树枝状酞菁酶激活光动力分子信标的合成与抗肿瘤活性研究

Photodynamic therapy (PDT) is an emergying cancer treatment modality involving the combination of light，a photosensitizer and an oxygen. It offer unique control the photosensitizers（PS） 's action because the key cytotoxic agent, singlet oxygen is produced only in situ upon irradiation. However, PDT can't achieve a high level of selectivity because of the limited tumor localization of the exsiting PS, which in turn cause treatment-related toxicity to surrounding normal tissue as well as sunlight-induced skin toxicity. A new direction for PDT is to exert control of the PS' s ability to produce 1O2. This level control has potential to achieve ultimate PDT selectivity to cancer cell while leaving normal cells unharmed. Photodynamic molecular beacons is an extension of the well known molecular beacons (MB) that use the FRET principle for controlling fluorescence emission in response to target activation. By combining MB with PDT, we seek to enable cancer biomarker-controlled 1O2 production to achieve unprecedented PDT selectivity. In this proposal, we plane to synthesis and characterization a serials of novel dendrimer phthalocyanine based protease control activation photodynamic molecular beacons (PPMB), and demonstrated its utility for image and therapy of neuroglioma in vitro and in vivo. The PPMB comprises three elements, (i)dendrimer phthalocyanine as the PS;(ii)a dual 1O2 and fluorescence quencher; (iii)a brain tumor-specific linker; PS's photoactivity is silence before the linker interacted with a neurogliome associate protease. The protease induces specific cleavage of the linker and removes the PS from vicinity of quencher restoring the PS's fluorescence and 1O2 production. The synthetic methods for both "folding" and "zipper" structure photodynamic molecular beacons are studied. The structures , photophysical properties，specific and sensitivity of protease activation are characterized. The time-dependent cell uptake and photodynamic efficacy of PPMB in glioma cell are evaluated. PPMB are applied as a molecular imaging agent to real time monitor subcellular localization, molecular pathways involved in PDT-induced apoptosis and biomarkes of therapeutic response to PDT. The PPMB mediate PDT molecular mechanism are explained. The nude mouse models of U251 human glioma are build, in vivo photodynamic efficacy of PPMB for neurogliomas are evaluated. The structure and photodynamic efficacy relationship are reviewed. These results will provide detail data for exploiting new target delivery strategies and new activation mechanism for phtosensitizers.

光敏剂的非特异性定位和激活是目前PDT面临的主要问题。为了提高光敏剂的肿瘤选择性，本课题合成系列具有靶向运输和靶分子特异性激活功能的树枝状酞菁酶激活光动力分子信标(PPMB)，用于诊断和治疗神经胶质瘤。PPMB由树枝状酞菁光敏剂，荧光猝灭剂和胶质瘤特异性连接体三部分耦联合成。胶质瘤过度表达酶为标靶分子，无标靶时，光敏剂荧光猝灭；有标靶时，标靶特异性识别和剪切连接体, 光敏剂产生荧光和单线态氧。研究"折叠"和"拉链"结构PPMB的合成方法，表征其结构、光物理性质和酶激活特异性和灵敏度。离体和动物模型实验研究PPMB对胶质瘤细胞的摄取规律，评价其治疗胶质瘤光动力活性。研究PPMB介导PDT诱导细胞凋亡的分子通道和PPMB－PDT诱导基质蛋白酶活性变化的高分辨时空成像，阐述PPMB介导的PDT治疗胶质瘤的分子机制。总结构效关系，为光敏剂新的靶向运输途径和特异性激活机制开发提供理论和实验依据。

针对光动力治疗面临的非特异性定位和激活问题，本课题合成一系列具有靶分子特异性激活功能的树枝状酞菁基激活纳米光敏剂，用于离体和在体诊断和治疗神经胶质瘤和不同亚型乳腺癌。第一部分，我们设计合成和表征了一系列新型功能基取代树枝酞菁配合物，包括甲基化吗啉取代树枝酞菁锌（硅）、金刚烷取代树枝酞菁锌（硅）、氟代树枝酞菁锌（硅）和噻吩、吡咯烷酮和炔基取代树枝酞菁锌（硅）配合物，并研究其光物理性质及其光诱导分子内电子转移和能量转移。第二部分，构建了一系列新型树枝酞菁基激活光敏剂。酶激活羧基树枝酞菁锌-PLGLAG-单壁碳纳米管分子信标（纳米复合）体系；一系列光激活/药物控制释放金刚烷树枝酞菁锌/氟代树枝酞菁锌（硅）-芘-环糊精-单壁碳纳米管纳米复合体系;光激活甲基化吗啉取代树枝酞菁锌（硅）/噻吩和炔基取代树枝酞菁锌-纳米金（棒）纳米复合体系。功能取代树枝酞菁的荧光（单线态氧）在纳米体系中基于荧光共振能量转移被碳纳米管或纳米金（棒）猝灭，当纳米激活体系到达肿瘤细胞后，通过酶剪切激活或光（药物）控制激活光敏剂，树枝酞菁荧光恢复，产生单线态氧，产生光动力活性。第三部分，将以上合成的一系列激活功能的树枝状酞菁基激活光敏剂分别用于离体和在体实验治疗神经胶质瘤细胞和离体治疗不同亚型的乳腺癌、黄斑变性等非肿瘤性疾病和多重耐药性菌，均有很好光动力活性。双光子成像评价了激活光敏剂的毒性和细胞亚定位、激光共聚焦荧光成像其在细胞中的代谢和治疗过程，激活纳米光敏剂通过胞吞作用进入细胞，定位线粒体和溶酶体，破坏线粒体分裂融合过程，导致细胞凋亡。本研究为开发靶向和特异性治疗肿瘤激活纳米光敏剂提供新思路和新方法。

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