Chitosan-modified porous silicon microparticles for enhanced permeability of insulin across intestinal cell monolayers

Porous silicon (PSi) based particulate systems are emerging as an important drug delivery system due to its advantageous properties such as biocompatibility, biodegradability and ability to tailor the particles' physicochemical properties. Here, annealed thermally hydrocarbonized PSi (AnnTHCPSi) and undecylenic acid modified AnnTHCPSi (AnnUnTHCPSi) microparticles were developed as a PSi-based platform for oral delivery of insulin. Chitosan (CS) was used to modify the AnnUnTHCPSi microparticles to enhance the intestinal permeation of insulin. Surface modification with CS led to significant increase in the interaction of PSi microparticles with Caco-2/HT-29 cell co-culture monolayers. Compared to pure insulin, the CS-conjugated microparticles significantly improved the permeation of insulin across the Caco-2/HT-29 cell monolayers, with ca. 20-fold increase in the amount of insulin permeated and ca. 7-fold increase in the apparent permeability (P_app) value. Moreover, among all the investigated particles, the CS-conjugated microparticles also showed the highest amount of insulin associated with the mucus layer and the intestinal Caco-2 cells and mucus secreting HT-29 cells. Our results demonstrate that CS-conjugated AnnUnTHCPSi microparticles can efficiently enhance the insulin absorption across intestinal cells, and thus, they are promising microsystems for the oral delivery of proteins and peptides across the intestinal cell membrane.     

Intestinal mucosa permeability following oral insulin delivery using core shell corona nanolipoparticles

Chitosan nanoparticles (NC) have excellent capacity for protein entrapment, favorable epithelial permeability, and are regarded as promising nanocarriers for oral protein delivery. Herein, we designed and evaluated a class of core shell corona nanolipoparticles (CSC) to further improve the absorption through enhanced intestinal mucus penetration. CSC contains chitosan nanoparticles as a core component and pluronic F127-lipid vesicles as a shell with hydrophilic chain and polyethylene oxide PEO as a corona. These particles were developed by hydration of a dry pluronic F127-lipid film with NC suspensions followed by extrusion. Insulin nested inside CSC was well protected from enzymatic degradation. Compared with NC, CSC exhibited significantly higher efficiency of mucosal penetration and, consequently, higher cellular internalization of insulin in mucus secreting E12 cells. The cellular level of insulin after CSC treatment was 36-fold higher compared to treatment with free insulin, and 10-fold higher compared to NC. CSC significantly facilitated the permeation of insulin across the ileum epithelia, as demonstrated in an ex vivo study and an in vivo absorption study. CSC pharmacological studies in diabetic rats showed that the hypoglycemic effects of orally administrated CSC were 2.5-fold higher compared to NC. In conclusion, CSC is a promising oral protein delivery system to enhance the stability, intestinal mucosal permeability, and oral absorption of insulin.     

Cellular evaluation of insulin transmucosal delivery

P(MAA-g-EG) microparticles have been extensively investigated as carriers for oral delivery of proteins such as insulin. In this study, we investigated the effect of the molecular weight of the PEG tethered chains in the copolymer network and of the microparticle size on the transepithelial electrical resistance (TEER) and insulin epithelial permeability, using monolayers of human intestinal epithelial Caco-2 cells. Two molecular weights of the PEG chains, 400 and 1000, were investigated, as well as three different dry microparticle sizes: 25-90, 90-150 and 150-212 μm. Their effect on the cell monolayer integrity was studied by monitoring TEER as a fraction of time and determining insulin permeability. The presence of insulin-loaded P(MAA-g-EG) microparticles decreases the TEERs value by 50% with respect to the control. This disruption of the cell monolayer was recovered in 3 h after the removal of the polymer microparticles. Within the range of PEG molecular weights studied, there was no significant change of the TEER values. However, decreased microparticle sizes and short PEG chains systems led to higher permeability values. Insulin-loaded P(MAA-g-EG) microparticles enhanced the transport of insulin through the Caco-2 cell monolayers.     

