Recent Advances in Protein and Peptide Drug Delivery: A Special Emphasis on Polymeric Nanoparticles

Recent advancements in biotechnology have resulted in the development of novel therapeutically active proteins and peptides. Some of the recently approved proteins and peptides are listed in Table 1 . These potent therapeutics are indicated for several chronic conditions such as cancer, hepatitis, diabetes, rheumatoid arthritis, and leukemia [ 1 ]. However, several pharmaceutical and biopharmaceutical challenges limit their clinical application. Parenteral administration is the major route for administration of therapeutic proteins and peptides [ 2 ]. However, requirement of frequent injections due to short in vivo half-life of proteins and peptides is a major concern. Moreover, in some chronic conditions, frequent injections are required for a longer period of time leading to poor patient compliance [ 3 ]. Administration of proteins and peptides by oral, pulmonary, transdermal and nasal routes are few of the potential alternatives to parenteral injections. Among non-invasive routes, oral delivery is the primary route for administration of small molecules however; it is not preferable for proteins and peptides. Oral delivery of proteins and peptides is highly challenging due to presence of proteolytic enzymes in gastrointestinal tract (GIT) and their intrinsic physicochemical and biological properties like poor stability in lower pH of gastric fluid, large molecular size and poor permeation across gastrointestinal membrane [ 4 ]. Nasal and pulmonary administration received considerable attention because of low proteolytic activity relative to oral route, highly vascularized and large absorptive surfaces especially in the lungs resulting in improved absorption. Large size and proteolytic instability are major factors for poor absorption of therapeutic proteins across nasal and pulmonary mucosal surfaces. Moreover, physiological barriers such as mucociliary clearance may further limit protein and peptide absorption [ 4 ]. Similarly, hydrophilicity and large molecular size also limit transdermal protein delivery [ 4 ]. To overcome these challenges, proteins and peptides can be delivered efficiently by encapsulating in carrier systems such as microparticles, polymeric NPs, liposomes and solid lipid NPs. In this aspect, polymeric NPs offer unique advantages over other carrier systems. Smaller size relative to microspheres renders polymeric NPs a suitable drug carrier for parenteral administration. Moreover, it has been generally observed that NPs can translocate efficiently across epithelial surfaces relative to microparticles [ 5 , 6 ]. Polymeric NPs also exhibit high stability in biological fluids compared to liposomes and solid lipid NPs. In addition, versatility of formulation, sustained release, subcellular size, protection of encapsulated proteins and peptides from enzymatic degradation and tissue biocompatibility render NPs as a promising delivery system for protein and peptide delivery [ 6 ]. Moreover, physicochemical properties (e.g. hydrophobicity, surface charge), drug release profile and biological behavior (e.g. bioadhesion, targeted drug delivery, improved cellular uptake) can be modulated by application of various polymeric materials and targeting ligands [ 7 , 8 ]. In this review, we have made an attempt to summarize few of the recent (five to six years) development and application of polymeric NPs for the delivery of proteins and peptides via oral, transdermal, ocular, parenteral, pulmonary and nasal routes. Barriers to proteins and peptides delivery by various routes and advantages of polymeric NPs in overcoming delivery barriers have been summarized in Table 2 .

In this method, polymers and drugs are dissolved in a polar, water-miscible solvent (DMSO, acetone, or ethanol). The solution is added in a drop by drop manner into an aqueous solution with surfactant. NPs are formed instantaneously by rapid solvent diffusion. Main advantage of the method is that the drug molecules can be encapsulated without subjecting to shearing stress and high temperatures. The method was originally developed for encapsulation of hydrophobic molecules [ 7 , 33 ]. Later on, Bilati et al . have modified this technique to encapsulate hydrophilic proteins and peptides into PLGA NPs [ 24 ]. NPs with small size and narrow size distribution can be easily obtained by this method. Similar to s/o/w method, protein in HIP complex form can also be incorporated into NPs by this technique. Combination of HIP complexation with nanoprecipitation have demonstrated enhancement in encapsulation of proteins such as human IgG-Fab fragment and lysozyme [ 31 , 34 ]. In a recent study, Gaudana et al . have hydophobically ion-paired lysozyme with dextran sulfate and then the complex was loaded into PLGA NPs by nanoprecipitation [ 34 ]. Higher lysozyme encapsulation was achieved and released lysozyme maintained its biological activity.

