Journal: Bioactive Materials

Article Title: Bioengineered extracellular vesicles escape lysosomal degradation and deliver Tet-PKM2 for macrophage immunometabolic reprogramming and periodontitis treatment

doi: 10.1016/j.bioactmat.2026.01.002

Figure Lengend Snippet: Generation of PKM2-overexpressing cells and generation of Tet-PKM2-enriched LEVs. ( A ) Schematic illustration depicting the overexpression of PKM2 in HEK293T cells by lentiviral transduction and subsequent allosteric activation of Tet-PKM2 via TEPP-46 treatment. Cells transfected with ov-NC or ov-PKM2 were named the NC or OV, respectively. ( B ) Relative mRNA expression levels of PKM2 in the NC and OV groups (qRT‒PCR, n = 4). ( C ) Representative immunoblot bands of PKM2 in the NC and OV groups. ( D ) Semiquantitative analysis of the expression levels of PKM2 in the NC and OV groups ( n = 6). ( E ) PK activity in PKM2-overexpressing HEK293T cells in response to treatment with various concentrations of TEPP-46 (0, 10, 20, 40, and 70 μM) ( n = 6). ( F ) Representative immunoblot bands of PKM2 conformational states in PKM2-overexpressing cells after treatment with TEPP-46 (40 μM) for 0, 8, and 24 h (DSS cross-linking assay). ( G ) Semiquantitative analysis of the expression levels of Tet-PKM2 in PKM2-overexpressing cells after treatment with TEPP-46 (40 μM) for 0, 8, and 24 h ( n = 4). ( H ) Schematic illustration of the isolation of LEVs and SEVs by differential velocity centrifugation. ( I ) Representative TEM images showing the morphology of SEVs and LEVs. ( J ) Size distributions of SEVs and LEVs (NTA). ( K ) Particle counts of SEVs and LEVs (NTA). ( L ) Particle-to-protein ratios of SEVs and LEVs. ( M ) Cellular uptake of SEVs and LEVs (labeled with PKH67; green) by macrophages (immunofluorescence assay); cell skeletons were stained with phalloidin (red), and nuclei were stained with DAPI (blue). ( N ) Expression of CD63, HSP70, TSG101, calnexin, and GM130 in whole-cell lysates (Cells), SEVs, and LEVs (Western blot). ( O ) Representative immunoblot bands of PKM2 conformational states in LEVs and SEVs. ( P ) Semiquantitative analysis of the expression levels of Tet-PKM2 in LEVs and SEVs ( n = 3) ( Q ) Conformational states of PKM2 in LEVs Tet−PKM2 in response to TEPP-46 treatment. ( R ) Semiquantitative analysis of the expression levels of Tet-PKM2 in LEVs following TEPP-46 treatment ( n = 3). The data are expressed as the mean ± SEM. Statistical analysis was performed by one-way ANOVA ( E and G ) and Student's t -test ( B , D , K , L , P , and R ). ns indicates no significant difference between the indicated columns; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 indicate significant differences between the indicated columns.

Article Snippet: Tet-PKM2 in HEK293T cells was allosterically activated by TEPP-46 (MCE, HY-18657, USA).

Techniques: Over Expression, Transduction, Activation Assay, Transfection, Expressing, Western Blot, Activity Assay, Isolation, Centrifugation, Labeling, Immunofluorescence, Staining

Journal: Bioactive Materials

Article Title: Bioengineered extracellular vesicles escape lysosomal degradation and deliver Tet-PKM2 for macrophage immunometabolic reprogramming and periodontitis treatment

