整合素受体介导的SiRNA和多柔比星靶向耐药肿瘤的研究

 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。

Integrin Receptor-Mediated Strategy for Tumor Targeting Delivery of siRNA and Doxorubicin

OBJECTIVE To investigate the possibility of using integrin receptor-mediated tumor targeted delivery for siRNA and antitumor drugs to overcome tumor multi-drug resistance. METHODS Small interference RNA(siRNA) lipoplex and doxorubicin(DOX) liposomes modified with arginine-glycine-aspartic acid (RGD) peptide were prepared by electrostatic complexing and ammonium sulfate gradient method, respectively. Cellular uptake and distribution of siRNA and DOX were observed by confocal laser microscope. Then the cellular accumulation and cytotoxicity of DOX against drug resistant human breast cancer cells (MCF7/A) were measured by flow cytometry and SRB (sulforhodamine B) assay, respectively. Finally tissue distribution of siRNA in RGD-modified or unmodified liposomes were investigated by in vivo imaging with drug resistant tumor model. RESULTS The liposomes were all of narrow size distribution below 200 nm. After being incubated with cells for 6 h, siRNA and DOX mainly located in cytoplasm and nucleus, respectively. RGD-modified siRNA lipopex promoted faster cellular uptake and even-distribution in cytoplasm. The results of cytotoxicity showed 1% molar RGD-modified liposomes had the highest transfection efficiency of siRNA, meanwhile RGD-modified liposomes enhanced cellular accumulation of DOX. Finally the in vivo image exhibited stronger signal in tumor than other tissues, and especially RGD-modified siRNA lipoplexes had stronger signal in tumor than unmodified lipoplexes. CONCLUSION The RGD-modified stealth liposomes could be used as the carriers to facilitate the targeted delivery of MDR-1 siRNA and DOX to the same tumor site and further improve the therapeutic efficiency of multi-drug resistant tumors.