Design,Tumor-targeting And Pharmacodynamics Of RGD-siRNA Conjugate For Inhibiting Intracranial Glioblastoma In Vitro And In Vivo

Introduction:Small interfering RNA(siRNA)can specifically silence the expression of target mRNA(Nobel Prize in Physiology and Medicine in 2006),and siRNA drugs have completed phase III clinical trials and have the prospect of treating diseases.Compared with nanoparticles,siRNA conjugates have the advantages of easy preparation,easy targeting,and low side effects.The integrin αvβ3 receptor is only highly expressed in neovascular and some solid tumors,such as gliomas.The cRGD(arginine-glycine-aspartate)can carry its molecules into the cell via the αvβ3 receptor.The abnormal activation of PI3K/Akt/mTOR pathway in gliomas blocks this pathway and inhibits tumor growth.Objectives:The bivalent biRGD group and siRNA backbone were optimized,and PI3K was used as a target to construct a siRNA for treating gliomas.Methods:1.Use RT-qPCR,serum incubation experiments,cell cycle and apoptosis experiments to screen the best PI3K subunits and their siRNA sequences for glioma activity,and use 2’-methoxy to modify its backbone structure.2.Use of c(RGDfk)with 6-aminocaproic acid(Ahx),glutamic acid(E)(and/or without)arginine(R),8-amino-3,6-dioxaoctanoic acid(PEG)Different biRGD intermediates were synthesized and conjugated to the 5’-end of the sense strand of siRNA by maleimidopropionate-thiol to obtain different biRGD-siRNAs and identify their structures.3.The biRGD-PIK3CB siRNA was synthesized by using flow cytometry and confocal microscopy to screen the biRGD-siRNA with the best cell uptake activity.4.The in vitro biological activity of biRGD-siPIK3CB was evaluated by RT-qPCR,Western blot,cell cycle assay,apoptosis assay and transwell assay.5.The in vivo dynamic distribution of biRGD-siPIK3CB in subcutaneous and intracranial in situ glioma nude mice was observed using a small animal imaging system.6.Establish an in situ glioma model to investigate the dose-dependent pharmacodynamics and mechanism of biRGD-siPIK3CB.The optimal dose of biRGD-siPIK3CB was combined with Gelofusine(succinyl gelatin injection)to compose the compound preparation,and its influence on the survival time of tumor-bearing nude mice was examined.Serum ALT levels were measured to evaluate hepatotoxicity,IL-6 and IFN-β levels to assess immunogenicity,and HE staining was used to analyze nephrotoxicity.Results:1.The PI3K P110β subunit(PIK3CB)is the best subunit that affects the growth activity of gliomas.Its framework sequence is:sense strand(5’-3’):AC(mC)(mU)G(mU)GGAUACUCAU(mU)A(mA)dTdT;antisense strand(5’-3’):U(mU)AA(mU)GAGUAUCCA(mC)AG(mG)(mU)dTdT.2.Obtain E-2Ahx-[c(RGDfk)]2-PEG-MPA-siRNA(biRGD 1-siRNA)and E-2R-2Ahx-[c(RGDfk)]2-PEG-MPA-siRNA(biRGD2-siRNA)The molecule,biRGD2,has two more arginines than biRGD,with a purity of more than 90%.The biRGD 1-siRNA was the most active structure for tumor uptake and obtained biRGD1-PIK3CB siRNA(biRGD-siPIK3CB).3.In vitro,biRGD-siPIK3CB can silence PIK3CB expression(P<0.05),block cell cycle progression(P<0.05),promote apoptosis(P<0.05),and inhibit cell invasion(P<0.05).4.Subcutaneous glioma model:The tumor accumulation of biRGD-siRNA was higher than that of cRGD-siRNA and up to 72 hours,while the naked siRNA did not accumulate.In situ glioma model:biRGD-siRNA targets intracranial and is taken up by gliomas.The composition of Jiale Shi compound preparation can reduce the accumulation of biRGD-siRNA in the kidney,but does not affect its tumor targeting.5.In situ glioma model:The dose of biRGD-siPIK3CB higher than 1 nmol/20g can significantly inhibit the growth of glioma(P<0.05),and significantly inhibit PIK3CB expression and cell proliferation activity and promote apoptosis(P<0.05),to extend survival.The molecule has no hepatotoxicity and systemic immune response.The composition of compound preparation with Gelofusine can reduce the renal interstitial edema caused by biRGD-siPIK3CB,but does not affect its efficacy.Conclusion:E-2Ahx-[c(RGDfk)]2-PEG-MPA-siPIK3CB(biRGD-siPIK3CB)can target in situ gliomas,inhibit tumor growth by inhibiting the expression of PIK3CB,prolong survival,and have no hepatotoxicity or immunogenicity.The composition of the compound preparation with Gelofusine can reduce its kidney side effects.