Fig. 5: HDDA NBs display high antitumor therapeutic efficacy and strong intratumoral immunostimulatory effect in the 4T1 tumor model.

a Experimental outline showing the treatment steps and the procedures for evaluating the therapeutic and immunostimulation outcomes of different groups in 4T1 tumor-bearing BALB/c mice. b Individual 4T1 tumor growth curves of the mice after different treatments. c Average tumor growth curves of the 4T1 tumor-bearing BALB/c mice after various treatments and d corresponding heat map of the P values between the control group and other groups. The mice treated with PBS were set as the control group. Data are presented as mean + SD. n = 9 mice per group. e Survival rates of the 4T1 tumor-bearing BALB/c mice after various treatments. f, g Confocal microscopic images of representative H&E staining results (scale bar: 300 μm) (f) and TUNEL assay results (scale bar: 100 μm) (g) of the tumor tissue slices from the 4T1 tumor-bearing BALB/c mice sacrificed at day 7 after different treatments. Before imaging, the cell nuclei were stained with Hoechst 33342 (Hoechst). h Representative confocal fluorescence images of the CD44 (green) immunofluorescence staining results of the tumor tissue slices from the 4T1 tumor-bearing BALB/c mice sacrificed at day 3 after PBS (control) or HDDA treatment. CD44 was stained with CD44-FITC antibody. Before imaging, the cell nuclei were stained with Hoechst. i Representative TEM images of the tumor tissue from a 4T1 tumor-bearing BALB/c mouse at day 3 after HDDA treatment. The blue dashed circles indicate the positions of HMVs. Note that the enlarged images without the blue dashed circles have also been given in Supplementary Fig. 33. j Representative confocal fluorescence images of the immunofluorescence staining results (scale bars: 100 μm) of CRT, CD206, Gr-1, FoxP3, and granzyme B of the tumor tissue slices from the 4T1 tumor-bearing BALB/c mice sacrificed at day 7 after different treatments. For f–j, n = 3 mice per group. k Intratumoral expression levels of IL-1β, IL-6, TNF-α, IFN-γ, and IL-10 analyzed by ELISA. Data are presented as mean ± SD. n = 6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical significance in d, k was calculated via one-way ANOVA with a Tukey’s post-hoc test. Source data are provided as a Source Data file.