动态监测线粒体自噬进程的pH比率响应型近红外荧光探针

Mitophagy is a spontaneous selective process of cells for removing the damaged or excess mitochondria. It plays critical roles in maintaining cell homeostasis. Mitophagy comprises key processes such as the decrease of mitochondrial membrane potential, ubiquitination of the mitochondrial outer membrane, formation of autophagosome followed the fusion with lysosomes. It has been reported that the continuous acidification in mitochondria is prerequisite for promoting the mitophagy processes. This study aims to establish a series of pH ratiometric near-infrared fluorescence probes. These probes are capable of crossing multiple physiological barriers after intravenous injection and then covalently anchor with the mitochondrial inner membrane. The probes will be protonated due to continuous acidification during the mitophagy, which results in the inhibition of internal charge transfer (ICT) and formation a dual absorption peaks with a ratiometric pH response. Measurement of the fluorescence intensity ratio upon excitation at the dual absorption wavelength will determine the mitochondrial pH and reveal its relationship to mitophagy processes. With the histopathological results as the gold standard, this study intends to compare the timeliness and accuracy of mitophagy probes with apoptotic or metabolic probes in evaluating the therapeutic responses of cytotoxic and neuroprotective drugs. In summary, this project not only provides new tools for real-time monitoring mitophagy process, but also creates a novel strategy for noninvasive evaluating therapeutic efficacy of innovative drug candidates.

线粒体自噬是细胞自发选择性清除受损或过量线粒体的过程，在维持细胞稳态等方面发挥重要作用。线粒体自噬包含受损线粒体膜电位降低、自噬体形成、溶酶体融合等关键进程，而线粒体内液持续性酸化是推动上述进程发展的必要条件。本项目拟构建一类pH比率响应型近红外荧光探针。该探针能够跨越多重生理屏障靶向锚定细胞线粒体内膜。线粒体自噬相伴随的酸化过程中，探针质子化抑制分子内电荷转移，形成强度呈比率型变化的双重吸收峰。拟合pH相关双波长激发荧光强度比值获得校正曲线，为实时测定线粒体pH值提供准绳。拟研究线粒体pH演变与线粒体自噬进程间的对应关系，以病理结果为金标准，运用光学、磁共振和核医学分子成像技术，比较pH比率响应型线粒体自噬探针与凋亡类、代谢类探针在评价化疗和神经保护药物疗效的时效性和准确率。由于近红外光高组织穿透性，本项目不但为活体监测线粒体自噬提供新的工具，还为创新药物疗效无创评估开辟新的途径。

线粒体自噬是细胞自发性、选择性清除受损或过量线粒体的过程，对调控线粒体数量和质量、减轻细胞负荷、避免细胞凋亡等方面发挥重要作用。包括神经退行性疾病在内多种疾病均表现为线粒体功能障碍和线粒体自噬过程受阻。对胞内线粒体自噬过程的动态、定量监测不但有助于探究疾病发生、发展机制，还可以为线粒体靶向药物筛选提供新工具。线粒体自噬包含受损线粒体膜电位降低、自噬体形成、溶酶体融合等关键进程并伴随pH值逐渐降低。因此我们提出监测线粒体自噬小体pH评估线粒体自噬进程的科学假说。本项目主要进展包括：（1）构建了一系列深红pH比率响应型探针，不但实现了生理环境pH定量读取，而且对活细胞线粒体表现很好靶向性。（2）实现了神经元细胞线粒体自噬过程的动态监测。发现了正常线粒体内液pH为8.0左右，线粒体自噬体pH酸化至6.0‒6.8，自噬体与溶酶体融合后pH下降至5.0‒5.5。（3）由于阿尔茨海默症等中枢神经系统疾病通常表现线粒体自噬水平缺失或下调。该探针有望通过监测线粒体自噬水平为线粒体靶标药物的筛选提供新工具。（4）拓展了pH响应探针在脑疾病诊疗方面医学应用。构建了pH响应表面增强拉曼（SERS）探针，发现了脑胶质瘤浸润组织恶性程度与组织间液酸化程度呈正相关性，提出了酸性“代谢边界”导航手术的新策略，提高了胶质瘤模型的切除率及生存时间，在胶质瘤病人手术切除组织上验证了酸化代谢边界的存在。总之，项目围绕活细胞线粒体自噬进程可视化的科学问题，构建了系列pH比率响应荧光/SERS探针，初步实现了神经元细胞中线粒体自噬过程的动态监测，揭示了线粒体pH变化与线粒体自噬进程间的对应关系，为以细胞线粒体为靶标药物的筛选提供新工具。项目执行期间申请人获得国家杰出青年基金，国家重点研发计划专项各1项，发表标注有本基金号的SCI收录论文10篇，其中IF > 10的文章3篇，申请发明专利3项，授权3项。培养博士研究生3名，获博士后创新人才支持计划1名，培养硕士研究生3名。