fibroin particles of controllable size were obtained in anall-aqueous process by salting out with potassium phosphate andcould be loaded with small molecule model drugs by simple adsorp-tion based on electrostatic interactions between the negativelycharged particles and the positively charged small molecules [72]. Ascanning electron micrograph of silk particles produced by saltingout was shown in Fig. 3B.

Stable negatively charged silk fibroin nanoparticles were pre-pared via a desolvation technique using dimethyl sulfoxide as adesolvating agent [73]. The active amino groups and tyrosine resi-dues of silk fibroin favored its bioconjugation with VEGF (vascularendothelial growth factor) providing a sustained release of VEGFover three weeks [73]. Similarly, a phase separation method usingpolyvinyl alcohol as a continuous phase was utilized to separatesilk solution into micro- and nanospheres [74]. The porous interiorspace and amphiphilic nature of silk spheres facilitated the entrap-ment of drugs with different molecular weights and hydrophobic-ities [74]. A capillary-microdot technique was used to fabricatenanoparticles via noncovalent blending of silk fibroin (SF) and chito-san (CS) to encapsulate curcumin [75]. Curcumin-polymer con-jugates were frozen, lyophilized, crystallized and suspended inphosphate-buffered saline. Pure SF curcumin nanoparticles showedthe highest curcumin entrapment, release, intracellular uptake andefficacy towards breast cancer cells as compared to SF–CS curcuminnanoparticles [75]. Mandal and Kundu [76] successfully preparedself-assembled micellar nanostructures capable of carrying both hy-drophilic (FITC-insulin) and hydrophobic (paclitaxel) drugs byblending silk sericin with pluronic. Paclitaxel-loaded nanoparticlesshowed significant apoptosis against breast cancer cells comparableto free paclitaxel [76].

2.6. Elastin

Elastin is the dominant extracellular matrix protein deposited in thearterial wall conferring the properties of extensibility and elastic recoilto many human tissues such as large blood vessels, lung, ligaments andskin [77]. Elastin undergoes a self-aggregation process in its natural envi-ronment where it is produced from a water soluble precursor, tropo-elastin, which spontaneously aggregates into a covalently crosslinkedfibrillar polymeric structure [78]. Recombinant protein technology hasallowed the synthesis of elastin-like polymers (ELPs) whose basic struc-ture is the repeating sequences found in the mammalian elastic proteinelastin, or some modifications of those sequences. The most strikingand longest sequence between crosslinks in pig and cow elastin is theundecapentapeptide (VPGVG)11 [79]. A prime reason as to why geneti-cally engineered, protein-based biopolymers are even used is the DNAblueprint constructed to produce the biopolymers. This allows for modu-lar exchange, deletion, truncation or rearrangement with di-, tri-, orhigher order polymers. ELPs retain all the advantages of polymeric drug

image of Fig.�3


delivery systems, but also provide a number of additional benefits thatare unique to genetically engineered biopolymers. First, because ELPsare composed of amino acids, they are non-toxic and biodegradablewith excellent biocompatibility [80]. Apparently, the immune system ig-nores these polymers because it does not distinguish them from the nat-ural elastin. Second, ELPs have a favorable pharmacokinetic profile [81].Third, because ELPs are designed and synthesizedusing genetic engineer-ing techniques, the molecular weights of ELPs can be precisely specifiedresulting in monodisperse polymers. Furthermore, some of the maincharacteristics of these ELPs are derived from the natural protein theyare based on such as elasticity [82,83].

Another advantage of ELPs is that they may be engineered to un-dergo a rapid phase transition in response to temperatures suitablefor adjuvant, clinical therapies such as microwave thermal ablation[84,85]. At temperatures above the transition temperature (Tt), re-combinant thermo-sensitive ELPs undergo a reversible phase transi-tion where they hydrophobically self-assemble into an insolubleaggregate, forming nano- and micro-particles which could be appliedas controlled release devices [84,85]. Herrero-Vanrell et al. [84] pre-pared self-assembled nano- and micro-particles of poly(VPAVG)which showed a sustained release of dexamethasone phosphate forabout 30 days. The particles, once formed, were stable either atroom or body temperature as they did not redissolve until a strongundercooling of ~12–15 °C was achieved. The temperature turbidityprofiles are illustrated in Fig. 4 [84].

