Hybrid macrophage-mitochondria extracellular vesicles for mitochondrial ROS regulation in diabetic wounds

Domain: chemistry
Published: 2026-02-28
Editorial outcome: major_revision

Abstract
Precise regulation of mitochondrial reactive oxygen species (mtROS) in macrophages is essential for reducing inflammation and promoting diabetic wound healing. However, achieving targeted and responsive mtROS modulation in specific cell remains challenging. Herein, we report a macrophage-mitochondria hybrid membrane derived artificial extracellular vesicle platform (C@AH-EV). C@AH-EV integrates biomimetic dual-targeting membranes with an mtROS activatable prodrug. It leverages membrane protein-mediated targeting of macrophage mitochondria to offer highly selective intracellular delivery. The payload is a ROS sensitive antioxidant prodrug, which incorporates lipid tails that enhance its drug loading efficiency in vesicles. Upon mtROS-triggered cleavage of the boronate ester bond, potent antioxidants were locally released within the mitochondria to restore redox balance. In vitro and in vivo studies demonstrate that C@AH-EVs effectively reprogram macrophages from an inflammatory toward an anti-inflammatory phenotype, thereby attenuating inflammation and accelerating diabetic wound repair. This work offers a promising direction for treating chronic inflammatory conditions.

Introduction
Impaired wound healing driven by diabetes is becoming a global concern, affecting 18.6 million people worldwide and posing a significant challenge to global healthcare system1. Current treatments for diabetic wounds include aggressive debridement, antibiotic medication, and revascularization2-4. However, these strategies often fall short of complete effectiveness. Statistics indicated that more than half of diabetic wounds still persist after three months of treatment5, leading to potential amputation. Additionally, individuals with diabetic foot ulcers face a one-year mortality risk of 23%, comparable to that of cancer6. Therefore, there is an urgent need to develop more effective therapies for the treatment of diabetic wounds.

Macrophages, as the predominant inflammatory cells in a high-glycemic microenvironment, are recognized as key drivers of inflammation that contribute to non-healing diabetic wounds7,8. Under normal wound conditions, macrophages transition from the inflammatory M1-like phenotype to the anti-inflammatory M2-like phenotype. However, in a high-glucose microenvironment, excessive production of mitochondrial reactive oxygen species (mtROS) results in mitochondria dysfunction by damaging various molecular components, including DNA, lipids, proteins, and carbohydrates. Consequently, the transformation of macrophages from an M1-like to an M2-like phenotype is severely disrupted, hindering proper wound healing9-11. While scavenging mtROS can alleviate mitochondrial dysfunction, moderate levels of mtROS are essential for activating antioxidant pathways and promoting M2-like macrophage polarization, therefore, precisely modulating mtROS levels in macrophages is crucial for promoting diabetic wound healing12,13.

To accomplish this goal, our primary task was to develop a smart macrophage mtROS modulation system, which remains a significant pharmacological challenge. In our previous study, we achieved preliminary success in macrophage mtROS modulation by combining mitochondrial-targeting elements with natural extracellular vesicle (EV)14. However, EV-based therapeutics still face challenges of low yield and heterogeneity15. In recent years, nanovesicles constructed by cell membrane and subcellular organelle membranes have attracted growing attention. These nanovesicles exhibit a striking resemblance to EVs in size, morphology, and protein composition, and are thus widely regarded as artificial EVs16. They offer the distinct advantages of being more amenable to large-scale production and efficiently encapsulating diverse types of therapeutic agents, making them excellent substitutes for natural EVs17. Importantly, artificial EVs exhibit targeting capabilities since they retain structural and biological information about the donor cells or subcellular organelles18,19. Thus, cell and mitochondrial hybrid membrane based artificial EVs hold great potential for dual-targeting to both the cells and their mitochondria. But such hybrid membrane-derived vesicles remain unreported.

In terms of effective yet controllable scavenging of mtROS, covalent conjugation of antioxidants with ROS-responsive linkers offers a well-established strategy. Caffeic acid (CA) and its phenethyl ester (CAPE), natural phenylpropanoids with potent antioxidant activity20,21, contain ortho-dihydroxyphenyl (catechol) groups that readily form dynamic boronic acid esters with phenylboronic acid derivatives22. However, direct conjugation often results in poor aqueous stability, limiting their utility for precise ROS modulation23,24. Anslyn et al. reported a robust, irreversible three-component reaction in aqueous media involving 2-formylphenylboronic acid, catechols, and N-alkyl hydroxylamines25, an approach now recognized as a form of click chemistry due to its efficiency and simplicity22,26. Applying this strategy to conjugate CA/CAPE with boronic acids enables the construction of water-stable, ROS-responsive prodrugs.

As a result, we synthesised the prodrugs via three-component click reactions of CA or its phenyl ethyl ester CAPE with 2-formylphenyl boronic acid (2-FPBA) and N-dodecyl hydroxylamine (DO-HA), denoted as LCAPE-FPBA-DO and CA-PFBA-DO, respectively. Among them, CAPE-FPBA-DO exhibited higher stability in aqueous solution and lower cytotoxicity. The dodecyl moiety of CAFE-FPBA-DO is designed to enhance drug hydrophobicity, enabling high-efficiency payload encapsulation via membrane adsorption27,28. By loading CAPE-FPBA-DO into artificial EVs derived from macrophage-mitochondria hybrid membrane, we engineered a macrophage mtROS on-demand scavenging nanovesicle, referred to as C@AH-EV (artificial hybrid membrane extracellular vesicle loaded with CAPE-FPBA-DO) (Fig. 1a). When applied to in vitro inflammatory cell models and in vivo diabetic wound mouse models, C@AH-EVs were taken up by macrophages through calthrin- and lipid raft-mediated endocytosis. Subsequently, these vesicles targeted the mitochondria via mitofusin (MFN) and fused with the mitochondrial membrane to release antioxidant prodrugs. In the presence of high levels of mtROS, CAPE-FPBA-DO degraded to release the potent antioxidants CAPE, which improved the mitochondria function by eliminating excessive mtROS, thereby reprogramming macrophage phenotypes and accelerating diabetic wound healing (Fig. 1b-c). This study presents a mitochondria-targeted, cell-specific nanotherapeutic that mimics endogenous communication mechanisms, providing a robust and precise mtROS scavenging strategy for wound healing and broader therapeutic application.

Results
Synthesis and analysis of ROS responsive/scavenging prodrugs.
To construct ROS responsive/scavenging prodrugs, we covalently conjugated caffeic acid (CA) or its phenyl ethyl ester (CAPE) with 2-formylphenyl boronic acid (2-FPBA) and N-dodecyl hydroxylamine (DO-HA) via three-component click reactions, denoted as CAPE-FPBA-DO and CA-FPBA-DO, respectively (Supplementary Fig.1, 2). The conjugates are anticipated to enable on-demand release of CAPE/CA in response to different ROS levels (Fig. 2a)23. Their successful syntheses were confirmed by nuclear magnetic resonance (NMR) spectroscopy (Supplementary Fig.3-10) and UV-Vis analysis (Fig. 2b, c). To assess their drug-likeness, a structure-property relationship analysis was performed using Oprea's rules29. The results indicated that the ratio of CAPE-FPBA-DO and CA-FPBA-DO are as follow: the number of rings 5/4 (for CAPE-FPBA-DO and CA-FPBA-DO, respectively), the number of rigid bonds 30/25, the number of rotatable bonds 17/13, all of which are acceptable according to Oprea’s rules (Supplementary Table 1). Then, we evaluated the cytotoxicity of CAPE-FPBA-DO and CA-FPBA-DO using the CCK-8 assay. The results showed that CAPE-FPBA-DO exhibit the significantly lower cytotoxicity compared to CA-FPBA-DO, with IC values of 106.6 μM for CAPE-FPBA-DO and 28.95 μM for CA-FPBA-DO 50 (Fig. 2d). This may be attributed to its ester moiety for their modulation of pharmacokinetic parameters30,31. We next conducted stability analyses on CAPE-FPBA-DO and CA-FPBA-DO. UV-Vis analysis indicated that CA-FPBA-DO undergoes significant hydrolysis at concentrations near the non-cytotoxic level (25 μM), whereas CAPE-FPBA-DO exhibits relatively good hydrolytic stability at this concentration (Fig. 2e, Supplementary Fig. 11).

Studies have shown that mitochondrial dysfunction can lead to a significant increase in mtROS concentration, with localized levels (e.g., in the mitochondrial matrix) reaching up to 1 mM32. Thus, we investigated the ROS responsive behavior of CAPE-FPBA-DO and CA-FPBA-DO under varying H2O2 levels. High-performance liquid chromatography (HPLC) results showed released CAPE/CA and by-products at different retention times and UV absorption wavelengths, indicating that H₂O₂ triggered the decomposition of CAPE-FPBA-DO and CA-FPBA-DO (Supplementary Fig. 12, 13). The extent of drug release was concentration dependent, with values for CAPE and CA rising from 51.32% and 37.14% at 0.1 mM H2O2 to 77.77% and 74.47% at 1 mM H2O2 after 480 min of incubation (Fig. 2f, g, Supplementary Fig. 14, 15). These results confirm the potential on-demand release capabilities of CAPE-FPBA-DO and CA-FPBA-DO in dysfunctional mitochondrial environments. Notably, CAPE-FPBA-DO exhibited a slightly higher release capacity than CA-FPBA-DO during the 480 min detection period. This divergence may be attributed to the greater propensity of CA-FPBA-DO to form micellar aggregates in aqueous environments, which sequester the reactive boronic ester groups within a hydrophobic core, thereby shielding them from H₂O₂ solution and attenuating the reaction-driven release33 (Supplementary Fig. 16). Subsequently, ABTS•+ (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation) was incubated with the CAPE, CA, 2-FPBA, CAPE-FPBA-DO, or CA-FPBA-DO alone to assess the ROS scavenging ability (Fig. 2h). The results showed that CAPE and CA possessed comparable antioxidant capacity. Notably, CAPE/CA-FPBA-DO also decolorized ABTS•+ to some extent, which may be attributed to the production of ROS in the generation of ABTS•+ (Fig. 2i) 34,35.

In summary, both CAPE-FPBA-DO and CA-FPBA-DO demonstrating rapid responsiveness to ROS and effective ROS scavenging capabilities. Compared to CA-FPBA-DO, CAPE-FPBA-DO showed higher hydrolytic stability and lower cytotoxicity, making it a more promising antioxidant prodrug.

