Preparation And Characterization Of Chitosan - Based Oral Insulin Preparations

Insulin is the main drug for treating diabetes. Subcutaneous injections of insulin remains to be the primary approach for the treatment of diabetic patients. Nevertheless, the subcutaneous administration of insulin often lead to inconvenient and pain for patients, which results in poor patient compliance. For its beneficial and reasonable, orally administered insulin is accepted by patients easily, therefore, it is becoming an ideal mode of administration. Because of its biological macromolecular structure, oral administration of insulin is encountered with a lot of difficulties, such as various barriers in the gastrointestinal tract and the poor physical and chemical stability of insulin. This results in the applications of drug delivery system with biocompatible, non-cytotoxic carrier materials, which not only can avoid the damage and degradation to insulin in gastric conditions and enhance bioavailability, but also can delay the time of insulin in the body, reduce the frequency of administration.Firstly, water-soluble polyethylene glycol chitosan (PEG-CS) was synthesized using hydrophilic polyethylene glycol (PEG) grafted onto chitosan (CS) by covalent coupling method, To study the interaction between insulin and PEG-CS, it is detected by ultraviolet spectrum (UV), fluorescence spectrum and circular dichroism (CD). All the data gave one result that, although the addition of PEG-CS caused the change of microenvironment, secondary structure of insulin did not alter. Then, the PEG-CS microspheres loaded insulin prepared by emulsion cross-linking and diffusion filling method. The morphology of the PEG-CS microspheres observed by scanning electron microscope (SEM), and the controlled release in vitro was also investigatived; NOD diabetic mice were used to evaluate the hypoglycemic effects of microspheres loaded insulin. The results indicated that the loading content of insulin in microspheres was 35 U/100 mg. It is obviously that the insulin-loaded microspheres has the property of sustained release in simulated intestinal fluid, furthermore, the insulin released from microspheres maintained the original biological activity. The relative pharmacological bioavailability of insulin was found to be approximately 5.82%. Thus the insulin-loaded microspheres had a significant hypoglycemic effect and prolonged over 24 hours. Therefore, it is believed that the microspheres might be employed as a potential carrier for the oral delivery of insulin.polylactic glycolic acid (PLGA) was selected to be encapsulated insulin because of the advantages of good biocompatibity and biodegradation, and the PLGA microspheres loaded insulin were prepared by the Double emulsion-solvent evaporation method (W/O/W). Orthogonal experimental of L16 (44) was designed to investigate encapsulation efficiency and drug loading affecting by various factors during the preparation of microspheres. The optimal prepareation conditions obtained as follows:Water-oil ratio of 1:10; PLGA concentration of 40 mg/ml; insulin concentration of 20 mg/ml; PEG-CS concentration of 0.1 mg/ml. The optimized drug-loaded microspheres charactered by transmission electron microscopy (TEM) and controlled release in vitro was explored in simulated gastrointestinal fluids. Relative cell viability of Caco-2 cells incubated with insulin-loaded microspheres by MTT assay, and the cellular uptake of the microspheres of Caco-2 cells was used inverted fluorescence microscope. The results indicated that the microspheres had smooth surface without holes, and released slowly in simulated intestinal fluid, howerer, which had a burst seriously in simulated gastric fluid. Insulin released from the microspheres had the original biological activity. Cell experiments showed that PLGA had no toxicity to cells, and the microspheres can be uptaked by Caco-2 cells.To improve the unreasonable release of insulin in simulated gastrointestinal fluid, the natural halloysite nanotubes (HNTs) with hollow tubular structure was chosen as a carrier material for drug-loading. Insulin loading was studied by impacted of the mass ratios between insulin and HNTs, the pH of the insulin solution. Then, HNTs/PEG-CS complex was prepared due to electrostatic interaction. Moreover, HNTs, HNTs/PEG-CS was enteric coated with hydroxypropyl methylcellulose phthalate (HPMCP). The structure of the HNTs/PEG-CS, HNTs /HPMCP, HNTs/PEG-CS/HPMCP complexes were characterized by FT-IR, EDS, SEM, TEM. Controlled release behaviors in vitro of theirs in simulated gastrointestinal fluid were measured by Folin-phenol reagent or UV absorption method. The result presented that, HNTs loaded insulin (INS/HNTs) had a poor sustained release in simulated gastrointestinal fluids; INS/HNTs/PEG-CS had a certain sustained release in simulated intestinal fluid; and INS/HNTs /HPMCP, INS/HNTs/PEG-CS/HPMCP can continued release in simulated intestinal fluid, while with small drug burst in simulated gastric fluid.