Study On Soluble Microneedles And Its Transdermal Delivery Of Loaded Glucose-responsive Insulin Drugs For The Treatment Of Diabetes Mellitus

Diabetes is a clinical high-risk disease.At present,oral sulfonylurea,biguanide hypoglycemic drugs or injection of insulin,exenatide and other means are generally used to reduce blood glucose,in which injection of insulin is the most effective treatment to control blood glucose.However,the injection process of 2-4 times a day makes diabetic patients suffer a lot,making them prone to resistance.One time administration is also easy to cause hypoglycemia and other symptoms,and at the same time,patients have the risk of weight gain.In this paper,we tried to explore a new way of treatment of diabetes mellitus through the mode of controlled release of insulin loaded microneedles.Firstly,the mineralized insulin（m-INS）amorphous aggregate was prepared by using insulin as the nucleation site of calcium phosphate crystal to simulate the natural biomineralization process in vitro.The diameter of the aggregate is about 1μm.It can keep stable in the physiological environment of pH=7.4,and release insulin rapidly in the environment of pH less than 4.5 due to the dissolution of calcium phosphate,with the release rate close to 100%.The suspension of mineralized insulin can be stored at 4℃for more than one month without degradation.According to the pH response characteristics of mineralized insulin,an intelligent insulin release system based on blood glucose concentration was designed.The system is mainly composed of mineralized insulin and mineralized glucose oxidase（m-GOx）.Mineralized glucose oxidase（m-GOx）is a kind of flower like copper phosphate crystal particles with glucose oxidase as the crystal nucleus,which is composed of many"petals"and the particle size of about 1-2μm.It has a high specific surface area.After mineralization,the stability of enzyme activity was improved,and more than 90%of enzyme activity could be maintained after 10 days at room temperature.In a high concentration of glucose environment,glucose is oxidized by glucose oxidase to produce gluconic acid and hydrogen peroxide.Gluconic acid can cause a local drop in pH,which leads to the dissolution of inorganic salt Ca₃（PO₄）₂ in mineralized insulin and the release of insulin.In low concentration glucose solution,the decrease of pH caused by a small amount of gluconic acid was not obvious,and the release of insulin was little or almost No.The results of in vitro experiments show that the insulin intelligent release system has good pH response,low cytotoxicity and high drug stability,which has great potential in the application of diabetes on-demand intelligent drug delivery.Secondly,a soluble sodium hyaluronate microneedle patch was prepared for the transdermal delivery of insulin intelligent release system.Through the design and manufacture of a new microneedle molding mold,the molding process of microneedles was optimized,and the commonly used soluble biomaterials were screened.Finally,sodium hyaluronate（HA）reagent with 300 000 nominal molecular weight and 30 000 nominal molecular weight was selected as raw materials,the preparation process was optimized,Therefore,25 pieces of sodium hyaluronate microneedle patches can be prepared at one time.Each microneedle patch contained 289 needles with a length of 730μm.The bottom diameter of microneedles is 356μm.The mechanical strength of each microneedle reached 0.33N,which can penetrate the skin of pig in vitro and completely dissolve and release the loaded drug within 1 hour.Fluorescence microscope analysis showed that FITC labeled mineralized insulin and Cy5labeled mineralized glucose oxidase were successfully integrated into the microneedles,and the mechanical strength of the drug-loaded microneedles was increased to 0.43N/needle.Each piece of microneedle patch could load 0.33U insulin at least.If the dose was 0.5U/kg,it would be enough to provide insulin for diabetic rats weighing about 300g for two days.The effectiveness and reliability of insulin intelligent release system loaded with HA microneedles for blood glucose control trials were further verified by animal experiments in vivo.The isletβcells of SD rats were selectively destroyed with streptozotocin（STZ）at 55mg/kg body weight to establish theⅠtype diabetic rat model.The microneedles patch loaded with insulin intelligent release system can reduce the blood glucose level of diabetic rats to less than 7 mmol/L within 1 hour,and maintain the blood glucose level at a low level（11.1 mmol/L）for 10 hours,which is significantly better than that of simple insulin administration for 2 hours.In addition,the system does not play a hypoglycemic role in healthy rats and diabetic model rats after the recovery of normal blood glucose,and will not cause hypoglycemia.The soluble microneedle patch based intelligent insulin delivery system can provide a safe,effective,on-demand treatment and painless drug delivery for diabetic patients.It has a constructive significance in improving patients’compliance and quality of life.It also broadens the way for the application of biomimetic mineralization technology and microneedle technology in clinic.