Mitochondrial Transplantation via Magnetically Responsive Artificial Cells Promotes Intracerebral Hemorrhage Recovery by Supporting Microglia Immunological Homeostasis

Abstract

The immune-inflammatory responses in the brain represent a key therapeutic target to ameliorate brain injury following intracerebral hemorrhage (ICH), where pro-inflammatory microglia and its mitochondrial dysfunction plays a pivotal role. Mitochondrial transplantation is a promising strategy to improve the cellular mitochondrial function and thus modulate their immune properties. However, the transplantation of naked mitochondria into the brain has been constrained by the peripheral clearance and the difficulty in achieving selective access to the brain. Here, a novel strategy for mitochondrial transplantation via intravenous injection of magnetically responsive artificial cells (ACs) are proposed. ACs can protect the loaded mitochondria and selectively accumulate around the lesion under an external magnetic field (EMF). In this study, mitochondria released from ACs can effectively improve microglial mitochondrial function, attenuate their pro-inflammatory attributes, and elevate the proportion of immunosuppressive microglia. In this way, microglia immune homeostasis in the brain is reestablished, and inflammation is attenuated, ultimately promoting functional recovery. This study presents an effective approach to transplant mitochondria into the brain, offering a promising alternative to modulate the immune-inflammatory cascade in the brain following ICH.

Keywords: artificial cells; immune homeostasis; intracerebral hemorrhage; magnetic drug targeting; mitochondrial transplantation.

Scheme 1

Schematic illustration of the preparation of ACs and its application for the treatment of ICH.

Figure 1

Donor mitochondria screening and isolation and construction of magnetically responsive ACs. A) Schematic diagram of donor mitochondria screening and isolation. B) Bar blots show the calculated mitochondrial respiratory functions in different cell lines, including basal respiration, maximal respiration, and ATP production (data presented as mean ± SD, n  = 6). **P < 0.01, ***P < 0.001, ****p < 0.0001, One‐way ANOVA. C) Bar blots show the production of mitochondrial superoxide (MitoSox) and the abundance of mitochondria (MitoTracker Green, MTG) and the relative mitochondrial superoxide production per mitochondrion (MitoSox/MTG) in different cell lines (data presented as mean ± SD, n = 3; 10000 events per run). ns ≥ 0.5, *p < 0.05, **p < 0.01, ****p < 0.0001, One‐way ANOVA. D) Representative TEM images of isolated mitochondria. Scale bar = 1 µm. E, F) Representative flow cytometry contour plots show the proportion of MitoSox⁺ BV2 after receiving different mitochondrial transplants and its quantitative analysis. Tmito: isolated mitochondria from 4T1 cells; Mmito: isolated mitochondria from MSCs; Bmito: isolated mitochondria from BV2 cells; Hmito: isolated mitochondria from HL‐1 cells (data presented as mean ± SD, n = 3; 10000 events per run). ns ≥ 0.5, *p < 0.05, ****p < 0.0001, One‐way ANOVA. G) Schematic diagram of mitochondrial loading and release by ACs. H) Representative scanning electron microscope (SEM) image and fluorescence microscopy image of microspheres (ACs scaffolds). Scale bar = 2 µm. I) Hydrodynamic diameter distribution of ACs scaffolds in water. J) Representative SEM image and fluorescence microscopy image of self‐healed ACs following the loading of Hmito. Scale bar = 2 µm. K) Line chart shows the phagocytosis efficiency of ACs and the Hmito by RAW 264.7 cells (data presented as mean ± SD, n = 3). ***p < 0.001, Two‐way ANOVA. L) Representative SEM and fluorescence microscopy image of the collapsed ACs scaffolds. Scale bar = 2 µm. M) Release kinetics of Hmito at varying temperatures over time (data presented as mean ± SD, n = 3). ***p < 0.001, Two‐way ANOVA.

