| Objective:To observe the degradation process of TN4003 thermosensitive gel in vivo and in vitro and to study its drug sustained release performance and biocompatibilityMethod:Chitosan thermosensitive gel was prepared by mixing 2.2%chitosan solution and 56%?-glycerophosphate solution at 5:1 in a 37? water bath for 15 minutes,and 27 SD rats were divided into 9 groups with 3 rats in each group.Each group was subcutaneously injected with 2ml blank chitosan thermosensitive gel on the back,and the rats in each group were killed at 6h,Id,4d,10d,13d,19d and 21 d after injection of chitosan thermosensitive gel,and the weight and volume of the gel were measured to determine the degradation in vivo.There were twenty-one 20ml plug test tubes were selected and divided into 7 groups with three 2ml gel in each group.2ml gel was put into the plug test tube of PBS sterilized by high pressure steam.Three groups of thermosensitive gels were taken out at the time point of 6h,Id,4d,7d,13d,16d,respectively,and the degradation solution was determined by calibrated pH meter.Three samples were measured in each group,and the changes of weight and volume were determined to determine its degradation in vitro.The TN14003 freeze-dried powders of lmg,2mg,3mg,4mg and 5mg were respectively weighed and added to the 2mL chitosan thermosensitive gel solution,which were placed in the 50mL centrifuge tube,bathed in water at 37? and placed for 15 minutes,and the TN14003 thermosensitive gels with different drug loading concentrations were prepared.The PBS solution of 4mL pH7.4 was added as the release medium on the upper part of the gel,and the in vitro release experiment was carried out at 37? in a constant temperature shaker and 100r/min.The release medium of 4mL was taken out at 6h,12h,24h,48h,4d,8d,16d,respectively,and the release concentration was detected by high performance liquid chromatography(HPLC),and the standard curve was made.Twenty-four SD rats were randomly divided into four groups.Normal saline,blank gel,TN14003 solution and TN14003/gel of 2ml were subcutaneously injected into the back of each group.Two rats in each group were killed at ld,3d and 7d after injection,and the back specimens were taken out and fixed with 4%paraformaldehyde.Paraffin embedding and HE staining were used to observe the inflammatory reactions such as necrosis and proliferation,vascular invasion and inflammatory cell infiltration around the gel.Immunohistochemistry was used to detect TNF-? and IL-1 of the specimens and to determined the biocompatibility of TN14003 thermosensitive gel.The data were processed and statistically analyzed by SPSS20.0 software.Result:In the process of degradation of thermosensitive gel in vivo,the gel mass in 6 hours was 78.11±1.62%of the initial mass,the volume was 73.50±6.00%of the initial volume,the gel mass was 39.14±11.97%of the initial mass,and the volume was 39.50±1.02 cm3 of the initial volume.The gel was completely degraded in 3 weeks in vivo.In the process of degradation in vitro,the gel mass in 6 hours was 87.68± 1.93%of the initial mass,the volume was 70.00±0.005%of the initial volume,the gel quality was 76.26%±0.66%of the initial volume,and the volume was 59.50±1.50%of the initial volume.The pH value of the degradation solution was basically maintained at about 6.8.The results of sustained release in vitro showed that with the increase of drug concentration,the drug release increased,but the drug release rate decreased continuously.the drug release peak was in the first 6 hours of each group,the cumulative release rate was more than 30%,and the total release rate was more than 95%in 16 days.Considering the side effects and release characteristics of the drug,the best concentration of TN14003 thermosensitive gel was lmg/ml.HE staining results of histological sections showed that chitosan thermosensitive gel could induce a large number of inflammatory cell infiltration,TN14003 thermosensitive gel inflammatory cell infiltration was improved,and there were a large number of granulation tissue and fibroblasts.Immunohistochemical results showed that the expression rates of IL-1 and TNF-? were higher in chitosan thermosensitive gel group,while in TN14003 thermosensitive gel group,the expression rates of IL-1 and TNF-? positive cells were higher at first,then decreasedConclusion:TN14003 thermosensitive gel could be mostly degraded in animals within 3 weeks,does not produce acid or alkaline substances,and had good biocompatibility.TN14003 thermosensitive gel with different drug concentrations has a sudden release site at the initial stage,and could release more than 95%of the total amount within 16 days.With the increase of drug loading,the slow release rate continues to decrease.Chitosan gel loaded with TN14003 had a certain anti-inflammatory effect and promote tissue repair. |
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