RCEES OpenIR  > 环境化学与生态毒理学国家重点实验室
钌/锇络合物与细胞核 DNA可逆结合机制及光-电成像研究
Alternative TitleMechanism of Reversible Binding with Nuclear DNA and Light– Electron Microscopy Imaging by Ru/Os Polypyridyl Complexes
黄蓉
Subtype博士
Thesis Advisor朱本占
2019-12
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学博士
Degree Discipline环境科学
Keyword钌(Ii)/锇(Ii)多吡啶络合物,氯代酚,细胞核摄取,光电关联成像,荧光 寿命成像 Ruthenium(Ii)/osmium(Ii) Polypyridyl Complex, Chlorophenols, Nuclear Uptake, Correlative Light And Electron Microscopy (Clem), Enantioselective Flim Imaging
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.

Pages160
Language中文
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/42215
Collection环境化学与生态毒理学国家重点实验室
Recommended Citation
GB/T 7714
黄蓉. 钌/锇络合物与细胞核 DNA可逆结合机制及光-电成像研究[D]. 北京. 中国科学院生态环境研究中心,2019.
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