Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: The schematic illustrates the nebulized inhalation of an integrated nanovesicle system (Res-PD-L1@nmEVs) alleviated inflammation, oxidative stress injury, neutrophil activation, and promote mitochondrial integrity to mitigate lung ischemia-reperfusion injury and MRSA-induced bacterial pneumonia.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Activation Assay

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Characterization of Res-PD-L1@nmEVs . (A) Schematic illustration of the Res-PD-L1@nmEVs synthesis procedure. (B-D) Representative transmission electron microscopy (TEM) images, dynamic light scattering (DLS) size distributions, and zeta potential measurements of nEVs, PD-L1@mEVs, PD-L1@nmEVs, and Res-PD-L1@nmEVs. (E) PD-L1 expression in PD-L1-overexpressing MSCs (OE-PD-L1) and negative control (NC) MSCs, and CD11b expression in HL60 cells before and after DMSO stimulation, as determined by Western blot. (F) Expression levels of neutrophil membrane markers (CD11b, CXCR2, RAGE, TLR2) and the exosomal marker CD63 in the four EV types. (G) Fluorescence co-localization images of DiO-labeled nEVs (green) and DiL-labeled PD-L1@mEVs (red) after fusion, demonstrating hybrid vesicle formation. (H) Size stability of Res-PD-L1@nmEVs stored at 4 °C and 37 °C for 7 days. (I-K) Binding and neutralization capacity of Res-PD-L1@nmEVs against inflammatory cytokines (TNF-α, IL-6, IL-1β) in vitro. ∗ vs. 0ug/ml; # vs. 100 μg/ml, p < 0.05, n = 5.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Transmission Assay, Electron Microscopy, Zeta Potential Analyzer, Expressing, Negative Control, Western Blot, Membrane, Marker, Fluorescence, Labeling, Binding Assay, Neutralization, In Vitro

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Attenuate Inflammation and Oxidative Damage in Lung Epithelial Cells In Vitro . (A-B) Flow cytometric analysis and quantification (B) of DiO-labeled Res-PD-L1@nmEVs uptake by BEAS-2B cells under H/R conditions after pretreatment with different endocytic inhibitors (chlorpromazine, chloroquine, and filipin) or incubation at 4 °C. (C) mRNA expression levels of IL-6, TNF-α, and IL-1β in BEAS-2B cells with or without H/R injury following pretreatment with Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs. (D-E) Representative fluorescence images (D) and quantitative analysis (E) of cell proliferation assessed by BrdU incorporation (red; nuclei stained with DAPI, blue). Scale bar: 50 μm. (F-G) Apoptosis rates detected by flow cytometry (F) and flow cytometric analysis of Annexin V-positive BEAS-2B cells under the indicated conditions (G). (H–K) Fluorescence microscopy images and quantitative analysis of intracellular nitric oxide (NO, green) (H-I) and reactive oxygen species (ROS, red) (J-K). Scale bar: 100 μm. (L) Flow cytometry analysis of intracellular ROS levels. (M − O) Levels of malondialdehyde (MDA) (M), superoxide dismutase 2 (SOD2) activity (N), and glutathione (GSH) content (O) in cells. (P-Q) Cell migration ability evaluated by wound healing assay under different treatments. ∗ vs. Control; # vs. H/R; & vs. H/R + PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: In Vitro, Labeling, Incubation, Expressing, Fluorescence, BrdU Incorporation Assay, Staining, Flow Cytometry, Microscopy, Activity Assay, Migration, Wound Healing Assay, Control

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Restores Mitochondrial Homeostasis and Improves Energy Metabolism BEAS-2B cells were pretreated with Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs followed by H/R stimulation for subsequent analysis. (A) Representative immunofluorescence images showing the expression and localization of PINK1 (green) and the mitochondrial marker TOMM20 (red), indicating activation of mitophagy. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (B) Quantitative analysis of PINK1 fluorescence intensity. (C) Expression and localization of autophagy-related proteins LC3B and Beclin-1 detected by immunofluorescence. (D-E) Quantitative analysis of LC3B (D) and Beclin-1 (E) fluorescence intensity. (F) Mitochondrial membrane potential assessed by JC-1 staining and flow cytometry. (G) Oxygen consumption rate (OCR) profiles of lung epithelial cells under different treatments. (H-K) Key mitochondrial respiration parameters: basal respiration (H), maximal respiration (I), proton leak (J), and ATP production (K). (L) Representative confocal microscopy images of mitochondria stained with MitoTracker (green) and lysosomes stained with LysoTracker (red), demonstrating mitochondrial-lysosomal colocalization. Scale bar: 5 μm ∗ vs. Control; # vs. H/R; & vs. H/R + PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Immunofluorescence, Expressing, Marker, Activation Assay, Staining, Fluorescence, Membrane, Flow Cytometry, Confocal Microscopy, Control

