脂质体包裹的反义寡核苷酸逆转耐甲氧西林金黄色葡萄球菌耐药性的研究

Reversal of antibiotic resistance in methicillin-resistant Staphylococcus aureus by antisense phosphothioate oligodeoxynucleotides liposome

目的:用人工合成磷脂二棕榈酰磷脂酰胆碱(dipalmitoyl phosphatidylcholine,DPPC),二肉豆蔻酰磷脂酰甘油(dimyristoyl phosphatidylglycerol,DMPG)制备反义寡核苷酸阴离子脂质体并研究脂质体包裹的抑制耐甲氧西林金黄色葡萄球菌(methicillin resistantstaphylococcus aureus,MRSA)耐药基因表达信号传导通路中BlaR1 mRNA表达的反义寡核苷酸(antisensephosphothioate oligodeoxynucleotides,AS-ODNs)对MR-SA耐药性的影响。方法:设计合成AS-ODNs;薄膜分散冻干法制备其脂质体;透射电镜观察脂质体的形态;离心纯化脂质体并用紫外分光光度计测定包封率、渗漏率;振荡法检测体外释放度;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。结果:反义寡核苷酸阴离子脂质体大小均匀,为圆球体,包封率为77.38%,冷冻条件下保存1月后渗漏率为0.18%,体外释放度实验表明24 h后约60%的药物从脂质体中释放,反义寡核苷酸脂质体可显著抑制MRSA生长,脂质体包裹的不同剂量的AS-ODNs中MRSA的菌落形成单位(CFU)与空白对照组比较明显减少,具有剂量依赖性,且效果明显优于未被脂质体包裹的AS-ODNs。结论:采用薄膜分散冻干法制备反义寡核苷酸阴离子脂质体,包封率较高,质量稳定,反义寡核苷酸脂质体能逆转MRSA的耐药性,效果明显优于单用AS-ODNs,可作为反义寡核苷酸进入细菌的载体。

AIM： This study was using man-made dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol to prepare antisense oligodeoxynucleotide （AsON） anionic liposomes and to investigate the inhibitory effect of an antisense phosphothioate oligodeoxynucleotides （AS-ODNs） liposome targeting BlaR1 mRNA in methicillin-resistant Staphylococcus aureus （MRSA）. METHODS： Designed and synthesized AS-ODNs by software. Prepare liposome by thin film-dispersion, lyophilized technique. The appearances of liposomes were observed by transmission electron microscope. The liposomes were purified by centrifuge. The encapsulation efficiencies and the leaking efficiencies were determined by UV methods. The release properties in vitro were determined by agitation in PBS. The total colony forming unit （CFU） was counted. The bacteria growth curve was drawn by microplate reader. RESULTS： The liposomes were in spherical shape with uniform size. The encapsulation efficiency was 77.38 % and the leaking efficiency was 0.18% after 1 month in lyophilized condition. The liposomes released 60% drug after 24h when incubated in PBS with mild agitation. The AS-ODNs liposome could significantly inhibit the growth of MRSA compared with control group and those AS-ODNs didn＇t encapsulated in liposome. Liposome encapsulated different concentration AS-ODNs could significantly decrease the CFU of MRSA, which showed a concentration dependent manner （ P 〈 0.05）, while AS-ODNs without liposome didn＇t show this effect. CONCLUSION： The liposomes prepared by this method were stable with a high encapsulation efficiency. The AS-ODNs liposome could reverse the antibiotic resistant in MRSA. The effect was better than those ASODNs without tipesomes. This liposome could be used as a delivery system for AS-ODNs entrapped to bacteria.