Construction and characterization of on-demand mitochondrial ROS scavenging nanovesicles
Next, we prepared artificial hybrid membrane extracellular vesicles (AH-EVs) to load the prodrug CAPE-FPBA-DO (Fig. 3a). Briefly, macrophage membrane (MM) and mitochondrial membrane (MitoM) from RAW 264.7 cells and its mitochondria were collected via membrane lysis and subsequently purified. AH-EVs were formed using the ultrasonic approach followed by extrusion to control vesicles size. CAPE-FPBA-DO was loaded into AH-EVs through a co-incubation and extrusion process to prepare C@AH-EVs (AH-EVs loaded with CAPE-FPBA-DO). The successful fusion of MM and MitoM was confirmed by confocal laser scanning microscopy (CLSM) (Fig. 3b). Scanning electron microscope (SEM) images revealed the spherical morphology of the AH-EVs and C@AH-EVs (Fig. 3c), while transmission electron microscopy (TEM) images displayed a single membrane of phospholipid bilayer for both AH-EVs and C@AH-EVs (Fig. 3d).

To further investigate the membrane protein retention of C@AH-EVs, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. As shown in Supplementary Fig. 17, the protein profiles of C@AH-EVs were similar to those of AH-EVs, indicating good retention of MM and MitoM proteins. Furthermore, western blot analysis confirmed the presence of the internal reference of cell membranes Na+/K+ ATPase, macrophage membrane protein F4/80, and mitochondria membrane protein Bcl-2 in C@AH-EVs, suggesting that the membrane of C@AH-EVs was comparable to those on macrophage and mitochondria (Fig. 3e). Then the nanovesicles were further characterized by means of nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS). The NTA showed that C@AH-EVs had an average size of 196 nm, which is similar to AH-EVs (197 nm) (Fig. 3f). In addition, there was no significant difference in the negative zeta potential between AH-EVs and C@AH-EVs (Fig. 3g). The drug loading capacity is crucial for determining the final therapeutic effect. Therefore, we utilized the highest safe concentration of 1 μM CALPE-FPBA-DO for C@AH-EVs preparation, achieving a 69% encapsulation efficiency for CAPE-FPBA-DO and a loading capacity of 403 ng/1010 particles (Fig. 3h, Supplementary Fig. 18). Subsequent CCK-8 assays and histological analysis further confirmed the biosafety of the prepared C@AH-EVs (Supplementary Fig. 19-21). Furthermore, C@AH-EVs maintained size stability for at least 72 h in both PBS and RPMI medium (Fig. 3i).

C@AH-EVs efficiently targeted the mitochondria of macrophage in vitro and in vivo.
Targeted delivery to mitochondria of macrophage is crucial for mtROS responding and scavenging. To this end, we first evaluated the cellular uptake of both DiI-labeled C@AH-EVs and C@AM-EVs (prepared by MM) by RAW 264.7 cells. The results indicated that the DiI signals in macrophages treated with C@AM-EVs and C@AH-EVs were similar. Additionally, we stimulated macrophages with lipopolysaccharide (LPS) to induce M1-like macrophages. In LPS-stimulated macrophages, we observed comparable uptake efficiency for both C@AM-EVs and C@AH-EVs. These results indicate that the addition of mitochondrial membranes did not affect the uptake efficiency of macrophages (Supplementary Fig. 22).

To further investigate mitochondrial homologous targeting of C@AH-EVs, MitoTracker (magenta) and DiO (green) were used (Fig. 4a). RAW 264.7 cells with C@AH-EVs produced enhanced white fluorescence resulting from the overlap of magenta and green, whereas RAW 264.7 cells treated with C@AM-EVs did not produce marked white fluorescence. Co-localization line scanning profiles also supported this conclusion, suggesting that the mitochondria membrane is necessary for mitochondria targeting. Furthermore, C@AH-EVs demonstrated favorable targeting in the mitochondria of macrophages stimulated with LPS (Fig. 4b, c). These results suggest that the mitochondrial targeting of C@AH-EVs is membrane potential-independent, compensating for the limitations of membrane potential-dependent targeting elements like lipophilic cations36.

In the subsequent experiment, we observed the mitochondrial targeting properties of C@AH-EVs in macrophages within diabetic wounds using CLSM (Supplementary Fig. 23). As shown in Figure 4d and Figure 4e, C@AH-EVs effectively targeted mitochondria of macrophages in both common and diabetic wounds. Notably, a small quantity of C@AH-EVs could potentially be internalized by other phagocytes and parenchymal cells. This is beneficial for wound treatment, as overproduction of mtROS in other cell types also contributes delayed wound healing37,38. Together, these data suggest that C@AH-EVs exhibits highly efficient and precise mitochondrial targeting in macrophages, holding significant superiority compared to membrane potential-dependent mitochondrial targeting materials.

Mechanism of cellular uptake and mitochondria targeting
Next, we elucidated the molecular mechanism underlying membrane-mediated homotypic recognition and delivery. Trypsin, a serine protease that can digest membrane proteins, was used to treat the MM for 6 h at 37℃, generating AH-EVs deficient in MM proteins, denote as C@AH-EVsMM trypsin (Supplementary Fig. 24). CLSM images showed that the uptake of C@AH-EVsMM trypsin by RAW 264.7 cells were decreased compared to the C@AH-EVs group (Fig. 5a, b). Similarly, C@AH-EVsMitoM trypsin, deficient in MitoM protein, exhibited reduced co-localization with mitochondria (Supplementary Fig. 25, Fig. 5c-e), confirming that certain membrane proteins play critical roles in homotypic targeting.

We next used various blockers of specific cellular endocytosis pathways to investigate the mechanisms underlying C@AH-EVs internalization. The endocytosis pathways can be categorized into four types: macro-pinocytosis, lipid raft-mediated endocytosis (LrME), clathrin-mediated endocytosis (CME), and caveolae-mediated endocytosis (CvME)39. Thus amiloride (5-(N, N-dimethyl)-amiloride hydrochloride, inhibitor for macro-pinocytosis), MβCD (methyl-β-cyclodextrin, inhibitor of LrME), CPZ (chlorpromazine hydrochloride, inhibitor of CME), and Gen (genistein, inhibitor of CvME) were utilized to pretreat the RAW 264.7 cells (Fig. 5f). The CLSM images revealed the significant reduction in macrophage uptake of MβCD-treated group and CPZ-treated group, with uptake levels decreasing to 52.8% and 66.2% of the control group, respectively (Fig. 5g, h). Lipid rafts require membrane proteins (e.g., integrins) as functional anchors, critical for membrane localization40. CME is associated with signals from transmembrane receptors, a specialized class of membrane-embedded proteins41. Therefore, these results further demonstrate the potential of membrane proteins in the homotypic cell targeting. Additionally, the uptake of C@AH-EVs in RAW 264.7 cells incubated at 4°C dropped to 4.5% of control levels, which is likely associated with suppressed cellular energy metabolism and the reduction in membrane fluidity induced by low temperature42.

As highly dynamic organelles, mitochondria possess remarkable membrane fusion capabilities. Consequently, nanoparticles prepared from mitochondrial membranes are highly likely to exhibit similar characteristics, which subsequently detected by a mitochondria-nanovesicle fusion assay (Fig. 5i). In Figure 5j and 5k, we observed that the membrane of C@AH-EVs were fused with the outer mitochondrial membranes (OMM). In contrast, C@AM-EVs prepared with solely RAW 264.7 cell membranes did not fuse with mitochondria. Studies have shown that the fusion of the OMM is mediated by the dynamin-like protein mitofusin (MFN)43. The small molecule MFI8, which inhibits this process by binding MFN's HR2 domain, was utilized to investigate subcellular targeting mechanisms. The results showed that mitochondria pretreated with MFI8 were unable to fuse with C@AH-EVs, indicating that MFN is involved in mitochondrial targeting (Fig. 5j, k). This is consistent with previous study44. Altogether, these results suggest that membrane proteins are an important basis for homotypic targeting.

C@AH-EVs improve mitochondrial function by eliminating mtROS.
To further evaluate the therapeutic efficacy of C@AH-EVSs, we first quantified CAPE release within macrophage mitochondria using HPLC, comparing LPS-stimulated macrophages with PBS controls. The results exhibited obvious increased mitochondrial CAPE accumulation in LPS-stimulated macrophages relative to PBS controls (Pp < 0.05), indicating that elevated mtROS concentrations effectively triggered boronate ester bond cleavage and promoted CAPE release (Supplementary Fig. 26). Then LPS-stimulated macrophages were divided into four treatment groups: PBS, AH-EVs, CAPE-FPBA-DO, and C@AH-EVs, with untreated macrophages serving as the control. mtROS levels were quantified using MitoSOX™ Red. As depicted in the Figure 6a and 6b, the mtROS level in the C@AH-EVs group was remarkably diminished compared to the PBS group, while the mtROS level in the AH-EVs group remained relatively unchanged. This phenomenon was primarily attributed to the antioxidant CAPE released by CAPE-FPBA-DO. In addition, although a slight visual decrease in mtROS was observed in cells treated with CAPE-FPBA-DO, this change was not statistically significant. This results further confirming that C@AH-EVs specifically target mtROS scavenging.

Functionally, excessive mtROS triggers mitochondrial lipid peroxidation, compromises membrane stability, leads to abnormal mitochondrial morphology, and disrupt ATP synthesis. Therefore, MitoPerOX was used to detect mitochondrial lipid peroxidation. The results revealed that C@AH-EVs significantly suppressed LPS-induced mitochondrial lipid peroxidation (Fig. 6c). To further evaluate the impact of mtROS on mitochondrial membrane potential (Δψm), we performed JC-1 staining. JC-1 accumulates in mitochondria in a potential-dependent manner, forming J aggregates (red) when inside. Conversely, upon mitochondrial depolarization, JC-1 persists as monomers (green) (Fig. 6d). The results demonstrated that LPS treatment induced Δψm depolarization, as indicated by enhanced green fluorescence from JC-1 monomers. When treated with C@AH-EVs, cells exhibited increased red fluorescence from J-aggregates, indicating that C@AH-EVs effectively restored the Δψm (Fig. 6e, Supplementary Fig. 27). It is noteworthy that CAPE-FPBA-DO, despite exhibiting antioxidant activity, demonstrated lesser regulatory effects on mitochondrial lipid peroxidation and Δψm than C@AH-EVs.

The effect of C@AH-EVs on mitochondrial morphology was observed by electron microscopy. In normal cells, the mitochondrial cristae were distinctly visible and elongated. Upon LPS treatment, the mitochondria underwent significant swelling and vesicular cristae, while C@AH-EVs administration significantly attenuated the damaged morphology (Fig. 6f, g). For ATP production, C@AH-EVs treatment resulted in a 2.72-fold, 2.94-fold, and 1.77-fold improvement compared with PBS, AH-EVs, and CAPE-FPBA-DO treatments, respectively (Fig. 6h). Taken together, these results suggests that C@AH-EVs have the capacity to improve mitochondrial function by effectively scavenging mtROS (Fig. 6i).