Recently, NPs are also prepared utilizing supercritical fluids. In this technique, drug and polymer are first dissolved in supercritical fluid and the solution is expanded through a nozzle. The supercritical fluid is evaporated using spraying process which eventually leads to precipitation of solute particles. Advantages of this technique include processing of biolabile pharmaceuticals under mild operating conditions, flexibility in procedures, and elimination of organic solvent in the final product. Elvassore et al . produced insulin-loaded PLA NPs with high product yield and maintenance of >80% of the insulin hypoglycemic activity using this method [ 37 ]. The major problems observed with this method are requirement for special equipment, poor solubility of high molecular mass (10,000) polymers and strong polar substances in supercritical CO 2 [ 38 ].

Oral administration is the most preferred route due to its advantages such as patient convenience and compliance, avoidance of contamination and infections caused by the use of injections [ 39 ]. However, proteins and peptides exhibit poor oral bioavailability due to lower permeation across intestinal epithelium, aggregation and denaturation. Hindrance to oral administration of protein and peptide can be categorized as physical, chemical and enzymatic barriers. The physical barrier is attributed mainly to the continuous monolayer of intestinal epithelial cells which highly expresses intercellular tight junctions. Fig. 1 depicts the structure of intestinal epithelia. The intercellular tight junctions provide high physical integrity to intestinal epithelial cells [ 40 ]. It comprises of various transmembrane integral proteins, which form a continuous seal between adjacent intestinal cells. The porous structure of tight junctions contains fenestrate with the dimension of 3 to 10 Å [ 41 ]. Large molecular size and hydrophilic nature of proteins and peptides pose a challenge to passing through cellular membrane and also limit the passage through paracellular pathway [ 42 , 43 ]. Physicochemical properties of polymeric NPs can be optimized to facilitate transport across intestinal epithelial cells [ 44 ]. Efflux proteins such as P-glycoprotein (P-gp) may act as a barrier and limit intestinal transport [ 45 ]. Polymeric NPs can be an excellent drug delivery systems to evade the efflux route as Pgp is incapable of recognizing NPs [ 46 ]. Moreover, rapid pH variations in GIT can induce degradation of orally administered proteins and peptides by oxidation, deamidation or hydrolysis. Proteins and peptides are vulnerable to acidic pH in the stomach [ 44 ]. Enzymatic degradation by pepsin in the stomach and/or pancreatic proteases in the intestine, and aminopeptidases existing in the brush-border membrane are other major barriers to oral protein and peptide delivery. In addition, pre-systemic degradation of peptides and proteins due to extensive first-pass metabolism may result in low dose fraction to enter the systemic circulation [ 47 , 48 ]. Consequently, most proteins and peptides exhibit low absorption via oral administration. Polymeric NPs have been thoroughly explored to overcome these barriers and improve the oral absorption of proteins and peptides. NP Transport Mechanisms Polymeric NPs can offer an alternative strategy to improve bioavailability of encapsulated proteins and peptides by providing protection towards degradation in gastrointestinal environment, enhancing cellular contact with intestinal membrane, and promoting absorption in the small intestine [ 49 ]. NPs could be trapped in the adherent mucus layer or translocated by intestinal epithelial cells. Possible interactions of NPs with the intestinal barrier have been depicted in ( Fig. 2 ). There are two distinct mechanisms for NPs transport across the intestinal epithelium: paracellular pathway (between adjacent cells) and transcellular route.

Transcellular route for proteins and peptides includes passive transcellular diffusion, carrier-mediated transport, transcytosis by normal enterocytes (with/without receptor-mediated) and phagocytosis by M cells (with/without receptor-mediated). Transcellular transport of NPs across intestinal epithelium generally involves the latter two pathways ( Fig. 2 ).

M cells represent only 1% of the total intestinal cells and approximately 5% of the human follicle-associated epithelium (FAE) [ 53 ]. M cells mediated phagocytosis is a major route of translocation for orally administered proteins and peptides as well as NPs due to their high transcytotic activity. Several research studies have reported that the majority of particles are translocated in the FAE [ 54 ].

