Journal: Advanced Science

Article Title: Copper‐Doped Prussian Blue Nanozymes With Hyaluronic Acid‐Mediated Targeting Alleviate Oxidative Stress and Regulate Cholesterol Handling for Atherosclerosis Therapy

doi: 10.1002/advs.76976

Figure Lengend Snippet: Preparation and anti‐atherosclerotic mechanisms of the CuPB@HA nanozyme. (A) Synthesis procedure of CuPB@HA. PB nanoparticles were prepared through a PVP/HCl‐assisted thermal reaction using K 3 [Fe(CN) 6 ] as the precursor, followed by Cu incorporation to obtain CuPB and subsequent HA functionalization to form CuPB@HA. (B) Therapeutic mechanisms. Systemically administered CuPB@HA selectively targets lesional CD44 + macrophages. Upon internalization, it synergistically remodels the plaque microenvironment by scavenging ROS to promote a shift toward an anti‐inflammatory macrophage phenotype and improving macrophage lipid‐handling profiles by downregulating CD36 and upregulating ABCA1/ABCG1‐related cholesterol transport mediators, thereby attenuating foam‐cell lipid accumulation. Some elements in the image were sourced from BioRender ( https://app.biorender.com/illustrations/69c95a7d8bdf29a2ebf6bea4 ).

Article Snippet: Subsequently, the samples were incubated overnight at 4°C with specific primary antibodies targeting the M1 marker iNOS (Proteintech, 22226‐1‐AP,1:400), the M2 marker ARG1 (Proteintech, 16001‐1‐AP, 1:400), or the lipid uptake receptor CD36 (MedChemExpress, HY‐P86458, 1:400).

Techniques:

Journal: Advanced Science

Article Title: Copper‐Doped Prussian Blue Nanozymes With Hyaluronic Acid‐Mediated Targeting Alleviate Oxidative Stress and Regulate Cholesterol Handling for Atherosclerosis Therapy

doi: 10.1002/advs.76976

Figure Lengend Snippet: Single‐cell transcriptomics identifies CD44 as a potential targeting receptor on pathogenic macrophages in atherosclerotic lesions. (A) UMAP projection of the human carotid plaque single‐cell transcriptomic dataset ( GSE253903 ), illustrating the distinct clustering of major immune and stromal cell lineages. (B) Dot plot depicting the expression profiles of cell‐type‐specific marker genes across all identified clusters. (C) Density Plot showing the high expression of CD44. (D) Violin plots demonstrate significantly elevated CD44 expression in macrophages from symptomatic patients compared to asymptomatic patients. (E) UMAP sub‐clustering of the macrophage population into distinct functional subsets. (F) Bar graph showing an increased proportion of inflammatory macrophages and a decreased proportion of Foamy_Trem2 macrophages in symptomatic lesions. (G) Violin plots detailing the differential expression of CD44 across macrophage subtypes between the two clinical groups. (H) Density Plot illustrating the strong co‐expression of CD44 with pathogenic markers (IL1B, NFE2L2, and CD36).

Article Snippet: Subsequently, the samples were incubated overnight at 4°C with specific primary antibodies targeting the M1 marker iNOS (Proteintech, 22226‐1‐AP,1:400), the M2 marker ARG1 (Proteintech, 16001‐1‐AP, 1:400), or the lipid uptake receptor CD36 (MedChemExpress, HY‐P86458, 1:400).

Techniques: Single-cell Transcriptomics, Single Cell, Expressing, Marker, Functional Assay, Quantitative Proteomics

Journal: Advanced Science

Article Title: Copper‐Doped Prussian Blue Nanozymes With Hyaluronic Acid‐Mediated Targeting Alleviate Oxidative Stress and Regulate Cholesterol Handling for Atherosclerosis Therapy

doi: 10.1002/advs.76976

Figure Lengend Snippet: CuPB@HA nanozymes accumulate in atherosclerotic plaques and synergistically remodel lipid metabolism, oxidative stress, and inflammatory polarization in macrophages. (A) Representative in vivo fluorescence images of HFD‐fed ApoE −/− atherosclerotic mice after intravenous administration of Cy5.5‐labeled CuPB or CuPB@HA at 12 and 24 h post‐injection. (B) Ex vivo fluorescence images of major organs, including heart, liver, spleen, lung, and kidney, harvested at corresponding time points after nanozyme administration. (C) Representative confocal fluorescence images of atherosclerotic plaque sections from HFD‐fed ApoE −/− mice showing the spatial association of Cy5.5‐labeled CuPB@HA with CD68‐positive macrophage‐rich regions and CD44‐positive regions. Cy5.5‐labeled CuPB@HA is pseudo‐colored red, CD68 or CD44 is shown in green. (D) Fluorescence microscopy images showing the time‐dependent cellular uptake of FITC‐labeled CuPB and CuPB@HA by macrophages, with or without excess free HA pre‐incubation. FITC‐labeled nanozymes are shown in green, and nuclei are stained with DAPI in blue. (E) Western blot analysis of proteins related to lipid metabolism, oxidative stress, and inflammatory polarization in RAW264.7 macrophages after different treatments. (F) RT‐qPCR analysis of genes related to lipid metabolism, oxidative stress, and inflammatory polarization in RAW264.7 macrophages after different treatments. (G) Representative Oil Red O staining images showing intracellular lipid accumulation in RAW264.7 macrophages after different treatments. (H–J) Representative immunofluorescence images showing the expression of ARG1 (H), iNOS (I), and CD36 (J) in RAW264.7 macrophages after different treatments. (K) Quantitative analysis of cellular uptake fluorescence intensity in Figure 4D. (L) Quantitative analysis of Oil Red O‐positive areas in Figure 4G (n = 3). (M–O) Quantitative fluorescence analysis of ARG1 (M), iNOS (N), and CD36 (O) staining in Figure 4H–J ( n = 5). Quantitative data are presented as the mean ± SD. Statistical significance was assessed via one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Subsequently, the samples were incubated overnight at 4°C with specific primary antibodies targeting the M1 marker iNOS (Proteintech, 22226‐1‐AP,1:400), the M2 marker ARG1 (Proteintech, 16001‐1‐AP, 1:400), or the lipid uptake receptor CD36 (MedChemExpress, HY‐P86458, 1:400).

