Journal: Bioactive Materials

Article Title: M2 macrophage-derived exosomes delivering haptoglobin and interleukin-10 plasmids for synergistic therapy of intracerebral hemorrhage

doi: 10.1016/j.bioactmat.2026.01.047

Figure Lengend Snippet: Schematic illustration of the preparation of M2-exo@HI and its mediated therapeutic mechanisms and signaling pathways. (a) RAW264.7 macrophages were polarized to M2 phenotype using DEX, followed by M2-exo isolation from cell supernatant via differential centrifugation. M2-exo@HI was prepared through encapsulating HI into M2-exo by electroporation. (b) M2-exo@HI crossed the BBB and localized to microglia in the hemorrhagic brain, delivering the HI plasmid into the nucleus. This prompted expression and secretion of Hp and IL-10 by microglia. The released Hp inhibited Hb toxicity by binding to Hb. IL-10 shifted microglial polarization from the pro-inflammatory M1 phenotype toward the reparative M2 phenotype, removing hematoma by engulfing erythrocyte and Hb-Hp complex, and decreasing pro-inflammatory cytokine levels—collectively enhancing neuroprotection and BBB repair. These beneficial outcomes were linked to the inhibition of the IL-17 and NF-κB signaling pathways and activation of the PI3K-Akt and ferroptosis pathways.

Article Snippet: For CLSM observation, 100 μL ICG and M2-exo@ICG were incubated with M1 microglia for 0.25 h, 0.5 h, 1 h and 2 h. Then cells were washed, fixed, stained with DAPI and observed by CLSM (A1R+, Nikon, Japan).

Techniques: Protein-Protein interactions, Isolation, Centrifugation, Electroporation, Plasmid Preparation, Expressing, Binding Assay, Inhibition, Activation Assay

Journal: Bioactive Materials

Article Title: M2 macrophage-derived exosomes delivering haptoglobin and interleukin-10 plasmids for synergistic therapy of intracerebral hemorrhage

doi: 10.1016/j.bioactmat.2026.01.047

Figure Lengend Snippet: Validation of M2-exo targeting, Hp/IL-10 transfection expression, and Hp/Hb binding. (a) Fluorescence imaging showing cellular uptake of ICG and M2-exo@ICG by M1 microglia. (b,c) Flow cytometry and corresponding quantification of RhB and M2-exo@RhB internalized by M1 microglia (n = 3). (d,e) Schematic illustration and quantitative analysis of the in vitro phagocytosis-release kinetics of M2-exo@RhB in BV2 under ICH-mimicking stimulation (n = 6). (f) Fluorescence images showing Hp and IL-10 expression in M1 microglia treated with M2-exo@HI for 12, 24, 48, 72 h. (g) Mean fluorescence intensity (MFI) quantification of Hp and IL-10 expression (n = 3). (h,i) ELISA measurements of secreted Hp and IL-10 protein levels (n = 3). (j,k) qPCR analysis of relative Hp and IL-10 mRNA expression (n = 3). (l) Western blot detection of Hp and IL-10 protein expression. (m) Densitometric quantification of Hp and IL-10 protein levels from Western blot (n = 3). (n) Co-immunoprecipitation assay confirming the formation of Hp-Hb complex. Data are presented as mean ± SD. Statistical significance was calculated by unpaired Student's t -test (c and e), and one-way ANOVA with Tukey's multiple comparisons test (g-k and m).

Article Snippet: For CLSM observation, 100 μL ICG and M2-exo@ICG were incubated with M1 microglia for 0.25 h, 0.5 h, 1 h and 2 h. Then cells were washed, fixed, stained with DAPI and observed by CLSM (A1R+, Nikon, Japan).

Techniques: Biomarker Discovery, Transfection, Expressing, Binding Assay, Fluorescence, Imaging, Flow Cytometry, In Vitro, Enzyme-linked Immunosorbent Assay, Western Blot, Co-Immunoprecipitation Assay

Journal: Bioactive Materials

Article Title: M2 macrophage-derived exosomes delivering haptoglobin and interleukin-10 plasmids for synergistic therapy of intracerebral hemorrhage

doi: 10.1016/j.bioactmat.2026.01.047

Figure Lengend Snippet: M2-exo@HI promotes in vitro microglia polarization, BBB repair and neuroprotection. (a) Flow cytometry analysis of M1-type (CD86 + ) and M2-type microglia (CD163 + ) following treatment with different formulations. (b,c) Percentages of CD86 + and CD163 + microglia populations (n = 3). (d–g) The cytokine levels of IL-10, TGF-β, TNF-α, and IL-1β in treated microglia (n = 3). (h) Fluorescence microscopy images showing erythrophagocytosis by microglia across treatment groups. (i) Schematic of the in vitro BBB model assessing FITC-dextran permeability using a transwell assay. (j) Quantitative analysis of FITC-dextran penetration (n = 7). (k) Flow cytometry analysis of neuronal apoptosis across treatments (n = 3). (l) Quantitative analysis of neuronal apoptosis (n = 3). Data are presented as mean ± SD. Statistical significance was tested by one-way ANOVA with Tukey's multiple comparisons test.

