Oral Delivery Of Protein Drugs Research Articles

Enhanced oral bioavailability of insulin using PLGA nanoparticles co-modified with cell-penetrating peptides and Engrailed secretion peptide (Sec)

Biodegradable polymer nanoparticle drug carriers are an attractive strategy for oral delivery of peptide and protein drugs. However, their ability to cross the intestinal epithelium membrane is largely limited. Therefore, in the present study, cell-penetrating peptides (R8, Tat, penetratin) and a secretion peptide (Sec) with N-terminal stearylation were introduced to modify nanoparticles (NPs) on the surface to improve oral bioavailability of peptide and protein drugs. In vitro studies conducted in Caco-2 cells showed the value of the apparent permeability coefficient (Papp) of the nanoparticles co-modified with Sec and penetratin (Sec-Pen-NPs) was about two-times greater than that of the nanoparticles modified with only penetratin (Pen-NPs), while the increase of transcellular transport of nanoparticles modified together with Sec and R8 (Sec-R8-NPs), or Sec and Tat (Sec-Tat-NPs), was not significant compared with nanoparticles modified with only R8 (R8-NPs) or Tat (Tat-NPs). Using insulin as the model drug, in vivo studies performed on rats indicated that compared to Pen-NPs, the relative bioavailability of insulin for Sec-Pen-NPs was 1.71-times increased after ileal segments administration, and stronger hypoglycemic effects was also observed. Therefore, the nanoparticles co-modified with penetratin and Sec could act as attractive carriers for oral delivery of insulin.

Characterization of particulate drug delivery systems for oral delivery of Peptide and protein drugs.

Oral drug delivery is a preferred route because of good patient compliance. However, most peptide/ protein drugs are delivered via parenteral routes because of the absorption barriers in the gastrointestinal (GI) tract such as enzymatic degradation by proteases and low permeability acrossthe biological membranes. To overcome these barriers, different formulation strategies for oral delivery of biomacromolecules have been proposed, including lipid based formulations and polymer-based particulate drug delivery systems (DDS). The aim of this review is to summarize the existing knowledge about oral delivery of peptide/protein drugs and to provide an overview of formulationand characterization strategies. For a better understanding of the challenges in oral delivery of peptide/protein drugs, the composition of GI fluids and the digestion processes of different kinds of excipients in the GI tract are summarized. Additionally, the paper provides an overview of recent studies on characterization of solid drug carriers for peptide/protein drugs, drug distribution in particles, drug release and stability in simulated GI fluids, as well as the absorption of peptide/protein drugs in cell-based models. The use of biorelevant media when applicable can increase the knowledge about the quality of DDS for oral protein delivery. Hopefully, the knowledge provided in this review will aid the establishment of improved biorelevant models capable of forecasting the performance of particulate DDS for oral peptide/protein delivery.

Enhanced permeability across Caco-2 cell monolayers by specific mannosylating ligand of buserelin acetate proliposomes

Context: Oral delivery of peptide and protein drugs still remains the area of challenges due to their low stability and permeability across GI tract. Among numerous attempts, the receptor-mediated drug targeting is a promising approach to enhance GI permeability.Objective: The aim of this study was to prepare mannosylated buserelin acetate (MANS-BA) proliposome powders grafted with N-octadecyl-d-mannopyranosylamine (SAMAN) as targeting moiety and evaluate their permeability across Caco-2 cell monolayers.Materials and methods: The MANS-BA proliposome powders were prepared by coprecipitation method. The targeting moiety SAMAN was synthesized in-house and confirmed by characterization using Fourier transform infrared (FTIR) and differential scanning calorimeter (DSC).Results: The MANS-BA liposomes reconstituted from proliposome powders exhibited the oligolamellar vesicular structure of phospholipid bilayer. Their size, zeta potential and entrapment efficiency were in the ranges of 93.11–218.95 nm, −24.03 to −37.15 mV and 21.12–33.80%, respectively. The permeability of reconstituted MANS-BA liposomes across Caco-2 cell monolayers was significantly enhanced to about 1.2- and 2.2-fold over those of conventional BA liposomes and solution, respectively.Discussion: Increase in dicetylphosphate, cholesterol and SAMAN contents resulted in significant increase in size and zeta potential of reconstituted MAN-BA liposomes. The entrapment efficiency was increased with increasing dicetylphosphate and mannitol contents in liposomes containing cholesterol.Conclusions: The significantly enhanced permeability across Caco-2 cell monolayers of MANS-BA liposomes might be due to the role of mannose receptor on intestinal enterocytes.

