EGFR单链抗体介导紫草素纳米颗粒逆转肺癌EGFR-TKIs耐药作用及其机制的研究

Non-small cell lung cancer (NSCLC) patients with EGFR mutations is sensitive to EGFR TKIs (kinase inhibitor), but the primary or secondary gene mutations can lead to drug resistance. Single-target, multi-targets, or combination strategies with chemotherapeutic drugs often failed, due to compensatory activation mechanisms leading to relapse and progression. Shikonin has a strong and mutiple anti-cancer effect but has significant side-effects. With high efficiency and low side-effects, antibody-mediated shikonin nanomedicine may be an effective method to solve this secondary drug resistance and reduce toxic side-effects. Earlier, we constructed a single-chain antibody (scFv) targeting EGFR, successfully delivered siRNA and Fe3O4 nanoparticles into EGFR TKIs resistant NSCLC cells, which reversed drug resistance and served as a tumor tracer. In this study, we suppose to deliver shikonin into TKI-resistant NSCLC cells by constructing scFv-shikonin nanoparticles composites to reverse drug resistance, reduce overall toxicity and trace the tumor cells both in vitro and in vivo on the base of specific recognition and internalization of scFv, and the anti-tumor effect of shikonin. Moreover, we suppose to explore the anti-cancer mechanism of shikonin to provide a new strategy to overcome EGFR TKIs resistance in NSCLC.

表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKIs）对EGFR敏感突变肺癌患者治疗有效，继发耐药导致疾病进展。单靶点、多靶点或与化疗药联合的策略，多因代偿性激活机制导致病情复发和进展。紫草素存在多重抗肺癌效应、作用强大，但副作用明显。抗体介导的紫草素纳米药物可能是解决靶向药物继发耐药、减轻紫草素毒副作用的有效方法。我们前期制备的EGFR单链抗体（EGFR scFv）携带siRNA与EGFR-TKIs联合，逆转肺癌TKI耐药，并成功将纳米颗粒靶向性地导入裸鼠移植瘤细胞。本项目在前期研究基础上，拟制备EGFR-scFv-紫草素纳米颗粒复合物，借助scFv特异识别和内化功能，将紫草素靶向输送至动物肺癌移殖瘤细胞内，以增加紫草素抗癌的靶向性、减轻毒副反应，探讨紫草素抗肺癌机制、抗体介导紫草素纳米抗癌效果及其逆转TKIs耐药机理，为克服肺癌EGFR-TKI耐药探索新机制、新方法。

紫草素存在多重抗肺癌效应、作用强大，但副作用明显。抗体介导的紫草素纳米药物可能是解决靶向药物继发耐药、减轻紫草素毒副作用的有效方法。本研究目的：制备EGFR-scFv-紫草素纳米颗粒复合物，借助scFv特异识别和内化功能，将紫草素靶向输送至动物肺癌移殖瘤细胞内，增加紫草素抗癌的靶向性、减轻毒副反应，实现动物体内高效低毒的药物治疗，逆转TKI耐药。内容：紫草素在EGFR-TKIs耐药NSCLC肿瘤细胞中的抗癌机制；抗EGFR单链抗体紫草素纳米颗粒靶向输送体系的建立；scFv-SHK-SIONPs体外药物靶向输送及其抗肿瘤效应测定；scFv-SHK-SIONPs体内靶向药物输送及抗肿瘤效应。结果：紫草素和吉非替尼联合用药，导致耐药细胞H1975增殖活性下降、克隆形成能力下降、细胞凋亡水平增加，分子水平上耐药细胞株EGFR及下游ERK磷酸化水平下调；成功制备scFv-SHK-SIONPs纳米颗粒复合物，载有脂溶性药物的纳米颗粒在PBS中呈水溶状态，径粒大小控制好，分散性佳，符合实验要求；scFv-SHK-SIONP能够被EGFR阳性PC9、SPC-A1肺癌细胞摄取，并能够抑制耐药细胞株的增殖，促进其凋亡；scFv-SHK-SIONP经尾静脉注射至裸鼠移植瘤体内，T2MRI核磁成像显示移植瘤内有低密度影聚集，活体成像显示瘤内有荧光聚集。结论：成功构建了EGFR单链抗体偶联的紫草素纳米颗粒，建立了动物体内单链抗体介导的特异性EGFR分子靶向递药系统，EGFR单链抗体携带紫草素逆转肺癌细胞EGFR-TKI耐药，实现动物体内高效低毒药物治疗目的，为克服EGFR-TKI耐药提供一条新思路。

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