Figure 2

Mitochondrial transplantation improves microglial mitochondrial function and restores cellular energy production. A) Representative fluorescence microscopy images of the dynamic internalization process of R‐Hmito. Green: MTG labeled R‐Hmito; Red: MTDR labeled endogenous microglial mitochondria; Blue: Hoechst, Nuclei; White arrows: Co‐localization of R‐Hmito with endogenous mitochondria, Scale bar = 20 µm. B) Line chart shows the ratio of BV2 that internalized F‐Hmito or R‐Hmito and the ratio of PC12 cells that internalized R‐Hmito at different time points (data presented as mean ± SD, n = 3). ns ≥ 0.5, ***p < 0.001, Two‐way ANOVA. C) Representative fluorescence microscopy images of MMP of BV2 after different treatments under H₂O₂ exposure. Blue: Hoechst, Nuclei; Green: JC‐1 monomers, representing damaged mitochondria with loss of MMP; Red: JC‐1 aggregates, representing mitochondria with normal MMP. Scale bar = 20 µm. D) Representative flow cytometry histograms of MitoSox levels in BV2 after different treatments under H₂O₂ exposure (n = 3; 10000 events per run). E) Representative flow cytometry histograms of ROS levels in BV2 after different treatments using DCFH‐DA fluorescent probe labeling (n = 3; 10000 events per run). F) Ultrastructure of mitochondria in BV2 cells demonstrated by TEM from different treatment groups (n = 15). Scale bar = 0.5 µm. G) Representative OCR profiles of BV2 in different treatment groups determined using the Seahorse XF Mitochondrial Stress Assay Kit (data presented as mean ± SD, n  = 6). Oligomycin: an inhibitor of mitochondrial ATP synthase; FCCP: trifluoromethoxy carbonylcyanide phenylhydrazone, an uncoupler in OXPHOS; Rot + Ant A: rotenone and antimycin A, an inhibitor of the mitochondrial electron transport chain. H) Bar blots show the mitochondrial respiratory functions in BV2 from different treatment groups, including basal respiration, maximal respiration, and ATP production (data presented as mean ± SD, n  = 6). ns ≥ 0.5, *p < 0.05, **P < 0.01, One‐way ANOVA. I) Representative flow cytometry scatter plots show the proportion of viable (Annexin V⁻, PI⁻), early phase apoptotic (Annexin V⁺, PI⁻), and late phase apoptotic (Annexin V⁺, PI⁺) BV2 cells from different treatment groups and quantitative analysis (data presented as mean ± SD, n = 3; 10000 events per run). ns ≥ 0.5, *p < 0.05, ****p < 0.0001, Two‐way ANOVA.

Figure 3

R‐Hmito transplantation ameliorates microglia inflammation. A) Representative immunofluorescence images of CD206 (green) and iNOS (red) in BV2 cells under different treatments. Blue: DAPI, Nuclei. Scale bar = 20 µm. Mean fluorescence intensities (MFI) of CD206 and iNOS were quantified for each cell separately (data presented as mean ± SD, n = 15). ns ≥ 0.5, *p < 0.05, **p < 0.01, One‐way ANOVA. B) Representative histograms show the expression levels of CD206 and iNOS in BV2 cells by flow cytometry in different treatment groups (n = 3; 10000 events per run). C) Bar blots show the relative quantitative analysis of CD86 and ARG1 expression levels of BV2 in different treatment groups (data presented as mean ± SD, n = 15). ns ≥ 0.5, **p < 0.01, One‐way ANOVA. D) Bar blots show the relative quantitative analysis of TNF‐α and IL‐6 secreted by BV2 cells in each group (data presented as mean ± SD, n = 3). ns ≥ 0.5, *p < 0.05, **p < 0.01, One‐way ANOVA. E) Schematic of the effect of R‐Hmito transplantation on the pro‐inflammatory and immunosuppressive properties of BV2. F) Schematic representation of BV2 supernatant from each group applied to PC12 cells. G) Representative fluorescence images of live/dead cell staining of PC12 cells incubated with supernatants of BV2 and its quantitative analysis (data presented as mean ± SD, n = 3). Scale bar = 100 µm. ns ≥ 0.5, *p < 0.05, **p < 0.01, One‐way ANOVA.