doi: 10.1016/j.bioactmat.2026.01.002

Figure Lengend Snippet: Generation of PKM2-overexpressing cells and generation of Tet-PKM2-enriched LEVs. ( A ) Schematic illustration depicting the overexpression of PKM2 in HEK293T cells by lentiviral transduction and subsequent allosteric activation of Tet-PKM2 via TEPP-46 treatment. Cells transfected with ov-NC or ov-PKM2 were named the NC or OV, respectively. ( B ) Relative mRNA expression levels of PKM2 in the NC and OV groups (qRT‒PCR, n = 4). ( C ) Representative immunoblot bands of PKM2 in the NC and OV groups. ( D ) Semiquantitative analysis of the expression levels of PKM2 in the NC and OV groups ( n = 6). ( E ) PK activity in PKM2-overexpressing HEK293T cells in response to treatment with various concentrations of TEPP-46 (0, 10, 20, 40, and 70 μM) ( n = 6). ( F ) Representative immunoblot bands of PKM2 conformational states in PKM2-overexpressing cells after treatment with TEPP-46 (40 μM) for 0, 8, and 24 h (DSS cross-linking assay). ( G ) Semiquantitative analysis of the expression levels of Tet-PKM2 in PKM2-overexpressing cells after treatment with TEPP-46 (40 μM) for 0, 8, and 24 h ( n = 4). ( H ) Schematic illustration of the isolation of LEVs and SEVs by differential velocity centrifugation. ( I ) Representative TEM images showing the morphology of SEVs and LEVs. ( J ) Size distributions of SEVs and LEVs (NTA). ( K ) Particle counts of SEVs and LEVs (NTA). ( L ) Particle-to-protein ratios of SEVs and LEVs. ( M ) Cellular uptake of SEVs and LEVs (labeled with PKH67; green) by macrophages (immunofluorescence assay); cell skeletons were stained with phalloidin (red), and nuclei were stained with DAPI (blue). ( N ) Expression of CD63, HSP70, TSG101, calnexin, and GM130 in whole-cell lysates (Cells), SEVs, and LEVs (Western blot). ( O ) Representative immunoblot bands of PKM2 conformational states in LEVs and SEVs. ( P ) Semiquantitative analysis of the expression levels of Tet-PKM2 in LEVs and SEVs ( n = 3) ( Q ) Conformational states of PKM2 in LEVs Tet−PKM2 in response to TEPP-46 treatment. ( R ) Semiquantitative analysis of the expression levels of Tet-PKM2 in LEVs following TEPP-46 treatment ( n = 3). The data are expressed as the mean ± SEM. Statistical analysis was performed by one-way ANOVA ( E and G ) and Student's t -test ( B , D , K , L , P , and R ). ns indicates no significant difference between the indicated columns; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 indicate significant differences between the indicated columns.

Article Snippet: Tet-PKM2 in HEK293T cells was allosterically activated by TEPP-46 (MCE, HY-18657, USA).

Techniques: Over Expression, Transduction, Activation Assay, Transfection, Expressing, Western Blot, Activity Assay, Isolation, Centrifugation, Labeling, Immunofluorescence, Staining

Journal: Bioactive Materials

Article Title: Bioengineered extracellular vesicles escape lysosomal degradation and deliver Tet-PKM2 for macrophage immunometabolic reprogramming and periodontitis treatment

doi: 10.1016/j.bioactmat.2026.01.002

Figure Lengend Snippet: Generation of bioengineered LEVs (LEVs@TA) through in situ TA modifications. ( A ) Schematic illustration showing the interaction of TA with the phospholipid bilayer of LEVs via hydrogen bonding and the uptake of LEVs@TA by macrophages. ( B ) The percentages of CY5-TA-modified cells following incubation with gradient concentrations of CY5-TA (0, 0.1, 1, 5, and 10 μM) for 24 h (flow cytometry assay). ( C ) Colocalization of CY5-TA on HEK293T cells following incubation in 10 μM CY5-TA for 24 h (fluorescence microscopy). The nuclei were stained with DAPI (blue). ( D ) The percentages of CY5-TA-modified LEVs following incubation with gradient concentrations of CY5-TA (0, 10, 20, 50, and 100 μM) for 24 h (flow cytometry assay). ( E ) Colocalization of CY5-TA and PKH67-labeled LEVs (green) following incubation with 100 μM CY5-TA for 24 h (fluorescence microscopy). ( F ) Snapshots of CGMD simulations depicting the uptake of LEVs and LEVs@TA by macrophages at 0, 5, 10, 15, and 20 ns. ( G ) Representative in vivo fluorescence images showing good stability of DIO-labeled-LEVs@CY5-TA in vivo .

Article Snippet: Tet-PKM2 in HEK293T cells was allosterically activated by TEPP-46 (MCE, HY-18657, USA).

Techniques: In Situ, Modification, Incubation, Flow Cytometry, Fluorescence, Microscopy, Staining, Labeling, In Vivo