Protein and peptide brain delivery via nasal route has gained considerable attention as it can bypass blood-brain barrier (BBB) and first-pass metabolism and provides rapid brain absorption due to high blood flow and porous endothelial membrane structures [ 114 ]. BBB is considered as the most formidable barrier for drug delivery into the brain from systemic circulation [ 115 ]. Hence, direct brain delivery through intranasal route offers a more promising and viable strategy to improve absorption of therapeutic agents which have short plasma half-life. Nasal cavity can be distinguished into nasal vestibular, respiratory and olfactory regions. Olfactory region has been proposed as the major route for brain transport following intranasal administration ( Fig. 3 ) [ 116 - 124 ]. Several polymeric NPs have been investigated for direct and sustained release of proteins and peptides into the brain following intranasal administration. Cheng et al . demonstrated brain delivery of neurotoxin-I with polylactic acid NPs (NT-I-NPs) following intranasal administration [ 125 ]. Intranasal administration of NT-I-NPs demonstrated shorter time to reach maximum concentration (T max ) relative to intravenous (i.v.) administration of NT-I-NPs and NT-I solution. T max values observed for intranasally and i.v. administered NT-I-NPs was 65 and 95 min, respectively. T max value displayed by NT-I solution was 145 min. Moreover, AUC generated by intranasal administration of NT-I-NPs was significantly higher relative to i.v. administration of NT-I-NPs and NT-I solution. Absolute bioavailability produced by intranasal administration was about 160 and 196% higher relative to i.v. administration of NT-I-NPs and NT-I solution. These results indicate that the brain delivery of peptide loaded NPs can be significantly improved by intranasal administration relative to i.v. administration. Xia et al . have demonstrated the efficacy of low-molecular-weight protamine (LMWP)-modified PEG-PLA NPs to deliver therapeutic agents to CNS via olfactory and trigeminal nerve pathways [ 126 ]. Such nanoparticulate formulation could be employed for improving brain protein or peptide absorption. PLGA NPs loaded with thyrotropin-releasing hormone (TRH) of size 108 ± 12 nm have been developed by Veronesi et al . [ 127 ]. Intranasal administration of TRH-loaded NPs significantly raised the stimulations required to reach stage V seizures in rats. Moreover, seizure after duration discharge was observed to be significantly reduced in rats treated with TRH-loaded NPs intranasally. These observations clearly indicate the neuroprotective potential of intranasally administered TRH-loaded NPs. A recent report discussed brain concentrations of neurotoxin-I (NT-I) peptide after intranasal administration as solution and PLA NPs coated with polysorbate-80 [ 128 ]. Investigators observed an approximately 3-fold higher accumulation of NT-I with NPs compared to solution. Concentrations of NT-I observed with NPs and solutions were 18.23 ± 3.30 ng/mL and 6.26 ± 0.23 ng/mL, respectively. Moreover, NPs generated about 2-fold higher brain concentration relative to i.v. administration (8.56 ± 0.33 ng/mL). The same group of investigators developed neurotoxin-II (NT-II)-loaded PLA NPs coated with polysorbate-80 [ 129 ]. Brain concentrations of NT-II observed after intranasal administration of solution and NPs were 0.26 ±0.03 and 8.66 ± 0.30 ng/g, respectively. NPs generated more than 30-fold higher brain concentrations of NT-II relative to solution. Moreover, NPs generated about 3-fold higher brain concentration relative to i.v. administration (2.50 ±0.21 ng/g). These observations clearly indicate that PLA NPs coated with polysorbate-80 have significant potential to enhance the antinociceptive activity of these peptides after intranasal delivery. Recently, Liu et al. demonstrated the transport of wheat germ agglutinin-modified PEG-PLA NPs into the brain following intranasal administration [ 130 ]. Ex vivo imaging analysis clearly displayed that olfactory and trigeminal nerve pathways were primarily responsible for transport of NPs to brain. Investigators also proposed that the brain transport was possibly due to extracellular transport along the nerve fibers. Lactoferrin receptor has been reported to be overly expressed on the luminal surface of respiratory epithelium, brain capillary endothelial cells and neurons [ 131 ]. Moreover, it has been observed to be highly expressed in CNS neurodegenerative conditions such as Alzheimer's, Parkinson's and Huntington's disease. Such high expression could be useful in generating lactoferrin receptor targeted delivery of polymeric NPs to promote absorption of proteins and peptides in these tissues. Lactoferrin functionalized PEG-co-PCL NPs exhibited significantly higher brain accumulation of coumarin-6 relative to unmodified NPs [ 131 ]. AUC 0-8h of coumarin-6 generated by lactoferrin-modified NPs in rat cerebrum, cerebellum, olfactory tract,

