PD-1 Highly Expressed Macrophage Membrane Biomimetic Modified Nanoparticles Induced Immune Regulation And Synergistic Anti-glioblastoma Studies

Glioblastoma(GBM)is one of the most common primary malignant tumor in central nervous system(CNS),which gravely threatens human health owing to their rapid development,poor diagnosis and fast recurrence.The blood brain barrier(BBB)is composed of capillary endothelial cells and tight junctions.It is a extremely selective permeability barrier that segregates the circulating blood from the extracellular fluid in the CNS.However,the BBB is also a substantial obstacle to the delivery of therapeutic drugs in the brain.Therefore,traditional chemotherapy can not effectively deliver drugs to the brain.The development of nano-drugs has the huge potential to ameliorate the prognosis of CNS diseases,particularly in the diagnosis and treatment of glioma.Biomimetic modification of cell membrane is an emerging way that enables nanoparticles new biological functions by coating different cell membranes on the surface of the nanoparticles.Many studies have shown that cell membrane biomimetic modified nanoparticles retain the complex biological functions of natural cell membranes while display physicochemical properties that are suitable for effective drug delivery.It can achieve prolonged blood circulation time and exhibit lower immunogenicity compared to traditional synthetic nano-drug delivery systems.If the cells are engineered to make them have certain characteristics,it is possible to further improve biomimetic modification of cell membrane effectiveness in tumor therapy.Programmed death receptor-1(PD-1)is a co-inhibitory receptor induced on T cells by antigenic stimulation.PD-1 expression is related with T cell exhaustion.Binding of PD-1 to its ligands PD-L1 can inhibit T cell effector function and enhance immune escape.PD-1 and PD-L1 interplay,creating an immunoregulatory axis in glioma.The expression of PD-L1 on glioblastoma surface promotes PD-1 receptor activation in microglia,which negatively regulated the T cell responses and promoting invasion of glioblastoma in the brain tissue.GBM mouse model studies have confirmed that PD-1 or PD-L1 monoclonal antibodies can significantly regress tumor mass and prolong the survival time of mice,indicating that blocking of the interaction between PD-1 and PD-L1 can re-establish proper immunity against GBM.Based on this,We guess that we can use the PD-1/PD-L1 signaling pathway to treat glioma through immune checkpoint blockade combined with chemotherapy.So we stably transferred the PD-1 gene into macrophage,and constructed an engineering macrophage strain with high expression of PD-1.Then we use PD-1 high expression engineered macrophage membrane biomimetic modified of rapamycin-loaded PLGA nanoparticles to prepare nano drugs PD-1-MM@PLGA/RAPA,which has immunotherapy function.Next,the ability of PD-1-MM@PLGA to penetrate BBB and target PD-L1 was be explored.And the effect of PD-1-MM@PLGA/RAPA on glioma was evaluated by using a mouse in situ glioma model.And explore whether PD-1-MM@PLGA/RAPA can regulate the immune microenvironment and enhance the anti-tumor effect.The main research contents and corresponding results of this topic are as follows:(1)Construction and characterization of an engineering macrophage strain with high expression of PD-1(Raw264.7-PD-1):Construct a macrophage cell line with high expression of PD-1 through lentiviral vector transfection.Real-time fluorescence quantitative PCR characterizations showed that the stable transfected strain PD-1 m RNA was 173 times higher than that of untransfected macrophage strain.Both the immunofluorescence and flow cytometry showed that the stable ransgenic strain highly expressed PD-1 protein.(2)Preparation and characterization of PD-1-MM@PLGA/RAPA:PLGA/RAPA were prepared by the nano-precipitation method using PLGA as the carrier.Subsequently,PD-1-MM@PLGA/RAPA were prepared by the co-extrusion method.And the properties of PD-1-MM@PLGA/RAPA were characterized in vitro by size and zeta potential analysis,TEM detection and so on..The results indicated that the PD-1-MM@PLGA/RAPA could be successfully created by co-extrusion method.PD-1-MM@PLGA/RAPA had an typical“core-shell”structure,good dispersibility,uniform nano-scale size and good stability.In addition,PD-1-MM@PLGA/RAPA retained the membrane proteins and characteristic functional proteins of Raw264.7-PD-1(integrin CD11c,F4/80,CD206 and PD-1).