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Suppresses Neutrophil Activation HL60 cells were differentiated into neutrophil-like cells using DMSO and subsequently stimulated with TNF-α to induce activation under conditions simulating IRI. The effects of Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, and Res-PD-L1@nmEVs on neutrophil activation were evaluated. (A) Cell surface PD-1 expression analyzed by flow cytometry. (B) Representative immunofluorescence images of CD206 expression (red). Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (C) Flow cytometric analysis of cell surface CD206 expression. (D) Flow cytometric analysis of cell surface CD95 expression. (E-G) Levels of myeloperoxidase (MPO) (E), neutrophil elastase (NE) (F), and MMP-9 (G) in neutrophil culture supernatants, measured by ELISA. (H-J) BEAS-2B cells were co-cultured with neutrophils in the presence or absence of TNF-α stimulation. Apoptosis levels (I) and migration capacity (J) of BEAS-2B cells were assessed under different treatment conditions. ∗ vs. Control; # vs. TNF-a; & vs. TNF-a+PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Activation Assay, Expressing, Flow Cytometry, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture, Migration, Control

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Nebulized Res-PD-L1@nmEVs Target and Attenuate Lung Ischemia-Reperfusion Injury (A) Experimental timeline: rats undergoing lung IRI received nebulized treatments (Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs) before ischemia and after reperfusion, with sample collection 2 h post-reperfusion. (B) Ex vivo organ fluorescence imaging 24 h after intravenous or bronchial nebulization of DiR-labeled Res-PD-L1@nmEVs. (C) In vivo lung distribution of nebulized DiL-labeled PD-L1@mEVs and PD-L1@nmEVs evaluated using a small animal dynamic imaging system. Blue: CD31 (vascular marker), Red: DiL. (D-E) Quantitative fluorescence intensity in ex vivo organs (heart, liver, spleen, lungs, kidneys) at 0–24 h after bronchial nebulization of DiR-labeled Res-PD-L1@nmEVs in Sham and IRI groups. (F-G) Representative H&E-stained lung sections (F) and corresponding lung injury scores (G). (H) Lung wet/dry weight ratio. (I-K) Levels of inflammatory cytokines in lung tissue. (L-N) Pulmonary oxidative stress markers: T-SOD2 activity (L), GSH/GSSG ratio (M), and MDA content (N). (O) Representative fluorescence images of ROS in lung tissue. Scale bar: 50 μm. (P-R) Immunofluorescence staining and co-localization of tight junction proteins Occludin-1 (green) and ZO-1 (red) in lung tissues (DAPI: blue). Scale bar: 50 μm. Quantitative analysis of ZO-1 (Q) and Occludin-1 (R) fluorescence intensity. ∗ vs. Sham; # vs. IRI; & vs. IRI + PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Ex Vivo, Fluorescence, Imaging, Labeling, In Vivo, Marker, Staining, Activity Assay, Immunofluorescence

