双探针共载肿瘤渗透性纳米荧光检测系统的构建及其在乳腺癌PDT精准治疗中的应用

Reactive oxygen species (ROS) produced by tumor photodynamic therapy (PDT) may up-regulate the expression of vascular endothelial growth factor (VEGF) in hypoxic environment, leading to angiogenesis and metastasis, which seriously affects the therapeutic effect of PDT. In this study, we prepared two fluorescent probes to detect hydrogen peroxide and VEGF through aggregation-induced emission effect and fluorescence quenching effect, respectively. And they were loaded in a mesoporous silica nanoparticles/graphene oxide (MSN@GO) composite carrier. The tumor-penetrating nano fluorescence detection system with the abilities of tumor penetrating and targeting, intracellular precise release and simultaneous detection of hydrogen peroxide and VEGF was established through the connection between MSN@GO and iRGD-poly(2-ethyl-2-oxazoline)-hyaluronic acid (iRGD-PEOz-HA, iPPH), a tumor-penetrating and precise targeting group. This system provided feedback for PDT treatment through real-time detection of changes in the level of hydrogen peroxide/VEGF in hypoxic parts of the tumor. We first synthesized and tested the properties of the two probes, and loaded them into MSN@GO through different ways. Then, the surface of the carrier was modified by iPPH. Through the tumor-penetrating study, in vitro cell experiments and in vivo experiments, we investigated the system's ability to penetrate into hypoxic tissues of breast cancer and attempted to judge the extent of PDT treatment by real-time changes in hydrogen peroxide and VEGF levels. We intend to provide references for the optimization of PDT treatment of breast cancer through controlling the development trend of tumors during PDT.

肿瘤光动力治疗（PDT）产生的活性氧可在缺氧环境中上调血管内皮生长因子（VEGF）表达，导致血管新生和肿瘤转移，从而严重影响治疗效果。本项目采用介孔二氧化硅纳米粒/氧化石墨烯复合载体（MSN@GO）共载分别通过聚集诱导发光和GO荧光猝灭效应检测过氧化氢及VEGF的探针，同时连接兼具肿瘤穿透及靶向能力的iRGD-PEOz-HA（iPPH）基团构建具肿瘤渗透、胞内精确释放及双因子同时检测的纳米荧光检测系统，通过对肿瘤缺氧部位过氧化氢/VEGF水平变化实时监测为PDT治疗提供反馈。首先合成探针并测试性能，再经不同方式装载于MSN@GO中，并用iPPH修饰载体表面。经肿瘤渗透性能测试、体外细胞及体内实验考察系统对乳腺癌缺氧组织渗透能力并尝试通过过氧化氢和VEGF水平变化对PDT治疗程度做出判断，从而实现对乳腺癌PDT治疗过程中肿瘤发展趋势的控制，并为其治疗方案优化提供参考。

光动力疗法通过特定波长光源照射靶体内光敏剂，并在氧气参与下发生光化学反应，产生治疗性ROS导致肿瘤细胞死亡和组织损伤。然而，PDT的耗氧特性会加剧肿瘤局部缺氧，不仅严重影响治疗效果，而且可能诱发血管新生及肿瘤转移。因此，迫切需要监控PDT治疗过程中的肿瘤发展趋势，从而为PDT的个体化精准治疗方案的制定提供参考。本项目创新性地设计了一种基于介孔二氧化硅（MSN）/聚多巴胺纳（PDA）米复合载体MSN@PDA，分别经不同途径装载通过聚集诱导发光（AIE）和PDA荧光猝灭效应检测过氧化氢（H2O2）及血管内皮生长因子（VEGF）的探针，通过对乳腺癌细胞中H2O2/VEGF水平变化实时监测为其PDT治疗提供反馈。主要结果包括：1）构建了一种“turn-on”型荧光探针TPE-2HPro，凭借其AIE效应实现对细胞内H2O2的高效检测；2）采用荧光素标记的VEGF适配体（FAM-AptamerVEGF）作为VEGF检测探针，并利用聚多巴胺的强吸附性及荧光猝灭效应构建PDA-Aptamer检测系统用于对细胞内VEGF的精确检测；3）制备MSN@PDA复合纳米载体，并通过自由扩散和静电吸附分别高效封装TPE-2HPro和FAM-AptamerVEGF构建最终纳米系统MSNTH@PDAApt，并实现两者的pH响应性释放；4）进行MSNTH@PDAApt的细胞水平研究，分别考察其在二氯乙酸（DCA）刺激及二氢卟吩（Ce6）的PDT治疗过程中，H2O2及VEGF水平变化与细胞存活率的关系，结果表明，在不同氧含量的条件下，相同浓度DCA可导致细胞存活率发生显著差异；此外，随着PDT治疗过程中Ce6浓度的增加，细胞内存活率也出随H2O2/VEGF的波动呈现“先增加，后下降”的趋势。本项目的实施为肿瘤PDT治疗过程监测和疗效评价提供了一种新思路。项目执行期间发表SCI论文3篇，申请国家发明专利1项。