Study On Influencing Factors Of Hypoglycemic Effect Of Folate Targeted Polymersomes Encapsulating Insulin

Oral insulin carrier containing folic acid(FA)can promote the absorption of insulin-oaded nanoparticles and thus improve the hypoglycemic effect.Nanoparticles containing targeted FA uptake by small intestinal epithelial cells through folate receptor-mediated endocytosis,which depends on the density of FA on the surface of nanoparticles.In this thesis,nano-sized polymersomes containing FA as oral insulin carriers were prepared,and the hypoglycemic effects of polymersomes formulations with different FA contents were investigated.In addition,D-?-tocopherol polyethylene glycol 1000 succinate(TPGS)is a kind of pharmaceutical adjuvant,which is commonly used as emulsifier,penetrant,absorption promoter,P glycoprotein inhibitor,etc.It has been reported that TPGS can enhance the hypoglycemic effect of oral insulin formulations.On the basis of better FA content,the influence of mixed polymersomes formulations with different TPGS content on hypoglycemic effect was explored.In this thesis,poly(lactic-co-glycolic acid)with FA molar content of 53.41%(FA-P85-PLGA)and poly(lactic-co-glycolic acid)without FA(PLGA-P85-PLGA)were synthesized using Pluronic(P85),glycolide(GA),lactide(LA)and FA as raw materials.Polymersomes were prepared by the nano-precipitation method.The morphology of polymersomes observed by transmission electron microscope(TEM)was spherical and closed bilayer structure.The particle size of polymersomes measured by dynamic light scattering(DLS)is between 50-70 nm,and?potential of polymersomes is negative.Caco-2 cells were cultured with FA-P85-PLGA with the highest FA content for 24 h,and the cell survival rate was determined as high as 96%by MTT colorimetric assay.FA-P85-PLGA and PLGA-P85-PLGA were used to prepare polymersome formulations with FA content of 10%,20%,40%and 50%,namely,10%FA-Ps,20%FA-Ps,40%FA-Ps,and 50%FA-Ps.The effects of polymersomes with different FA contents on oral hypoglycemic effect were analyzed in vivo and in vitro.In vitro release experiments of polymersomes under different pH conditions were simulated.The results showed that polymersome formulations could protect insulin to some extent.Using FITC-insulin as a fluorescent agent,Caco-2 cells uptake of FITC-insulin-loaded polymersomes with different FA contents was detected by fluorescence spectrophotometer.The order of Caco-2 cells uptake was 10%FA-Ps>20%FA-Ps>40%FA-Ps>50%FA-Ps>Free insulin.The diabetic rat model induced by alloxan was used to investigate the hypoglycemic effect of insulin-loaded polymersomes with different FA contents in vivo.The experiment showed that with the increase of FA content,the hypoglycemic effect decreased,and 10%FA-Ps had a better hypoglycemic effect.On the basis of screening out the better FA content,the hypoglycemic effect of the mixed polymersome formulations with the mass ratio of polymer to TPGS of 5:1,5:3 and 5:5 was investigated.The corresponding insulin-loaded mixed polymersomes containing TPGS were named as FA-Ps/TPGS5:1,FA-Ps/TPGS5:3,and FA-Ps/TPGS5:5,respectively.With the increase of TPGS content,the encapsulated efficiency increases.From the in vitro drug release results,the cumulative release of FA-Ps/TPGS5:1,FA-Ps/TPGS5:3 and FA-Ps/TPGS5:5 was less than 47%.Compared with the polymersome formulations group without TPGS,the release amount of insulin from the mixed polymersome formulations adding TPGS reduced in gastrointestinal tract.The cellular uptake tests of FITC-insulin by Caco-2 cells showed that the uptake of mixed polymersome formulations increased initially and then decreased.FA-Ps/TPGS5:1 had the highest cellular uptake.From in vivo hypoglycemic results,10%FA-Ps and FA-Ps/TPGS mixed polymersome formulations showed hypoglycemic activity for 24 h,and serum insulin level reached the maximum within 6 h.The BA_R%of FA-Ps/TPGS5:1 is 2.04 times,1.40 times and 2.09 times that of 10%FA-Ps,FA-Ps/TPGS5:3,and FA-Ps/TPGS5:5 respectively.Therefore,FA-PS/TPGS5:1 has better hypoglycemic effect than other polymersome formulations,and is a better oral insulin carrier.To sum up,FA-PS/TPGS5:1 mixed polymersome formulation has certain application value in oral delivery of insulin,and the carrier is expected to become a carrier for other peptides and proteins.