Experimental Studies On Math1 Gene Transfected HEK293 Cells Mediated By Activated-PAMAM/CM-β-CD Nanoparticles

Most of the deafness mainly due to noise or ototoxic drugs which cause the damage of cells and structure of the inner ear. The process of damage always begins with the hair cell of inner ear(including the inner hair cell and the outer hair cell),then the organ of corti. Fundamental method of hearing loss treatment is hair cells regeneration or repearment of hair cell in specific pathological periods.Gene therapy is most hopeful way in all sorts of research of this field. The low-volume, fluid-filled chambers of the inner ear makes it become ideal system for gene therapy. Mathl gene is basic helix-loop-helix transcription factor (Mammalian atonal homolog 1), a mouse atonal homolog, Mathl gene is 1180bp which is sufficient for development of hair cells in certain types of cells. Meanwhile, Math1 gene play the key role in regeneration of hair cells.The gene vector is divided into two categories:virus vestor and the non-viral vestor, choosing a ideal carrier which efficiently take gene into cells of inner ear is key steps for gene therapy. Many researches demonstrated that virus vestor and non-viral delivery systems including cationic liposomes could bring gene into inner ear and express. There are, however, many disadvantages restrict the futher clinical application. Polyamidoamine(PAMAM), highly branched, well-control nanoparticle, has recently emerged as an alternative non-viral gene delivery system. Polyamidoamine has been studied for gene delivery processes in vitro and in vivo. Previously reported that researchers could require higher thansfection efficiency and lower cell toxicity of PAMAM dendrimer through modification.Our present study contains two parts:firstly, nanoparticles (Activated-PAMMA/CM-β-CD) was obtained by heating and modified with CM-β-CD Meanwhile, nanoparticles connected with pRK5-Math1-EGFP. Particle morphology was examined by transmission electron microscopy (TEM), Dynamic light scattering (DLS) was used to measure the hydrodynamic diameter and size distribution, The zeta potential of nanoparticles was measured with a zeta analyzer. DNA binding test were carried out with SDS and EcoR I. Secondly, We investigated the transfection efficiency of Activated-PAMMA/CM-β-CD-pRK5-Mathl-EGFP in HEK 293 cells. At the same time, the PAMAM, heating-PAMAM, Superfect, Ad--Mathl-EGFP and naked pRK5-Mathl-EGFP plasmid are sellected as positive controls. The green fluorescent protein (GFP) reported transfection test.RT-PCR and Western blot measured the expression of Math1. The cytotoxicity of Activated-PAMMA/C-β-CD-pRK5-Mathl-EGFP nanoparticles was evaluated via methylthiazolyl tetrazolium assay (MTT) test with cultured HEK293 cells. Conclusion:Activated-PAMAM/CM-β-CD-pRK5-Mathl-EGFP nanoparticles were spherical in shape, their size were 125nm, their zeta potential is 49Mv. The highest transfection efficiency of Active-PAMAM/CM-β-CD-pRK5-Mathl-EGFP nanoparticles in HEK293 cells was above 38%.The results of our studies showed that:Activated-PAMAM/CM-β-CD-pRK5-Mathl-EGFP nanoparticles can mediate Mathl-EGFP gene transfection in vitro cells. It is worthy of further fully researches in living animals and the possibility of future clinical application.