| Abstract | 我们先前发现PCP可与钌多吡啶络合物如[Ru(phen)2(dppz)]Cl2（phen：1,10-邻菲咯啉；dppz：多吡啶并吩嗪）形成离子对复合物，通过被动运输的方式促进其细胞核摄取。但尚并不清楚除PCP之外，其它氯代酚（CPs）是否也能够促进其细胞核摄取？如果能，决定细胞核摄取的主要物理化学因素是什么？本研究发现其它两类氯代酚包括三种四氯酚（ TeCPs）和六种三氯酚（ TCPs）也能促进[Ru(phen)2(dppz)]2+的细胞核吸收；2,3,4,5-四氯酚和3,4,5-三氯酚是其同分异构体中能够最有效地促进[Ru(phen)2(dppz)]2+的细胞核摄取的，甚至比含氯原子数最高的PCP的效果更好。结构-性能研究发现[Ru(phen)2(dppz)]2+的细胞核摄取量与其和CPs形成的离子对复合物的结合力呈正相关，而与其亲脂性呈负相关。
2.钌(II)多吡啶络合物与活细胞核DNA的可逆结合及机制研究
近年来，能够和 DNA可逆结合的金属络合物逐渐引起了人们广泛的兴趣。然而鲜有文献报道金属络合物可与活细胞核 DNA发生可逆结合。本研究发现钌(II)络合物通过氯代酚与活细胞 DNA可逆且可控的结合。当清洗细胞去除氯代酚后，已进入细胞核的[Ru(phen)2(dppz)]2+会排出到细胞质；更有趣的是，当重新加入氯代酚后细胞质的[Ru(phen)2(dppz)]2+又能够重新返回到细胞核。通过使用特异性的抑制剂、si-RNA和过表达ATP结合盒（ABC）转运蛋白，我们发现[Ru(phen)2(dppz)]2+可通过三种 ABC转运蛋白（ABC-B1/C1/G2）排出细胞。不仅如此，在两个纯化的手性形式的 [Ru(phen)2(dppz)]Cl2异构体上也观察到了类似的，且具有手性选择性的细胞核 DNA可逆结合。本研究首次发现 DNA“光开关”钌(II)络合物通过氯代酚可与活细胞 DNA发生可逆且可控结合，这为今后利用离子对的形成作为一个有效的方法来将具有生物活性的金属络合物可逆且可控地带入细胞内的靶点提供了新思路。
3.锇多吡啶络合物在细胞内的光电关联成像研究
具有高分辨率的 TEM是细胞生物学和医学领域不可或缺的工具，OsO4被作为传统的固定和染色剂。尽管 OsO4被广泛使用，但其并不完美，因为它具有较高挥发性和毒性。锇(II)多吡啶络合物如[Os(phen)2(dppz)]2+不仅是公认的 DNA“光开关”，也是 TEM研究潜在的理想染色剂。本研究中，我们发现氯代酚抗性阴离子能够通过形成中性亲脂离子对将不能进入活细胞的阳离子 [Os(phen)2(dppz)]2+带入活细胞核，使其作为具有手性选择特性的细胞核 DNA成像试剂尤其适用于活细胞和固定细胞的光电关联（CLEM）成像，能让我们清楚地观察到细胞在有丝分裂期染色质到染色体的变化。我们建议手性 Os(II)多吡啶络合物可作为一类新颖的具有手性选择特性的高分辨率CLEM成像探针应用于活细胞核DNA研究。
4.手性锇多吡啶络合物对核仁和细胞核的荧光寿命成像（FLIM）研究
核仁是一个重要的亚细胞结构，然而却只有很少的染料能对其成像。我们发现通过 FLIM成像，[Os(phen)2(dppz)]Cl2的两个手性对映异构体均能够在固定细胞中对核仁和细胞核进行成像，其中 Λ对映异构体和氯代酚共同孵育还能够在活细胞中从细胞核区域区分出核仁。进一步利用三种能够特异性抑制核仁蛋白生成的新陈代谢抑制剂，发现两个对映异构体在核仁内的荧光寿命变化能够反映出
细胞内微环境的变化，而这又和细胞核仁的总体病理状况有关。在此基础上，通过两个手性异构体观察到细胞有丝分裂不同时期核仁、染色质和纺锤体的动态变化。与商业化核仁荧光探针 Syto 9相比，本研究发现的手性锇(II)络合物具有许多优点：更大的 Stokes位移、近红外发射、更长的荧光寿命、能够对细胞有丝分裂期的纺锤体成像、更重要的是它具有手性选择性。这是首次利用手性锇 (II)络合物对细胞核和核仁进行手性选择性 FLIM成像，这使得在活细胞中使用一种探针同时对多个细胞器进行 FLIM成像成为可能。
总之，本论文研究了钌盐络合物与活细胞核 DNA的可逆结合机制：氯代酚促进[Ru(phen)2(dppz)]2+的细胞摄取量与氯代酚和钌盐络合物形成的离子对复合物的结合力正相关，与其亲脂性负相关；当清洗细胞后，细胞核内的[Ru(phen)2(dppz)]2+可以通过三种 ABC转运蛋白排出细胞。氯代酚能够通过形成亲脂性离子对复合物的方式促进[Os(phen)2(dppz)]Cl2的细胞核吸收，使其不仅能够作为细胞核 DNA双染色剂应用与光电联用成像，还能够通过 FLIM对核仁和细胞核进行手性区分成像。 |
| Other Abstract | Ru/Os(II)-polypyridyl complexes with dppz ligand have high binding ability with DNA and show DNA “light switch” effect. However, most of previous studies are in vitro studies because it is difficult to control their cellular uptake and distribution. We found recently that nuclear uptake of Ru(II)-polypyridyl complexes were remarkably enhanced by pentachlorophenol (PCP) via forming lipophilic ion-pair. However, it is not clear whether the enhanced nuclear uptake of [Ru(phen)2(dppz)]2+ (phen = 1,10-phenanthroline; dppz = dipyridophenazine) is only limited to PCP, or it is a general phenomenon for other highly chlorinated phenols (CPs), and if so, what are the major physicochemical factors in determining nuclear uptake. An in-depth understanding on the mechanisms of cellular uptake and efflux would facilitate the design of Ru complexes with not only better functionality and targeted imaging effect and therapeutic efficiency, but also with controlled toxicity. We still don’t know ehether [Ru(phen)2(dppz)]2+ already delivered to the nucleus viaion-pairing can get oput out of the cell, and if so, what is the underlying mechanism. Os(II) polypyridyl complexes have some distinguished characteristics as compared to Ru(II) complexes, but is it not clear whether CPs can also facilitate nuclear uptake of Os(II) polypyridyl complexes, and whether they can be used for correlative light and electron microscopy studies. Therefore, we plan to address the above questions in this dissertation, which contains four major parts：
1. The Major Physicochemical Factors in Determining the Preferential Nuclear Uptake of the DNA “Light-switching” Ru(II)-Polypyridyl Complex in Live-Cells via Ion-Pairing with Chlorophenolate Counter-Anions
We have found recently that nuclear uptake of the cell-impermeable DNA light-switching Ru(II)-polypyridyl cationic complexes such as [Ru(phen)2(dppz)]Cl2 was remarkably enhanced by PCP by forming ion-pairing complexes via a passive diffusion mechanism. However, it is not clear whether the enhanced nuclear uptake of [Ru(phen)2(dppz)]2+ is only limited to PCP, or it is a general phenomenon for other highly chlorinated phenols (CPs); and if so, what are the major physicochemical factors in determining nuclear uptake? Here, we found that the nuclear uptake of Ru(phen)2(dppz)]2+ can also be facilitated by other two groups of CPs including three tetrachlorophenol (TeCP) and six trichlorophenol (TCP) isomers. Interestingly and unexpectedly, 3,4,5-TCP was found to be the most effective one for nuclear delivery of [Ru(phen)2(dppz)]2+, which is even better than the most-highly chlorinated PCP, and much better than its five other TCP isomers. Further studies showed that the nuclear uptake of [Ru(phen)2(dppz)]2+ was positively correlated with the binding stability, but to our surprise, inversely correlated with the lipophilicity of the ion-pairing complexes formed between [Ru(phen)2(dppz)]Cl2 and CPs.
