| Objectives: The tumor microenvironment refers to the localized environment in which infiltrating interstitial cells(including macrophages,fibroblasts,and endothelial cells)and their secreted active media which surrounds tumor cells.Among these cells,TAMs are the most abundant interstitial cells.TAMs can be divided into two categories based on their phenotypes and functions.Many studies have shown that most TAMs express an M2-like phenotype,which promotes tumor growth,tumor invasion,neovascularization,and lymphangiogenesis,as well as participates in tumor immunosuppression.Given the effects of TAMs on tumors,the removal of TAMs from the tumor microenvironment can be used as a treatment for tumors.CD163 is a hemoglobin scavenger receptor that is specifically expressed in mononuclear macrophage systems,and is considered to be a highly specific M2 macrophage marker.Gold nanoparticles(Au NPs)can be used as photoacoustic materials because of their high stability,low toxicity,and good light absorption characteristics.The surface plasmon resonance(SPR)of gold nanoparticles after exposure to near-infrared(NIR)laser irradiation not only produces strong light scattering,but also causes phase change of liquid fluorocarbon.Therefore,gold particles have been extensively studied in the imaging and treatment of tumors.Gold nanorods(GNRs)are rod-shaped gold nanoparticles with a range of useful physicochemical properties,making them widely used in biomedicine.The association of GNPs with polypeptides,folic acid,and antibodies enhances their targeting ability.Increasing attention has been paid to photothermal therapy(PTT),which mainly utilizes the high capacity of NIR laser to penetrate tissues.Gold nanoparticles can effectively kill tumor cells under NIR laser.Therefore,functionalized gold nanorods can kill TAMs targetedly under photothermal conditions,and play a role in the treatment of tumors.Methods: 1.Preparation of gold nanoparticles targeting TAMs: Gold nanoparticles(m Ab-CD163/Au)targeting tumor associated macrophages were prepared using CD163 monoclonal antibody and gold nanorods.m Ab-CD163/Au was physically characterized.The coupling of gold nanorods and CD163 monoclonal antibody was verified by flow cytometry.Different concentrations of m Ab-CD163/Au solution were irradiated by NIR laser,and the temperature of each solution was recorded at each time point.2.The anti-tumor effect of targeted killing TAMs in vitro: Human monocyte line THP-1 cells were induced to differentiate into M2 macrophages by Phorbol-12-myristate-13-acetate(PMA)and interleukin-4(IL-4)in this study,and the phenotype was verified by flow cytometry.During the transformation from M1 macrophages to M2 macrophages,the m RNA and protein levels of tumor necrosis factor-?(TNF-?)and CD163 were detected by quantitative real-time PCR(q PCR)and Western blot.M2 macrophage climbing slices were prepared,and the aggregation of m Ab-CD163/Au in macrophages was observed by two-photon microscopy.Different concentrations of m Ab-CD163/Au solution were added into M2 macrophages,and the cytotoxicity of m Ab-CD163/Au solution with different concentrations was detected by CCK-8 assay.CCK-8 assay was also used to detect the killing effect of m Ab-CD163/Au solution with different concentrations on M2 macrophages under NIR laser irradiation;Transwell chamber was used for co-culture of tumor cells and macrophages.Exposed to NIR laser,M2 macrophages were killed by m Ab-CD163/Au,and then the change of invasion ability of human breast cancer MCF-7cells was analyzed.3.Anti-tumor effect of targeted killing TAMs in vivo: The xenograft model of human breast cancer MCF-7 cells was established in BALB/c nude mice.When the mean diameter of xenograft tumors reached 5 mm,0.1 m L of m Ab-CD163/Au solution was injected into the upper,lower,left and right parts of each tumor in the experimental group,and then irradiated by NIR laser.The tumor size was measured every 2 days,and the tumor growth curve was drawn.After 15 days,the mice in all groups were euthanized.The xenograft tumor tissues of each group were collected for immunohistochemical analysis and CD163+ cells on the periphary of tumors were counted.Results: 1.Preparation of gold nanoparticles targeting TAMs: The results showed that the average particle size of gold nanoparticles was 363 nm and the zeta potential of the solution was-6.4 m V by Malvern analyzer,and the solution had an absorption peak near 1064 nm detected by UV-Vis spectrophotometer.The results of flow cytometry showed that the gold nanoparticles were coated with CD163 antibody.The longer the NIR laser irradiation time,the higher the solution temperature;when the irradiation time is equal,the higher the concentration of m Ab-CD163/Au,the higher the solution temperature.2.The anti-tumor effect of targeted killing TAMs in vitro: THP-1 cells were round,with transparent inside,and grew in suspension.After PMA treatment,the cells aggregated and adhered to the wall.After IL-4 treatment,the cells were spindle shaped and pseudopodia could be seen.The results of flow cytometry showed that the THP-1 cells induced by PMA and IL-4 highly expressed CD163.q PCR and Western blot results showed that during the transformation from M1 macrophages to M2 macrophages,the expression of TNF-? decreased at m RNA and protein levels,while the expression of CD163 increased at m RNA and protein levels.The results of two-photon microscopy showed that more m Ab-CD163/Au particles were accumulated in M2 macrophages.CCK-8 results showed that the cytotoxicity of m Ab-CD163/Au solution increased with the increase of concentration;under the irradiation of NIR laser,the higher the concentration of m Ab-CD163/Au,the greater the killing effect on M2 macrophages.Transwell results showed that under NIR laser irradiation,the number of MCF-7 cells passing through the chamber decreased by killing M2 macrophages with m Ab-CD163/Au,indicating that the invasion ability of MCF-7 cells decreased.3.Anti-tumor effect of targeted killing TAMs in vivo: Human breast cancer MCF-7 cell suspension was injected into the armpit fat pad of BALB/c nude mice,and the xenograft tumor model was successfully established in about 5 days.The tumor volume of the experimental group(injected with m Ab-CD163/Au solution in the upper,lower,left and right parts of the tumor respectively and irradiated by NIR laser)was significantly smaller than that of the control group.Immunohistochemical results showed that the number of CD163+ cells around tumor in experimental group was significantly less than that in control group.Conclusions: 1.gold nanoparticles modified by CD163 monoclonal antibody are targeted to TAMs.2.In vitro,targeted killing of TAMs under photothermal conditions can reduce the invasive ability of tumor cells.3.In vivo,targeted killing of TAMs under photothermal conditions can inhibit tumor growth. |
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