(3)Evaluation of PD-1-MM@PLGA penetrating the BBB in Vitro and Vivo:On the transwell model both PD-1-MM@PLGA/Di I and MM@PLGA/Di I could be absorbed by rat glioma C6 cells,indicating that PD-1-MM@PLGA/Di I and MM@PLGA/Di I could effectively penetrates BBB in vitro.On the other hand,in vivo distribution experiments showed that PD-1-MM@PLGA loaded with Di R could penetrate BBB and target brain glioma of tumor-bearing mice compared with PLGA/Di R and free Di R.In vitro imaging of brain sections showed that PD-1-MM@PLGA mainly accumulated in PD-L1 high expression areas of glioma,indicating that PD-1-MM@PLGA could penetrate the BBB.(4)Evaluation of PD-1-MM@PLGA targrting PD-L1 in vitro and vivo:The cell uptake results showed that the fluorescence intensity of PD-1-MM@PLGA/Di I in cells progressively increases with time,and C6 cells showed strongest uptake capacity,indicating that PD-1-MM@PLGA could target PD-L1.The inhibition rate of C6 cells(37.54%)by PD-1-MM@PLGA/RAPA was significantly higher than that of 4T1 cells(22.42%)after24 h incubation.And the inhibition rate of the 4T1(24.21%)and C6(27.38%)cells by MM@PLGA/RAPA was no significant difference,which indicated that PD-1-MM@PLGA/RAPA had the potential to target PD-L1.On the other hand,the results of in vivo experiments showed that after PD-1-MM@PLGA/RAPA treatment,the growth of glioma in the 4T1/C6 co-bearing models increase slowly compared to the Glucose,PLGA/RAPA and MM@PLGA/RAPA groups.And the expression levels of cytokines IFN-γand TNF-αof PD-1-MM@PLGA/RAPA gruop are also higher than that of other groups.In addition,the tumor infiltrating CD45~+CD8~+T cells of C6-tumor was significantly higher than 4T1-tumor.It indicated that the PD-1-MM@PLGA/RAPA could target PD-L1 and regulated the immune microenvironment to enhance the anti-tumor effect.(5)Pharmacodynamic experiments in vivo and modulates immune responses in the brain tumor microenvironment:The results showed that after PD-1-MM@PLGA/RAPA treatment,the fluorescence intensity of C6-Luc cells in the brain of tumor-bearing mice increased slowly compared to the Glucose,PLGA/RAPA and MM@PLGA/RAPA groups,indicating that PD-1-MM@PLGA/RAPA could significantly inhibit brain tumor growth.And the survival curve showed that the median survival time of the PD-1-MM@PLGA/RAPA group was 29 days,which was significantly higher than the other three groups.H&E staining of brain sections also showed that glioma cells were sparsely arranged in the PD-1-MM@PLGA/RAPA group.At the same time,immunohistochemical results showed that the expression of GFAP was higher in PD-1-MM@PLGA/RAPA group compared to Glucose group,PLGA/RAPA group and MM@PLGA/RAPA group,while Ki-67 expression was relatively low,indicating that PD-1-MM@PLGA/RAPA could helpfully restrain the malignant hyperplasia of tumor cells and delay the malignant development of glioma.Flow cytometry characterizations showed that tumor infiltrating CD45~+CD8~+T cells of PD-1-MM@PLGA/RAPA group was significantly higher than that of the other three groups.In addition,the expression levels of cytokines IFN-γand TNF-αof PD-1-MM@PLGA/RAPA group are also higher than that of the other groups.It indicated that the PD-1-MM@PLGA/RAPA could regulate the immune microenvironment to enhance the anti-tumor effect.In this study,we successfully prepared an engineering macrophage strain with high expression of PD-1,and then prepared a biomimetic modified nano-drug PD-1-MM@PLGA/RAPA.The PD-1-MM@PLGA/RAPA could efficient package the anti-tumor drug RAPA and retained the membrane proteins of engineered macrophages.At the same time,PD-1-MM@PLGA could penetrate the BBB and target PD-L1 in the glioma area.Finally,in vivo studies have showed that PD-1-MM@PLGA/RAPA could not only helpfully restrain brain tumor growth,but also regulate the immune microenvironment to enhance the anti-tumor effect.This research helps to provide new options for non-invasive targeted drug delivery of glioma,as well as provides experimental data and theoretical basis to explore immunotherapy based on PD-1/PD-L1 immune checkpoint signaling pathway.