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Suppresses Neutrophil Activation and Preserves Mitochondrial Integrity via PD-L1 Delivery (A-B) Rats subjected to lung IRI received nebulized administration of different formulations (Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs) before ischemia and after reperfusion. Lung tissues were collected 2 h post-reperfusion. (A) Representative immunofluorescence images showing the expression and localization of CD11b (green), MPO (red), and PD-1 (yellow) in lung sections across treatment groups. (B) Enlarged view of the IRI group from (A). (C-D) mRNA levels of CD95 (C) and CD206 (D) in lung tissues. (E-F) Levels of myeloperoxidase (MPO) (E) and matrix metalloproteinase-9 (MMP-9) (F) in bronchoalveolar lavage fluid (BALF). (G-I) (G) Representative transmission electron microscopy (TEM) images of lung tissues (scale bar: 2 μm). (H) Proportion of damaged mitochondria. (I) Average number of mitophagic events per cell. (J) Immunofluorescence co-localization of mitochondrial marker TOMM20 (red) and EpCAM (green) in lung tissues (nuclei stained with DAPI, scale bar: 50 μm). (K-L) Protein expression levels of Beclin-1 (K) and LC3 (L) in lung tissues, with insets showing immunofluorescence co-localization of Beclin-1 (green) and LC3 (red) across treatment groups (nuclei stained with DAPI, scale bar: 50 μm). ∗ vs. Sham; # vs. IRI; & vs. IRI + PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Activation Assay, Immunofluorescence, Expressing, Transmission Assay, Electron Microscopy, Marker, Staining

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Transcriptomic Analysis Reveals the Mechanism of Res-PD-L1@nmEVs Against IRI-Induced Lung Injury (A-B) Transcriptome sequencing of lung tissues from the Res-PD-L1@nmEVs-treated IRI group (N = 3) and the IRI-only group (N = 3). (A) Volcano plot and (B) heatmap display differentially expressed genes (DEGs) between the IRI + Res-PD-L1@nmEVs and IRI groups. (C-D) GO term and KEGG pathway enrichment analyses of upregulated DEGs after Res-PD-L1@nmEVs treatment. (E-F) GO term and KEGG pathway enrichment analyses of downregulated DEGs following Res-PD-L1@nmEVs treatment. (G-J) Gene Set Enrichment Analysis (GSEA) revealed enrichment in energy metabolism pathways (G) (TCA cycle and oxidative phosphorylation), biosynthetic pathways (H) (ribosome, amino acid biosynthesis, DNA replication), immune pathways (I) (allograft rejection, PD-L1 expression and PD-1 checkpoint pathway), and inflammatory responses (J) (chemokine signaling pathway, ECM-receptor interaction, cytokine-cytokine receptor interaction).

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Sequencing, Phospho-proteomics, Expressing

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Effectively Attenuates MRSA-Induced Pneumonia (A-B) Rats with MRSA-induced pneumonia received three bronchial nebulization treatments over one week with different formulations (Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs). (A) Representative H&E-stained lung sections and (B) corresponding lung injury scores are shown (n = 5). (C) TUNEL staining of lung tissues to assess apoptosis. (D) Representative micro-CT images of anesthetized rats. (E-G) Flow cytometric analysis of immune cell proportions in lung single-cell suspensions: CD8 + T cells (E), neutrophils (F), and classical monocytes (G). (H-J) Plasma levels of inflammatory cytokines IL-6 (H), IL-1β (I), and TNF-α (J) (n = 5). (K) Immunofluorescence staining of tight junction proteins Occludin (green) and ZO-1 (red) in lung tissues (nuclei stained with DAPI). Scale bar: 50 μm. (L-N) Pulmonary function parameters: lung compliance (L), airway resistance (M), and oxygenation index (N) (n = 4). ∗ vs. Sham; # vs. MRSA; & vs. MRSA + PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Staining, TUNEL Assay, Micro-CT, Single Cell, Clinical Proteomics, Immunofluorescence

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: The schematic illustrates the nebulized inhalation of an integrated nanovesicle system (Res-PD-L1@nmEVs) alleviated inflammation, oxidative stress injury, neutrophil activation, and promote mitochondrial integrity to mitigate lung ischemia-reperfusion injury and MRSA-induced bacterial pneumonia.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Activation Assay

