Journal: Bioactive Materials

Article Title: ADGRG1-targeted hypoxia preconditioned extracellular vesicles ameliorate intervertebral disc degeneration by delivering taurine to disrupt the oxidative stress feedback loop-driven ferroptosis in nucleus pulposus cells

doi: 10.1016/j.bioactmat.2026.02.029

Figure Lengend Snippet: Preparation and characterization of engineered ADGRG1-targeting and hypoxia-treated EVs. (A)Induced fit docking analysis of ADGRG1-binding peptide (A1TP, 7 peptides) and extracellular domain of ADGRG1 protein (PDB database: 7SF8). (B) Analysis of the binding of the A1TP to purified ADGRG1 proteins using a microscale thermophoresis (MST) binding assay. (C) Induced fit docking analysis of A1TP-PEG and extracellular domain of ADGRG1 protein. (D) The binding free energy between A1TP or A1TP-PEG and ADGRG1 were calculated using molecular dynamics simulations. Lower values indicate more stable interactions, with values less than or equal to −20 considered as stable binding modes. (E) Schematic illustration of the conjugating reaction between DSPE-PEG-Alkyne and A1TP. Schematic illustration of the fabrication of A1TP-HX-EVs through external modification by A1TP anchoring. Specific steps for the synthesis of DSPE-PEG-A1TP (DPA) are shown in . (F) FT-IR analysis showed the characteristic peaks of the DSPE-PEG-A1TP. The new triazole ring itself showed a characteristic C=N stretching vibration, a peak at 1538 cm −1 revealed the successful conjugation of A1TP. (G) H Nuclear magnetic resonance (NMR) spectra of DSPE-PEG-A1TP in D2O. The hydrogen signatures of the phenyl and phenol groups at 7.5-8.0 ppm confirmed the successful conjugation of DSPE to A1TP. (H) Western blot analysis verified the presence of three EV marker proteins (ALIX, TSG101, and CD81) and one EV negative marker (GM130) in EVs, HX-EVs, and A1TP-HX-EVs. (I) Transmission electron microscopy (TEM) images of EVs, HX-EVs and A1TP-HX-EVs. Scale bar, 200 nm. (J) Zeta potentials of EVs, HX-EVs and A1TP-HX-EVs, n = 3. Two-tailed unpaired Student's t-test was used for statistical analysis. ns, not significant. A two-tailed unpaired Student's t-test was used for statistical analysis. (K) Representative images of the spherical morphology and dispersion states of EVs, HX-EVs and A1TP-HX-EVs. Scale bar, 500 nm. (L) Size distributions of EVs, HX-EVs and A1TP-HX-EVs.

Article Snippet: Finally, the presence of the characteristic EV markers Alix (92880, Cell Signaling Technology), CD81 (56039, Cell Signaling Technology) and TSG101 (sc-7964, Santa Cruz Biotechnology) was confirmed by Western blot analysis.

Techniques: Binding Assay, Purification, Microscale Thermophoresis, Modification, Conjugation Assay, Nuclear Magnetic Resonance, Western Blot, Marker, Transmission Assay, Electron Microscopy, Two Tailed Test, Dispersion

Journal: Bioactive Materials

Article Title: Cord blood natural killer cell-derived extracellular vesicles inhibit Zika virus infectivity through ITGB2/perforin-mediated envelope disruption in vitro and in vivo

