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: The primary antibodies used included: ZO-1 ( GB111402 , Servicebio), Occludin (111401, Servicebio), PINK1 ( GB114934 , Servicebio), TOMM20 ( GB151481 , Servicebio), LC3B ( GB153801 , Servicebio), Beclin-1 ( GB115741 , Servicebio), CD206 ( GB113497 , Servicebio), PD-1 ( GB153744 , Servicebio), MPO ( GB150006 , Servicebio), and CD11b (GB15058, Servicebio).

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 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: The primary antibodies used included: ZO-1 ( GB111402 , Servicebio), Occludin (111401, Servicebio), PINK1 ( GB114934 , Servicebio), TOMM20 ( GB151481 , Servicebio), LC3B ( GB153801 , Servicebio), Beclin-1 ( GB115741 , Servicebio), CD206 ( GB113497 , Servicebio), PD-1 ( GB153744 , Servicebio), MPO ( GB150006 , Servicebio), and CD11b (GB15058, Servicebio).

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

Journal: Bioactive Materials

Article Title: Spatiotemporally engineered microneedle for microenvironment remodeling propels mucosal regeneration after tracheal mucosal injury

doi: 10.1016/j.bioactmat.2026.01.026

Figure Lengend Snippet: Gel-AgNA/MgGA MN promote mucosal regeneration. (a) HE and Safranin O staining of rabbit tracheal samples harvested at Day 10 post-operation after treated with Gel, Gel-AgNA, Gel-MgGA, and Gel-AgNA/MgGA MN. (b) IF staining of CK14 (marker of basal cells, red) and AC-Tub (marker of cilia cell, green). (c) IF staining of ZO-1 (marker of tight junctions, orange). (d, e) Quantitative analysis of regenerated epithelial coverage and thickness (n = 9). (f) Masson and Sirius Red staining for collagen evaluation after various treatments (n = 5). Quantitative analysis of collagen volume fraction (g) and fiber orientation (h) . The pentagram indicates luminal side of trachea.

Article Snippet: Immunofluorescence staining of CK14 (Abcam, ab181595), AC-Tub (Proteintech, 66200-1-Ig), ZO-1 (Proteintech, 21773-1-AP), and Immunohistochemical (IHC) staining for CD31 (Servicebio, S1002) were conducted to reveal the conditions of mucosal regeneration, according to previous literature [ ].

Techniques: Staining, Marker

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from hair follicle neural crest stem cells enhance perineurial cell proliferation and migration via the TGF-β/SMAD/HAS2 pathway

doi: 10.4103/NRR.NRR-D-25-00127

Figure Lengend Snippet: hfNCSC-sEVs are taken up by PCs in vitro and enhance their proliferation and migration. (A) Primary cultures of hfNCSCs were established from male Sprague–Dawley rats. (B) Immunofluorescence staining of the neural crest cell marker p75 (red) and the stem cell marker nestin (green) in hfNCSCs, with 4′,6-diamidino-2-phenylindole (DAPI) staining indicating the nuclei. (C) Western blot analysis demonstrated the presence of surface markers (cluster of differentiation [CD]9, CD81, and tumor susceptibility gene 101 protein [TSG101]) and the absence of an endoplasmic reticulum marker (calnexin) in hfNCSC-sEVs. (D) Nanoparticle tracking analysis was used to quantify the concentration and size distribution of hfNCSC-sEVs. (E) Transmission electron microscopy was used to visualize the characteristic morphology of hfNCSC-sEVs. (F) Immunofluorescence staining indicated that the third-generation PCs cultured in vitro were positive for claudin-1, zonula occludens 1 (ZO1), and glucose transporter 1 (GLUT1) but negative for S100, with DAPI staining marking the nuclei. (G) The internalization of PKH26-labeled hfNCSC-sEVs (red) by ZO1-positive PCs (green) was visualized using immunofluorescence staining, with DAPI staining to mark the nuclei. (H) The Cell Counting Kit-8 assay was used to evaluate the cell viability of PCs across concentrations of 0, 2 × 10 8 , 5 × 10 8 , and 10 × 10 8 particles/mL hfNCSC-sEVs at 3, 5, and 7 days of in vitro culture ( n = 5 per group). (I) The Transwell assay was used to quantify the number of migrating PCs at 6, 12, and 18 hours post-treatment with the aforementioned concentrations of hfNCSC-sEVs, in in vitro culture ( n = 6 per group). (J) Western blot and (K) statistical analyses revealed the relative protein expression levels of proliferating cell nuclear antigen (PCNA) and vimentin in PCs from the phosphate-buffered saline (PBS) and hfNCSC-sEVs groups on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s multiple comparison test for H and I; Student’s t -test for K). The data were from at least three separate and independent studies. CCK-8: Cell counting kit-8; GLUT1: glucose transporter 1; hfNCSCs: hair follicle neural crest stem cells; ns: not significant; PCNA: proliferating cell nuclear antigen; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-p75 neurotrophin receptor (p75) antibody (1:100, Cat# 55014-1-AP, Proteintech), mouse monoclonal anti-nestin antibody (1:100, Cat# MAB353, Sigma), rabbit polyclonal anti-claudin-1 antibody (1:250, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-ZO1 antibody (1:200, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-glucose transporter 1 (GLUT1) antibody (1:500, Cat# 21829-1-AP, Proteintech), rabbit monoclonal anti-S100 antibody (1:800, Cat# MAB353, Abcam), mouse monoclonal anti-neurofilament 200 (NF200) antibody (1:800, Cat# N5389, Sigma), rabbit polyclonal anti-myelin basic protein (MBP) antibody (1:400, Cat# 10458-1-AP, Proteintech), mouse monoclonal anti-β-tubulin antibody (1:1000, Cat# M20005 , Abmart), and rabbit polyclonal anti-HAS2 antibody (1:200, Cat# DF13702, Affinity).

