Metal-Polybenzimidazole Complexes As A Nonviral Gene Carrier:Synthesis And Their Roles In Gene Delivery

Gene therapy is a medical technology that can treat a disease by introducing an exogenous gene into the patient’s cells. As a new treat means, gene therapy attracts more and more attention of researchers. How to transfer exogenous gene into the patient’s cells is the key during gene therapy. Designing safe, effective and targeted gene carriers can solve the trouble. There are mainly two types of gene carrier:viral gene carriers and nonviral gene carriers. Viral carriers are more often applied for the clinical therapy currently. However, most viral carriers are highly toxic. Research efforts are currently focused on nonviral carriers, including mainly cationic polymers, lipids and peptides. The lower transfection efficiency and poor targeting of nonviral gene carriers limit their applications. But more and more their potential will be found by the researchers.Metal-Polybenzimidazole complexes we synthesized can induce DNA condensation under certain conditions. The DNA condensates can be transfer into cells, and the transfection efficiency is moderate.In this work, four Ca2+-Polybenzimidazole complexes with CTB, NTB, EDTB and two Mn2+-Polybenzimidazole complexes with DTPB have been synthesized. The properties on promoting DNA condensation of these complexes have been investigated. The four Ca2+ complexes have been selected as DNA-condensing agent to transfer the DNA into cells.The DNA-binding affinity of the complexes and their interaction mode were first examined, and the formation of condensates at different reaction condition was examined by UV-visible spectroscopy, viscosity experiments, gelose gel electrophoresis, RALS and DLS. The results show that the six complexes can condense DNA into the condensates with certain size, morphology and surface charge, and the size of the condensates gradually increased with the increase of the molar ratio of complexe to DNA and reaction time. Based on viscosity experiments and UV-visible spectra, it is speculated that the molecules of complexes close DNA and bind themselves to DNA by electrostatic interaction and π…π interaction is produced by the benzimidazole groups in the the molecules of complexes intercalating into base pairs of DNA when they close enough, and the complexes and DNA are bound close together this moment. Then, the morphology of the condensates was observed by TEM. We found that the loose aggregates got into homogeneous spherical and compact nanoparticles, and the spherical nanoparticles aggregated into significant dendritic aggregates further with the increase of the complexe concentration. With the existence of KCl the significant aggregates disaggregated into small ones, and KCl could inhibit DNA aggregation.The cytotoxicity of four Ca2+ complexes and their condensates have been detected by MTT method. The MTT data show that compared with [Cu(NTB)(H2O)]2+ complexe-DNA condensates and [Co(NTB)Cl] complexe-DNA condensates the cytotoxicity of Ca2+ complexe-DNA condensates is obviously reduced, and lower than it of the Ca2+ complexe. The cytotoxicity of both Ca2+ complexes and their DNA condensates is enhanced with the the increase of the molar ratio of complexe to DNA, and the cytotoxicity for normal cells (COS-7 cells) is lower than for cancer cells (HeLa cells and Hep G2 cells). Under inverted fluorescence microscope, that Ca2+ complexe/DNA condensate got into cell across the cell membrane was observed. The particles formed at molar ratio 1:2 of the complexe to DNA are in favor of transmembrane because of their appropriate size. Based on the cell transfection experiment by the luciferase (pGL3) and green fluorescent protein (pEGFP) expression method, we draw the conclusions that the genes transferred by the condensates can be expressed, although it is much less efficient than PEI under the same experiment conditions. The genes transferred by the condensate formed at molar ratio 1:2 of the complexe to DNA can be more efficiently expressed in the cells by adding the DOPE or appropriate amount of CQ. With or without the adding of DOPE or CQ, the genes transferred by the condensates are more efficiently expressed in normal cells than in cancer cells.