Photo-thermal Effect Mediated Gene Therapy Research Of Polyethyleneimine Modified Hollow Gold Nanospheres

Hollow gold nanospheres(HAuNs)have a low toxicity,large surface area,good biocompatibility,easy to synthesize particle size from 30nm to 100nm,and ease of combining biological macromolecules.In this study we connect lipoic acid(TA)and polyethyleneimine(PEI 2kDa)which has a proton sponge effect to HAuNs to construct a HAuNs-PEI/pDNA complex nanoparticles gene delivery system.Since hollow gold nanospheres can efficiently convert light energy into heat by the Au-S covalent bond breakage in the near-infrared region,thus achieving efficient gene transfection and in vivo anti-tumor efficacy.Oxidation-reduction method using sodium borohydride(NaBH4)as a reducing agent,sodium citrate(CitrateNa)as a stabilizer prepared hollow gold nanoparticles of different size in the protection of argon(Hollow gold nanospheres,HAuNs).Carbodiimide(EDC)as a crosslinking agent for the synthesis of polyethyleneimine was coupled to lipoic acid(PEI-TA),and H1-NMR and infrared spectroscopy identified the chemical structure of graft,then through co-covalent bond to get the HAuNs-PEI.We obtained the measured particle size was about 100 nm,the surface potential of HAuNs-PEI was about 40mV;MTT Assay measured the IC50 of the vector was 203.1839ug/ml.Fluorescent plasmid DNA(pEGFP-C1)as a reporter gene detected the adhesion ability of the carrier with DNA and then selected a best ratio for in vitro transfection.In vitro transfection was carried in HEK 293,HeLa cell.PEI(2kDa)and PEI(25kDa)were as negative and positive control respectively,we also set the laser group and the non-irradiated laser group at the same time.Transfection experiments revealed that the transfection efficiency of HAuNs-PEI/pDNA containing GFP plasmid after 48h incubation significantly stronger than PEI(2kDa),and fluorescence expression of laser irradiation group which was close to PEI25000 at best transfection ratio was obviously higher than non-irradiated laser group.Cytotoxicity assay showed toxicity of HAuNs-PEI/pDNA exposed to laser irradiation was smaller than positive control,and it did not cause majority of cell death as well.We observed that whether near-infrared laser irradiation mediated endolysosomal escape and laser irradiation activated the release of DNA or not by confocal microscopy(CLSM)using rhodamine B(RhodamineB,RITC B)marked HAuNs-PEI,FAM labeled DNA,Hoechst33342 marked nucleus,Lysotracker labeled lysosomes.The research showed HAuNs-PEI/pDNA when exposing to laser irradiation can promote endolysosomal escape and accelerate the release DNA from vector.