Fig. 4: In vitro regulatory effect of RG@M-γ-CD upon macrophage.

Cell internalization of CNPs by macrophages. a Representative CLSM images of the peritoneal macrophages cultured with Rho@M-γ-CD and Rho@γ-CD CNPs (equivalent Rho, 5.0 μM) for 2 h. Blue fluorescence indicated nuclear staining with DAPI; green fluorescence indicated mannose receptors staining with FITC-antibody; red fluorescence presented the internalization of CNPs. Scale bar, 20 μm. b FACS analysis and statistical result of intracellular fluorescence intensities induced by internalized Rho@M-γ-CD and Rho@γ-CD (equivalent Rho, 5.0 and 10.0 μM) in peritoneal macrophages (2 × 10⁴ cells/sample). N = 3 biological replicates in each group. Data were presented as means ± SD. Statistical significance was calculated using two-tailed unpaired t-test (Rho@M-γ-CD vs. Rho@γ-CD at the same Rho concentration, the exact p values were indicated in the figure). c Schematic illustration of peritoneal macrophages isolation, stimulation, and phenotype evaluation. Peritoneal macrophages were treated with different formulations for 24 h, followed by incubation with LPS, IL-4, or tumor supernate for 2 h. Then the cells and culture media were collected for phenotype evaluation. d mRNA expression levels of pro-inflammatory cytokines by peritoneal macrophages treated with different formulations (equivalent RG, 0.5 μM). N = 3 biological replicates in each group. Data were presented as means ± SD. e Western blot analysis of p-p65, p65, and abca1 expressions in macrophages under different treatments. Images were representative of three independent experiments. f mRNA expression levels of M2-associated markers by macrophages under different treatments (equivalent RG, 0.5 μM). N = 3 biological replicates in each group. Data were presented as means ± SD. g mRNA expression levels of VEGF-B, PDGF-α, and MMP-9 by macrophages under different treatments (equivalent RG, 0.5 μM). N = 3 biological replicates in each group. Data were presented as means ± SD. Statistical significance for d, f, and g was calculated using one-way ANOVA followed by Dunnett’s multiple comparison test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, treatment group vs. control, the exact p values were indicated in the Source Data file). Source data are provided as a Source Data file.