双级靶向的原位生物发光激发纳米光敏剂的构建及其用于深层肿瘤的光动力治疗研究

Photodynamic therapy (PDT) is considered as a promising minimally invasive treatment for various premalignant and neoplastic diseases. Owing to the shallow light penetration in tissues and the drawbacks of the organic PDT agents, including poor water dispersibility, low photostability, as well as low singlet oxygen quantum yield, the clinical applications of light-activated PDT have been limited. In order to obtain a new type of photosensitizer with high tumor specificity and therapeutic efficiency, in this study, a tumor microenvironment and mitochondria dual-targeted nanophotosensitizer with in-situ bioluminescence excitation will be designed and constructed, which can perform efficient bioluminescence energy-induced PDT of macroscopic tumors in deep tissues. The mutant Renilla luciferases (Rluc) will be conjugated on the surface of a novel photosensitizer based on graphene quantum dots (GQDs). When the conjugates are exposed to the coelenterazine (CTZ), the substrate of Rluc, the produced light is transferred to GQDs via bioluminescence resonance energy transfer (BRET), thereby efficiently generating singlet oxygen from GQDs. The mitochondria-targeted ligand triphenylphosphine (TPP) will be modified to the Rluc-GQDs conjugate, which empowers the nanophotosensitizer with capability to specifically localize in the mitochondria of cancer cells. Then, the mitochondria targeted nanophotosensitizers will be loaded into pH Low Insertion Peptide (pHLIP) modified PEGylated-liposomes. pH Low Insertion Peptide has the capability in enhancing membrane fusion and lipid exchange of pHLIP modified liposomes at low pH of tumor microenvironment, leading to the increase of cellular uptake and payload release. Upon the addition of CTZ, intracellular nanophotosensitizers can selectively trigger the mitochondrial ROS burst and induce a series of cascade reactions, thereby leading to the apoptosis of tumor cells and activation of the host immune response, which could destroy tumor cells ultimately and show a long-term tumor control. It is expected to reveal the general mechanism for the construction of novel nanophotosensitizer with dual-targeting to both tumor acidic pH microenvironment and mitochondria based on Rluc-CTZ bioluminescence excitation. This study will open a new direction for developing novel type of nanophotosensitizers with superior antitumor effect, and promote the photodynamic therapy and synergistic therapy to the deep tumors.

为了克服临床光动力治疗（PDT）中外源性激光对生物组织穿透深度浅及有机光敏剂的不足，获得高肿瘤选择性和高效的光敏剂，实现深层肿瘤有效的PDT，本项目设计和制备一种双级靶向的原位生物发光激发的纳米光敏剂。在合成的新型纳米光敏剂（石墨烯量子点，GQDs）表面偶联海肾荧光素酶（Rluc），利用Rluc-腔肠素生物发光系统代替传统的外源光照，通过生物发光能量共振转移直接激发GQDs高效生成单线态氧。pH敏感肽修饰的脂质体装载三苯基膦修饰的Rluc-GQDs，实现肿瘤微酸环境和细胞内线粒体的双级靶向，使光动反应直接作用于肿瘤细胞的线粒体，激活细胞凋亡通路加速细胞死亡，并激活免疫系统，在提高光动治疗效率的同时增强机体的抗肿瘤免疫效应，实现对活体深层肿瘤的长效治疗。本研究有望揭示构建靶向的原位生物发光激活的纳米光敏剂的一般规律和内在机理，为深层组织的PDT或协同治疗提供理论基础和实验方法。

为了克服传统光动力治疗中外源性光源对生物组织穿透深度浅及有机光敏剂的不足，获得高肿瘤选择性和高效的光敏剂，实现对深层肿瘤的有效治疗，本项目基于肿瘤微环境（TME）的特征和催化反应，研究了不需要外源性光源激发生成活性氧（ROS）的治疗方法，发展了多种治疗策略：1）利用适配体AS1411形成的G4结构，不仅作为肿瘤靶向配体，也和氯化血红素作用作为DNA酶催化鲁米诺在肿瘤细胞中原位发光，进而激发所负载光敏剂生成ROS；2）利用TME中高浓度的葡萄糖和过氧化氢，通过催化级联反应在肿瘤细胞生成ROS，进一步协同材料的光热效应和化疗药物实现对高转移性肿瘤的有效抑制和预防转移；3）制备了响应型铪基放疗增敏协同治疗系统，通过X射线的辐照，该系统可以不受深度限制的在肿瘤细胞中生成ROS，实现放疗/热疗/化疗协同对耐药肿瘤的有效治疗。此外，我们还构建了几种靶向肿瘤的药物递送系统，有效地将基因药物或化疗药物递送至肿瘤细胞中，实现对肿瘤细胞中目标基因的激活或敲除，抑制肿瘤生长，减少肿瘤免疫抑制，这些药物递送系统同样可以用于光敏剂的靶向递送。在本项目基金的支持下，共发表学术论文12篇，其中SCI论文8篇，EI论文4篇（第一标注3篇，第二标注5篇，第三标注4篇），申请发明专利1项。本研究为构筑不受外源光穿透深度限制的、能够特异性靶向肿瘤的光敏剂递送载体和协同治疗系统提供了一些设计思路和实验基础。