Similarly, by thermoresponsive self-assembly of the biologicallyproduced (VPAVG)220, elastin-like nanoparticles (~237 nm) wereprepared for delivering both BMP-2 and BMP 14 in a sustained way[85]. The activity of these growth factors was retained, as shown bythe induction of alkaline phosphatase activity and osteogenic miner-alization in C2Cl2 cells [85]. In another study, stable nanoparticleswere successfully obtained by the thermosensitive aggregation ofthe elastin model polypeptide, (GVGVP)251 [86]. Three differentheating processes, “slow heating,” “fast heating” and “heat shock,”were used for the aggregation of the peptide, followed by gamma-ray crosslinking. Only the “heat shock” process successfully yieldedstable nanoparticles [86]. Furthermore, ELPs have been used to phys-ically entrap viruses for local gene therapy. Hatefi et al. [87] prepareda hydrogel system composed of silk-elastin‐like protein polymers(SELPs) which controlled the release rate of adenoviruses over a peri-od of four weeks while preserving their bioactivity. After intratumoralinjection into xenograft tumor models of breast and head and neckcancer in mice, a prolonged and localized expression of adenoviruseswas observed.

ELP-diblock copolymers have been reported with an ELP block andrepeat amino acid block for drug and gene delivery [88–90]. Chen et

Fig. 4. Temperature profiles of aggregation (heating) and dissolution (cooling) of a30 mg/mL ELP solution. Inserts show a polymer solution below Tt (5 °C) and above Tt(40 °C), showing the formation and segregation of polymer nanoparticles [84].

al. [88] successfully constructed new ELP-based diblock copolymersfeaturing poly glutamic/aspartic acids for facile drug conjugationand an ELP block for thermo-targeted chemotherapy of hyperthermictumor margins. The resultant biopolymer-conjugates were used as amacromolecular solubilizer for geldanamycin, a potent HSP90 inhibi-tor. The diblock copolymer formed stable nanoconstructs and dis-played tunable, acute phase transitions at high temperatures [88]. Inanother study, biodegradable p[Asp(DET)]53ELP(1–90) was synthe-sized as a hybrid recombinant block copolymer for thermo-sensitivegene transfection that possess a thermo-responsive ELP segmentand a diethylenetriamine (DET) modified poly-L-aspartic acid seg-ment [89]. The polyplexes formed by the copolymer and pGL4 plas-mid (90–100 nm) retained the thermal phase transition behaviorconferred by the copolymer and showed appreciable transfection ef-ficiency with low cytotoxicity [89].

Krishna et al. [91] modified two recombinant elastin-like proteinsin order to self-crosslink through hydrazone bonding for increasingtheir mechanical strength. Hydrazone crosslinking allowed the for-mation of biocompatible in situ chemically and physically crosslinkedELP hydrogels that may have potential applications in drug delivery,tissue engineering or as components of implanted medical devices[91,92].

Recently, chimeric polypeptides derived from ELPs with conjugatedhydrophobic drugs could self-assemble into nanoparticles [93]. ELP[VA8G7]160 was selected to form the corona of the micelle then a shortpeptide consisting of Cys-(Gly-Gly-Cys)7 was appended to the C-terminus of the ELP to provide multiple, unique sites for drug attach-ment (Cys residues) thus forming a chimeric peptide (CP) [80,93].Doxorubicin (Dox) was sequestered into the core of the micelle uponattachment to the Cys residues in the CP thus limiting the toxicity tohealthy tissues while targeting the drug to tumor via the enhanced per-meation and retention effect. The conjugation of multiple Dox mole-cules to the CP imparted the necessary amphiphilicity to drive self-assembly of the CP-Dox conjugate into nanoparticles. The nanoparticlesshowed a 3.5-fold increase in tumor concentration after 24 h over freedrug and a 2.6-fold decrease in heart concentration after 24 h [80,93].

