Journal: Infection and Drug Resistance

Article Title: Exosomal miR-148a-3p from LPS-Activated Macrophages Promotes M1 Polarization and Ferroptosis-Related Characteristics of Recipient Macrophages by Reducing SLC7A11

doi: 10.2147/IDR.S590998

Figure Lengend Snippet: Macrophages stimulated by LPS secrete exosomes enriched with miR-148a-3p. ( A ) A representative TEM image showing the morphology of isolated exosomes (scale bar = 100 nm). ( B ) Exosome size distribution and concentration were analyzed by NTA. ( C ) Exosomal markers, specifically TSG101 and CD63, along with the negative control marker Calnexin were detected by Western blotting. ( D ) Fluorescence imaging of RAW264.7 cells after a 24-hour incubation with 20 μg of PKH67-labeled exosomes (green: PKH67-labeled exosomes; blue: DAPI-stained nuclei; scale bar = 50 μm). ( E ) The absolute quantification of candidate miRNAs in exosomes isolated from RAW264.7 cells treated with PBS or LPS was performed using ddPCR (n = 3). ( F ) The relative expression levels of candidate miRNAs in recipient RAW264.7 cells post-exosome uptake were determined via qRT-PCR (n = 3). ( G ) The absolute quantification of candidate miRNAs in exosomes isolated from BMDMs treated with PBS or LPS was performed using ddPCR (n = 3). For ( E and G ), statistical analysis was performed using Student’s t -test. Significance levels, in comparison to the PBS-exo group, are indicated as follows: * p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Article Snippet: Figure 2 Macrophages stimulated by LPS secrete exosomes enriched with miR-148a-3p. ( A ) A representative TEM image showing the morphology of isolated exosomes (scale bar = 100 nm). ( B ) Exosome size distribution and concentration were analyzed by NTA. ( C ) Exosomal markers, specifically TSG101 and CD63, along with the negative control marker Calnexin were detected by Western blotting. ( D ) Fluorescence imaging of RAW264.7 cells after a 24-hour incubation with 20 μg of PKH67-labeled exosomes (green: PKH67-labeled exosomes; blue: DAPI-stained nuclei; scale bar = 50 μm). ( E ) The absolute quantification of candidate miRNAs in exosomes isolated from RAW264.7 cells treated with PBS or LPS was performed using ddPCR (n = 3). ( F ) The relative expression levels of candidate miRNAs in recipient RAW264.7 cells post-exosome uptake were determined via qRT-PCR (n = 3). ( G ) The absolute quantification of candidate miRNAs in exosomes isolated from BMDMs treated with PBS or LPS was performed using ddPCR (n = 3).

Techniques: Isolation, Concentration Assay, Negative Control, Marker, Western Blot, Fluorescence, Imaging, Incubation, Labeling, Staining, Quantitative Proteomics, Expressing, Quantitative RT-PCR, Comparison

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: PKH67-labeled SEVs and LEVs resuspended in complete medium were used to treat RAW 264.7 cells for 24 h. The cells were then fixed with 4 % PFA (Coolaber), permeabilized, and stained for cytoskeletal visualization using fluorescein phalloidin (1:1000 dilution; MCE) for 30 min.

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

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: Endo/lysosomal escape capacity of bioengineered LEVs@TA following uptake by macrophages. ( A ) Schematic illustration showing the endo/lysosomal escape process of LEVs@TA within the cytoplasm of macrophages. After uptake by macrophages, LEVs@TA were entrapped within endo/lysosomes, and then TA underwent protonation and disassembled from LEVs in an acidic environment, leading to rupture of the endo/lysosomal structure. ( B ) Snapshots of CGMD simulations showing the disassembly of TA and LEVs in the lysosomal environment. ( C ) Colocalization of LysoTracker-labeled endo/lysosomes (violet) and PKH67-labeled LEVs or LEVs@TA (green) (fluorescence microscopy). The nuclei were stained with Hoechst (blue). ( D ) Quantification of the colocalization of endo/lysosomes and LEVs or LEVs@TA using the Pearson correlation coefficient ( n = 12). ( E ) Schematic illustration showing the leakage of calcein into the cytosol when TA diffused from LEVs@TA and destabilized the endo/lysosomal membranes. ( F ) The distribution of calcein (green) in macrophages treated with PBS, LEVs, and LEVs@TA (fluorescence microscopy). (G) Representative TEM images of macrophages showing the structure of lysosomes in macrophages treated with LEVs and LEVs@TA. The data are expressed as the mean ± SEM. Statistical analysis was performed with Student's t -test ( D ). ∗∗∗ p < 0.001 indicates significant differences between the indicated columns.

Article Snippet: PKH67-labeled SEVs and LEVs resuspended in complete medium were used to treat RAW 264.7 cells for 24 h. The cells were then fixed with 4 % PFA (Coolaber), permeabilized, and stained for cytoskeletal visualization using fluorescein phalloidin (1:1000 dilution; MCE) for 30 min.

Techniques: Labeling, Fluorescence, Microscopy, Staining