The Design Of Spermine-grafted Pullulan-PLGA Nanoparticles Conjugated With CD3 And The Study Of The Anti-hepatoma Activities

BackgroundAlthough surgery,radiotherapy,chemotherapy and interventional therapy have obvious therapeutic effect on hepatocellular carcinoma,the survival time of patients has not been significantly prolonged.A single treatment method can not achieve good therapeutic effect.Therefore,it is of great significance to explore a more safe and efficient comprehensive treatment of hepatocellular carcinoma.The multi-function of nanocarriers is expected to achieve the comprehensive treatment of hepatocellular carcinoma such as chemotherapy and immunotherapy.We prepared spermine pullulan polysaccharide-PLGA-CD3 nanoparticles（PS-PLGA-CD3 NPs）with surface-coupled specific antibodies,then modified Fe₃O₄ to prepare magnetic nanoparticles MNPs,in order to construct a multifunctional nanotherapy system,and to investigate its feasibility in combination chemotherapy/immunotherapy/magnetic targeting therapy for hepatocellular carcinoma.Objective1.Polymer PS-PLGA was synthesized and surface-coupled CD3 antibody nanoparticles PS-PLGA-CD3 NPs were prepared.The effects of PS-PLGA-CD3 NPs on T cell viability,cytokine secretion and uptake were elucidated.2.To explore the feasibility of PS-PLGA-CD3 modified Fe₃O₄ magnetic nanoparticles（MNPs）to construct magnetotactic T cells after incubation with T cells,and to preliminarily study the magnetotactic targeting effect of magnetotactic T cells in vitro and in vivo.Method1.Preparation and characterization of PS-PLGA-CD3 NPs.Polymer PS-PLGA was synthesized and characterized by FTIR and 1H NMR.PS-PLGA NPs were prepared by ultrasound dialysis method and PS-PLGA-CD3 NPs were prepared by surface coupling with CD3 antibody.The particle size and surface potential of the nanoparticles were measured by particle size analyzer,the morphology of the nanoparticles were observed by TEM,and the content of CD3 antibody on the surface of the nanoparticles was determined by fluorescence quantitative method.2.CCK-8 assay was used to detect the effects of two nanoparticles（PS-PLGA NPs and PS-PLGA-CD3 NPs）on the viability of T cells and Hep G2 cells in vitro.The toxicity of PS-PLGA-CD3 NPs was evaluated by hemolysis test and acute toxicity test in mice.3.The effects of PS-PLGA-CD3 NPs on the secretion of IFN-γ,IL-2,and TNF-βby T cells were determined by ELISA kit.The uptake efficiency of nanoparticles by T cells and Hep G2 cells was measured by Image Xpress confocal high-content imaging system and flow cytometry,respectively.4.PS-PLGA-CD3 NPs modified Fe₃O₄ nanoparticles were used to prepare modified magnetic nanoparticles（MNPs）and incubation method was used to construct magnetotactic T cells;MTT method was used to investigate the effect of MNPs on T cell viability;ELISA kit was used to determine the effect of MNPs on the secretion of IFN-γ,IL-2,and TNF-βby T cells;Flow cytometry was used to detect the uptake of MNPs by T cells.Under the action of magnetic field,the magnetotactic properties of magnetotactic T cells in vivo and in vitro were measured by Image Xpress confocal high-content imaging system and animal photoacoustic imager,respectively.Results1.The synthesis of PS-PLGA was confirmed by FTIR and 1H NMR.The morphologies of PS-PLGA NPs and PS-PLGA-CD3 NPs were regular spherical with uniform particle sizes of 121.9±20.3 nm and 174.3±27.5 nm,respectively.The antibody content of PS-PLGA-CD3 NPs with Spermine substitution of 9.7%was 52.1±9.4μg/mg.2.When the concentration of PS-PLGA NPs was between 1～200μg/m L,there was no significant effect on T cell viability in vitro,but PS-PLGA-CD3 NPs could significantly enhance T cell viability,and with the increase of nanoparticle concentration,the enhancement of cell viability was more obvious.PS-PLGA NPs and PS-PLGA-CD3 NPs had no significant effect on the cell viability of Hep G2 cells in the concentration range of5～1000μg/m L.The results of hemolysis showed that PS-PLGA-CD3 NPs had no hemolysis effect.Compared with the control group,there were no significant differences in body weight,food intake and pathological sections of main organs.3.PS-PLGA-CD3 NPs can be effectively uptaked by T cells and Hep G2 cells.Compared with uncoupled antibody group（PS-PLGA NPs）,PS-PLGA-CD3 NPs at the same concentration significantly promoted the secretion of IFN-γ,IL-2 and TNF-βby T cells.4.In a certain concentration range,MNPs can be absorbed by T cells and enhance the cell viability of T cells.MNPs can significantly promote the secretion of IFN-γ,IL-2 and TNF-βby T cells.Under the action of magnetic field,magnetotactic T cells exhibit certain magnetotactic properties in vitro and in vivo.Conclusion1.PS-PLGA-CD3 NPs and MNPs with rounded morphology were successfully prepared,which had no obvious cytotoxicity in a certain concentration range,and could effectively enhance the cell viability of T cells and secrete cytokines.2.PS-PLGA-CD3 NPs and MNPs can be efficiently absorbed by T cells.The constructed magnetotactic T cells show certain magnetotacticity in vitro and in vivo.3.In this study,PS-PLGA-CD3 NPs and MNPs,which can effectively enhance the activity of T cells,have been successfully prepared,and are expected to become a new multifunctional nanosystem of chemotherapy/immunotherapy/magnetic targeting for the treatment of hepatocellular carcinoma.