Research On The Biosafety Of Immortalized MSCs And Study Of Their Cell Membrane-modified Biomimetic Nanomedicine Lipo-PM@shCD73@DOX Targeting For Glioma Treatment

Background and Objective:Research into new therapies for gliomas has focused on molecular targeted therapy,gene therapy and novel drug carriers.The tumor tropism makes mesenchymal stem cells(MSCs)and their cell membranes as potential drug carriers for tumors treatment.However,limited passaging ability,cellular senescence,and cellular heterogeneity in vitro culture limit the use of MSCs as a long-term cellular source for tumors treatment.Therefore,it is necessary to immortalize MSCs to obtain a stable,adequate and homogeneous source of cells or cell products.After clarifying the biosafety of immortalized MSCs(im-MSCs)and the tumor-homing ability of their cell membranes,a biomimetic liposome nanomedicine based on immortalized MSC plasma membranes(im-MSCs-PM)was designed for glioma-targeted chemotherapy and gene therapy.Methods:Two lines of im-MSCs were obtained by co-transfection of BMI1 and TERT into MSCs,and the molecular characterization of im-MSCs was detected by flow cytometry.Detection of differentiation capacity of im-MSCs by directed induction.The pro-angiogenic capacity of im-MSCs was assessed using angiogenesis assay.The immunosuppressive functional properties of im-MSCs were investigated by indirect co-culture with peripheral blood mononuclear cells(PBMC).The proliferative capacity of the immortalized cells was assessed using CCK-8 proliferation curves and EdU assays.The safety of im-MSCs was evaluated by soft agar cloning assay,karyotype analysis assay,subcutaneous tumorigenesis assay,and in situ tumorigenesis assay.Expression of homing molecules of different generations of im-MSCs was detected by PCR.Uptake of labeled im-MSCs-PM by gliomas and aggregation toward in situ tumors was detected by flow cytometry,laser confocal fluorescence imaging,and small animal live imaging.Bioinformatics was used to analyze CD73 expression in gliomas,and CD73 expression in glioma cell lines was detected by PCR and WB.After down-regulation of CD73 expression in U87 cells,the effects on proliferation and cell cycle of U87 were assessed by CCK-8 cell proliferation assay and cell cycle assay.The signaling pathways that might be affected by CD73 were also predicted by GSEA enrichment analysis,and the mRNA expression of relevant pathway molecules was detected by PCR.Bionic nanodrug carriers based on im-MSCs-PM were synthesized by thin-film method combined with sonication,and the characterization of the syntheses was detected by DLS.The drug was loaded onto the carriers by reverse evaporation to finally obtain Lipo-PM@shCD73@DOX,and cytotoxicity assay was used to detect the in vitro anti-glioma cell capacity of the drug,as well as to assess its anti-glioma efficacy and targeting by tail vein injection.An intracranial glioma resection mouse model was constructed to simulate a recurrent clinical scenario,and the synthesized drug was used for treatment in the model of glioma recurrence.Results:In this study,immortalized human umbilical cord MSCs(im-hUCMSCs)and immortalized human adipose-derived MSCs(im-hADSCs)were constructed.im-hUCMSCs have stronger proliferative ability and higher culturable number of generations.im-hUCMSCs and im-hADSCs were cultured to more than 35 and 60 generations,respectively.im-MSCs retain the molecular expression profile of primary MSCs ana can differentiate into osteoblasts,adipocytes and chondrocytes.im-MSCs retained the properties of primary MSCs that promote angiogenesis and immunosuppression.im-MSCs consistently expressed the tumor homing molecules CXCR4 and SELPG mRNA,which enabled their cell membranes to be taken up by glioma cells in vitro and also to migrate towards the tumor site.Bioinformatics analysis suggested that CD73 was highly expressed in glioma tissues,qRT-PCR and WB assays confirmed that CD73 was highly expressed in glioma cells.In vitro cell proliferation assay and cell cycle assay showed that CD73 downregulation reduced the proliferation ability of glioma U87 cells and the cell cycle was arrested.CD73 downregulation resulted in diminished proliferative capacity of U87 cells,cell cycle arrest,and decreased expression of cell cycle-related molecules CCND,CCNB1/2,and CDK1.Biomimetic nanodrug carriers based on im-MSCs-PM were successfully synthesized.And shCD73 and DOX were successfully loaded onto the carrier to obtain the bionic nanodrug Lipo-PM@shCD73@DOX.Lipo-PM@shCD73@DOX inhibited the growth of glioma cells in vitro,and also inhibited the progression of glioma in vivo.The therapeutic effect was also observed in the mouse model of glioma recurrence.Conclusions:In the present study,two lines of im-MSCs were constructed,which retained the molecular characteristics,directed differentiation,pro-angiogenic and immunosuppressive properties of the primary MSCs.im-MSCs have well biological safety in vitro and in vivo,and can be uptaken by glioma cells in vivo and in vitro,due to the continuous expression of tumor homing molecules.We successfully synthesized Lipo-PM@shCD73@DOX,a biomimetic nanomedicine with dual gene therapy and chemotherapy functions,which can inhibit the growth of glioma cells in vitro.It can also inhibit the disease progression of glioma in both tumor-bearing mice and tumor-recurrent mice models.