Cellular evaluation of insulin transmucosal delivery

P(MAA-g-EG) microparticles have been extensively investigated as carriers for oral delivery of proteins such as insulin. In this study, we investigated the effect of the molecular weight of the PEG tethered chains in the copolymer network and of the microparticle size on the transepithelial electrical resistance (TEER) and insulin epithelial permeability, using monolayers of human intestinal epithelial Caco-2 cells. Two molecular weights of the PEG chains, 400 and 1000, were investigated, as well as three different dry microparticle sizes: 25-90, 90-150 and 150-212 microm. Their effect on the cell monolayer integrity was studied by monitoring TEER as a fraction of time and determining insulin permeability. The presence of insulin-loaded P(MAA-g-EG) microparticles decreases the TEERs value by 50% with respect to the control. This disruption of the cell monolayer was recovered in 3 h after the removal of the polymer microparticles. Within the range of PEG molecular weights studied, there was no significant change of the TEER values. However, decreased microparticle sizes and short PEG chains systems led to higher permeability values. Insulin-loaded P(MAA-g-EG) microparticles enhanced the transport of insulin through the Caco-2 cell monolayers.     

Thiol functionalized polymethacrylic acid-based hydrogel microparticles for oral insulin delivery

In the present study thiol functionalized polymethacrylic acid–polyethylene glycol–chitosan (PCP)-based hydrogel microparticles were utilized to develop an oral insulin delivery system. Thiol modification was achieved by grafting cysteine to the activated surface carboxyl groups of PCP hydrogels (Cys-PCP). Swelling and insulin loading/release experiments were conducted on these particles. The ability of these particles to inhibit protease enzymes was evaluated under in vitro experimental conditions. Insulin transport experiments were performed on Caco-2 cell monolayers and excised intestinal tissue with an Ussing chamber set-up. Finally, the efficacy of insulin-loaded particles in reducing the blood glucose level in streptozotocin-induced diabetic rats was investigated. Thiolated hydrogel microparticles showed less swelling and had a lower insulin encapsulation efficiency as compared with unmodified PCP particles. PCP and Cys-PCP microparticles were able to inhibit protease enzymes under in vitro conditions. Thiolation was an effective strategy to improve insulin absorption across Caco-2 cell monolayers, however, the effect was reduced in the experiments using excised rat intestinal tissue. Nevertheless, functionalized microparticles were more effective in eliciting a pharmacological response in diabetic animal, as compared with unmodified PCP microparticles. From these studies thiolation of hydrogel microparticles seems to be a promising approach to improve oral delivery of proteins/peptides.     

Goblet cell-targeting nanoparticles for oral insulin delivery and the influence of mucus on insulin transport

The present study was to demonstrate the effects of goblet cell-targeting nanoparticles on the oral absorption of insulin in vitro, ex vivo and in vivo, and identify the targeting mechanism as well as the influence of mucus. The insulin loaded nanoparticles were prepared using trimethyl chitosan chloride (TMC) modified with a CSKSSDYQC (CSK) targeting peptide. Compared with unmodified nanoparticles, the CSK peptide modification could facilitate the uptake of nanoparticles in villi, enhance the permeation of drugs across the epithelium, meanwhile, induce a significantly higher internalization of drugs via clathrin and caveolae mediated endocytosis on goblet cell-like HT29-MTX cells. In transport studies across Caco-2/HT29-MTX co-cultured cell monolayer (simulating intestinal epithelium), the CSK peptide modification also showed enhanced transport ability, even if the targeting recognition was partially affected by mucus. Moreover, it was found the existence of mucus was propitious to the transport of insulin from both modified and unmodified nanoparticles. In the pharmacological and pharmacokinetic studies in diabetic rats, the orally administrated CSK peptide modified nanoparticles produced a better hypoglycemic effect with a 1.5-fold higher relative bioavailability compared with unmodified ones. In conclusion, CSK peptide modified TMC nanoparticles showed sufficient effectiveness as goblet cell-targeting nanocarriers for oral delivery of insulin.     

