Study On The In Vivo And In Vitro Properties Of Targeted Polymersomes As The Oral Delivery Carriers Of Insulin

The oral delivery of insulin provides a new approach for the treatment of diabetes.Different from subcutaneous insulin injection,oral administration could improve the life quality of diabetic.In recent years,various oral insulin preparations have been widely studied,such as nanoparticles,polymersomes,microcapsules,microspheres,liposomes and so on.In this thesis,polymersomes were chosen as oral insulin preparations and in vivo and in vitro properties of polymersomes were studied.Unlike core-shell spherical nanoparticles,polymersomes have a double-layered structure that are able to load hydrophilic drugs as well as hydrophobic drugs,similar to liposomes.But polymersomes are more stable than liposomes due to their low critical micelle concentration(CMC).And nano-scale paticles could be internalized by cells easily.Moreover,based on folate receptor existing around the gastrointestinal tract,nanoparticles can be modified with folic acid to enhance the cellular uptake efficiency and bioavailability of loaded drugs.In this thesis,novel folate-targeted Pluronic P85/poly(lactic-co-glycolic)block copolymer FA-P85-PLGA and non-targeted Pluronic P85/poly(lactic-co-glycolic)block copolymer PLGA-P85-PLGA were synthesized.And nanoparticles were prepared by nano precipitation method.The inner morphologies and size of nanoparticals were measured by transmission electron microscope and laser particle size analyzer.The morphologies of FA-P85-PLGA and PLGA-P85-PLGA nanoparticles were polymersomes with spherical bilayer structure.Their diameter is between 70-110 nm.The in vitro release characteristics of the insulin-loaded polymersomes were studied with Micro BCA protein assay.The release behaviors of insulin from FA-P85-PLGA polymersomes and PLGA-P85-PLGA polymersomes showed that the initial burst release,followed by slow release.In vitro targeting effect of FA-P85-PLGA polymersomes were studied with Caco-2 cells through exploring the MTT assays,the uptake behavior of polymersomes and the internalization mechanism of polymersomes.MTT assays showed that the blank folate-targeted polymersomes and non-targeted polymersomes were nontoxic.The targeting effect of FA-P85-PLGA polymersomes was studied by the qualitative and the quantitative cellular uptake.FITC-insulin as a fluorescent probe was loaded into FA-P85-PLGA polymersomes and PLGA-P85-PLGA polymersomes.Results showed that both polymersomes and FITC-insulin were mainly located in the cytoplasm of Caco-2 cells.The results of qualitative and quantitative cellular uptake verified that the uptake of polymersomes by Caco-2 cells is time-dependent.Furthermore,the amount of the cellular uptake of FA-P85-PLGA polymersomes was higher than that of PLGA-P85-PLGA polymersomes,and that of free FITC-insulin was the lowest.The results of the internalization mechanism of polymersomes showed that the internalization of FA-P85-PLGA and PLGA-P85-PLGA polymersomes consist of multiple uptake mechanisms,including clathrin and caveolae mediated endocytosis,macropinocytosis.In addition,the folate receptor mediated endocytosis also effect the cellular uptake of targeted FA-P85-PLGA polymersomes.The fasting diabetic rats were administered orally with a single dose of insulin-loaded FA-P85-PLGA(50 IU/kg)and insulin-loaded PLGA-P85-PLGA polymersomes(50 IU/kg).For comparison,the free insulin at a dose of 5 IU/kg was also injected subcutaneously to the diabetic rats.In the pharmacodynamics study,both insulin-loaded FA-P85-PLGA polymersomes and insulin-loaded PLGA-P85-PLGA polymersomes exhibited lasting hypoglycemic effects and did not appear hypoglycemia.And in the pharmacokinetics study,the group administered free insulin subcutaneously exhibited highest concentration in one hour,whereas the groups administered orally the insulin-loaded FA-P85-PLGA polymersomes and insulin-loaded PLGA-P85-PLGA polymersomes revealed a maximum insulin concentration at 6 h after treatment.Insulin-loaded FA-P85-PLGA exhibited significant higher plasma insulin concentration(35.8 ± 5.27 ? IU/mL),compared with insulin-loaded PLGA-P85-PLGA polymersomes(27.6 ± 3.67 ? IU/mL).The AUC(area under the curve)and relative bioavailability of insulin-loaded FA-P85-PLGA polymersome is almost 1.27 and 1.3 times of those of insulin-loaded PLGA-P85-PLGA polymersome,respectively.In summary,targeted folate-conjugated FA-P85-PLGA polymersomes have potential applications in oral delivery of insulin.