近年来，随着铂类化疗药物在临床应用中的毒副作用渐渐浮现，科学家将目光转向其他金属基（如钌、铱、铼、铑等）抗癌药物以及新型肿瘤治疗手段的开发上。钌(II)配合物因具有突出的光物理、光化学性质和良好的生物相容性，可作为生物成像探针和光敏剂应用于光动力学肿瘤治疗（PDT）领域而备受关注。此外，由于钌(II)配合物具有较大且可调节的共轭平面，作为DNA键合试剂和DNA分子光开关表现出广泛的应用前景。本论文涉及2种噻吩基单核钌配合物和本课题组已报道的2种具有近红外（NIR）发光特性双核钌配合物的DNA键合、生物成像以及光动力学肿瘤治疗活性研究，主要研究工作及创新点如下：

（1）合成了2个钌(II)配合物[Ru(bpy)₂(dtdpq)](ClO₄)₂（Ru1）和[Ru(dtdpq)₃](ClO₄)₂（Ru2），其中bpy = 联吡啶，dtdpq = 2,3-二-(2-噻吩)-吡嗪-[2,3-f][1,10]邻菲咯啉，利用核磁共振氢谱、高分辨质谱和元素分析对它们进行了表征。利用紫外-可见吸收光谱和荧光光谱测试了二者的溶剂变色性质。两个钌配合物在不同溶剂中均具有较高发光量子效率和明显的溶剂效应，其中Ru2的溶剂效应更显著，在水溶液中的最大发射峰在NIR光区(～700 nm)，比在CH₂Cl₂溶剂中的磷光发射红移了112 nm。

（2）采用紫外-可见吸收光谱滴定、发光光谱滴定、EB竞争实验和琼脂糖凝胶电泳实验研究了Ru1和Ru2与DNA的相互作用。结果表明两个配合物都通过嵌入方式与DNA键合，并且Ru1具有很强的光裂解pUC18质粒DNA活性。琼脂糖凝胶电泳实验探究了两个已知具有较强DNA键合活性的双核钌配合物[(bpy)₂Ru(Hdip)Ru(H₂bimp)](ClO₄)₄（Ru3）和[(bpy)₂Ru(Hdip)Ru(H₂bipp)](ClO₄)₄（Ru4），其中Hdip = 2-([2,2':6',2''-三联吡啶]-4'-基)-1H-咪唑[4,5-f][1,10]邻菲罗啉，H₂bimp = 2,6-二-1H-咪唑-2-吡啶，H₂bipp = 2,2'-(2,6-吡啶)二[1-甲基-1H-苯并咪唑]，在不同pH（5.0，6.5，7.4和8.0）条件下的光裂解pUC18质粒活性，结果表明Ru3和Ru4均具有显著光裂解质粒DNA活性，且裂解活性强弱受溶液pH影响，生理pH（5.0 - 8.0）范围内，酸性比碱性条件下的光裂解活性更强。三个钌配合物都是通过活性氧机制发挥光裂解DNA活性的。

（3）研究了Ru1、Ru3和Ru4在细胞和活体中的成像性质。流式细胞术结果表明，Ru1具有较好的细胞膜穿透能力，2 h内可达到最大细胞摄取量。3个钌配合物均为亲水性分子且亲水性适中，均适合用于生物成像。激光共聚焦显微镜研究亚细胞定位结果表明，Ru1定位在HeLa细胞的线粒体，而在生理微酸性pH发光响应的Ru3和Ru4靶向定位在细胞的溶酶体。小鼠活体成像实验结果表明，3个钌配合物在小鼠体内的代谢都主要通过肝脏和肾脏途径，NIR发光的Ru3在细胞和活体水平均具有肿瘤靶向识别能力，尾静脉注射后各离体器官中肿瘤组织的磷光信号最强，有潜力成为肿瘤靶向识别探针。

（4）研究了Ru1、Ru3和Ru4的PDT活性和活性作用机制。三个钌(II)配合物在光照下均具有较高的¹O₂量子产率。Ru3虽不具有PDT活性，但在生理pH区具有显著的pH诱导“¹O₂产生开关”活性，在生理pH范围酸性条件（pH = 5.0）和碱性条件（pH = 8.0）的开关比为10，可作为一种水溶液中高效的pH诱导¹O₂产生开关。采用MTT法、Annexin V-FITC凋亡检测、IncuCyte实时细胞成像、激光共聚焦显微镜、流式细胞术和倒置荧光显微镜探究Ru1和Ru4的体外PDT活性，结果表明2个钌(II)配合物均具有显著的PDT活性，且活性作用机制均为光诱导产生¹O₂。研究Ru4对荷瘤小鼠的活体PDT活性，结果表明与对照组相比注射Ru4且光照处理的荷瘤小鼠肿瘤组织生长受到明显抑制，Ru4可作为一种很有研究意义和应用前景的PDT光敏剂。

（5）通过构-效关系探究发现，配体上取代基对调控钌(II)配合物的DNA键合、生物成像和PDT治疗肿瘤等生物活性发挥重要作用。与母体配合物Ru(bpy)₂(dpq)](ClO₄)₂相比，噻吩基团的引入显著增强了Ru1的DNA键合强度、质粒DNA光裂解活性、光诱导¹O₂产生以及PDT治疗肿瘤活性。通过比较Ru3和Ru4的构-效关系发现，对配合物桥联配体上的咪唑基进行结构修饰会减弱配合物的质子化/去质子化过程，进而降低pH响应特性，但对PDT活性的增强有利。本文的研究结果将为设计高效的pH诱导¹O₂产生开关、肿瘤成像探针、PDT治疗肿瘤光敏剂提供理论依据和重要的参考数据。

