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.     

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.     

Remarkably enhanced stability and function of core/shell nanoparticles composed of a lecithin core and a pluronic shell layer by photo-crosslinking the shell layer: In vitro and in vivo study

A core/shell nanoparticle system with a lecithin core and a pluronic shell has been previously reported, and it was shown to act as an effective sustained release system for positively charged proteins. Here, to provide improved stability of the core/shell nanoparticle system in a physiological environment, we prepared the core/shell nanoparticle system with a photo-crosslinked shell layer by using a lecithin liposome as the core and pluronic F 127 diacrylate (DA-PF 127) as the shell layer. The DA-PF 127 was then photo-polymerized. Compared with a purely physical system, chemical crosslinking of the shell layer resulted not only in significantly increased structural stability of the core/shell nanoparticles in both an organic co-solvent and in serum but also several remarkably enhanced functioning as a protein delivery system. First, the chemically crosslinked systems were resuspended in aqueous solution after lyophilization without using a cryo-protectant. Second, target proteins were efficiently loaded into the nanoparticles by simple co-incubation in aqueous solution at a low temperature (4 °C) and the dried powder form of the protein-loaded nanoparticles was obtained. The loading capacity of the system was increased by more than 10 times compared with that of a purely physical system. Most importantly, the chemically crosslinked system showed more sustained release of the loaded proteins, and the release rate was not noticeably affected by the presence of serum proteins, whereas sustained release of loaded vascular endothelial growth factor (VEGF) in a purely physical system was greatly reduced by serum proteins. In an in vivo corneal angiogenesis assay the chemically crosslinked system loaded with VEGF resulted in more efficient new blood vessel formation than the physical system.     

The effect of complexation hydrogels on insulin transport in intestinal epithelial cell models

A novel class of pH-sensitive complexation hydrogels composed of methacrylic acid and functionalized poly(ethylene glycol) (PEG) tethers, referred to as P(MAA-g-EG) WGA, was investigated as an oral protein delivery system. The PEG tethers were functionalized with wheatgerm agglutinin (WGA), a lectin that can bind to carbohydrates in the intestinal mucosa, to improve residence time of the carrier and absorption of the drug at the delivery site. The ability of P(MAA-g-EG) WGA to improve insulin absorption was observed in two different intestinal epithelial models. In Caco-2 cells P(MAA-g-EG) WGA improved insulin permeability 9-fold as compared with an insulin only solution, which was similar to the improvement by P(MAA-g-EG). P(MAA-g-EG) and P(MAA-g-EG) WGA were also evaluated in a mucus-secreting culture that contained Caco-2 and HT29–MTX cells. Insulin permeability was increased 5-fold in the presence of P(MAA-g-EG) and P(MAA-g-EG) WGA. Overall, it is clear that P(MAA-g-EG) WGA enhances insulin absorption and holds great promise as an oral insulin delivery system.     

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.     

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.     

Co-delivery of doxorubicin and paclitaxel by PEG-polypeptide nanovehicle for the treatment of non-small cell lung cancer

Despite progress, combination therapy of different functional drugs to increase the efficiency of anticancer treatment still remains challenges. An amphiphilic methoxy poly(ethylene glycol)-b-poly(l-glutamic acid)-b-poly(l-lysine) triblock copolymer decorated with deoxycholate (mPEsG-b-PLG-b-PLL/DOCA) was synthesized and developed as a nanovehicle for the co-delivery of anticancer drugs: doxorubicin (DOX) and paclitaxel (PTX). The amphiphilic copolymer spontaneously self-assembled into micellar-type nanoparticles in aqueous solutions and the blank nanoparticles possessed excellent stability. Three different domains of the copolymer performed distinct functions: PEG outer corona provided prolonged circulation, middle biodegradable and hydrophilic PLG shell was designed for DOX loading through electrostatic interactions, and hydrophobic deoxycholate modified PLL served as the container for PTX. In vitro cytotoxicity assays against A549 human lung adenocarcinoma cell line demonstrated that the DOX + PTX co-delivered nanoparticles (Co-NPs) exhibited synergistic effect in inducing cancer cell apoptosis. Ex vivo DOX fluorescence imaging revealed that Co-NPs had highly efficient targeting and accumulation at the implanted site of A549 xenograft tumor in vivo. Co-NPs exhibited significantly higher antitumor efficiency in reducing tumor size compared to free drug combination or single drug-loaded nanoparticles, while no obvious side effects were observed during the treatment, indicating this co-delivery system with different functional antitumor drugs provides the clinical potential in cancer therapy.     