3. Plant proteins

Nanoparticulate carriers from vegetal proteins represent a new ap-proach which presents some advantages [94]. In contrast to drug deliv-ery using hydrophilic animal proteins, hydrophobic plant proteins suchas zein and gliadin have the capability of yielding sustained drug release[95]. Due to their high hydrophobicity, the nanoparticles may not needany further chemical or physical treatment to harden them thusavoiding the toxic chemical crosslinkers [94]. They are less expensivethan animal proteins and also possess functional groups which can beeasily used either to adsorb or to covalently couple molecules capableof modifying the targeting properties of nanoparticles. Moreover,plant proteins reduce the risk of spreading diseases such as bovinespongiform encephalitis (mad cow disease) [94,95].

3.1. Zein

Zein is a water-insoluble but alcohol-soluble protein with a molec-ular weight of about 40 kDa that is predominantly present in the en-dosperm of corn kernels [95]. It contains three quarter of lipophilicand one quarter of hydrophilic amino acid residues. Commercialzein is currently separated from corn gluten meal, a co-product ofcorn wet milling and is a mixture of at least four types of proteins:α-, β-, γ- and δ-zein, each with a different amino acid sequence, mo-lecular weight and solubility [96]. Being a natural protein having agood biodegradability in vivo, zein is considered as GRAS and food-grade ingredient by FDA. Zein has been employed as an edible coatingfor foods and pharmaceuticals because it shows low water uptakevalues, high thermal resistance and good mechanical properties [97].

image of Fig.�4


Zein and its degraded products showed good cell compatibility for tis-sue engineering [98]. Because of its high hydrophobicity, zein hasbeen successfully applied as a promising carrier for encapsulationand controlled release of fat-soluble compounds (e.g. gitoxin) [97].In addition, the protein structure allows zein to function as a poly-meric amphiphile where it exists as small globules with diameters be-tween 150 and 550 nm in aqueous ethanol solution. The zeinmolecule has a very special bricklike shape and thus has a potentialto carry other molecules inside them. It can also overcome the draw-back of hydrophilic polymeric systems in order to achieve sustaineddrug release [95]. Sustained release of water-soluble drugs such asivermectin and heparin was observed in vitro over 9 and 20 days, re-spectively, from the films made of zein microspheres [99,100].

Liquid–liquid dispersion process was used to produce zeinnanoparticles. In this process, zein was dissolved in 70% v/v aqueousethanol, followed by shearing zein solutions into deionized waterusing a high-speed homogenizer [96]. When the alcohol concentra-tion in the emulsified droplets decreases below a level required fordissolving zein, zein becomes insoluble and precipitates to formnanoparticles. If a nonpolar compound can be co-dissolved in aque-ous alcohol together with zein, the compound may be encapsulatedin zein nanoparticles [97]. This process is advantageous over conven-tional emulsification methods that require strong shear forces to re-duce droplet sizes to sub-micrometers. Also, formation of particlesis fast and the process is simple and scalable [96,97]. This processwas later used to microencapsulate spice essential oils-oregano, redthyme and cassia [101]. Zhong and Jin [96] showed sustained releaseof water-soluble lysozyme from zein nanocapsules at neutral pH.Similarly, zein nanoparticles were used to encapsulate fish oil insolid as an alternative to emulsions [97]. The lyophilized samplesshowed good oxidative stability of fish oil during storage. Lai andGuo [95] demonstrated the targeting potential of zein nanoparticlesto the liver. Following i.v. injection of 5-fluorouracil loaded zeinnanoparticles, the nanoparticles were mostly accumulated in liverand adequately remained in blood for at least 24 h due to its relativelyhigher molecular weight and smaller particle size [95]. In anotherstudy, doxorubicin-loaded zein nanoparticles showed a sustaineddrug release for 4 days and an enhanced cytotoxicity in doxorubicin-resistant breast cancer cells [102].