In vitro orchestration of macrophage phenotype by C@AH-EVs
Considering the regulatory effect of mitochondria on macrophage activation, we speculated that C@AH-EVs would modulate mitochondria-dependent macrophage activation. Subsequently, the phenotype of LPS-stimulated macrophages treated with PBS, AH-EVs, CAPE-FPBA-DO, and C@AH-EVs was analyzed by flow cytometry assay (Fig. 7a). As expected, C@AH-EVs demonstrated an inhibitory effect on CD86, a marker for M1-like macrophages (Fig. 7b). Additionally, qRT-PCR results showed that C@AH-EVs inhibited the expression of proinflammatory genes including Tnfα, Nos2, Il6, and Cxcl1 (Fig. 7c). Given the necessity of M2-like macrophages for anti-inflammatory responses and diabetic wound healing, we assessed the effect of C@AH-EVs on M2-like macrophage activation by co-administering them with Interleukin-4 (IL-4), a key inducer of M2-like macrophage polarization (Fig. 7d). The results showed that IL-4 and C@AH-EVs synergistically upregulated expression of CD206, a hallmark of M2-like macrophages, in LPS-stimulated macrophages (Fig. 7e), suggesting a conversion from an M1-like to an M2-like phenotype. Furthermore, these cells exhibited obvious upregulation of the anti-inflammatory genes including Arg1, Mrc1, Ym1, and Fizz1 (Fig. 7f).

In contrast, AH-EVs did not effectively regulate the genes and surface markers related to M1-like or M2-like phenotypes mentioned above in RAW 264.7 cells. Notably, while CAPE-FPBA-DO suppressed the expression of CD86 and inflammatory gene, and induced the expression of CD206 and anti-inflammatory genes, their effectiveness were reduced compared with C@AH-EVs. Altogether, these results indicate that C@AH-EVs can effectively regulate the phenotype of macrophages.

Besides, we also prepared hybrid membrane vesicles loaded with an equivalent mass of CAPE (CAPE@AH-EVs) as a control to evaluate the potential advantages of C@AH-EVs. The results demonstrated that whereas both AH-EVs effectively attenuated mtROS levels and reduced the proportion of M1-like macrophages, only C@AH-EVs significantly promoted M2-like macrophage polarization (Supplementary Fig. 28, 29). This discrepancy is likely attributable to the excessive scavenging of mtROS by CAPE@AH-EVs, which may obliterate the physiological levels of mtROS essential for driving M2-like macrophage polarization.

C@AH-EVs promote the M2-like macrophage polarization in vivo
The wound healing process consists of three stages: the inflammatory stage, the proliferative stage, and the remodeling stage. The inflammatory stage, typically lasting 2-3 days post-injury but often chronically prolonged in diabetic wounds, is characterized by sustained elevation of proinflammatory factors mediated through excessive M1-like macrophage polarization. Therefore, we initially assessed the capability of C@AH-EVs to scavenge mtROS in macrophages and orchestrate macrophage phenotypes within a diabetic mouse full-thickness wound model.

First, the diabetic wound model was created by injecting streptozotocin at a dose of 50 mg kg-1 day-1 for 5 days, followed by surgical removal of 10-mm-diameter full-thickness skin after consistent blood glucose levels over 16.65 mmol L-1 for one week. The persistent hyperglycemia and impaired weight gain collectively demonstrate that the mice maintained a sustained diabetic state throughout the experimental period (Supplementary Fig. 30). Subsequently, PBS, AH-EVs, CAPE-FPBA-DO, and C@AH-EVs were subcutaneously administrated at the wound sites, respectively. Tracking of DiI-labeled C@AH-EVs exhibited an efficient distribution throughout the full thickness of the wound tissue (Supplementary Fig. 31). Considering the prolonged inflammation associated with diabetic wounds, in vivo macrophage mtROS levels and macrophage phenotypes were determined on the day 3, 7, and 14 (Supplementary Fig. 32). Macrophages were gated using the markers F4/80 and CD11b (Supplementary Fig. 33). With in the CD11b⁺ F4/80⁺ macrophage population, the expression level of mtROS, CD86, and CD206 were evaluated.

The results showed that in diabetic mouse model with full-thickness skin defect, mtROS levels and the proportion of proinflammatory M1-like macrophages in wound tissues exhibited a persistent high-state throughout the observation period. Upon intervention with C@AH-EVs in diabetic wounds, the mtROS levels decreased to 32.4%, 22.6%, and 26.4% on day 3, 7, and 14, respectively (Fig. 8a, c, e). Concurrently, the proportion of M1-like macrophages decreased to 43.1% at day 3, 38.1% at day 7, and 19.1% at day 14, while the proportion of M2-like macrophages, which are associated with tissue repair, increased to 39.7%, 56.9%, and 72.2% at the respective timepoints (Fig. 8b, d, f, Supplementary Fig. 34). ELISA analysis of cytokines further validated the macrophage phenotypic shift, showing a reduction in proinflammatory cytokines and an elevation in anti-inflammatory cytokines across all time points. Specifically, levels of TNF-α and IL-1β in the C@AH-EV-treated group were lower than those in the control group at each time point (Supplementary Fig. 35). Conversely, the anti-inflammatory cytokines TGF-β and IL-10 were significantly increased (Supplementary Fig. 36). In contrast, the AH-EV group demonstrated unresolved inflammation, characterized by a failure to downregulate CD86+ macrophages and persistently high levels of proinflammatory cytokines. While CAPE-FPBA-DO exhibited some capacity of macrophage phenotypic regulation, its overall therapeutic efficacy in wound repair is significantly inferior to that of C@AH-EVs. These results suggest that C@AH-EVs-mediated suppression of mtROS levels promotes a microenvironment conducive to M2-like macrophage activation.

C@AH-EVs enhance diabetic wound healing in full-thickness skin defect model
We next investigated the therapeutic effects of C@AH-EVs in diabetic wounds (Fig. 9a). Representative macroscopic wound images (Fig. 9b), along with the wound closure traces at different time intervals (Fig. 9c), and the corresponding quantitative data (Fig. 9d, Supplementary Fig. 37) collectively demonstrate the enhanced therapeutic efficacy of C@AH-EVs. On day 5, the wounds in the C@AH-EVs group were noticeably smaller compared to those in the other groups. By day 10, a remarkable reduction in the wound area was observed in the C@AH-EVs group, achieving a wound healing rate of 78.2%. In contrast, the wounds healing rate in the control group, AH-EVs group, and CAPE-FPBA-DO group remained at 58.6%, 58.8%, and 68.3%, respectively. On day 14, the C@AH-EVs-treated diabetic wounds showed substantial healing (86.3% wound healing rates), while the control group, AH-EVs group, and CAPE-FPBA-DO group exhibited persistently lower healing rates (72.9%, 75.5%, 77.7%, respectively).

H&E staining was performed to investigate the histological changes in wounds. On day 3, the inflammatory cells in the wounds of the C@AH-EVs group were significantly lower than those in the other three groups, with no epidermal formation observed in any group (Fig. 9e, Supplementary Fig. 38). By day 7, the C@AH-EVs group exhibited epidermal regeneration, while the other groups did not show this progression. On day 14, all groups completed re-epithelialization and displayed mature stratified epithelial cells. However, the regeneration of skin appendages, such as hair follicles and sebaceous glands was the most pronounced in the C@AH-EVs group. Organized formation of granulation tissue further substantiated its therapeutic superiority in wound healing (Fig. 9e, Supplementary Fig. 39).

Neovascularization is a key characteristic during the proliferative stage of wound healing. During tissue repair, M2-like macrophages regulate the expression and function of platelet endothelial cell adhesion molecule-1 (CD31) in endothelial cells by secreting vascular endothelial growth factor (VEGF), thereby stimulating the formation of new blood vessels and accelerating wound healing. Therefore, during the proliferative phase, immunostaining of CD31 and VEGF was performed to evaluate neovascularization in the granulation tissue. As shown in Supplementary Figure 40, the wound sections from the C@AH-EVs group exhibiting the highest level of CD31 and VEGF, with the relative coverage areas of VEGF and CD31 being 5.59 times and 3.80 times higher compared to the PBS group, respectively. This suggests that C@AH-EVs hold great potential in facilitating wound healing by augmenting angiogenesis.

Proper collagen deposition and remodeling during the remodeling phase are vital for restoring wound biomechanics. Thus, we employed Masson's trichrome staining to evaluate collagen architecture in this critical stage. As shown in Supplementary Fig. 41, the C@AH-EVs group displayed denser collagen fibers with a collagen volume fraction of 51.86%. In contrast, the PBS group, AH-EVs group, and CAPE-FPBA-DO group had lower collagen volume fractions of 39.56%, 40.95%, and 40.67%, respectively. These fibers demonstrated a more mature phenotype characterized by well-organized alignment and an intricate collagen network topology, especially in comparison to the PBS and AH-EVs group. These results verified the improved collagen deposition ability of C@AH-EVs.

Besides, macrophage membrane-derived artificial EVs (AMM-EVs), mitochondria membrane-derived artificial EVs (AMitoM-EVs), CAPE, CAPE@AH-EVs, and clinically used agent Beifuji were also served as controls to evaluate the therapeutic efficacy of C@AH-EVs. The results demonstrated that diabetic wounds treated with C@AH-EVs exhibited accelerated healing than other groups (Supplementary Fig. 42). Consistent with this, the C@AH-EVs group formed more mature granulation tissue, accompanied by accelerated re-epithelialization, skin appendage regeneration, and neovascularization relative to all controls (Supplementary Fig. 43). In summary, C@AH-EVs can effectively accelerate the healing process of diabetic wounds.

Methods
Ethical regulations
All experiments were authorized by the Animal Care and Ethic Committee of Fourth Military Medical University (Approval NO. IACUEC-20240917).

Materials
All organic solvents were used as received. CA, CAPE and 2-FBPA were purchased from Ambeed USA. Dodecyl aldehyde, hydroxyamine hydrochloride, pyridine borane complex and acetyl chloride were purchased from Sigma Aldrich Australia. 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) kits were obtained from Yuanye Bio-Technology (China). Mitochondria isolation kit, cell counting kit-8 (CCK-8), MitoTracker Deep RedFM, and ATP assay kit were purchased from Beyotime Biotechnology (China). RPMI (Roswell Park Memorial Institute) and fetal bovine serum were purchased from Excell Bio (China). Penicillin streptomycin was supplied by Solarbio (China). 3,3'-Dioctadecyloxacarbocyanine perchlorate (DiO), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), Hoechst 33342, 4′,6-diamidino-2-phenylindole (DAPI), MitoSOXTM, and JC-1 were purchased from Invitrogen (US). MitoPerOx, MFI8 was supplied by MedChemExpress (US). Amiloride (5-(N,N-dimethyl)-amiloride hydrochloride), MβCD (methyl-β-cyclodextrin, 5 mM), Gen (genistein), and CPZ (chlorpromazine) were purchased from Sigma-Aldrich (China). Mouse ELISA kits were obtained from Elabscience (China). Antibodies used in flow cytometry: FITC-conjugated Rat Anti-Mouse CD86 (105006), PE-conjugated Rat Anti-Mouse CD206 (141706), and APC-conjugated Rat Anti-Mouse F4/80 (123116) were purchased from Biolegend (US), PerCP-conjugated Rat Anti-Mouse CD11b (E-AB-F1081F) was purchased from Elabscience (China). Antibodies used in western blot: rabbit anti-F4/80 (29414-1-AP), rabbit anti-Bcl-2 (26593-1-AP), rabbit anti-Na+/K+ ATPase (14418-1-AP) were obtained from Proteintech (China). Antibodies used in immunofluorescence and immunohistochemistry: rabbit anti-Tom20 (11802-1-AP) was purchased from Proteintech (China), rabbit anti-F4/80 (GB113373), rabbit anti-CD31 (GB15063), and rabbit anti-VEGF (GB15165) were purchased from Servicebio (China).

