PEI Derivatives-mediated P53 And MiRNA Delivery To Achieve Anti-tumor Efficacy

Background: National cancer statistics released by the National Cancer Center show that malignant tumor deaths account for 23.91% of the national deaths.The morbidity and mortality of malignant tumors are continuously rising,and the medical expenses incurred exceed 220 billion yuan annually.The treatment of cancer has become a worldwide problem to be solved urgently.As an emerging anti-tumor technology,gene therapy uses gene transfer technology to(gene transfection is used to)introduce foreign normal genes into target cells to correct gene defects and abnormalities and restore normal expression.However,currently anti-tumor gene drugs are unstable during biological circulation and are easily degraded by nucleases in the blood.Nucleic acid molecules in the drugs are easily eliminated by macrophages in the blood circulation system,and meanwhile the tissue barrier penetration and selective enrichment of target positions are weak.Therefore,it is necessay to develop an effective and safe gene carrier for realizeing the efficient and stable delivey of therapetic genes in vivo.Common gene transfer vectors are mainly divided into two categories,viral gene vectors and non-viral gene vectors.Viral gene vectors can carry foreign genes and can be packaged into infectious virus particles to mediate the transfer and expression of foreign genes.However,viral gene vectors have serious immunogenicity,which greatly limits their application in the field of gene delivery.Common materials for non-viral gene carriers include liposomes or lipid complexes,cationic polymers,chitosan carrier,inorganic nanoparticles,and so on.Among them,polyethyleneimine has received extensive attention due to its unique advantages,including the following points: First,a large number of positive charges can easily assemble with negatively charged genes to form stable nanocomposites,protecting gene drugs from the degradation and being conducive to enter the cell.Second,it contains a large number of amino groups,which is convenient for the further modification and the improvement of transfection effiency.Third,it could easily achive the endosomal escape owing to the proton sponge effect,thereby releasing the loaded genes into the cytoplasm to perform their functions.However,polyethyleneimine still has many problems,such as low transfection efficiency and high cytotoxicity.Method: In response to the above problems,this article first uses carbodiimide chemistry to modify the low-molecular-weight polyethyleneimine(OEI1800)with lipoic acid to construct a class of lipoic acid modified polyethyleneimine derivatives with good biocompatibility,named LA-OEI.a derivative LA-OEI with good biocompatibility.The introduction of disulfide bonds not only realizes the reduction-responsive genes release releasing,but also achieves the intermolecular cross-linking,thereby increasing the molecular weight and positive charge density of the vector and greatly improving the gene transfection efficiency of the vector.The derivative LA-OEI was then used as a carrier to deliver the anti-tumor genes p53 and mi R-34 a.Using the human cervical cancer cell line He La as a model,the inhibitory effect and the mechanism of tumor cell proliferation,migration and invasion after gene delivery were systematically evaluated.Meanwhile,Michael addition reaction was employed to modify PEI25 K with N-isopropylacrylamide to synthesize the derivative PEN.The introduction of N-isopropylacrylamide can significantly improve the hydrophobicity of PEI25 K without reducing the positive charge density.Next,the anti-tumor efficacy was evaluated after the mi R-29 a transfection using human non-small cell lung cancer cell line A549 as a model,including the inhibition of cell proliferation,migration and invasion.Result: 1.LA-OEI-mediated delivery of p53 gene for cervical cancer treatment In this study,we first delivered the p EGFP-N3 and p GL-3 plasmids using LA-OEI as a vector,and this vector demonstrated favorable gene transfection efficiency.Using He La cell line(p53 wild type)as a model,the antitumor effect and the mechanism of LA-OEI/p53 transfection were systematically evaluated.Fluorescence Quantitative real-time PCR andWestern blot analysis showed that after the delivery of p53 gene was mediated by the vector LA-OEI,the up-regulation of p53 expression levels can be detected at the m RNA and protein levels.The vector LA-OEI mediated p53 gene delivery could detect the up-regulation of p53 expression levels at m RNA and protein levels.MTT cell proliferation evaluation,Live/Dead cell staining analysis and cell colony formation experiments have all shown that LA-OEI/p53 transfection can significantly inhibit the tumor cell proliferation,which was significantly better than that OEI/p53 group.The mechanism analysis has shown that it is closely related to the induction of apoptosis and S-phase arrest of the cell cycle,and that the induction of apoptosis depends on the activation of mitochondrial apoptotic signaling pathways.Finally,cell scratch healing and Transwell invasion experiments showed that LA-OEI-mediated p53 gene transfection can efficiently induce tumor cell migration and invasion infiltration.2.LA-OEI-mediated delivery of mi R-34 a for cervical cancer therapy In this study,we used LA-OEI as a carrier for mi R-34 a delivery,and evaluated the effect and mechanism of this nanocomplex on tumor cell proliferation,migration and invasion inhibition at the cellular level.After verifying the efficiency of mi R-34 a delivery,the transfection of LA-OEI/mi R-34 a nanocomplex can effectively induce the cell apoptosis and arrest the cell cycle at the G1 phase.The above two mechanisms are the key factors for tumors to produce significant anti-proliferative effects.In addition,LA-OEI-mediated mi R-34 a delivery can significantly down-regulate the expression levels of MMP-9 and Notch-1 and inhibit the migration and invasion of tumor cells.3.PEN-mediated mi R-29 a delivery for lung cancer therapy In this study,Michael addition technology was used to modify PEI25 k material by N-isopropylacrylamide,and a kind of derivative named PEN was constructed.In this modification,the introduction of N-isopropylacrylamide fragment significantly improved the hydrophobicity of the carrier material,while the amino density was not affected,so it was helpful to enhance the interaction of other cell membranes and improve the transfection efficiency.Taking low expression mi R-29 a in malignant tumor as the research object,efficient and stable transfection of mi R-29 a in cells was achieved by pen mediated mi R-29a delivery.The results showed that mi R-29 a could also inhibit the proliferation,migration and invasion of tumor,and its mechanism of action was studied in depth.Conclusion: We successfully synthesized two PEI derivatives through chemical modification,and constructed a class of cationic gene delivery vectors LA-OEI and PEN with high transfection efficiency and low cytotoxicity which were then employed to realize the successful delivery of p53 gene,mi R-34 a and mi R-29 a.This study provided a new strategy for the design and development of delivery vectors for malignant tumor gene therapy,and constructed a good foundation for the basic research and clinical application of PEI derivetives-mediated gene delivery.