Techniques: In Vitro, Migration, Immunofluorescence, Staining, Marker, Western Blot, Concentration Assay, Transmission Assay, Electron Microscopy, Cell Culture, Labeling, Cell Counting, Transwell Assay, Expressing, Saline, Comparison, CCK-8 Assay

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from hair follicle neural crest stem cells enhance perineurial cell proliferation and migration via the TGF-β/SMAD/HAS2 pathway

doi: 10.4103/NRR.NRR-D-25-00127

Figure Lengend Snippet: hfNCSC-sEVs enhance tube formation and barrier function in PCs and promote tight junction protein expression. (A) Optical micrographs of the tube formation assay and (B) statistical analyses demonstrated the number of junctions and total length of tubes in PCs in both the phosphate-buffered saline (PBS) and hfNCSC-sEVs groups ( n = 5 per group). (C) Measurements of transmembrane resistance ( n = 3 per group) and (D) cell monolayer permeability assays ( n = 9 per group) indicated the barrier formation ability of PCs in both the PBS and hfNCSC-sEVs groups. (E) Western blot and (F) statistical analyses revealed the relative protein expression levels of the tight junction proteins zonula occludens 1 (ZO1) and claudin-1 in PCs from the PBS and hfNCSC-sEVs groups on day 7 of in vitro culture (normalized to β-actin, n = 3 per group). (G, H) Immunofluorescence staining (G) and statistical analyses (H) showed the integrated optical density (IOD) of ZO1 (green) and the expression of β-tubulin (red) in PCs from the PBS and hfNCSC-sEVs groups on day 7 of in vitro culture ( n = 3 per group). (I) Schematic illustration of the rat sciatic nerve defect model: a 5-mm defect was surgically created in the rat sciatic nerve, which was then bridged using a silicon tube, followed by an orthotopic injection procedure. (J) Immunofluorescence staining revealed the expression of claudin-1 (red) in the proximal end of regenerated tissue in both the PBS and hfNCSC-sEVs groups on day 7 post-operation, with 4′,6-diamidino-2-phenylindole (DAPI) staining indicating the nuclei. Data are expressed as the mean ± SEM. * P < 0.05, *** P < 0.001 (Student’s t -test for B, C, D, F, and H). The data were from at least three separate and independent studies. hfNCSCs: Hair follicle neural crest stem cells; IOD: integrated optical density; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-p75 neurotrophin receptor (p75) antibody (1:100, Cat# 55014-1-AP, Proteintech), mouse monoclonal anti-nestin antibody (1:100, Cat# MAB353, Sigma), rabbit polyclonal anti-claudin-1 antibody (1:250, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-ZO1 antibody (1:200, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-glucose transporter 1 (GLUT1) antibody (1:500, Cat# 21829-1-AP, Proteintech), rabbit monoclonal anti-S100 antibody (1:800, Cat# MAB353, Abcam), mouse monoclonal anti-neurofilament 200 (NF200) antibody (1:800, Cat# N5389, Sigma), rabbit polyclonal anti-myelin basic protein (MBP) antibody (1:400, Cat# 10458-1-AP, Proteintech), mouse monoclonal anti-β-tubulin antibody (1:1000, Cat# M20005 , Abmart), and rabbit polyclonal anti-HAS2 antibody (1:200, Cat# DF13702, Affinity).

Techniques: Expressing, Tube Formation Assay, Saline, Permeability, Western Blot, In Vitro, Immunofluorescence, Staining, Injection

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from hair follicle neural crest stem cells enhance perineurial cell proliferation and migration via the TGF-β/SMAD/HAS2 pathway