doi: 10.1016/j.bioactmat.2026.01.030

Figure Lengend Snippet: Characterization and anti-ZIKV activity of CBNK-EVs. (a) Isolation of CBNK cells by fluorescence-activated cell sorting. (b) Collection of CBNK-EVs and NTA for size distribution and concentration. (c) Morphological observation of CBNK-EVs by transmission electron microscopy, scale bar:100 nm. (d) WB analysis of specific markers for CBNK-EVs. (e) Anti-ZIKV activity of CBNK-EVs assessed by immunofluorescence. Vero-E6 cells were seeded in 12-well plates at 1 × 10 5 cells per well one day prior to experiments. Cells were pretreated with varying volumes of CBNK-EVs (stock concentration: 3 × 10 11 particles/mL) for 1 h to achieve the indicated final particle counts, followed by infection with ZIKV (MOI = 1) for 24 h in the presence of fresh medium containing the same EVs. ZIKV-infected cells were quantified by counting E protein-positive cells in three random fields by two independent investigators. Scale bar, 40 μm. (f) Statistical analysis of the data presented in (e) panel. (g) Vero-E6 cells were treated as in (e) and infected with ZIKV (MOI = 1) for 24 h. (h) ZIKV E and NS1 RNA levels were detected by quantitative RT-PCR. Data were normalized to GAPDH, log10-transformed, and presented as fold change compared to the virus control group (set to 1). Negative values indicate inhibition of viral replication. (i) Plaque reduction assay assessing the antiviral activity of CBNK-EVs against ZIKV. (j) Statistical analysis of the percentage of plaque reduction. (k) ZIKV NS5 protein levels in (g) were analyzed by Western blot. Data in panels (f, h, j and k) are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: Finally, CBNK-EVs were diluted with exosome-free phosphate-buffered saline (PBS) to the desired concentration and stored at −80 °C for subsequently experiments.

Techniques: Activity Assay, Isolation, Fluorescence, FACS, Concentration Assay, Transmission Assay, Electron Microscopy, Immunofluorescence, Infection, Quantitative RT-PCR, Transformation Assay, Virus, Control, Inhibition, Western Blot

Journal: Bioactive Materials

Article Title: Cord blood natural killer cell-derived extracellular vesicles inhibit Zika virus infectivity through ITGB2/perforin-mediated envelope disruption in vitro and in vivo

doi: 10.1016/j.bioactmat.2026.01.030

Figure Lengend Snippet: Functional components and mechanisms of CBNK-EVs in combating ZIKV infection. (a) Schematic of three treatment modalities: 1) pre-treatment of cells with CBNK-EVs, 2) pre-treatment of virus with CBNK-EVs, and 3) co-incubation of CBNK-EVs with ZIKV. (b) Vero-E6 cells were seeded at 1 × 10 5 cells per well in 12-well plates and subjected to the treatments outlined in (a), followed by infection with ZIKV (MOI = 1) for 1 h. After 48 h, ZIKV E and ZIKV NS5 protein levels were analyzed by Western blot (left). Densitometric analysis of the protein bands is shown (right, n = 3). (c) Schematic workflow for functional component inactivation in CBNK-EVs (e.g., by heat, protease, or nuclease treatment). (d) Vero-E6 cells were treated with inactivated CBNK-EVs as per the scheme in (c), followed by infection with ZIKV (MOI = 1) for 1 h. ZIKV E and ZIKV NS5 protein levels were analyzed by Western blot after 24 h. Densitometric analysis of the protein bands is shown (right, n = 3). (e) Represent images of the co-localization of CBNK-EVs, ZIKV E and host cells by confocal microscope, scale bar: 5 μm. (f) Fluorescence intensity of CBNK-EV and β-actin co-localization signals (white arrows) in the EVs group. (g) Fluorescence co-localization signals (yellow arrows) of CBNK-EVs and ZIKV in the co-culture group. (h) TEM images of CBNK-EVs, ZIKV particles, and their interactions. Scale bar: 100 nm. (i) Membrane disruption assay. ZIKV particles were co-incubated with CBNK-EVs, followed by treatment with micrococcal nuclease to digest unprotected RNA. Intact viral RNA, indicative of membrane integrity, was then measured by RT-qPCR targeting the ZIKV E. Mean ± SD (n = 3). ∗∗P < 0.01, ∗∗∗P < 0.001 (one-way ANOVA).