Article Snippet: For CLSM observation, 100 μL ICG and M2-exo@ICG were incubated with M1 microglia for 0.25 h, 0.5 h, 1 h and 2 h. Then cells were washed, fixed, stained with DAPI and observed by CLSM (A1R+, Nikon, Japan).

Techniques: In Vitro, Flow Cytometry, Fluorescence, Microscopy, Permeability, Transwell Assay

Journal: Bioactive Materials

Article Title: M2 macrophage-derived exosomes delivering haptoglobin and interleukin-10 plasmids for synergistic therapy of intracerebral hemorrhage

doi: 10.1016/j.bioactmat.2026.01.047

Figure Lengend Snippet: In vivo therapeutic effects of M2-exo@HI in hemorrhagic stroke. (a) The schematic diagram illustrates the construction of mouse cerebral hemorrhage model and treatment regimens. (b) Digital photos showing cerebral hematoma of ICH mice in different groups. (c) Quantitative measurements of hemoglobin concentration in different groups (n = 3). (d) Cerebral edema quantification by brain water content measurements (n = 3). (e) CLSM images showing M1 microglia (CD86 + , green) and M2 microglia (CD163 + , red) in different groups. Nucleus were stained with DAPI (blue). (f) Immunofluorescence staining showing co-localization of Hb/Hp with microglia in different groups. (g) Representative images of HE, Nissl, and TUNEL staining. Data are presented as mean ± SD. Statistical significance was tested by one-way ANOVA with Tukey's multiple comparisons test.

Article Snippet: For CLSM observation, 100 μL ICG and M2-exo@ICG were incubated with M1 microglia for 0.25 h, 0.5 h, 1 h and 2 h. Then cells were washed, fixed, stained with DAPI and observed by CLSM (A1R+, Nikon, Japan).

Techniques: In Vivo, Concentration Assay, Staining, Immunofluorescence, TUNEL Assay

Journal: Journal of Neuroinflammation

Article Title: Disruption of Midkine gene reduces traumatic brain injury through the modulation of neuroinflammation

doi: 10.1186/s12974-020-1709-8

Figure Lengend Snippet: Effect of MK-deficiency on M1 and M2 microglia/macrophages phenotype marker after TBI. The M1 and M2 phenotype markers (CD16/32 and arginase-1, respectively) were expressed in the perilesional site of Mdk +/+ and Mdk −/− mice at 3 days ( a ). The immunohistochemical staining was performed through serial sections of mice (corresponding to * and + in a ). The ratios of the CD16/32-immunoreactive area were significantly reduced in Mdk −/− mice compared to Mdk +/+ mice at 3 days. The CD16/32- and arginase-1-immunoreactive areas were significantly decreased at 7 days ( b ). RT-qPCR analysis revealed the mRNA levels of the M1 phenotype markers (TNF-α, CD11b) to be significantly downregulated in Mdk −/− than in Mdk +/+ mice ( c ). Data are presented as mean ± SE ( n = 5 mice/group in immunohistochemistry, n = 3–4 mice/group in RT-qPCR). * p < 0.05, ** p < 0.01 (comparison with MK +/+ and Mdk −/− ). ## p < 0.01 (comparison with 3 days and 7 days). Scale bar = 50 μm (all panels)

Article Snippet: TBI, traumatic brain injury The coronal sections were immunostained with the following antibodies: rabbit anti-glial fibrillary acidic protein (GFAP; a marker of activated astrocytes) (Cosmo Bio Co., Japan; RO1003), rabbit anti-ionized calcium-binding adaptor molecule1 (Iba1; a marker of resting microglia/macrophage) (Wako, Osaka, Japan; 019-19741), rat anti-CD16/32 (a marker of M1 microglia/macrophage) (BD Bioscience, US; #553142), rabbit anti-arginase-1 (a marker of M2 microglia/macrophage) (Cell Signaling Technology, Inc., US; #93668), rabbit anti-activated caspase-3 (a marker of apoptotic activity) (Proteintech Group, Inc.; USA; 19677-1-AP), and mouse anti-neuronal nuclei (NeuN; a marker of neuron) (Abcam plc, Cambridge, UK; ab104224).

Techniques: Marker, Immunohistochemical staining, Staining, Quantitative RT-PCR, Immunohistochemistry