用于跟踪细胞溶酶体酸度变化及小鼠癌组织成像的一类触发型荧光探针的研究

Eukaryotic cells are featured by the occurring of organelle specific pH.For example, the cytoplasma is slightly alkaline at about pH 7.2 while the luminal pH of lysosomes is in the range of 6.0-4.0. The acidity of lysosomes is essential for a variety of cellular events ranging from endocytosis, autophagy, to apoptosis, etc. The organelle specific intracellular pH gradients is elaborately controlled and yet dynamically regulated. Changes of the parameters of lysosomes have been associated with cell maturation, cancer metastasis, etc. The morphology of lysosomes in cancer cells are significantly altered relative to normal cells. Dyes that could be used for noninvasive imaging of lysosomes are valuable for in vivo cancer diagnosis and evaluation of lysosome-targeted cancer therapy.. Rhodamines attracted considerable interest in biological studies due to their properties of high fluorescent quantum yields, bioorthogonal fluorescence spectra, and stability against photo-bleaching. Rhodamine-lactams, a group of non-fluorescent and colorless compounds hallmarked by intra-molecular spirolactams, are poised to form highly fluorescent rhodamine derives via ring opening of the spirolactam. Although it has long been documented that rhodamine lactams display strong fluorescence in acidic media, applications of the lactams as intracellular acidic pH sensors have been largely unexplored. .We recently developed low background and selective staining of lysosomes in live cells with a rodamine-lactam based sensor that is activated in lysosomes to give a highly fluorescent species via proton triggered ring opening of the lactam. Long retention time of the sensor in lysosomes and its exceptional photostability allowed full time tracking of lysosome morphology changes in TNF-α triggered cell death, suggesting its broad utility for acidic vesicle studies.Herein, a large number of rhodamine derivatives with tuned affinity for proton will be synthesized and screened for optimal and selective responses to lysosomal pH in the range of pH 6-5. The new sensors will be extensively modified with various functional functionalities or incorporated into nanoparticles. The resultant sensors will be used for ratiometric sensing of lysosomal pH changes in selected cell signaling pathways in live cells, evaluation of the efficacy lysosome-targeted anti-cancer drugs, and fluorescent imaging of cancerous tissues in mice.

溶酶体不仅消化细胞吞噬的外源物质，还参与细胞自吞噬、凋亡、以及癌症发展等过程。在上述过程中，溶酶体的pH、体积、形态、位置等会发生显著的变化。低背景的、能灵敏测量溶酶体酸度、并能标记溶酶体形态与位置的荧光探针对研究溶酶体相关细胞信号传导，以及癌组织成像等领域都有重要意义。本课题利用有机化学的理论方法，对一类具有分子内5员酰胺环的罗丹明衍生物进行结构优化与功能化。得到对溶酶体酸度区间灵敏响应的多种溶酶体探针。将利用具有双发射荧光光谱的pH探针研究溶酶体酸度在一些细胞生物信号传导过程中的变化规律、研究一些抗癌药物对癌细胞溶酶体酸度的影响。制备pH响应的荧光纳米材料，用于对老鼠体内癌细胞溶酶体进行成像研究，为发展低背景的动物体内癌组织成像技术进行前期研究。

细胞溶酶体除了消化细胞吞噬进的物质的功能以外，还与细胞的自吞噬，凋亡, 癌症发展等过程有直接的关联。在上述过程中,溶酶体参数，如pH，体积，形态，位置等会发生显著的变化。低背景的、能够灵敏测量溶酶体pH并能够表标记溶酶体的荧光探针，对研究与溶酶体相关的细胞信号传导，以及癌症的诊断具有重要的意义。本课题利用有机化学的方法，对具有分子内5员酰胺环的罗丹明衍生物进行结构优化，获得通过新型发光机理的具有双发射荧光光谱的、pH敏感的探针或纳米材料，用于准确测量溶酶体pH变化，研究溶了酶体pH变化在一些细胞生物信号传导过程中的作用（细胞凋亡、坏死、自吞噬等）；最后利用pH敏感探针的功能化（糖缀合）实现了对老鼠体内小尺寸的肿瘤病灶、炎症组织的溶酶体激活荧光的成像（荧光成像、光声成像、光热疗法）。糖靶向小分子成像制剂有望成为新一代低背景的、用于辅助手术过程癌症定位技术。