Dual-responsive Nanoparticles For Targeted Delivery Of Gene And Chemotherapeutic Drug To Treat Hepatocellular Carcinoma

ObjectiveHepatocellular carcinoma(HCC)is one of the most common malignant tumors.Chemotherapy plays an important role in the treatment of HCC.In recent decades,it has been well established that cancer occurrence and development is a multi-factor,multi-gene,and multi-step process involving structural and expression abnormalities of many related genes.Therefore,gene therapy is becoming increasingly known as a potent strategy for cancer treatment.In this research,a dual-responsive nanoparticle system was designed based on the oxidized pullulan(oxPL)and disulfide-containing poly(β-amino)ester(ssPBAE)for efficient delivery of gene and chemotherapeutic agent through polymer degradation responding to the tumor intracellular pH and redox state.ContentsThe main content of this research were divided into three parts.The first part was the preparation and characterization of polymeric prodrug of ssPBAE-oxPLDOX,including the synthesis and chemical structure confirmation of ssPBAE,oxPL and ssPBAE-oxPL-DOX.The second part was the in vitro study of ssPBAE-oxPLDOX/pDNA nanoparticles,including the preparation and characterization of ssPBAE-oxPL-DOX/pDNA,the evaluation of pH-and redox-responsive releases of DOX and pDNA from nanoparticles,and their cytotoxicity,cellular internalizations,cell apoptosis and cell cycle arrest in human hepatoma HepG2 cells.The third part was the in vivo study of ssPBAE-oxPL-DOX/FAM-DNA nanoparticles,including their biodistribution,and hepatoma targeting in HepG2 tumor-bearing mice.Methods1.The preparation and characterization of polymeric prodrug of ssPBAE-ox PLDOX nanoparticles: ox PL was synthesized through the sodium periodate oxidation of pullulan and characterized by IR and 1H NMR,the aldehyde contents of ox PLs were determined by the hydroxylamine hydrochloride/ potentiometric titration method.The average molecular weights of pullulan and oxPLs relative to PEG standards were determined by the GPC method.2,2’-Dithiodiethanol diacrylate(DSEA)was firstly synthesized by the reaction of 2,2’-dithiodiethanol with acryloyl chloride,and DSEA was reacted with 5-amino-1-pentanol to synthesize ssPBAE,the structure of DSEA and ssPBAE was characterized by the analysis of 1H NMR.ssPBAE was modified with diethylenetriamine at end-alkene groups to form amino-modified ssPBAE(NH-ssPBAE),and ssPBAE-oxPL was synthesized by the Schiff’s base reaction between NH-ssPBAE and oxPL,the structure of NH-ssPBAE snd ssPBAE-ox PL were characterized by the analysis of IR and 1H NMR,ssPBAE-oxPL-DOX was synthesized through doxorubicin conjugated to the ssPBAE-oxPL,and sodium cyanoborohydride was used to selectively reduce the carbon–nitrogen double bonds in ssPBAE-ox PL-DOX to obtain the reduced ssPBAE-oxPL-DOX(R-ssPBAE-oxPL-DOX),the loading contents of DOX in polymeric prodrug were detected by ultraviolet spectrophotometry,the structure of ssPBAE-ox PL-DOX and R-ssPBAE-oxPL-DOX were characterized by the analysis of IR.2.The in vitro study of dual-responsive nanoparticle system of ssPBAE-oxPL-DOX/pDNA nanoparticles: the DNA condensing and protecting capacities of ssPBAE and ssPBAE-oxPL-DOX were assessed by agarose gel electrophoresis,the size and surface charge character of ssPBAE/pDNA and ssPBAE-oxPL-DOX/pDNA were measured by dynamic laser scattering method and zeta potential,and the morphology of ssPBAE/pDNA and ssPBAE-ox PL-DOX /pDNA nanoparticles were observed using transmission electron microscope(TEM),and the redox-responsive releases of pDNA from ssPBAE/pDNA and ssPBAE-oxPLDOX/pDNA nanoparticles were also measured by agarose gel electrophoresis;the in vitro drug releases of DOX from ssPBAE-ox PL-DOX/pDNA nanoparticles were studied in buffer solutions with different pHs using dynamic dialysis method.The cellular uptake and intracellular localization of ssPBAE-oxPL-DOX in HepG2 cells and A549 cells were analyzed by laser scanning confocal microscope.The cytotoxicities of ssPBAE-oxPL/pDNA and ssPBAE-oxPL-DOX/pDNA nanoparticles in HepG2 cells were measured by CCK-8 assay;the effects of free DOX,ssPBAE-oxPL/p DNA and ssPBAE-ox PL-DOX/pDNA on the apoptosis and cell cycle of HepG2 cells were analyzed by the flow cytometry.3.The in vivo study of dual-responsive nanoparticle system of ssPBAE-ox PLDOX/FAM-DNA nanoparticles: HepG2 cells were injected subcutaneously into the nude mice to construct HCC mouse model.The biodistribution of Cy5.5(an infrared fluorescent dye)labeled ssPBAE-ox PL-DOX/FAM-DNA nanoparticles in HepG2 tumor-bearing mice was evaluated by the in vivo imaging technique.In addition,the accumulations of ssPBAE-ox PL-DOX/FAM-DNA/Cy5.5 nanoparticles in the liver,kidney and tumor tissues were further assessed using the frozen section technique and fluorescence microscope.Results1.Ox-PL was synthesized and its chemical structure was confirmed by the IR and 1H NMR,the aldehyde contents and the average molecular weights of oxPLs were determined.DSEA snd ssPBAE were successfully synthesized and their chemical structures were confirmed by the 1H NMR.The ssPBAE-oxPL-DOX and R-ssPBAE-oxPL-DOX was successfully synthesized and its chemical structure was confirmed by the IR,DOX content in polymeric prodrug was about 3.6%.2.SsPBAE and ssPBAE-ox PL-DOX exhibited relatively high condensation capability for pDNA and displayed redox-responsive properties,and ssPBAE/pDNA nanoparticles were prepared and had spherical shapes and relatively uniform size,The ssPBAE-oxPL-DOX/pDNA nanoparticles had non-spherical shapes and relatively uniform size.The results of in vitro releases showed that DOX displayed significant pH-sensitve release from ssPBAE-ox PL-DOX/pDNA.The ssPBAE-ox PL-DOX /pDNA nanoparticles were evidently more stable than ssPBAE/pDNA nanoparticles in 10% calf serum.In addition,ssPBAE-oxPL-DOX/FAM-DNA significantly induced the cell apoptosis and cell cycle arrest at the S-phase.3.At 24 h after administration via intravenous injection,ssPBAE-oxPL-DOX/ FAM-DNA/Cy5.5 nanoparticles were mostly accumulated in the tumor tissues,and partially in the liver and kidney of HepG2 tumor-bearing nude mice.ConclusionsIn this study,a dual-responsive nanoparticle system was designed based on oxidized pullulan(oxPL)and a disulfide containing poly(β-amino)ester(ssPBAE)for the efficient delivery of genes and chemotherapeutic agents through polymer degradation responding to the tumor intracellular pH and redox state.In hepatoma HepG2 cells,ssPBAE-oxPL-DOX/pDNA nanoparticles effectively inhibited cell proliferation,and induced cell apoptosis and cell cycle arrest at the S-phase.In summary,this novel dual-responsive nanoparticle system showed great potential for combined gene therapy and chemotherapy on hepatoma.