Recent years, as some undesirable toxic and side effects of platinum chemotherapy drugs have gradually been revealed in clinical application, scientists are turning their attention to the development of other metal-based anticancer drugs (eg. ruthenium, iridium, rhenium, etc.) and new tumor treatment methods. Ruthenium(II) complexes have attracted much attention as bioimaging probes and photosensitizers in photodynamic tumor therapy (PDT) due to their outstanding photophysical and photochemical properties and good biocompatibility. In addition, ruthenium (II) complexes have shown wide application prospects as DNA binding reagents and DNA molecular luminescence switches due to their large and adjustable conjugated planes. This Ph.D. thesis involves the synthesis and studies of DNA binding, biological imaging and PDT properties of two mononuclear thiophene-containing ruthenium (II) complexes and two known binuclear ruthenium(II) complexes with near-infrared (NIR) luminescence properties. The main work and innovations of this thesis are described as follows:

(1) Two ruthenium (II) complexes [Ru(bpy)₂(dtdpq)](ClO₄)₂ (Ru1) and [Ru(dtdpq)₃](ClO₄)₂ (Ru2) were synthesized, in which bpy = bipyridine, dtdpq = 2,3-di-2-thienylpyrazino[2,3-f][1,10]phenanthroline, and were characterized by ¹H NMR, high resolution mass spectrometry and elemental analysis. The solvatochromism of the Ru1 and Ru2 was studied by UV-visible absorption and emission spectroscopy. Both of the complexes have been found to exhibit high luminescence quantum efficiency and obviously solvatochromic effect, especially for the Ru2. The maximum emission peak in aqueous solution is in NIR window of around 700 nm, which is red shifted by 112 nm as compared to that in CH₂Cl₂.

(2) The interaction of Ru1 and Ru2 with DNA was studied by UV-visible absorption and emission spectrophotometric titrations, EB competition test and agarose gel electrophoresis. The results revealed that both of them bind to DNA through intercalative mode and the Ru1 has strong photocleavage activity of pUC18 plasmid DNA. The DNA photocleavage properties of the two known binuclear ruthenium (II) complexes [(bpy)₂Ru(Hdip)Ru(H₂bimp)](ClO₄)₄ (Ru3) and [(bpy)₂Ru(Hdip)Ru(H₂bipp)](ClO₄)₄ (Ru4) were investigated by agarose gel electrophoresis at different pH values (5.0, 6.5, 7.4 and 8.0). The results indicated that both of Ru3 and Ru4 had strongly pH-dependent plasmid DNA photocleavage activity. The DNA photocleavage mechanism of Ru1, Ru3 and Ru4 involves a pathway of photogeneration of reactive oxygen species (ROS).

(3) The bioimaging applications of Ru1, Ru3 and Ru4 in vitro and in vivo were studied. Flow cytometry results showed that Ru1 had good cell membrane penetration property and could achieve the maximum cell uptake within 2 h. All of the Ru1, Ru3 and Ru4 are moderately hydrophilic, which is suitable for biological imaging. The subcellular localization study was conducted by confocal laser fluorescence microscopy, and the results indicated that Ru1 could localize in mitochondria of HeLa cells, while previously reported and physiologically pH responsive Ru3 and Ru4 were found to localized in lysosomes. In vivo bioimaging studies showed that three ruthenium (II) complexes were excreted from the body of nude mouse mainly through the liver and kidney systems. The NIR emitting Ru3 was found to have a capability to recognize tumor cells both in vitro and in vivo. After intravenous injection, the tumor tissues displayed the highest luminescence signal among the main ex vivo organs. Therefore, Ru3 has the potential to be an outstanding tumor targeted probe.

(4) The PDT activity and mechanism of Ru1, Ru3 and Ru4 were studied. All of the three ruthenium complexes have high ¹O₂ photogeneration quantum yields. Although Ru3 has no PDT activity, it has significant physiological pH-induced “¹O₂ generation switching” properties, namely, ¹O₂ photogeneration quantum yield in acidic condition (pH = 5.0) was 10 folds as great as that in alkaline condition (pH = 8.0). Therefore, Ru3 in aqueous solution could be used as a highly efficient pH-induced ¹O₂ generation switch. In vitro PDT activities of Ru1 and Ru4 were investigated by MTT assay, Annexin V-FITC apoptosis assay, IncuCyte real-time cell imaging, laser confocal fluorescence microscopy, flow cytometry and inverted fluorescence microscopy. The results showed that both Ru1 and Ru4 had significant PDT activities and the PDT mechanism involved photogeneration of ROS. The results of in vivo PDT studies showed that the tumor growth of tumor-bearing nude mouse was significantly inhibited after injection and irradiation of Ru4 as compared with the control group. Therefore, Ru4 would act as a promising PDT photosensitizer.

(5) By investigating the structure-activity relationship we found that the substituents on ligands play an important role in regulating the DNA binding, bioimaging and PDT properties of Ru(II) complexes. Compared with the parent Ru(II) complex Ru(bpy)₂(dpq)](ClO₄)₂, the introduction of thiophene groups can significantly enhance the DNA binding strength, plasmid DNA photocleavage activity, photoinduced ¹O₂ production and PDT tumor therapy activity of Ru1. Comparing the structure-activity relationship of Ru3 and Ru4, it was found that modification on the imidazole groups of the bridge ligand can significantly weaken the protonation/deprotonation process of the complex, thus reducing the pH response characteristics, but be benefical to the enhancement of PDT activity.