多功能智能化MR纳米探针的构建及肝癌诊疗一体化实验研究

The emergence of “Nanotheranostics” will be expected to solve the problem of early detction, inefficiency of chemotherapy and delay of therapeutic effect evaluation for liver carcinoma. Traditional nanoparticles probe for targeted molecular imaging always had only single treatment and low targeted intake of tumor due to the unstable expression of receptors on the surface of tumor cells. In previous studies, we had developed a new molecular probe based on hyaluronic acid (HA) loaded with a negative magnetic resonance imaging (MRI) contrast agent (superparamagnetic iron oxide, SPIO) and an antitumor drug (doxorubicin, DOX) for integrated diagnosis and treatment targeting against HCC,which demonstrated with promising effect in animal mode. Herein, We try to design and synthesize a CD44 and αvβ3 double-targeted MR nano-theranostic agent, integrated with an acid- and reductional- sensitive "double drug loaded" theranostic system. In this multifunctional MR nano-theranostic agent, DOX and VDAs are conjugated covalently on HA through a acid sensitive hydrazone bond and disulfide bond, then tumor-targeted group c(RGD) is connected on the surface of HA, and SPIO is also loaded inside the HA for targeted imaging.Physicochemical properties of this integrated MR nano-theranostic agent will be measured. In vitro and in vivo tests will be performed to systematically evaluate the effectiveness of targeted MR imaging and treatment for early detection and therapy for liver carcinoma based on MR nanotheranostic agent. If successful, it will be possible for one-stop early detection, targeting personalized treatment and dynamic monitoring of the efficacy of liver carcinoma and it will play a vital role in the assurance of the treatment integrity and recurrence prevention of liver carcinoma.

“纳米治疗诊断学”的出现有望解决肝癌诊治中的早期诊断困难、化疗效果欠佳及疗效预测滞后等问题。传统的纳米诊疗一体化分子探针治疗方式单一，同时由于肿瘤细胞表面受体表达不稳定而造成肿瘤靶向摄取量低。本研究设计一种基于CD44和整合素ανβ3受体的“双靶向”、“双载药”多功能智能化MR纳米探针，该探针以透明质酸（HA）为结构主体，内部装载超顺磁性氧化铁（SPIO）纳米颗粒，以具有pH敏感性腙键及生物还原性二硫键分别连接肿瘤治疗药物阿霉素（DOX）与血管靶向剂（VDAs）,表面连接c(RGD)靶向基团，实现靶向MR成像、细胞毒性药物与血管靶向药物协同作用的目的。我们将对该多功能智能化MR纳米探针行条件优化、理化性质检测，设计体内、外实验考察在肝癌诊疗一体化中的应用。本研究一旦成功，有望实现肝癌的一站式早期检出、靶向个性化给药及动态监测疗效，对确保肝癌患者治疗完整性和预防术后复发有着至关重要的作用。

尽管基于MR 的多功能肿瘤纳米诊治体系在原发性肝癌的诊治中具有相当大的发展前景，但高效靶向输送、有效治疗的联合及其如何协同等问题尚未解决。本项目旨在研究研究多种集医学影像诊断和治疗于一体的多功能智能化MR纳米分子影像探针，了解不同靶向分子、结构的纳米探针的性能，在细胞、动物水平评价其靶向输送药物及肿瘤微环境响应智能药物释放的功效，证明其作为药物、MR成像对比剂载体的能力，同时评价其功效，为肝癌的早期准确诊断，实现靶向个性化给药和实时疗效监控提供实验和理论依据。主要成果包括：（1）以透明质酸为亲水高分子及利用其靶向CD44受体的特性，并通过简单的化学偶联反应成功偶联抗癌药物阿霉素及MR成像对比剂SPIO，制备得到具有肿瘤主动靶向及肿瘤酸性微环境控制阿霉素智能释放的诊疗一体化纳米探针DOX-HA-SPIO，实现了对肝癌的靶向诊断与高效治疗；（2）制备了以透明质酸为亲水链段及靶向分子、以二硫键连接的聚己内酯为内核的核-壳结构高分子纳米载体，初步实现了纳米粒子所包载的药物在肿瘤还原性微环境的选择性药物释放，并提出利用肿瘤细胞高表达CD44受体及还原性微环境，构建能特异性靶向肿瘤CD44受体及GSH响应型的高分子纳米作为抗肿瘤药物及SPIO载体，实现化疗药物及成像对比剂的选择性输送；（3）利用哺乳动物肝细胞表面存在的半乳糖(乳糖)受体只能与半乳糖(乳糖)存在特异性的识别和结合这一特性，通过开环聚合及点击化学反应首次制备得到由二硫键连接亲水糖聚肽及疏水聚己内酯链段的两亲性嵌段聚合物。通过该嵌段聚合物的自组装包裹作用成功包载抗癌药物及MR成像对比剂，实现化疗药物及成像对比剂的选择性输送。（4）充分发挥透明质酸的分散稳定性及肿瘤靶向特性，以此发展了基于聚集诱导发光及磁共振对比增强特性相结合的荧光/MR双模态成像诊疗一体化纳米探针SPIO/AIE@HA-g-PZLL，在细胞及动物水平实现了对肿瘤的有效诊断与光动力治疗；在RSC Advances、International Journal of Nanomedicine、Materials Science & Engineering C、International Journal of Biological Macromolecules等期刊上表SCI论文6篇，在中文核心期刊上发表论文3篇，顺利完成了预期目标。