Additional file 1 of “Two-birds-one-stone” colon-targeted nanomedicine treats ulcerative colitis via remodeling immune microenvironment and anti-fibrosis

Additional file 1: Figure S1. Effect of PA on macrophage repolarization and synergistic effect with SV. (A–C) The mRNA levels of M1-associated pro-inflammatory cytokines (e.g., IL-1β, IL-6, and TNF-α) in PA-treated RAW264.7 macrophages, as measured by qPCR. (D, E) Western blot analysis of Akt/MAPK/NF-κB pathway-related biomarkers and M2-related MR expression after PA treatment. (F) The mRNA levels of the M1-related pro-inflammatory molecules (e.g., TNF-α, iNOS, and COX-2) and M2-related Arg1 in SV-treated RAW264.7 macrophages, as measured by qPCR. (G) IL-6 mRNA levels in LPS-induced peritoneal macrophages treated with PA (10 μM) and SV (0, 1, and 2 μM). Figure S2. Cytotoxicity study of PA and SV on (A) Caco-2 cells, (B) RAW264.7 cells, and (C) L929 cells. Cytotoxicity of the NPs in (D) L929 cells and (E) M2Φ. Figure S3. Anti-colitis treatment of synergistic drugs. (A) Schematic diagram of DSS-induced colitis and treatment. (B) Changes in daily bodyweight of each group during the trial period. (C) Statistical analysis and (D) images of colon lengths in each group (n = 4). Figure S4. Preliminary biosafety assessment of PA and SV. (A) Organ coefficients. (B) H&E staining of the major organs (Scale bar: 100 μm). Figure S5. Fluorescence images of (A) M1Φ and (B) L929 after incubation with the coumarin 6-labeled NPs (scale bar: 50 µm). (C, E) Histogram and (D, F) mean fluorescence intensity of the NPs-internalized M1Φ (LPS-induced RAW264.7 cells) and L929 cells were analyzed by flow cytometry (n = 3). Figure S6. Specific accumulation of CS-PLGA NPs in inflamed colons. Ex vivo imaging and radiant efficiency of (A, B) organs and (C) colons at 3 h. Ex vivo imaging and radiant efficiency of (D, E) organs and (F) colons at 5 h (n = 3). Figure S7. (A–E) Individual bodyweight curves and (F–J) DAI curves in CMC-Na, DSS, PA/SV, PLGA NPs, or CS-PLGA NPs groups (n = 6). Figure S8. Preliminary biosafety assessment. (A) Organ coefficients. (B) H&E staining of the major organs (Scale bar: 100 μm). Figure S9. The dot plots of M2Φ and DCs in the colon tissue. Figure S10. The dot plots of neutrophils, inflammatory monocytes, and G-MDSCs in the colon tissue. Figure S11. The dot plots of Tregs in the colon tissue. Table S1. The primer sequence used in qPCR. Table S2. Disease activity index (DAI) scoring. Table S3. Characterization of the NPs. Table S4. Drug encapsulation efficiency and drug loading efficacy