Research On The Water-soluble Chitosan Insulin Nanosphere Drug Delivery System

Diabetes mellitus is a common endocrine and metabolic disease caused by abnormal metabolism of protein and fat.Because the main clinical manifestation is due to the relative or absolute deficiency of insulin in the body leading to hyperglycemia.Injecting insulin is one of the important means for the treatment of diabetes in clinical practice.Although insulin has obvious efficacy and the hypoglycemic rate is fast,it will cause hypoglycemia to occurand patient's inflammation at the administration site to cause trouble to the patient.Chitosan is a kind of natural polycationic polysaccharide with the features of mucous membrane adsorption,good compatibility and biodegradability.However,the poor water solubility of chitosan limits its widespread use.Therefore,the chitosan is modified to solve the problem that it can only be dissolved in a small amount of dilute acid,and the preparation of the drug carrier into a drug carrier is the current research direction.Using the modified chitosan as a carrier,there are few reports on the preparation of the nanospheres by the ion crosslinking method,and the modified chitosan-loaded insulin has not yet been reported as a nanosphere.The main research contents are:preparation and in vitro release of chitosan nanospheres;modification of chitosan;preparation and in vitro release of modified chitosan nanospheres;and modification of chitosan-loaded insulin Preparation of nanospheres and in vitro and in vivo experimental studies.1.Construct and characterize chitosan nanospheres to prepared with bovine serum albumin as a model drug and ionically crosslinked method,and investigate the TEM images,encapsulation efficiency and drug loading of nanospheres and release conditions in vitro.The optimum conditions obtained by orthogonal experiments were??CS?=2.0 mg/ml,??TPP?=1.2 mg/ml,30 h,pH 4.5.The maximum entrapment efficiency and drug loading measured under these conditions reached 89.14%and4.02%,respectively.The drug-loaded nanospheres were placed in a pH 1.2hydrochloric acid solutionand the release degree reached about 80%at 10 hours.Then the release rate reached 90%at 48 hours,and sustained release was achieved.2.AmphiphilicchitosangraftcopolymerN-octyl-N'-?2-carboxycyclohexylcarbonyl?was prepared by introducing hydrophobic long-chain octyl and hydrophilic carboxyl groups on chitosan 2-NH₂-Chitosan,which characterized by FT-IR and ¹H-NMR.The infrared spectrum shows that the chitosan derivative?OCCC?increases the-CH₂-stretching vibration of alkanes by 2927 cm^-1and 2860 cm^-1 with respect to chitosan.According to NMR spectroscopy,??ppm?2.14was hydrogen on the acetyl group?NHCOCH₃?,??ppm?was 1.82,1.94 was attributed to hydrogen on the cyclohexane,and the alkyl hydrogen appeared on the octyl group was??ppm?.0.87-1.48.By carbon spectroscopy,the newly emerging??ppm?180 and163 are assigned to carbonyl carbon signals,and the strong signal peaks appearing at??ppm?44,28,and 25 are attributed to the carbon of cyclohexane and long-chain alkanes.The study of its physical properties shows that the ability to form hydrogen bonds after the introduction of hydrophilic substituents is reduced by X-ray diffractograms and DSC diagrams.A part of the crystal form is transformed into amorphous,and the ability to crystallize is reduced to make it disorderly from order,indicating the successful preparation of water-soluble chitosan.3.Using ion-crosslinking method,water-soluble chitosan BSA nanospheres were prepared using modified water-soluble chitosan as carrier.A systematic study was conducted on the encapsulation efficiency and sustained-release effects of drug loading.It was found that with the increase of the initial concentration of water-soluble chitosan,the modified water-soluble chitosan BSA nanospheres increased their drug loading and entrapment efficiency.With the increase of drug concentration,drug loading and entrapment efficiency also increased.According to the orthogonal experimental design analysis,the optimal conditions are:??CS?:??TPP?=10:1,??TPP?=1.2 mg/ml,??BSA?=0.8 mg/ml,40 h.The maximum entrapment efficiency and drug loading measured under these conditions reached91.16%and 3.18%,respectively.The drug-loaded nanospheres were placed in a pH=7.4 hydrochloric acid solution.The release degree reached 80%at 8 hours.Then the release reached 90%at 48 hours,whichachieving a sustained release effect.The results show that the drug-loaded nanospheres have higher release rate in pH 7.4 PBS than in pH 1.2 hydrochloric acid solution,which proves that drug-loaded nanospheres are more suitable for release in body fluids.4.OCCC-Ins-NPs was prepared by ion-crosslinking method with using modified chitosan:water-soluble chitosan as carrier material and sodium tripolyphosphate as cross-linker.Studies have shown that strong acid environments result in lower insulin release efficiency.In the release medium pH 6.8 and pH 7.4,the cumulative release of insulin can reach 50%,70%,respectively.For mice injected subcutaneously with water-soluble chitosan insulin nanospheres,the blood glucose level decreased steadily within 1-5 hours,and the blood glucose level could eventually be maintained at a reasonably stable level.It shows that the water-soluble chitosan insulin nanospheres have obvious hypoglycemic effect,and compared with insulin,the sustained-release effect obviously takes longer.The experimental results show that water-soluble chitosan also has a certain degree of hypoglycemic effect.In summary,N-octyl-N'-?2-carboxylcyclohexylcarbonyl?-Chitosan-Insulin nanospheres have higher entrapment efficiency,which can effectively inhibit the burst release of insulin,and not only has a significant hypoglycemic effect in diabetic mice,but also have significant hypoglycemic effect in diabetic mice.Compared to simple injections of insulin,the effect is longer and achieves a sustained release effect.It shows that the modified chitosan can be used as an effective carrier for insulin administration.