Electrospraying technique was used by Torres-Giner et al. [103] tostabilize docosahexaenoic acid (DHA) via encapsulation within zeinultrathin capsules (490 nm). The DHA encapsulated within zeinultrathin capsules was more stable against degradation across relativehumidity and temperature. Luo et al. [104,105] successfully developedchitosan (CS)/zein nanocomplex system to encapsulate hydrophilic(selenite) and hydrophobic (α-tocopherol) nutrients to protect themagainst GI conditions and enhance their releasing property. The posi-tively charged CS molecules could interact with the negatively chargedzein nanoparticles; thus forming a CS coating around the surface of zeinnanoparticles [104,105]. In a recent study, zein nanoparticles coatedwith carboxymethyl chitosan provided better controlled release of vita-min D3 and improved its photostability against UV light compared touncoated nanoparticles [106].

3.2. Gliadin

Wheat gluten is a protein carbohydrate complex of which proteinsare the major component. Two main fractions are present: gliadin,which is soluble in neutral 70% ethanol, made of single chain polypep-tides with an average molecular weight of 25–100 kDa linked by intra-molecular disulphide bonds and glutenin, an alcohol-insoluble fractionconsisting of gliadin-like subunits stabilized by intermolecular di-sulphide bonds with molecular weight greater than 106 kDa [94].Thus, the term gliadin defines a group of proteins extracted from glutenby 70% ethanol. These proteins are polymorphic and can be classified onthe basis of their electrophoretic mobility into four fractions, named α

(25–35 kDa), β (30–35 kDa), γ (35 40 kDa) andω (55–70 kDa), respec-tively [94]. All fractions have remarkably low solubility in aqueous solu-tion except at extreme pH. This lowwater solubility has been attributedto the presence of disulphide bonds and to the cooperative hydrophobicinteractions which cause the protein chains to assume a folded shape.The amino acid composition shows that gliadin has equal amounts ofpolar and neutral amino acids, mainly glutamine (about 40%) in addi-tion to high proline content (14%) [107]. As biopolymers, gliadins donot present the common drawbacks of synthetic materials, related tothe presence of monomer or initiator residues. As plant proteins, theyare recognized as prion-free unlike animal proteins [108]. Besides, glia-dins are hydrophobic and slightly polar. Consequently, they are able tointeract with epidermal keratin due to their richness in proline. Thus,gliadin systems seem to have promise in the development of topical for-mulations [107].

Gliadin has been used to elaborate nanoparticles as carriers for all-trans-retinoic acid (RA) by a desolvation method [94]. In this method,gliadin was dissolved in an organic solvent/water phase then pouredinto a constantly magnetically stirred physiological saline containingpluronic as a stabilizer. A biphasic pattern of RA release was observedwith an initial burst effect followed by zero-order diffusion [94]. Gli-adin nanoparticles were shown to be suitable controlled release sys-tems for hydrophobic and amphiphilic drugs [108]. The entrappedamounts of the hydrophobic vitamin E and the slightly polar mixtureof linalool and linalyl acetate in gliadin nanoparticles were higherthan that of the the cationic benzalkonium chloride, confirming astrong interaction between gliadins and apolar compounds, due tothe apolarity of the proteins [108].

