基于给受体型近红外核酸探针的合成及体内外肿瘤细胞长循环示踪

Organic near-infrared (NIR) nucleic acid-selective fluorescent probes have emerged as the most promising alternatives for real-time tracking and detection tumor cells due to their advanced features including low excitation energy, strong penetrability and high sensitivity. At present, they still suffer from poor luminescence properties, weakened or quenched fluorescence in aggregated state, poor cell membrane permeability and high cytotoxicity. In view of this, this project focuses on the design and synthesis of high performance donor-acceptor (D-A) based NIR nucleic acid-selective fluorescent probes, which containing the structure of benzothiadiazole and pyridinium salt. In order to achieve long wavelength absorption and emission, reduction of the energy gap (Eg) of donor-acceptor structure in fluorogens by regulation of D, A groups and their substituented groups and extended the conjugated length via introduction of thiophene and furan ring. On the other hand, the substituents of aggregation induced emission (AIE) active part, including triphenylamine and phenothiazine units, will improve the luminescence properties of fluorogens. Apart from this, the tumor cells targeted units (e.g. pyrimidine, purine, maleimide, etc) will promote the selectivity and targeting properties for applications in long-term tumor cells tracking in vitro and in vivo. This project investigated the relationship about the luminescence properties of probes between the molecular band gap and structure. It also obtained the mechanism of AIE active units on the morphology of the probes and that of long-term tumor cells tracking in vitro and in vivo. The results should provide high guidance for the design and synthesis of high performance NIR nucleic acid-selective fluorescent probes and accelerate the application in disease diagnosis and clinical treatment for our country.

有机近红外（NIR）荧光核酸探针具有激发能量低、穿透力强、探测灵敏度高等优点，在肿瘤细胞的实时示踪和检测领域具有较大应用前景。现该类探针存在发光性能不好，在聚集态下荧光易淬灭，对细胞膜通透性差且毒性较大等缺点。鉴此，本项目设计合成含苯并噻二唑和吡啶鎓盐的电子给受体（A-D）型NIR荧光探针，通过调控D、A基团及其取代基来降低分子能级带隙，引入噻吩、呋喃环等提高探针的共轭链长，引入三苯基胺和吩噻嗪等赋予探针聚集诱导发光（AIE）活性，提高探针的发光性能,实现NIR发光；引入嘧啶、嘌呤及马来酰亚胺等靶向基团，提高探针对核酸的选择性及靶向性，实现体内外肿瘤细胞的长效示踪；探明分子能级带隙与构型对探针发光性能的调控规律，获得AIE基团对探针分子形态学的作用机制，阐明荧光探针体内外长循环示踪机理。研究结果为开发高性能NIR荧光探针的设计合成提供必要的理论指导，加快核酸探针在癌症诊疗一体化的应用进程。

项目针对近红外（NIR）荧光探针在肿瘤细胞的实时长效示踪方面仍存在发光性能不好，细胞膜通透性差和毒性较大等缺陷。开发性能优异的荧光探针，探明其发光性能的调控规律。主要研究内容：（1）以噻吩基团修饰二甲基芴，合成一种阳离子型荧光探针FTI，表征其结构，研究其光物理性能。（2）设计合成一种D-A-D型的双三苯胺基-苯并噻二唑类荧光探针TCNT，发现探针TCNT能制成试纸用于饮用水中CN−的检测，且能于HepG-2细胞中标识氰离子浓度水平和器官组织层次的成像；（3）设计合成了一种以三苯胺和苯并噻二唑为电子供体、吲哚鎓盐为电子受体的具有D-π-A结构的荧光探针TBZTI。发现TBZTI能明显抑制小鼠肿瘤的生长，有潜力作为光敏剂应用到PDT的临床研究。（4）设计合成了一种以9-苯基咔唑为电子供体、吡啶鎓盐为电子受体的具有D-π-A结构的荧光探针PCTP。PCTP的NIR发射波长为710 nm，斯托克斯位移高达285 nm。PCTP能有效地透过细胞膜进入细胞实时成像，表现出较高的光稳定性，能够示踪4T1细胞体外增殖最高到11代，示踪小鼠肿瘤生长至22天以上。主要创新点：（1）以苯并噻二唑作为电子受体，合成一种双电子给体三苯胺类红光发射（595 nm）AIE荧光探针TCNT，能在99%水溶液中实现对于CN−的特异性检测，检测限为0.35 μM。（2）合成了一种具有D-π-A结构和ICT的阳离子型荧光探针TBZTI。TBZTI具有近红外发射，最大发射波长为730 nm，斯托克斯位移达到180 nm，具有AIE效应，能在体内外高效地产生ROS，在细胞水平上具有理想的光动力效应，能通过EPR靶向肿瘤，对小鼠肿瘤的PDT效果显著；（3）设计合成了一种具有D-π-A结构和ICT的阳离子型荧光探针PCTP。PCTP具有近红外发发射，最大发射波长为710 nm，斯托克斯位移高达285 nm，能够识别体内外的RNA，对RNA表现出高选择性并且检测极限为2.17 μg/mL，具有良好的生物相容性和较高的光稳定性，能示踪4T1细胞体外增殖最高到11代，示踪小鼠肿瘤生长到22天以上。上述研究已发表论文11篇，申请发明专利3项。

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