CREKA肽修饰的载多西他赛脂质体抗前列腺癌靶向研究

CREKA peptide-modified docetaxel loaded liposomes for targeted treatment of prostate cancer in vitro

目的构建以CREKA肽修饰的包载多西他赛脂质体(CREKA-lipo-DTX),评价该药物传递系统体外靶向性和体外抗肿瘤效应。方法首先采用迈克尔加成反应合成靶向部分DSPE-PEG2000-CREKA,随后使用薄膜水化-超声法制备CREKA-lipo-DTX并对其基本表征进行评价;将PC-3细胞和RWPE-1细胞分别分为3组(n=3):空白对照组、PEG-lipo-DiI组和CREKA-PEG-DiI组,脂质体与细胞共孵育后,用激光共聚焦显微镜和流式细胞术分别对脂质体的细胞摄入进行定性和定量的分析,从而评价CREKA修饰脂质体对PC-3细胞的靶向性;将PC-3细胞分为4组(n=3):空白对照组、游离DTX组、PEG-lipo-DiI组和CREKA-PEG-DiI组,通过CCK-8法和Annexin V/PI双染色法评价该药物载体对PC-3细胞的增殖抑制效应。结果成功制备出CREKA肽修饰的载药脂质体(CREKA-lipo-DTX),其粒径为(130.37±1.44)nm,包封率为(91.93±4.64)%。该药物载体稳定性良好,体外释放结果显示其具有较好的缓释性。激光共聚焦显微镜和流式细胞术分析结果显示,与PEG-lipo-DiI组相比,PC-3细胞对CREKA修饰脂质体的摄取显著增加(P<0.05),而RWPE-1细胞对CREKA修饰脂质体的摄取差异无统计学意义(P>0.05);体外抗肿瘤实验结果显示,与游离DTX组和PEG-lipo-DTX组相比,CREKA-lipo-DTX组增强了多西他赛对PC-3细胞的杀伤作用(P<0.05),而且使PC-3细胞早期凋亡和晚期凋亡的数量均显著增加(P<0.05)。结论成功制备出一种具有长循环和肿瘤特异靶向特性的高效低毒的脂质体载药系统,体外可以特异靶向于PC-3细胞,提高了该载体的体外抗肿瘤效应。

Objective To prepare CREKA peptide-modified docetaxel(DTX)loaded liposomes(CREKA-lipo-DTX)and to evaluate their targeted binding ability and anti-proliferative activity in vitro.Methods Firstly,Michael addition reaction was used to synthesize the targeted part DSPE-PEG2000-CREKA,and then CREKA-lipo-DTX was prepared by thin film hydration-ultrasound method,and its basic characterization was evaluated.Both PC-3 cells and RWPE-1 cells were divided into 3 groups respectively(n=3):blank control group,PEG-lipo-DiI group and CREKA-lipo-DiI group.After co-incubation with the cells,the cellular uptake of liposomes was analyzed qualitatively and quantitatively by laser confocal microscopy and flow cytometry respectively,so as to evaluate the targeting effect of CREKA modified vector on PC-3 cells.The cellular uptake of liposomes was analyzed qualitatively and quantitatively by laser confocal microscopy and flow cytometry respectively.The anti-proliferative activity of CREKA-lipo-DTX was evaluated by CCK-8 assay and Annexin V-PI apoptosis detection kit on blank control group,free DTX group,PEG-lipo-DiI group and CREKA-PEG-DiI group of PC-3 cells.Results CREKA-lipo-DTX was successfully prepared,with an average particle size of 130.37±1.44 nm,and encapsulation rate of(91.93±4.64)%.The obtained drug carrier had good stability and showed good slow-release behavior in vitro.The results of laser confocal microscopy and flow cytometry analysis indicated that the uptake of CREKA-modified liposomes by PC-3 cells was significantly increased(P<0.05)when compared with the unmodified liposome group(PEG-lipo-DiI),while no such difference was seen in RWPE-1 cells(P>0.05).In vitro anti-tumor experimental results suggested that enhanced killing effect of DTX on PC-3 cells and significantly increased numbers of early and late apoptotic PC-3 cells were observed in the CREKA-lipo-DTX group when compared with the free DTX and PEG-lipo-DTX groups(P<0.05).Conclusion A high-efficiency and low-toxicity lipoid drug delivery system with long cycle and tumor specific targeting characteristics has been successfully prepared,which can specifically target PC-3 cells in vitro,and thus can improve the anti-tumor effect of the carrier in vitro.