Article Snippet: Finally, CBNK-EVs were diluted with exosome-free phosphate-buffered saline (PBS) to the desired concentration and stored at −80 °C for subsequently experiments.

Techniques: Functional Assay, Infection, Virus, Incubation, Western Blot, Microscopy, Fluorescence, Co-Culture Assay, Membrane, Disruption, Quantitative RT-PCR

Journal: Bioactive Materials

Article Title: Cord blood natural killer cell-derived extracellular vesicles inhibit Zika virus infectivity through ITGB2/perforin-mediated envelope disruption in vitro and in vivo

doi: 10.1016/j.bioactmat.2026.01.030

Figure Lengend Snippet: Protective effects of CBNK-EVs against ZIKV infection in fetal mice. (a) In vivo imaging of pregnant mice at 4 h post-intravenous injection of DiD-labeled CBNK-EVs (6 × 10 12 particles/mL, 200 μl per mouse). (b) Relative quantitative analysis of fluorescence intensity in the liver and lower abdomen from (a). (c) Representative image of CBNK-EV signals enriched in uterine tissues at 4 h time point. (d) Representative image showing transplacental migration and distribution of CBNK-EVs in fetus at 4 h time point. (e) Quantitative analysis of CBNK-EV fluorescence intensity in embryonic tissues from (d). (f) CBNK-EVs reduced ZIKV E RNA copies in placental tissues as measured by RT-qPCR. (g) CBNK-EVs significantly suppressed ZIKV E RNA levels in fetal heads. (h) CBNK-EVs ameliorated ZIKV-induced microcephaly, assessed by measuring the maximum head circumference. (i) Immunofluorescence staining of ZIKV E protein (red) in fetal hippocampal and cortical brain regions. Cell nuclei were stained with DAPI (blue). Scale bar: 100 μm. (j) Quantification of ZIKV E-positive cells in the hippocampus and (k) cortex from at least three random fields per sample. Data are presented as mean ± SD; n = 3 for (b, e, j, k), n = 14 for (f, g). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (one-way ANOVA).

Article Snippet: Finally, CBNK-EVs were diluted with exosome-free phosphate-buffered saline (PBS) to the desired concentration and stored at −80 °C for subsequently experiments.

Techniques: Infection, In Vivo Imaging, Injection, Labeling, Fluorescence, Migration, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Bioactive Materials

Article Title: Cord blood natural killer cell-derived extracellular vesicles inhibit Zika virus infectivity through ITGB2/perforin-mediated envelope disruption in vitro and in vivo

doi: 10.1016/j.bioactmat.2026.01.030

Figure Lengend Snippet: CBNK-EVs rapidly impair ZIKV infectivity and protect host cells. (a) Multiplex immunohistochemical analysis of ZIKV infection in neural progenitor cells. Co-staining for Nestin (neural progenitor cells, green) and ZIKV E protein (red) was performed, and double-positive cells were quantified across six random fields, mean ± SD, ∗P < 0.05. Scale bar, 20 μm. (b) KEGG pathway enrichment bubble plot from RNA-seq of HTR-8/Svneo cells treated with CBNK-EVs (3 × 10 11 particles/mL, 10 μl) and ZIKV (MOI = 1) for 1 h. (c) GSEA analysis of transcriptomic data from HTR-8/Svneo cells. (d) Western blot analysis of ZIKV E and ZIKV NS5 protein levels after co-incubation of ZIKV particles with CBNK-EVs for the indicated time periods (0–30 min) prior to infection. (e) The abundance of NK cell-associated proteins in CBNK-EVs. ∗P < 0.05 (one-way ANOVA).

Article Snippet: Finally, CBNK-EVs were diluted with exosome-free phosphate-buffered saline (PBS) to the desired concentration and stored at −80 °C for subsequently experiments.

