Construction Of Aptamer-functionalized Nanoparticles For Codelivery Of Fasudil/miRNA-195 And The Study On The Treatment Of Hepatocellular Carcinoma

Part one:Construction and characterization of aptamer-functionalized nanoparticles for codelivery of fasudil and miRNA-195Aim:Angiogenesis and vasculogenic mimicry(VM)play an important role in the development and progression of hepatocellular carcinoma(HCC).In this study,we used a new cationic peptide H3R5 as a reducible vector,aptamer ST21 with specific binding to HCC cells to conjugate to peptide H3R5 as the targeting probe,miRNA-195(miR195)as a powerful gene drug to inhibit VEGF,and Fasudil to suppress VM via blocking Rho kinase,all of which were simultaneously encapsulated in the same NPs(FasudilST21-H3R5-PEGmiR195.FasudilSHPmiR195).In this study,the physiologic characteristic was summarized.Methods:H3R5 and stearyl-H3R5 were synthesized by solid phase peptide synthesis.After the purification of high performance liquid chromatography(purity>95%),the final product was identified by mass spectrometry and the molecular size was determined.The-SH groups of stearyl-H3R5 were oxidized to disulfide linkage to get SHP with the oxidation of low concentration of H2O2.Different proportions of aptamers ST21 and mPEG-MAL was used to controlled bridging degree.1H NMR was used to identify the synthesis results of SHP s.Subsequently,fasudil was encapsulated by the method of ammonium sulfate gradient,and miR195 was condensed via electrostatic force.The particle size and potential of different nanocomposites were determined by Zeatsizer Nano.The encapsulation ability and serum stability were investigated by agarose gel electrophoresis.The in vitro release of nanocomposites was evaluated with the treatment of different concentrations of DTT.Results:A stable nanocomposite can be formed with different proportions of fasudil and miR195,which are wrapped in SHP.The particle size of FasudilSHPmiR195 was dynamically distributed.The average particle size was 133±9.9 nm(polydispersity coefficient(PDI)=0.297)and the zeta potential was 14.4±1.8 mV at N/P=7.5.The nanocomposites are intact and nearly spherical.The appropriate potential and particle size ensures that it can be assimilated largely.Due to DTT has a strong reducing capacity,SHP will be reduced to monomer and the ability to wrap miR195 has declined.Conclusion:Disulfide bridged SHP can effectively encapsulate fasudil and miR195 to the cells,but also to ensure that the drug quickly dissociated to play a curative effect when nanocomposite into the cell.Part two:In vitro study of aptamer-functionalized nanoparticles for codelivery of fasudil and miRNA-195 targeting HCCAim:To investigate the effect of targeted,non-targeted nanocomposites on anti-hepatocarcinoma in vitro.Methods:The uptake of ST21 in vitro was determined by normal cells L02 and hepatocellular carcinoma cells SK-Hep-1 with the treatment of targeted and non-targeted nanocomposites.pDNA(pGL3 and pEGFP)were used as reporter genes to evaluate the transfection efficiency of SHPpDNA in HCC cells(SK-Hep-1).The mechanism of FasudilSHPmiR195 was studied under 4?,37? and different inhibitors.The effect of different nanocomposites on cell migration ability was investigated by wound healing and on cell formation ability was evaluated by test of formation of tubular network structures on matrigel in vitro.Western blot was used to investigate the blocking effect of nanocomposites on the related proteins ROCK2 and VEGF.Resuluts:Compared with non-targeting vectors,the uptake of the targeting vector in HCC cells was significantly increased(*P<0.05).The uptake of FasudilSHPmiR195 in 37 ?was significantly higher than that of 4 ?.FasuailSHPmiR195 was almost not taken up by SK-Hep-1 due to the competitive inhibition with excess aptamer ST21.The uptake of nanocomposites was significantly reduced with the treatment of CPZ?Filip and NaN3.Compared with FasudilHPmiR195,FasudilSHPmiRi95 can inhibit cell migration and tube formation in a greater degree.Compared with FasudilHPmiR195,FasudilSHPmiR195 had a greater effect on the inhibition of ROCK2 and VEGF protein expression(**P<0.01).Conclusion:The modification of aptamer ST21 enhance the anti-HCC effect.The dominant pathway of cellular uptake of FasudilSHPmiR195 was clathrin-mediated endocytosis and caveolae-mediated endocytosis,while micropinocytosis contributed little to the cellular uptake.Part three:In vivo study of aptamer-functionalized nanoparticles for codelivery of fasudil and miRNA-195 targeting HCCObjective:To investigate the distribution of FasudilHPmiR195 and FasandilSHPmiR195 ta in SK-Hep-1 tumor-bearing nude mice model,and evaluate the pharmacodynamics of these system.Methods:SK-Hep-1 cells were inoculated in nude mice by subcutaneous injection to construct tumor-bearing mice.The in vivo distribution of nanocomposites in nude mice was observed by using near infrared fluorescent dye Cy7 labeled HP and SHP.FasuailHPmiR195 and FasandilSHPmiR195 were administered consecutively four times for every four days via tail vein and the body weight,major diameter and minor diameter were recorded every three days.Cervical dislocation was used to sacrifice mice on day 21,the tumor was separated and weighted.Results:Compared with the non-targeted nanocomposite FasudilHPmiR195,the targeted nanocomposite FasandilSHPmiR195 can accumulate in the tumor site for a long time.The fluorescence of the tumor is the strongest in the isolated organs.The change of tumor volume and weight in control group,HP group and SHP group is most obvious and tumor growth is almost not inhibited.FasandilSHPmiR195 had the most significant reduction in tumor volume(#p<0.01 vs FasudilHPmiRi95)and tumor weight(#p<0.05 vs FasudilHPmiR195),and had the strongest inhibitory effect on tumor growth.Conclusion:SHP is biocompatible and the nanocomposite FasandilSHPmiR195 has high safety in vivo.FasandilSHPmiR195 has better effect on anti-HCC with the modification of aptamer ST21.