Nitrogen-doped Graphene Quantum Dot Complex Targeting M1 Macrophages By FR-β

The purpose of this study is to explore the quantum dot complex by FR-βthe target effect on M1 macrophages.It is mainly divided into the following three partsThe first part : Preparation and characterization of quantum dot complexObjective: The compound of quantum dot complex(FA-N-GQDs)was prepared by nitrogen dopped graphene quantum dots(N-GQDs)and folate(FA).Methods: 1.N-hydroxysuccinimide(NHS)and 1-(3-dimethylaminopro-pyl)-3-ethylcarbodiimide hydrochloride(EDC)were used to mix with FA.N-GQDs were added into 2-morpholine ethanesulfonic acid(MES)buffer for reaction,and then purified by dialysis to obtain FA-N-GQDs complex solution with high purity.A part of the solution and N-GQDs aqueous solution were taken to mix FA-N-GQDs complex with N-GQDs by vacuum freeze dryer The freeze-dried powder was prepared and stored in a refrigerator at-20 ℃ for subsequent test.2.The FA-N-GQDs complex solution was characterized by ultraviolet spectrophotometer,and its characteristic absorption peak was determined.At the same time,the characteristic absorption peaks of FA solution and N-GQDs aqueous solution were determined and compared to verify the synthesis of FA-N-GQDs complex.3.The FA-N-GQDs freeze-dried powder was mixed with KBr to make pressed tablets.The structure of the tablets was analyzed by infrared spectroscopy.The FA powder pressed tablets and N-GQDs freeze-dried powder pressed tablets were prepared at the same time.The structure was analyzed and compared with FA-N-GQDs complex to verify the synthesis of FA-N-GQDs complex.4.Scanning N-GQDs and FA-N-GQDs complexes under transmission electron microscope to observe the microstructure and synthesis of FA-N-GQDs complexes.Results: 1.By comparing the UV Vis absorption spectra,the UV characteristic absorption peaks of FA-N-GQDs complex contained the characteristic absorption peaks of N-GQDs and FA.2.By comparing the infrared spectra,the infrared characteristic absorption peaks of FA-N-GQDs complex contain both the characteristic absorption peaks of N-GQDs and FA.3.Electron microscopy showed that FA-N-GQDs complex N-GQDs adhered to the background of FA formation.Conclusion: The characterization confirmed that FA-N-GQDs complex was successfully prepared.The second part : Induction of THP-1-M1 macrophages and expression of FR-βObjective: The expression of FR-β in THP-1-M0(M0),THP-1-M1(M1)and THP-1-M2(M2)macrophages was detected.Methods: 1.The THP-1 cells were changed,subcultured and cryopreserved.The images were taken under the light microscope to observe the morphology,and compared with the reference images.2.2.Two 6-well plates were labeled as A plate and B plate respectively,and the same treatment was done: the holes on the 6-well plate were divided into 3 groups,2 holes in each group,which were divided into M0 group,M1 group and M2 group respectively.The treated THP-1 cells were seeded into M1 group and M2 group,and a certain concentration of phorbol myristate acetate was added to each hole,After 24 hours of induction,THP-1 cells were induced to differentiate into M0 macrophages.After morphological changes were observed under light microscope,lipopolysaccharide(LPS)and interferon(IFN)-γ were added into each well of M1 group to induce M0 macrophages to polarize into M1 macrophages.In M2 group,interleukin-6 was added into each well In M0 group,THP-1 cells were seeded into THP-1 cells and induced by PMA for 24 hours to differentiate into M0 macrophages.After induction,pictures were taken under light microscope to observe the morphological differences among the three kinds of macrophages.3.A plate was taken out and one hole in each group was taken for flow cytometry.M0 group was stained with CD14 antibody by flow cytometry;M1 group was stained with CD80 antibody and CD86 antibody by flow cytometry;M2 group was stained with CD163 antibody and cd206 antibody by flow cytometry.4.The m RNA of cells in each group was extracted.The expression of folr2(FR-β)gene was detected by PCR.The above experiment was repeated three times.5.Using B plate,the total protein of each group was extracted.Western blot was used to detect the expression of folr2 protein.The above experiment was repeated three times.6.The 24 well