Fluorescence Imaging And Antitumor Activity Of Novel Ruthenium Complexes

In this paper, a novel ligand MNAIP?2-?5-Methoxy-1-naphthyl?imidazo[4,5-f][1,10] phenanthroline? and their corresponding ruthenium?II? complexes [Ru?bpy?₂MNAIP]²⁺, [Ru?phen?₂MNAIP]²⁺?bpy=2,2?-dipyridyl,phen=1,10-phenanthroline? were synthesized and characterized. Further research work on the interactions between [Ru?phen?₂ODBIP]²⁺?ODBIP=3,4-dibromo-imidazo[4,5-f][1,10]phenanthroline? and DNA, tumor cell was carried out.The binding modes of these ruthenium?II? complexes to different types of DNA?including CT-DNA?pBR322-DNA?poly?dA-d T?-DNA?poly?d G-dC?-DNA?HTG-21 and bcl-2 DNA? were investigated by spectroscopic methods of UV/vis spectra, flourescence spectra, and agarose gel electrophoresis. The cellular imaging, anti-tumoractivities, apoptosis and cytotoxicity?in vitro? of these complexes on several cell lines?including BEL-7402, Hela, MCF-7, Hep-2 and HBMEC as contrast? were investigated in detail by H&E staining, Hoechst 33258 staining, flow cytometry and western blot analysis essays.The main contents are as follows:1. A novel ligand and its corresponding ruthenium?II? complexes [Ru?bpy?₂MNAIP]²⁺, [Ru?phen?₂MNAIP]²⁺ were synthesized and characterized by EA, IR and 1H NMR.2. We have studied and published some of the research results of interaction between [Ru?phen?₂ODBIP]²⁺?ODBIP=3,4-dibromo-imidazo[4,5-f][1,10] phenanthroline? and DNA. The complex was found to bind to G4-DNA by intercalation and it can cause the flourescence quenching severely in the following investigations. We also found out that Cu2+ can reduce the ability intercalation binding and the flourescence quenching with CT-DNA in the bimetalic system with complex and Cu2+. Cell experiments indicated that the complex showed good flourescence for cellular imaging, obviously inhibited the migration and movement of Hep-2 and demonstrated good antitumor activities on Hep-2. Western Blot analysis revealed that the expression of BAX increased while Bcl-2 protein decreased which induce the apoptosis of Hep-2 cells. [Ru?phen?₂ODBIP]²⁺ showed good cell uptake, flourescence imaging and antitumor activity.3. The results indicated that [Ru?bpy?₂MNAIP]²⁺ preferentially bound to DNA at adenine-thymine base pairs by intercalation and DNA-binding did not cause the flourescence quenching. The complex exhibited higher DNA cleavage efficiency under visible light irradiation than under UV light irradiation. The hydroxyl radical may play an important role in the DNA photocleavage. The complex with ligand bpy also showed good antitumor activities on BEL-7402 and it obviously inhibited the migration and movement of BEL-7402. By observing the Hoechst 33258 stainning, we found that complex could be gathered around the nucleus in low concentration, and it could enter into the nucleus with increasing concentration. It showed good flourescence for cellular imaging and apoptosis morphological characteristics can be founded obiviously. We speculated that the complex could induce the apoptosis of BEL-7402 cells by interacting with DNA in the nucleus. We found when the expression of BAX protein increased, the expression of Caspase-3 protein decreased, which induced the apoptosis of BEL-7402 cells.4. The results showed that [Ru?phen?₂MNAIP]²⁺ preferentially bound to DNA at guanine-cytosine base pairs by intercalation which strongly caused the flourescence quenching. The complex also exhibited higher DNA cleavage efficiency under visible light than UV light irradiation. Machenism experiments indicated that the superoxide anion radical may be responsible for the DNA photocleavage. Compared with [Ru?bpy?₂MNAIP]²⁺, [Ru?phen?₂MNAIP]²⁺ with the ancillary ligand phen showed good absorbing ability and more cytotoxicity to BEL-7402 cells. Almost all the complex concentrated in the cytoplasm which was observed by confocal laser scanning microscopy, indicating the complex with hydrophobic ligand phen can easily enter into the cells and induced the subsequent cell apoptosis. The pathway of apoptosis in BEL-7402 cells was due to the increased expression of BAX protein.