Delivery of platinum(IV) drug to subcutaneous tumor and lung metastasis using bradykinin-potentiating peptide-decorated chitosan nanoparticles

Selectively activating tumor vessels to increase drug delivery and reduce interstitial fluid pressure of tumors is actively pursued. Here we developed a vasoactive peptide-decorated chitosan nanoparticles for enhancing drug accumulation and penetration in subcutaneous tumor and lung metastasis. The vasoactive peptide used here is bradykinin-potentiating peptide (BPP) containing 9 amino acid residues and the drug is bioreductively sensitive platinum(IV) compound which becomes cisplatin in intracellular reductive environments. Both peptide and drug are covalently linked with chitosan nanoparticles with a diameter of 120 nm. We demonstrate that BPP-decorated chitosan nanoparticles increase the tumorous vascular permeability and reduce the interstitial fluid pressure of tumor simultaneously, both of which improve the penetration of nanoparticles in tumor tissues. The in vivo biodistribution and tumor inhibition examinations demonstrate that the BPP-decorated nanoparticle formulation has more superior efficacy in enhancing drug accumulation in tumor, restraining tumor growth and prolonging the lifetime of tumor-bearing mice than free drug and non-decorated nanoparticle formulation. Meanwhile, the drug accumulation in the lung with metastasis reaches 17% and 20% injected dose per gram of lung for the chitosan nanoparticles without and with BPP decoration, respectively, which is 10-fold larger than that of free cisplatin. The examination of lung metastasis inhibition further indicates that BPP-decorated chitosan nanoparticle formulations can more effectively inhibit lung metastasis.     

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.     

Gross and histologic effects of topical misoprostol on canine gastric mucosa.

This study reports the histological effects of topical misoprostol, a synthetic PGE1 analog, administered in varying dosages on the resting canine gastric mucosa. Misoprostol did not macroscopically or microscopically damage the mucosa but its presumed permeability effects on the gastric vasculature induced marked edema of the mucosa and submucosa. Consistent features included increased thickness of both layers, dilated interglandular regions of the lamina propria, marked subepithelial edema, reduced depth and width of gastric foveolae, vasodilation of the vascular channels, reduced height of surface epithelial cells, swelling of their basolateral intercellular spaces, and increased amounts of surface adherent mucus. It is speculated that the mucosal edema, in addition to an increased mucus layer, may be important in the mechanism of gastric cytoprotection by increasing the distance of penetration or absorption for a mucosal-damaging agent, diluting its concentration, and disseminating any focal accumulations of red blood cells.     

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.     

LPS-binding IgG arrests actively motile Salmonella Typhimurium in gastrointestinal mucus

The gastrointestinal (GI) mucosa is coated with a continuously secreted mucus layer that serves as the first line of defense against invading enteric bacteria. We have previously shown that antigen-specific immunoglobulin G (IgG) can immobilize viruses in both human airway and genital mucus secretions through multiple low-affinity bonds between the array of virion-bound IgG and mucins, thereby facilitating their rapid elimination from mucosal surfaces and preventing mucosal transmission. Nevertheless, it remains unclear whether weak IgG-mucin crosslinks could reinforce the mucus barrier against the permeation of bacteria driven by active flagella beating, or in predominantly MUC2 mucus gel. Here, we performed high-resolution multiple particle tracking to capture the real-time motion of hundreds of individual fluorescent Salmonella Typhimurium in fresh, undiluted GI mucus from Rag1^−/− mice, and analyzed the motion using a hidden Markov model framework. In contrast to control IgG, the addition of anti-lipopolysaccharide IgG to GI mucus markedly reduced the progressive motility of Salmonella by lowering the swim speed and retaining individual bacteria in an undirected motion state. Effective crosslinking of Salmonella to mucins was dependent on Fc N-glycans. Our findings implicate IgG-mucin crosslinking as a broadly conserved function that reduces mucous penetration of both bacterial and viral pathogens.     

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.     

