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

Journal: Journal of Extracellular Vesicles

Article Title: Neutrophils Promote Metabolic Dysfunction‐Associated Steatotic Liver Disease Through Extracellular Vesicle‐mediated Lipid Transfer

doi: 10.1002/jev2.70350

Figure Lengend Snippet: Neutrophils develop a lipid‐laden state through free fatty acid uptake. (A–C) Neutrophils uptake free fatty acids (FAs). Human neutrophils were exposed to free fatty acids (PA, palmitic acid, 50 µM; OA, oleic acid, 50 µM; LA, linoleic acid, 50 µM). Lipid droplets (LDs) were visualized by BODIPY staining and immunofluorescence microscopy. SSO, sulfo‐ N ‐succinimidyl esters (CD36 inhibitor, 100 µM); Lipo, lipofermata (FATP2 inhibitor, 5 µM). (A) Schematic illustration of free FAs uptake by neutrophils. Representative immunofluorescence images (B) and quantification of LDs (C) in neutrophils exposed to free FAs. (D–F) Neutrophils acquire lipids from adipocytes. Mouse neutrophils were co‐cultured with differentiated 3T3‐L1 (d3T3‐L1) in the presence of TNF‐α, and LDs within the neutrophils were examined using immunofluorescence microscopy. (D) Schematic illustration of adipocyte‐to‐neutrophil lipid transfer. (E) Representative immunofluorescence images of neutrophils co‐cultured with adipocytes. Adipocytes are delineated by dashed lines, and neutrophils surrounding the adipocytes are indicated by arrowheads. (F) Quantification of LDs in neutrophils co‐cultured with adipocytes. Neutrophils co‐cultured with adipocytes were harvested, and intracellular LDs were measured using BODIPY staining. (G and H) Metabolic pathways in lipid‐laden neutrophils. (G) qPCR analysis of lipid metabolism‐related genes in vehicle‐ and OA‐treated neutrophils. (H) Heatmap showing the relative mRNA expression levels in OA‐treated neutrophils compared with the vehicle‐treated neutrophils. (I) Intracellular concentrations of FAs and TGs in OA‐treated neutrophils. (J–M) Lipidomics analysis in neutrophils exposed to OA. (J) Principal component analysis of glycerolipids in neutrophils. (K) Differential metabolite analysis in OA‐treated neutrophils. Scatter plot showing the log2 fold change of metabolites between OA‐ and vehicle‐treated neutrophils, with upregulated DG and TG species marked. (L and M) Heatmap showing the relative expression levels of selected DG and TG species enriched in OA‐treated neutrophils. Each heatmap annotates lipid species and their fatty acid composition. ABHD5, Abhydrolase domain containing 5; ACAA2, Acetyl‐CoA acyltransferase; ACACA, Acetyl‐CoA carboxylase alpha; ACAT, Acetyl‐CoA: cholesterol acyltransferase; ACLY, ATP citrate lyase; AGPAT, 1‐acylglycerol‐3‐phosphate O‐acyltransferase; ACSL, Acyl‐CoA synthetase long‐chain family member; CD36, Cluster of differentiation 36; CPT, Carnitine palmitoyltransferase; DGAT, (Diacylglycerol O‐acyltransferase); FABP, Fatty acid‐binding protein; FASN, Fatty acid synthase; G0S2, G0/G1 switch gene 2; HADHA, Hydroxyacyl‐CoA dehydrogenase / 3‐ketoacyl‐CoA thiolase / enoyl‐CoA hydratase alpha subunit; HILPDA, Hypoxia‐inducible lipid droplet‐associated protein; LIPE, Lipase E; MGAT, Mannosyl‐glycoprotein beta‐1,2‐N‐acetylglucosaminyltransferase; MGLL, Monoglyceride lipase; PLIN, Perilipin; PNPLA, Patatin‐like phospholipase domain containing; SLC27A4, Solute carrier family 27 member 4. All results are expressed as mean ± SEM. * p < .05; ** p < .01; *** p < .001.

Article Snippet: To examine the functional contribution of EV biogenesis and uptake to neutrophil‐mediated lipid transfer HepG2 cells (1 × 10 6 cells) were co‐cultured with lipid‐laden neutrophils (1 × 10 7 cells) for 2 h in the presence of ROCK inhibitor Y‐27632 (50 μM, MedChemExpress, #HY‐10071) to suppress EV formation, or cytochalasin D (50 μg/mL) to inhibit EV uptake.