The high bioadhesive capacity of gliadin nanoparticles may beexplained by gliadin composition being rich in neutral and lipophilicresidues where neutral amino acids can promote hydrogen bondinginteractions with the mucosa, while the lipophilic components caninteract with the biological tissue by hydrophobic interactions [109].It was observed that gliadin nanoparticles dramatically increased thecarbazole oral bioavailability up to 49% and provided sustained releaseproperties related to the bioadhesive capacity of gliadin nanoparticleswith the stomachmucosa after oral administration [110]. The carbazolerelease rate from the nanoparticles was found to be of the same orderas the elimination rate of the adhered fractions of nanoparticles inthe stomach mucosa [110]. As a consequence, mucoadhesive gliadinnanoparticles bearing amoxicillin were developed for eradicatingHelicobacter pylori in stomach [111]. The nanoparticles eradicatedHelicobacter pylori from the GI tract more effectively than freeamoxicillin because of the prolonged GI residence time attributedto mucoadhesion [111]. Similarly, Ramteke et al. [112] preparedsustained release mucoadhesive gliadin nanoparticles of clarithromycin,omeprazole and triple therapy (amoxicillin, clarithromycin and omepra-zole) in order to improve patient compliance. A greater eradication effectof triple therapy entrapped formulations on isolated culture ofHelicobacter pylori was shownwhen comparedwith single therapy con-taining formulations and plain drugs [112].

3.3. Soy proteins

Soybean, from the most cultivated plant in the world, is rich in pro-teins (40–50%), lipids (20–30%) and carbohydrates (26–30%) [113]. Soyprotein is abundant, renewable, inexpensive, biodegradable andexpected to present a tailorable degradation profile varying with thecrosslinking degree. The use of soy-based polymers for drug deliveryand tissue engineering applications was proposed where soy-basedthermoplastics andmembranes are described and characterized as bio-materials and carriers for drug delivery applications [114]. Soy proteinisolates (SPI) are also used as emulsifiers in food emulsions due to thesurface active properties of their constitutive proteins; the storage glob-ulins 7 S (β-conglycinin) and 11 S (glycinin) [115].

Fig. 5. The targeting potential of bioadhesive lectins at epithelial barriers: binding, internalization and intracellular transport of lectins and lectin-conjugates depends on the pro-tein–sugar interaction [118].


Electrospun nanofibers (200 nm to 2 μm) fabricated from soy pro-tein isolate (SPI)/poly(ethylene oxide) (PEO) blend and poly(lacticacid) were used for controlled release of a naturally occurring antimi-crobial compound, allyl isothiocyanate (AITC) [114]. Release of AITCwas negligible under dry conditions, but increased dramatically asrelative humidity increased. This interactive effect was likely causedby the plasticizing effect of water on SPI which increased the molec-ular motion in the fiber matrix that triggered the release of AITC[114]. Legumin, one of the main storage proteins in the seeds of thepea (Pisum sativum L.), belongs to the 11S globulin group with a mo-lecular mass of 359±25 kDa and isoelectric point of 4.8 [116].Legumin nanoparticles with a size of about 250 nm were preparedby a pH-coacervation method and chemical crosslinking with glutar-aldehyde [116]. The nanoparticles were quite stable in pH conditionsclose to neutrality whereas nanoparticles stored under acidic condi-tions showed a rapid degradation and this fact may be of interestfor pharmaceutical applications like cutaneous or transdermal admin-istration of drugs.

3.4. Lectins

Lectins comprise structurally diverse class of carbohydrate-binding natural nonenzymatic and non‐immunogenic proteins/glyco-proteins [117,118]. Wheat germ agglutinin (WGA) is one of the mostextensively studied plant lectins for its high stability, low toxicity, re-sistance to proteolytic degradation in addition to specific recognitionand binding to glycosylated membrane components on the intestinalmucosal surface [119,120]. In the last two decades, lectins openedtwo attractive areas for drug delivery: improving absorption of poorlyavailable drugs and glycotargeting of anticancer drugs. Moreover,several lectins such as WGA were found to have remarkable anti-tumor activity by inducing apoptosis or autophagy in cancer cells[117–120].

