Drug Resistance Reversal By Interventing Cancer Bioenergetics With Spherical Helical Polypeptide-Potented Gene Silencing

Part Ⅰ:Preparation and characterization of spherical helical polypeptideObjective:To construct polypeptide(DPP)with a helical structure,which was loaded with doxorubicin(DOX)and combined with PKM2-siRNA to form nanocomplexes(NCs),further surface-decorated with hyaluronic acid(HA),and characterized its physical and chemical properties.Methods:DPP was obtained by chemical synthesis and lyophilization.Gel permeation chromatography(GPC)was used to determine molecular weight(MWs)and dispersity of polypeptides,Circular dichroism(CD)was used to evaluate the helical structure of the polypeptides in the aqueous solution.DOX was first encapsulated into the inner cavity of DPP,thus obtaining the drug loaded composites DD,which formed a nanocomposite DD/P through electrostatic adsorption with PKM2-siRNA,the outer surface of the composite was covered with a layer of HA to form the final nanocomposite DD-H/P.The diameter and zeta potential of several NCs at various DPP/siPKM2 weight ratios(5,10,15,20,and 25)and various HA/DPP weight ratios(0:4,1:4,1:2,1:1,2:1,and 4:1)were measured by the Malvern Zetasizer.Detected the amount of DOX contained in the nanocomposite by spectrofluorimetry and calculated determination of drug loading capacity(DLC)and drug loading efficiency(DLE).The stability of various NCs in serum was evaluated by dynamic laser scattering(DLS)and gelose gel electrophoresis.Detected the drug release of DD-H/P nanocomposite in different time periods and different pH(pH 7.4,6.5,5.0)by spectrofluorimetry.The siRNA release from DD-H/P NCs were evaluated by gel electrophoresis in the presence of heparin at various concentrations(0.005,0.01,0.05,0.1,0.5 mg/mL).Results:GPC analysis showed that the MWs of the polypeptide was 82400 g/mol,the relative degree of polymerization(DP)was 276,and the polydispersity index(PDI)was 1.31.The CD spectra of DPP in the aqueous solution revealed double minima at 208 and 222 nm,which indicated the α-helix conformation.At the DPP/HA/siPKM2 weight ratio of 20/20/1,DD-H/P NCs with a diameter of 127 nm and slightly negative zeta potential(-8.1 mV)were obtained.The DLC and DLE of the DD-H/P nanocomposite were 6.1%and 75.3%respectively.After exposure to 10%FBS,the siRNA loaded in DD-H/P NCs can maintain its band intensity and was not degraded,and DD-H/P NCs exhibited desired stability.The loaded siRNA could be effectively released in the presence of 0.1 mg/mL heparin,and DOX release pattern at different pH were plotted a typically biphasic and pH-dependent.Conclusion:DPP possessed the stable α-helix conformation.DLC and DLE of DOX in the DD-H/P NCs were relatively high.DD-H/P NCs exhibited desired stability after exposure to serum,successfully released DOX and siRNA under certain conditions.Part Ⅱ:Effects of PKM2 silencing caused by DD-H/P on drug resistance and apoptosis of cancer cells in vitroObjective:To investigate the effect of PKM2 silencing caused by DD-H/P on drug resistance and apoptosis of cancer cells in vitro.Methods:The capability of NCs to mediate trans-membrane siRNA delivery into DOX-resistant cancer cells was first probed by monitoring the cellular uptake levels of NCs containing FAMsiScr using flow cytometry.The endolysosomal escape of NCs was explored by confocal laser scanning microscopy(CLSM).DOX-resistant cancer cells were incubated with various NCs before determination of PKM2 mRNA levels using real-time PCR with specific primers,and the PKM2 protein expression levels were determined using Western blot,thus the PKM2 silencing efficiencies were investigated.The capability of siPKM2 to inhibit DOX efflux in DOX-resistant cells was evaluated by CLSM and flow cytometry.The cellular ATP level was measured using an ATP bioluminescent assay kit.Cells were then stained using the mitochondrial membrane potential assay kit(with JC-1)or the reactive oxygen species(ROS)assay kit(with DCFH-DA)followed by observation with CLSM,and the cytoplasmic levels of cytochrome C(Cyto C)and caspase 3 were determined by Western blot,to probe the mechanism of DD-H/P lead to apoptosis of cancer cells.A live/dead double-staining assay,MTT assay and Annexin-V-FITC/PI double staining analysis were adopted to further assess the anti-cancer efficacy.Results:Flow cytometry exhibited significantly high intracellular siRNA