Facribaction And Characterization Of Starch-based Nanocomposites For Oral Insulin Delivery

Oral delivery of insulin is highly desired because of its convenience,safety and good patient compliance.However,the oral bioavailability of insulin is always low and limited by many factors,such as the occurrence of presystematic enzymatic degradation and poor intestinal absorption severely.With the rapid development of nanotechnology,nano-delivery systems provide new ideas and hopes for oral antidiabetic therapy.Natural biopolymers have been the main materials for preparing nano-delivery due to their good biocompatibility,easy decoration and easy degradation.Natural starch has its own several advantages including low cost,good biocompatibility and low side effects for the construction of nanocarrier.In this research,short-chain amylose（SCA）prepared from natural starch using pullulanase was used as the main material to fabricate starch-based nanocomposites which were used to encapsulate a model drug（insulin）.Firstly,the binary SCA-insulin nanocomposites were synthetized using self-assembly method,which exhibited hypoglycemic effect.However,the binary nanocomposites possessed low embedding efficiency and pharmacological bioavailability for insulin.Subsequently,zein was selected as the auxiliary material using its self-assembly capability and the complexation characteristics with starch to co-encapsulate insulin,which possessed high encapsulation efficiency.On this basis,the IN-Z-CSA nanocomposites were coated with chitosan by electrostatic interactions to further improve the oral bioavailability of insulin.Finally,the cells biological characteristics in vitro and in vivo safety assessment of nanocomposites were further studied.The main results are shown as follows:（1）The insulin loaded SCA were synthetized with self-assembly method to form binary SCA-insulin nanocomposites.The flurescence spectroscopy,FTIR and XRD were used to explore the interaction between SCA and insulin.The particle size and morphology of SCA-insulin nanocomposites were characterized by DLS and TEM.The loading capacity and delivery for insulin and MTT assay were extensively evaluated.The in vivo hypoglycemic effects and bioavailability after oral administration of binary nanocomposites in diabetic rats were investigated.The results showed that the diameter of SCA-insulin nanocomposites ranged from 60 nm to 200 nm with a narrow distribution,nearly spherical shape.The encapsulation efficiency（EE）and loading capacity（LC）of insulin was 53.9%and 2.5%for binary nanocomposites,respectively.The formation of binary nanocomposites was mainly driven by hydrogen bonding and hydrophobic interactions.The binary nanocomposites showed nontoxicity and had good biocompatibility.The pharmacological bioavailability after oral administration of SCA-insulin nanocomposites was found to be 4.59%in diabetic rats without any sharp fluctuations in 8 h.The SCA-insulin nanocomposites had a long-lasting hypoglycemic effect.（2）The carboxymethyl short-chain amylose（CSA）was assembled with zein to form tertiary（IN-Z-CSA）nanocomposites which were used to encapsulate insulin.The particle size,PDI,Zeta-potential and morphology of IN-Z-CSA nanocomposites were characterized by DLS and TEM.The flurescence spectroscopy,XRD and FTIR were used to explore the interaction among CSA,zein and insulin.The loading capacity and delivery for insulin and MTT assay were extensively evaluated.The in vivo hypoglycemic effects and bioavailability after oral administration of IN-Z-CSA nanocomposites in diabetic rats were investigated.The results showed that the IN-Z-CSA_1.0 nanocomposites were homogeneous,generally exhibiting sizes of<200 nm with a narrow distribution（PDI<0.200）,spherical shape,and strong negative charge（-40 mV）.The insulin EE was up to 90.5%,and the drug LC was 9.2%.The formation of IN-Z-CSA_1.0 nanocomposites was mainly driven by electrostatic and hydrophobic interactions.The IN-Z-CSA_1.0 nanocomposites showed nontoxicity and had good biocompatibility.The pharmacological activity and relative bioavailability of IN-Z-CSA_1.0 nanocomposites were 10.74%and 11.01%,respectively,which produced gently hypoglycemic effect during 8 h.（3）Based on foregoin section,we introduced positively charged chitosan covering the formed IN-Z-CSA nanocomposites through electrostatic interaction.The morphology and structure of IN-Z-CSA/CS nanocomposites were characterized.The in vivo hypoglycemic effects after oral administration of IN-Z-CSA/CS nanocomposites in diabetic rats were investigated.The results showed that the IN-Z-CSA/CS_0.2 nanocomposites have a smaller particle size（311.32 nm）,well-defined spherical shape,a narrow distribution（0.227）,and strong positive charge（+40 mV）.The formation of the IN-Z-CSA/CS_0.2 nanocomposites was mainly driven by hydrogen-bond and electrostatic interactions.The IN-Z-CSA/CS_0.2nanocomposites were nontoxicity and biocompatibility.The pharmacological activity and relative bioavailability of IN-Z-CSA/CS_0.2 nanocomposites were 14.12%and 15.19%,respectively.The diabetic rats showed more apparent hypoglycemic effect after oral administration of the IN-Z-CSA/CS_0.2 nanocomposites.（4）The Caco-2 cell models were used to evaluate the transepithelial transport of insulin in IN-Z-CSA_1.0 and IN-Z-CSA/CS_0.2 nanocomposites in vitro.The CLSM and flow cytometry were selected to quantificationally and qualitatively analyze the transmembrane transport in vitro of insulin loaded nanocomposites.The results showed that the Papp of insulin in the presence of IN-Z-CSA_1.0 and IN-Z-CSA/CS_0.2 nanocomposites increased by 5-fold to 12-fold compared with the insulin solution in the transport studies.The IN-Z-CSA/CS_0.2nanocomposites produced a TEER reduction within 2 h.However,after removal of the incubated IN-Z-CSA/CS_0.2 nanocomposites,the TEER mainly returned to the initial value.The IN-Z-CSA_1.0 nanocomposites and free insulin solution has no noticeable influence on the TEER value.The uptake behavior of IN-Z-CSA_1.0 and IN-Z-CSA/CS_0.2 nanocomposites showed the time-dependent manner.The IN-Z-CSA/CS_0.2 nanocomposites increased the uptake amout of insulin.All those studies indicated that the IN-Z-CSA_1.0 nanocomposites were transported only by endocytosis route,while the IN-Z-CSA/CS_0.2 nanocomposites were transported by both endocytosis and paracellular route.（5）The ICR mice were administrated with IN-Z-CSA/CS_0.2 nanocomposites for safety evaluation.The acute toxicity experiment and subacute toxicity experiments were carried on.The acute toxicity experiment results showed that IN-Z-CSA/CS_0.2 nanocomposites belonged to non-toxic substances.The subacute toxicity experiments results showed no significantly toxic effects on body weights,organ weihgts ratio,hacmatology paractcrs,blood biochcmistrics and histopathological findings of mice compared with the normal control during 30 days.These results demonstrated that the IN-Z-CSA/CS_0.2 nanocomposites exhibited no toxicity and good biocompatibility in vivo.