Construction Of Nano Oral Insulin Delivery System And Study On Intestinal Absorption Mechanism

In recent years,diabetes has increased rapidly and has become the disease with the third morbidity and mortality rate worldwide.Insulin is a biological macromolecular protein and has been used clinically to treat typeⅠdiabetes and typeⅡdiabetes in which oral hypoglycemic agents show no promises.To avoid the biodegradation of insulin by pepsase,insulin is always administered via injection;however,subsequent long-term and frequent injections typically casue lots of pains and inconveniences to patients.Therefore,administration of insulin via oral has attracted scientific and clinical interests since oral administration of insulin can offer higher convenience and patient compliance.In this paper,an insulin nanodelivery system co-assembled with chitosan derivatives and hyaluronic acid was deveploed and the absorption mechanism of oral administration was systematically studied.The developed insulin nanoparticles(NPs)contained a core insulin coated with cell-penetrating octaarginine(r8)and dicyandiamide modified chitosan(DCDA-CS),and were further coated of hydrophilic hyaluronic acid(HA)by electrostatic self-assembly technology.Reversed-phase HPLC was used to study the content of insulin in nanoparticles;the cell viability of prepared nanocercarriers at different concentrations was investigated by MTT method;artificial mucus layers were constructed to investigate the mucus permeability in different insulin nanoparticles.Moreover,circular dichroism spectrum was used to test the preparation stability and enzyme stability of prepared NPs in simulated gastric and intestinal fluids.The capacity of cellular uptake and mechanism of insulin nanoparticles were analyzed by Confocal Laser Scanning Microscope(CLSM)and Flow Cytometry.In addition,the Caco-2 cell monolayer model was constructed and the process of tight junctions opening caused by the nanoparticles was reflected by the determination of trans-epithelium electrical resistant(TEER)and detection of tightly coupled morphological changes.The absorption capacity of different insulin nanoparticles were investigated in situ for intestinal loop models.Type 1 diabetes mice models were built by intraperitoneal injection of streptozotocin to study the hypoglycemic effect of insulin nanoparticles in vivo.TEM results showed that the HA-DCDA-CS-r8-INS NPs had a regular sphere with a very uniform size distribution.Specifically,the average size was 187.17±2.31 nm and the average Zeta potential was-15.33±0.45 mV.Moreover,Reversed-phase HPLC results showed that the entrapment efficiency and drug loading of HA-DCDA-CS-r8-INS NPs were 59.91±1.53%and 16.21±1.08%,respectively.In the study of mucus penetration,we found that the double-coated insulin nanoparticles of HA and DCDA-CS showed the higher permeability than without HA.In addition,results of circular dichroism spectrum revealed that HA-DCDA-CS-r8-INS NPs had mainly retained more desirable measures of biological activity in simulated gastric and intestinal fluids while the secondary structure of free insulin without carrier was destroyed.Besides,MTT results demonstrated that prepared HA-DCDA-CS-r8-INS NPs had no obvious cytotoxicity and excellent cytocompatibility.We further investigate cell-uptake mechanism of the HA-DCDA-CS-r8-INS NPs.Results demonstrated that cellular uptake were mainly achieved through active transport via lipid rafts and the macropinocytosis pathway.Moreover,in transmembrane transport experiments we found that the caveolae-mediated transport and the DCDA-CS-mediated paracellular pathway both contributed to the increased efficiency of avoidance of lysosomal embedding and sequential transportation from the apical to basolateral areas.And the apparent permeability coefficient(Papp)of HA-DCDA-CS-r8-INS NPs was1.25×10~-66 cm/s,which was 3.56 times and 2.12 times of that of insulin solution and HA-DCDA-CS-INS NPs group,respectively.Therefore,the higher efficiency of absorption of NPs in situ for intestinal loop models were realized and there was a strong hypoglycemic effect in diabetic mice of NPs in which the blood glucose was reduced to38%of the initial blood glucose level within 6 hours.In conclusion,we developed an oral insulin delivery system based on chitosan derivatives and hyaluronic acid by the electrostatic self-assembly method.The prepared HA-DCDA-CS-r8-INS NPs show high stability in the gastric and intestinal fluids and have good permeability in the mucus.And transmembrane transport in the small intestinal epithelial cells could be realized through the DCDA-CS-mediated paracellular pathway thus inducing a slow hypoglycemic effect in vivo.HA-DCDA-CS-r8-INS NPs have great potentials in improving the oral absorption of insulin,which provide a new method and platform for the development of oral preparations of protein drugs.