Tumor Targeting Effect And Biological Distribution Of CRGD-PEG-siRNA Modified By Targeting Peptide And Polyethylene Glycol

Introduction:Small interfering RNA(small interfering RNA,siRNA)is a double-stranded RNA molecule with a length of about 19-23 base pairs that naturally exists in various organisms or artificially synthesized.siRNA has been shown to effectively downregulate gene expression in human cells through the mechanism of RNA interference(RNAi).Integrin αvβ3 receptors are only highly expressed in neovascularization and some solid tumors,such as gliomas.Meanwhile,abnormal expression of Epidermal growth factor receptor(EGFR)is a relevant gene for abnormal proliferation and differentiation of glioma.Our group used RGD cyclic peptide to bind specifically to integrin αvβ3 receptor to carry EGFR siRNA molecules into tumor cells and exert anti-tumor therapeutic effects.The preliminary results suggest that cRGD-siEGFR specifically targets glioma cells U87MG and is effectively taken up by U87 cells and plays a role in silencing EGFR mRNA and protein expression,but still faces the challenges of insufficient delivery efficiency,poor biodistribution and low concentration in tumor sites.Therefore,optimization and improvement of cRGD-siRNA molecules are needed to achieve the effect of improving its biodistribution,increasing the concentration of siRNA tumor target sites,and inhibiting the metabolic distribution in the kidney.Objectives:(1)Optimize the structure of siRNA molecules co-modified with targeting peptides and polyethylene glycol,and target EGFR to construct siRNA drugs;(2)Verify the biodistribution of cRGD-PEG-siRNA molecules;(3)In vitro functional validation of cRGD-PEG-siRNA molecules.Methods:1.The monovalent cyclic targeting peptide cRGDfk-Mal was synthesized from monovalent c(RGDfk),8-amino-3,6-dioxanoic acid(PEG)and 3-maleimidopropionic acid(MPA),The acid-sensitive mPEG-hdy-Mal macromolecule was synthesized from polyethylene glycol-aldehyde group(mPEG-CHO),p-aminobenzoic hydrazide,and 3-maleimidopropionic acid(MPA),.and then passed The maleimido propionic acid-sulfhydryl group is conjugated with the 5’ end of the sense strand and the 3’ end of the antisense strand of the siRNA to obtain a cRGD-PEG-siRNA molecule.Identification of its structure,pH responsiveness and serum stability by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and agarose gel electrophoresis.2.The cellular uptake kinetics and cytotoxicity of cRGD-PEG-siRNA were examined by flow cytometry,confocal microscopy and CCK-8,and these results were compared with the uptake efficiency and toxicity of cRGD-siRNA.3.Established a mouse orthotopic glioma model,and used a small animal imaging system to observe the dynamic distribution of cRGD-PEG-siRNA in intracranial orthotopic glioma nude mice.4.The in vitro biological activity of cRGD-PEG-siRNA wes evaluated by RT-PCR,Western blot,cell apoptosis assay,CCK-8 and EdU assay.Results:1.The two single-stranded synthetic siRNAs(righteous strand:A-siEGFR-SH;antisense strand:AS-siEGFR-SH)and cRGD-PEG-siRNA were synthesized using the EGFR sequences validated by the group in the previous stage,which were purified by HPLC,characterized by MALDI-TOF-MS mass spectrometry and agarose gel electrophoresis.The results showed that the constructed siRNA delivery system was structurally well-defined,stable in vitro,pH-responsive and suitable for subsequent experiments.2.In vivo toxicity and uptake assays and in vivo distribution showed that the acid-sensitive PEGylated cRGD-PEG-siRNA molecules could reduce the toxicity to renal tubular epithelial cells in vitro and could be effectively taken up by tumor cells in a low pH microenvironment.PEGylated modifications can also reduce the renal accumulation of siRNA molecules and prolong the circulation in vivo,allowing more molecules to accumulate in tumor tissues through the circulation and exert gene silencing effects.3.In vitro biofunctional validation experiments showed that cRGD-PEGsiEGFR molecule can effectively reduce the expression of EGFR mRNA and protein,which can inhibit the proliferation of tumor cells and promote the apoptosis of tumor cells.Conclusion:The cRGD-PEG-siRNA molecule was successfully synthesized by covalently coupling the cyclic peptide cRGD and acid-sensitive PEG molecules(molecular weight:2000)to the ends of the sense and antisense strands of EGFR siRNA.Our experiments show that this molecule can be effectively taken up by cells in the weak acid microenvironment of tumors,improve the stability of siRNA molecules,prolong the circulation time in vivo,inhibit the metabolic accumulation of siRNA in the kidney,and improve the efficiency of tumor-targeted delivery.