Abstract: BACKGROUND: Local antibiotic slow-release system can solve the problems of total toxicity caused by systemic antibiotics and short half-life of short-term local antibiotics.  
OBJECTIVE: To prepare polylactic acid-glycolic acid copolymer-chitosan-hyaluronic acid composite sustained-release microspheres loaded with vancomycin and evaluate its performance.
METHODS:  Vancomycin-loaded polylactic acid-glycolic acid copolymer-chitosan-hyaluronic acid composite sustained-release microspheres and unloaded polylactic acid-glycolic acid copolymer-chitosan-hyaluronic acid composite microspheres were prepared by emulsion method. Mass concentrations of vancomycin in the drug-loaded microspheres were 25, 50, and 100 g/L. The drug-loading amount, encapsulation efficiency, and in vitro sustained release properties of the drug-loaded microspheres were detected. The three kinds of drug-loaded microspheres were co-cultured with Staphylococcus aureus bacteria separately, and the antibacterial rate was detected within the corresponding time points. The four kinds of microsphere extracts were co-cultured with MC3T3-E1 cells and MG-63 cells, and the cytotoxicity was detected by CCK-8 method after 1, 3, and 7 days of culture.  
RESULTS AND CONCLUSION: (1) The encapsulation efficiencies of 25, 50, and 100 g/L drug-loaded microspheres were (79.70±5.11)%, (86.41±3.91)%, and (63.18±1.96)%, and the drug loading was (3.98±0.26)%, (8.64±0.39)%, and (12.63±0.39)%. The encapsulation efficiency of 50 g/L drug-loaded microspheres was higher than that of 100 g/L drug-loaded microspheres (P < 0.05). The drug loading of 100 g/L drug-loaded microspheres was higher than that of the other two groups (P < 0.05). (2) Three kinds of drug-loaded microspheres had no obvious burst release within 24 hours, of which the drug release rate of 50 g/L drug-loaded microspheres at different time points was faster than that of the other two groups. The drug release amount of 100 g/L drug-loaded microspheres at different time points was higher than that of the other two groups, and the drug mass concentration of the three groups was higher than minimum antibacterial concentration of vancomycin at 56 days. (3) All three kinds of drug-loaded microspheres could effectively kill Staphylococcus aureus within a certain period of time. From 14 to 28 days, the relative colony rate of the three kinds of microspheres was lower than 3%, indicating that three kinds of drug-loaded microspheres can continuously and effectively kill Staphylococcus aureus. (4) The 25 and 50 g/L drug-loaded microspheres have no obvious cytotoxicity to MC3T3-E1 cells and MG-63 cells, and 100 g/L drug-loaded microspheres have certain cytotoxicity. (5) The results show that the VA@PLGA-CS-HA microspheres have good sustained-release performance, antibacterial ability and biological tissue compatibility.

Key words: bone, material, microspheres, vancomycin hydrochloride, chitosan, hyaluronic acid, polylactic acid-glycolic acid copolymer, biocompatibility