The rationale behind lectin-mediated drug targeting is very sim-ple. Most cell surface proteins and many lipids in cell membranesare glycosylated and these glycans are binding sites for lectins [118].Different cell types express different glycan arrays and in particular,diseased cells, such as transformed or cancerous cells, often expressdifferent glycans compared with their normal counterparts. There-fore, lectins could be used as carrier molecules to target drugs specif-ically to different cells and tissues. The concept of bioadhesion vialectins may be applied not only for the GI tract but also for other bio-logical barriers like the nasal mucosa, lung, buccal cavity, eye and the

blood–brain barrier [117]. Lectins can be considered as bioadhesives ofthe second generation that offer the main advantage of mediating cel-lular uptake after diffusion through the intestinal mucus layer [118].The targeting potential of bioadhesive lectins at epithelial barriers:binding, internalization and intracellular transport of lectins andlectin-conjugates was represented in Fig. 5 [118]. By encapsulatingquantum dots as fluorescent probes into the core of WGA-conjugatedpoly(ethylene glycol)–poly(lactic acid) (PEG-PLA) nanoparticles, Gaoet al. [121] showed that the cellular uptake begins with binding ofWGA to its receptor at the cell surface then endocytosis happened bymeans of clathrin and caveolae-mediated mechanisms [121]. Liu et al.[122] revealed that the endocytic and exocytic processes of WGAwere both actin- and microtubule-dependent. The vesicle fusionevent occurred near the cytomembrane, followed by two destinieswith WGA: shedding to the extracellular or reversing to the cytoplasm[122].

The lectin-coated nanoparticles could bind to intestinal epithelialcells which may be sufficient to stimulate the enterocytes toendocytose the ‘coated nanoparticles’ and transport the particles acrossthe cell and into the circulation [123]. Moreover, the oral delivery ofpeptides and proteins within lectin-coated nanoparticles appears tobe an ideal method for protecting these molecules against digestionas well as increasing their uptake [124]. Lectins have been conjugatedto different types of nanoparticles including protein [117,125–127],poly(lactic-co-glycolic acid) (PLGA) [128–130], PEG–PLA [131–133]and solid lipid nanoparticles (SLNs) [134–136]. Ulex europaeus lectin(UE lectin) conjugated to vicilin (storage protein from Pisum sativum)nanoparticles increased the interaction with mucin three timesmore than the control vicilin nanoparticles. Moreover, the presence ofL-fucose strongly inhibited their interaction with mucin indicatingthat UE lectin–vicilin nanoparticles kept the lectin specificity aftercoupling [125]. Similarly, Ezpeleta et al. [126] used lectin-conjugatedgliadin nanoparticles bearing acetohydroxamic acid as ameans of locat-ing and anchoring a drugdelivery systemon the carbohydrate receptorsof Helicobacter pylori [127]. In another study, the eradication rate oflectin-conjugated gliadin nanoparticleswith triple therapy (amoxicillin,clarithromycin and omeprazole) against Helicobacter pylori was 94.83%compared to 88.28% for gliadin nanoparticles with triple therapy [117].This may be due to the increased residence time of the lectinizednanoparticles to the GI mucosa.

WGA-conjugated paclitaxel-loaded PLGA nanoparticles showed su-perior anti-proliferation activity against the malignant pulmonary andcolon cancer cells compared with conventional paclitaxel formulations

image of Fig.�5


[128]. The authors attributed this to a more efficient intracellular inter-nalization of paclitaxel via WGA receptor-mediated endocytosis [128].Lectin-conjugated PLGA nanoparticles were successfully used for oraldelivery of thymopentin. The nanoparticles effectively improved its in-testinal absorption due to specific bioadhesion on GI cell membrane[129]. In another study, lectin from Arachis hypogaea was anchoredonto the surface of the Hepatitis B surface antigen (HBsAg)-loadedPLGA nanoparticles as an oral immunization against Hepatitis B inorder to enhance their affinity towards the antigen presenting cells ofthe Peyer's patches. The lectinized nanoparticles demonstrated approx-imately four folds increase in degree interaction with the bovine sub-maxillary mucin as compared to plain nanoparticles [130].

