Preparation and in vitro characterization of chitosan nanoparticles containing Mesobuthus eupeus scorpion venom as an antigen delivery system

Hydrophilic nanoparticles have been widely investigated in recent years as delivery systems for therapeutic macromolecules such as antigens. In the present study Mesobuthus eupeus venom-loaded chitosan nanoparticles were prepared via ionic gelation of tripolyphosphate (TPP) and chitosan. The optimum encapsulation efficiency (91.1 percent) and loading capacity (76.3 percent) were obtained by a chitosan concentration of 2 mg/mL, chitosan-to-TPP mass ratio of 2 and M. eupeus venom concentration of 500 µg/mL. The average nanoparticle size at optimum conditions was determined by Zetasizer (Malvern Instruments, UK). The nanoparticle size was about 370 nm (polydispersity index: 0.429) while the zeta potential was positive. Transmission electron microscope (TEM) imaging showed a spherical, smooth and almost homogenous structure for nanoparticles. Fourier transform infrared (FTIR) spectroscopy confirmed tripolyphosphoric groups of TPP linked with ammonium groups of chitosan in the nanoparticles. The in vitro release of nanoparticles showed an initial burst release of approximately 60 percent in the first ten hours, followed by a slow and much reduced additional release for about 60 hours. It is suggested that the chitosan nanoparticles fabricated in our study may provide a suitable alternative to traditional adjuvant systems.(AU)

Ex vivo evaluation of insulin nanoparticles using chitosan and arabic gum.

Polymeric delivery systems based on nanoparticles have emerged as a promising approach for peroral insulin delivery. The aim of the present study was to investigate the release of insulin nanoparticulate systems and ex vivo studies. The nanoparticles were prepared by the ion gelation method. Particle size distribution, zeta potential, and polydispersity index of the nanoparticles were determined. It was found that the nanoparticles carried positive charges and showed a size distribution in the range of 170-200 nm. The electrostatic interactions between the positively charged group of chitosan and negatively charged groups of Arabic gum play an important role in the association efficiency of insulin in nanoparticles. In vitro insulin release studies showed an initial burst followed by a slow release of insulin. The mucoadhesion of the nanosystem was evaluated using excised rat jejunum. Ex vivo studies have shown a significant increase in absorption of insulin in the presence of chitosan nanoparticles in comparison with free insulin.

Development of a Gas Empowered Drug Delivery system for peptide delivery in the small intestine.

The aim of this investigation was to design a novel Gas Empowered Drug Delivery (GEDD) system for CO(2) forced transport of peptide drugs together with mucoadhesive polymers to the surface of the small intestine. The GEDD effect of the core tablet was achieved using CO(2) gas to push insulin together with the mucoadhesive excipients poly(ethyleneoxide) (PEO) and the permeation enhancer trimethyl chitosan (TMC) to the surface of the small intestine. The in-vitro insulin release showed that almost 100% of the insulin was released from enterically coated tablets within 30 min at pH 6.8. The designed GEDD system was shown to increase the insulin transport by approximately 7 times in comparison with the free insulin across sheep's intestine ex-vivo. Three different peroral formulations were administered to male rabbits: F1 containing no TMC or PEO, F2 containing PEO but no TMC and F3 containing both PEO and TMC. The administrations of insulin using the formulation F1 resulted in a low FR value of 0.2%+/-0.1%, while the formulations F2 and F3 resulted in a much higher FR values of 0.6+/-0.2% and 1.1%+/-0.4%, respectively. Hence, the insulin permeation achieved by the GEDD system is primarily due to the enhancing effect of TMC and the mucoadhesive properties of PEO both of which synergistically increase the bioavailability of insulin.

Permeation enhancer effect of chitosan and chitosan derivatives: comparison of formulations as soluble polymers and nanoparticulate systems on insulin absorption in Caco-2 cells.

