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Synthesis and Characterization of Polyethyleneimine-dextran Ferric Oxide NanoparticlesObjective:We attempted to synthesize a cationic gene carrier with polyethyleneimine(PEI),dextran and iron oxide magnetic nanoparticle(PDI)and characterize the carrier.Methods:A chemical co-precipitation method was used to synthesize dextran coated iron oxide nanoparticles(DI),then underwent oxidization by sodium periodate for synthesis of aldehyde functionised magnetic nanoparticles(ADI).The PEI and ADI were conjuncted through Schiff base formation.Fourier transform infrared spectrometer(FTIR)was used to detect functional groups such as aldehyde groups.Transmission electron microscopy(TEM)was for observation of morphology and size.Laser particle analyzer(LPA)was for detection of hydrodynamic diameter and zeta potential.Thermogravimetric analyzer(TGA)was applied to detect thermal weight loss of the materials.Magnetic property was detected by vibrating sample magnetometer(VSM).For detection of the plasmid DNA concentration and estimation of the encapsulation rate,ultraviolet spectrophotometer(UVS)was used.Agarose gel electrophoresis(AGE)was used to evaluate the binding ability of PDI to the plasmid.Results:We synthesized superparamagnetic dextran and polyethyleneimine-dextran coated iron oxide nanoparticles.TEM images showed that the diameter of DI was about 5.11±1.17 nm and a part of the particles were agglomerated.FTIR data showed that ADI had an absorption peak at 1737.6 cm-1,which confirmed that the hydroxyl group(-OH)of the dextran coating was oxidized to aldehyde group(-CHO).Laser particle sizer data show that the hydrodynamic diameters of Fe3O4and DI were83.55±2.09 nm,105.33±1.17 nm,the relative zeta potentials was-5.23±1.01 m V,-1.76±0.13 m V.And the optimal mass ratio of PEI/ADI was confirmed to be 30.The hydrodynamic diameter of PDI was 148.67±1.52 nm,and the zeta potential was32.50±0.62 m V.TGA showed that the weight loss rates of DI,ADI,and PDI were71.80%,61.66%,and 59.16%accordingly,confirmed the successful grafting of polyethyleneimine.According to the hysteresis loop,the saturation magnetizations of DI and PDI were 6.63 emu/g,5.31 emu/g,respectively.And the residual magnetization and coercivity were both zero,showing superparamagnetism of the DI and PDI.This indicated that the magnetic properties of magnetic nanoparticles were not significantly effected by the PEI grafting.AGE showed that PDI possess strong binding ability to the plasmid DNA.The MTT results showed that while the concentration of PDI was 10?g/m L,the relative cell viability of 293T,143B,and MG63 cells was 86.0±2.6%,91.3±5.0%,and 88.3±6.7%,respectively.The relative cell viability of 293T,143B,and MG63 cells were 75.33±7.37%,69.33±3.22%,and 47.0±4.58%at the highest PDI's concentration of 300?g/m L.These confirmed that PDI exhibited certain cell toxicity at very high concentrations,while showed low cytotoxicity at 10?g/m L.Therefore,10?g/m L was selected for the subsequent experiments.Conclusion:The cationized polyethylenimine-dextran coated iron oxide magnetic nanoparticles were successfully synthesized,which possessed qualities of ultra small particle size,superparamagnetism,low cytoxicity and strong binding ability to the mi RNA plasmid.This may be applied for gene therapy,such as mi RNA.Part ? Anti-osteosarcoma effect of PDI/mi R-302b magnetic nanoparticles in vitroObjective:To explore the transfection efficiency of PDI,the anti-osteosarcoma effects of PDI/mi R302b on proliferation,migration and apoptosis,and uncover the underling molecular mechanism.Methods:After incubation of PDI/mi R-302b and osteosarcoma cells for 24 h,the proliferation,the apoptosis,cell migration ability of osteosarcoma cells was detected by MTT method,flow cytometry,wound healing method,respectively.The magnetic field was applied to evaluate the targeting ability of PDI/mi R302b.The transfection efficiency of PDI/mi R-302b was detected by fluorescence microscopy and q RT-PCR.And the internalization of PDI/mi