Overcoming the diffusion barrier of mucus and absorption barrier of epithelium by self-assembled nanoparticles for oral delivery of insulin.

Nanoparticles (NPs) have demonstrated great potential for the oral delivery of protein drugs that have very limited oral bioavailability. Orally administered NPs could be absorbed by the epithelial tissue only if they successfully permeate through the mucus that covers the epithelium. However, efficient epithelial absorption and mucus permeation require very different surface properties of a nanocarrier. We herein report self-assembled NPs for efficient oral delivery of insulin by facilitating both of these two processes. The NPs possess a nanocomplex core composed of insulin and cell penetrating peptide (CPP), and a dissociable hydrophilic coating of N-(2-hydroxypropyl) methacrylamide copolymer (pHPMA) derivatives. After systematic screening using mucus-secreting epithelial cells, NPs exhibit excellent permeation in mucus due to the "mucus-inert" pHPMA coating, as well as high epithelial absorption mediated by CPP. The investigation of NP behavior shows that the pHPMA molecules gradually dissociate from the NP surface as it permeates through mucus, and the CPP-rich core is revealed in time for subsequent transepithelial transport through the secretory endoplasmic reticulum/Golgi pathway and endocytic recycling pathway. The NPs exhibit 20-fold higher absorption than free insulin on mucus-secreting epithelium cells, and orally administered NPs generate a prominent hypoglycemic response and an increase of the serum insulin concentration in diabetic rats. Our study provides the evidence of using pHPMA as dissociable "mucus-inert" agent to enhance mucus permeation of NPs, and validates a strategy to overcome the multiple absorption barriers using NP platform with dissociable hydrophilic coating and drug-loaded CPP-rich core.

Development and evaluation of mucoadhesive nanoparticles based on thiolated Eudragit for oral delivery of protein drugs

The objective of this study was to develop pH-sensitive Eudragit L100–cysteine/reduced glutathione (Eul–cys/GSH) nanoparticles (NPs), which provided the mucoadhesion and protection for protein drugs against enzymatic degradation. Insulin was chosen as a model biomolecule for testing this system. The Eul–cys conjugate, which was obtained by grafting cysteine onto the carboxy group of Eudragit L100, was analyzed by HNMR and SEM, and the swelling degree (SD), cation binding, and enzymatic inhibition were also determined. The results obtained showed that the Eul–cys conjugate represent a pH-sensitive delivery system which effectively protected the insulin from being degraded by the proteases, and this is related to the mechanism of Ca2+ binding. Insulin-loaded Eul–cys/GSH NPs were prepared by a diffusion method involving an electrostatic interaction between the network structure of the polymer and the embedded proteins, including insulin and GSH. TEM images indicated that Eul–cys/GSH existed as smooth and spherical NPs in aqueous solution with particle sizes of 260 ± 20 nm. The X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) findings showed the presence of amorphous insulin in thiolated NPs and higher free thiol oxidation than the result obtained by Ellman’s reagent method. In addition, thiolated NPs showed excellent binding efficiency to the mucin in rat intestine, indicating that Eul–cys/GSH NPs have great potential to be applied as safe carriers for the oral administration of protein drugs.