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Characterization of Res-PD-L1@nmEVs . (A) Schematic illustration of the Res-PD-L1@nmEVs synthesis procedure. (B-D) Representative transmission electron microscopy (TEM) images, dynamic light scattering (DLS) size distributions, and zeta potential measurements of nEVs, PD-L1@mEVs, PD-L1@nmEVs, and Res-PD-L1@nmEVs. (E) PD-L1 expression in PD-L1-overexpressing MSCs (OE-PD-L1) and negative control (NC) MSCs, and CD11b expression in HL60 cells before and after DMSO stimulation, as determined by Western blot. (F) Expression levels of neutrophil membrane markers (CD11b, CXCR2, RAGE, TLR2) and the exosomal marker CD63 in the four EV types. (G) Fluorescence co-localization images of DiO-labeled nEVs (green) and DiL-labeled PD-L1@mEVs (red) after fusion, demonstrating hybrid vesicle formation. (H) Size stability of Res-PD-L1@nmEVs stored at 4 °C and 37 °C for 7 days. (I-K) Binding and neutralization capacity of Res-PD-L1@nmEVs against inflammatory cytokines (TNF-α, IL-6, IL-1β) in vitro. ∗ vs. 0ug/ml; # vs. 100 μg/ml, p < 0.05, n = 5.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Transmission Assay, Electron Microscopy, Zeta Potential Analyzer, Expressing, Negative Control, Western Blot, Membrane, Marker, Fluorescence, Labeling, Binding Assay, Neutralization, In Vitro

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Attenuate Inflammation and Oxidative Damage in Lung Epithelial Cells In Vitro . (A-B) Flow cytometric analysis and quantification (B) of DiO-labeled Res-PD-L1@nmEVs uptake by BEAS-2B cells under H/R conditions after pretreatment with different endocytic inhibitors (chlorpromazine, chloroquine, and filipin) or incubation at 4 °C. (C) mRNA expression levels of IL-6, TNF-α, and IL-1β in BEAS-2B cells with or without H/R injury following pretreatment with Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs. (D-E) Representative fluorescence images (D) and quantitative analysis (E) of cell proliferation assessed by BrdU incorporation (red; nuclei stained with DAPI, blue). Scale bar: 50 μm. (F-G) Apoptosis rates detected by flow cytometry (F) and flow cytometric analysis of Annexin V-positive BEAS-2B cells under the indicated conditions (G). (H–K) Fluorescence microscopy images and quantitative analysis of intracellular nitric oxide (NO, green) (H-I) and reactive oxygen species (ROS, red) (J-K). Scale bar: 100 μm. (L) Flow cytometry analysis of intracellular ROS levels. (M − O) Levels of malondialdehyde (MDA) (M), superoxide dismutase 2 (SOD2) activity (N), and glutathione (GSH) content (O) in cells. (P-Q) Cell migration ability evaluated by wound healing assay under different treatments. ∗ vs. Control; # vs. H/R; & vs. H/R + PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: In Vitro, Labeling, Incubation, Expressing, Fluorescence, BrdU Incorporation Assay, Staining, Flow Cytometry, Microscopy, Activity Assay, Migration, Wound Healing Assay, Control

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Restores Mitochondrial Homeostasis and Improves Energy Metabolism BEAS-2B cells were pretreated with Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs followed by H/R stimulation for subsequent analysis. (A) Representative immunofluorescence images showing the expression and localization of PINK1 (green) and the mitochondrial marker TOMM20 (red), indicating activation of mitophagy. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (B) Quantitative analysis of PINK1 fluorescence intensity. (C) Expression and localization of autophagy-related proteins LC3B and Beclin-1 detected by immunofluorescence. (D-E) Quantitative analysis of LC3B (D) and Beclin-1 (E) fluorescence intensity. (F) Mitochondrial membrane potential assessed by JC-1 staining and flow cytometry. (G) Oxygen consumption rate (OCR) profiles of lung epithelial cells under different treatments. (H-K) Key mitochondrial respiration parameters: basal respiration (H), maximal respiration (I), proton leak (J), and ATP production (K). (L) Representative confocal microscopy images of mitochondria stained with MitoTracker (green) and lysosomes stained with LysoTracker (red), demonstrating mitochondrial-lysosomal colocalization. Scale bar: 5 μm ∗ vs. Control; # vs. H/R; & vs. H/R + PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Immunofluorescence, Expressing, Marker, Activation Assay, Staining, Fluorescence, Membrane, Flow Cytometry, Confocal Microscopy, Control