2. Targeted and Reversible Nuclear Delivery of the DNA“Light-switching”Ru(II)-Polypyridyl Complex in Live-Cells via Ion-Pairing with Chlorophenolate Counter-Anions Coordination complexes that reversibly bind to DNA are becoming of increasing interest. However, the reversible-binding of metal-complexes with nuclear DNA in living-cells has not been observed so far. Here we show, unexpectedly, that [Ru(phen)2(dppz)]2+ already delivered to the nucleus can efflux to cytoplasm when cells were washed and then incubated with fresh culture-medium; and more interestingly, [Ru(phen)2(dppz)]2+ in cytoplasm can be redirected back to nucleus when CPs were reintroduced again. By using specific inhibitors, si-RNA and overexpression of ATP-Binding Cassette (ABC) transporter protein, we found that the efflux of nuclear [Ru(phen)2(dppz)]2+ is mediated mainly via three ABC transporter proteins (ABC-B1/C1/G2). Analogous reversible, but enantio-selective nuclear DNA-binding were observed with the two pure chiral forms of [Ru(phen)2(dppz)]Cl2. This represents the first report of reversible and controllable binding of DNA “light-switching” Ru(II)-complexes with nuclear DNA in living-cells via ion-pairing with chlorophenolate counter-anions. These findings should provide new perspectives for future investigations on using ion-pairing as an effective method for reversibly and controllably delivering other bio-active metal-complexes into their intended cellular targets.
3. Chiral Os(II) Polypyridyl Complexes as Enantioselective Nuclear DNA Imaging Agents Suitable for CLEM Studies
The high-resolution technique TEM, with OsO4 as the traditional fixative, is an essential tool for cell biology and medicine. Although OsO4 has been extensively used, it is far from perfect due to its high volatility and toxicity. Os(II) polypyridyl complexes like [Os(phen)2(dppz)]2+ are not only the well-known molecular DNA “light-switches”,but also the potential ideal candidates for TEM studies. Here we report that the cell-impermeable cationic [Os(phen)2(dppz)]2+ can be preferentially delivered into live-cell nucleus through ion-pairing with chlorophenlolate counter-anions, where it functions as an unparalleled enantioselective nuclear DNA imaging reagent especially suitable for correlative light and electron microscopy (CLEM) studies in both living and fixed cells, which can clearly visualize chromosome aggregation and de-condensation during mitosis simultaneously. We propose that the chiral Os(II) polypyridyl complexes can be used as a distinctive group of enantioselective high-resolution CLEM imaging probes for live-cell nuclear DNA studies.
4. Enantioselective and Differential FLIM Imaging of Nucleus and Nucleolus by the Two Chiral Enantiomers of Os(II) Polypyridyl Complex
The nucleolus is an important sub-nuclear structure, but very few dyes are available for nucleolar imaging. Here we show that the Λ-enantiomer of [Os(phen)2(dppz)]Cl2 can differentially distinguish the nucleolus from nucleus in living-cells with tetrachlorophenolate as counter-anion, while Δ-enantiomer can do so in fixed-cells by FLIM-imaging. Further studies with three specific metabolic inhibitors for nucleolar proteins synthesis found that the lifetime changes of the two enantiomers in nucleolus can reflect the alteration of cellular microenvironment which is related to the general pathological status of nucleolus. We then observed dynamical architecture changes of nucleolus, chromosome and spindle apparatus during cell differentiation by these two enantiomers. The chiral Os(II) complex shows many advantages as compared to the commercially available nucleolus dye Syto 9: it displays a much larger Stokes shift value with a near-red emission and a longer lifetime, it can image spindle apparatus during mitosis, and more importantly, it is enantioselective. To the best of our knowledge, this is the first report of the enantioselective and differential FLIM imaging of nucleus and nucleolus by the chiral Os(II)-dppz complex, which may hold much promise for live cell imaging of different organelles.
In conclusion, this dissertation studied the mechanism of reversible binding of ruthenium complex with live cell nuclear DNA, and we found that the nuclear uptake of [Ru(phen)2(dppz)]2+ was positively correlated with the binding-stability, but inversely correlated with the lipophilicity of the formed ion-pairing complexes; the efflux of nuclear [Ru(phen)2(dppz)]2+ is mediated mainly via three ABC transporter proteins (ABC-B1/C1/G2). We also found that the cell-impermeable cationic [Os(phen)2(dppz)]2+ can be preferentially delivered into live-cell nucleus through ion-pairing, where it acts not omly as an unparalleled enantioselective nuclear DNA dual-contrast agent suitable for correlative light and electron microscopy studies, but also as an enantioselective and differential FLIM imaging probe for nucleus and nucleolus. |
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