Techniques: Staining, Immunofluorescence, Microscopy, Cell Culture, Expressing, Binding Assay

Journal: Journal of Extracellular Vesicles

Article Title: Neutrophils Promote Metabolic Dysfunction‐Associated Steatotic Liver Disease Through Extracellular Vesicle‐mediated Lipid Transfer

doi: 10.1002/jev2.70350

Figure Lengend Snippet: Lipid‐laden neutrophils transfer lipids to the hepatocytes. (A–C) Lipid transfer from lipid‐laden neutrophils (LNs) to hepatocytes. Mouse neutrophils were co‐cultured with either undifferentiated 3T3‐L1 or differentiated 3T3‐L1 (d3T3‐L1) and subsequently co‐cultured with AML12 cells for 2 h. Neutrophils were removed and intracellular fat levels in AML12 cells were quantified using BODIPY staining. NNs, normal neutrophils; LNs, lipid‐laden neutrophils; AML12+OA, AML12 cells treated with oleic acid (OA, 50 µM); AML12+LA, AML12 cells treated with linoleic acid (LA, 50 µM); AML12+d3T3L1, AML12 cells co‐cultured with d3T3‐L1 cells at the ratio of 1:1; AML12+NNs, AML12 cells co‐cultured with NNs; AML12+LNs, AML12 cells co‐cultured with LNs. (A) Schematic representation of the experiment. (B) Left, representative immunofluorescence images. Right, quantification of BODIPY fluorescence in AML12 cells. (C) qPCR analysis of the metabolic genes in AML12 cells. (D–F) Lipid transfer from neutrophils exposed to free FAs to hepatocytes. Human neutrophils exposed to OA were subsequently co‐cultured with human HepG2 cells for 2 h. Neutrophils were removed and intracellular fat levels were quantified using BODIPY staining. HepG2+Veh; HepG2 cells treated with vehicle, HepG2+OA; HepG2 cells treated with OA (50 µM), HepG2+NNs, HepG2 cells co‐cultured with NNs; HepG2+LNs, HepG2 cells co‐cultured with LNs. (D) Schematic representation of the experiment. (E) Quantification of BODIPY fluorescence in HepG2 cells (F) qPCR analysis of metabolic pathway genes in HepG2 cells. Dgat1, Diacylglycerol O‐Acyltransferase 1; LPIN, Lipin family member; Scd, Stearoyl‐CoA Desaturase. All results are expressed as mean ± SEM. * p < .05; ** p < .01; *** p < .001.

Article Snippet: To examine the functional contribution of EV biogenesis and uptake to neutrophil‐mediated lipid transfer HepG2 cells (1 × 10 6 cells) were co‐cultured with lipid‐laden neutrophils (1 × 10 7 cells) for 2 h in the presence of ROCK inhibitor Y‐27632 (50 μM, MedChemExpress, #HY‐10071) to suppress EV formation, or cytochalasin D (50 μg/mL) to inhibit EV uptake.

Techniques: Cell Culture, Staining, Immunofluorescence, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Neutrophils Promote Metabolic Dysfunction‐Associated Steatotic Liver Disease Through Extracellular Vesicle‐mediated Lipid Transfer

doi: 10.1002/jev2.70350

Figure Lengend Snippet: Lipid‐laden neutrophils transfer lipids to hepatocytes via extracellular vesicles. (A, B) LNs transfer lipids to HepG2 cells in the absence of direct cell to cell contact. (A) HepG2 cells were co‐cultured with either NNs or LNs in a transwell chamber, and intracellular BODIPY fluorescence was quantified. (B) Conditioned media from LNs mediate lipid transfer to HepG2 cells. HepG2 cells were treated with supernatants derived from either NNs or LNs, and intracellular BODIPY fluorescence was quantified. (C) Extracellular vesicles (EVs) mediate lipid transfer from LNs to hepatocytes. HepG2 cells were treated with EVs isolated from either NNs (NNEVs) or LNs (LNEVs), and intracellular BODIPY fluorescence was quantified. Veh, HepG2 cells treated with vehicle; +NNEVs, HepG2 cells treated with NNEVs; +LNEVs, HepG2 cells treated with LNEVs. (D) LNEVs exhibit increased lipid content. Left, representative immunofluorescence images. Right, quantification of BODIPY fluorescence in neutrophil‐derived EVs. (E) Quantification of lipid contents in neutrophil‐derived EVs. (F) In vivo distribution of fluorescence‐labeled neutrophil‐derived EVs. Mice were intravenously injected with fluorescence‐tagged neutrophil‐derived EVs, and bioluminescence was assessed 24 h after administration using IVIS spectrum in vivo imaging system. All results are expressed as mean ± SEM. * p < .05; ** p < .01; *** p < .001.

