生物自发光触发高荧光碳量子点基复合光敏剂的合成及其光动力治疗性能研究

The present imperfect photosensitizers and the limitation of light penetration in living tissues for external excitation light are the major obstacles for the wide application of photodynamic therapy (PDT). Nanocarriers provide an efficient way to deliver the photosensitizers. Compared with the upconversion nanoparticles and semiconductor quantum dots, the good biocompatibility, surface modification, and many other advantages of carbon quantum dots (CDs), besides the comparable fluorescent properties, make it easy to win out in the field of PDT. While the bioluminescence is generally in the visible regions, which is adjacent to or overlapped with the main absorption bands of present photosensitizers. Furthermore, the bioluminescence could served as a internal light to activate the photosensitizer and then the limitation of light penetration is of no concern. Therefore, in this project we propose to synthesize high luminescent CDs with folic acid (Fa) decorated in the surface (named Fa-N-Mg-CDs) by a Mg/N double doping strategy using suitable carbon source with Fa mixed, surface passivation agent containing nitrogen and magnesium salts through a hydrothermal method. The growth process and tunable photoluminescence mechanism of Fa-N-Mg-CDs will be illuminated. On this basis, the photosensitizer chlorin E6 (Ce6) and Renilla luciferase 8 (Rluc8) are immobilized on the surface of Fa-N-Mg-CDs through chemical coupling reactions to form a new bioluminescence triggered photosensitizer (called Rluc8-CDs-Ce6). The fluorescence resonance energy transfer between Rluc8, CDs and Ce6 will be studied. Meanwhile, the photodynamic fluorescence imaging and PDT effeciency will be explored. The bioluminescence triggered RLuc8-CDs-Ce6 is expected to be an ideal photosensitizer for PDT of tumors.

目前，光敏剂及其激发光源的组织穿透限制是影响光动力疗法（PDT）发展的关键。纳米载体传输是一种有效途径，碳量子点（CDs）优异的荧光性能、良好的生物相容性、表面易修饰等特点使其在PDT领域更具优势。而生物荧光可触发现有光敏剂，且可避免组织穿透障碍。因此，本项目拟结合前期工作基础，选择适宜的有机物与叶酸混合前躯体、含氮钝化剂以及镁盐，水热合成具有高荧光性能、表面叶酸修饰和氮-镁双杂化的CDs，剖析CDs的生长规律及荧光调控机制；在此基础上，将光敏剂二氢卟吩e6（Ce6）和海肾萤光素酶（Rluc8）通过化学偶联反应结合在CDs表面，形成生物自发光触发的复合光敏剂Rluc8-CDs-Ce6，分析Rluc8、CDs和Ce6之间荧光共振能量转移机制，探索Rluc8-CDs-Ce6对肿瘤细胞的荧光成像诊断与PDT效果。这种生物自发光触发的RLuc8-CDs-Ce6有望成为一种较理想的肿瘤PDT光敏剂。

目前，光敏剂及其激发光源的组织穿透限制是影响光动力疗法（PDT）发展的关键。纳米载体传输是一种有效途径，碳量子点（CDs）优异的荧光性能、良好的生物相容性、表面易修饰等特点使其在PDT领域更具优势。而生物荧光/化学发光可触发现有光敏剂，且可避免组织穿透障碍。首先，本项目选择短链钝化剂合成表面富含氨基的高荧光碳量子点（CDs），CDs的相对荧光量子产率达84.6%（以硫酸奎宁为标准参照物，量子产率54%），通过经典的EDC/NHS反应将二氢卟吩E6（Ce6）与CDs结合，构建高荧光共振能量转移（FRET）效率的CDs-Ce6体系。利用HepG2细胞进行了初步的光动力治疗（PDT）效果评价，结果显示，660nm激光照射30s后，CDs-Ce6组的HepG2细胞死亡率明显高于纯Ce6组。其次，将荧光虫荧光素酶引发的生物荧光来作为PDT光源，根据其发光特性构建了碳量子点-原卟啉（CDs-PIX）体系。体外细胞检测显示，生物自荧光激发下的CDs-PIX体系可以产生大量的活性氧，满足癌症治疗的条件。细胞试验结果也显示，生物荧光激发的CDs-PIX体系对SMMC-7721肝癌细胞有显著的致死作用。最后，制备了黄色荧光碳量子点（y-CDs）和Ce6的复合光敏剂y-CDs-Ce6，并利用鲁米诺化学发光来激发y-CDs-Ce6体系，通过CRET和CRET-to-FRET双重途径直接和间接激发复合光敏剂。化学发光激发y-CDs-Ce6后显著增加SMMC-7721肝癌细胞内活性氧产量，细胞死亡率达到90%左右。这种不需要外部光源、以高荧光CDs为载体、基于能量共振转移原理的复合光敏剂体系有望成为一种较为理想的肿瘤PDT光敏剂。