| Metallothionein ? are naturally a class of low molecular proteins,which can have properties of biodegradability,immunogenicity and binding heavy metals,and also act as a scavenger of free radicals.At present,there are no reports that metallothionein ? as nano-carrier for targeting drug delivery system at home and abroad,it makes it possible as good nano-carrier for targeting drug delivery system because of special spatial structure and composition of amino acid,thus,this project focused on metallothionein ? as nano-carrier for targeting drug delivery system,which load two typical anticancer drugs:one is docetaxel which is fat-soluble,the other is vinblastine sulfate which is water-soluble,and then make drugs-loaded metallothionein ? to couple with folate,in the way prepared drug-loaded metallothionein ? as nano-carrier with folate-decoration which could act as anticancer cells,this provide more well treatment protocols for anticancer in the clinical.In this paper,firstly we have made metallothionein ? from rabbit liver as nano-carrier for targeting drug delivery system,and then carried on further research.The results were as follows:1.MT?-NPS were prepared by a coacervation method and chemical cross-linking,preparation procedure of MT?-NPS was optimized with central composite design of Design Expert 7.0.The particle size and Zeta potential of MT?-NPS were considered as the most important parameters.The results of optimization scheme was as follows:concentration of MT?-NPS with 1mg/mL,ethanol solvent ratio with 3.36,cross-linking ratio with 0.2 and reaction time with 8h.To characterize optimized MT 11-NPS:round sphere by SEM,stenosis distribution of average particle size with 122.1nm,average Zeta potential with-26.62mV,freeze-dried powder of MT?-NPS with 10%mannitol with well dispersion.To characterize optimized FA-MT?-NPS:round sphere but a little adhesion by SEM,stenosis distribution of average particle size with 133.7nm,average Zeta potential with-22.13mV,freeze-dried powder of FA-MT?-NPS with 15%mannitol with well dispersion.Make sure folate-NHS could couple with MT?-NPS for 132.4nmol/g.FITC could couple with FA-MT?-NPS by fluorescence microscope.2.MT?-DTX-NPS were prepared by a coacervation method and chemical cross-linking,preparation procedure of MT?-DTX-NPS was optimized with factorial design of Design Expert 7.0.The particle size,Zeta potential,entrapment efficiency and drug loading of MT?-DTX-NPS were considered as the most important parameters.The results of optimization scheme was as follows:concentration of MT?-DTX-NPS with lmg/mL,addition ratio of DTX with 1.06,ethanol solvent ratio with 1,cross-linking ratio with 0.5 and reaction time with 10.8 h.To characterize optimized MT?-DTX-NPS:round sphere by SEM,stenosis distribution of average particle size with Z15.4nm,average Zeta potential with-26.13mV,average entrapment efficiency with 53.1%and average drug loading with 36.89%.To characterize optimized FA-MT?-DTX-NPS:round sphere but a little adhesion by SEM,stenosis distribution of average particle size with 224.5nm,average Zeta potential with-21.36Mv.Make sure folate-NHS could couple with MT?-DTX-NPS for 101.3nmol/g.FA-MT?-DTX-NPS have well property of sustained release,and in vitro its cumulative release rate was 73.70%during 72 h.3.MT ?-VBL-NPS were prepared by a coacervation method and chemical cross-linking,preparation procedure of MT?-VBL-NPS was optimized with factorial design of Design Expert 7.0.The particle size,Zeta potential,entrapment efficiency and drug loading of MT?-VBL-NPS were considered as the most important parameters.The results of optimization scheme was as follows:concentration of MT?-VBL-NPS with 1.27mg/mL,addition ratio of VBL with 1,ethanol solvent ratio with 1,cross-linking ratio with 0.5 and reaction time with 8 h.To characterize optimized MT?-VBL-NPS:round sphere by SEM,stenosis distribution of average particle size with 237.2nms average Zeta potential with-23.89 mV,average entrapment efficiency with 64.3%and average drug loading with 41.25%.To characterize optimized FA-MT?-VBL-NPS:round sphere but a little adhesion by SEM,stenosis distribution of average particle size with 248.8nm,average Zeta potential with-20.47 mV.Make sure folate-NHS could couple with MT?-VBL-NPS for 104.2nmol/g.FA-MT?-VBL-NPS have well property of sustained release,and in vitro its cumulative release rate was 82.65%during 72 h.4.Researches on in vitro antitumor cells activity of prepared FA-MT ?-DTX-NPS and FA-MT?-VBL-NPS to PC-3 cells,the results were as follows:(1)Growth inhibition was analyzed by MTT assay.It was found that FA-MT?-DTX-NPS and FA-MT?-VBL-NPS markedly inhibited proliferation of PC-3 cells for 72h(FA-MT?-DTX-NPS,IC50:10.23±0.31?g/mL;FA-MT?-VBL-NPS,IC50:62.97±0.28?g/mL.(2)Growth inhibition was analyzed by flow cytometry using AnnexinV-FITC/PI.It was found that FA-MT?-DTX-NPS and FA-MT?-VBL-NPS markedly inhibited proliferation of PC-3 cells for 72h,the most of PC-3 cells are necrosis or late apoptosis cells,and it showed that the apoptotic population increased in a concentration-dependent manner with increasing concentrations after the cells treated with FA-MT?-DTX-NPS and FA-MT?-VBL-NPS,and markedly inhibited proliferation of PC-3 cells with increasing concentrations.(3)Targeting ability of prepared drugs to PC-3 was observed using laser scanning confocal microscope,it was found that FA-MT?-DTX-NPS and FA-MT?-VBL-NPS well targeting ability to PC-3 cells,markedly compared with MT?-DTX-NPS and MT?-VBL-NPS.(4)Prepared drugs effect on the morphology of PC-3 cells using inverted microscope,it was found that FA-MT?-DTX-NPS(IC50:10.23 ±0.31 ?g/mL)and FA-MT?-VBL-NPS(IC50:62.97±0.28?g/mL)markedly effect on the morphology of PC-3 cells for 72h,thus,it was proved that FA-MT?-DTX-NPS and FA-MT?-VBL-NPS markedly inhibited proliferation of PC-3 cells.In this study,it was proved that metallothionein ? as nano-carrier for targeting drug delivery system is feasible,and prepared FA-MT?-DTX-NPS and FA-MT?-VBL-NPS have high quality and ability to antitumor cells in vitro,it is believed that it has more application prospect on metallothionein? as nano-carrier for targeting drug delivery system with further research. |
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