Cell lines and animals
The murine cell line RAW 264.7 was obtained from the American Type Culture Collection (TIB-71, ATCC). Cells were cultured in RPMI medium supplemented with the 10% FBS and 1% antibiotics at 37℃, 5% CO2. Cells were changed with fresh medium every other day. BALB/c mice (male, 8-10 weeks) were obtained from the Animal Center of Fourth Military Medical University. All mice were housed at a constant temperature and humidity in a room with an artificial 12 h light/dark cycle and allowed free access to water and diet. The animal experimental procedures were performed strictly following the guidelines approved by the Institutional Animal Experiment Administration Committee of the Fourth Military Medical University.

Synthesis of CAPE-FPBA-DO and CA-FPBA-DO
(E, Z)-Dodecanal oxime S2: Hydroxylamine hydrochloride (2.8 g, 40 mmol) was dissolved in a NaOH solution in water (4 N, 12 mL) and the resulted solution was added to dodecylaldehyde S1 solution (17.5 mmol in 15 mL EtOH). The mixture was heated at 70 ºC overnight. After cooling to room temperature, water (30 mL) was added to the mixture resulting in precipitation. Solid was collected by filtration to get an off-white solid as a mixture of isomers (E/Z 1:0.8, 86% yield).
N-dodecylhydroxylamine S3: In one flask, to MeOH (5 mL) was added AcCl (12.5 mmol, 0.9 mL) at 0ºC under N2. After stirring for 10 min, the generated HCl solution in MeOH was added to a solution of oximes (5 mmol) and pyridine borane complex (Py·BH3, 2.2 equiv., 11 mmol) in MeOH dropwise at 0ºC via syringe. The reaction was stirred for 5 h at rt followed by quenching with Na2CO3 solutions. The mixture was then extracted with EtOAc (40 mL  3) and then the combined organic phase was washed with brine. After drying over MgSO4, the solution was filtered and concentrated before purification via flash chromatography. The title compound was obtained as a white solid (75% yield).
CAPE-FPBA-DO: To the suspension of CAPE (1.0 mmol) and 2-FPBA (1.0 mmol) in CHCl3 (5 mL) was added hydroxyamine S3 (1.05 mmol) powder, the reaction was stirred at rt for 15 min as it turned bright yellow. The mixture was then loaded to a column packed with silica (230–400 mesh) and eluted with acetone. The yellow fractions were collected and concentrated to obtain a bright yellow solid (83% yield).
CA-FPBA-DO: To the suspension of CA (1.0 mmol) and 2-FPBA (1.0 mmol) in CHCl3 (5 mL) was added hydroxyamine S3 (1.05 mmol) powder, the reaction was stirred at rt for 15 min as it turned bright yellow. The mixture was then loaded to a column packed with silica (230–400 mesh) and eluted with acetone. The yellow fractions were collected and concentrated to get yellow solids (78% yield).

IC50 assay of CAPE-FPBA-DO and CA-FPBA-DO to RAW 264.7 cells
The IC50 (half-maximal inhibitory concentration) assay was analyzed by CCK-8 as instructed. Briefly, 5×103 RAW 264.7 cells were seeded in 96-well plates and cultured for 12 h. Subsequently, the media were replaced with fresh media containing different concentrations of CAPE-FPBA-DO and CA-FPBA-DO, respectively. After incubation for another 24 h, replaced the medium and added 10 ul CCK-8 to each well. Absorbance at 450 nm was measured using a microplate reader (Tecan infinite M200 Pro, Switzerland) after 1 h.

Stability assay
The complexes (CAPE-FPBA-DO and CA-FPBA-DO) used for the testing were dissolved in the mixture of PBS /DMSO (8:2 v/v) with 0.5% (w/v) surfactant. UV-vis spectra were recorded for determining the stability of the complexes.

Synthesis of SA-DO
Dodecyl hydroxylamine (402 mg, 2 mmol) and salicylaldehyde (244 mg, 2 mmol) were mixed in dichloromethane (5 mL). The mixture was stirred overnight at rt before purification via flash chromatography over silica gel. The compound was isolated as a white solid.

ROS-responsive of CAPE-FPBA-DO and CA-FPBA-DO
To determine the ROS-responsive ability of CAPE-FPBA-DO and CA-FPBA-DO, the in vitro drug release tests were evaluated in the absence/presence of H2O2 conditions. Specifically, the CAPE-FPBA-DO and CA-FPBA-DO were incubated with PBS (pH = 7.4, 0.1 M) or H2O2 (0.1 mM H2O2, 1 mM H2O2) at 37 ℃ with orbital shaking at about 100 rpm. At specified time intervals (0, 40, 80, 120, 240, 480 min), the solution was withdrawn and replaced with the same volume of fresh media. The released CAPE and CA were determined by high performance liquid chromatography (ACQUITY UPLC H-Class water s, US).

ABTS free radical scavenging analysis
The antioxidant activity of CAPE, CA, 2-FPBA, CAPE-FPBA-DO, and CA-FPBA-DO were assessed by the 2,2’- azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical scavenging assays and quantified by microplate reader. Specifically, the working solution is prepared by mixing ABTS (7 mM) and potassium persulfate (2.45 mM) solutions in a 1:1 ratio, followed by incubation in the dark at room temperature (25°C) for 12 h. Then the solution was diluted 30-fold and combined with CAPE, CA, 2-FPBA, CAPE-FPBA-DO, and CA-FPBA-DO at varying concentrations (0, 0.025, 0.05, 0.10, 0.20, 0.40 mM). After incubation in the dark for 30 min, the values of OD samples and OD control were measured using a microplate reader at 734 nm. The ABTS radical scavenging ratios were obtained using the following formulae:
ABTS ROS scavenging ratio (%) = (OD control - OD sample)/OD control×100%

Preparation of macrophage and mitochondria membrane
Macrophage membrane (MM) fragments collected from RAW 264.7 cells. Briefly, RAW 264.7 cells were collected and suspended at a density of 2.0 × 107 cells mL−1 in hypotonic lysis buffer (pH 7.4, 10 mM Tris, 1 mM MgCl2) containing Complete Tablets mini protease inhibitor and disrupted via homogenization with Dounce homogenizer (Sigma, US). Then the obtained mixture was centrifuged (700 g, 10 min, 4 °C) and further centrifuged (10,000 g, 10 min, 4 °C) to remove unbroken cells and organelles. Finally, the resulting supernatant was centrifugated at 100,000 g (70 min, 4 °C) to obtain the MM. The resulting membrane pellets were stored at − 80 °C for future study.
Mitochondria membrane (MitoM) was prepared through mitochondrial isolation and subsequent disruption of the mitochondria. In brief, the collected cells were resuspended in mitochondria isolation buffer for 15 min at 4 ℃, then subjected to Dounce homogenization to break up the cells. Next, the cell homogenate was centrifuged at 11,000 × g for 10 min at 4 °C to obtain mitochondria. The MitoM was obtained via breaking up by lysis buffer and ultracentrifuging at 100,000 g, 4 °C for 70 min, which was stored at −80 °C for further use.

Preparation of AH-EVs and C@AH-EVs
Combine the MM solution and MitoM solution in a 1: 1 membrane protein mass ratio and the samples were sonicated putted into ultrasonic disrupter (Sonics, US) an ice water bath to facilitate membrane fusion (on 2 s/off 2 s, 5 min, 20% amplitude). Then the 5.8 μg CAPE-FPBA-DO was added into the hybrid membrane suspension (100 μg of membrane protein), followed by co-incubation for 30 minutes and subsequent sequential extruded through 800 nm, 400 nm, and 200 nm polycarbonate porous membranes using an Avanti mini extruder to facilitate drug-laden vesicle formation. The resulting hybrid membrane vesicles were subsequently isolated at 100,000 g (4 °C, 90 min) to remove the unencapsulated drugs, followed by PBS resuspension to acquire C@AH-EVs. While AH-EVs were generated under identical experimental conditions excluding drug administration.

Characterization of AH-EVs and C@AH-EVs
The membrane hybridization was characterized by confocal laser scanning microscopy (CLSM, Nikon A1R, Japan). Scanning electron microscopy (ThermoFisher Quattro S, US), transmission electron microscopy (JEM-2000 EX TEM, Japan), western blot, Nanoparticle Tracking Analysis (Particle Metrix, GER), and dynamic light scattering (Mastersizer 2000, Malvern, US) were performed to characterize AH-EVs and C@AH-EVs morphology, classic membrane markers (F4/80, Bcl-2, Na+/K+ ATPase), size distribution, and zeta potential. Loading capacity and encapsulation efficiency of CAPE-FPBA-DO were quantified by using HPLC. Encapsulation efficiency (EE) was calculated using the following formulae:
EE (%) = (Amount of CAPE-FPBA-DO in AH-EVs/Amount of total CAPE-FPBA-DO) × 100

In vitro and in vivo safety evaluation
In vitro cytotoxicity was assessed with RAW264.7 cells by CCK-8 assays as instructed. Briefly, 2×103 cells were seeded in 96-well plates. After incubation for 12 h, the media were replaced with fresh media containing PBS and C@AH-EVs (40 μg mL-1), respectively. After incubation for another 24 h, replaced the medium and added 10 ul CCK-8 to each well. Absorbance at 450 nm was measured to quantify the cytotoxicity.
The biocompatibility of C@AH-EVs in vivo was also evaluated. Eight-week-old BALB/c mice were randomly divided into two groups receiving subcutaneous injections of either PBS or C@AH-EVs (100 μg cm-2) around the wounds. Skin tissues were harvested after 3 days continuous injection. Major organs and blood samples were harvested from 14 days of continuous injection. Organ samples underwent hematoxylin and eosin (H&E) staining for histological assessment. Serum was collected from the blood for the measurement of biochemical markers (ALT, AST, Cr, CK).