Lectins opened a novel pathway to improve the brain uptake ofdrugs loaded by biodegradable nanoparticles following intranasal ad-ministration [131]. Ulex europeus agglutinin I (UEA I) modified PEG–PLA nanoparticles facilitated the absorption of 6-coumarin into thebrain following intranasal administration [131]. Similarly, Gao et al.[132] showed that WGA modification on the surface of nanoparticlesfacilitated the uptake of vasoactive intestinal peptide (VIP) by brainand improved its therapeutic effect on cholinergic inhibited rats fol-lowing intranasal administration. Odorranalectin, the smallest lectin,is an exciting newcomer in the lectin family with much less immuno-genicity and able to specifically recognize L-fucose which is widely lo-cated on the olfactory epithelium of nasal mucosa [120]. It was shownthat Odorranalectin modification increased the nose-to-brain deliv-ery of PEG–PLGA nanoparticles and enhanced the therapeutic effectsof urocortin peptide-loaded nanoparticles on Parkinson's disease[120]. A slight excitotoxicity and oxidative stress was observed afterrepeated intranasal administration of WGA-conjugated PEG–PLAnanoparticles [133].

After oral administration, WGA-modified SLNs protected insulinagainst degradation by digestive enzymes with the stabilizing effectof WGA-modified SLNs was greater than that observed in unmodifiedSLNs [134]. In another study, WGA enhanced the cellular uptake ofthe SLNs loaded with bufalin (a hydrophobic active componentextracted from the traditional Chinese medicine Chan'su) throughCaco-2 monolayers compared withWGA-free SLNs [135]. The oral ad-ministration of the WGA-grafted nanoparticles to fasted rats showedmuch larger AUC and Cmax and a 2.7-fold improvement in oral bio-availability as compared with suspensions [136].

4. Advantages and pharmaceutical applications

Proteins are posed as the natural counterpart to synthetic poly-mers for the development of nanoparticles. They offer several advan-tages over synthetic polymers being GRAS drug delivery devices withhigh nutritional value and abundant renewable sources. As related tosafety, they are metabolizable in vivo by digestive enzymes into in-nocuous peptides whereas synthetic polymers may give harmful deg-radation products. Additionally, protein nanoparticles exhibit highloading capacity of various drugs due to multiple binding sites pre-sent in their molecules. They exhibit a variety of possible drug loadingmechanisms including electrostatic attractions, hydrophobic interac-tions and covalent bonding. Moreover, protein-based nanoparticlesoffer various possibilities for surface modification due to the presenceof functional groups (i.e. carboxylic and amino groups) on the surfaceof the nanoparticles thus enabling specific drug targeting to the site ofaction [7]. In vitro studies revealed that protein nanoparticles suc-cessfully controlled the release rate of drugs for prolonged periodssuggesting protein nanoparticles as efficient controlled release vehi-cles. Concerning the cost; proteins are much less expensive comparedto synthetic polymers.

From the studies reviewed, it can be seen that protein nanocarrierspossess various applications in the field of drug delivery: proteinnanoparticleswere found to sustain drug release for prolonged durations,enhance the stability of sensitive drugs and neutraceuticals and increase

the solubility and hence the absorption of poorly soluble drugs. More-over, collagen nanoparticles could enhance transdermal drug delivery.Protein nanoparticles also enhanced the drug uptake into the brain, mac-rophages and liver and enabled targeted delivery of anticancer drugs totumor cells. Additionally, gliadin and lectin-coated nanoparticlesshowed a potential mucoadhesive capacity. In gene delivery, proteinnanoparticles could protect the oligonucleotides from nuclease di-gestion and facilitated their transportation into the nucleus.