In this study four quaternized derivatives of chitosan: trimethyl chitosan (TMC), dimethylethyl chitosan (DMEC), diethylmethyl chitosan (DEMC) and triethyl chitosan (TEC) with degree of substitution of approximately 50+/-5% were synthesized and their effect on the permeability of insulin across intestinal Caco-2 monolayers was studied and compared with chitosan both in free-soluble form and in nanoparticulate systems. Transepithelial electrical resistance (TEER) studies revealed that all four chitosan derivatives in free-soluble forms were able to decrease the TEER value in the following order TMC>DMEC>DEMC=TEC>chitosan, indicating their abilities to open the tight junctions. Recovery studies on the TEER showed that the effect of the polymers on Caco-2 cell monolayer is reversible and proves the viability of cells after incubation with all polymers. A similar rank order was also observed when measuring the zeta-potentials of the various polymers in solution form. Transport studies of insulin together with the soluble polymers across Caco-2 cell layers showed the following ranking: TMC>DMEC>DEMC>TEC>chitosan which is in agreement with the strength of the cationic charge of the polymer. In comparison to the free-soluble polymers, the nanoparticles prepared by ionic gelation of the chitosan and its quaternized derivatives had much lower effect on decreasing the TEER by opening of the tight junctions. This can be explained by the reduced available amount of positive charge at the surface of the nanoparticles. In accordance with these results, the insulin loaded nanoparticles showed much less permeation across the Caco-2 cell monolayer in comparison to the free-soluble polymers. Mass balance transport studies revealed that a substantial amount of the nanoparticles has been entrapped into the Caco-2 monolayer or attached to the cell surface. It can thus be stated that while free-soluble polymers can reversibly open the tight junctions and increase the permeation of insulin, the nanoparticles had basically only a low effect on the opening of the tight junction and the paracellular transport of insulin across the Caco-2 cell monolayer. These data convincingly show that nanoparticles consisting of chitosan and its quaternary ammonium derivatives loaded with insulin are less effective in facilitating paracellular transport across Caco-2 cell monolayers than the corresponding free polymers.

Preparation, characterization and antibacterial activities of chitosan, N-trimethyl chitosan (TMC) and N-diethylmethyl chitosan (DEMC) nanoparticles loaded with insulin using both the ionotropic gelation and polyelectrolyte complexation methods.

TMC and DEMC, quaternized derivatives of chitosan, have been shown to have penetration enhancement properties and able to open the tight junctions of the intestinal epithelia at neutral and alkaline pH environments. The use of the nanoparticulate systems has the advantage of protecting the peptidic drugs from the harsh environment of the gastrointestinal tract. Hence, the aim of this study was to synthesize and characterize TMC and DEMC, both with quaternization degrees of 50+/-5%, which were then used to prepare insulin nanoparticles with two different methods: ionotropic gelation and the polyelectrolyte complexation (PEC) techniques. The obtained nanoparticles were then characterized for size, zeta potential, insulin loading and release as well as antibacterial activities. The results showed that nanoparticles prepared by the PEC method had higher insulin loading efficiency and zeta potential than those made by the ionotropic gelation method and may subsequently be used for further in vitro, ex vivo and in vivo studies. Moreover, the antibacterial studies suggest that the polymers in free form have higher antibacterial activity against Gram-positive bacteria than in the nanoparticulate form.

Diethyl methyl chitosan as an intestinal paracellular enhancer: ex vivo and in vivo studies.

Chitosan exhibits favorable biological properties such as no toxicity, biocompatibility and biodegradability; therefore, it has attracted great attention in both pharmaceutical and biomedical fields. Chitosan exhibits poor solubility at pH values above 6 that prevents enhancing effects at the sites of absorption of drugs. In the present work, N-diethyl methyl chitosan (DEMC) was prepared and the enhancing effect of this polymer was investigated. Ex vivo studies have shown a significant increase in absorption of brilliant blue in the presence of diethyl methyl chitosan in comparison with chitosan. DEMC with positive charges is able to interact with tight junctions of colon epithelial cells and hence increases permeability of brilliant blue across the tight junctions. In vivo investigations have exhibited the absorption enhancer effects of DEMC on the colon absorption of insulin in normal and diabetic rats. The insulin absorption from the rat's colon was evaluated by its hypoglycemic effect. A significant decrease in blood glucose was observed, when mixture of insulin and DEMC was introduced in ascending colon of rats.