Design and evaluation of solid lipid nanoparticles modified with peptide ligand for oral delivery of protein drugs

Designing feasible and effective peptide ligand modified solid lipid nanoparticles (SLNs) to improve oral bioavailability of protein drugs and evaluating the influence of mucus remains important. In the present work, two kinds of peptide ligand modified SLNs loaded with salmon calcitonin (sCT), namely, sCT CSK-SLNs and sCT IRQ-SLNs, were prepared by coupling the peptide ligand CSKSSDYQC (CSK) which was reported to show affinity with goblet cells, or IRQRRRR (IRQ), a cell penetrating peptide, to polyoxyethylene (40) stearate (SA-PEG2000). Compared with unmodified SLNs, CSK or IRQ modified SLNs with better drug protection ability could facilitate the internalization of drug on Caco-2/HT29-MTX co-cultured cells and permeation in excised rat duodenum mucosa. The internalization mechanism of two kinds of peptide ligand modified SLNs was mainly active transport via both clathrin- and caveolae-dependent endocytosis. Although mucus was an impediment to the transport of SLNs, the peptide ligand modified SLNs still showed improved drug absorption. The absolute bioavailability of sCT CSK-SLNs (12.41±3.65%) and sCT IRQ-SLNs (10.05±5.10%) raised to 2.45-fold and 1.98-fold compared with unmodified SLNs (5.07±0.54%), implying the feasibility and effectiveness of CSK and IRQ peptide modification for the enhancement of the oral bioavailability of protein drugs. In summary, the nanoparticles modified with CSK or IRQ peptide ligand could be the potential carriers for the transport of protein drugs across intestinal barriers.

Structure-Toxicity Relationship of Chemically Modified Chitosan as an Oral Protein Drug Delivery Carrier

Structure-Toxicity Relationship of Chemically Modified Chitosan as an Oral Protein Drug Delivery Carrier

Solid lipid particles for oral delivery of peptide and protein drugs III - the effect of fed state conditions on the in vitro release and degradation of desmopressin.

The effect of food intake on the release and degradation of peptide drugs from solid lipid particles is unknown and was therefore investigated in vitro using different fed state media in a lipolysis model. Desmopressin was used as a model peptide and incorporated into solid lipid particles consisting of trimyristin (TG14), tripalmitin (TG16), and tristearin (TG18), respectively. Fasted state and fed state media with varying phospholipid and bile salt concentrations, as well as fed state media with milk and oleic acid glycerides, respectively, were used as the release media. The presence of oleic acid glycerides accelerated the release of desmopressin significantly from all solid lipid particles both in the presence and absence of lipase. The presence of oleic acid glycerides also reduced the degradation rate of desmopressin, probably due to the interactions between the lipids and the protease or desmopressin. Addition of a medium chain triglyceride, trilaurin, in combination with drug-loaded lipid particles diminished the food effect on the TG18 particles, and trilaurin is therefore proposed to be a suitable excipient for reduction of the food effect. Overall, the present study shows that strategies to reduce food effect, such as adding trilaurin, for lipid particle formulations should be considered as drug release from such formulations might be influenced by the presence of food in the gastrointestinal tract.

Solid Lipid Particles for Oral Delivery of Peptide and Protein Drugs II – The Digestion of Trilaurin Protects Desmopressin from Proteolytic Degradation

To investigate the in vitro release and degradation of desmopressin from saturated triglyceride microparticles under both lipolytic and proteolytic conditions. The release of desmopressin from different solid lipid microparticles in the absence and presence of a microbial lipase and protease was determined. Trilaurin (TG12), trimyristin (TG14), tripalmitin (TG16), and tristearin (TG18) were used as lipid excipients to produce solid lipid microparticles. In the presence of lipase, the rate of drug release from different lipid particles was in the order of TG14 > TG16 > TG18, which is the same rank order as the lipid degradation rate. A reverse rank order was found for the protection of desmopressin from enzymatic degradation due to spatial separation of desmopressin from the protease. TG12 accelerated the release of desmopressin from all lipid particles when added as either drug-free microparticles to the lipolysis medium or incorporated in TG16 particles. Additionally, TG12 particles protected desmopressin from degradation when present in the lipolysis medium with the other lipid microparticles. TG12 is a very interesting lipid for oral lipid formulations containing peptides and proteins as it alters release and degradation of the incorporated desmopressin. The present study demonstrates the possibility of bio-relevant in vitro evaluation of lipid-based solid particles.