Article Snippet: To examine the functional contribution of EV biogenesis and uptake to neutrophil‐mediated lipid transfer HepG2 cells (1 × 10 6 cells) were co‐cultured with lipid‐laden neutrophils (1 × 10 7 cells) for 2 h in the presence of ROCK inhibitor Y‐27632 (50 μM, MedChemExpress, #HY‐10071) to suppress EV formation, or cytochalasin D (50 μg/mL) to inhibit EV uptake.

Techniques: Cell Culture, Fluorescence, Derivative Assay, Isolation, Immunofluorescence, In Vivo, Labeling, Injection, In Vivo Imaging

Journal: Journal of Extracellular Vesicles

Article Title: Neutrophils Promote Metabolic Dysfunction‐Associated Steatotic Liver Disease Through Extracellular Vesicle‐mediated Lipid Transfer

doi: 10.1002/jev2.70350

Figure Lengend Snippet: Transcriptomic profiling of HepG2 cells exposed to extracellular vesicles derived from lipid‐laden neutrophils. HepG2 cells were treated with extracellular vesicles (EVs) derived from NNs (+NNEVs) or LNs (+LNEVs) for 2 h and subsequently subjected to bulk RNA sequencing. (A) Schematic representation of the experimental procedure. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis highlighting enrichment of human disease‐associated pathways in EV‐treated HepG2 cells. (C) KEGG pathway analysis of metabolic processes altered by neutrophil‐derived EVs. (D) Gene Ontology (GO) Biological Process enrichment analysis of differentially expressed genes. (E) Metabolic pathways modulated by LNEVs in HepG2 cells. Upper panel, a heatmap depicting differentially expressed genes (DEGs) in +LNEVs relative to vehicle‐treated HepG2 cells. Lower panel, a schematic summary illustrating alterations in lipid metabolic genes in +LNEVs based on KEGG metabolic pathway mapping.

Article Snippet: To examine the functional contribution of EV biogenesis and uptake to neutrophil‐mediated lipid transfer HepG2 cells (1 × 10 6 cells) were co‐cultured with lipid‐laden neutrophils (1 × 10 7 cells) for 2 h in the presence of ROCK inhibitor Y‐27632 (50 μM, MedChemExpress, #HY‐10071) to suppress EV formation, or cytochalasin D (50 μg/mL) to inhibit EV uptake.

Techniques: Derivative Assay, RNA Sequencing

Journal: Journal of Extracellular Vesicles

Article Title: Neutrophils Promote Metabolic Dysfunction‐Associated Steatotic Liver Disease Through Extracellular Vesicle‐mediated Lipid Transfer

doi: 10.1002/jev2.70350

Figure Lengend Snippet: Lipid‐laden neutrophils contribute to hepatic fat accumulation in vivo. (A–E) Effect of neutrophil depletion on hepatic fat accumulation in a murine model of diet‐induced obesity. (A) Schematic representation of the experimental design. C57BL/6J mice were fed either a high‐fat diet (HFD) or normal chow (NC). Neutrophils were depleted via intraperitoneal administration of an anti‐Ly6G antibody. (B) Representative immunofluorescence images showing BODIPY staining of peripheral neutrophils isolated from mice. ( C ) Immunofluorescence analysis of liver sections, showing the presence of BODIPY + Ly6G + lipid‐laden neutrophils in livers of HFD‐fed mice. (D and E) Visualization of hepatic lipid accumulation using Oil Red O staining (D) and BODIPY immunofluorescence staining (E). (F–J) Effects of adoptive transfer of LNs on hepatic fat accumulation. (F) Schematic illustration of the experimental design. C57BL/6J mice were intravenously injected with either LNs or NNs at two‐day intervals for 10 days. (G and H) Representative images and quantification of hepatic lipid accumulation assessed by Oil Red O staining. (I and J) Representative images and quantification of hepatic lipid accumulation assessed by BODIPY fluorescence. All results are expressed as mean ± SEM. * p < .05; ** p < .01; *** p < .001.

Article Snippet: To examine the functional contribution of EV biogenesis and uptake to neutrophil‐mediated lipid transfer HepG2 cells (1 × 10 6 cells) were co‐cultured with lipid‐laden neutrophils (1 × 10 7 cells) for 2 h in the presence of ROCK inhibitor Y‐27632 (50 μM, MedChemExpress, #HY‐10071) to suppress EV formation, or cytochalasin D (50 μg/mL) to inhibit EV uptake.