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Suppresses Neutrophil Activation HL60 cells were differentiated into neutrophil-like cells using DMSO and subsequently stimulated with TNF-α to induce activation under conditions simulating IRI. The effects of Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, and Res-PD-L1@nmEVs on neutrophil activation were evaluated. (A) Cell surface PD-1 expression analyzed by flow cytometry. (B) Representative immunofluorescence images of CD206 expression (red). Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (C) Flow cytometric analysis of cell surface CD206 expression. (D) Flow cytometric analysis of cell surface CD95 expression. (E-G) Levels of myeloperoxidase (MPO) (E), neutrophil elastase (NE) (F), and MMP-9 (G) in neutrophil culture supernatants, measured by ELISA. (H-J) BEAS-2B cells were co-cultured with neutrophils in the presence or absence of TNF-α stimulation. Apoptosis levels (I) and migration capacity (J) of BEAS-2B cells were assessed under different treatment conditions. ∗ vs. Control; # vs. TNF-a; & vs. TNF-a+PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Activation Assay, Expressing, Flow Cytometry, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture, Migration, Control

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Nebulized Res-PD-L1@nmEVs Target and Attenuate Lung Ischemia-Reperfusion Injury (A) Experimental timeline: rats undergoing lung IRI received nebulized treatments (Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs) before ischemia and after reperfusion, with sample collection 2 h post-reperfusion. (B) Ex vivo organ fluorescence imaging 24 h after intravenous or bronchial nebulization of DiR-labeled Res-PD-L1@nmEVs. (C) In vivo lung distribution of nebulized DiL-labeled PD-L1@mEVs and PD-L1@nmEVs evaluated using a small animal dynamic imaging system. Blue: CD31 (vascular marker), Red: DiL. (D-E) Quantitative fluorescence intensity in ex vivo organs (heart, liver, spleen, lungs, kidneys) at 0–24 h after bronchial nebulization of DiR-labeled Res-PD-L1@nmEVs in Sham and IRI groups. (F-G) Representative H&E-stained lung sections (F) and corresponding lung injury scores (G). (H) Lung wet/dry weight ratio. (I-K) Levels of inflammatory cytokines in lung tissue. (L-N) Pulmonary oxidative stress markers: T-SOD2 activity (L), GSH/GSSG ratio (M), and MDA content (N). (O) Representative fluorescence images of ROS in lung tissue. Scale bar: 50 μm. (P-R) Immunofluorescence staining and co-localization of tight junction proteins Occludin-1 (green) and ZO-1 (red) in lung tissues (DAPI: blue). Scale bar: 50 μm. Quantitative analysis of ZO-1 (Q) and Occludin-1 (R) fluorescence intensity. ∗ vs. Sham; # vs. IRI; & vs. IRI + PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Ex Vivo, Fluorescence, Imaging, Labeling, In Vivo, Marker, Staining, Activity Assay, Immunofluorescence

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Suppresses Neutrophil Activation and Preserves Mitochondrial Integrity via PD-L1 Delivery (A-B) Rats subjected to lung IRI received nebulized administration of different formulations (Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs) before ischemia and after reperfusion. Lung tissues were collected 2 h post-reperfusion. (A) Representative immunofluorescence images showing the expression and localization of CD11b (green), MPO (red), and PD-1 (yellow) in lung sections across treatment groups. (B) Enlarged view of the IRI group from (A). (C-D) mRNA levels of CD95 (C) and CD206 (D) in lung tissues. (E-F) Levels of myeloperoxidase (MPO) (E) and matrix metalloproteinase-9 (MMP-9) (F) in bronchoalveolar lavage fluid (BALF). (G-I) (G) Representative transmission electron microscopy (TEM) images of lung tissues (scale bar: 2 μm). (H) Proportion of damaged mitochondria. (I) Average number of mitophagic events per cell. (J) Immunofluorescence co-localization of mitochondrial marker TOMM20 (red) and EpCAM (green) in lung tissues (nuclei stained with DAPI, scale bar: 50 μm). (K-L) Protein expression levels of Beclin-1 (K) and LC3 (L) in lung tissues, with insets showing immunofluorescence co-localization of Beclin-1 (green) and LC3 (red) across treatment groups (nuclei stained with DAPI, scale bar: 50 μm). ∗ vs. Sham; # vs. IRI; & vs. IRI + PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Activation Assay, Immunofluorescence, Expressing, Transmission Assay, Electron Microscopy, Marker, Staining