Dissecting stromal-epithelial interactions in a 3D in vitro cellularized intestinal model for permeability studies

Absorption evaluation plays an increasingly important role at the early stage of drug discovery due to its potential to scan the ADME (absorption, distribution, metabolism and excretion) properties of new drug candidates. Therefore, a new three-dimensional (3D) in vitro model replicating the intestinal functioning is herein proposed aiming to dissect the stromal-epithelial interactions and evaluate the permeation of a model drug, insulin. Inspired on the intestinal mucosal architecture, the present model comprises intestinal myofibroblasts (CCD18-Co cells) embedded in Matrigel, onto which epithelial enterocytes (Caco-2 cells) and mucus-producing cells (HT29-MTX cells) were seeded. CCD18-Co myofibroblasts showed to have a central role in the remodeling of the surrounding matrix confirmed by the production of fibronectin. Subsequently, this matrix revealed to be essential to the maintenance of the model architecture by supporting the overlying epithelial cells. In terms of functionality, this model allowed the efficient prediction of insulin permeability in which the presence of mucus, the less tight character between Caco-2 and HT29-MTX epithelial cells and the 3D assembly were critical factors. Concluding, this model constitutes a robust tool in the drug development field with potential to bridge the traditional 2D cell culture models and in vivo animal models.     

MyD88 adaptor-like (Mal) functions in the epithelial barrier and contributes to intestinal integrity via protein kinase C

MyD88 adapter-like (Mal)-deficient mice displayed increased susceptibility to oral but not intraperitoneal infection with Salmonella Typhimurium. Bone marrow chimeras demonstrated that mice with Mal-deficient non-hematopoietic cells were more susceptible to infection, indicating a role for Mal in non-myeloid cells. We observed perturbed barrier function in Mal^−/− mice, as indicated by reduced electrical resistance and increased mucosa blood permeability following infection. Altered expression of occludin, Zonula occludens-1, and claudin-3 in intestinal epithelia from Mal^−/− mice suggest that Mal regulates tight junction formation, which may in part contribute to intestinal integrity. Mal interacted with several protein kinase C (PKC) isoforms in a Caco-2 model of intestinal epithelia and inhibition of Mal or PKC increased permeability and bacterial invasion via a paracellular route, while a pan-PKC inhibitor increased susceptibility to oral infection in mice. Mal signaling is therefore beneficial to the integrity of the intestinal barrier during infection.     

Novel complexation hydrogels for oral peptide delivery: in vitro evaluation of their cytocompatibility and insulin-transport enhancing effects using Caco-2 cell monolayers

Poly[methacrylic acid-grafted-poly(ethylene glycol)] [P(MAA-g-EG)] is a complexation hydrogel molecularly designed for oral peptide delivery. In this work, the cytotoxicity and insulin-transport enhancing effect of P(MAA-g-EG) microparticles on intestinal epithelial cells were evaluated using Caco-2 cell monolayers. A series of P(MAA-g-EG) microparticles with different polymer compositions were prepared by a photo-initiated free radical solution polymerization and subsequent pulverization. The hydrogel microparticles were preswollen in either Ca2+-containing (CM+) or Ca2+-free medium (CM-; pH 7.4) and applied to the apical side of the Caco-2 monolayers. No significant cytotoxic effects, as determined by a calorimetric assay with P(MAA-g-EG) microparticles preswollen in the CM+, were observed at doses ranging from 3 to 31 mg/cm2 of cell monolayer. Transepithelial electrical resistance (TEER) measurements showed that the P(MAA-g-EG) microparticles induced a Ca2+ concentration-dependent lowering in TEER values. The reduction effect in CM- media was greater than that in CM+ media (17 +/- 2% reduction in CM+ and 45 +/- 3% reduction in CM-, respectively). Insulin transport in the presence of the preswollen P(MAA-g-EG) microparticles was also strongly depended on the Ca2+ concentration in the medium. The respective estimated permeability for insulin alone and the insulin with hydrogels in CM+ were 0.77 and 1.16 x 10(-8) cm/s, whereas those in CM- were 1.18 and 24.78 x 10(-8) cm/s. The results demonstrate that the P(MAA-g-EG) hydrogel microparticles could be used as a cytocompatible carrier possessing the transport-enhancing effect of insulin on the intestinal epithelial cells.     

