Fig. 8: siRNA lipid nanoparticles targeting Gpnmb in lesional macrophages alleviated atherosclerosis in mice.

A Schematic diagram of targeted siRNA-LNPs design. Created in BioRender. Wang, M. (2025) https://BioRender.com/39a6n06. B Quantitative PCR analysis of Gpnmb expression in BMDMs treated with the indicated concentration of siGpnmb-LNPs or siNC-LNPs, N = 3 independent experiments. Representative images of ApoE^−/− BMDMs treated with siGpnmb-LNPs or siNC-LNPs followed by Dil-OxLDL-treatment for 12 h (right, C) or OxLDL-treatment for 24 h and Nile Red staining (left, D). N = 3 independent experiments. The quantitative analysis of total fluorescence intensity of Dil-OxLDL (C, left, n = 30 images, Scale bar, 10 μm) and Nile Red (C, right, n = 50 images, Scale bar, 15 μm), mean ± SEM, N = 3 independent experiments. E Representative confocal microscopic images of ApoE^−/− BMDMs after incubation for 1, 3, or 12 h with Cy5.5-siRNA-LNPs (red). Nuclei were stained with Hoechst 33342 (blue), and late endosomes were stained with LysoTracker Red (green), N = 3 independent experiments. Scale bar, 2 or 3 μm. F At the fifth day after the whole eight injections of Cy5.5-siRNA-LNPs, in vivo MSOT imaging of Cy5.5-siRNA-LNPs in the aortic arch, descending thoracic aorta, thoracic abdominal aorta, and abdominal aorta from 16-week WD-fed ApoE^−/− mice (Late Group, male). Aortic arch: White dotted lines frame, N = 3 independent experiments. G At 12 h after whole eight injections of Control-LNPs or CD36 + TREM2-LNPs, In Vivo Animal Live-Cell Imaging of Cy5.5-LNPs in 16-week WD-fed ApoE^−/− mice (male), mean ± SEM, N = 3 independent experiments, n = 5/12/6 images for plaque/ liver/ kidney). H, At 24 h after whole eight injections of Control-LNPs or CD36 + TREM2-LNPs, ex vivo fluorescence imaging of isolated organs from 12-week WD-fed ApoE^−/− mice (Early Group, male), mean ± SEM, N = 4 independent experiments). I Representative images of cross sections of the aortic sinus from immunofluorescence of F4/80⁺ macrophages (Green), Nile Red⁺ foam cells (Red), and Cy5.5-siRNA-LNPs (Magenta) in atherosclerotic plaque from 16-week WD-fed ApoE^−/− mice (Late Group, male), N = 3 independent experiments. J, At the seventh day after the whole eight injections of Cy5.5-siRNA-LNPs, ex vivo fluorescence imaging of isolated aortas from 12-week WD-fed ApoE^−/− mice (Early Group) and 16-week WD-fed ApoE^−/− mice (Late Group), mean ± SEM, n = 3. K The increased body weight (= final weight – WD-starting weight, n = 5/7/3/4) and the levels of total triglyceride, TCHO, and LDL-C in serum from 12-week WD-fed ApoE^−/− mice (Early Group, male, n = 7) and 16-week WD-fed ApoE^−/− mice (Late Group, male, n = 6), mean ± SEM. L, On the sixth day after the whole eight injections of Cy5.5-siRNA-LNPs, the representative images of ultrasound examinations on the aortic arch and abdominal aorta from 12-week WD-fed ApoE^−/− mice (male, n = 3). M, Representative photomicrographs and the quantification of en face lesion area in the whole aortas from 12-week WD-fed ApoE^−/− mice (Early Group; left upper, male, n = 7; left lower, female, n = 3) and 16-week WD-fed ApoE^−/− mice (Late Group, male, n = 5), mean ± SEM. N Representative flow cytometric expression of CD64 and BODIPY levels in aortic macrophages (left) and the quantitative percentages of flow cytometric BODIPY^hi CD64^hi foam cells in aortic macrophages (right, mean ± SEM, male, n = 3/group). O The quantification of plaque area percentages of Oil-Red-O-stained cross sections of aortic sinus from 12-week WD-fed ApoE^−/− mice (Early Group; n = 5; female, n = 3) and 16-week WD-fed ApoE^−/− mice (Late Group, male, n = 5), mean ± SEM. For each mouse, the average of the staining results from three consecutive sections was calculated for statistical analysis. P, Representative images of cross sections of the aortic sinus from immunofluorescence of F4/80⁺ macrophages (Green) and Nile Red⁺ foam cells (Red) in atherosclerotic plaque from 12-week WD-fed ApoE^−/− mice (Early Group; n = 5; female, n = 3) and 16-week WD-fed ApoE^−/− mice (Late Group, male, n = 5), mean ± SEM. For each mouse, the average of the staining results from three consecutive sections was calculated for statistical analysis. P values were calculated by unpaired Student t-test (B, C, G, H, J, K, M–P). Differences are significant for *P  <  0.05, **P  <  0.01, ***P  <  0.001, ****P  <  0.0001.