PLGA–lipid liposphere as a promising platform for oral delivery of proteins

The main challenge in the oral delivery of protein drugs is to enhance their oral bioavailability. Herein, we report the uniform-sized liposphere prepared by premix membrane emulsification combined with W1/O/W2 double-emulsion method as a potential oral carrier for proteins. The protein-loaded liposphere was composed of a hydrophobic poly (d, l-lactide-co-glycolide) (PLGA) core and the lipid molecules self-assembled at the interface of W1/O and O/W2. During the preparation, the protein structure was effectively maintained. Compared with PLGA microsphere, the liposphere achieved a higher loading capacity (LC, 20.18%), entrapment efficiency (EE, 90.82%) and a lower initial burst (24.73%). Importantly, the lipospheres also showed high transcytotic efficiency with human microfold cell (M cell) model, leading to a potential enhancement of intestinal absorption. This result, together with the above studies supported that the PLGA–lipid liposphere could be a promising platform for enhancing the proteins oral bioavailability.

PH-responsive release of proteins from biocompatible and biodegradable reverse polymer micelles

A reverse polymer micelle with a diameter of 100nm was prepared for a protein carrier releasing payloads in a pH-dependent manner. The reverse polymer micelle was made from an amphiphilic diblock copolymer of biodegradable poly(d,l-lactic-co-glycolic acid) (PLGA) and biocompatible poly(ethylene glycol) (PEG). PLGA having a terminal carboxyl group was additionally embedded in the micelle's PLGA layer via hydrophobic interaction. The micelles encapsulating bovine serum albumin and streptavidin released the proteins under neutral and basic conditions, whereas the proteins remained in the interior at acidic pH. Using erythropoietin as a protein drug, it was also exemplified that the released protein retained its cell proliferation activity even after rigorous formulation processes, including water-in-oil emulsion. The present reverse polymer micelle could potentially find application as an oral protein drug delivery carrier.

Synthesis and evaluation of a novel β-cyclodextrin derivative for oral insulin delivery and absorption

In this paper, we describe the synthesis and testing of a novel β-cyclodextrin derivative, carboxymethy-hydroxypropyl-β-cyclodextrin (CM-HP-β-CD). After synthesis, we verified that carboxymethyl and hydroxypropyl groups were successfully substituted onto β-cyclodextrin to form CM-HP-β-CD. Then, we performed in vitro experiments to investigate (1) the ability of CM-HP-β-CD to bind insulin, (2) the transportation of insulin-CM-HP-β-CD complexes across a Caco-2 cell monolayer, and (3) the cytotoxicity of CM-HP-β-CD. CM-HP-β-CD showed the strongest insulin-binding ability and the best transport properties compared with the other β-cyclodextrin derivatives we evaluated. In addition, CM-HP-β-CD showed no cytotoxicity up to 200mM (IC50>200mM). To examine the effectiveness of the cyclodextrin complexes in delivering insulin in vivo, we administered different insulin-cyclodextrin complexes to diabetic rats. In these studies, we found that the insulin-CM-HP-β-CD complex provided a significant and sustained (5h) reduction in the blood glucose levels of diabetic rats. Overall, our study showed that CM-HP-β-CD may be a promising protein carrier for use in oral protein drug delivery.

Solid lipid particles for oral delivery of peptide and protein drugs I – Elucidating the release mechanism of lysozyme during lipolysis

The mechanism of protein release from solid lipid particles was investigated by a new lipolysis model in a biorelevant medium containing both bile salts and phospholipids. Lysozyme, a model protein, was formulated into solid lipid particles using four different types of lipids, two triglycerides with different chain-length of fatty acyl groups i.e. trimyristin (TG14) and tristearin (TG18), and two lipid blends dominated by diglycerides and monoglycerides, respectively. The release of lysozyme from the solid lipid particles and the lipid hydrolysis process were assessed in the lipolysis model, while the change in particle surface during the lipolysis process was evaluated using scanning electron microscopy. The lysozyme release profiles from TG14 and TG18 as well as diglyceride particles correlated well with the release of free fatty acids from the lipid particles during the lipolysis and therefore exhibited a lipase-mediated degradation-based release mechanism. The release of lysozyme from monoglyceride particles was independent on lipase degradation due to the instability of the lipid matrix in the lipolysis medium. In conclusion, the established lipolysis model is successfully used to elucidate the drug release mechanism from solid lipid particles and can potentially be used in rational selection of lipid excipients for oral delivery of peptide/protein drugs.

