| Objective:Pancreatic carcer is characterized by a dense matrix rich with activated fibroblasts and tumor-associated macrophages(TAMs).This inhibits delivery of chemotherapeutic agents and induces an immunosuppressive microenvironment to protect cancer cells and accelerate their growth.TAM-released deoxycytidine inhibit gemcitabine(GEM)therapy in pancreatic cancer.Recent studies have shown that the bilayer membrane structure of cell membrane nanovesicles has the advantages of good biocompatibility and low toxicity,which can load chemotherapeutic agents and deliver drugs to tumor cells by membrane fusion.CD47/SIRPα immune checkpoint plays an important role in the treatment of pancreatic cancer.The aim of this study was to construct GEM & SIRPα NVs.This nanomaterial enables precise delivery of GEM into tumor tissue and enhances the tumor-killing effect of GEM.At the same time,this nanomaterial is able to reduce treatment resistance and can enhance the ability of macrophages to engulf pancreatic cancer cells.Methods:(1)GEPIA2 database analysis,immunohistochemistry(IHC)and RT-qPCR were used to investigate the expression of CD47 in pancreatic cancer cells;CCK8 kit assay was used to investigate the effect of tumor-associated macrophages on pancreatic cancer proliferation and drug resistance.(2)We constructed cells stably overexpressing SIRPα by Lentiviral Guide,and the efficiency of stable rotation was verified by laser scanning confocal microscope(LSCM)and Western Blot.Membrane vesicles of cells overexpressing SIRPα were prepared by differential centrifugation.To prepare GEM&SIRPα NVs,we loaded cell membrane vesicles with GEM by electroporation.(3)Transmission Electron Microscope,dynamic light scattering and ultraviolet and visible spectrum were used to understand the SIRPα NVs characterization and drug loading efficiency.(4)Validation of the targeting of SIRPα NVs on pancreatic cancer cells and tumors with the help of LSCM and IVIS spectrum.(5)Using Flow cytometry and CCK8 kit to verify the effect of GEM&SIRPα NVs on phagocytosis of tumor-associated macrophages and the improvement of tumorassociated macrophages causing drug resistance in pancreatic cancer treatment.Results:(1)GEPIA2 database analysis,IHC and RT-qPCR showed that pancreatic cancer cells expressed higher CD47 than adjacent normal tissue,and CCK8 kit showed that tumorassociated macrophages promoted pancreatic cancer proliferation and led to GEM resistance in pancreatic cancer cells by releasing deoxycytidine.(2)We successfully constructed HEK 293 T cells which stably expressing SIRPα and obtained cell membrane vesicles overexpressing SIRPα by differential centrifugation of ground cells.(3)The prepared cell membrane vesicles were bilayer structure which showed by Transmission Electron Microscope.Vesicle measured by dynamic light scattering showed that the particle size is about 100 nm and the potential is about-8 m V.The loading GEM efficiency is about 30%.(4)LSCM and IVIS spectrum showed that SIRPα NVs can target binding to pancreatic cancer cells and tumor tissue.(5)GEM&SIRPα NVs promoted phagocytosis of pancreatic cancer cells by tumorassociated macrophages,reduced GEM treatment resistance caused by tumor-associated macrophages.GEM&SIRPα NVs also promoted the killing of tumor cells by GEM.Conclusion:Our study revealed that GEM&SIRPα NVs can target binding to pancreatic cancer cells and tumor tissue and is highly effective in killing pancreatic cancer cells.At the same time,this nanomaterial can enhance TAMs phagocytosis of pancreatic cancer cells,which has the advantages of reducing the dose of chemotherapy drugs,reducing the resistance of chemotherapy drugs and improving the precision targeting of chemotherapy drugs to tumors,and has good clinical transformation value for the treatment of pancreatic cancer. |
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