Intestinal patches with an immobilized solid-in-oil formulation for oral protein delivery

Oral administration of biomolecular drugs such as peptides, proteins, and DNA is an attractive delivery method because of the safety and convenience of delivery in contrast to injection administration. However, oral delivery of biomolecules has several potential barriers such as enzymatic degradation in the gastrointestinal tract and low permeability across an intestinal membrane. In this study, we proposed an intestinal patch system that included surfactant-coated insulin for oral delivery. The intestinal patches, which have mucoadhesive and drug-impermeable layers, induced sustained unidirectional insulin release toward intestinal mucosa and inhibition of insulin leakage from the patches. Moreover, the surfactant-coated insulin, which has high compatibility with cell membranes, enhanced insulin transport across the intestinal membrane. This study demonstrates that the intestinal patches might improve protein permeability in the intestinal mucosa, thereby offering an innovative therapeutic strategy.     

Self-assembling bubble carriers for oral protein delivery

Successful oral delivery of therapeutic proteins such as insulin can greatly improve the quality of life of patients. This study develops a bubble carrier system by loading diethylene triamine pentaacetic acid (DTPA) dianhydride, a foaming agent (sodium bicarbonate; SBC), a surfactant (sodium dodecyl sulfate; SDS), and a protein drug (insulin) in an enteric-coated gelatin capsule. Following oral administration to diabetic rats, the intestinal fluid that has passed through the gelatin capsule saturates the mixture; concomitantly, DTPA dianhydride produces an acidic environment, while SBC decomposes to form CO₂ bubbles at acidic pH. The gas bubbles grow among the surfactant molecules (SDS) owing to the expansion of the generated CO₂. The walls of the CO₂ bubbles consist of a self-assembled film of water that is in nanoscale and may serve as a colloidal carrier to transport insulin and DTPA. The grown gas bubbles continue to expand until they bump into the wall and burst, releasing their transported insulin, DTPA, and SDS into the mucosal layer. The released DTPA and SDS function as protease inhibitors to protect the insulin molecules as well as absorption enhancers to augment their epithelial permeability and eventual absorption into systemic circulation, exerting their hypoglycemic effects.     

Protease inhibition and absorption enhancement by functional nanoparticles for effective oral insulin delivery

Complexing agents such as diethylene triamine pentaacetic acid (DTPA) are known to disrupt intestinal tight junctions and inhibit intestinal proteases by chelating divalent metal ions. This study attempts to incorporate these benefits of DTPA in functional nanoparticles (NPs) for oral insulin delivery. To maintain the complexing agent concentrated on the intestinal mucosal surface, where the paracellular permeation enhancement and enzyme inhibition are required, DTPA was covalently conjugated on poly(γ-glutamic acid) (γPGA). The functional NPs were prepared by mixing cationic chitosan (CS) with anionic γPGA-DTPA conjugate. The γPGA-DTPA conjugate inhibited the intestinal proteases substantially, and produced a transient and reversible enhancement of paracellular permeability. The prepared NPs were pH-responsive; with an increasing pH, CS/γPGA-DTPA NPs swelled gradually and disintegrated at a pH value above 7.0. Additionally, the biodistribution of insulin orally delivered by CS/γPGA-DTPA NPs in rats was examined by confocal microscopy and scintigraphy. Experimental results indicate that CS/γPGA-DTPA NPs can promote the insulin absorption throughout the entire small intestine; the absorbed insulin was clearly identified in the kidney and bladder. In addition to producing a prolonged reduction in blood glucose levels, the oral intake of the enteric-coated capsule containing CS/γPGA-DTPA NPs showed a maximum insulin concentration at 4 h after treatment. The relative oral bioavailability of insulin was approximately 20%. Results of this study demonstrate the potential role for the proposed formulation in delivering therapeutic proteins by oral route.     