Computer simulation of the role of protein corona in cellular delivery of nanoparticles

Understanding the role of serum protein in the process of nanoparticle delivery is of great importance in biomedicine. Here, by using dissipative particle dynamics simulations, we systematically investigate the interactions between the nanoparticle-protein corona complex and cell membranes of different types. It is found that the human serum albumin (HSA) will just adsorb onto charged (especially for positively charged) and hydrophobic nanoparticle surface. More importantly, we also provide specific insights into the effect of HSA adsorption on the in vivo transportation of nanoparticle (i.e., immune response and targeted cellular uptake). Our results show that the protein corona can change the interaction modes of hydrophobic nanoparticles and enhance the interaction of charged nanoparticles with macrophage cell membranes, while it may also cause the failure of insertion of hydrophobic nanoparticles and the loss of targeting specificity of charged nanoparticles with cancer cell membranes. These results can help better understand the biological significance of protein corona and may give some useful suggestions on better design of future nanoparticles in drug delivery.     

Topical pluronic F-127 gel application enhances cutaneous wound healing in rats

Pluronic F-127 gel is used as vehicle for various topical applications. In the present study, effects of topical application of pluronic F-127 gel were evaluated in cutaneous wound healing in Wistar rats. Normal saline solution and pluronic F-127 gel (25%) were applied topically on open excision wounds for 14 days. Photography, determination of percentage wound contraction, and collection of granulation tissue were done on days 3, 7, 11 and 14 post-wounding. Topical application of gel (once daily) significantly increased the wound closure on days 11 and 14. The gel application increased the expressions of vascular endothelial growth factor (VEGF) and transforming growth factor-beta1 (TGF-β₁) on days 3 and 7. Histopathologically, more leukocyte infiltration followed by well formed granulation tissue with marked fibroblast proliferation was evident in the gel-treated group, as compared to the saline-treated control group. Immunohistochemistry of CD31 on day 7 revealed significant higher microvessel density in gel-treated wounds. Picrosirius staining demonstrated higher collagen fraction in gel-treated wounds. Thus, from the results, it could be concluded that pluronic F-127 gel has a mild inflammatory nature and enhanced the healing by stimulating expression of VEGF and TGF-β₁.     

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.     

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.     

Epithelial-specific blockade of MyD88-dependent pathway causes spontaneous small intestinal inflammation

Accumulating evidence suggests a role for Toll-like receptor (TLR) signaling at the intestinal epithelial cells (IECs) level for intestinal protection against exogenous injury or pathogenic infection. We hypothesized that MyD88 dependent TLR signaling at intestinal epithelium is critical for mucosal immune homeostasis. In the current study, a transgenic mouse model was generated in which a dominant-negative mutant of MyD88 (dnMyD88) was driven by an intestinal epithelial-specific murine villin promoter. Aged transgenic mice spontaneously developed chronic small intestinal inflammation, as revealed by increased CD4+ and CD8+ lymphocytes, neutrophil and macrophage infiltration, increased production of cytokines as TNF-α, IFN-γ, IL-1β, and IL-17, crypt abscesses, lymphedema, and Goblet cell depletion. The chronic inflammation was not due to increased epithelial apoptosis or permeability, but to a decreased Paneth cell-derived α-defensins (cryptdins) and RegIII-γ and increased commensal bacteria translocation. Thus, epithelial MyD88-dependent pathway plays an essential role in limiting mucosal microflora penetration and preventing mucosal immunoregulation disturbance in vivo.     

Doxorubicin loaded singlet-oxygen producible polymeric micelle based on chlorine e6 conjugated pluronic F127 for overcoming drug resistance in cancer

Drug resistance remains one of the primary obstacles to the success of cancer chemotherapy. In this work, we demonstrate a singlet-oxygen producible polymeric (SOPP) micelle based on photosensitizer (PS, chlorin e6 (Ce6)) conjugated amphiphilic copolymer (pluronic F127^®, PF127) for overcoming drug resistance in cancer by applying photochemical internalization (PCI). The doxorubicin (DOX)-loaded SOPP micelles were self-assembled from Ce6-PF127 conjugates, which have a spherical shape with a uniform size of ∼30 nm. Compared with free Ce6, enhanced singlet-oxygen generation efficiency in the DOX-loaded SOPP micelles have been demonstrated in aqueous environments due to their increased water-dispersibility. Under low dose of laser power and anti-cancer drug (DOX) conditions, in vitro and in vivo studies on drug-resistant cancer cells demonstrated that singlet-oxygen-mediated cellular membrane damage (caused by lipid peroxidation) significantly increased the cellular uptake of drug (DOX), which led to overcoming the drug resistance in cancer cells without undesirable side effects. We believe this approach could represent a promising platform for drug-resistant cancer treatment.