Diagnostic Value Of Mannose Contrast Agent Targeting TAM In Lymph Node Metastasis Of Breast Cancer

Objective: To explore the value of multi-functional fluorescent contrast agent in breast cancer lymph node micrometastasis imaging.The mannose fluorescent contrast agent MR780 NPs was prepared,and the metastasis lymph nodes were imaged.Methods: 1.preparation and characterization of materials:MR780 nanoparticles(MR780 NPs)were prepared by ethanol injection method.The signal of fluorescent dye was blocked by disulfide bond.FITC-MR780 NPs were prepared by self-assembly of lipid soluble fluorescent probe DSPE-FITC on the surface of MR780 NPs.FITC-DSPC NPs without mannose were prepared by the same method.Dynamic light scattering was used to detect the particle size;transmission electron microscope to observe its morphology;ultraviolet spectrophotometer and fluorescence spectrophotometer to detect the ultraviolet absorption and fluorescence emission of nanoparticles;and MTT method and hemolytic experiment to test its biosafety.2.The targeting of MR780 NPs to M2 macrophages was detected at cell level:IL-4 was added to RAW264.7 to induce M2 phenotype,and the expression of CD206 was detected by flow cytometry;FITC-MR780 NPs and FITC-DSPC NPs were added into M2 macrophages,and the phagocytosis of M2 macrophages was detected by flow cytometry;After CD206 was pre blocked by free mannose,FITC-MR780 NPs was added to M2 macrophages to detect their phagocytic capacity;The dynamic process of phagocytosis of FITC-MR780 NPs by M2 macrophages was observed by confocal microscopy.3.4T1 nude mice were randomly divided into two groups(n = 10).BALB/C nude mice bearing 4T1-Fluc tumor were established by subcutaneous plantar model.The normal control group was injected with the same amount of PBS.The popliteal lymph nodes of tumor bearing nude mice and normal nude mice were detected by ultrasound every 5 days;After subcutaneous injection of MR780 NPs into the plantar,the popliteal lymph nodes of tumor bearing BALB/C nude mice and normal mice were imaged every 5 days by in vivo fluorescence imaging system.After 20 days,the metastatic lymph nodes were resected under fluorescence guidance for H&E staining and immunohistochemical analysis;One day,10 days and 20 days after injection of MR780 NPs,the heart,liver,spleen,lung and kidney of each group were taken for H&E staining.Results: 1.The average diameter of dynamic light scattering MR780 NPs was 72.81 ±4.39 nm.Under electron microscope,it was spherical structure,uniform size and good dispersion.The UV absorption spectrum of MR780 NPs moved forward compared with IR780,and the absorption waveform of MR780 NPs did not change after the addition of reduced glutathione;the fluorescence spectrum showed that the fluorescence signal of MR780 NPs was successfully blocked,gradually recovered after the addition of 10 m M reduced glutathione,and the peak moved forward compared with IR780.MTT showed that the cytotoxicity of MR780 NPs to HUVEC,RAW264.7 and 4T1 was very low,and the cell survival rate was more than 80%.Hemolysis test showed that the hemolysis of MR780 NPs was less than 5% in the safe range.2.RAW264.7 polarized to M2 type after 24 hours of IL-4induction.CD206 was highly expressed by 81.22%±0.64,while the expression of CD206 in M1 type macrophages induced by LPS was only 1.25% ± 0.23.The difference was statistically significant(t=21.21,P < 0.05);Flow cytometry showed that M2 macrophages had high sensitivity to FITC-MR780 NPs,and phagocytosis was dose-dependent and time-dependent,while the phagocytosis of FITC-DSPC NPs was weak,and did not change with time and dose,the difference was statistically significant(P < 0.05).3.After injection of MR780 NPs(2mg / ml)into the plantar of normal mice for 24 hours,no obvious fluorescence signal was detected in the popliteal fossa.After injection of 4T1-Fluc breast cancer cells into the plantar of normal mice,MR780 NPs(2mg/ml)was injected every 5 days and observed for24 hours.The signal of metastatic lymph nodes was detected by fluorescence imaging,and the difference was statistically significant(P<0.05).Under the guidance of fluorescence,lymph nodes were resected and H&E staining was performed to confirm the existence of tumor metastasis.Immunohistochemical analysis showed that CD206 was highly expressed in metastatic lymph nodes,and KI67 was highly proliferative.After 1 、 10 and 20 days of injection,there was no obvious damage to the important organs.Conclusion: The infiltration of tumor associated macrophages is significantly increased in metastatic lymph nodes,but not in normal lymph nodes.MR780 NPs first targeted CD206 on the surface of tumor associated macrophages in metastatic lymph nodes,and enriched in a large number of metastatic lymph nodes.Reduced glutathione is highly expressed in the tumor microenvironment,which reacts with the disulfide bond of MR780 NPs,resulting in the disintegration of particles and the responsive fluorescence recovery of tumor microenvironment.Because MR780 NPs belongs to nano scale contrast agent,with the characteristics of small particle size,it can pass through lymphatic vessels smoothly.Our study shows that the mannose contrast agent targeting Tam can diagnose lymph node metastasis in a mouse breast cancer model.