Development Of Intestinal Delivery Systems Of Insulin By Electrohydrodynamics Technique And Their Blood Glucose Regulation Mechanism

Insulin（INS）known as a bioactive peptide has been the preferred drug for the treatment of diabetes mellitus.The subcutaneous injection of INS has good therapeutic efficacy;however,frequent injections are needed due to the short biological half-life of INS,which thus results in pains and other serious side effects.In this case,oral administration can not only improve the patient compliance,but also be beneficial to the function of INS based on the physiological advantages.However,it is essential to overcome the critical problems that hamper the development of oral INS systems,such as maintaining INS stability during the passage through the gastrointestinal environment,targeted delivery,and high absorption efficiency.Existing studies have shown that the bioavailability of INS can be effectively improved by developing various small intestinal delivery systems.Nervertheless,as functions of the colon and colonic flora have become increasingly clear,the colon can also be used as an absorption site for the active proteins or polypeptides,and the colonic delivery system has thus progressively received broad attention.Currently,only a few studies have preliminarily confirmed the feasibility of colonic delivery of INS.Despite this,no comparison evaluation of the real hypoglycemic effect of different INS intestinal delivery systems has been reported,nor has the influence mechanism of intestinal flora on the efficacy of colonic delivery system.Electrohydrodynamic technique,including electrospinning and electrospraying,is a simple and effective technique to encapsulate active substances to efficiently improve their stability,and therefore has a good application prospect.Herein,in this study,electrohydrodynamic technique was used to develop different multi-unit vehicles for INS using natural polysaccharides and p H-sensitive material,and their feasibilities for small intestinal and colonic delivery and release of INS were confirmed through the in vitro and in vivo experiments.Meanwhile,the colonic INS delivery system was creatively developed by incorporating prebiotic and the probable mechanisms of prebiotic on the pharmacy efficacy of the colonic INS delivery system were preliminarily analyzed according to the influences of prebiotic on the intestinal flora,colon tissue,etc.,and the results of the in vitro and in vivo experiments.The main research contents and results are as follows:（1）Preparation and properties of small intestine delivery system for INSDifferent small intestine delivery systems for INS,that is uniaxial nanofiber mat（IFM）and composite beads（CISB）loaded with INS nanoparticles（CITN）,were successfully prepared for the first time by electrospinning and electrospraying,respectively.First,CITN was prepared by ionic gelation method.IFM was prepared by electropun the blending of CITN,sodium alginate（SA）polyvinyl alcohol（PVA）and hydroxypropyl methyl cellulose（HPMC）.For the CISB,it was fabricated by coaxial electrospraying with CITN/SA and SA as the core layer and the shell layer,respectively,and the chitosan solution（p H 5.5）containing Ca Cl₂was used as the curing liquid.The results of in vitro release experiments showed that both carriers can effectively decrease the burst release of INS in simulated gastric fluid（SGF）.The amount of INS released from the IFM in SGF was less than 40%,and the release was controlled by the Fickian diffusion mechanism.The release of INS in simulated intestinal fluid（SIF）followed the Case II release mechanism;In particular,less than 20%of INS was released from CISB in SGF,and the release conformed to the Fickian diffusion mechanism,while the release in SIF was based on a complex mechanism of diffusion and swelling.In summary,CISB was selected as the small intestine delivery system for INS based on the comparison of carrier preparation and INS release,and used for the following in vivo studies.（2）Preparation and properties of colonic delivery system for INSThe core-shell nanofiber mat（CF-IFM）and Eudragit S-100（ES100）coated beads（EF-CISB）loaded with CITN and prebiotic were creatively prepared by using two electrostatic fluid technologies.First,fructo-oligosaccharide（FOS）was selected as the prebiotic for the preparation of colonic delivery system due to its better probiotic effect.The CF-IFM was fabricated by coaxial electrospinning with PVA/SA（PS）and FOS/PS/HPMC/CITN as the shell and core solutions,respectively.In addition,the EF-CISB was developed by adding FOS to the shell solution of CISB,and the beads obtained were further coated with ES100.The results of in vitro release experiments showed that the cumulative release of INS from CF-IFM in the upper gastrointestinal simulated fluid（SGF+SIF）was less than 40%;however,the cumulative amount of INS from EF-CISB released in the SGF+SIF was less than 20%.The release mechanism of INS from these two colonic delivery systems in SGF and SIF were both based on Fickian diffusion,while the release in simulated colon fluid（SCF）followed the Case II release mechanism,including swelling and erosion of the substrate.In summary,EF-CISB was selected as the colonic delivery system for INS based on the comparison of carrier preparation and INS release,and used for the following in vivo studies.（3）Comparative study on blood glucose regulation and pharmacokinetics of two different INS intestinal delivery systemsThe type I diabetic rat model was established,and the hypoglycemic effect of these two INS intestinal delivery systems were investigated by analyzing the pharmacological bioavailability,relative bioavailability of INS.Results indicated that subcutaneous injection of INS can quickly and greatly reduce blood glucose within 1 h.In contrast,a slow and long-lasting hypoglycemic effect were achieved for the CISB and EF-CISB groups,which could effectively reduce the occurrence of hypoglycemia.In addition,CISB was more effective in blood glucose control than CITN,and its pharmacological bioavailability（PA）was significantly higher than that of the CITN group（P<0.05）,and the relative bioavailability of INS（RB）was increased by 1.8 times.Besides,for the colonic INS delivery systems,EF-CISB had better hypoglycemic effect due to the incorporation of prebiotic.The blood glucose level at 6 h was reduced to 62.7%of the original blood glucose value,Moreover,the hypoglycemic effect of EF-CISB was longer and its PA and RB values were significantly higher than the colonic delivery system without prebiotic（P<0.05）.In addition,it was found from the comparison results that the addition of prebiotic into the colonic delivery system could significantly improve the oral bioavailability and blood glucose control level of INS,and its overall application effect was better than that of the small intestinal delivery system.（4）Preliminary study on the internal influence mechanism of prebiotic to improve the efficacy of colonic INS delivery systemThe results of flora analysis showed that the diversity of intestinal flora,the ratio of Firmicutes/Bacteroides,and the abundance of beneficial bacteria in diabetic rats treated with prebiotic increased.In addition,the observation results of colon tissue morphology suggested that prebiotic could effectively alleviate the damage to the mucosa of the colon tissue and decrease the degree of tissue ulcers,and therefore protect the intestinal structural integrity of diabetic rats to a certain extent.It was comprehensively analyzed that changes in the intestinal flora of diabetic rats,the increase in SCFAs-producing bacteria,and repairment of colon tissue were the possible mechanisms of prebiotic to improve the efficacy of the colonic INS delivery system.This study will provide the theoretical base and scientific methods for the development of oral intestinal delivery systems for INS,and is of great significance to promote the application of electrohydrodynamic technique in the encapsulation and stable delivery of active substances.