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Transcriptomic Analysis Reveals the Mechanism of Res-PD-L1@nmEVs Against IRI-Induced Lung Injury (A-B) Transcriptome sequencing of lung tissues from the Res-PD-L1@nmEVs-treated IRI group (N = 3) and the IRI-only group (N = 3). (A) Volcano plot and (B) heatmap display differentially expressed genes (DEGs) between the IRI + Res-PD-L1@nmEVs and IRI groups. (C-D) GO term and KEGG pathway enrichment analyses of upregulated DEGs after Res-PD-L1@nmEVs treatment. (E-F) GO term and KEGG pathway enrichment analyses of downregulated DEGs following Res-PD-L1@nmEVs treatment. (G-J) Gene Set Enrichment Analysis (GSEA) revealed enrichment in energy metabolism pathways (G) (TCA cycle and oxidative phosphorylation), biosynthetic pathways (H) (ribosome, amino acid biosynthesis, DNA replication), immune pathways (I) (allograft rejection, PD-L1 expression and PD-1 checkpoint pathway), and inflammatory responses (J) (chemokine signaling pathway, ECM-receptor interaction, cytokine-cytokine receptor interaction).

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Sequencing, Phospho-proteomics, Expressing

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Effectively Attenuates MRSA-Induced Pneumonia (A-B) Rats with MRSA-induced pneumonia received three bronchial nebulization treatments over one week with different formulations (Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs). (A) Representative H&E-stained lung sections and (B) corresponding lung injury scores are shown (n = 5). (C) TUNEL staining of lung tissues to assess apoptosis. (D) Representative micro-CT images of anesthetized rats. (E-G) Flow cytometric analysis of immune cell proportions in lung single-cell suspensions: CD8 + T cells (E), neutrophils (F), and classical monocytes (G). (H-J) Plasma levels of inflammatory cytokines IL-6 (H), IL-1β (I), and TNF-α (J) (n = 5). (K) Immunofluorescence staining of tight junction proteins Occludin (green) and ZO-1 (red) in lung tissues (nuclei stained with DAPI). Scale bar: 50 μm. (L-N) Pulmonary function parameters: lung compliance (L), airway resistance (M), and oxygenation index (N) (n = 4). ∗ vs. Sham; # vs. MRSA; & vs. MRSA + PD-L1@nmEVs, p < 0.05.

Article Snippet: For assessing PD-L1 overexpression efficiency, the primary antibodies included PD-L1 (ET1701-4, HUABIO, China) and β-actin (66009-1, Proteintech, China).

Techniques: Staining, TUNEL Assay, Micro-CT, Single Cell, Clinical Proteomics, Immunofluorescence

Journal: Molecular Therapy Oncology

Article Title: CAR-T cells directed toward PD-L1 demonstrate potent, antigen-specific activity against cholangiocarcinoma: A proof of concept study

doi: 10.1016/j.omton.2026.201209

Figure Lengend Snippet: Generation of PD-L1 CAR-T and validation of PD-L1 expression in target cells (A) Schematic of the second-generation PD-L1 CAR construct containing an anti-PD-L1 scFv, CD4 transmembrane domain, and 4-1BB/CD3ζ signaling domains and tEGFR safety switch. (B) Flow cytometry results of PD-L1 expression in HuCCT1, HuCCT1-PD-L1 KO, and SNU1079 cells. (C) Characterization of non-CAR-T and CAR-T showing 98.8% and 98.6% CD3 expression and 1.03% and 28% EGFR expression, respectively.

Article Snippet: Cas9 was combined with multi-guide RNA targeting PD-L1 (Synthego, Redwood City, CA, USA) in NEB buffer (New England Biolabs) at a 12:1 ratio and incubated to form RNP complexes.