In vitro cellular uptake and in vivo distribution of C@AH-EVs
The cellular uptake tests were conducted in RAW 264.7 cells (with or without LPS pretreated). The cells were incubated with C@AM-EVs and C@AH-EVs (labeled with DiI, red) for 12 h, respectively. Then the cells were washed with PBS for three times and subsequent counterstained with Hoechst (blue) for 15 min. After PBS washed again, the cells were observed via CLSM.
For mitochondrial localization studies, a similar protocol was used. Cells were incubated with C@AM-EVs and C@AH-EVs (labeled with DiO, green) for 12 h, then washed three times with PBS and sequentially stained with MitoTracker Deep RedFM for mitochondria (magenta) and Hoechst for nuclei (blue), and finally visualized under CLSM. Image J software was used to calculate a Pearson’s coefficient.
Immunofluorescence was used to analyze the in vivo distribution of C@AH-EVs. Specifically, normal and diabetic BALB/c mice were subcutaneously injected with DiI-labeled C@AH-EVs (red) at a dose of 100 μg cm-2 around the wound site. At 12 h post-injection, the mice were euthanized and skin tissues were harvested for fixation. The penetration depth of C@AH-EVs in vivo was observed using CLSM following DAPI staining. Besides, Cryosectioned specimens were subjected to triple immunofluorescence staining using anti-F4/80 antibody (macrophage marker, magenta), anti-TOM20 antibody (mitochondrial marker, green), and Hoechst (nuclear counterstain, blue). The in vivo biodistribution of C@AH-EVs was subsequently analyzed by CLSM. Image J software was used to calculate a Pearson’s coefficient.

Endocytic pathway study of C@AH-EVs
The impact of membrane protein integrity on endocytic pathways was evaluated according to established trypsin digestion protocol46. Briefly, isolated macrophage membranes and mitochondrial membranes were separately incubated with 50 μg mL-1 trypsin at 37°C for 6 h, followed by C@AH-EVs constitution using the established membrane fusion methodology. Purified C@AH-EVs from both trypsin-treated and untreated groups were resuspended in PBS. After labeling C@AH-EVs with lipophilic fluorescent dyes (DiI for C@AH-EVsMM trypsin, DiO for C@AH-EVsMitoM trypsin), they were co-cultured with macrophages for 12 h. Cells were subsequently stained with Hoechst and MitoTracker Deep Red. C@AH-EVs internalization and mitochondrial co-localization were quantitatively analyzed by CLSM.
Pharmacological inhibitors were used to study endocytosis of C@AH-EVs. Briefly, RAW 264.7 cells were pretreated with amiloride (5-(N, N-dimethyl)-amiloride hydrochloride, 200 μM), MβCD (methyl-β-cyclodextrin, 5 mM), Gen (genistein, 200 μM), and CPZ (chlorpromazine, 10 μM) for 30 min, and 4 ℃ for 1 h, respectively. Subsequently, the cells were co-cultured with DiI-labeled C@AH-EVs for 12 h followed with Hoechst staining. Fluorescence images were acquired with CLSM.

In vitro fusion assay
To evaluate the mitochondrial uptake mechanism of C@AH-EVs, MFI8 (Mitochondrial Fusion Inhibitor 8) was utilized. In brief, mitochondria were isolated according to manufacturers’ instructions, followed by treatment with MFI8 (20 μM) for 30 min. Then C@AH-EVs and mitochondria were mixed in equal amounts and incubated at 37°C for 60 min. Mitochondria treated with C@AM-EVs (lacking mitochondrial membrane components) served as the negative control. Mitochondria-nanovesicle interactions were then evaluated by TEM.

ROS-responsiveness of C@AH-EVs in mitochondria of RAW 264.7 cells
RAW 264.7 cells were incubated with media containing 100 ng mL-1 LPS and 40 μg mL-1 C@AH-EVs for 12 h. Subsequently, the cells were collected, and the mitochondria were isolated using mitochondria extraction kit. Prepared mitochondrial lysates, and the concentration of CAPE in mitochondria were detected by HPLC. RAW 264.7 cells treated with C@AH-EVs alone served as control.

Detection of mitochondrial reactive oxygen species
Mitochondrial reactive oxygen species (mtROS) were detected by MitoSOX. Briefly, RAW 264.7 cells were pretreated with 100 ng mL-1 LPS (lipopolysaccharide, typical ROS inducer) for 12 h and subsequently subjected to different treatments (PBS, AH-EVs, CAPE-FPBA-DO, C@AH-EVs). After 24 h of treatment, the culture medium was discarded and cells were incubated with 5 mM MitoSOX working solution at 37℃ for 20 min in the dark. Then cells were washed thrice with PBS and stained with Hoechst. mtROS were quantitatively analyzed by CLSM.

Mitochondria lipid peroxidation measurement
RAW 264.7 cells were seeded in 6-well plates and pretreated with LPS for 12 h. After treated with PBS, AH-EVs (40 μg mL-1), CAPE-FPBA-DO (1 μM), C@AH-EVs (40 μg mL-1) for 24 h, respectively, cells were harvested and incubated with 100 nM MitoPerOx for 30 min at room temperature. Mitochondria lipid peroxidation was evaluated using Beckman CytoFlex (US) and the data were analyzed with Flowjo VX software.

Detection of mitochondrial membrane Potential (ΔΨm)
Mitochondrial membrane potential (ΔΨm) was studied using the JC-1 fluorescence according to the manufacturer’s instructions. Briefly, RAW 264.7 cells were cultured in 6-well plates and pretreated with LPS for 12 h. Then the cells were treated with PBS, AH-EVs, CAPE-FPBA-DO, C@AH-EVs for 24 h, respectively. Subsequently, cells were incubated with 1 mg mL-1 JC-1 at 37°C in the dark for 20 min. The fluorescence signals were analyzed by means of flow cytometry.

Detection of mitochondria structure
RAW 264.7 cells were cultured in 10 cm plates and pretreated with LPS for 12 h. After treated with PBS, AH-EVs, CAPE-FPBA-DO, C@AH-EVs for 24h, respectively, cells were fixed using 2.5% glutaraldehyde solution at 4℃ for 4 h followed by post-fixed in 1% osmium tetroxide solution at room temperature for 1 h. Then the cells were washed three times with PBS and dehydrated in a graded series of ethanol (50%, 70%, 90%, 95%, and 100%) to acetone and embedded in epoxy resin that was polymerized in 60℃ thermostats for 48 h. Then the cell blocks were cut into 70 nm thick ultrathin sections and dual-stained with 2% uranyl acetate and 0.4% lead citrate for 15 min at room temperature. The ultra-thin section was imaged and analyzed by TEM. Mitochondrial morphology was quantified by analyzing the length/width ratio of all mitochondria within three randomly selected cells per group.

ATP measurement
ATP levels were measured using ATP Assay Kit according to the manufacturer’s instructions. Briefly, the collected cells were lysed with a lysis buffer and then centrifuged at 12000 g for 10 min at 4 ℃. The ATP content was determined by chemiluminescence of the supernatant using a microplate reader.

Evaluation of macrophage polarization in vitro
After treatment with PBS, AH-EVs, CAPE-FPBA-DO, and C@AH-EVs, RAW 264.7 cells cultured in 6-wells plate were harvested and incubated with fluorochrome-conjugated antibodies (M1-like macrophages were labeled with CD86-FITC, and M2-like macrophages were labeled with CD206-PE) for 30 min at 4℃. Then cells were washed twice with flow-cytometry buffer. The fluorescence signals were analyzed by flow cytometry and the data were analyzed with Flowjo VX software.
mRNA expression of proinflammatory genes (Tnfα, Nos2, Il6, and Cxcl1) and anti-inflammatory genes (Arg1, Mrc1, Ym1, and Fizz1) were performed by Quantitative PCR. Relative gene expression was normalized to β-actin quantified with the 2-ΔΔCt method for comparison. The PCR primers were obtained from PrimerBank and synthesized by AuGCT Biotech. Their sequences are provided in Supplementary Table S2.

In vivo therapeutic efficacy study in diabetic wounds
A mouse model of diabetic wounds was created to evaluate the therapeutic efficacy. Male BALB/c mice (8-10 weeks) were fasted for 12 h, followed by a single intraperitoneal injection of 50 mg kg-1 streptozotocin for 5 consecutive days. Blood glucose levels were monitored after 7 days, and those exhibiting levels >16.65 mM were classified as diabetic. Subsequently, full thickness wounds were established by 10 mm-diameter skin biopsy punches.
After the diabetic wound model was established, mice were subcutaneously injected with PBS (100 μL), AH-EVs (100 μg), CAPE-FPBA-DO (100 μL of 1 μM), CAPE (100 μL of 1 μM), AMitoM- EVs (100 μg), AMM-EVs (100 μg), CAPE@AH-EVs (100 μg), Beifuji (100 μL), and C@AH-EVs (100 μg) around the wounds at 4 injection sites (25 μL per site). The healing process of the wounds were recorded by taking the digital images of wounds and the wound area was measured using Image J software.
At indicated time points, wound tissues were harvested and homogenized for assessing the macrophage mtROS levels and phenotypes using flow cytometry. Hematoxylin & eosin (H&E) and Masson's trichrome staining were performed to histopathological analysis. The levels of TNF-α, IL-1β, TGF-β, and IL-10 were determined through ELISA kits. Immunofluorescence staining was conducted to detect CD31 and VEGF expression.

