HER2靶向新型纳米载体荷载BCRP-siRNA经UTMD逆转乳腺癌耐药性的实验研究

Drug resistance of breast cancer is one of the most causes of failure in clinical chemotherapy. Currently there is no effiective means to reverse it. Recent researches show the expression of breast cancer resistance protein (BCRP) gene is directly related to breast cancer cell's drug resistance. RNA interference (RNAi) is suggested to be used to block BCRP gene expression, which could help to reverse breast cancer's drug resistance and improve prognosis. what kind of method of transfering target gene is a determining factor to the result of treatment. More or less, most of the methods of transfering target gene have shortcomings of low efficiency, having side effects and short period of expression. Our previous study have found that ultrasound targeted microbubbles destruction (UTMD) has advantages of targeted transfection, security and high efficiency. Meanwhile, we have successfully set up a new nano delivery system that carries siRNA. Preliminary experiment has proven UTMD can facilitate the uptake of loaded siRNA nanoparticles (NPs) by resistant cell of breast cancer. On this basis, our study will aim at HER2-positive breast cancer, prepare mPEG-PLGA-PLL NPs carrying anti-HER2 and encapsulate BCRP-siRNA. Through UTMD, the composite carrier can go through blood vessel and target HER2-positive breast cancer cells, and BCRP is silenced as a result. This new technology features multiple targeting is expected to reverse breast cancer cell's drug resistance in a safe, highly efficient, stable and long term manner.

乳腺癌细胞耐药性的产生是化疗失败的主要原因，目前尚缺乏有效手段逆转。最近的研究发现，乳腺癌耐药蛋白(BCRP)的表达与乳腺癌细胞耐药性直接相关，提示采用RNAi阻断BCRP的表达有望逆转乳腺癌耐药细胞的耐药性、改善预后。使用何种手段导入目的基因直接决定治疗效果。目前绝大多数导入方法不同程度存在低效、副作用大、表达时间短的缺陷。我们的前期研究发现，超声靶向破坏微泡(UTMD)具有靶向辐照定向转染、安全高效的优势，并成功构建了载siRNA的新型纳米递送系统，预实验已证实UTMD能促进乳腺癌耐药细胞对载siRNA纳米粒的摄取。本项目将在此基础上，针对HER2阳性乳腺癌，制备携抗HER2的mPEG-PLGA-PLL，包裹BCRP-siRNA，通过UTMD作用使复合载体穿过血管壁，靶向HER2阳性乳腺癌细胞，沉默BCRP。这种"多重靶向"新技术有望实现安全、高效、长期稳定逆转乳腺癌耐药性的目的。

乳腺癌细胞耐药性的产生是化疗失败的主要原因，乳腺癌耐药蛋白（BCRP，又称ABCG2）基因的表达可直接导致细胞耐药，阻断其表达可逆转耐药细胞的耐药性、改善预后。超声靶向破坏微泡技术 (UTMD)、RNAi及纳米技术均是目前的研究热点。我们的前期研究发现，UTMD能够安全有效地促进细胞对载siRNA纳米粒的摄取。在此基础上，我们进行了一系列的深入研究。. 首先，制备了siRNA/PEAL复合纳米粒，并对其理化性质进行了表征。细胞毒性实验证明其生物相容性好，安全、无毒。其次，筛选出最优的UTMD超声条件：微泡比例20%；超声功率1.0 W；超声时间40s ；占空比20%。然后，体外细胞实验证实UTMD协同PEAL复合纳米粒可促进siRNA入胞并降解ABCG2 mRNA，阻断ABCG2蛋白的合成，从而减少细胞对ADR的外排作用，增加其对ADR的敏感度，较低浓度的药物即可达到相对较强的细胞杀伤效果，为ABCG2-siRNA/PEAL复合纳米粒的体内应用奠定了基础。最后，建立了乳腺癌移植瘤模型，考察UTMD协同siRNA/PEAL复合纳米粒增敏ADR的体内抗肿瘤效果。结果同样证实，PEAL NPs携带ABCG2-siRNA进入裸鼠体内后，通过EPR效应被动靶向到肿瘤组织，然后在UTMD的协助下进入癌细胞，下调ABCG2基因的表达，逆转小鼠对ADR的耐药性，从而提高机体对ADR的敏感性，在相同的ADR浓度下，可以达到更好的肿瘤抑制效果。此外，本研究还初步考察了该递送系统的体内毒性，结果表明：siRNA/PEAL复合纳米粒对心、肝、脾、肺和肾等主要器官均无明显的毒副作用。. 根据上述实验结果，PEAL NPs协同UTMD是一种良好的siRNA递送系统，有望为耐药性乳腺癌的治疗提供一种全新的治疗策略。项目研究成果已在Oncotarget，International Journal of Nanomedicine等期刊发表。