Techniques: Infection, Multiplex Assay, Immunohistochemical staining, Staining, RNA Sequencing, Western Blot, Incubation

Journal: Bioactive Materials

Article Title: Cord blood natural killer cell-derived extracellular vesicles inhibit Zika virus infectivity through ITGB2/perforin-mediated envelope disruption in vitro and in vivo

doi: 10.1016/j.bioactmat.2026.01.030

Figure Lengend Snippet: Perforin mediates the direct virion-disrupting activity of CBNK-EVs. (a) Quantification of perforin and granzyme B in CBNK-EVs by ELISA. (b) Vero-E6 cells were infected with ZIKV (MOI = 1) after pre-incubation of viral particles with 40 ng/L of recombinant perforin or granzyme B for 2 h. ZIKV E protein levels were assessed by Western blot after 24 h, densitometric analysis of the protein bands is shown (right, n = 3). (c) CBNK-EVs were pre-incubated with ZIKV in the presence or absence of 10 mM EGTA, followed by micrococcal nuclease digestion. Protected ZIKV E RNA was quantified by RT-qPCR to assess virion integrity. (d) Western blot analysis of perforin expression in CBNK cells after transfection with the indicated siRNAs. (e) Quantification of perforin levels from (d). (f) Perforin levels in CBNK-EVs collected from control or perforin-knockdown cells, measured by ELISA and normalized to particle count (per 10 11 particles, n = 6). (g) Vero-E6 cells were infected with ZIKV (MOI = 1) that had been pre-incubated with control or perforin-knockdown CBNK-EVs. ZIKV E protein levels were evaluated by Western blot. (h) Analysis of ZIKV E protein levels from (g). (i) Characterization of ITGB2-EVs and control EVs from 293T cells by NTA and TEM. Scale bar, 100 nm. (j) Zeta potential measurements of ITGB2-EVs and control EVs. (k) Western blot analysis of EV markers and ITGB2 expression in ITGB2-EVs and control EVs. (l) Antiviral activity of CBNK-EVs and ITGB2-EVs evaluated by cell-based ZIKV E protein ELISA. Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗P < 0.001 (one-way ANOVA).

Article Snippet: Finally, CBNK-EVs were diluted with exosome-free phosphate-buffered saline (PBS) to the desired concentration and stored at −80 °C for subsequently experiments.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Infection, Incubation, Recombinant, Western Blot, Quantitative RT-PCR, Expressing, Transfection, Control, Knockdown, Zeta Potential Analyzer

Journal: Bioactive Materials

Article Title: Cord blood natural killer cell-derived extracellular vesicles inhibit Zika virus infectivity through ITGB2/perforin-mediated envelope disruption in vitro and in vivo

doi: 10.1016/j.bioactmat.2026.01.030

Figure Lengend Snippet: ITGB2 facilitates CBNK-EVs binding to Zika virions and enhances cellular susceptibility to ZIKV. (a) Multicolor immunofluorescence staining of 293T cells transfected with ITGB2, showing co-localization (yellow) between ITGB2 (green) and ZIKV E protein (red). Scale bar: 20 μm. (b) Quantitative analysis of ZIKV E-positive 293T cells from (a). (c) Fluorescence intensity profile along white arrows in (a), indicating sites of ITGB2 and ZIKV E co-localization. (d) 293T cells were transfected with increasing amounts (0.5, 1, 2 μg) of ITGB2 plasmid and infected with ZIKV (MOI = 1) for 1 h. ZIKV RNA levels were measured by qPCR to assess infection susceptibility (mean ± SD, n = 3). (e) Molecular docking model predicting the interaction interface between ITGB2 and ZIKV E protein. (f) Co-IP assay in 293T cells, followed by immunoblotting with anti-ZIKV E protein antibody. (g) CBNK-EVs were pre-incubated with ITGB2 mAb (0, 5, 20 μg/mL) before being applied to Vero-E6 or BHK-21 cells. Cells were then infected with ZIKV (MOI = 1) for 1 h, and antiviral activity was assessed by measuring ZIKV RNA levels at 24 h post-infection. Data are presented as mean ± SD (n = 3 for d and g; n = 6 for b). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (one-way ANOVA).

