| Objective:1.According to the method provided by our research group in the previous work,the multi-layer sodium alginate-chitosan microspheres loading vascular endothelial growth factor（VEGF）and vancomycin（VAN）are prepared in a sterile environment.After observing their morphology,determining the drug loading and entrapment efficiency of VEGF and VAN,the microspheres were served as a blank control for the further experiments.2.In order to evaluate whether the microspheres have anti-infective ability in vitro,the inhibitory effect that the microspheres against Staphylococcus aureus is tested by paper stripe method.3.To explore the ideal storage conditions of the microspheres,the structural stability of the microspheres and the effective release of the loaded drug are detected after treating in different physical conditions.Methods:1.Refer to previous experiments,select the optimized reagent ratio and prepare VEGF/VAN multi-layer sodium alginate-chitosan microspheres in a sterile environment.The surface and cross-sectional morphology of multi-layer drug-loaded microspheres are observed by scanning electron microscopy（SEM）.The prepared multi-layer drug-loaded microspheres were completely dissolved.The VEGF drug loading and entrapment efficiency are determined by the VEGF ELISA kit.The VAN drug loading and entrapment efficiency are determined by UV spectrophotometer.2.First,inoculate the resuscitated Staphylococcus aureus（ATCC25923）on agar medium.Meanwhile,place the microspheres in sodium citrate solution to obtain the supernatant.Next,place the filter paper that was added by the microsphere supernatant in the medium and incubate it in a 37℃incubator for 24h.Observe whether there is a bacteriostatic ring around the filter paper and determine the diameter of the bacteriostatic ring.3.The prepared microspheres are placed under the conditions of-20℃,4℃,25℃,37.5℃and 4500±500Lx for 5 days and 10 days.Observe the surface and cross-sectional morphology of microspheres by SEM.Place the microspheres treated with different conditions in sodium citrate solution for 24h to obtain the supernatant.Determining the effective release of VEGF and VAN by VEGF ELISA kit and UV spectrophotometer respectively.Results:1.The multi-layer sodium alginate-chitosan microspheres are prepared after optimization of the reagent ratio.Their morphology are spherical,their diameters between 900-1100μm,their surfaces are complete and smooth.There are some slight folds on the surfaces.The cross-section morphology of the microspheres is a dense network structure.The drug loading of VEGF is 6.63×10-5%and the entrapment efficiency is 72.1%.The drug loading of VAN is 1.39%and the entrapment efficiency is 3.37%.2.The results of the paper stripe method experiment show that the antibacterial rings around the filter paper are clearly visible,and the diameters between12.61±1.01mm.3.The surface and cross-section morphology of the microspheres have no change after treating in different conditions.The effective release of VEGF and VAN in microspheres have decreased.The results of effective release of VEGF are significantly different between the experimental groups and the control group（P<0.05）.The effective release of VAN shows no significant difference between the-20°C group and the control group（P>0.05）,and there is a significant difference between the other groups and the control group（P<0.05）.Conclusions:1.The multi-layer sodium alginate-chitosan microspheres loading VEGF/VAN with optimized reagent ratios are successfully prepared in a sterile environment.2.The antibacterial ability of microspheres against Staphylococcus aureus（ATCC25923）in vitro was proved by paper stripe method.3.It is proved that the microspheres are structurally stable by controlling the variables of different physical factors,but the increase of storage temperature and light irradiation will cause the effective release of VEGF and VAN contained in the microspheres decrease,and the decrese degree of VEGF is more obvious than VAN. |
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