Fig. 2: Epacasome strengthened IDO1 inhibition and enhanced cellular uptake mainly via clathrin-mediated endocytosis.

a A table illustrating the physicochemical properties of Epacasome composed of SM/SM-EPA/Cholesterol/DSPE-PEG_2K with various molar ratios. d.nm diameter values in nanometres, DLS dynamic light scattering. b DLS size distribution by intensity. c Cryo-electron microscopy (cryo-EM) of Epacasome-2 (n = 3 independent experiments, similar results were observed). Scale bar, 100 nm. d IDO1 enzymatic inhibitory activity via measuring the Kyn in supernatants in HeLa cells treated with IFN-γ along with free EPA, Lipo-SM/Chol, EPA/Lipo-SM/Chol and Epacasome-2 at equivalent (eq.) EPA concentration (0, 1, 5, 10, 20, 50, 100, 200 and 10,000 nM.) for 48 h. e T-cell proliferation by co-culture^{46, 65}. B16–F10 cells were stimulated by IFN-γ to induce IDO1 expression, then treated by Mitomycin C prior to mixing with splenocytes. Free EPA, Lipo-SM/Chol, EPA/Lipo-SM/Chol and Epacasome-2 were added to co-culture cells at eq. dose of EPA (0, 8, 40, 200 and 1000 nM). To evaluate T-cell proliferation, anti-CD3 and IL-2 were added to co-cultures. Three days later, CD8⁺ T-cell proliferation was assessed by FACS analysis. f Cellular uptake levels and EPA release of Epacasome-2 in B16–F10 cells after 24- and 48-h incubation measured by HPLC, respectively (Supplementary Fig. 3). The release ratios of EPA from Epacasome-2 were presented as percentage values in the figures. g Representative confocal laser scanning microscopy (CLSM) for visualising the intracellular trafficking of Cy5/Epacasome-2 in B16–F10 cells after incubating with Cy5/Epacasome-2 for 2 h. Hoechst 33342 (blue) and Lysotracker (green) were used to stain cell nuclei and lysosome, respectively (n = 3 independent experiments, similar results were observed). Scale bar, 10 µm. h, i Mechanistic investigation of cellular internalisation. B16–F10 cells were treated with various endocytotic inhibitors first for 0.5 h then incubated with Epacasome-2 labelled by DSPE-Cy5 for another 2 h. h Representative Cy5 intensity histogram by flow cytometry. i Cellular uptake by cell count percentage analysis. Data in (a, right portion, d–f, i) are expressed as mean ± s.d. (n = 3 biologically independent samples). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Source data are provided as a Source Data file.