Techniques: In Vivo, Fluorescence, Labeling, Injection, Ex Vivo, Microscopy, Incubation, Staining, Western Blot, Quantitative RT-PCR, Immunofluorescence, Expressing

Journal: Advanced Science

Article Title: Copper‐Doped Prussian Blue Nanozymes With Hyaluronic Acid‐Mediated Targeting Alleviate Oxidative Stress and Regulate Cholesterol Handling for Atherosclerosis Therapy

doi: 10.1002/advs.76976

Figure Lengend Snippet: Transcriptomic reprogramming of pathogenic macrophages by CuPB@HA nanozymes. (A) Differential expression scatter plot of Model vs. Control, highlighting upregulated DEGs (red, Fold Change > 1.5, FDR < 0.05). (B) GO biological process enrichment of the upregulated DEGs from (A). (C) Differential expression scatter plot of Treat vs. Model, highlighting downregulated DEGs (blue, Fold Change > 1.5, FDR < 0.05). (D) GO biological process enrichment of the downregulated DEGs from (C). (E) Heatmap of representative DEGs for lipid uptake, cholesterol efflux, oxidative stress, and inflammation. (F) Quantitative expression profiles of essential genes selected from (E). Data are mean ± SD ( n = 3). (G) UpSet plot showing the intersection of DEGs between the disease progression and treatment sets. (H) Protein‐protein interaction (PPI) network of the key intersected DEGs. (I) Core PPI sub‐network of highly interconnected hub genes (Cd36, Il1b, Tnf, Il10, Nos2, Arg1, Mmp9).

Article Snippet: Subsequently, the samples were incubated overnight at 4°C with specific primary antibodies targeting the M1 marker iNOS (Proteintech, 22226‐1‐AP,1:400), the M2 marker ARG1 (Proteintech, 16001‐1‐AP, 1:400), or the lipid uptake receptor CD36 (MedChemExpress, HY‐P86458, 1:400).

Techniques: Quantitative Proteomics, Control, Expressing, Biomarker Discovery

Journal: Advanced Science

Article Title: Copper‐Doped Prussian Blue Nanozymes With Hyaluronic Acid‐Mediated Targeting Alleviate Oxidative Stress and Regulate Cholesterol Handling for Atherosclerosis Therapy

doi: 10.1002/advs.76976

Figure Lengend Snippet: CuPB@HA attenuates atherosclerotic plaque burden and promotes plaque stability in HFD‐fed ApoE −/− mice. (A) Schematic of the in vivo experimental design and treatment timeline. (B–E) Serum lipid profiles of mice in different treatment groups, including (B) total cholesterol (TC), (C) triglycerides (TG), (D) low‐density lipoprotein cholesterol (LDL‐C), and (E) high‐density lipoprotein cholesterol (HDL‐C). Data are mean ± SD ( n = 6). Significance was assessed via one‐way ANOVA with Tukey's post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (F–J) Representative histological and immunohistochemical images of aortic root cross‐sections (scale bars: 100 µm): (F) Representative Oil Red O (ORO) staining of aortas. (G) ORO staining for lipid accumulation; (H) H&E staining for necrotic core and plaque morphology; (I) Masson's trichrome staining for collagen deposition; and (J) IHC staining for CD36 expression. (K–O) Quantification of lesional characteristics across treatment groups: (K) relative plaque area ( en face ORO), (L) lipid area (aortic root ORO), (M) necrotic core area (H&E), (N) collagen‐to‐plaque ratio (Masson's trichrome), and (O) CD36‐positive area (IHC). Data are presented as the mean ± SD ( n = 6). Significance was assessed via one‐way ANOVA with Tukey's post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Subsequently, the samples were incubated overnight at 4°C with specific primary antibodies targeting the M1 marker iNOS (Proteintech, 22226‐1‐AP,1:400), the M2 marker ARG1 (Proteintech, 16001‐1‐AP, 1:400), or the lipid uptake receptor CD36 (MedChemExpress, HY‐P86458, 1:400).

Techniques: In Vivo, Immunohistochemical staining, Staining, Immunohistochemistry, Expressing