Tumor Acidity-sensitive PEG-PEI Copolymer For Targeted Delivery Of SiRNA

RNA interference(RNAi)is a sequence-specific gene regulation mechanism,which is triggered by double stranded RNA(ds RNA)and inhibits gene expression by endogenous micro RNA(mi RNA)or synthetic small interfering RNA(siRNA).RNAi has become an effective means of cancer treatment,which can inhibit almost all genes.However,several characteristics of naked siRNA such as large molecular weight and volume,unable to effectively penetrate the cell membrane,sensitive to ribozyme,easy to be cleared by the kidney,potential immunogenicity and off-target effect,seriously hinder its application in tumor therapy.Therefore,researchers have developed a variety of delivery vectors for the effective delivery of siRNA.Among them,cationic polymers can bind and compress nucleic acid molecules to form stable nanocomplexes,exhibit excellent transfection efficiency,and have become the most widely used nonviral gene vector.In this study,the gold standard cationic polymer polyethylene imine(PEI)with high transfection efficiency was selected and conjugated with polyethylene glycol(PEG)to obtain the copolymer PEG-PEI,which electrostatically compact siRNA targeting S100A4 into stable PEG-PEI@siS100A4nanocomplex,and its antitumor activity was verified at cellular level.This study mainly includes the following three parts:(1)PEG-PEI copolymer was synthesized,and its molecular weight and structure were characterized by GPC,FTIR and ~1H NMR.~1H NMR results showed that the benzoic imine bond of PEG-PEI copolymer would break under weakly acidic environment(p H 6.5),which made PEG detach from PEG-PEI copolymer,indicating that the copolymer has good p H-responsive ability.And the cytotoxicity of PEG-PEI against human colon cancer cell line(HCT116)and human ovarian cancer cell line(SKOV3)was determined by MTT assay.The results showed that the introduction of PEG reduced the cytotoxicity of PEI and improved its biocompatibility.(2)Gel retardation test suggested that complete binding of siRNA to PEG-PEI copolymer was observed when the N/P ratio was higher than 5:1,and the particle size and zeta potentialmeasurements showed that PEG-PEI@siRNA nanocomplex was successfully prepared.After incubation with the cell medium containing 10%or 50%serum for 72 h,the siRNA band did not degrade significantly,indicating that the nanocompplex has good serum stability and could significantlyprolong the circulation time of siRNA.The Cy5-labled siRNA was employed to fabricate the nanocomplex,and its cellular uptakeby HCT116 and SKOV3 cells was monitored by high-resolution living cell imaging system and flow cytometry.The results showed that the nanocomplex could effectively deliver siRNA into the cells at p H 6.5,strong red fluorescence was detected in both tumor cells,and the cellular uptake of the nanocomplex by two tumor cells was significantly enhanced.Moreover,the nanocomplex dramatically downregulated the expression of S100A4 at m RNA and protein levels under weakly acidic condition as determined by RT-PCR and Western blot.(3)Firstly,the inhibitory effect of PEG-PEI@siS100A4 nanocomplex on proliferation of SKOV3 cells was analyzed by Calcein AM/PI double staining,Ed U staining and CFDA-SE labeling.The results showed that the nanocomplex significantly suppressed the proliferation of SKOV3 cells under weakly acidic condition.Secondly,Annexin V-FITC/PI double staining results indicated that PEG-PEI~@siS100A4 nanocomplex markedly elevated the apoptotic rate of SKOV3 cells at p H 6.5.In addition,western blot analysis suggested that PEG-PEI@siS100A4 nanocomplex significantly reduced the expression of Ki67 and PCNA(cell proliferation marker),and remarkably upregulated the expression of p53(pro-apoptotic protein)under weakly acidic condition.The results indicated that the nanocomplex exerted antitumor effect via inhibiting tumor cell proliferation and inducing tumor cell apoptosis.Finally,wound healing and transwell assays confirmed that the PEG-PEI@siS100A nanocomplex notably inhibited SKOV3 cell migration in a p H-dependent manner.To sum up,we successfully synthesized PEG-PEI copolymer by conjugating PEG and PEI via acidity-responsive benzoic imine bond.PEG-PEI@siS100A4 nanocomplex was fabricated by complexation between PEG-PEI copolymer and siRNA targeting S100A4.This strategy is favorable for overcoming the“PEG dilemma”,and improving the stability of the nanocomplex and the transfection efficiency into tumor cells,which is of great significance for stable and efficient siRNA delivery in vivo.