Injectable Thermosensitive Hydrogel PLGA-PEG-PLGA Loaded With Vascular Endothelial Growth Factor To Promote Angiogenesis In Ischemic Lower Limbs Of Rats

Objective A suitable concentration was selected in the preparation of thermosensitive hydrogel polylactic acid glycol-polyethylene glycol-polylactic acid glycol polymer(PLGA-PEG-PLGA)with different concentrations,and test its cytotoxicity to confirm its safety as a biological material.Then it is loaded with vascular endothelial growth factor(VEGF)in a physical mixed manner inside it to establish a drug-loading system with a sustained release function.A standardized ischemic model of lower limbs of wistar rats was established,and a thermosensitive hydrogel loaded with VEGF was administered.The blood flow recovery of the lower limb ischemic limbs was observed by laser Doppler.Method:(1)Dissolve PLGA-PEG-PLGA in PBS,configure thermosensitive hydrogels PLGA-PEG-PLGA with different concentrations,and load VEGF at an appropriate concentration according to the solution-gel transition temperature.(2)The selected PLGA-PEG-PLGA was co-cultured with umbilical vein endothelial cells,and the absorbance of the microplate reader was measured at different time points to analyze the cytotoxicity of PLGA-PEG-PLGA.(3)The establishment of a standardized lower limb ischemia model in Wistar rats.The left lower limb common femoral artery,superficial femoral artery and saphenous artery were ligated and the arterial branches were cut off.(4)Twenty wistar rats with successfully modeled lower limb ischemia were randomly divided into Gel / VEGF group,Gel group,VEGF group,and Control group of saline.On the first day after the ischemia model was established,Blood gastrocnemius muscles were injected with 200 ul of Gel / VEGF,Gel,VEGF and saline.Moor LTD laser Doppler flowmeter was used to record the blood flow of the lower limbs of rats in each group at 0d,1d,3d,7d,10,and 14 d,and the blood perfusion ratio was used(ischemic /non-ischemic).Reflects the recovery of blood flow in the lower limbs of rats.(5)Wistar rats were sacrificed after laser Doppler perfusion ratio analysis,and ischemic lower limb gastrocnemius muscle specimens were collected.HE staining and immunofluorescence were used to analyze the number of capillaries in the ischemic tissue.Result:(1)Obtained through research on three different concentrations of PLGA-PEG-PLGA(1654-1500-1654).When the hydrogel concentration is 15% wt,20% wt,and 25% wt,the gel transition temperatures exhibited by the hydrogel solution are: 29.1o C,26.9o C,and 23.1o C,respectively.The precipitation transition temperatures are : 37.7o C,39.3o C,42.9o C.It can be seen that the higher the concentration of the hydrogel polymer,the lower the temperature required for its transition from solution to gel,but the higher the temperature required for its transition to precipitation.VEGF and PLGA-PEG-PLGA hydrogel can build a sustained-release system in a physically mixed manner,and can release VEGF in vitro for about 9 days.(2)CCK8 cytotoxicity test and tissue HE staining show that PLGA-PEG-PLGA hydrogel has good biosafety at the cellular and tissue levels,respectively,and can be used as a biodegradable material for experiments.(3)Laser Doppler blood flow perfusion analysis.The ischemic lower limbs of rats injected with Gel / VEGF group recovered blood flow perfusion ratio to 87.61% after14 days.Compared with the control group,P < 0.05,the results were statistically significant.significance.Observing the HE sections of the gastrocnemius muscles on the ischemic side of the Gel / VEGF group,we can see that the morphology of the muscle cells on the ischemic side is closer to the non-ischemic side,and the tissue gap is smaller than that of the other three groups.The immunofluorescence double-labeled CD31 and ?-SMA tests of the gastrocnemius muscle on the ischemic side proved that the Gel / VEGF group had more microangiogenesis than the other three groups.Conclusion:(1)The thermosensitive hydrogel PLGA-PEG-PLGA can be used as a sustained-release material to build a drug sustained-release system because of its good biocompatibility and degradability.(2)Constructing a successful thermosensitive hydrogel sustained-release system has a certain promotion effect on ischemic lower limb blood flow recovery.