Techniques: In Vivo, Immunofluorescence, Staining, Isolation, Adoptive Transfer Assay, Injection, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Neutrophils Promote Metabolic Dysfunction‐Associated Steatotic Liver Disease Through Extracellular Vesicle‐mediated Lipid Transfer

doi: 10.1002/jev2.70350

Figure Lengend Snippet: Neutrophils from patients with MASLD exhibit a lipid‐laden phenotype. (A) Schematic overview of the experimental workflow for the isolation and analysis of neutrophils and EVs from patients with MASLD and healthy volunteers. (B–E) Phenotypic and metabolic characterization of neutrophils isolated from patients with MASLD. Neutrophils were isolated from healthy volunteers and patients with MASLD and subjected to BODIPY staining, intracellular TG quantification, and qPCR analysis of the lipid metabolic genes. (B) Representative immunofluorescence images of neutrophils isolated from healthy volunteers and patients with MASLD stained with BODIPY to visualize LDs, with LDs indicated by arrows. (C) Quantification of LD counts per neutrophils from healthy volunteers and patients with MASLD. (D) Intracellular TG levels in neutrophils isolated from healthy volunteers and patients with MASLD. (E) qPCR analysis of lipid metabolism‐related genes in neutrophils, comparing expression levels between patients with MASLD and healthy volunteers. (F and G) Characterization of circulating EVs isolated from patients with MASLD. EVs were isolated from serum of healthy volunteers (HV EVs) and patients with MASLD (MASLD EVs) and analyzed for miRNAs expression and TG content. (F) qPCR analysis of selected miRNAs in HV EVs and MASLD EVs. (G) Quantification of TG concentrations in HV EVs and MASLD EVs. (H) qPCR analysis of selected miRNAs in neutrophils isolated from HV (HV Neu) and MASLD patients (MASLD Neu). (I) Spearman correlation analysis between miRNA levels in circulating EVs and neutrophils from patients with MASLD. All results are expressed as mean ± SEM. * p < .05; ** p < .01; *** p < .001.

Article Snippet: To examine the functional contribution of EV biogenesis and uptake to neutrophil‐mediated lipid transfer HepG2 cells (1 × 10 6 cells) were co‐cultured with lipid‐laden neutrophils (1 × 10 7 cells) for 2 h in the presence of ROCK inhibitor Y‐27632 (50 μM, MedChemExpress, #HY‐10071) to suppress EV formation, or cytochalasin D (50 μg/mL) to inhibit EV uptake.

Techniques: Isolation, Staining, Immunofluorescence, Expressing

Journal: International Journal of Nanomedicine

Article Title: Targeted Delivery of Bilirubin to Pulmonary Endothelium Mitigates Paraquat-Induced Lung Injury

doi: 10.2147/IJN.S617608

Figure Lengend Snippet: Cellular uptake kinetics of BR@Lipo and BR@Lipo-CerTP in PMVECs. ( A – C ) Dose-response of PMVECs treated with gradient concentrations of free BR, BR@Lipo or BR@Lipo-CerTP for 24 h. Cell viability was quantified by CCK-8 assay. ( D ) Hydrodynamic size distribution measured by DLS. ( E ) Quantitative DLS analysis: average diameter and PDI. ( F ) Zeta potential measurement. ( G ) Fluorescence microscope images of PMVECs incubated with DiI-labeled BR@Lipo and BR@Lipo-CerTP (BR@Lipo and BR@Lipo-CerTP, 2 μM BR equivalent) for 1, 2, and 4 h. Nuclei were counterstained with DAPI (blue). Red fluorescence indicates DiI localization. Scale bar, 100 μm. ( H and I ) Flow cytometric quantification of time-dependent nanoparticle internalization. Values in (A-C, I) are shown as mean ± SEM and in (D-F) are shown as mean ± SD, * P < 0.05; ** P < 0.01.

Article Snippet: Lipid nanoparticles encapsulating the bilirubin (MedChemExpress, NJ, USA, HY-N0323) were fabricated via a nanoprecipitation technique., In brief, bilirubin was first dissolved in DMSO, while a blend of lipids—consisting of egg phosphatidylcholine (Egg-PC) (AVT Pharmaceutical, Shanghai, China), cholesterol (MeloPEG Technology Inc., Shenzhen, China), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)2000] (DSPE-PEG2k) (MeloPEG Technology Inc., Shenzhen, China) (BR@Lipo) or DSPE-PEG2k-CGSPGWVRC (MeloPEG Technology Inc., Shenzhen, China) (BR@LipoCerTP) at a mass ratio of 35:5:8—was separately dissolved in ethanol.

Techniques: CCK-8 Assay, Zeta Potential Analyzer, Fluorescence, Microscopy, Incubation, Labeling