Preparation Of Oral Insulin-loaded Microspheres By The Method Of Electrostatic Deposition

Objectives:Insulin(INS)is a kind of biological macromolecular protein.It was used to treat type 1 diabetes and type 2 diabetes that were ineffective through oral anti-hyperglycemic agents in clinical practice.The bioavailability of insulin was extremely low through directly oral administration.Subcutaneous injection was the main drug delivery route of insulin,but frequent injections at long-term brought a lot of pain and inconvenience to the patients.A new oral insulin-loaded microsphere(INS-MP)was preparaed in the study by the method of electrostatic deposition to improve the efficacy of oral insulin and benefit people with diabetes.The follow studies were conducted to the INS-MP: quality evaluation,stability and hypoglycemic effect.Methods:1 The preparation of INS-MP: The blank microspheres were prepared by conventional solvent evaporation method and used as carrier material.The insulin was loaded on the surface of blank microspheres by alternate deposition of positive ion and negative ion through electrostatic force of attraction to prepare the INS-MP with the right number of film layers that we need.The factors that have influence to the preparation were confirmed through the single factor investigation.Orthogonal experiment was conducted on the basis of these influential factors(each factor with three levels)to optimize the preparation technology and loading quantity was chosen as the main evaluation index.At the same time,3 batches of INS-MP were prepared to evaluate the reproducibility of formulation.2 The identifications of INS-MP: The morphology and particle size of blank microspheres and INS-MP was separately determined by scanning electron microscope and laser particle size analyzer,then the results was compared to identify the formation of INS-MP.3 The evaluation of quality of INS-MP: The HPLC method was established to determine the insulin content of the INS-MP and the methodology was evaluated.To study the relationship between loading quantity,entrapment efficiency and film layers,the INS-MP with different film layers were prepared and the loading quantity and entrapment efficiency were calculated.4 Study on the stability and dissolution rate of INS-MP in vitro: The dissolution apparatus was used to study the in vitro release of INS-MP in HCl solution(p H1.0)and phosphate buffered solution(p H6.8)and then the insulin concertration was determined to calculate the cumulative release rate.The INS-MP was placed in different surroundings(25?and 4?).Then loading quantity and particle size was measured at different time points(1,3,6 months)to study the stability of microspheres.5 The hypoglycemic effect of oral INS-MP: Diabetic rats of type 1 were induced by a single intraperitoneal injection of streptozotocin to rats.All diabetic rats were randomly divided into six groups.They were blank control group,positive control group,negative control group and experimental groups of oral INS-MP at low,middle,high dose respectively.Blood samples were withdrawn from the orbital venous plexus of diabetic rats of all the groups prior to drug dosing and at different times after drug administration to observe changes in blood sugar levels.Results:1 Three factors that weight of blank microspheres,volume of solution and centrifugal speed were chosen to conduct orthogonal test through the results of single factor study and each with three levels.The best preparation conditions were 25 mg blank microspheres,0.8 mL solution and centrifugation at 6000 r/min.And the reproducibility of optimal technology was excellent.2 The results of identifications of INS-MP showed that: The INS-MP produced under optimum technological conditions with round appearance and uniform particle size.The balnk microspheres were with smooth surfaces and the mean particle size was(3.6±0.46)?m.However,the INS-MP was covered with apparently rough surfaces and the mean particle size is(5.25±0.15)?m.Both the changes of morphology and particle size proved the formation of INS-MP.3 HPLC method was established successfully to determine the insulin content of microspheres.According to the results of methodology investigation,there were good linear relationship between the concentration and peak area of insulin within the scope of 20~100 ?g/mL.The recovery,stability and precision were meeting the requirements of the methodology.Film layers were positively related to loading quantity and negatively related to encapsulation efficiency through the measurement of loading quantity and encapsulation efficiency of INS-MP with different film layers.The loading quantity of INS-MP could be regulated through changing the number of film layers.4 The dissolution rate result of INS-MP in vitro illustrated that: The INS-MP was stable in HCl solution(p H1.0),but the amount of insulin release reached nearly 90% in phosphate buffered solution(p H6.8).The changes of drug loading and particle size of INS-MP after placement at different surroundings showed that: The loading quantity and particle size were stable in 6 months at 4? better than 25?.Therefore,the INS-MP should be stored in cold storage conditions.5 The results of hypoglycemic effect of INS-MP indicated that: The stable diabetic rat models were successfully established through subcutaneous injection of streptozotocin.The experimental rats which were gavaged INS-MP had obviously hypoglycemic effect compared with the blank and negative control group at the same dose and the difference was statistically significant(P < 0.05).Meantime,the hypoglycemic effect was dose dependent.Conclusions:The INS-MP prepared by alternate deposition of positive ion and negative ion on the surface of blank microspheres with mild preparation conditions,good quality and stability.The loading quantity of INS-MP could be regulated through changing the number of film layers.Meantime,INS-MP had a significantly dose-dependent hypoglycemic effect as well as a good application prospect.