Statistical analysis
All data are given as mean ± SD. Results were analyzed by GraphPad Prism software 10.1.2 and Microsoft Excel (2024). Two-tailed Student’s t test was utilized for differences between two groups. One-way ANOVA with Tukey’s multiple comparison test and Two-way ANOVA with Dunnett’s multiple comparisons were utilized for statistical significance of multiple comparisons.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript by Fan et al. is a very interesting and well-executed study. The strategy of creating a hybrid membrane derived from both mitochondria and macrophages is a novel approach that represents an important development in the field of targeted drug delivery. The authors performed meticulous and well-thought-out experiments to validate their design and thoroughly investigate the proposed mechanisms. The level of detail and the logical progression of the work make it a strong contribution to the literature of this field. Moreover, while boronic acids have been extensively studied as ROS-responsive functional groups, typically in the form of iminoboronates, this work employs the use of a boronated oxime complex. This scaffold offers improved stability over traditional systems, adding an extra layer of translational relevance to the platform. While the article is a strong and promising contribution, a few points must be addressed before it can be recommended for publication.\n-Major point: The interpretation of the ABTS assay requires further clarification. Since no ROS are added to the system, the CA/CAPE moieties should not be released from the complex, and therefore should not act as scavengers. This suggests that another mechanism may be contributing to ABTS decolorization and must be discussed. One possibility is that ABTS is reacting directly with the boronic acid. A suggested control would be incubating ABTS with FPBA or PBA alone to evaluate any intrinsic reactivity. This point is crucial for understanding the actual mode of antioxidant action in this system and clarifying all the biological data obtained afterwards.\n-CAPE demonstrates better performance than CA, but the structural difference lies only in the ester moiety, which is distant from the reactive center. Could the authors provide a possible explanation? Is it due to differences in uptake, stability, or redox properties?\n-Figure 2d shows that CAPE-FPBA-DO and CA-FPBA-DO are fairly cytotoxic to RAW 264.7 macrophages (IC50 <100 uM). Since this cell line is often used to model inflammation, it would be helpful to clarify whether this cytotoxicity is intended or an undesirable off-target effect. Assuming the latter, if the aim is not to kill macrophages, this level of toxicity should be discussed.\n- The diabetic wound healing model shows a relatively high baseline healing rate in the control group, which is somewhat unexpected. While the treatment does lead to statistically significant improvements, the magnitude of the effect observed should be discussed in the context of the model’s standard responsiveness.\n-Supplementary Figures 11 & 12 are currently very difficult to interpret. The experimental conditions appear inconsistent (why is only CA/CAPE incubated for 72h if the goal is to study CA/CAPE-FPBA-DO stability?). Panel (a) is not described in the legend, and if (b-d) are meant to show CA/CAPE spectra, the aromatic protons are surprisingly absent. This figures and respective legends must be revised to provide a clearer explanation.\n-In the oxidation studies (HPLC Analysis), besides the released CA or CAPE, oxidation of the boronic acid should generate a phenol. This product should also be visible in HPLC traces, which is not. Is there an explanation for this?\n-In the UV-Vis stability assay in PBS, a concentration of 500 µM was used, which is significantly higher than the 1 µM employed in biological experiments (and even higher than the determined IC50). Since stability and other reactions may be dependent on the concentration, this discrepancy should be acknowledged and discussed in terms of relevance and translational interpretation.\n-The conclusion that uptake is energy-dependent based on reduced internalization at 4 °C is not entirely obvious. At low temperature, membrane fluidity is also reduced, which can impair passive diffusion and non-endocytic mechanisms. A more cautious interpretation is advised.\n-Supplementary Fig. 3c: The 1H NMR signals are misassigned. The peak labeled D should correspond to signal C.\n-Figure 2a: The hydroxylamine oxygen is missing in the first structure.\n-Typo in Figure 2 Title: The word “Synthesis” is missing an “n.”", "reviewer_2": "Fan et al present an innovative nanotherapeutic platform based on macrophage–mitochondria hybrid membrane-derived artificial extracellular vesicles for mtROS regulation in diabetic wounds. The concept is attractive and the work demonstrates encouraging in vitro and in vivo results. However, the manuscript in its current form is not yet suitable for publication, as several critical issues need to be addressed.\n1. “Notably, CAPE-FPBA-DO exhibited a higher release capacity than CA-FPBA-DO during the 480 min detection period, favoring faster ROS clearance (Supplementary Fig. 16)”. The authors should clarify the reason why CAPE-FPBA-DO exhibited a higher release capacity than CA-FPBA-DO. In addition, no experimental evidence was provided to substantiate the claim that CAPE-FPBA-DO achieves faster ROS clearance.\n2. The authors demonstrated the excellent ROS scavenging ability of CAPE-FPBA-DO and CA-FPBA-DO using the ABTS·⁺ assay. However, a direct comparison with free CAPE and free CA is needed to evaluate how conjugation affects the intrinsic ROS scavenging capacity of these molecules. Moreover, since CAPE-FPBA-DO and CA-FPBA-DO already exhibit strong ROS scavenging ability prior to drug release, the authors should clarify the rationale and added benefit of incorporating a ROS-responsive drug release design. Since both processes involve ROS depletion, the authors should clarify the conceptual distinction between ROS responsive and ROS scavenging.\n3. “As shown in Fig. 4d, C@AH-EVs effectively targeted mitochondria of macrophages in both common and diabetic wounds”. In the arrow-indicated regions, no clear overlap between Dil red fluorescence and Tom20 green fluorescence is observed. Instead, the yellow signal appears to colocalize with F4/80 fluorescence. The authors should provide further clarification to substantiate mitochondrial targeting.\n4. “In addition, cells treated with CAPE-FPBA-DO showing no obvious mtROS reduction compared to the LPS group, further confirming that C@AH-EVs specifically target mtROS scavenging”. In Fig. 6a, the data appear to show that the CAPE-FPBA-DO group exhibits a noticeable reduction in mtROS relative to the LPS (PBS) group. The authors should clarify this discrepancy.\n5. In both in vitro and in vivo assyas on mtROS clearance and M1 repolarization, the authors did not demonstrate that loading CAPE-FPBA-DO into AH-EVs offers a clear advantage over loading free CAPE. It is therefore recommended to include a CAPE@AH-EVs group as an additional control in both cell-based and animal studies. Furthermore, in the animal experiments, a clinically used wound dressing for diabetic wound healing should be included as a reference control.", "reviewer_3": "This manuscript presents a novel hybrid membrane-derived artificial extracellular vesicle (C@AH-EV) system, integrating macrophage and mitochondrial membranes with a ROS-responsive prodrug (CAPE-FPBA-DO), for the treatment of diabetic wounds. The study is well-designed, with a systematic evaluation that spans from material characterization to in vivo therapeutic effects. The manuscript is generally well-structured. However, several critical issues remain and must be properly addressed before the manuscript can be considered further.\nComments:\n1. The authors should more clearly clarify the unique innovations of this study.\n2. Please explain in greater detail why nanoscale therapeutics provide advantages over currently applied small-molecule antioxidants in diabetic wound healing.\n3. The authors claim that the mechanism of macrophage and mitochondrial targeting is attributed to membrane proteins, but the key proteins involved have not been validated.\n4. Tissue penetration depth and mechanism should be addressed. As extracellular vesicles generally do not offer inherent advantages in tissue penetration, further clarification is needed.\n5. The control groups could be improved. Free drug and single-membrane vesicle groups should be included, and comparisons with clinically used medicines would better demonstrate the specific advantage of C@AH-EVs.\n6. The authors should clarify whether blood glucose levels remained within the diabetic range throughout the wound healing study, to ensure that the model reflects a persistent diabetic state.\n7. The therapeutic administration in this study was performed via subcutaneous injection at the wound site. While this approach ensures local delivery in the animal model, the authors should provide an evaluation of the overall safety of the system under this administration route. In addition, it would be valuable for the authors to clarify whether alternative delivery methods, such as topical application or wound dressings, could be feasible, as these are more commonly used in clinical diabetic wound management."}, "author_response": "We sincerely appreciate the reviewer’s encouraging feedback and valuable suggestions, which have greatly contributed to the improvement of our manuscript. In response to the comments provided, we have carefully revised the paper to the best of our ability. Below is a point-by-point response to the specific recommendations offered.\n1. Major point: The interpretation of the ABTS assay requires further clarification. Since no ROS are added to the system, the CA/CAPE moieties should not be released from the complex, and therefore should not act as scavengers. This suggests that another mechanism may be contributing to ABTS decolorization and must be discussed. One possibility is that ABTS is reacting directly with the boronic acid. A suggested control would be incubating ABTS with FPBA or PBA alone to evaluate any intrinsic reactivity. This point is crucial for understanding the actual mode of antioxidant action in this system and clarifying all the biological data obtained afterwards.\nResponse: Thanks very much for your insightful suggestion. In the ABTS•+ assay, no exogenous ROS were added, yet we observed that CA/CAPE-FPBA-DO exhibit free radical scavenging ability, which is indeed intriguing. Clarifying this mechanism is highly meaningful. Following your advice, we included a separate 2-FPBA group (along with CA and CAPE groups, as recommended by Reviewer #2) and found that it did not cause ABTS•+ decolorization, indicating that the boronic acid likely does not directly react with the ABTS•+ (Fig. 2i). Given that a lower pH can degrade the conjugates, we further tested the ABTS•+ solution and confirmed its neutrality. Additionally, studies indicates that K₂S₂O₈ (used to generate the ABTS•+) can decompose in aqueous solution to produce H₂O₂, SO₄•-, and other radical species1,2. This could lead to the partial release of CAPE/CA, thereby resulting in ABTS•+ decolorization. Therefore, we utilized HRP/TMB assay to detect and confirmed the presence of a certain level of H₂O₂ in the ABTS•+ solution (Response Fig. 1). We suppose that this may be the reason why CA/CAPE-FPBA-DO causes ABTS•+ decolorization. All corresponding modifications have been incorporated into the revised manuscript.