多功能智能化壳聚糖超声纳米泡介导siRNA-p23/MDV3100治疗耐药CRPC的作用及机制研究

As a safe, high-efficiency and controllable drug delivery system, ultrasonic nanobubbles have advanced the accurate and visual treatment for tumor. We have previously observed that the ultrasonic nanobubbles prepared with chitosan not only showed a good ultrasound imaging ability, but also promoted the uptake rate of doxorubicin; the nanobubbles with two fold content of folate showed better cellular targeted ability to tumor cells than their controls; and the optimum sonoporation caused by UTMD technology could obtained through orthogonal array design, and further improve the effect of gene or drug. Based on our previous achievement, and considering the good biocompatibility and biodegradability of chitosan, as well as the influence of in vivo circulation and tumor microenvironment on nanobubbles, we will design and construct a novel multifuntional nanobubbles with the characteristic of high targeting, pH-dependent charge-reversal capability, controlled release and ultrasonic imaging. The nanobubbles also posess the abilities of loading anti-p23 gene (siRNA-p23) and chemotherapeutics (MDV3100). Folate mediates the active targeting of this nanobubble. The purposes of this project is to reduce the side effect of chemotherapeutics and reverse the drug-resistance of CRPC with the help of our developed nanobubbles. The accomplishment of this project will provides a new strategy for targeting drug delivery/gene delivery, and also provides a new way for the theranostic of malignant tumors such as CRPC.

超声纳米泡可实现药物/基因的安全、高效、可控靶向传递，可极大地促进肿瘤的精准化及可视化治疗。课题组前期工作发现，壳聚糖纳米泡显影能力优良，并能够有效负载阿霉素，促进肿瘤细胞的药物摄取率；双倍叶酸修饰纳米泡具有更强的肿瘤靶向结合能力；优化超声参数组合可获得肿瘤细胞的最佳声孔效应，从而进一步实现基因或药物的最有效递送。本课题基于前期研究基础，结合壳聚糖材料良好的生物相容性与可修饰性，以及体内循环及肿瘤微环境对纳米泡的影响，拟进一步制备具有高靶向性、表面电荷可逆转、可控释，兼具超声监测的多重功能壳聚糖纳米泡，同时负载siRNA-p23与化疗药MDV3100，以耐药CRPC肿瘤为模型，通过双倍叶酸介导的主动靶向运输及肿瘤组织的EPR效应，联合UTMD技术，以达到联合靶向诊疗耐药CRPC的目的。本项目的成功不仅为靶向超声纳米泡的药物/基因递送提供新的策略，而且为CRPC等恶性肿瘤的治疗提供新的思路。

多功能生物相容性纳米泡的研制是实现超声影像引导的肿瘤可视化靶向诊疗的关键，本课题利用壳聚糖衍生物O-羧甲基壳聚糖材料自身的两性特征，以及全氟己烷的液气相变特征，采用高速均质法制备超声纳米泡，并对其在可视化药物/基因递送及恶性肿瘤（包括前列腺CRPC肿瘤）诊疗应用方面进行了研究。我们的结果：（1）通过单因素分析法获得纳米泡的最优制备条件组合，优化后纳米泡的平均粒径为184.7nm（PDI：0.219），zeta电位为-7.01mv，无明显溶血效应，同时具备体外显影与pH响应能力；（2）有效携载并递送了化疗药物（阿霉素、多西紫杉醇、羟氯喹）、过表达基因（DKK-2）以及小干扰siRNA（siCAT-1）；（3）评价了纳米泡联合超声辐照对前列腺肿瘤细胞的抑制作用，发现超声靶向微泡破裂（UTMD）显著提高肿瘤细胞活性氧（ROS）水平，并通过ROS/miR-200c/ZEB1信号通路抑制肿瘤细胞的上皮间质转换（EMT）；（4）体内动物实验进一步分析了纳米泡的生物相容性、超声对比增强显影、药物/基因靶向递送能力，并验证了纳米泡联合UTMD可显著抑制前列腺移植肿瘤的生长。综合上述结果，本研究制备获得了pH响应、液气相变、携载药物/基因的多功能靶向超声纳米泡，实现了药物/基因的可视化精准递送并显著抑制了前列腺肿瘤的生长，为探讨基于多功能超声纳米泡的肿瘤可视化靶向诊疗奠定了坚实的基础。