小分子功能化的金纳米粒子对革兰氏阴性多药耐药细菌的抗菌特性

Antibacterial properties of small molecule-functionalized gold nanoparticles against Gram-negative multidrug-resistant bacteria

摘要:

[目的] 研究大肠杆菌tRNA合成底物类似物4，6-二氨基-2-巯基嘧啶功能化的金纳米粒子（gold nanoparticles，AuNPs）对革兰氏阴性多药耐药细菌的抗菌特性。[方法] 以4，6-二氨基-2-巯基嘧啶为表面配体合成AuNPs，采用肉汤稀释法测定其对4种临床分离的革兰氏阴性多药耐药细菌的最低抑菌浓度（MIC）。通过不同浓度AuNPs处理后经平板计数绘制不同菌株的时间-杀菌动力学曲线。以铜绿假单胞菌为代表菌株，采用激光共聚焦显微镜、透射电子显微镜和凝胶电泳分析AuNPs对细菌细胞组分的损伤。通过亚致死浓度反复诱导评估细菌对AuNPs的耐药性演化。并以MTT实验初步评估了AuNPs对哺乳动物细胞的生物相容性。[结果] 4，6-二氨基-2-巯基嘧啶介导的AuNPs平均粒径为6.8 nm，zeta电位为+38.4 mV。该AuNPs对4种临床分离的革兰氏阴性多药耐药细菌均表现出时间和浓度依赖的抗菌活性，MIC值介于4-8 μg/mL之间。抗菌机制研究显示AuNPs主要通过诱导细菌细胞膜损伤和DNA断裂导致细菌死亡。耐药性演化评估发现细菌在为期30 d的反复诱导下也基本不会对该AuNPs产生耐药性。细胞毒性结果显示AuNPs对哺乳动物细胞具有良好的生物相容性，在浓度达到256 μg/mL时正常肝细胞L02和正常肺细胞AT II的存活率仍高达85%以上。[结论] 小分子介导的AuNPs对临床分离的革兰氏阴性多药耐药细菌具有较好的抗菌活性，在应对当前严峻的多药耐药细菌感染方面具有潜在的应用价值。

Abstract:

[Objective] In this study, we investigated the antibacterial properties of 4,6-diamino-2mercaptopyrimidine-functionalized gold nanoparticles (AuNPs) against Gram-negative multidrug-resistant (MDR) bacteria. [Methods] Minimum inhibitory concentration (MIC) of AuNPs against 4 clinically isolated Gram-negative MDR bacteria was determined by the broth dilution method. Time-kill curves within 24 h were drawn by plate counting after treatment with different concentrations of AuNPs. Employing Pseudomonas aeruginosa as a representative strain, the damage of AuNPs to bacterial cell components was studied using laser scanning confocal microscopy, transmission electron microscopy, and gel electrophoresis. The evolution of potential drug resistance of bacteria to AuNPs was evaluated by repeatedly exposed to AuNPs at a sublethal concentration. The biocompatibility of AuNPs to mammalian cells was evaluated by MTT analysis. [Results] The average size of synthesized AuNPs was 6.8 nm, and the zeta potential was +38.4 mV. The AuNPs showed timeand concentration-dependent antibacterial activity against the 4 Gram-negative MDR bacteria, with MIC values ranging from 4 to 8 μg/mL. The AuNPs killed these MDR bacteria through a combined mechanism including cell membrane destruction and DNA damage. No resistance development toward the AuNPs was demonstrated even after continuous passage for 30 d. Preliminary cytotoxicity assay results showed that AuNPs have excellent biocompatibility to mammalian cells, and the survival rate of normal L02 liver cells and normal AT II lung cells is still more than 85% when the concentration reaches 256 μg/mL. [Conclusion] Small molecule-mediated AuNPs have excellent antibacterial activity against Gram-negative MDR bacteria, and potential application value in dealing with severe MDR bacterial infection.