Cetuximab介导、共载siRNA-Ras和顺铂的纳米药物制备及其对单抗耐药性鼻咽癌的治疗研究

Cetuximab as the representative of the EGFR molecular target therapy is in the ascendant in the treatment of nasopharyngeal carcinoma, but the clinical effect is not ideal, Cetuximab-resistance has been the key factor in clinical treatment. Our previous study found that : the abnormal expression of Ras gene could activate compensatively EGFR downstream signaling pathways which blocked by Cetuximab, Ras would be the main cause leading to Cetuximab-resistance. Inhibition of the Ras expression is expected to reverse drug resistance and beneficial in the treatment of resistant nasopharyngeal carcinoma. Therefore, based on our early research of construction of EGFR targeted gene nano-carrier, we intends to construct Cetuximab mediated nanomedicine (CRas-DDP-LPD) which co-deliverys siRNA-Ras and cisplatin (DDP) in this study. The target property, drug distribution, activity and mechanism of nanomedicine in vitro and in vivo reversal of Cetuximab-resistant nasopharyngeal carcinoma would be studied in this subject. It would be proved that the nanomedicine could delivery siRNA-Ras and cisplatin into targeted nasopharyngeal carcinoma cell. Thus, siRNA-Ras inhibits Ras gene, enhance sensitivity of resistant cells to Cetuximab, and cisplatin kill tumor cells collaboratively. Research is not only beneficial to improve the clinical therapeutic effect of Cetuximab-resistant nasopharyngeal carcinoma, will also provide new ideas and ways for the treatment of other antibody-resistance tumors.

以西妥昔单抗（Cetuximab）为代表的EGFR分子靶向治疗鼻咽癌等恶性肿瘤的研究方兴未艾，但临床疗效不甚理想，单抗耐药是影响其效果的关键。我们前期研究发现：Ras基因异常表达可补偿激活被Cetuximab阻断的EGFR下游信号通路，是引发Cetuximab耐药的主要原因。抑制Ras表达有望逆转单抗耐药，利于对Cetuximab耐药性鼻咽癌的治疗。为此，我们在前期构建EGFR靶向基因纳米载体研究的基础上，制备Cetuximab介导\共载siRNA-Ras和化药顺铂（DDP）的纳米药物CRas-DDP-LPD，研究其体内外靶向性、抗耐药肿瘤活性、药物分布及作用机制。证实纳米药物可靶向进入耐药鼻咽癌细胞中，siRNA抑制异常Ras基因，增强耐药细胞的Cetuximab敏感性，化药协同杀伤肿瘤细胞。本研究不仅为提高单抗耐药性鼻咽癌的治疗效果，也将为其他抗体耐药肿瘤的治疗提供新思路、新方法。

鼻咽癌EGFR受体阳性表达率在80-100％，Cetuximab无疑是解决其局部复发和远处转移的重要手段，然而， 临床中Cetuximab药物可产生继发性耐药现象往往导致鼻咽癌的治疗失败。采用siRNA信号抑制耐药信号Ras基因，有望提高Cetuximab药物的鼻咽癌治疗效果。本项目制备一种同时包裹针对Ras基因的siRNA和治疗药物DDP的阳离子脂质载体系统，经Cetuximab修饰，构建一种靶向鼻咽癌EGFR受体的递药系统CRas-DDP-LPD，以实现对耐药鼻咽癌的治疗。本项目利用化学共价反应将Cetuximab与脂质体进行连接修饰。电镜观察证实纳米药物系统存在纳米脂质体结构，载药纳米粒的平均粒径在120 nm左右，Zeta电位约为+30 mv，脂质体形态圆整，粒径均匀；siRNA包封率达90%，脂质体中药物呈现缓释效果，单抗修饰对药物释放无显著影响。经单抗耐药鼻咽癌细胞摄取、动物活体成像及裸鼠组织分布实验结果均显示，Cetuximab修饰可显著增加鼻咽肿瘤的摄取。通过RT-PCR机制研究表明， 单抗耐药鼻咽癌CNE1/Cetuxi、HNE-2/Cetuxi细胞株中EGFR/P-EGFR以及EGFR下游Ras/MAPK(ERK)与PI3-K/AKT信号通路中相关H-Ras基因是单抗耐药的关键，而CRas-DDP-LPD可以有效干扰H-Ras基因，达到逆转单抗耐药的目的。另外，通过药动学研究证实Cetuximab修饰对纳米脂质体可提高药物的AUC显著增加，t1/2显著延长（P<0.05)，这将促进纳米粒进入肿瘤部位。采用体外耐药细胞杀伤实验，荷瘤裸鼠模型治疗实验证明，复合靶向载体药物组合制成CRas-DDP-LPD，可提高抗体药物、化疗药物、逆转基因药物对靶部位的分布，抑制单抗耐药鼻咽癌的生长，具有良好的动物模型治疗效果。综上所述，本项目所构建的Cetuximab修饰、siRNA-Ras与药物顺铂包载于阳离子脂质体CRas-DDP-LPD可有效靶向EGFR肿瘤组织，对单抗耐药鼻咽癌具有良好治疗效果。该研究结果实现了本项目的设计目标。