Parenteral route has attracted considerable attention for nanoparticulate delivery as it can avoid first-pass metabolism, provide highest bioavailability and active targeting for drug delivery [ 143 ]. However, NPs properties such as size, charge, dissolution rate and surface coating can significantly alter the in vivo fate [ 143 , 144 ]. Moreover, NPs with size greater than 200 nm or slow dissolution rate can be rapidly cleared by mononuclear phagocytic system [ 145 ]. This process might significantly enhance nanoparticulate uptake in liver and spleen. PEGylation has been proposed as a promising approach to evade reticuloendothelial system uptake [ 146 - 148 ]. In addition, the use of poly (N-vinyl-2-pyrrolidone) as a coating material to improve blood circulation time of particulate carriers has also been reported [ 149 ]. Interestingly, NPs with size between 100-300 nm can highly accumulate in tumors due to enhanced permeability and retention effect [ 143 , 150 ]. For systemic to brain administration, BBB poses a formidable barrier for brain transport of proteins and peptides ( Fig. 4 ). Brain capillary endothelial cells (BCECs) express tight intracellular junctions and occlude paracellular spaces. Moreover, BCECs are also encircled by astrocytic end feet and pericytes which further forms an implausible barrier for protein and peptide transport. Hence, there is an imperative need for the development of drug delivery systems capable of overcoming BBB following systemic administration. In recent years, several polymeric NPs have been investigated to target poorly permeable tissues such as brain and lung following systemic administration. Lactoferrin-modified PEG-PLGA NPs have been investigated for their efficacy to treat Parkinson's disease [ 151 ]. Urocortin-loaded NPs were spherical in shape and exhibited size of 120 nm. In vivo studies clearly demonstrated that i.v. injection of urocortin-loaded NPs significantly reduced striatum lesions caused by 6-hydroxydopamine. Angiopep-2 peptide and EGFP-EGF1 protein functionalized PEG-PCL NPs have been developed by Huile et al. [ 152 ]. These functionalized NPs demonstrated remarkable potential in penetrating BBB (angiopep-2) and binding to neuroglial cells (EGFP-EGF1). Moreover, these dual functionalized NPs generated significantly higher brain accumulation relative to unmodified NPs. These targeting potential of angiopep-2 peptide and EGFP-EGF1 protein represents a novel promising formulation for the treatment of neuroglial related diseases following systemic administration. A novel CS based NPs loaded with caspase inhibitor N-Benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone (Z-DEVD-FMK) or basic fibroblast growth factor have been observed to rapid transport across BBB following systemic circulation [ 153 , 154 ]. Recently, Bao et al. developed OX26 functionalized hyperbranched polyglycerol-conjugated PLGA to improve brain delivery of endomorphins [ 155 ]. NPs were spherical in shape with an average size of 170 ± 20 nm. Drug loading and entrapment efficiency observed were 8.65 ± 1.27% and 83.96 ± 2.60 %, respectively. Nearly, 65% of endomorphin was released in first 72 h. Moreover, OX26-modified NPs generated significantly higher analgesic activity in chronic constriction injured rats relative to unmodified NPs and endomorphin itself, following i.v. injection. Xia et al. demonstrated penetratin (CPP with low cationic amino acid content) modified polyethylene glycol)-poly(lactic acid) NPs as an excellent brain drug delivery system compared to protamine (high arginine content) modified NPs [ 156 ]. Such systems can be utilized for improving brain protein or peptide delivery following systemic administration. RVG29- and CS-conjugated pluronic-based nanocarrier has been proposed as a promising formulation for improving brain delivery of proteins such as β-galactosidase [ 157 ]. B6 peptide (CGHKAKGPRK) functionalized PEG-PLA NPs have been demonstrated as an excellent delivery system for the brain delivery of neuroprotective peptide-NAPVSIPQ (NAP) [ 158 ]. NAP-loaded B6 NPs were 118.3 ± 7.8 nm in size with entrapment and loading efficiency of 0.65 ± 0.021% and 0.57 ± 0.023%, respectively. NAP-loaded B6 NPs demonstrated remarkable improvement in learning impairment recovery, loss of hippocampal neurons, and cholinergic disruption at significantly lower doses. The shape of NPs has been observed to affect the hydrodynamic properties and interactions with target tissues significantly [ 159 ]. Moreover, size, surface charge and modification (targeting ligand) can significantly influence the uptake mechanism at BBB [ 160 ]. This process in turn affects the endocytotic pathway mechanisms and therefore, intracellular fate of NPs. Such information might be valuable for brain specific delivery of NPs following i.v. administration.