\nResponse Figure 1. Detection of H₂O₂ in the ABTS•+ solution by the HRP/TMB assay. The HRP-catalyzed oxidation of TMB in the presence of H₂O₂ was measured by detecting the absorbance of the colored product at 450 nm. The assay was conducted in a 96-well plate. Reagents were added sequentially: first the samples, followed by HRP, and finally TMB. A solution of 10 μmol/L H₂O₂ and PBS were used as the positive and negative controls, respectively. To validate specificity, samples were pre-incubated with CAT prior to the addition of HRP. Data are presented as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test was used. HRP, Horseradish peroxidase. TMB, 3,3’,5,5’- teramethylbencidine. CAT, Catalase.\nBefore revision: “To assess the ROS scavenging ability of CAPE-FPBA-DO and CA-FPBA-DO, the stable radical cation ABTS·+ (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation) was utilized (Fig. 2h). The results demonstrated that CAPE-FPBA-DO and CA-FPBA-DO scavenge 73.30 % and 80.62 % (p>0.05) of ABTS·+ after incubation at 200 μg mL-1 for 30 min, respectively (Fig. 2i).”\nFig. 2i ROS scavenging activities of CAPE-FPBA-DO and CA-FPBA-DO were assessed by ABTS assay (n = 3 independent samples).\nAfter revision (Results section, page 5): “Subsequently, ABTS•+ (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation) was incubated with the CAPE, CA, 2-FPBA, CAPE-FPBA-DO, or CAPE-FPBA-DO alone to assess the ROS scavenging ability (Fig. 2h). The results showed that CAPE and CA possessed comparable antioxidant capacity. Notably, CAPE/CA-FPBA-DO also decolorized ABTS•+ to some extent, which may be attributed to the production of ROS in the generation of ABTS•+1,2 (Fig. 2i).”\nFig. 2i ROS scavenging activities of CAPE, CA, 2-FPBA, CAPE-FPBA-DO, and CA-FPBA-DO were assessed by ABTS assay (n = 3 independent samples).\nReferences\n1. Wacławek, S., et al. Chemistry of persulfates in water and wastewater treatment: A review. Chem Eng J 330, 44-62 (2017).\n2. Wu, X.J., et al. A catalyst-free co-reaction system of long-lasting and intensive chemiluminescence applied to the detection of alkaline phosphatase. Mikrochim Acta 189, 181 (2022).\n2. CAPE demonstrates better performance than CA, but the structural difference lies only in the ester moiety, which is distant from the reactive center. Could the authors provide a possible explanation? Is it due to differences in uptake, stability, or redox properties?\nResponse: We appreciate your constructive suggestion. Both CA and CAPE exhibit substantial antioxidant activity. However, the CA is highly reactive and chemically unstable, resulting in poor retention of its bioavailable active form1. Furthermore, Low lipophilicity of CA limits its capacity to traverse cell membranes, thereby restricting its potential for direct pharmaceutical application2. In contrast, CAPE demonstrates more stability and enhanced lipophilicity due to esterification, enabling more efficient intracellular delivery. Following our synthesis of their respective boronic ester, we observed that CAPE-based compounds exhibit lower cytotoxicity. This may also be attributed to the ester moiety, which modulate the pharmacokinetic parameters thereby mitigating toxicity while preserving bioactivity3,4. We have included corresponding changes in the revision.\nBefore revision: “The results showed that CAPE-FPBA-DO exhibit the significantly lower cytotoxicity compared to CA-FPBA-DO, with IC values of 106.6 μM for 50 CAPE-FPBA-DO and 28.95 μM for CA-FPBA-DO (Fig. 2d). We next conducted stability analyses on CAPE-FPBA-DO and CA-FPBA-DO.”\nAfter revision (Results section, Page 5): “The results showed that CAPE-FPBA-DO exhibit the significantly lower cytotoxicity compared to CA-FPBA-DO, with IC 50 values of 106.6 μM for CAPE-FPBA-DO and 28.95 μM for CA-FPBA-DO (Fig. 2d). This may be attributed to its ester moiety for their modulation of pharmacokinetic parameters5,6. We next conducted stability analyses on CAPE-FPBA-DO and CA-FPBA-DO.”\nReferences\n1. Nicola Celli, L.K.D., Stefania Murzilli, Tommaso Pagliani, Andreina Poggi. In vitro and in vivo stability of caffeic acid phenethyl ester, a bioactive compound of propolis. J Agric Food Chem 55, 3398-3407 (2007).\n2. Shao, B., et al. Caffeic Acid Phenyl Ester (CAPE) Protects against Iron-Mediated Cellular DNA Damage through Its Strong Iron-Binding Ability and High Lipophilicity. Antioxidants (Basel) 10(2021).\n3. Dekant, W., Fujii, K., Shibata, E., Morita, O. & Shimotoyodome, A. Safety assessment of green tea based beverages and dried green tea extracts as nutritional supplements. Toxicol Lett 277, 104-108 (2017).\n4. C. Magnani, V.L.B.I., M. A. Correa and H. R. N. Salgado. Caffeic acid: a review of its potential use in medications and cosmetics. RSC 6, 3203-3210 (2014).\n3. Figure 2d shows that CAPE-FPBA-DO and CA-FPBA-DO are fairly cytotoxic to RAW 264.7 macrophages (IC50 <100 uM). Since this cell line is often used to model inflammation, it would be helpful to clarify whether this cytotoxicity is intended or an undesirable off-target effect. Assuming the latter, if the aim is not to kill macrophages, this level of toxicity should be discussed.\nResponse: We thank the reviewer for this insightful comment and for raising this important point. The observed cytotoxicity against RAW 264.7 cells was not the intended purpose of these compounds. The primary objective of this study was to develop an on-demand mtROS regulating delivery system aimed at scavenging mtROS levels and improving mitochondrial function in macrophages. The RAW 264.7 cell line was utilized as an in vitro model to evaluate the cytocompatibility of these synthesized conjugates, with the goal of identifying appropriate non-damaging treatment doses. We fully acknowledge that the cytotoxicity of CAPE/CA-FPBA-DO requires thorough validation and discussion, particularly in the context of wound healing where macrophages play a critical role. This point has been addressed in the revised manuscript.\nBefore revision: “The drug loading capacity is crucial for determining the final therapeutic effect. Based on CCK-8 assay results demonstrating minimal cytotoxicity at 1 μM of CAPE-FPBA-DO, we selected this concentration for C@AH-EVs preparation, achieving a 69% encapsulation efficiency for CAPE-FPBA-DO and a loading capacity of 403 ng/1010 particles.”\nAfter revision (Results section, Page 7): “The drug loading capacity is crucial for determining the final therapeutic effect. Therefore, we utilized the highest safe concentration of 1 μM CAPE-FPBA-DO for C@AH-EVs preparation, achieving a 69% encapsulation efficiency for CAPE-FPBA-DO and a loading capacity of 403 ng/1010 particles (Fig. 3h, Supplementary Fig. 20). Subsequent CCK-8 assays and histological analysis further confirmed the biosafety of the prepared C@AH-EVs (Supplementary Fig. 21-23).”\n4. The diabetic wound healing model shows a relatively high baseline healing rate in the control group, which is somewhat unexpected. While the treatment does lead to statistically significant improvements, the magnitude of the effect observed should be discussed in the context of the model’s standard responsiveness.\nResponse: We thank the reviewer for this insightful and important observation regarding the relatively high baseline healing rate in our control group. The control group did indeed exhibit a relatively high baseline healing rate, which we consider may be related to the blood glucose levels of the diabetic mice. Studies have shown that blood glucose concentrations significantly correlating with wound healing rates1,2. In this study, diabetic mice exhibited random blood glucose levels below 30 mmol/L, indicating a non-severe diabetic state3,4. This trend is consistent with findings reported in multiple previous studies5-8. Furthermore, we have provided the blood glucose values and body weight changes of the diabetic mice (Supplementary Fig. 32). These data confirm the successful induction of diabetes in the mice, with hyperglycemia maintained below the threshold for severe diabetes.\nBefore revision: “First, the diabetic wound model was created by injecting streptozotocin at a dose of 50 mg/kg/day for 5 days, followed by surgical removal of 10-mm-diameter full-thickness skin after consistent blood glucose levels over 16.65 mmol/L for one week. Subsequently, PBS, AH-EVs, CAPE-FPBA-DO, and C@AH-EVs were subcutaneously administrated at the wound sites, respectively.”\nAfter revision (Results section, Page 18): “First, the diabetic wound model was created by injecting streptozotocin at a dose of 50 mg/kg/day for 5 days, followed by surgical removal of 10-mm-diameter full-thickness skin after consistent blood glucose levels over 16.65 mmol/L for one week. The persistent hyperglycemia and impaired weight gain collectively demonstrate that the mice maintained a sustained diabetic state throughout the experimental period (Supplementary Fig. 32). Subsequently, PBS, AH-EVs, CAPE-FPBA-DO, and C@AH-EVs were subcutaneously administrated at the wound sites, respectively.”\nSupplementary Figure 32. Successful creation of diabetic model. Records of blood glucose (a) and body weight (b) in mouse models within two weeks (n = 6 biologically independent samples). Data are presented as mean ± SD.\nReferences\n1. Kruse, C.R., Singh, M., Sorensen, J.A., Eriksson, E. & Nuutila, K. The effect of local hyperglycemia on skin cells in vitro and on wound healing in euglycemic rats. J Surg Res 206, 418-426 (2016).\n2. Christman, A.L., Selvin, E., Margolis, D.J., Lazarus, G.S. & Garza, L.A. Hemoglobin A1c predicts healing rate in diabetic wounds. J Invest Dermatol 131, 2121-2127 (2011).\n3. Furman, B.L. Streptozotocin‐Induced Diabetic Models in Mice and Rats. Current Protocols 1(2021).\n4. Yu, B., et al. Effects of gradient high-field static magnetic fields on diabetic mice. Zool Res 44, 249-258 (2023).\n5. Qian, Y., et al. Immunoregulation in Diabetic Wound Repair with a Photoenhanced Glycyrrhizic Acid Hydrogel Scaffold. Adv Mater 34, e2200521 (2022).\n6. Cong, R., et al. Dimeric copper peptide incorporated hydrogel for promoting diabetic wound healing. Nature Communications 16(2025).\n7. Jiankang He, Y.L., Nianqiang Jin, Wenqiang Xie, Lingling Ou, Lu Sun, Chufeng Li, Jiayi Luan, Longquan Shao. Nb2C MXene functionalized janus amphiphilic membrane manipulates antioxidant, antibacterial and proangiogenic properties to accelerate diabetic wound healing. Chem Eng J 515, 163899 (2025).\n8. Xue, Y., et al. LNP-RNA-engineered adipose stem cells for accelerated diabetic wound healing. Nat Commun 15, 739 (2024).\n5. Supplementary Figures 11 & 12 are currently very difficult to interpret. The experimental conditions appear inconsistent (why is only CA/CAPE incubated for 72h if the goal is to study CA/CAPE-FPBA-DO stability?). Panel (a) is not described in the legend, and if (b-d) are meant to show CA/CAPE spectra, the aromatic protons are surprisingly absent. These figures and respective legends must be revised to provide a clearer explanation.