Targeting Gene Therapy Of NHE1 Antisense Gene Magnetic Nanoparticies On Gastric Cancer In Vivo

Background and Aims: Most traditionary gene therapies are not targeting therapy,which acutely holds back the rapid development of gene therapy.The key question is how to develop novel genetic carrier.With the development of nanotechnology, magnetic iron oxide nanoparticies provid new way for genetic carrier. The NHE1(Na+-H+ exchanger1) as a ubiquitous, integral membrane protein in human cell, is primarily responsible for the regulation of pHi (intracellular pH, pHi). Our earlier studies showed that NHE1 gene was relative to the occurrence and growth of gastric cancer. NHE1gene could be a good target for antisense gene therapy of gastric cancer. The study aims to construct NHE1 antisense gene magnetic nanoparticies with biological ,magnetic targeting and genetic therapy function,and explore the effects of the human NHE1 antisense gene transefection on the biological behaviours of SGC-7901 human gastric carcinoma cells.Then we will explore the feasibility of targetic gene therapy with magnetic nanoparticies by observing the targetic function and the depressant effect on the growth of gastic transplantation tumor in nude mice. Our study want to develop a new type of gene carrier and probe into a new gene therapeutic strategy for gastric cancer. Methods:The dextran coated iron oxide nanoparticles (DCIONP) was synthesized with deposition.The configuration and diameter of DCIONP was detected by transmission electron microscope and zetasizer.The potency of adsorbing NHE-1 antisense gene and resisting DNase I digestion of DCIONP was analyzed by agarose gel electrophoresis.The potency of orientation movement of iron oxide nanoparticles binding NHE-1 antisense gene was analyzed by attracting of magnetic field. In order to definite the best condition of transfection, the EGFP(Enhanced Green Fluorescent Protein) gene eukaryotic expression plasmid(pEGFP-N3) was transfected into SGC-7901 gastric carcinoma cells by DCIONP. NHE1 antisense gene eukaryotic expression plasmid and pcDNA3.1-Zeo was transfected into SGC-7901 gastric carcinoma cells by DCIONP. PCR was used to confirm the transfection.The effect of gene transefection on the expression of NHE1 protein, cells cycle distribution and apoptotic rates were measured.After the model of transplanted gastric cancer has been established in nude mice,magnetic field was applied to investigate the target distribution of NHE1 antisense gene magnetic nanoparticies in transplanted gastric cancer in nude mice. NHE1 antisense gene magnetic nanoparticies was injected through the caudal vein in nude mice.The tumor was exposed to the magnetic filed for 45 min. The tumor were taken out to calculate the tumor weight and the inhibitory effect on tumor growth. Results :(1)Transmission electron microscope showed that shape of nanoparticies is irregular.There is a core of ferric oxide inside nanoparticies. Under zetasizer ,the mean diameter of the DCIONP is 47 nm. (2)Agarose gel electrophoresis of binding experiment showed that pll-DCIONP has the satisfactory potential to adsorb DNA.(3)Protection experiment showed that pll-DCIONP can effectively protect DNA from DNaseI digestion.(4)Deposition experiment showed that the directional movement of pll-DCIONP binding plasmid under magnetic field in liquid.(5)Transfection experiment in vitro showed that the transfection efficiency was the highest(22.86 % ) when pll-DCIONP-DNA complexes prepared at a w/w ratio of 5:1.(6) The expression of NHE1 protein in Anti-7901 cells decreased in contrast to Zeo-7901 cells and SGC-7901 cells.(7) Apoptotic rates in Anti-7901 cells(11.751%) was markedly higher than that in Zeo-7901 cells(4.40%) and SGC-7901 cells(3.85%). Flow cytometric analysis displayed a slight decreased percentage of cells in proliferative index.(8)The Fe concentration in tumor tissue (79.38±8.64) was remarkably higher than that in control group(38.13±9.37,p<0.01).This showed that NHE1 antisense gene magnetic nanoparticies could directionally move and concentrate in tumor tissue with the magnetic field added.(9)The tumor weight of NHE1 antisense gene magnetic nanoparticies added magnetic field group(1.78±0.80) was less than those of NS and NHE1 antisense gene magnetic nanoparticies groups(2.65±0.62, 2.63±0.71),but there was not statistics difference. Conclusion: (1)We succeedly synthesized the dextran coated iron oxide nanoparticles,and the mean diameter is 47 nm.(2)We succeedly constructed NHE1 antisense gene magnetic nanoparticies.The dextran coated iron oxide nanoparticles modified with Poly-L-Lysine have the satisfactory potential to adsorb DNA and protect DNA from DNaseI digestion effectively.We also found the directional movement of pll-DCIONP bindingplasmid under magnetic field in liquid.(3)NHE1 antisense gene eukaryotic expression plasmid was transfected into SGC-7901 gastric carcinoma cells by DCIONP.(4) The NHE1 antisense gene therapy could significantly suppress the growth of SGC-7901 gastric carcinoma cells,partially reverse malignant phenotypes of SGC-7901 cells and induce cells apoptosis.(5) Guided by magnetic field, NHE1 antisense gene magnetic nanoparticies directionally moved and concentrated in tumor tissue.(6) Guided by magnetic field, NHE1 antisense gene magnetic nanoparticies has some anticancer effect on the tumor growth.(7) The results would open a new perspective for targeting and gene therapy of gastric cancer.