Ocular route has been utilized for both systemic and localized delivery of proteins and peptides. For systemic delivery, ocular route provides advantages such as ease of administration, avoidance of first-pass metabolism and comparatively rapid rate of absorption (over oral administration) [ 164 ]. Systemic absorption of several proteins and peptides including insulin, calcitonin, and enkephalins has been studied following ocular administration [ 165 - 167 ]. Currently, ocular route is mainly used for the delivery of proteins and peptides for the treatment of local ocular disorders such as age related macular degeneration, dry eye disease, or proliferative diabetic retinopathy. Lucentis ® and Eylea ® are the recently marketed proteins intended for the treatment of ocular diseases [ 9 , 168 ]. The physiological and anatomical barriers and enzymatic degradation within the ocular environment limit the efficacy of proteins and peptides administered by ocular route [ 169 ]. Polymeric NPs have been successfully designed to overcome these barriers and improve ocular bioavailability of proteins and peptides. Polymeric NPs can improve localized ocular delivery by providing sustain release, protection from enzymatic degradation and enhancing precorneal residence time compared to aqueous eye drops. However, NPs may be eliminated rapidly from precorneal pocket. Hence, for topical administration it is highly desirable to formulate NPs with mucoadhesive properties to increase their retention time in the cul-de-sac. PEG, carbopol and hyaluronic acid have been utilized to improve NPs precorneal residence time [ 170 - 172 ]. The prolonged residence time allows optimal contact between the formulation and mucosa and thereby sufficient drug concentration in external ocular tissues can be achieved. In a recent study, coating of cyclosporine A (CsA)-loaded poly caprolactone (PCL)/benzalkonium chloride (BKC) nanospheres with hyaluronic acid resulted in high concentrations of cyclosporine A into the cornea compared with non-coated NPs [ 172 ]. Cationic polymers such as Eudragit ® have also been employed to increase precorneal residence time. It was observed that Eudragit ® can prolong the residence time of NPs by interacting with the anionic mucins present in the mucus layer at the eye surface. Aksungur et al. evaluated efficiency of Eudragit ® in improving effectiveness of CsA NPs formulations against inflammation of the eye surface [ 171 ]. The NPs were prepared using either PLGA alone or a mixture of Eudragit ® RL with PLGA or were coated with Carbopol ® . Tear kinetic parameters were determined following topical application of CsA-loaded NPs suspension and RestasisA ® (drug-loaded emulsion) in rabbit eye ( Table 4 ). Cellular uptake, tear film concentration of the drug and AUC 0→24h were significantly higher for PLGA: Eudragit ® RL (75:25)-CsA NPs (cationic NPs) and Carbopol ® coated PLGA-CsA NPs (adhesive) formulations compared to Restasis A ® . hese results clearly demonstrate the effect of surface characteristics of NPs in the improvement of ocular retention and bioavailability. Even though topical administration is the major route for the anterior segment conditions, it is difficult to deliver proteins and peptides to posterior ocular tissues via topical route. Currently, intravitreal injection is the most commonly employed technique to treat posterior ocular diseases. However, short vitreal half-life of proteins and peptides and chronic nature of most of the posterior ocular diseases lead to requirement of frequent intravitreal injections. These administrations are usually associated with potential undesired side effects such as increased risk of cataract development, vitreous hemorrhage, retinal detachment, and endophthalmitis leading to poor patient acceptance. Several research studies have demonstrated suitability of intravitreally injected NPs in the treatment of posterior segment ocular diseases. In a recent study, Kim et al . have developed polylactic acid/polylactic acid-polyethylene oxide NPs (PLA/PLA-PEO) for the delivery of C16Y (an integrin-antagonist peptide) to the sub-retinal space for the treatment of choroidal neovascularization (CNV) [ 173 ]. Efficacy of C16Y-NPs was evaluated in laser-induced CNV in rats. A single intravitreal administration of both C16Y peptide and C16Y-NPs inhibited CNV at 5 and 9 days post laser photocoagulation. However, CNV inhibition was significantly higher for C16Y-NPs than the C16Y peptide solution on day 5. Because of short intravitreal half-life of C16Y peptide, inhibition of the experimental CNV was less with the peptide solution as compared to C16Y-NPs. On the contrary, the prolonged C16Y peptide release from C16Y-NPs aided to more CNV inhibition following intravitreal injection. These results demonstrate the potential of intravitreally injected biodegradable polymer-based NPs for treating choroidal neovascularization related to age-related macular degeneration.