Fabrication And Antitumor Efficacy Of A PEG-detachable Magnetic SiRNA Delivery Vector

Gene therapy treats gene-related diseases by introducing normal genes（exogenous）into targeted cells.Therefore,as a new tumor treatment modality,gene therapy has attracted extensive attention.In recent years,siRNA-based RNA interference（RNAi）technology has developed rapidly,which can specifically reduce or shut down the expression of target genes.Therefore,this technology has been widely used in the field of gene therapy.However,the naked siRNA is highly unstable,sensitive to serum degradation,and cannot penetrate the cell membrane.Therefore,researchers have developed various delivery systems to achieve efficient delivery of siRNA.Polymer-based delivery vectors have the advantages of high safety and ease of structural modification,making them the most investigated siRNA delivery vectors.Polyethyleneimine（PEI）can effectively bind siRNA,and its“proton sponge effect”can efficiently escape the endosome.However,the high cytotoxicity of PEI severely limits its biomedical application.PEGylation is a widely used strategy to improve the biocompatibility of the delivery systems and prolong their circulation time,but this process will hinder the cellular internalization,which is known as the“PEG dilemma”.Fe₃O₄nanoparticles have the advantages of ease of synthesis,small in size,ease of chemical functionalization,excellent biocompatibility and stability,and good magnetic responsiveness,so they display great application prospects in biomedical field.Based on the above research background,this paper designed and constructed a PEG detachable magnetic si PKM2nanodelivery system,which can significantly enhance the stability and delivery efficiency of si PKM2 under weakly acidic conditions,and exert antitumor efficacy by downregulating the expression of PKM2.This paper mainly includes the following three parts.（1）Synthesis and characterization of PEG-PEI/Fe₃O₄complex.Firstly,PEG-PEI copolymer was synthesized,and then PEG-PEI/Fe₃O₄complex was prepared by electrostatic interaction between PEG-PEI copolymer and negatively charged Fe₃O₄nanoparticles,and was characterized by TEM,SQUID and DLS,proving the successful synthesis of PEG-PEI/Fe₃O₄complex.Then,the results of RT-PCR and Western Blot indicated that PKM2 was significantly upregulated in colon cancer cell HCT116 at both m RNA and protein levels as compared to colon epithelial cell FHC.Therefore,HCT116cells with high PKM2 expression were employed in the following study.The cytotoxicity of PEG-PEI/Fe₃O₄complex was determined by MTT assay,and the result indicated that the incoporation of PEG significantly reduced the cytotoxicity of PEI.（2）Study on the complexation of PEG-PEI/Fe₃O₄complex with siRNA and its delivery efficiency.The complexation of PEG-PEI and PEG-PEI/Fe₃O₄complex with siRNA was determined by gel retardation assay.When the N/P ratio is greater than or equal to 5:1,the siRNA migration was completely retarded by PEG-PEI copolymer and PEG-PEI/Fe₃O₄complex.Agarose gel electrophoresis demonstrated that PEG-PEI/Fe₃O₄@siRNA complex had good serum stability.The cellular uptake of PEG-PEI/Fe₃O₄@siRNA nanocompplex by HCT116 cells was measured by confocal laser scanning microscope and flow cytometry.The results showed that the cellular internalization ability of PEG-PEI/Fe₃O₄@si PKM2 nanocomplex was significantly enhanced under magnetic field guidance and weakly acidic conditions,and the delivery efficiency of siRNA was markedly improved.In addition,RT-PCR and Western Blot confirmed that PEG-PEI/Fe₃O₄@si PKM2 nanocomplex could effectively downregulate PKM2 expression at both m RNA and protein levels under magnetic field guidance and weakly acidic conditions.（3）Evaluation of the in vitro antitumor activity of PEG-PEI/Fe₃O₄@si PKM2nanocomplex.The in vitro antitumor activity of PEG-PEI/Fe₃O₄@si PKM2 nanocomplex was investigated systematically by MTT assay,crystal violet staining,Annexin V-FITC/PI double staining apoptosis detection kit,glucose and lactic acid detection kit,wound healing and Transwell assays.In the presence of external magnetic field at p H 6.5,PEG-PEI/Fe₃O₄@si PKM2 nanocompplex could effectively inhibit the proliferation of HCT116 cells and trigger extensive tumor cell apoptosis.Moreover,the nanocomplex could significantly inhibit the glucose uptake of HCT116 cells and reduce the production of lactic acid,thereby suppressing glycolysis of tumor cells.In addition,PEG-PEI/Fe₃O₄@si PKM2 nanocomplex could inhibit the migration of HCT116 cells with high efficacy.To sum up,PEG-PEI/Fe₃O₄@siPKM2 nanocomplex was successfully synthesized in this study,and the nanocomplex displayed good serum stability;under weakly acidic conditions,PEG will detach from the nanocomplex and enter tumor cells effectively under the guidance of external magnetic field,thus promoting efficient delivery of si PKM2.Thus it is expected to solve the“PEG dilemma”.PEG-PEI/Fe₃O₄@si PKM2 nanocomplex exerted antitumor activity by downregulating the expression of PKM2.This study may provide a new strategy for the construction of p H and magnetic dual-responsive delivery systems,as well as a new idea for metabolism-targeted tumor therapy.