Study On A Novel Multifunctional Fe₃O₄@SiO₂-Glu-GNR Nanocomposite Carrier-mediated SiRNA Targeted Delivery And Tumor Therapy

Objectives:This study was designed to construct a novel multi-functional composite nanomaterial（Fe₃O₄@SiO₂-Glu-GNR）:the core is silica-coated ferroferric oxide@silicon dioxide（Fe₃O₄@SiO₂）,and the outer layer is Glu（p-aminophenyl-β-D-glucopyranoside）targeting molecule and is linked to gold nanorods（GNRs）.To explore the potential of this nano-targeting complex to be loaded with si RNA（small interfering ribonucleic acid）or protein,and as a multi-model targeted therapy for tumors.The aim of this study was to research the ability of the novel targeting drug-loading system to silence the targeting gene BRAF from melanoma（B16F10）,to select the mode of administration of the C57BL/6tumor-bearing mouse model,and to evaluate the effect of RNAi technology combined with photothermal therapy（PTT）on tumor treatment.Methods:1.Construction and characterization of Fe₃O₄@SiO₂-Glu-GNR composite nanocarriers:firstly,GNR with surface positively charged was prepared by seed solution growth method,which further modified by 11-mercaptoundecanoic acid（MUA）to make it be negatively charged as GNR-MUA;secondly,silica-coated ferroferric oxide（Fe₃O₄@SiO₂）was synthesized by template method,which was then modified by ethylene imine polymer（PEI）and polyethylene glycol derivative（maleimide-PEG-SCM）to conjugate glucose targeting molecule（Glu）.Finaly,a multifunctional nanocomposite carrier（Fe₃O₄@SiO₂-Glu-GNR）was constructed.The morphology of the material was observed by transmission electron microscopy;and the surface properties of the material was detected by potential and particle size analysis;and the plasmon resonance absorption of the material was observed by UV-visible absorption spectrum;and the si RNA loading capacity of the material determined by the gel retardation experiment.2.The quantity of BSA loaded on Fe₃O₄@SiO₂-Glu-GNR was detected by BCA method and B16F10 cells were transfected with Fe₃O₄@SiO₂-Glu-GNR-FITC-BSA.3.qPCR was used to detect the silencing effect of Fe₃O₄@SiO₂-Glu-GNR/cy3-siGAPDH complex on GAPDH,the silencing effect of Fe₃O₄@SiO₂-Glu-GNR/si BRAF complex on BRAF,and the fluorescence microscopy to detect the efficiency of Fe₃O₄@SiO₂-Glu-GNR/si BRAF complex entering the cell.4.Frozen section and fluorescence microscope were used to observe the targeted effect of Fe₃O₄@SiO₂-Glu-GNR/cy3-siGAPDH complex entering the tumor site under the action of external magnetic field by transdermal administration and intravenous administration.5.To establish a C57BL/6 B16F10 tumor-bearing mouse model,and Fe₃O₄@SiO₂-Glu-GNR/si BRAF was administered by transdermal administration and intravenous injection.The tumor site of the experimental groups of C57BL/6tumor-bearing mice was treated by external magnetic field and near-infrared excitation light of 808 nm,and the tumor growth conditions and treatment effect of these mice were observed regularly.Conclusion:1.The morphology of the material in the synthesis process was not changed obviously under the transmission electron microscope.And the potential of the material also changed with the synthesis stage;and the overall particle size of the final product was increased;and the UV absorption of the peak of the final product composite nanocarrier had a blue shift;and the si RNA loading was a mass ratio of material to si RNA of 20:1.2.The BCA method detected that the average loading of BSA on Fe₃O₄@SiO₂--Glu-GNR was about 12.47%,and Fe₃O₄@SiO₂-Glu-GNR-FITC-BSA could enter the cells.3.The results of qPCR showed that the efficiency of gene silencing was the same as that of lipo2000 when the concentration of Fe₃O₄@SiO₂-Glu-GNR/cy3-siGAPDH was 40μg/ml.The same concentration of Fe₃O₄@SiO₂-Glu-GNR/si BRAF was transfected into B16F10 cells,whose transfection efficiency under the fluorescence microscope was higher compared with lipo2000 and Fe₃O₄@SiO₂-PEI--GNR/si BRAF,and the m RNA silencing of BRAF was about 70%.4.The fluorescence microscopy results of the tumor sections of the two modes of administration showed that the external magnetic field can guide the enrichment of Fe₃O₄@SiO₂-Glu-GNR/cy3-siGAPDH at the tumor site,which has a certain targeting effect.5.Animal experiments had shown that Fe₃O₄@SiO₂-Glu-GNR/si BRAF is better for the treatment of melanoma under light and external magnetic field treatment.