Fig. 2: RILO maintains a stable nanostructure in macrophages and RILO@MG maintains the M1 phenotype in an immunosuppressed environment.

a Schematic illustration of the preparation process of RILO@MG. b Micromorphological characterizations of RIL, OMV and RILO (n = 3 biologically independent experiments). Scale bar, 50 nm. c, d Dynamic light scattering (c) and zeta potential (d) analyses of RIL, OMV and RILO (n = 3 biologically independent experiments). e SDS–PAGE protein analysis in OMV, RIL and RILO (n = 3 biologically independent experiments). f TEM images of RILO@M at 0 and 48 h after preparation (n = 3 biologically independent experiments). Scale bar, 200 nm. g Representative confocal images showing the stability of the nanostructure of fluorescently labelled RILO in M1-type macrophages from RAW264.7 cells (M1-type macrophages^RAW) (n = 3 biologically independent experiments). The cell membrane was stained with anti-F4/80 antibody (blue). DSPE-Rhodamine B and C6 were selected to label lipids (red) and replace the drug (green), respectively. Scale bar, 10 µm. h Representative confocal images of RILO@M, RILO@M-free GTP and RILO@MG (n = 3 biologically independent experiments). Scale bar, 10 µm. RAW264.7 cells were used in (h). i, j Representative confocal images (i) and flow cytometric analysis (j) of RILO@MG^RAW after preparation for 24 and 48 h (n = 3 biologically independent experiments). Scale bar, 10 µm. FITC was selected to label GTP in (h–j). All cells were stained with anti-F4/80 antibody (red) and Hoechst 33342 (blue) in (h and i). k, l Phenotype analysis of RILO@MG including the percentage of macrophages with different phenotypes (k) and M1/M2 ratio (l) in different culture environments (n = 3 biologically independent experiments). Data are expressed as the mean ± SD and were processed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (l). ns no significance. BMDMs were used in all experiments involving macrophages unless marked RAW superscript.