Neutrophil Membrane Engineered Hucmscs SEV In Targeted Anti-tumor Progression

Objective:Human umbilical cord mesenchymal stem cells derived small extracellular vesicle(HucMSC-sEV)play an important role in the intervention of malignant tumor progression.However,the clinical application is limited because that natural hucMSC-sEV pursue short circulating retention in vivo and short target to tumor tissue.In the cause of breaking through these challenges,this study aims to establish an engineering strategy for targeted modification of natural hucMSC-sEV with the neutrophil membrane to improve its targeting and medical virtue,thereby enhancing its anti-tumor efficacy and providing a novel method and significant proof their clinical application.Methods:Primary culture of hucMSC by employ tissue culture.The purity of hucMSC was identified through lipogenesis and osteogenesis experiments and flow cytometry characterization of surface labeled molecules.Obtain 48-hour hucMSC serum free medium,extract hucMSC-sEV by ultracentrifugation,and identify its morphology,particle size distribution and marker molecules through TEM,AFM,NTA and Western blot.The two gastric cancer cell lines were coped with36-hour hucMSC-sEV,and the changes in cell cloning and proliferation ability were detected by clone formation assay and CCK-8 assay.The changes in cell migration and invasion ability were detected by migration and invasion assay.Cell immunofluorescence and Western blot assay were used to detect the changes of protein molecules related to proliferation,apoptosis,and migration.Isolate human peripheral blood neutrophils by density gradient centrifugation.The purity and integrity of neutrophils were identified by cell morphological characteristics,RayleighGiemsa staining,and fluorescence double staining experiments.The membrane of neutrophils was extracted by differential centrifugation and was compressed into about 150 nm neutrophil membrane derived nanovesicles(Neu-NV)using a liposome extruder.Then,a liposome extruder was used to fuse Neu-NV with hucMSC-sEV to form a chimeric nanovesicle(Neu/MSC-sEV).Their morphology,particle size distribution,and protein distribution were identified by TEM,AFM,NTA and Coomassie brilliant blue experiment.Neu-NV,hucMSC-sEV,and Neu/MSC-sEV were labeled with DIL and incubated with gastirc cancer cells for respective times.Cell uptake efficiency was measured by fluorescence.Two gastric cancer cell lines were treated with three types of vesicles for 36 hours.Clonogenesis and CCK-8 assay were used to detect the changes in cell cloning and proliferation,and migration and invasion experiments were used for cell migration and invasion.Cell immunofluorescence and Western blot assay were used to detect the changes of protein molecules related to proliferation,apoptosis,and migration.The reactive oxygen species detection kit detected the production of reactive oxygen species(ROS).In vivo experiment,tumor observation and tumor volume of nude mice were used for evaluation on the anti-neoplastic effect of continuous administration of three types of vesicles through the caudal vein for 15 days.The safety of three types of vesicles was evaluated by HE staining of important organs in animals and functional experiments of normal gastric mucosal cell lines.Mass spectrometry was used to detect significant protein molecules in hucMSC-sEV by contrast with human embryonic lung fibroblasts derived small extracellular vesicles(HFL1-sEV).Western blot showed that the protein molecule PTX3(Pentraxin related protein)was significantly expressed in hucMSC,hucMSC-sEV,and Neu/MSC-sEV,and express low in gastric cancer cell lines and tissues.Two gastric cancer cell lines were transfected with overexpressed PTX3 plasmids,and the changes in cell cloning,migration,and invasion ability were detected through cellular functional experiments.Results:Primary culture of hucMSC and identified.The isolated hucMSC-sEV showed a typical cup-shaped structure,with a particle size analysis showing a normal distribution,with a peak value of about 180 nm.HucMSC-sEV expressed characteristic proteins such as HSP70,CD9,CD81,Alix,and TSG101 positively,while negatively expressed Calnexin and Apolipoprotein B proteins.HucMSC-sEV treatment significantly inhibited the proliferation,cloning,migration,and invasion of SNU-1 and HGC-27 cells.The purity of neutrophils isolated from peripheral blood attained 95%.Neu-NV and Neu/MSC-sEV exhibit a typical cup-shaped structure,and the particle size analysis presented a normal distribution,with peaks around 150 nm and 160 nm,respectively.Neu/MSC-sEV had a protein component common to Neu-NV and hucMSC-sEV.Neu/MSC-sEV boosted the uptake ability of gastric cancer cells and Neu/MSC-sEV treatment exhibited a more significant anti-tumor effect.In the subcutaneous tumor model of nude mice,Neu/MSC-sEV apparently inhibited tumor proliferation and had no significant impact on major organs in nude mice.The high expression of PTX3 in hucMSC-sEV was screened from the mass spectrometry results.After overexpression of PTX3 in gastric cancer cells,the power of cell cloning,migration,and invasion decreased,demonstrating that PTX3 had a tumor inhibiting effect.Conclusions:HucMSC-sEV can bar the gastric cancer cells progression.Mass spectrometry screening shows the overexpression of a tumor suppressor protein PTX3 in hucMSC-sEV,which is able to restrain the cloning,migration,and invasion of gastric cancer cells.Neutrophil membrane engineered hucMSC-sEV significantly improves the targeting of hucMSC-sEV to tumors and inhibits the progression of tumor.This engineering method is due to be an inspiring strategy for enhancing the tumor suppression efficacy of hucMSC-sEV,and afford a new propose for promoting the clinical translation and application of hucMSC-sEV.