A review of the prospects for polymeric nanoparticle platforms in oral insulin delivery

Success in the oral delivery of therapeutic insulin can significantly improve the quality of life of diabetic patients who must routinely receive injections of this drug. However, oral absorption of insulin is limited by various physiological barriers and remains a major scientific challenge. Various technological solutions have been developed to increase the oral bioavailability of insulin. Having received considerable attention, nano-sized polymeric particles are highly promising for oral insulin delivery. This review article describes the gastrointestinal barriers to oral insulin delivery, including chemical, enzymatic and absorption barriers. The potential transport mechanisms of insulin delivered by nanoparticles across the intestinal epithelium are also discussed. Finally, recent advances in using polymeric nanoparticles for oral insulin delivery and their effects on insulin transport are reviewed, along with their future.     

The use of deoxycholic acid to enhance the oral bioavailability of biodegradable nanoparticles

Oral delivery of nanoparticles encapsulating drugs and proteins remains a challenging route for administration due to the many barriers in the gastrointestinal tract that limit bioavailability. We hypothesized that bile salts could be used to improve the bioavailability of poly(lactide-co-glycolide) (PLGA) nanoparticles by protecting them during their transport through the gastrointestinal tract and enhancing their absorption by the intestinal epithelia. A deoxycholic acid emulsion is shown to protect PLGA nanoparticles from degradation in acidic conditions and enhance their permeability across a Caco-2 cell monolayer, an in vitro model of human epithelium. Oral administration of loaded PLGA nanoparticles to mice, using a deoxycholic acid emulsion, produced sustained levels of the encapsulant in the blood over 24-48 h with a relative bioavailability of 1.81. Encapsulant concentration was highest in the liver, demonstrating a novel means for targeted delivery to the liver by the oral route.     

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.     

In vivo evaluation of safety and efficacy of self-assembled nanoparticles for oral insulin delivery

A variety of approaches have been studied in the past to overcome the problems encountered with the oral delivery of insulin, but with little success. In this study, self-assembled nanoparticles (NPs) with a pH-sensitive characteristic were prepared by mixing the anionic poly-γ-glutamic acid solution with the cationic chitosan solution in the presence of MgSO₄ and sodium tripolyphosphate. The in vitro results found that the transport of insulin across Caco-2 cell monolayers by NPs appeared to be pH-dependent; with increasing pH, the amount of insulin transported decreased significantly. An in vivo toxicity study was performed to establish the safety of the prepared NPs after oral administration. Additionally, the impact of orally administered NPs on the pharmacodynamics (PD) and pharmacokinetics (PK) of insulin was evaluated in a diabetic rat model. The in vivo results indicated that the prepared NPs could effectively adhere on the mucosal surface and their constituted components were able to infiltrate into the mucosal cell membrane. The toxicity study indicated that the NPs were well tolerated even at a dose 18 times higher than that used in the PD/PK study. Oral administration of insulin-loaded NPs demonstrated a significant hypoglycemic action for at least 10 h in diabetic rats and the corresponding relative bioavailability of insulin was found to be 15.1 ± 0.9%. These findings suggest that the NPs prepared in the study are a promising vehicle for oral delivery of insulin.     