doi: 10.4103/NRR.NRR-D-25-00127

Figure Lengend Snippet: miR-21-5p in hfNCSC-sEVs augments cell proliferation and migration by enhancing HAS2 expression in PCs. (A, B) Western blot (A) and statistical analyses (B) revealed the relative protein expression levels of HAS2, proliferating cell nuclear antigen (PCNA), and vimentin in PCs across the –/–, –/si- Has2 , hfNCSC-sEVs/–, and hfNCSC-sEVs/si- Has2 groups on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). (C, D) The wound healing assay (C) and statistical analysis (D) demonstrated the migration rates of PCs in the aforementioned groups ( n = 3 per group). (E) The Cell Counting Kit-8 assay was used to assess cell viability in PCs across the same groups on day 5 of in vitro culture ( n = 5 per group). (F, G) Western blot (F) and statistical analyses (G) indicated the relative protein expression levels of HAS2, PCNA, and vimentin in PCs treated with phosphate-buffered saline (PBS), hfNCSC-sEVs, or hfNCSC-sEVs + miR-21-5p inhibitor on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). (H–J) Immunofluorescence staining visualized the expression of HAS2 (red) and 5-ethynyl-2′-deoxyuridine (EdU; green) in PCs (H), and statistical analysis revealed the integrated optical density (IOD) of zonula occludens 1 (ZO1; I) and the cell proliferation rates (J) in the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 5 of in vitro culture ( n = 3 per group). (K, L) Western blot (K) and statistical analyses (L) showed the relative protein expression levels of HAS2, PCNA, and vimentin in regenerated tissue from the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 5 post-operation (normalized to β-tubulin, n = 3 per group). Data are expressed as the mean ± SEM. ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s multiple comparison test for B, D, E, G, I, J, and L). The data were from at least three separate and independent studies. CCK-8: Cell counting kit-8; EdU: 5-ethynyl-2′-deoxyuridine; HAS2: hyaluronan synthase 2; hfNCSCs: hair follicle neural crest stem cells; IOD: integrated optical density; PCNA: proliferating cell nuclear antigen; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-p75 neurotrophin receptor (p75) antibody (1:100, Cat# 55014-1-AP, Proteintech), mouse monoclonal anti-nestin antibody (1:100, Cat# MAB353, Sigma), rabbit polyclonal anti-claudin-1 antibody (1:250, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-ZO1 antibody (1:200, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-glucose transporter 1 (GLUT1) antibody (1:500, Cat# 21829-1-AP, Proteintech), rabbit monoclonal anti-S100 antibody (1:800, Cat# MAB353, Abcam), mouse monoclonal anti-neurofilament 200 (NF200) antibody (1:800, Cat# N5389, Sigma), rabbit polyclonal anti-myelin basic protein (MBP) antibody (1:400, Cat# 10458-1-AP, Proteintech), mouse monoclonal anti-β-tubulin antibody (1:1000, Cat# M20005 , Abmart), and rabbit polyclonal anti-HAS2 antibody (1:200, Cat# DF13702, Affinity).

Techniques: Migration, Expressing, Western Blot, In Vitro, Wound Healing Assay, Cell Counting, Saline, Immunofluorescence, Staining, Comparison, CCK-8 Assay

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from hair follicle neural crest stem cells enhance perineurial cell proliferation and migration via the TGF-β/SMAD/HAS2 pathway

doi: 10.4103/NRR.NRR-D-25-00127

Figure Lengend Snippet: miR-21-5p in hfNCSC-sEVs enhances tight junction protein expression in PCs. (A, B) Immunofluorescence staining (A) and statistical analysis (B) demonstrated IOD of ZO1 (green) and the expression of β-tubulin (red) in PCs across the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 7 of in vitro culture ( n = 3 per group). (C) Western blot and (D) statistical analyses revealed the relative protein expression levels of the tight junction proteins ZO1 and claudin-1 in PCs from the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 7 of in vitro culture (normalized to β-actin, n = 3 per group). (E) Immunofluorescence staining depicted the expression of claudin-1 (red) at the proximal end of regenerated tissue in the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 7 post-operation, with DAPI staining highlighting the nuclei. (F, G) Western blot (F) and statistical analyses (G) indicated the relative protein expression levels of ZO1 and claudin-1 in regenerated tissue across the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 7 post-operation (normalized to β-actin, n = 3 per group). Data are expressed as the mean ± SEM. ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s multiple comparison test for B, D, and G). The data were from at least three separate and independent studies. DAPI: 4,6-Diamidino-2-phenylindole; hfNCSCs: hair follicle neural crest stem cells; IOD: integrated optical density; PBS: phosphate-buffered saline; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-p75 neurotrophin receptor (p75) antibody (1:100, Cat# 55014-1-AP, Proteintech), mouse monoclonal anti-nestin antibody (1:100, Cat# MAB353, Sigma), rabbit polyclonal anti-claudin-1 antibody (1:250, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-ZO1 antibody (1:200, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-glucose transporter 1 (GLUT1) antibody (1:500, Cat# 21829-1-AP, Proteintech), rabbit monoclonal anti-S100 antibody (1:800, Cat# MAB353, Abcam), mouse monoclonal anti-neurofilament 200 (NF200) antibody (1:800, Cat# N5389, Sigma), rabbit polyclonal anti-myelin basic protein (MBP) antibody (1:400, Cat# 10458-1-AP, Proteintech), mouse monoclonal anti-β-tubulin antibody (1:1000, Cat# M20005 , Abmart), and rabbit polyclonal anti-HAS2 antibody (1:200, Cat# DF13702, Affinity).

Techniques: Expressing, Immunofluorescence, Staining, In Vitro, Western Blot, Comparison, Saline