plates were divided into three groups with 2 holes in each group,which were divided into M0 group,M1 group and M2 group,respectively.THP-1 cells were evenly seeded to the top of cell climbing sheet in M0,M1 and M2 groups.THP-1 cells were polarized into M0,M1 and M2 macrophages by the above induction method.One well in each group was added with folr2 immunohistochemical first antibody as the experimental group,and the rest were used as blank control group.FR-β staining was observed and compared under the light microscope.Results: 1.There were morphological differences among THP-1 cells,M0 macrophages,M1 macrophages and M2 macrophages under light microscope.2.M0 macrophages were positive for CD14 by flow cytometry;M1 macrophages were positive for CD80 and CD86;M2 macrophages were positive for CD163 and cd206.3.The relative expression of folr2 gene was different among M0,M1 and M2 macrophages by PCR,and M1 macrophages had the highest relative expression level(p<0.05).4.The difference of folr2 protein expression among M0,M1 and M2 macrophages was detected by Western blot,and M1 macrophages had the highest expression level(p < 0.05).5.Immunohistochemistry showed that M1 macrophage test group had the deepest staining.Conclusion:1.THP-1 cells successfully polarized into M0,M1 and M2 macrophages.2.The content of FR-β in THP-1 cells,M0,M1 and M2 macrophages was different,and the content of FR-β in M1 macrophages was the highest.The third part : The study of FA-N-GQDs complex target by FR-β to M1 macrophagesObjective: FA-N-GQDs complex was observed to pass through FR-β on M1 macrophage targeting and the effect on M1 macrophage targeting.Methods: 1.The 6-well plates were divided into 5 groups with 1 hole in each group: M1-FA-N-GQDs complex group,M1-N-GQDs group,M1-FA-N-GQDs complex + FA group,M2-FA-N-GQDs complex group and M2-N-GQDs group.THP-1 cells were induced to differentiate into M1 and M2 macrophages.First,the M1-FA-N-GQDs complex + FA group was cocultured with FA solution in advance for 30 min,then the M1-FA-N-GQDs complex group,M1-FA-N-GQDs complex + FA group and M2-FA-N-GQDs complex group were cocultured with FA-N-GQDs complex solution;the M1-N-GQDs group and M2-FA-N-GQDs complex group were cocultured with FA-N-GQDs complex solution;the M1-N-GQDs group and M2-FA-N-GQDs complex group were cocultured with FA-N-GQDs complex solution.LPS and IFN-γ were added into the pores containing M1 macrophages to stabilize their polarization morphology,and IL-4 and IL-13 were added into the pores containing M2 macrophages to stabilize their polarization morphology.After co culture for 6hours,the medium was discarded and washed with PBS for 2-3 times.The pictures were taken under light microscope and fluorescence microscope to observe the fluorescence difference.2.Two 96 well plates were divided into A plate and B plate.THP-1 cells were seeded into A plate and polarized into M1 macrophages and divided into 7 groups,with 4 holes in each group as 4 parallel experiments,namely,M1 1000 group,M1 500 group,M1 250 group,M1 125 group,M1 62.5 group,M1 31.3 group and M1 group(control group).Cell culture medium was added into a circle of holes around the group to prevent liquid evaporation and served as blank group.VE cells were seeded into the B plate and divided into groups by the same method as plate A.The final concentrations of the complex were 1000,500,250,125,62.5,31.3 ug / ml and added into different groups.LPS and IFN-γ were added into the pores containing M1 cells to stabilize their polarization morphology.After co culture for 24 hours,CCK-8 experiment was performed.Results: 1.Under fluorescence microscope,M1-FA-N-GQDs complex group showed obvious fluorescence,while M1-FA-N-GQDs complex group,M1-FA-N-GQDs complex + FA group,M2-FA-N-GQDs complex group and M2-N-GQDs group showed no obvious fluorescence.2.With the increase of the concentration of FA-N-GQDs complex,the activity of VE cells and M1 macrophages were inhibited,and the activity of M1 macrophages was lower than that of VE cells(p<0.05).Conclusion: 1.Compared with M2 cells,the effect of FA-N-GQDs complex on the expression of FR-β on M1 macrophages was significant it has a good targeting effect.2.FA-N-GQDs complex has good biocompatibility,but has certain toxicity to M1 macrophages.