Figure 4

ACs enable brain‐targeted mitochondrial transplantation. A) A schematic showing an in vitro hemodynamic model used to test the ability of ACs to be retained under a magnetic field. Left: flowing magnetic ACs were clearly retained in a region with a magnetic field. Right: flowing non‐magnetic ACs passed rapidly through the magnetic field region with almost no retention. B) Representative in vivo fluorescence imaging images of mouse brain regions from each group at different time points after intravenous injection. C) Representative ex vivo fluorescence imaging maps and quantitative analyses of major organs from each group at 24 hours after intravenous injection. From top to bottom: brain, heart, liver, spleen, lung, kidney (data presented as mean ± SD, n = 3). *p < 0.05, **p < 0.01, Two‐way ANOVA. D) Representative ex vivo fluorescence imaging images and quantitative analyses of brain sections from each group 24 hours after intravenous injection. From top to bottom: brain sections from anterior to posterior (data presented as mean ± SD, n = 3). ns ≥ 0.5, *p < 0.05, One‐way ANOVA. E) Schematic diagram of ICH mouse brain slices used to aid in the interpretation of Figure 4F and G. Left: unaffected side of brain; right: ICH affected side of brain. F) Fluorescence imaging of brain sections showing the distribution of DiO labeled ACs. Blue: DAPI; green: ACs scaffolds. Scale bar = 1000 µm. Localized enlargements are observed on the right. Scale bar = 20 µm. G) Distribution of naked Hmito and R‐Hmito in the brain and their internalization by microglia 24 hours after intravenous injection. Blue: DAPI; red: naked Hmito or R‐Hmito; green: activated microglia marker IBA1; white arrows: co‐localization of naked Hmito or R‐Hmito with microglia. Scale bar = 20 µm. Localized enlargements are observed on both sides. Scale bar = 10 µm.

Figure 5

ACs promote recovery from ICH. A) Schematic flow of grouping, treatment, and assessment. B)Line graph of the change in body weight of each group of mice over the seven‐day postoperative period (data presented as mean ± SD, n = 8). *p < 0.05, **p < 0.01, Two‐way ANOVA. C) Composite Garcia scoring of each group of mice on day 3 and 7 after surgery. A lower score is indicative of a more severe injury (data presented as mean ± SD, n = 5). ns ≥ 0.05, **p < 0.01, Two‐way ANOVA. D) Grip strength test of mice in each group on day 3 and 7 after surgery. A lower strength value is indicative of a more severe injury (data presented as mean ± SD, n = 6). ns ≥ 0.05, *p < 0.05, **p < 0.01, Two‐way ANOVA. E) Normal step sequence ratio and affected side step base of mice in each group on day 3 and 7 after surgery, a decrease in the proportion of normal step sequences and an increase in step base represent impaired mobility of the affected limb (data presented as mean ± SD, n = 6). ns ≥ 0.05, **p < 0.01, ***p < 0.001, Two‐way ANOVA. F) Representative gait diagrams of each group of mice on the seventh day after surgery. G, H) Representative MRI images on day 3 after ICH. Sequence: A T2‐weighted image (T2WI), a high b‐value image of DWI and an ADC‐valued pseudo‐color image of DWI. The regions delineated by the dotted line or the arrow are indicative of cerebral edema. The extent of cerebral edema was quantified from the ADC‐valued pseudo‐color images of DWI (data presented as mean ± SD, n = 5). ns ≥ 0.05, *p < 0.05, One‐way ANOVA. I) Brain water content of mice on day 3 after surgery (data presented as mean ± SD, n = 3). *p < 0.05, **p < 0.01, One‐way ANOVA.

Figure 6

ACs promote microglia immune homeostasis and attenuate intracerebral inflammation. A) PCA of Saline group and ACs group. B) GO enrichment bar plot showing the top 25 significantly enriched pathways in GO‐Biological Process. The darker backgrounds represent the enriched pathways associated with immune and cell survival. C) GSEA showing DEGs associated with inflammatory response and leukocyte infiltration. D) GSEA showing DEGs associated with ATP production and OXPHOS. E) Venn diagram showing shared DEGs between ACs versus Saline and R‐Hmito versus PBS. F) Dendrogram showing enrichment pathways of shared DEGs in GO‐Biological Process. G) Representative flow cytometry contour plots revealing differences in the expression of CD206 of microglia, of the ICH affected hemibrains in each group of mice (n = 3; 100000 events per run). CD11b⁺/CD45int: microglia, CD11b⁺/CD45int/CD206⁺: immunosuppressive microglia. H) Representative flow cytometry dot plots showing the percentage of neutrophils (CD45high /Ly6G⁺) of the affected hemibrains in each group of mice. NEUT: neutrophils. I) Representative MPO immunofluorescence staining images of the region surrounding the ICH foci on day 7 after ICH in each group of mice. Blue: DAPI; red: MPO⁺ infiltrating neutrophils. Scale bar = 50 µm. J,K) Representative H&E and Nissl staining images of the region surrounding the ICH foci (Area right of dotted line) on day 7 after surgery in each group of mice. Scale bar = 200 µm. L) Representative TUNEL staining of the area around the ICH foci on day 7 after ICH in each group of mice. Blue: DAPI; green: TUNEL⁺ apoptotic cells. Scale bar = 50 µm.