Novel delivery system based on complexation hydrogels as delivery vehicles for insulin-transferrin conjugates

A variety of approaches have been investigated to address the problems associated with oral insulin delivery, but the bioavailability of oral insulin is still low. Insulin is rapidly degraded by the enzymes in the GI tract and is not transported across the epithelial barrier easily. The oral insulin formulation developed in this work makes use of complexation hydrogels for oral delivery of insulin bioconjugates. The insulin bioconjugates synthesized in this work consist of insulin bound to transferrin molecule which can be uptaken by the epithelial cells. The conjugates can increase the permeability of insulin across the epithelial barrier by receptor-mediated transcytosis. The transferrin in the conjugate is also shown to stabilize insulin in the presence of intestinal enzymes. Use of complexation hydrogels for delivery of insulin-transferrin conjugate may greatly increase the bioavailability of oral insulin. This is because, the complexation hydrogels are known to exhibit characteristics that make them ideal candidates for oral protein delivery. They can also inhibit the degradation of insulin in the GI tract. Thus, combination of these two approaches may provide an innovative platform for oral insulin delivery.     

Drug permeability and mucoadhesion properties of thiolated trimethyl chitosan nanoparticles in oral insulin delivery

Trimethyl chitosan-cysteine conjugate (TMC-Cys) was synthesized in an attempt to combine the mucoadhesion and the permeation enhancing effects of TMC and thiolated polymers related to different mechanisms for oral absorption. TMC-Cys with various molecular weights (30, 200, and 500 kDa) and quaternization degrees (15 and 30%) was allowed to form polyelectrolyte nanoparticles with insulin through self-assembly, which demonstrated particle size of 100–200 nm, zeta potential of +12 to +18 mV, and high encapsulation efficiency. TMC-Cys/insulin nanoparticles (TMC-Cys NP) showed a 2.1–4.7-fold increase in mucoadhesion compared to TMC/insulin nanoparticles (TMC NP), which might be partly attributed to disulfide formation between TMC-Cys and mucin as evidenced by DSC measurement. Compared to insulin solution and TMC NP, TMC-Cys NP induced increased insulin transport through rat intestine by 3.3–11.7 and 1.7–2.6 folds, promoted Caco-2 cell internalization by 7.5–12.7 and 1.7–3.0 folds, and augmented uptake in Peyer's patches by 14.7–20.9 and 1.7–5.0 folds, respectively. Such results were further confirmed by in vivo experiment with the optimal TMC-Cys NP. Biocompatibility assessment revealed lack of toxicity of TMC-Cys NP. Therefore, self-assembled nanoparticles between TMC-Cys and protein drugs could be an effective and safe oral delivery system.     

Anti-tumor efficacy of c(RGDfK)-decorated polypeptide-based micelles co-loaded with docetaxel and cisplatin

There are two important obstacles for the currently applied anti-cancer drug delivery systems. One is the conflict between long-circulation and cellular uptake while the other one is the achievement of ideal anti-cancer efficacy. To solve these problems, we designed a polypeptide-based micelle system that combined the advantages of receptor mediated endocytosis and multi-drug delivery. Firstly, an amphiphilic PLG-g-Ve/PEG graft copolymer was prepared by grafting α-tocopherol (Ve) and polyethylene glycol (PEG) to poly(l-glutamic acid) (PLG). Then docetaxel (DTX) and cisplatin (CDDP) were co-loaded into the PLG-g-Ve/PEG micelles via hydrophobic and chelation effect. After that, the surface of the dual-drug-loaded micelles was decorated with an α_vβ₃ integrin targeting peptide c(RGDfK). The targeted dual-drug-loaded micelles showed synergistic cytotoxicity and enhanced internalization rate in mouse melanoma (B16F1) cells. In vivo tests demonstrated that remarkable long circulation, anti-tumor and anti-metastasis efficacy could be achieved using this drug delivery system. This work revealed a strategy for the design and preparation of anti-cancer drug delivery systems with reduced side effect, enhanced anti-tumor and anti-metastasis efficacy.