Application Of Graphene Nanocarriers In Immunotherapy Of Breast Cancer

Objective Programmed death 1(PD-1)and programmed death 1 ligand 1(PD-L1)are important immunosuppressive effector molecules known to cause tumor immune escape mechanisms.The immunomodulatory effects of PD-1 and PD-L1 immunosuppressive agents targeting PD-1 and PD-L1,respectively,are of great value in anti-tumor and anti-inflammatory.Graphene oxide(GO)has particularly unique effect in the adhesion and transport of aromatic,poorly water-soluble drugs and other materials due to its high electrical conductivity,good chemical stability,and extremely large specific surface area and rich surface chemical groups.In this study,a new cancer therapeutic drug delivery system was constructed using graphene oxide(GO)as the carrier core and linking PD-1、PD-L1 antibody to explore its role in breast cancer targeted immunotherapy.Methods The PD-1 and PD-L1 antibody was attached to the surface of GO by an amide bond provided by PEG under mild conditions,and a fluorescent material(FITC)was used as a fluorescent probe to obtain a nanocarriers drug delivery system(GO-PEG-FITC / PD-1/ PD-L1 antibody).The surface structure of the pure GO sheet and the constructed delivery system was observed and compared by transmission electron microscopy(TEM).The drug delivery vector was scanned by atomic force microscopy to observe the binding of PD-1 and PD-L1 antibodies on the GO surface.Immunofluorescence assay was used to detect the expression of PD-1 and PD-L1 in the cytoplasm of MCF-7 cells.The MCF-7 cells were incubated with the drug delivery vehicle,and the uptake of the nanocarriers by the cells was studied by confocal fluorescence microscopy and thin layer scanning techniques.The cytotoxicity of GO,GO-PEG and the cytotoxicity of the delivery system at different drug concentrations and different incubation times were examined by MTT assay using MCF-7 cells.Establish a nude mouse tumor-bearing model,then,injected the different anti-tumor drugs into the tumor,and the tumor inhibition rate was calculated by tumor growth and tumor volume measurement,and the ability to inhibit tumor growth in nude mice was observed.Results TEM results showed a clear and smooth surface of pure GO flakes(<1 μm).However,after the delivery of the drug delivery vehicle,the surface of the GO showed a large amount of particles with a relatively uniform distribution of dot-like changes.AFM estimates that the thickness of the pure GO sheet is about 0.75 nm,the finished delivery carrier has a thickness of 1.5 nm,and has a rough surface with some protrusions on the surface compared to the pure GO sheet.Immunofluorescence assay showed that PD-L1 molecule expression was present in the cytoplasm of MCF-7 cells.MTT assay showedthat GO and GO-PEG have no cytotoxicity to MCF-7 cells cultured in vitro.The delivery vehicle carrying the PD-L1 antibody(GO-PEG-FITC / PD-L1,GO-PEG-FITC / PD-1PD-L1)compared to the PD-L1,PD-1 antibody alone and GO-PEG-FITC / PD-1 showed relatively strong cytotoxicity against MCF-7 cells.There is also a tendency to increase with increasing drug concentration and co-incubation time.Animal experiments showed that the delivery vehicle carrying the PD-L1 antibody had a relatively strong inhibitory effect on the growth of tumors in nude mice compared with the other experimental groups.Conclusion This study successfully prepared a novel cancer immunotherapy drug delivery system using PD-1 and PD-L1 antibodies with graphene oxide(GO)as the carrier core.It also confirmed that the PD-L1 antibody delivery system has a growth inhibitory effect on MCF-7 tumor cells and nude mice in vitro,providing a reference treatment method for targeted immunotherapy of breast cancer.