负载抗菌肽的聚乳酸纺丝纤维体外药物缓释性能及抗菌性研究

Study on drug controlled release and antibacterial activity of poly-l-lactic acid fibers loaded with HHC-36in Vitro

目的:研究负载抗菌肽(HHC-36)的聚乳酸纺丝纤维的体外药物缓释能力及抗菌性。方法:通过熔融纺丝技术和化学反应及物理吸附法合成载有HHC-36的聚乳酸纺丝纤维,采用扫描电子显微镜及XPS分析抗菌肽接枝情况,接触角表征其亲水性,采用分光光度法检测HHC-36体外释放情况。通过抑菌环法和细菌活/死染色检测其对大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)的体外抗菌性。通过细胞相容性实验检测其细胞毒性。结果:扫描电镜结果显示纤维直径较均匀且其表面光滑没有未熔融聚合物;XPS检测显示PLLA组N含量为3.68%,PLLA/PDA组N含量为14.61%,PLLA/DH-36组N含量为17.08%;亲水性检测显示经改性的纤维表面亲水性增强,润湿性较好,有利于细胞黏附。抗菌肽的释放实验结果显示,抗菌肽HHC-36在PBS溶液中能稳定释放,其释放量随时间变化而持续增加;释放速率初始时急速增大,在150 min时达至峰值,随后逐渐下降,3 d后速率趋于平稳。细菌与材料共培养结果显示,PLLA/DH-36组的细菌浓度始终保持在一个较低的水平。抑菌环实验中,PLLA/DH-36组并没有产生期望中的抑菌环,与其他两组并无明显差异;细菌的活/死染色实验中,PLLA/DH-36组材料表面的死细菌数量较PLLA组多,活细菌数较少。细胞相容性实验中,培养1 d时,与空白对照组相比,3组材料的细胞增殖率均较低;培养3 d时,与PLLA/PDA组相比,PLLA/DH-36组表现出细胞毒性,但它与PLLA组的细胞毒性无明显差异;培养3 d后,3组材料的细胞毒性均表现出下降趋势,恢复其良好的生物相容性,细胞增殖速率高于空白对照组。荧光显微镜观察经DAPI-FITC染色的材料发现,PLLA/DH-36组材料表面的细胞存活率与PLLA组并无明显差异。结论:负载HHC-36的聚乳酸纺丝纤维具有良好的体外药物缓释能力以及抗菌性,同时具备促进细胞增殖和控制感染两种潜能,为促进开窗减压术后创口愈合的填充材料的研究提供新思路。

Objective:To study the drug controlled release and antibacterial activity of Poly-l-lactic acid(PLLA) fibers loaded with Antimicrobial Peptides(HHC-36).Methods:PLLA containing HHC-36 was synthesized by melt spinning technology, chemical reaction and physical adsorption methods.Scanning electron microscopy and XPS were used to analyze the grafting of antimicrobial peptides, and the contact angle was used to characterize its hydrophilicity.The release of HHC-36 in vitro was detected by absorption spectrophotometry.The in vitro antibacterial activity against Escherichia coli(E.coli) and Staphylococcus aureus(S.aureus) was detected by bacteriostatic ring method and bacterial live/dead staining.Its cytotoxicity was detected by cytocompatibility experiment.Results:The results of scanning electron microscopy showed that the fiber diameter was relatively uniform and its surface was smooth without unmelted polymer.XPS test showed that the N content of the PLLA group was 3.68%,that of the PLLA/PDA group was 14.61%,and that of the PLLA/DH-36 group was 17.08%.The hydrophilicity test showed that the modified fiber surface had enhanced hydrophilicity and better wettability, which was conducive to cell adhesion.The release test results of antimicrobial peptides showed that the antimicrobial peptide HHC-36 could be stably released in PBS solution, and its release amount continued to increase with time.The release rate increased rapidly at the beginning, reached a peak at 150 min, then gradually decreased, and the rate stabilized after 3 days.The co-culture results of bacteria and materials showed that the bacterial concentration of the PLLA/DH-36 was always kept at a low level.In the antibacterial circle experiment, the PLLA/DH-36 group did not produce the expected antibacterial circle, and there was no significant difference with the other two groups, the live/dead staining experiment of bacteria in the PLLA/DH-36 group, the number of dead bacteria on the surface of the material was more than that of the PLLA group, and the number of live bacteria was less.In the cell compatibility experiment, the cell proliferation rate of the three groups was lower than that of the blank control group on the 1 day of culture.Compared with the PLLA/PDA group, the PLLA/DH-36 group showed cytotoxicity on the 3 rd day of culture, but it was not significantly different from the PLLA group.After 3 days of culture, the cytotoxicity of the three groups showed a downward trend, and its good biocompatibility was restored, and the cell proliferation rate was higher than that of the blank control group.The DAPI-FITC stained materials were observed by fluorescence microscopy, and it was found that the cells in the PLLA/DH-36 group survived on the surface of the material.The rate was not significantly different from the PLLA group.Conclusion:The PLLAfiber loaded with HHC-36 has the ability of drug sustained release and good antibacterial properties in vitro, as well as the potential of promoting cell proliferation and controlling infection, which can provide a new idea for the research of filling material for promoting wound healing after decompression.