Techniques: Biomarker Discovery, Expressing, Construct, Flow Cytometry

Journal: Molecular Therapy Oncology

Article Title: CAR-T cells directed toward PD-L1 demonstrate potent, antigen-specific activity against cholangiocarcinoma: A proof of concept study

doi: 10.1016/j.omton.2026.201209

Figure Lengend Snippet: PD-L1 CAR-T delay tumor progression and reduce tumor burden in vivo (A) Longitudinal bioluminescent imaging of mice with orthotopic HuCCT1 tumors treated with PBS as a control, Non-CAR-T, or CAR-T at 7 and 14 days. (B) Quantification of total bioluminescent signal confirming significantly reduced tumor burden in CAR-T treated animals compared with both control groups. Results are reported as mean ± standard deviation (SD). Two-way ANOVA with Tukey’s multiple comparisons between tumor control and CAR-T are represented by ( p values: ∗∗ ≤0.01), and between non-CAR-T and CAR-T by ( p values: # # ≤ 0.01). n = 6 biological replicates at all days and time points with the exception of non-CAR-T week 9, where n = 5 biological replicates.

Article Snippet: Cas9 was combined with multi-guide RNA targeting PD-L1 (Synthego, Redwood City, CA, USA) in NEB buffer (New England Biolabs) at a 12:1 ratio and incubated to form RNP complexes.

Techniques: In Vivo, Imaging, Control, Standard Deviation

Journal: Molecular Therapy Oncology

Article Title: CAR-T cells directed toward PD-L1 demonstrate potent, antigen-specific activity against cholangiocarcinoma: A proof of concept study

doi: 10.1016/j.omton.2026.201209

Figure Lengend Snippet: Antigen-specific degranulation and granzyme B released by PD-L1 CAR-T (A) CD8 + T cell degranulation in response to HuCCT1 wild-type (WT) or PD-L1 knockout (KO) cells by flow cytometry after CAR-T co-culture at 6 h. (B) CD4 + T cell degranulation under the same conditions, showing CAR-T-mediated activity against WT but not KO cells. (C) Degranulation of CD8 + and CD4 + T cells in response to SNU1079 cells by flow cytometry after CART co-culture at 6 h compared with non-CAR-T controls. (D) Granzyme B release in HuCCT1 WT and KO cells following co-culture by ELISA after 72 h n = 3 technical replicates. Two-way ANOVA with Tukey’s multiple comparisons test. ∗∗∗∗ p value ≤ 0.0001. Results are reported as mean ± standard deviation (SD).

Article Snippet: Cas9 was combined with multi-guide RNA targeting PD-L1 (Synthego, Redwood City, CA, USA) in NEB buffer (New England Biolabs) at a 12:1 ratio and incubated to form RNP complexes.

Techniques: Knock-Out, Flow Cytometry, Co-Culture Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Molecular Therapy Oncology

Article Title: CAR-T cells directed toward PD-L1 demonstrate potent, antigen-specific activity against cholangiocarcinoma: A proof of concept study

doi: 10.1016/j.omton.2026.201209

Figure Lengend Snippet: CAR-T release cytotoxic effector molecules and reduce tumor cell viability in an antigen-dependent manner (A and B) Granzyme B and perforin release from CAR-T and non-transduced T cells co-cultured with HuCCT1 (A) or SNU1079 (B) cells at 1:1 and 2:1 effector-to-target (E:T) ratios. n = 3 technical replicates. One-way ANOVA with Tukey’s multiple comparisons test. (C) Luciferase viability assay of HuCCT1, PD-L1 knockout HuCCT1, or SNU1079 cells at 1:1 and 2:1 effector-to-target after 24 and 48 h. n = 6 technical replicates. One-way ANOVA with Tukey’s multiple comparisons test. ∗ p value ≤ 0.05, ∗∗∗∗ p value ≤ 0.0001. Results are reported as mean ± standard deviation (SD).

Article Snippet: Cas9 was combined with multi-guide RNA targeting PD-L1 (Synthego, Redwood City, CA, USA) in NEB buffer (New England Biolabs) at a 12:1 ratio and incubated to form RNP complexes.