Eudragit® L100/N-Trimethylchitosan Chloride Microspheres for Oral Insulin Delivery

Effective oral delivery of protein and peptide drugs remains an active topic in scientific research. In this study, matrix type microspheres were prepared with Eudragit® L100 containing N-trimethylchitosan chloride to improve the permeation of insulin across the intestinal epithelium via the paracellular pathway. Insulin loaded microspheres were initially formulated in accordance with a factorial design (23) and manufactured by means of a single water-in-oil emulsification/evaporation method. Based on external and internal morphology two microsphere formulations were selected from the initial formulations for further investigation in terms of particle size, dissolution behaviour and in vitro insulin transport across excised rat intestinal tissue. The initial eight microsphere formulations exhibited drug loading capacities ranging from 27.9–52.4% with different shapes and internal structures. The two selected microsphere formulations had average particle sizes of 157.3 ± 31.74 µm and 135.7 ± 41.05 µm, respectively, and mean dissolution time values for insulin release of 34.47 and 42.63 min, respectively. In vitro transport of insulin across excised rat intestinal tissue from the two selected microsphere formulations was 10.67–fold and 9.68–fold higher than the control group (insulin alone). The microsphere delivery system prepared from Eudragit® L100 containing N-trimethylchitosan chloride is therefore a promising candidate for effective oral insulin delivery.

Synthesis of pH-responsive starch nanoparticles grafted poly (l-glutamic acid) for insulin controlled release

In this article, a novel oral protein drug delivery made of starch nanoparticles (SNPs) as backbone and poly (l-glutamic acid) (PGA) as graft chains was successfully synthesized by click reaction. The grafting efficiency and structure of the resultant copolymer SNP-g-PGA were verified by 1H NMR and FT-IR spectra. The copolymer could assemble into amphiphilic aggregates. Hydrodynamic radii (Rh) of the aggregate decreased obviously as increasing pH value due to its excellent pH-dependent property. To take advantage of this pH-responsive property, in vitro insulin release experiment was carried out. The loaded-insulin released from the copolymers more slowly in artificial gastric juice (pH=1.2) than that in artificial intestinal liquid (pH=6.8) due to the excellent stability in acidic condition. These results suggested that the natural starch based material with pH sensitivity could be a promising delivery for insulin controlled release.

Approaches for enhancing oral bioavailability of peptides and proteins

Oral delivery of peptide and protein drugs faces immense challenge partially due to the gastrointestinal (GI) environment. In spite of considerable efforts by industrial and academic laboratories, no major breakthrough in the effective oral delivery of polypeptides and proteins has been accomplished. Upon oral administration, gastrointestinal epithelium acts as a physical and biochemical barrier for absorption of proteins resulting in low bioavailability (typically less than 1-2%). An ideal oral drug delivery system should be capable of (a) maintaining the integrity of protein molecules until it reaches the site of absorption, (b) releasing the drug at the target absorption site, where the delivery system appends to that site by virtue of specific interaction, and (c) retaining inside the gastrointestinal tract irrespective of its transitory constraints. Various technologies have been explored to overcome the problems associated with the oral delivery of macromolecules such as insulin, gonadotropin-releasing hormones, calcitonin, human growth factor, vaccines, enkephalins, and interferons, all of which met with limited success. This review article intends to summarize the physiological barriers to oral delivery of peptides and proteins and novel pharmaceutical approaches to circumvent these barriers and enhance oral bioavailability of these macromolecules.

