基于体内定点自组装构建智能诊疗一体化探针用于改善光动力疗效及无创监测的实验研究

The EPR effect and drug-controlled releasing of nanodrugs could improve the poor targeting of small molecular drugs and maintain effective concentration. However, nanodrugs are facing the weak tissue penetration and hazardous accumulation in vivo, which tarnishes their clinical transformation. As suggested by literatures and our previous research, the in vivo self-assembly strategies could combine both advantages by transforming small molecules drugs into nanodrugs under specific pathological conditions. Thus, we propose a theranostic scheme based on self-assembly in mitochondria of tumor cells for improving photodynamic rfficacy and non-invasive monitoring. In this project, we design a multi-functional probe based on pyropheophorbide-a (Pyro) and phenylalanine peptide carriers, which could response to high-glutathione microenvironment in tumor cells and conduct the in vivo self-assembly in mitochondria. The formation of nanomicelles will improve photodynamic efficacy of Pyro on colon cancer by prolonging the effective time, increasing the effective concentration and optimizing the localization. Meanwhile, a Caspase 3 targeting molecule will be integrated to monitor and evaluate the enhanced efficacy through PET imaging. This project will construct a new molecular platform for the intelligent conversion of small molecule drugs into nanodrugs in situ, which is promoting the clinical transformation of nanomaterials hopefully. With the smart cooperation of therapy and imaging, visualized statistics will be provided for non-invasive, early accurate evaluation and guidance of photodynamic therapy.

纳米材料的EPR效应及缓控释有利于改善多数小分子药物靶向性差、难以维持有效浓度的缺陷，然而纳米材料体内蓄积及较差的组织穿透力极大阻碍其临床转化。文献及我们既往工作发现小分子在体内特定的环境下形成纳米材料的体内自组装策略可综合二者优势。因此，本项目提出一种基于肿瘤线粒体内自组装的诊疗一体化方案用于改善光动力治疗（PDT）并对其无创监测。拟以小分子PDT药物pyropheophorbide-a（Pyro）和苯丙氨酸多肽载体设计肿瘤细胞内高谷胱甘肽响应型探针，高选择性定位于肿瘤线粒体后自组装形成纳米胶束，通过延长作用时间、提高有效浓度及优化作用位点来改善Pyro对结肠癌的PDT疗效。同时整合靶向Caspase 3的PET成像监测并评价其疗效增强效应。本项目的成功将为小分子向纳米材料在体智能转变提供分子平台，同时集诊疗一体化，为临床实现无创、早期精确评价及指导PDT提供直观的影像学依据。

恶性肿瘤的发病率逐年递增，然而治疗效果并无显著改善。小分子药物在肿瘤治疗领域占有举足轻重的地位，具有组织穿透力强、体内代谢快等优势，但多数小分子药物靶向性及特异性较差，在肿瘤病灶的停留时间即疗效发挥时间也相对较短。纳米材料的EPR效应及缓控释有利于改善多数小分子药物靶向性差、难以维持有效浓度的缺陷，然而纳米材料体内蓄积及较差的组织穿透力极大阻碍其临床转化。小分子在体内特定的环境下形成纳米材料的体内自组装策略可综合二者优势。.本项目构建了新型体内自组装体系，以苯丙氨酸多肽PEP为核心，在关键位点以二硫键形式引入功能性基团吲哚菁绿（Indocyanine Green, ICG）和靶向基团（靶向整合素αvβ3的环形多肽, c[RGD]fk），构建智能自组装探针ICG-PEP-c(RGD)fk（IPR）。该分子探针可响应肿瘤高GSH环境启动体内自组装行程纳米胶束，延长探针局部滞留时间，同时进行近红外光学成像及光热治疗（PTT），达到靶向和显像治疗的双重目的。.本项目通过液相色谱及质谱鉴定IPR的成功合成。结果证明IPR能够在GSH条件下有效组装，在9.446 μ M时即可自组装成纳米胶束。体外实验证明IPR能够与人胶质母细胞瘤细胞系U87MG-luc特异性结合，并具有较好的PTT疗效。体内近红外显像证明IPR探针不仅具有更清晰的肿瘤图像，并且在肿瘤中滞留时间比对照组延长了2.2倍。在U87MG-luc荷瘤鼠光热治疗实验中，IPR能够显著抑制肿瘤生长，肿瘤体积比对照组小2.6倍，中位生存时间延长2.8倍。同时本项目成功合成68Ga-PEP2，可在体内外靶向凋亡蛋白酶Caspase 3，通过PET成像监测肿瘤治疗反应。.综上，本项目从体内自组装策略构建及靶向诊疗两个方面出发，最终实现肿瘤细胞治疗策略的提高，为小分子向纳米材料在体智能转变提供分子平台。