Techniques: Cell Culture, Luciferase, Viability Assay, Knock-Out, Standard Deviation

Journal: Molecular Therapy Oncology

Article Title: CAR-T cells directed toward PD-L1 demonstrate potent, antigen-specific activity against cholangiocarcinoma: A proof of concept study

doi: 10.1016/j.omton.2026.201209

Figure Lengend Snippet: PD-L1 CAR-T disrupt and kill tumor cells in multicellular CSFE spheroids (A) Brightfield images of HuCCT1 and SNU1079 CSFE spheroids following 24 h co-culture with non-CAR-T or CAR-T at 1:1 or 2:1 effector-to-target (E:T) ratios. Quantification of spheroid area is shown. n = 3 technical replicates. One-way ANOVA with Tukey’s multiple comparisons test. Scale bar is 300 µm (B) Live/dead staining (calcein-AM/propidium iodide) and luciferase viability assays of spheroids under the same conditions. n = 3 technical replicates. One-way ANOVA with Tukey’s multiple comparisons test. ∗ p value ≤ 0.05, ∗∗ p value ≤ 0.01, ∗∗∗ p value ≤ 0.001, ∗∗∗∗ p value ≤ 0.0001. Results are reported as mean ± standard deviation (SD). Scale bar is 100 µm.

Article Snippet: Cas9 was combined with multi-guide RNA targeting PD-L1 (Synthego, Redwood City, CA, USA) in NEB buffer (New England Biolabs) at a 12:1 ratio and incubated to form RNP complexes.

Techniques: Co-Culture Assay, Staining, Luciferase, Standard Deviation

Journal: Molecular Therapy Oncology

Article Title: CAR-T cells directed toward PD-L1 demonstrate potent, antigen-specific activity against cholangiocarcinoma: A proof of concept study

doi: 10.1016/j.omton.2026.201209

Figure Lengend Snippet: Gemcitabine upregulates PD-L1 and enhances CAR-T cytotoxicity in HuCCT1 cells (A) Schematic of experimental design for gemcitabine pretreatment. (B) Flow cytometry showing increased PD-L1 surface expression in HuCCT1 cells after Gem treatment, with maximal induction at 0.2 μM for 48 h. n = 3 technical replicates. One-way ANOVA with Tukey’s multiple comparisons test. (C) Luciferase-based viability assays at both effector-to-target (E:T) ratios and at 24 and 48 h time points. n = 6 technical replicates, two-way ANOVA with Šídák’s multiple comparisons test. (D) Representative live/dead staining of HuCCT1 CSFE spheroids under the same conditions. ∗∗ p value ≤ 0.01, ∗∗∗ p value ≤ 0.001, ∗∗∗∗ p value ≤ 0.0001. Results are reported as mean ± standard deviation (SD). Scale bar is 50 µm.

Article Snippet: Cas9 was combined with multi-guide RNA targeting PD-L1 (Synthego, Redwood City, CA, USA) in NEB buffer (New England Biolabs) at a 12:1 ratio and incubated to form RNP complexes.

Techniques: Flow Cytometry, Expressing, Luciferase, Staining, Standard Deviation

Journal: Molecular Therapy Oncology

Article Title: CAR-T cells directed toward PD-L1 demonstrate potent, antigen-specific activity against cholangiocarcinoma: A proof of concept study

doi: 10.1016/j.omton.2026.201209

Figure Lengend Snippet: Gemcitabine upregulates PD-L1 and enhances CAR-T cytotoxicity in SNU1079 cells (A) Schematic depicting Gemcitabine pretreatment. (B) Flow cytometry showing Gemcitabine-induced PD-L1 upregulation in SNU1079 cells, with maximal effect at 0.2 μM for 48 h. n = 3 technical replicates. One-way ANOVA with Tukey’s multiple comparisons test. (C) Luciferase-based viability assays at both effector-to-target (E:T) ratios and at 24 and 48 h time points. n = 6 technical replicates. Two-way ANOVA with Šídák’s multiple comparisons test. (D) Representative live/dead staining of SNU1079 CSFE spheroids under the same conditions. p value∗ ≤ 0.05, p value∗∗ ≤ 0.01, p value∗∗∗∗ ≤ 0.0001. Results are reported as mean ± standard deviation (SD). Scale bar is 50 µm.

Article Snippet: Cas9 was combined with multi-guide RNA targeting PD-L1 (Synthego, Redwood City, CA, USA) in NEB buffer (New England Biolabs) at a 12:1 ratio and incubated to form RNP complexes.

Techniques: Flow Cytometry, Luciferase, Staining, Standard Deviation