Dinuclear Metal(Ⅱ) Complexes Of Polybenzimidazole Ligands As Carriers For DNA Delivery

Gene therapy is the insertion of genes into an individual's cells and tissues to treat disease, which is highly dependent on the recent research results of modern medicine and molecular biology technologies. Although the technology is still in its infancy, it has been used with some success. For example, Rosenberg et al performed the first human gene therapy trial in 1989. It is believed that scientific breakthroughs will continue to move gene therapy toward mainstream medicine.The biology of human gene therapy is complex and the related techniques are required to be further developed. Providing effective and safe carriers remains a major challenge for gene delivery so far. There are mainly two types of gene vectors. They are viral vectors and nonviral vectors. Viral vectors exhibit high effciency at delivering both DNA and RNA to numerous cell lines. However, limitations with viral approaches include small cargo capacity, resistance to repeated infection, difficulty in production and quality control, and low safety. For these reasons, research attention has been attracted to nonviral approaches. Making use of synthetic chemical carriers has the potential to overcome many of inherent limitations or challenges of viral carriers.In this work, we made an attempt at designing new nonviral gene delivery systems. It comprise new classes of defined components which might overcome the fundamental barriers by using the dinuclear metal(II) complexes. The main results are as follows:1. Two polybenzimidazole ligands: EGTB(N,N,N',N'-tetrakis(2'-benzimidazol- 2-ylmethyl)-1,4-diethylene amino glycol ether) and DTPB(1,1,4,7,7-pentakis(2'-benzi midazol-2-ylmethyl)triazaheptane) have been prepared, and six metal complexes with EGTB or DTPB have been synthesized.2. The affinity of each complex for DNA was detected by UV-Visible absorption under neutral conditions. The observed results showed that the binding capability of different complexes for DNA is closely similar. It indicates that intercalation between the ligands of the complexes and base pairs of DNA may be the key force to drive the binding process. The conclusion was supported by two lines of evidence provided by both exclusion assays of EtBr bound to DNA and fluorescence quenching of complexes. In order to explore the different forces to drive the binding of the dinuclear metal(II) complexes to ctDNA, we measured the affinity of each complex and ssDNA. The results showed that the electrostatic interaction was prerequisite for the dinuclear complexes to induce DNA condensation.3. The condensation behavior of ctDNA induced by polybenzimidazole complexes was examined by gelose gel electrophoresis, RALS and DLS at different condition. The results showed that the DNA condensation inducing by complexes happened only at pH 6-8. As shown by TEM images, the size and morphology of the DNA condensate are abundant, and their polymorphism is significantly dominated by concentration of the reactant and incubation time. The DNA condensates are packed more and more compactly with prolonging incubation time or increasing complex concentration. Different condition leads to different morphology of DNA condensates. The condensates mainly include loose assemblies, globular nanoparticles, amorphous, web-like morphology.4. We proposed a reasonable model for the inductive role of the dinuclear metal(II) complexes of polybenzimidazoles in DNA condensation. Firstly, the complex molecules bind to DNA respectively. Then the DNA nature structure collapsed. It depended on the negative charge of DNA neutralized by the positive charge of complexes. Further more, the DNA complexes formed individual globular DNA condensates or conglomerate driven by the intermolecularπ–πcontacts. The conglomerates contain several DNA globular DNA condensates.5. By using the sameλDNA as the TEM experiments, cellular uptake experiment was carried out in Hep G2 cell line. The results showed that the DNA condensates could enter into the cytoplasm and distributed around the nuclei. Qualitatively, the cellular uptake efficiency of the DNA condensates provided by the dinuclear Co²⁺ complexes 3 and 4 is higher than that by the Cu²⁺ counterparts 1 and 2 at the same dose. We examined the expression of DNA condensates in Hep G2 cells. Hep G2 cells were incubated with the condensates containing luciferase-encoding plasmid pGL3 control vector. The results showed that the tested factors can significantly dominate the transfection efficiency of DNA condensates. However, the dinuclear Co²⁺ complexes 3 and 4 exhibited higher transfection efficiency at the high doses compared to the Cu²⁺ counterparts 1 and 2.