Article Snippet: Finally, CBNK-EVs were diluted with exosome-free phosphate-buffered saline (PBS) to the desired concentration and stored at −80 °C for subsequently experiments.

Techniques: Binding Assay, Multicolor Immunofluorescence Staining, Transfection, Fluorescence, Plasmid Preparation, Infection, Co-Immunoprecipitation Assay, Western Blot, Incubation, Activity Assay

Journal: Bioactive Materials

Article Title: Cord blood natural killer cell-derived extracellular vesicles inhibit Zika virus infectivity through ITGB2/perforin-mediated envelope disruption in vitro and in vivo

doi: 10.1016/j.bioactmat.2026.01.030

Figure Lengend Snippet: CBNK-EVs cross the blood–brain barrier and attenuate ZIKV infection in AG6 mice. (a) Schematic timeline of the AG6 mouse experiment. Mice were intravenously administered CBNK-EVs (6 × 10 12 particles in 200 μL PBS) via the tail vein, and viremia was monitored at indicated time points (n = 10). (b) In vivo imaging to monitor the biodistribution of CBNK-EVs in AG6 mice. (c) CBNK-EVs exhibited differential accumulation in the liver, kidneys, spleen and (d) brain. (e) Relative quantitative analysis of fluorescence intensity for EVs distribution in liver, kidneys, spleen and (f) brain (mean ± SD, n = 3). (g) Multicolor immunofluorescence staining showed the distribution of CBNK-EVs within the hippocampus. Scale bar: 100 μm. (h) Behavioral scoring analysis of AG6 mice post-ZIKV challenge (mean ± SD, n = 10). (i) Survival analysis of AG6 mice post-ZIKV challenge (n = 10). (j) Viremia at various time points and viral loads in different tissues were quantified by qPCR (mean ± SD, n = 10). ∗P < 0.05, ∗∗P < 0.01, log-rank test for (i) and one-way ANOVA for other panels.

Article Snippet: Finally, CBNK-EVs were diluted with exosome-free phosphate-buffered saline (PBS) to the desired concentration and stored at −80 °C for subsequently experiments.

Techniques: Infection, In Vivo Imaging, Fluorescence, Multicolor Immunofluorescence Staining

Journal: Bioactive Materials

Article Title: Cord blood natural killer cell-derived extracellular vesicles inhibit Zika virus infectivity through ITGB2/perforin-mediated envelope disruption in vitro and in vivo

doi: 10.1016/j.bioactmat.2026.01.030

Figure Lengend Snippet: CBNK-EVs reduce ZIKV infection and protect neuroglial cells from damage. (a) Multicolor immunofluorescence (scale bar: 50 μm) combined with H&E staining revealed the neuroprotective effects of CBNK-EVs in AG6 mice. In the magnified immunofluorescence panels (scale bar: 10 μm), yellow arrows indicate ZIKV E-positive signals. H&E staining of the cortical region (scale bar: 100 μm) revealed perivascular cuffing (black arrow) in virus group. (b) Compared to the Virus group, GO analysis revealed that CBNK-EV pre-treatment markedly attenuated the expression of genes associated with ZIKV induced neurodevelopmental impairment. (c) Compared to the Virus group, KEGG pathway analysis indicated a significant reduction in ZIKV-triggered activation of innate immune signaling and inflammatory cascades. (d) Compared to the Virus group, GSEA demonstrated a reversal of the aberrant expression profile for genes implicated in ZIKV-mediated central nervous system developmental disruption.

Article Snippet: Finally, CBNK-EVs were diluted with exosome-free phosphate-buffered saline (PBS) to the desired concentration and stored at −80 °C for subsequently experiments.

Techniques: Infection, Immunofluorescence, Staining, Virus, Expressing, Activation Assay, Disruption