双靶向载PTX-XBP1-siRNA金纳米光热治疗联合UTMD可视化增敏三阴性乳腺癌化疗的实验研究

It has been reported that the crucial reason of chemotherapy failure for triple negative breast cancer (TNBC) is resistance to chemoradiotherapy and absence of effective targeted treatment. In previous studies, the applicant research group had developed an novel and safe drug delivery system by the combination of ultrasound targeted microbubbles destruction (UTMD), RNA interference and Nanotechnology and reversed the drug resistance of HER2-positive breast cancer, which provides a promising tool for the targeted therapy of TNBC. However, specific treatment target, precise chemosensitization and imaging evaluation are needed for TNBC. Hereby, the applicant research group had prepared gold-nano-microcapsules, and the pilot experiments showed that photothermic therapy inhibited effectively TNBC cell; nanoscale ultrasound contrast agent had been prepared and used to observe dynamic changes of carriers. On this basis, our study will aim at X-box binding protein 1 (XBP1) that is the specific treatment target of TNBC, prepare gold-nano-microcapsules carrying anti-EGFR and RGD and encapsulate Paclitaxel (PTX) and XBP1-siRNA. Through UTMD, the composite carrier can go through blood vessel and target TNBC cells, and the gold-nano-microcapsules-mediated photothermic therapy, XBP1-siRNA and PTX combine to inhibit TNBC cells, the precise chemosensitization is realized as a result and ultrasound contrast imaging is conducted to monitor and assess the treatment. The purpose of this study is to establish a new mode of adjuvant therapy in TNBC, which integrates not only effective and safe cell targeted chemotherapy but also a new real time target imaging method.

化疗抵抗且缺乏有效靶向治疗是长期以来三阴性乳腺癌（TNBC）化疗失败的关键原因。申请人前期利用超声靶向破坏微泡（UTMD）、RNA干扰及纳米技术建立了一种新型靶向安全的载体递送平台，逆转了HER2阳性乳腺癌对阿霉素的耐药性，为TNBC的靶向治疗提供了可能手段。针对TNBC，仍需找到特异治疗靶点、实现精确定位的化疗增敏及进行影像化评估。据此，申请人预实验制备出金纳米微囊，其光热效应有效抑制了TNBC细胞生长；制备出纳米超声造影剂，超声成像可观察载体动态行为；本课题将继以TNBC特异性的X盒结合蛋白1（XBP1）为靶基因，制备内部载XBP1-siRNA及紫杉醇表面修饰EGFR及整合素αvβ3双重抗体的金纳米微囊，UTMD促进微囊更多进入肿瘤，双重抗体识别瘤细胞，纳米金联合微囊内基因及药物在肿瘤部位行强有力的抑制，实现精确定位的化疗增敏及进行超声成像，以期建立TNBC靶向辅助治疗的新模式。

三阴性乳腺癌（triple negative breast cancer, TNBC）的治疗是世界性的难题，化疗抵抗且缺乏有效靶向治疗是其化疗失败的关键原因。课题组前期利用超声靶向破坏微泡（ultrasound targeted microbubbles destruction, UTMD）、RNA干扰及纳米技术建立了一种新型靶向安全的载体递送平台，逆转了HER2阳性乳腺癌对阿霉素的耐药性，为TNBC的靶向治疗提供了可能手段。针对TNBC，仍需找到特异治疗靶点、实现精确定位的化疗增敏及进行影像化评估。据此，我们进行了一系列的深入研究。.首先，制备了双靶向载PTX载siRNA微囊EGFRGDMBs-PTX- XBP1-siRNA（A-MBs-PTX-XS），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、低毒。其次，筛选出最优的UTMD超声条件：声强1.5W/cm2，频率1MHz，连续超声作用时间60s。然后，体外细胞实验证实UTMD联合A-MBs-PTX-XS可促进更多的PTX和XBP1-siRNA进入细胞，XBP1-siRNA可阻断XBP1蛋白的合成，增加其对PTX的敏感度，达到更强的细胞杀伤效果，为A-MBs-PTX-XS的体内应用奠定了基础。最后，建立了TNBC乳腺癌移植瘤模型，考察UTMD联合A-MBs-PTX-XS增敏化疗的体内抗肿瘤效果。结果同样证实， UTMD能够增强A-MBs-PTX-XS的药物和基因释放，使其在肿瘤组织中蓄积，强有力地抑制肿瘤生长。此外，本研究考察了微囊的体内毒性，结果表明：A-MBs-PTX-XS对心、肝、脾、肺和肾等主要器官均无明显的毒副作用。同时，研究发现A-MBs-PTX-XS微囊在体内外均可获得满意的超声造影图像。.根据上述实验结果，A-MBs-PTX-XS联合UTMD技术大大增强了PTX和XBP1-siRNA体内外抗TNBC肿瘤的能力，明显减少了药物的毒副作用。同时它还是一种较好的超声对比增强剂，有助于TNBC肿瘤的诊断，从而实现三阴性乳腺癌的诊疗一体化。项目研究成果已在RSC Advances, Int J Mol Sci , Clin Hemorheol Microcirc等期刊发表。