Peptide-enhanced oral delivery of therapeutic peptides and proteins

Systemic therapy upon oral delivery of biologics, such as peptide and protein drugs is limited due to their large molecular size, their low enzymatic stability and their inability to cross the intestinal epithelium. Ways to overcome the epithelial barrier include the use of peptide-based excipients such as the cell penetrating peptides (CPPs) and the tight junction modulating peptides (TJMPs), which are able to translocate across the cellular membranes in a non-disruptive way or reversibly modulate the integrity of intercellular tight junctions (TJs), respectively. However, because of the harsh environment throughout the gastrointestinal (GI) tract, chemical stability is an inherent challenge when employing amino acid-based excipients for oral delivery, and multiple approaches have been investigated to improve this. The exact mechanisms of transepithelial translocation are discussed, and it is believed that CPP-mediated translocation involves transcytosis and/or direct translocation through the epithelial cells; whereas TJMP-mediated translocation is dependent on interaction with transmembrane or peripheral TJ proteins. This review focuses on the CPPs and the TJMPs currently employed as excipients for oral delivery of peptide and protein drugs highlighting recent studies and the most promising compounds from these classes of peptide excipients.

Phage selection of cyclic peptide antagonists with increased stability toward intestinal proteases

The oral delivery of protein and peptide drugs is limited by their proteolytic degradation and the poor absorption across the intestinal epithelia. In this work, we exposed a phage library of small bicyclic peptides (<1.5 kDa) to a pancreatic extract of proteases prior to affinity selection to enrich binders with higher stability in the intestinal environment. Panning with the therapeutic target plasma kallikrein yielded potent inhibitors (K(i)s between 5.6 and 336 nM) wherein bicyclic peptides isolated with proteolytic pressure were more stable. A proline residue found in a specific position of several resistant bicyclic peptides proved to be a 'protective mark', rendering the bicyclic peptides resistant to significantly higher concentrations of intestinal proteases while retaining essentially their inhibitory activity.

Chitosan-Carrageenan Polyelectrolyte Complex for the Delivery of Protein Drugs

A chitosan-carrageenan polyelectrolyte complex (PEC) was prepared by salt induced impeding of polyplex formation method and was encapsulated with bovine serum albumin (BSA) to study the potential to be tailored to the pH responsive oral delivery of protein drugs. The FTIR spectra showed the successful formation of the PEC under the experimental condition. The release kinetics of BSA from the PEC was studied in the simulated gastrointestinal fluids with and without digestive enzymes. The prepared PEC showed the nature of pH-sensitivity. A typical controlled release of BSA from the PEC (180 μg of BSA from 3 mg of PEC) was obtained in the simulated intestinal fluid (SIF, pH 7.5), which was due to the significant swelling and disintegration of PEC, but little amount of BSA was released (11 μg of BSA from 3 mg of PEC) in the simulated gastric fluid (SGF, pH 1.2), confirming acidic stability of the prepared PEC. The presence of digestive enzymes was found not to affect the response of PEC to ambient pH value, but to speed up the release of BSA from carriers.

Size-dependent absorption mechanism of polymeric nanoparticles for oral delivery of protein drugs

Polymeric nanoparticles have been widely applied to oral delivery of protein drugs, however, few studies focused on the systematical elucidation of the size-dependent oral absorption mechanism with well-defined polymeric nanoparticles. Rhodamine B labeled carboxylated chitosan grafted nanoparticles (RhB-CCNP) with different particle sizes (300, 600, and 1000 nm) and similar Zeta potentials (−35 mV) were developed. FITC labeled bovine serum albumin (FITC-BSA) was encapsulated into RhB-CCNP to form drug loaded polymeric nanoparticles (RhB-CCNP-BSA). RhB-CCNP-BSA with uniform particle size and similar surface charge possessed desired structural stability in simulated physiological environment to substantially guarantee the validation of elucidation on size-dependent absorption mechanisms of polymeric nanoparticles using in vitro, in situ, and ex vivo models. RhB-CCNP-BSA with smaller sizes (300 nm) demonstrated elevated intestinal absorption, as mechanistically evidenced by higher mucoadhesion in rat ileum, release amount of the payload into the mucus layer, Caco-2 cell internalization, transport across Caco-2 cell monolayers and rat ileum, and systemic biodistribution after oral gavage. Peyer’s patches could play a role in the mucoadhesion of nanoparticles, resulting in their close association with the intestinal absorption of nanoparticles. These results provided guidelines for the rational design of oral nanocarriers for protein drugs in terms of particle size.