R-302b by osteosarcoma cells was detected by Prussian blue staining and transmission electron microscopy(TEM).In order to explore the anti-osteosarcoma mechanism of mi R-302b,we predicted YOD1 as the potential target gene of mi R-302b through bioinformatics software and website.Then the dual luciferase reporting system was used to verify whether mi R-302b could be complementary binded by YOD1.QRT-PCR was used to detect the relative expression of YOD1 and Hippo pathway related genes in 143B cells after PDI/mi R-302b treatment.We designed a rescue experiment which consisted of four groups:up-regulation of mi R-302b(mimic+YOD1-ctrl),up-regulation of mi R-302b and YOD1(mimic+YOD1),up-regulation of YOD1(mimic-NC+YOD1),and the control group(mimic-NC+YOD1-ctrl).The proliferation,the apoptosis,cell migration ability of 143B cells were detected by MTT method,flow cytometry,wound healing method,respectively.Results:After incubation of PDI/mi R-302b and 143B,the fluorescence microscope images showed that the green fluorescence was gradually emerged after 3 h,and reached the peak at 48 h of incubation.QRT-PCR datas showed that the relative expression of mi R-302b in 143B,MG63,and U2OS cells were significantly lower in the group treated with magnetic field.Therefore,the magnetic field significantly enhanced the transfection efficiency of PDI.And MTT data showed that the cell viability of osteosarcoma cells were inhibited by PDI/mi R302b,especially with the help of the magnetic field.Besides,the group with magnetic field induced more apoptosis,slower cell scratch healing in 143B cell than that of the group without.We found YOD1 was highly expressed in 143B,MG63,and U2OS cell lines than in h FOB1.19 cells.Compared to the group without magnetic field,PDI/mi R302b suppressed the expression of YOD1,ITCH,and YAP and enhanced LATS1's expression,these effects could be strengthened with the help of magnetic field.The results of the dual luciferase reporter gene experiment showed that mi R-302b could complementary bind with the YOD1.Rescue assay confirmed that overexpression of YOD1 could counteract the effect of mi R-302b on osteosarcoma cells.Conclusion:We confirmed that PDI could deliver mi R-302b into osteosarcoma cells and exhibit anti-OS effects of mi R-302b,which can be enhanced by the magnetic field.And mi R-302b may regulate the proliferation,migration and apoptosis of osteosarcoma through the YOD1-Hippo axis.Part ? Anti-OS effect of PDI/mi R-302b magnetic nanoparticles in vivoObjective:To investigate the anti-OS effect and the magnetic responsibility of PDI/mi R-302b in vivo.Methods:We successfully constructed a subcutaneous osteosarcoma nude mouse model and randomly divided them into five groups for administration as follows:control group,PDI group,pmi R-302b group,PDI/mi R-302b group,PDI/mi R-302b+magnetic field group.The tumor volume was measured each day and the mice were administered every three days.After three weeks of administration,mice were sacrificed,the tumors,viscera and relative tissues were dissected for HE staining and Prussian blue staining.The content of iron was detected by ICP-OES.Results:The tumor volume of each group was as follows:the control group>the PDI group>the pmi R-302b group>the PDI/mi R-302b group>the PDI/mi R-302b+magnetic field group.HE staining results of the tumor tissue showed that the largest necrosis area emerged in the mi R-302b+magnetic field group,followed by PDI/mi R-302b group,pmi R-302b group,PDI group,and control group.The Prussian blue images showed that the iron content of the PDI/mi R-302b+magnetic field group was more than that of the PDI/mi R-302b group.Furthermore,the ICP-OES showed the iron in liver and spleen was significantly reduced after the magnetic field treatment,confirming the superior magnetic responsibility of PDI/mi R302b in vivo.Conclusion:We confirmed that PDI/mi R-302b could inhibit osteosarcoma growth in vivo,and magnetic field could enhance antitumor effect though magnetic targeting. |
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