Functionalized Dendrimer Delivers TRAIL Plasmid And Mdm2-siRNA In The Treatment Of Osteosarcoma

Osteosarcoma is the most common malignancy in primary bone tumors. It affects thechildren and adolescents most frequently. The standard therapy of extended tumor marginresection combining neoadjuvant chemotherapy has made the5-year survival rate ofpatients prolonged to50-65%. However, for patients with special-site osteosarcoma,metastatic tumor sites in early stage or multidrug resistant osteosarcomas, the prognosis ispoor. In addition, the side effects of traditional chemotherapy for patients are notneglectable.Tumor necrosis factor (TNF) related apoptosis inducing ligand (TRAIL) is able tospecifically induce apoptosis of tumor cells but not normal cells. However, TRAIL has ashort circulation time and could be quickly cleared in vivo. Therefore, constructing theplasmid DNA which expressing TRAIL (pTRAIL) and delivering the pTRAIL into tumorcells is able to continuously produce TRAIL in tumor cells and induce tumor apoptosismore effectively. Murine double mimute2(Mdm2) gene can negatively regulate theexpression of p53gene. Mdm2gene overexpression was found in almost all tumor cellswith p53gene deficiency or mutation. Therefore, reducing Mdm2gene expression intumor cells through gene interference will produce effective anti-tumor potency.Gene transfection vector plays an important role in an efficient gene therapy.Polyamidoamine dendrimers as a gene transfection vector has the advantages of wellcharged nano size, relatively high charge density, relatively low cytotoxity and abundantfunctional group for further innovation. Our research group had previously modified thegeneration5PAMAM (G5) with2,4-diamino-1,3,5-triazine (DAT) for synthesizeG5-DAT66, and G5-DAT66was proved to have a better gene transfection efficiency andserum resistance than commercial transfection products in several cell lines. Therefore, inthis research, the potency of G5-DAT66delivers pTRAIL and Mdm2-siRNA separately willbe further studied in osteosarcoma gene therapy. 1. Plasmid EGFP (pEGFP) transfection efficiency of G5-DAT66in osteosarcoma MG-63cells, preparation and Characterizationof G5-DAT66/pTRAILObjective: Understanding the gene transfection applicability of G5-DAT66vector onosteosarcoma MG-63cells and potential gene transfection ability of G5-DAT66/pTRAILcomplex.Methods: Synthesizing G5-DAT66with one-step process and the synthetic productwas confirmed by nuclear magnetic resonance (NMR) Characterization. Thenitrogen/phosphorus ratios (N/P) with optimal pEGFP transfection efficiency in MG-63cells of G5-DAT66/pEGFP, G5/pEGFP, SuperFectamineTM(SuperFect)/pEGFP andLipofectamine2000TM(Lipo2000)/pEGFP were screened, respectively. MG-63cells weretransfected with G5-DAT66/pEGFP, G5/pEGFP, SuperFect/pEGFP and Lipo2000/pEGFPcomplexes in optimal N/P, respectively. Then the EGFP positive percentage and meanfluorescence intensity of cells in each group were counted and compared through flowcytometry. G5-DAT66/pTRAIL complexes in different N/P were prepared, and size andZeta potential of which were examined with Dynamic Light Scattering.Results: synthetic product was confirmed by NMR1H NMR and COSY spectrum.The optimal transfection N/P of G5-DAT66/pEGFP, G5/pEGFP, SuperFect/pEGFP andLipo2000/pEGFP complexes were14、8、3μL/μg DNA and2.5μL/μg DNA, respectively.The percentage of EGFP positive cells and mean fluorescence intensity inG5-DAT66/pEGFP group were obvious higher than other three groups (P<0.01). The sizeof G5-DAT66/pTRAIL complexes was below400nm at N/P of above8and was around200nm at N/P of above16. Zeta potential of the complexes was around+45mV at N/P ofabove8.Conclusion: G5-DAT66is also able to deliver pEGFP efficiently in osteosarcomaMG-63cells. G5-DAT66/pTRAIL complexes that formed at N/P of above8~16are insuitable size and have a positive charge surface.2. Anti-tumor potency of G5-DAT66delivers pTRAIL in MG-63cellsObjective: We aimed to study the pTRAIL transfection efficiency of G5-DAT66inosteosarcoma MG-63cells as well as the apoptosis inducing and proliferation inhibitionpotency of pTRAIL delivering by G5-DAT66in MG-63cells.Methods: The N/P with optimal pTRAIL transfection efficiency in MG-63cells ofG5-DAT66/pTRAIL, G5/pTRAIL, SuperFect/pTRAIL and Lipo2000/pTRAIL werescreened, respectively. MG-63cells were transfected with above different complexes formed in their optimal N/P, respectively. Then the cells proliferation rates of each groupwere