Cell imaging mechanism of highly selective luminescent probe : cyclometalated iridium(III) complex (MIr-ITC) and enhanced cell growth on novel substrate : graphene oxides
一種高選擇性的環金屬化銥配合物探針 (MIr-ITC) 的細胞成像機制研究和氧化石墨烯促細胞生長檢測
Student thesis: Doctoral Thesis
The focus of interest in this thesis is the bioapplication of two promising materials, phosphorescent iridium(III) complex (MIr-ITC) and graphene oxides (GOs) that is currently one of the most popular carbon materials. This thesis also includes an investigation on the mechanism of MIr-ITC specifically labeling mitochondria of live cells, and a clear answer to the controversial question on the biocompatibility of GOs. Heavy-metal -based luminescent complexes, including iridium(III) complexes, have been widely used in the biological field in the past decade. As promising probe molecules, they can especially label almost all organelles, such as nuclei, lysosomes, and mitochondria. Such a fascinating feature has attracted more and more attention, but little information is available about the underlying mechanism of their particular accumulation inside cells, partially due to the limitation of chemical approaches. Herein, an ITC functionalized luminescent cyclometalated iridium(III) complex, [Ir(pq)2(phen-ITC)](PF6) (MIr-ITC) (Hpq = 2-phenylquinoline, phen-ITC = 1,10-phenanthroline-5-isothiocyanate), that efficiently and specifically labels mitochondria in living mammalian cells, is reported. Interestingly, a longer incubation with MIr-ITC results in significant mitochondrial shortening and fragmentation, but the release of cytochrome c associated with this event is not detected, which indicates the integrity of mitochondria. To better understand the mechanism of the interactions between MIr-ITC and intracellular molecules, gel electrophoresis for protein extracts in denaturing conditions is performed in combination with a gel scanner. The strong fluorescence confirms its covalent conjugating ability to its protein targets, which is rapid and highly selective, in a process that requires active cellular metabolism, as the conjugation is abolished at low temperature or in the presence of sodium azide (NaN3). Based on the measurements of the luminescence intensity, I have devised a biochemical fractionation procedure that allows the enrichment of the conjugated proteins, and their subsequent separation by two-dimensional gel electrophoresis (2DGE). Forty-six proteins have been identified by mass spectrometry. Many of the labeled proteins that are strongly luminescent are mitochondrial proteins, one of which is a voltage-dependent anion channel (VDAC1) that might be relative to mitochondria fission. An analysis of the secondary structures of the luminescent proteins reveals that lysine residues that border hydrophobic amino acids in both Helix and Strand structures are very important in conjugation with MIr-ITC. This finding demonstrates that the conjugation efficiency of MIr-ITC is not correlated with the frequency of lysine residues, but associated with the distribution of lysine residues on the protein. To the best of my knowledge, this is the first report on the molecular characterization of the interactions of a luminescent dye with its biological targets. As many biological dyes exhibit specific intracellular staining patterns, the identification of their molecular targets can help elucidate the mechanisms behind their staining specificities and cytotoxicity. I believe that this biochemical approach can be applied to identify the targets of a wide range of fluorescent and luminescent probes. With regards to another studied material; that is GOs, they have been extensively used as cell culture substrates for various types of cells due to their 2D nature with ultra-large surface areas. However, there exist multiple conflicting reports about the biocompatiblilty of GOs. To address this, I have conducted a study here to characterize their biocompatibility with mammalian cells. Cellular growth on GO-coated solid-state substrate grow better than on glass slide without GO. Also, GOs are shown to greatly enhance the attachment and proliferation of mammalian cells. HT-29 cells are able to grow on GO films to approximately 2.6 x 104 cells/well from an initial cell density of 1.0 x 104 cells/well in a 96-well plate, which indicates that the solid film-form of GO does not have intrinsic cytotoxic properties to mammalian cells, despite the fact that GO sheets in a more concentrated aqueous suspension are somewhat toxic to cell. GO aqueous solutions that induce the loss of cell viability are GO-size related in which larger sheets have better biocompatibility. This result significantly impels knowledge on the biological properties of GOs. Moreover, when stem cell cultures are compared on a glass surface, mesenchymal cell (MSC) on the GO-based composite coating adheres and proliferates at a faster rate and GOs expedite the MSCs differentiation by the formation of 3D aggregates known as embryoid bodies (EBs), which is an indicator of the beginning of differentiation. These data implicate the potentials of GOs as a platform for MSCs culture and diverse applications in tissue engineering and regenerative medicine.
- Iridium compounds, Oxidation, Luminescent probes, Graphene, Cell culture, Imaging systems in biology