Fig. 1: Hyaluronic acid-bilirubin nanomedicine (HABN) localizes in tumor cells and tumor-associated myeloid cells (TAMCs) in MC38 tumor-bearing mice.

a HABN-mediated chemoimmunotherapy. b–d MC38 tumor-bearing mice were administered IV on day 25 with 10 mg/kg of HABN-Cy5.5 (equivalent mass of free Cy5.5), or 0.25 mg/kg of free Cy5.5. Whole body fluorescence images (b) and organ images (c) of mice treated with HABN-Cy5.5 or free Cy5.5 by IVIS. d Comparison of uptake levels of HABN-Cy5.5 and free Cy5.5 among various immune cells and cancer cells in tumor tissues by flow cytometry. e Flow cytometry analysis and confocal microscopy images of CT26 and MC38 cells stained with anti-CD44 antibody. f Confocal microscopy images of MC38 cells incubated with HABN-Cy5.5 (20 µg/ml) for 1 h. For the competition study, MC38 cells were pre-treated for 1 h with free HA (2 mg/ml). g Confocal microscopy images of BMDM cells pre-incubated with LPS (100 ng/ml) and IFN-γ (10 ng/ml), IL-4 (20 ng/ml), or control medium for 20 h and stained with anti-CD44 antibody. h, Confocal microscopy images and HABN-Cy5.5 fluorescence intensities of BMDM cells pre-incubated with LPS (100 ng/ml) and IFN-γ (10 ng/ml), IL-4 (20 ng/ml), or control medium for 20 h, followed by 1 h treatment with HABN-Cy5.5 (20 µg/ml). For the competition study, BMDM cells were pre-treated for 1 h with free HA (2 mg/ml) before the antibody treatment. DAPI was used for nuclei counter staining. Scale bars = 20 μm. i MC38 tumor-bearing mice were administered IV with free Cy5.5 or HABN-Cy5.5, followed by flow cytometric analysis for Cy5.5 MFI among Cy5.5⁺ M0-, M1-, and M2-like macrophage subsets. J, k Pearson correlation between j the frequency of HABN-Cy5.5⁺ cells or k HABN-Cy5.5 MFI and CD44 MFI among the total macrophages. l, The expression level of CD44 among HABN-Cy5.5⁺ macrophage subsets. The data represent mean ± s.e.m., biological replicates with n = 3 (d), n = 7 (f, g, h), n = 4-5 (i), n = 15 (j, k), or n = 5 (l). *p < 0.05, ***p < 0.001, ****p < 0.0001, analyzed by one-way ANOVA with Tukey’s HSD multiple comparison post hoc test (d, f, h, l), two-sided Student’s T-test (i), or Pearson correlation (j, k). Source data are provided as a Source data file.