反义寡核苷酸-聚乙烯亚胺纳米微粒逆转耐甲氧西林金黄色葡萄球菌耐药性的研究

目的 以聚乙烯亚胺(polyethyleneimine, PEI) 为载体, 将硫代寡聚脱氧核苷酸(PS-ODNs) 转染到耐甲氧西林金黄色葡萄球菌(methicillin resistant staphylococcus aureus, MRSA) 体内, 通过药效学观察PS-ODNs 逆转MRSA 对β-内酰胺类抗生素的耐药性。方法 制备PEI 与PS-ODNs 结合的纳米微粒(PEI-ODNs 纳米微粒) ;PEI-ODNs 纳米微粒的粒径分析及结合率的测定;平板克隆形成实验计数菌落数(CFU) ;微量法测定细菌生长曲线;液体稀释法测定细菌的最小抑菌浓度(MIC)。结果 PEI-ODNs 纳米微粒的粒径为(85±22) nm, PEI与PS-ODNs 的结合率最高为(97.3±1.1) %。含苯唑西林(6 mg L) 的M-H 琼脂板上, 30 μg/mL 的PEI-ODNs 纳米微粒组MRSA 的菌落数为6.4×108 mL, 而空白对照组的菌落数为3.3×109 mL,二者相比给药组菌落数明显减少, 差异具有统计学意义(P<0.01), 而其他对照组与空白对照组比较差异无统计学意义。实验结果显示PEIODNs纳米微粒组的苯唑西林对MRSA 生长有抑制作用, 30 μg/mL 的PEI-ODNs 纳米微粒可将苯唑西林对MRSA 最小抑菌浓度由1 024 μg/mL 降低至16 μg/mL。结论 PEI 与PS-ODNs 的结合率很高且粒径较小, PS-ODNs 可以部分逆转MRSA 对β-内酰胺类抗生素的耐药性。药效学结果显示PEI 可以有效地将反义寡核苷酸转入MRSA 体内, 可考虑将其作为反义寡核苷酸进入细菌的载体。

Reversal of antibiotic resistance methicillanin-resistant staphylococcus aureus by polyethyleneimine-phosphothioate oligodeoxynucleotide nanometer particle

AIM: This study used polyethyleneimine (PEI) and phosphothioate oligodeoxynucleotide (PS-ODNs) to prepare PEI-ODNs nanometer particle and transmit it into methicillin resistant staphylococcus aureus (MRSA) in vitro by pharmacodynamic evaluation. METHODS: The PEI-ODNs nanometer particle was prepared and its diameter was analyzed. The PEI and PS-ODNs binding rate by fluorospectrophotometer was evaluated. The total colony forming unit (CFU) was counted. The cell growth curve was drawn by measuring A630 nm at different time points with microplate reader. The minimal inhibitory concentration (MIC) was determined by fluid dilution method. RESULTS: The diameter of PEI-ODNs nanometer particle was (85±22) nm and the highest binding rate was (97.3±1.1) %. In M-H agar plate(including 6 μg/mL oxacillin), the number of MRSA colonies was 6.4×108 mL in 30 μg/mL PEI-ODNs nanometer particle treated group, while the number of MRSA colonies in blank control was 3.3×109 mL. The number of MRSA colonies was significantly decreased in PEI-ODNs nanometer particle treated group (P<0.01), while CFU of MRSA was not influenced in control group (P >0.05). Significant growth inhibition of cells treated with PEI-ODNs nanometer particle was observed as compared with those cells in control. The MIC of oxacillin to MRSA was decreased from 1 024 μg/mL to 16 μg/mL when treated with 30 μg/mL PEI-ODNs nanometer particle. CONCLUSION: PEI binds PS-ODNs with high binding rate and has little diameter. The susceptibility of MRSA to β-lactam antibiotics is significantly restored. The results indicate that the PEI can transmit PS-ODNs into MRSA and it is considered as a new vector for PS-ODNs into bacteria.