5. Drawbacks and challenges

Proteins as natural polymers are heterogeneous mixtures of dif-ferent sizes with a wide range of molecular weights thus producingheterogeneous nanoparticle size distribution and exhibiting batch-to-batch variation [137]. This may hinder the scaling-up process ofprotein nanoparticle preparation for industrial application. An inter-esting strategy to overcome this drawback is the recombinant proteintechnology. The monodispersity and precisely defined properties of re-combinant proteins aswell as the predictable placement of crosslinkinggroups, binding moieties or their programmable degradation ratesmake themuseful for drugdelivery and tissue engineering [137].Mono-disperse nanoparticles based on recombinant HSA [138] and recombi-nant gelatin [28,139] were successfully prepared.

Among other drawbacks of the animal protein-based nanoparticlesis their inability to achieve a sustained drug release due to their hydro-philic nature and rapid solubilization in aqueous environments. Whenthe system absorbs water and swells, drugs may rapidly diffuse out.Chemical crosslinkers (e.g. glutaraldehyde and formaldehyde) usuallyused to harden protein nanoparticles suffer from the presence of resid-ual unreacted crosslinker inside the nanoparticles togetherwith the riskof formation of toxic products by reactionwith the tissues during in vivobiodegradation [140]. This problem could be overcome by using hydro-phobic plant proteins with no need for crosslinking. Moreover, plantproteins reduce the risk of spreading animal protein-related diseasessuch as bovine spongiformencephalitis. Concerning possible protein im-munogenicity, no antigenic reactionswere reported after i.v. administra-tion of albumin [49], gelatin [12], zein [95] and casein [6] nanoparticles.Protein nanoparticles are likely to be well tolerated in vivo without del-eterious side effects.

6. Future perspectives

Studies will continue to further improve delivery of drugs usingprotein nanocarriers. Special emphasis seems to be given for using re-combinant protein-based nanoparticles for drug delivery as alterna-tives to native ones. Few studies investigating such approach werereported for recombinant HSA [138], recombinant gelatin [28,139]and elastin-like polypeptides (ELPs) nanoparticles [82]. Additionally,studies will continue to further investigate new safe crosslinkers ofprotein nanoparticles as alternatives to the toxic chemical ones. Inour laboratory, a new protein nanosystem, ionically crosslinked caseinnanoparticles, composed of casein crosslinked with tripolyphosphateinstead of chemical crosslinking, was recently developed and investi-gated as a carrier for delivery of drugs [141]. Ionically crosslinked caseinnanoparticles loaded with the anti-androgen, flutamide (FLT), showedsatisfactory entrapment efficiency with a positive zeta potential, agood colloidal stability and prolonged in vitro drug release. After i.v. ad-ministration into rats, pharmacokinetic parameters revealed that FLT-loaded casein nanoparticles were well-tolerated without any side ef-fects and showed a longer circulation time relative to FLT free solution,suggesting that ionically crosslinked casein nanoparticles may havepromising future as carriers for hydrophobic drugs (data to be publi-shed soon) [141].

American Bioscience, Inc. has developed a unique albumin-basednanoparticle technology (nab-technology) that is ideal for encapsulatinglipophilic drugs into nanoparticles [45,46]. Abraxane® (nab-paclitaxel;


paclitaxel-albumin nanoparticle) with an approximate diameter of130 nm is the first FDA approved nanotechnology based chemothera-peutic that has shown significant benefit in treatment of metastaticbreast cancer. The market approval of Abraxane® can be viewed as alandmark not just for albumin-based drug delivery technology but alsofor nanomedicine [45,46]. The near future may also hold the emergenceof new commercial protein nanocarrier-based products. However, at thepresent stage, a better fundamental understanding of themechanisms ofaction of these vehicles and of the protein–drug interactions at the mo-lecular level will provide a basis for their further optimization to ensuredesign of ideal protein nanocarriers and open more exciting opportuni-ties for their use in the area of drug and gene delivery.

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Protein-based nanocarriers as promising drug and gene delivery systems

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