Targeted Multi-Gene Therapy Of Malignant Glioma Based On Superparamagnetic Fe₃O₄ Magnetic Nano-particles

Objective:To synthesize Fe_O4 magnetite nanoparticles(Fe₃O₄MNP) wich could be used in targeted glioma multi-gene therapy of malignant glioma.In this study,the newly nanotechnology and molecular biology were combined to develope a new glioma suicide gene therapy system base on superparamagnetic Fe₃O₄ magnetic nanoparticle gene vehicle and CD/5-FC system.Methods:Fe₃O₄MNP were prepared by thermal decomposition of Fe(acac)3 in 2-pyrrolidone,and 3-aminopropyltriethoxy-silane(APTTS) was used to modify the surface of the nanoparticles.Base on XRD,TEM, analysis,the nanoparticle size were tested.The Fe₃O₄MNP was evaluated as a kind of plasmid pCMVCD carrier and transfected into human glioma cell line U251 in vitro.The mRNA and protein expression of intracellular CD gene were tested by RT-PCR,Western blotting,and Immunofluorescent staining,respectively.Methods Methyl thiazolyl tetrazolium(MTT) method was used to detect the toxicity of Fe_O4MNP the proliferation of U251 cells in the presence of CD/5-FC system,and "bystand effect"of U251-CD.Results:APTTS-modified Fe3O4 magnetite nanoparticles prepared in this part,had a narrow particle size distribution(10±2nm),good crystallinity.DNA binding assay and co-sedimentation assay showed Fe3O4MNP could absorb most plasmid pCMVCD,when the m/m ratio of pCMVCD to Fe3O4 MNP was 4:1 at pH=7.4,the binding interaction also protected DNA against the digestion of DNase-â… and blood serum.The result of RT-PCR,Western blotting,and immunofluorescent staining revealed that intracellular CD gene levels continuously increased in a time-dependent manner after transfection of U251 cells with Fe3O4MNP-pCMVCD complex.MTT result showed Fe3O4MNP was safe to use in U251 gene tansfection in vitro,Fe3O4MNP/ CD/5-FC system could killed U251 cells effectively,the "bystand effect"show 10% U251-CD cells could killed 20-30%U251 and U251-CD mixed cells(p＜0.01 vs negtive control group),15%killed 50%mixed cells,35% killed 80%mixed cells,50%killed almost all cells in vitro.Conclusion:Fe₃O₄MNP could be used as one of the ideal gene carriers for CD gene delivery.U251-CD cells could transform 5-FC into 5-FU.Fe₃O₄MNP/CD/5-FC system can become a new method of brain glioma adjunctive therapy. malignant glioma.Try to increase chemotherapy effect of glioma by reversing the multi-drug resistance of glioma stem cells.Methods:pCDNA3.1-P16,pYD5-P53 and pCMVCD were transfected into U251 glioma cells by Fe3O4 magnetic nanoparticles. Methyl thiazolyl tetrazolium(MTT) method was used to detect the cell growth inhibition rate of U251 cells in the presence of 5-fluorocytosine, and growth inhibition rate of VM-26,VP-16,TMZ,5-FU to U251,U251-GCS cells.RT-PCR,Western blotting were used to detect target gene expression.G lioma stem cells was isolated from 30 different glioma tissue(WHO Gradeâ…¢orâ…£),and their apoptosis after different gene therapy was test by Annexinâ…¤-FITC/PI Apoptosis Detection Kits.Results:Glioma stem cells was isolated from 30 different glioma tissue sucessfully.wt-p53,wt-pl6 and CD gene were transfcted into U251 cells induced by Fe₃O₄ magnetic nanoparticles,and wt-p53,wt-p16 could increase the antitumor effect of CD/5-FC gene therapy system(p＜0.01), and the growth inhibition rate of VM-26,VP-16,TMZ,5-FU to U251 (p＜0.01),U251-GCS cells.Chemotherapy effect of TMZ was best. Apoptosis detection showed apoptotic rates increase in different gene therapy group,p＜0.01:CD/P53/P16 vs P53 and P16 group,CD /P53/P16/5-FC group(89.87±9.02%) better than other groups(p＜0.01).Conclusion:The combination of wt-p53,wt-p16 gene with CD/5-FC system had extremely good antitumor effect in vivo,reversed the multi-drug resistance of glioma stem cells and increased growth inhibition rate of VM-26,VP-16,TMZ,5-FU to U251 cells.Its antitumor Objective:To investigate the establishment of nude mice-subcutaneous glioma model induced by glioma stem cells,and its application feasibility in detecting the transfected effect of wt-p53,p16 and CD/5-FC gene therapy system base on Fe₃O₄MNP gene carrier in vivo,distribution of Fe₃O₄MNP in nude mice,and magnetic targeted localization in tumor.Methods:Human glioma stem cell- suspension isolated from different human glioma tissues,was inolulated to the 30 male nude mice subcutaneously.Distribution of Fe₃O₄MNP in nude mice was tested by atomic absorption Spectrometry.Pathological and immunohistochemical features of tumor were studied.TUNEL(TdT-mediated dUTP nick end labeling) was used to test cells apoptosis in tumor tissue after gene therapy.Results:Success rates of this methods were 100%,tumor grew in good condition,and the histological and immunohistochemical examination results of tumor were consistent with human glioma cell morphology.Glioma nodus could be found 14d after glioma stem cells were inolulated to nude mice subcutaneously.Under 4000 gauss magnetic field near the tumor,tumor Fer content(91.46±19.94μg/g) higher than control gruop(30.23±6.34μg/g)(p＜0.01),showed that Fe₃O₄MNP could lateralizaed to tumor under magnetic field guiding.Otherwise,Fer content of brain increased higher than control group(p＜0.01),showed that Fe₃O₄MNP could pass blood brain barrier without magnetic field guiding.Wt-p53,wt-p16 could decrease tumor volum and nude mice body weight lose.After intraperitoneal injection of 5-FC in glioma Bearing Nude Mice- BALB/c-CD/p53/p16,the tumor volume deflated grudually with 7d.TUNEL result showed glioma cells apoptosis increased in vivo after gene therapy.Conclusion:The combination of wt-p53,wt-p16 gene with CD/5-FC system may be more suitable for clinical therapeutic trials of suicide gene therapy for malignant gliomas.Its antitumor effect was correlation with increasing cell apoptosis just as the in vitro result.