Fig. 5: Analysis of in vitro macrophage repolarization and antitumor efficacy.

a Confocal images (Scale bar, 15 µm) and (b) flow cytometry analysis depicting the uptake of FITC by RAW 264.7 cells using various delivery nanoparticles (n = 3 independent experiments). c FITC⁺ ratio in M2Φs treated with various formulations (n = 3 independent experiments). d Mean fluorescence intensity (MFI) ratio of FITC⁺ M2Φs across different treatment groups (n = 3 independent experiments). e Polarized RAW264.7 cells (M2-like phenotype) were treated with different formulations for 24 h, followed by flow cytometry analysis to determine the percentages of M1-like and M2-like macrophages (n = 3 independent experiments). f The ratio of M1-like/M2-like macrophages after treatment of varioius formulations (n = 3 independent experiments). g Phagocytic capacity of M2-polarized RAW264.7 cells pretreated with various formulations, assessed by flow cytometry and MTT assay in a coculture with GFP-labeled Hepa1-6 cells. Created in BioRender. Y.Q. (2025) https://BioRender.com/tait9j9. h Flow cytometry analysis of the phagocytic rate of M2-like RAW264.7 cells in a coculture system with GFP-labeled Hepa1-6 cells following incubation with different formulations (n = 3 independent experiments). i Differences in phagocytic rates of Hepa1-6 cells by RAW264.7 cells treated with different formulations (n = 3 independent experiments). j Hepa1-6 cell viability investigation using a coculture system of polarized M2-like RAW264.7 and GFP-labeled Hepa1-6 cells, incubated with different formulations (n = 6 independent experiments). k Co-culture of tEiNS-treated M2-like macrophages, Hepa1-6 cells, and isolated CD3⁺ T cells to assess CD8⁺ T cells activation. Created in BioRender. Y.Q. (2025) https://BioRender.com/tait9j9. l Representative scatter plots of flow cytometry data showing the percentage of GzmB⁺ and IFNγ⁺ cells within the CD8⁺ T cell population following co-culture with EiNS, tDL, tDML or tEiNS-treated M2-like macrophages, Hepa1-6 cells, and CD3⁺ T cells, as indicated (n = 3 independent experiments). m Quantitative results showing the numbers of GzmB⁺ and IFNγ⁺ cells as a percentage of the total CD8⁺ T cell population (n = 3 independent experiments). Data are expressed as mean ± SD and were determined using one-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. ****P < 0.0001.