delivery levels of DD-H/P,and the internalized NCs could also efficiently escape from endolysosomal entrapment,thus demonstrated the potent membrane activity of the spherical helical polypeptide.DD-H/P NCs at a DPP/siPKM2 weight ratio of 20 induced the highest PKM2 mRNA knockdown efficiency,and similar trend was noted for the NCs-mediated reduction of the PKM2 protein level using Western blot analysis.The ATP level in A549/ADR cells was significantly reduced by 60%within 24 h after treatment with DD-H/P NCs.A549/ADR cells treated with DD-H/P NCs revealed extensive fluorescence of DOX in the nuclei.The result was further supported by the quantitative flow cytometric analyses.A549/ADR cells treated with DD-H/P NCs revealed green fluorescence exclusively,indicating notable mitochondrial exhaustion and collapse induced by DD-H/P NCs.The cytoplasmic green fluorescence in DD-H/P NCs treated A549/ADR cells evidenced the notably enhanced intracellular ROS level.DD-H/P NCs enhanced the intracellular levels of Cyto C and caspase 3.The results of MTT assay showed that DD-H/P NCs exhibited higher cytotoxicity.Such finding was consistently supported by the live/dead cell staining assay and cancer cell apoptosis assay using Annexin-V-FITC/PI double staining.Conclusion:DD-H/P NCs had the potent membrane activity,significantly high intracellular siRNA and DOX delivery levels,and induced PKM2 mRNA knockdown efficiency to reduce ATP level,thus inhibited DOX efflux and induced mitochondrial exhaustion and collapse,promoted cancer cell death,finally contributed to the anti-cancer efficacy.Part Ⅲ:In vivo anticancer efficacy of DD-H/P NCs against A549/ADR tumorsObjective:To investigate anticancer efficacy of DD-H/P NCs against A549/ADR tumors in vivo.Methods:DOX,DD/P NCs,or DD-H/P NCs were i.v.injected to mice,and the DOX content was determined by spectrofluorimetry for the pharmacokinetics study.Subsequently,at 1,6,12,and 24 h post injection,the biodistribution of the NCs was evaluated in A549/ADR tumor-bearing mice using live animal fluorescence imaging.Nude mice bearing A549/ADR tumors were randomly divided into five groups,and were i.v.injected with PBS,DOX,DD-H/N NCs,D-H/P NCs,or DD-H/P NCs.The tumor size was measured every three days for up to 21 d.Tumors as well as major organs were harvested,then stained with haematoxylin&eosin(H&E)and examined under an optical microscope.To further observe the apoptotic level of tumor cells,the harvested tumors were assayed with the TUNEL Apoptosis Assay kit.The accumulation of DOX in tumor cells in vivo was evaluated by both CLSM and flow cytometry.The PKM2 mRNA levels in tumors were determined by real-time PCR,and the PKM2 protein expression levels were evaluated by Western blot.In addition,the tumor tissues were subjected to immunofluorescence staining of PKM2.Results:DD-H/P NCs displayed prolonged blood circulation,conferring t1/2 of 4.21 h that was higher than those of free DOX and DD/P NCs.Mice injected with DD-H/P NCs exhibited much stronger DOX fluorescence intensity at the tumor sites than mice injected with free DOX or DD-H/N NCs.Others NCs didn’t inhibite the tumor growth,while in comparison,tumor growth was completely suppressed by DD-H/P NCs within the same observation period.In consistence,mice receiving DD-H/P NCs exhibited a survival rate of 80%within the 50 d observation period,remarkably higher than that of mice in other groups.Tumors treated with DD-H/P NCs showed the most pronounced nuclear condensation and fragmentation in the hematoxylin and eosin(H&E)-stained images,indicating massive tumor remission.Also,the highest tumor cell apoptotic level was noted for tumors treated with DD-H/P NCs after TUNEL staining.In addition,both DD-H/P NCs and D-H/P NCs induced>70%PKM2 mRNA down-regulation.Western blot and immunofluorescent staining analyses consistently revealed that the expression of PKM2 protein was notably down-regulated by DD-H/P NCs and D-H/P NCs.CLSM images of tumor tissues further manifested that DD-H/P NCs promoted the drug accumulation in the drug-resistant cancer cells in vivo and facilitated the nucleus delivery of DOX.Such an observation was further supported by the quantitative flow cytometry analysis.Conclusion:DD-H/P NCs induced expression of PKM2 down-regulation,promoted the drug accumulation in the drug-resistant cancer cells,and caused apoptosis of cancer cell,thus inhibited the tumor growth.