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目的采用整合素受体介导的靶向载体输送系统实现RNA干扰技术和抗肿瘤药物对耐药肿瘤的联合治疗。方法分别用静电复合法和硫酸铵梯度法制备载多药耐药基因(MDR1)相关的小干扰RNA(siRNA)的精氨酸-甘氨酸-天冬氨酸三肽(RGD)修饰阳离子脂质体复合物和载多柔比星(DOX)的RGD修饰长循环脂质体,采用激光共聚焦显微镜来观察siRNA和多柔比星的细胞摄取和细胞内分布情况;采用流式细胞术和SRB(磺酰罗丹明B)实验测定多柔比星的细胞累积量和多柔比星对人乳腺癌耐药细胞(MCF7/A)的毒性;采用活体成像技术考察了靶向和非靶向制剂输送siRNA到肿瘤模型裸鼠体内的组织分布情况。结果所制备的各种脂质体的平均粒径均在200 nm以内;与细胞孵育6 h后,观察发现siRNA和多柔比星各自主要分布在细胞质和细胞核中,与非靶向组相比,靶向脂质体组细胞摄取siRNA更快更多,且有利于siRNA在细胞质中均匀分布;细胞毒实验结果证实,1%摩尔比RGD修饰的阳离子脂质体对siRNA的转染效果最好,流式细胞术实验结果也同样证实RGD修饰脂质体组细胞中多柔比星累积量更高;活体成像结果显示荧光标记的siRNA经靶向脂质体输送后主要集中在肿瘤且比非靶向组蓄积量更高。结论采用RGD修饰脂质体载运技术可将MDR1 siRNA和多柔比星两种药物同位点输送到肿瘤部位提高靶向性,并有利于改善多药耐药肿瘤的治疗效果。
OBJECTIVE: To adopt the integrin receptor-mediated targeting vector delivery system to achieve RNA interference and antitumor drug-resistant tumor combination therapy. Methods The arginine - glycine - aspartic acid tripeptide (RGD) modified cationic liposomes containing small interfering RNA (siRNA) containing multidrug resistance gene (MDR1) were respectively prepared by electrostatic complex method and ammonium sulfate gradient method. The RGD-modified long-circulating liposomes loaded with doxorubicin (DOX), complex, and doxorubicin (DOX) were used to observe the cellular uptake and intracellular distribution of siRNA and doxorubicin by laser scanning confocal microscopy. Flow cytometry and SRB Sulfonyl rhodamine B) assay was used to determine the cellular accumulation of doxorubicin and the toxicity of doxorubicin on human breast cancer drug-resistant cells (MCF7 / A). The bioluminescence imaging of target and non-target agents Tissue distribution of siRNA into tumor model nude mice. Results The average diameters of all kinds of liposomes prepared were less than 200 nm. After incubation with the cells for 6 h, the siRNA and doxorubicin were mainly distributed in the cytoplasm and nucleus, respectively, compared with the non-target group , SiRNAs targeting liposomes could be taken up more quickly and more easily, and the siRNAs distributed evenly in the cytoplasm. Cytotoxicity results showed that 1% molar ratio RGD-modified cationic liposomes had the best siRNA transfection efficiency The results of flow cytometry also confirmed the higher amount of doxorubicin in the RGD-modified liposome group. The results of in vivo imaging showed that the fluorescent-labeled siRNAs were mainly localized in the tumor after targeted liposome delivery, The target group has a higher volume of accumulation. Conclusion RGD-modified liposome delivery technique can deliver MDR1 siRNA and doxorubicin to the tumor site at the same site to improve the targeting and help to improve the therapeutic effect of multidrug-resistant tumors.