Fabrication Of A Novel Tumor Acidity-Responsive Gene Delivery System And Evaluation Of Its SiRNA Delivery Efficiency

Since the discovery of RNA interference(RNAi)technique,drug research based on small interfering RNAs(si RNAs)has made great progress,which provides new ideas for gene therapy of cancer.However,the poor stability,ease of degradation by ribozyme in serum and short half-life in blood of naked si RNAs lead to their low transfection efficiency.The low tumor targeting efficiency and inability to rapidly and effectively escape from the endosomes of traditional gene delivery systems greatly limit the application of si RNAs in tumor therapy.Polyethylenimine(PEI)is a gold standard polymer with excellent transfection effect,but its severe cytotoxicity and non-degradability hinder its therapeutic application as a gene delivery carrier.In this study,by integration biodegradable polylactic acid(PLA)into PEI,a PEI-PLA copolymer was synthesized to improve the biocompatibility of PEI,and its si RNA delivery efficiency was verified at cellular level.Low pH insertion peptide(pHLIP)is a pH-dependent soluble polypeptide with transmembrane activity.As a nanosyringe,pHLIP could deliver membrane impermeable cargo molecules that attached to its C-terminus into tumor cells under weakly acidic conditions.In this study,pHLIP with specific cell membrane-penetration ability was conjugated to PEI-PLA copolymer,and pHLIP-PEI-PLA copolymer was synthesized for the first time.The si RNA delivery efficiency of pHLIP-PEI-PLA copolymer was verified at cellular level.This paper mainly includes the following two parts:In the first part,PEI-PLA copolymer was synthesized by incorporation of PLA into PEI.si RNA targeting PKM2 was bound to cationic PEI-PLA copolymer through an electrostatic interaction,resulting in the formation of PEI-PLA@si PKM2 polyplex.The delivery vehicle PEI-PLA copolymer was characterized by FTIR,GPC and 1H NMR,and the results showed that the copolymer was successfully synthesized.Gel retardation experiments indicated that si RNA completely bound with PEI-PLA copolymer when the N/P ratio is higher than 7.5.The serum stability test showed that PEI-PLA@si RNA could protect si RNA from nuclease degradation in serum within 72 h in 10% serum.The cytotoxicity of PEI and PEI-PLA copolymer was evaluated by MTT assay in three different cancer cell lines—human colon cancer cell line HCT116,human hepatocarcinoma cell line Hep G2,and human ovarian cancer cell line SKOV3.The introduction of PLA significantly improved the cytocompatibility of PEI and reduced its cytotoxicity.Cell internalization experiments,RT-PCR and Western blot tests confirmed that the PEI-PLA copolymer could effectively deliver si PKM2 into HCT116,Hep G2 and SKOV3 cells,and efficiently inhibit PKM2 expression at both m RNA and protein levels.The transfection efficiency of PEI-PLA@si PKM2 polyplex is equivalent to that of commercial transfection reagent Lipofectamine?2000.In the second part,pHLIP-PEI-PLA copolymer was synthesized by conjugation of pHLIP to PEI-PLA copolymer.GPC and UV-Vis spectrum analysis confirmed that pHLIP-PEI-PLA copolymer was synthesized successfully.pHLIP-PEI-PLA@si PKM2 polyplex was obtained by incubating pHLIP-PEI-PLA copolymer with si RNA targeting PKM2.Gel retardation experiments showed that si RNA completely bound with pHLIP-PEI-PLA copolymer when the N/P ratio is higher than 7.5.In addition,we investigated the in vitro stability of pHLIP-PEI-PLA@si PKM2 polyplex via serum stability tests.The results showed that pHLIP-PEI-PLA@si RNA polyplex could protect si RNA from nuclease degradation for 72 h and 12 h in 10% serum and 50% serum,respectively,indicating that pHLIP can further enhance the serum stability of PEI-PLA@si RNA polyplex.Delta Vision Elite high-resolution cell imaging system and flow cytometry analysis suggested that pHLIP-PEI-PLA copolymer could obviously improve the delivery efficiency of si PKM2 at pH 6.5.To sum up,we have successfully synthesized PEI-PLA copolymer and pHLIP-PEI-PLA copolymer,and they can effectively complexed with si RNA to form a polyplex.Cell experiments showed that both PEI-PLA and pHLIP-PEI-PLA copolymer could deliver si RNA to tumor cells with high efficiency.Introduction of pHLIP could remarkably improve the si RNA delivery efficiency of pHLIP-PEI-PLA copolymer under weakly acidic condition(pH 6.5).