compared through MTT assay; the apoptotic cells in each group were labeled byAnnexin V-FITC/PI for comparing apoptotic rates; the expression of protein TRAIL,protein Cleaved Caspase-8and protein p53in each group were determined and comparedthrough Western-blot experiments; the TRAIL mRNA, p53mRNA and p21mRNAexpression in each group was quantified and compared through RT-PCR experiments.Results: Comparing to other control groups, the MG-63cells transfected withG5-DAT66/TRAIL complexes had the lowest proliferation rate (P<0.001), the highestpercentage of apoptotic cells, the highest expression of protein TRAIL, protein CleavedCaspase-8and protein p53and the highest expression of TRAIL mRNA, p53mRNA andp21mRNA (P<0.05).Conclusion: G5-DAT66has higher pTRAIL transfection efficiency than G5、SuperFect and Lipo2000in MG-63cells. G5-DAT66/TRAIL transfection achieves strongerapoptosis inducing and proliferation inhibition potency through more expression of proteinTRAIL and related apoptosis markers.3. Anti-tumor potency of G5-DAT66delivers pTRAIL in MG-63multicellular tumor spheroids (MTS)Objective: MTS is a muticellular3D culture model in vitro which mimics thebiological characters of solid tumor in vivo. We aimed to study the gene transfectionefficiency of G5-DAT66in MG-63MTS as well as the apoptosis inducing and growthinhibition potency of pTRAIL delivering by G5-DAT66in MG-63MTS.Methods: G5-DAT66, G5, SuperFect and Lipo2000were separately mixed with pEGFP to form complexes. Then the complexes were transfected in MG-63MTS. Theamount and distribution of EGFP positive cells in each optical sections of different depthin MTS of each group were observed through Confocal laser scanning microscope (CLSM).G5-DAT66, G5, SuperFect and Lipo2000were separately mixed with pTRAIL whichlabeled by YOYO-1to form complexes. Then the complexes were mixed with MTS for2,6and24hours, respectively. The penetration depths of complexes in central section ofMTS were observed through CLSM. The apoptotic cells in MTS which transfected withG5-DAT66/pTRAIL, SuperFect/pTRAIL and Lipo2000/pTRAIL complexes were labeledby PI. Then the amount and distribution of PI labeled cells in each optical sections ofdifferent depth in MTS of each group were observed through CLSM. The volume changes of those MTS were monitored for comparing the growth rate of those groups.Results: There were more EGFP positive cells in every section of different depths inMTS of G5-DAT66/pEGFP group than that of any other groups.G5-DAT66/pTRAIL-YOYO-1and G5/pTRAIL-YOYO-1could penetrate more deeply incentral section of MTS than SuperFect/pTRAIL-YOYO-1andLipo2000/pTRAIL-YOYO-1. There were more PI labeled cells in every section ofdifferent depths in MTS of G5-DAT66/pTRAIL group than that of any other groups. Thevolume increasing rate of MTS in G5-DAT66/pTRAIL group was also smaller than that ofother groups (P<0.05).Conclusion: G5-DAT66has higher pEGFP transfection efficiency in MG-63MTSthan G5, SuperFect and Lipo2000. G5and G5-DAT66vectors have stronger penetrationability than SuperFect and Lipo2000. The apoptosis inducing and growth inhibitionpotency of G5-DAT66/pTRAIL is stronger than SuperFect/pTRAIL andLipo2000/pTRAIL.4. Anti-tumor potency of G5-DAT66delivers Mdm2-siRNA inMG-63cellsObjective: We aimed to study the Mdm2-siRNA transfection efficiency of G5-DAT66in osteosarcoma MG-63cells as well as the apoptosis inducing and proliferation inhibitionpotency of Mdm2-siRNA delivering by G5-DAT66in MG-63cells.Methods: MG-63cells were transfected with G5-DAT66/Mdm2-siRNA complexesformed in different N/P and Lipo2000/Mdm2-siRNA. Then the Mdm2mRNA expressionin each group was quantified and compared through RT-PCR experiments; the expressionof protein Mdm2, protein p53and protein Caspase-8in each group were determined andcompared through Western-blot experiments; the apoptotic cells in each group werelabeled by Annexin V-FITC/PI and counted through flow cytometry (FCM); the cellsproliferation rates of each group were compared through MTT assay.Results: The MG-63cells transfected with G5-DAT66/Mdm2-siRNA/82complexeswhich formed in N/P of82had the lowest expression of Mdm2mRNA and protein Mdm2,the highest expression of protein p53and protein Caspase-8, the highest percentage ofapoptotic cells and the lowest proliferation rate (P<0.01).Conclusion: Mdm2-siRNA delivering by G5-DAT66in MG-63cells provids strongerpotency of Mdm2gene interference as well as apoptosis inducing and proliferation inhibition.