偶联CD133核酸适体的载紫杉醇PLGA-PEG纳米载体靶向清除CD133阳性肺癌干细胞

Targeted killing of CD133+lung cancer stem cells using paclitaxel-loaded PLGA-PEG nanoparticles with CD133 aptamers

目的构建偶联CD133核酸适体载紫杉醇的聚乳酸-乙醇酸-聚乙二醇(PLGA-PEG)纳米载体(N-Pac-CD133)拟清除肺癌干细胞。方法采用乳液/溶剂蒸发的方法制备N-Pac-CD133,同时对N-Pac-CD133进行表征,利用磁珠分离法分离出CD133^(+)肺癌干细胞后并对该群体的肺癌干细胞特异性进行检测,同时对肺癌细胞的靶向性和杀伤活性进行检测。小鼠体内接种A549肿瘤后,肿瘤治疗分组:生理盐水,空纳米载体链接CD133核酸适体(N-CD133),紫杉醇,负载紫杉醇的纳米载体(N-Pac)和N-Pac-CD133,8只/组,5 mg/kg紫杉醇,分别于第10、15和20天进行注射。在第40天时,处死小鼠后,对肿瘤进行摘除并称重,同时测量小鼠的体质量。结果 N-Pac-CD133的粒径为100 nm左右,包封率>80%,载药量>8%,在48 h内都显示出持续的药物释放。肺癌细胞的CD133^(+)细胞群体表现出肺癌干细胞的特征:更快的肺癌生长速度(30 d,P=0.001)和更高的肿瘤干细胞基因表达:OV6(P<0.001)、CD133(P=0.001)、OCT3/4(P=0.002)、EpCAM(P=0.04)、NANOG(P=0.005)和CD44(P=0.02)。与非靶向N-Pac和紫杉醇相比,N-Pac-CD133对肺癌干细胞的靶向性(P<0.001)和细胞毒性作用显著增强。另外,N-PacCD133可显著减少肿瘤球的形成(P<0.001)。在治疗终点时,取小鼠肿瘤并对肿瘤进行称量,N-Pac-CD133治疗组和其他治疗组相比,肿瘤体质量显著减小(P<0.001)。结论 CD133核酸适体可以促进紫杉醇纳米载体靶向递送至CD133^(+)肺癌干细胞并杀伤肺癌干细胞。N-Pac-CD133可能是一种有效的靶向肺癌干细胞的治疗手段。

Objective To construct a polylactic acid-glycolic acid-polyethylene glycol(PLGA-PEG)nanocarrier(N-Pac-CD133)coupled with a CD133 nucleic acid aptamer carrying paclitaxel for eliminating lung cancer stem cells(CSCs).Methods Paclitaxel-loaded N-Pac-CD133 was prepared using the emulsion/solvent evaporation method and characterized.CD133^(+) lung CSCs were separated by magnetic bead separation and identified for their biological behaviors and gene expression profile.The efficiency of paclitaxel-loaded N-Pac-CD133 for targeted killing of lung cancer cells was assessed in vitro.SCID mice were inoculated with A549 cells and received injections of normal saline,empty nanocarrier linked with CD133 aptamer(N-CD133),paclitaxel,paclitaxel-loaded nanocarrier(N-Pac)or paclitaxel-loaded N-Pac-CD133(n=8,5 mg/kg paclitaxel)on days 10,15 and 20,and the tumor weight and body weight of the mice were measured on day 40.Results Paclitaxel-loaded N-Pac-CD133 showed a particle size of about 100 nm with a high encapsulation efficiency(>80%)and drug loading rate(>8%),and was capable of sustained drug release within 48 h.The CD133+cell population in lung cancer cells showed the characteristic features of lung CSCs,including faster growth rate(30 days,P=0.001)and high expressions of tumor stem cell markers OV6(P<0.001),CD133(P=0.001),OCT3/4(P=0.002),EpCAM(P=0.04),NANOG(P=0.005)and CD44(P=0.02).Compared with N-Pac and free paclitaxel,paclitaxel-loaded N-Pac-CD133 showed significantly enhanced targeting ability and cytotoxicity against lung CSCs in vitro(P<0.001)and significantly reduced the formation of tumor spheres(P<0.001).In the tumor-bearing mice,paclitaxel-loaded N-Pac-CD133 showed the strongest effects in reducing the tumor mass among all the treatments(P<0.001).Conclusion CD133 aptamer can promote targeted delivery of paclitaxel to allow targeted killing of CD133^(+)lung CSCs.N-Pac-CD133 loaded with paclitaxel may provide an effective treatment for lung cancer by targeting the lung cancer stem cells.