Research On Enzyme-sensitive Tumor Targeting SiRNA Delivery System

Objective:RNA interference technology(RNAi)exerts its therapeutic effect by regulating the expression of the target gene and provides a new method for cancer treatment.However,the physicochemical properties of siRNA make it difficult to penetrate the cell membrane to exert effective pharmacodynamic properties.Many delivery systems have been developed to improve the delivery of siRNA,but most of them still have problems such as low transfection efficiency and off-target effects.Therefore,we constructed a dual enzyme-sensitive prodrug-based targeted siRNA delivery system(siRNA-LMWP-Linker-Antibody),where cell penetrating peptide was introduced to facilitate transmembrane delivery,meanwhile,targeted delivery and site-specific release of siRNA are achieved by antibodies and tumor enzymes.Content:Our designed drug delivery system,siRNA-LMWP-Linker-Antibody,has chosen a highly effective macromolecular nucleic acid drug siRNA.Then,cell penetrating peptide(LMWP)was introduced to delivery siRNA into cell.After that,the prodrugs and antibodies are linked by a substrate peptide which is overexpressed on the surface of cancer cells.When antibody-mediated system targeting colon cancer cells,tumor cell surface-specific enzyme legumain can act as an activating factor to cut off the corresponding linker,release siRNA-LMWP,reactivate the cell penetrating properties of the CPP and finally improve target gene silencing efficiency.Methods:Firstly,a novel prodrug siRNA-S-S-PEG-LMWP was developed to effectively solve the problem of aggregation and precipitation of the system.After that,the prodrug was covalently coupled to the antibody via an enzyme-specific substrate peptide to construct siRNA-LMWP-Linker-Antibody drug delivery system.A series of characterizations of synthetic prodrugs and delivery systems were performed,and key scientific issues such as antibody specificity,tumor-enzyme responsiveness,and gene silencing efficacy of the delivered delivery systems were examined.1.Bifunctional PEG derivatives were used to covalently link cell penetrating peptides to siRNAs.The prodrug was prepared by three different synthetic and purification methods,and the yield of each route was analyzed to optimize the synthesis scheme.Prodrugs were investigated by mass spectrometry,gel electrophoresis,particle size and zeta potential.The cellular uptake capacity of the prodrug was also investigated.2.Fusion peptide,consisting of the CPP and substrate peptide,was covalently linked to a thiolated siRNA to obtain a prodrug.Then they were activated with EDC / NHS to react with the antibody to form the siRNA delivery system siRNA-LMWP--AANL-Antibody.The products were verified by gel electrophoresis.3.The serum stability,targeting,enzyme sensitivity and pharmacodynamics of the delivery system were studied.In vitro specific binding of antibody and cellular uptake of "prodrugs" were qualitatively tested in HCT116,SW620,HT1080 cells.The cleavage of the substrate peptide in the delivery system were also verified.The delivery system gene silencing ability was qualitatively observed with a laser confocal scanning microscope,while Western Blot was used for semi-quantitative validation.4.In vitro MTT assays examined the safety of the delivery system.The universality of the constructed system was examined by the enzyme-responsiveness test of the MMP-2 enzyme-sensitive delivery system.Results:1.Among three synthetic methods,the one using hetero-bifunctional NHS-PEG-OPSS as a crosslinker had the hightest yield of 48%.The molecular weight of prodrug detected by MALDI-TOF and its agarose gel electrophoresis behavior confirmed the successful synthesis of prodrugs.2.Agarose gel electrophoresis was performed to confirm th at the enzyme-sensitive prodrug targeted delivery systems of siRNA-LMWP-AANL--Antibody were successfully constructed.3.The drug delivery system can protect the siRNA from degrading by serum endonucleases without affecting the target performance of the antibody,and can specifically response to legumain.Good gene silencing after internalization of the drug delivery system were observed.4.The cytotoxicity results confirmed that the delivery system was relatively safe.The MMP-2 enzyme-sensitive delivery system produced good enzyme responsiveness,indicating that the constructed delivery system has certain universality.Conclusion:The enzyme-sensitive prodrug targeting delivery system was successfully constructed,which provides a platform for targeted delivery of nucleic acid macromolecular drugs such as siRNA.Moreover,a number of techniques adopted in the study are universal,which can be extended to other drug delivery systems and provide theoretical support and technical reference for the clinical transformation.