\nResponse: We sincerely apologize for the lack of clarity in Supplementary Figures 11 and 12 and their corresponding legends. In this experiment, the aqueous stability of CAPE/CA-FPBA-DO was monitored over 0 h, 24 h, and 72 h (b-d), while CAPE/CA served as control groups (a). We have clearly labeled the spectra corresponding to different treatment times and different drugs in the figure, while providing detailed descriptions for each panel in the legend to ensure clearer interpretation. We believe these revisions have significantly improved the clarity and interpretability of the data.\nBefore revision:\nSupplementary Figure 11. Stability of CAPE-FPBA-DO with PBS by 1H NMR. a 5 mg of CAPE in the mixture of PBS-D/DMSO-d6 (8:2 v/v, 1 mL) with 0.5 wt% brij 58 surfactant. 0 h (b), 24 h (c) and 72 h (d) after preparing the sample: 5 mg CAPE-FPBA-DO in the mixture of PBS-D/DMSO-d6 (8:2 v/v, 1 mL) with 0.5 wt% brij 58 surfactant. The NMR studies showed no noticeable degradation PBS-D in 72 h. Preparation of PBS-D: PBS tablet (Thermofisher Gibco™) 20 mg was dissolved in 1.6 mL D O to make a PBS-D solution (12.5 mM).\nSupplementary Figure 11. Hydrolytic Stability of CAPE-FPBA-DO in PBS by 1H NMR. Spectra of CAPE-FPBA-DO (5 mg in 1mL PBS-D/DMSO-d6, 8:2 v/v, with 0.5 wt% Brij-58) were acquired at 0, 24, and 72 h. Reference spectrum of CAPE (5 mg) prepared under identical conditions. Preparation of PBS-D (12.5 mM): A 20 mg PBS tablet was dissolved in 1.6 mL D₂O.\nSupplementary Figure 12. Hydrolysis stability of CA-FPBA-DO in PBS by 1H NMR. Spectra of CA-FPBA-DO (5 mg in 1mL PBS-D/DMSO-d6, 8:2 v/v, with 0.5 wt% Brij-58) were acquired at 0, 24, and 72 h. Reference spectrum of CA (5 mg) prepared under identical conditions. Preparation of PBS-D (12.5 mM): A 20 mg PBS tablet was dissolved in 1.6 mL D₂O.\nReferences\n1. Wang, M.M., et al. Elucidation of lipid nanoparticle surface structure in mRNA vaccines. Sci Rep 13, 16744 (2023).\n6. In the oxidation studies (HPLC Analysis), besides the released CA or CAPE, oxidation of the boronic acid should generate a phenol. This product should also be visible in HPLC traces, which is not. Is there an explanation for this?\nResponse: Thanks for pointing out this. Due to the difference of retention time and UV absorption wavelengths for these compounds, the byproduct phenol was not observed in our original HPLC method. To identify the byproduct, we synthesized it (denoted as SA-DO) from dodecyl hydroxylamine and salicylaldehyde (Supplementary Fig. 14). Using this reference compound, we successfully observed it at 300 nm in the HPLC chromatograms (Supplementary Fig. 15). These new results have been included in the Supplementary Material.\nBefore revision: “Thus, we investigated the ROS responsive behavior of CAPE-FPBA-DO and CA-FPBA-DO under varying H O levels. And the extent of drug release was concentration dependent, with values for CAPE and CA rising from 51.32 % and 37.14 % at 0.1 mM H O to 77.77 % and 74.47 % at 1 mM H O after 480 min of incubation (Fig. 2f, g, Supplementary Fig. 16, 17).”\nAfter revision (Results section, Page 5): “Thus, we investigated the ROS responsive behavior of CAPE-FPBA-DO and CA-FPBA-DO under varying H O levels. High-performance liquid chromatography (HPLC) results showed released CAPE/CA and byproducts at different retention times and UV absorption wavelengths, indicating that H₂O₂ triggered the decomposition of CAPE-FPBA-DO and CA-FPBA-DO (Supplementary Fig. 14, 15). And the extent of drug release was concentration dependent, with values for CAPE and CA rising from 51.32 % and 37.14 % at 0.1 mM H O to 77.77 % and 74.47 % at 1 mM H O after 480 min of incubation (Fig. 2f, g, Supplementary Fig. 16, 17).”\nSupplementary Figure 14. Synthetic procedure of SA-DO. a Synthetic procedure of SA-DO. b 1H NMR spectrum of the byproduct SA-DO. 1H NMR (401 MHz, CDCl ) δ 12.41 (s, 1H), 3 7.52 (s, 1H), 7.38 (ddd, J = 8.7, 7.2, 1.7 Hz, 1H), 7.05 (dd, J = 7.8, 1.7 Hz, 1H), 6.96 (dd, J = 8.4, 1.2 Hz, 1H), 6.84 (ddd, J = 8.0, 7.2, 1.2 Hz, 1H), 3.95 (t, J = 7.1 Hz, 2H), 1.99 (p, J = 7.1 Hz, 2H), 1.49 – 1.20 (m, 18H), 0.92 – 0.84 (m, 3H). c 13C NMR spectrum of the byproduct SA-DO. 13C NMR (101 MHz, CDCl ) δ 159.74, 140.80, 133.91, 131.97, 120.20, 118.92, 116.66, 3 65.18, 31.92, 29.60, 29.53, 29.40, 29.34, 29.09, 27.75, 26.34, 22.69, 14.13.\nSupplementary Figure 15. HPLC analysis of the decomposition products from CAPE-FPBA-DO (a) and CA-FPBA-DO (b). The HPLC analysis were representative data from three independent experiments.\n7. In the UV-Vis stability assay in PBS, a concentration of 500 µM was used, which is significantly higher than the 1 µM employed in biological experiments (and even higher than the determined IC50). Since stability and other reactions may be dependent on the concentration, this discrepancy should be acknowledged and discussed in terms of relevance and translational interpretation.\nResponse: We thank the reviewer for this insightful comment regarding the potential concentration dependence of stability. The discrepancy between the concentration used in in vitro stability assay (500 µM) and that employed in the biological experiments (1 µM) is indeed a critical factor to consider for data interpretation and translational application. To better approximate conditions used in biological experiments, we performed additional UV-Vis experiments at a concentration of 25 µM in PBS. The results revealed that CA-FPBA-DO undergoes hydrolysis at this lower concentration, whereas CAPE-FPBA-DO shows only minimal hydrolysis over three days (Supplementary Fig. 13), which suggested that CAPE-FPBA-DO remains a more suitable biological application. However, the hydrolytic stability of CAPE-FPBA-DO also potentially concentration dependent.\nBesides, the hydrolysis kinetics could be further altered within the complex in vivo environment. Although in this study, loading CAPE-FPBA-DO into AH-EVs may mitigate some direct influences from the bodily milieu (by limiting access to macromolecular bio-enzymes like serum esterases and proteases), the small molecules, such as ions can still diffuse through the membrane or utilize specific channels for their semi-permeable lipid bilayer. Thus, we provided a more rigorous interpretation of the results and this potential phenomenon. These findings and corresponding discussions have been incorporated into the revised manuscript.\nBefore revision: “According to NMR spectroscopic and UV-Vis results neither CAPE-FPBA-DO nor CA-FPBA-DO exhibited noticeable degradation after 72 h of incubation in PBS (Fig. 2e, Supplementary Fig. 11-13).”\nBefore revision: “In summary, we developed a novel drug delivery system capable of precise clearance of mtROS in macrophages.”\nSupplementary Figure 15. UV-vis Analysis of PBS effects on CAPE-FPBA-DO and CA-FPBA-DO Stability. UV-vis absorption spectra of 0.5 mM CAPE-FPBA-DO (a) and 0.5 mM CA-FPBA-DO (b) in the mixture of PBS /DMSO (8:2 v/v) with 0.5 % (w/v) Tween 20 at 0, 24, 48, 72 h. (c) Temporal variation of 346 nm absorbance for CAPE-FPBA-DO in PBS (n = 3 independent samples). (d) Temporal variation of 335 nm absorbance for CA-FPBA-DO in PBS (n = 3 independent samples). Data are presented as mean ± SD. Source data are provided as a Source data file.\nAfter revision (Results section, Page 5): “NMR results indicate that both CAPE-FPBA-DO and CA-FPBA-DO exhibit good hydrolytic stability at high concentrations. However, significant hydrolysis of CA-FPBA-DO is observed when the concentration is closer to the non-cytotoxic concentration (25 μM) (Fig. 2e, Supplementary Fig. 11-13).”\nSupplementary Figure 13. UV-Vis spectra of CAPE-FPBA-DO and CA-FPBA-DO in PBS. UV-Vis absorption spectra of 25 µM CAPE-FPBA-DO (a) and 25 µM CA-FPBA-DO (b) in the mixture of PBS/DMSO (8:2 v/v) with 0.5 % (w/v) Brij-58 at different time. The UV-Vis spectra were representative data from three independent experiments.\nAfter revision (Discussion section, Page 23) “Furthermore, although CAPE-FPBA-DO demonstrates good hydrolytic stability at low concentrations, it still potentially exhibits concentration dependence. While encapsulation within AH-EVs offers a degree of protection, the semi-permeable nature of the lipid bilayer membrane still allows small molecules such as ions to potentially enter the vesicles through diffusion or channel-mediated transport, thereby exposing the prodrug to more complex physiological conditions. Hence, elucidating their in vivo behavior will enable a more efficient path toward clinical translation.\nIn summary, we developed a novel drug delivery system capable of precise clearance of mtROS in macrophages.”\n8. The conclusion that uptake is energy-dependent based on reduced internalization at 4 °C is not entirely obvious. At low temperature, membrane fluidity is also reduced, which can impair passive diffusion and non-endocytic mechanisms. A more cautious interpretation is advised.\nResponse: We appreciate the reviewer for this valuable comment on our manuscript. The interpretation of 4°C experiment must consider the pleiotropic effects. Study indicates that hypothermia concurrently induces a drastic reduction in membrane fluidity and suppresses mitochondrial oxidative phosphorylation, leading to ATP depletion1. Therefore, the near-complete inhibition of C@AH-EVs uptake we observed at 4°C is likely a synergistic consequence of both impaired membrane dynamics and lack of ATP for endocytic machinery, rather than being attributable to a single mechanism. We have revised the manuscript and incorporated relevant references to adopt a more cautious and accurate interpretation of these results.\nBefore revision: “The uptake of C@AH-EVs in RAW 264.7 cells incubated at 4°C dropped to 4.5% of control levels, demonstrating that internalization is energy-dependent.”\nAfter revision (Results Section, Page 11): “The uptake of C@AH-EVs in RAW 264.7 cells incubated at 4°C dropped to 4.5% of control levels, which is likely associated with suppressed cellular energy metabolism and the reduction in membrane fluidity induced by low temperature1.”\nReference\n1. Bag, N., Yap, D.H.X. & Wohland, T. Temperature dependence of diffusion in model and live cell membranes characterized by imaging fluorescence correlation spectroscopy. Biochimica et Biophysica Acta (BBA) - Biomembranes 1838, 802-813 (2014).\n9. Supplementary Fig. 3c: The 1H NMR signals are misassigned. The peak labeled D should correspond to signal C.\nResponse: We thank the reviewer for the careful attention to identify this error in the assignment of the ¹H NMR signals. We have corrected the manuscript accordingly.\nBefore revision:\nSupplementary Figure 3. 1H NMR (CDCl ) of S2. 1H NMR (401 MHz, CDCl ) (E-isomer) δ 3 3 7.42 (t, J = 6.1 Hz, 1H), 2.19 (td, J = 7.5, 6.1 Hz, 2H), 1.54–1.44 (m, 2H), 1.36–1.22 (m, 16H), 0.88 (t, J = 6.7 Hz, 3H); (Z-isomer) δ 6.76 (brs, 1H), 2.35 (brs, 2H), 1.54–1.44 (m, 2H), 1.36–1.22 (m, 16H), 0.88 (t, J = 6.7 Hz, 3H).\nAfter revision (Supplementary section, Page 36):\nSupplementary Fig. 3 1H NMR (CDCl ) of S2. 1H NMR (401 MHz, CDCl ) (E-isomer) δ 3 3 7.42 (t, J = 6.1 Hz, 1H), 2.19 (td, J = 7.5, 6.1 Hz, 2H), 1.54–1.44 (m, 2H), 1.36–1.22 (m, 16H), 0.88 (t, J = 6.7 Hz, 3H); (Z-isomer) δ 6.76 (brs, 1H), 2.35 (brs, 2H), 1.54–1.44 (m, 2H), 1.36–1.22 (m, 16H), 0.88 (t, J = 6.7 Hz, 3H).\n10. Figure 2a: The hydroxylamine oxygen is missing in the first structure.\nResponse: Thank you for your careful review and for pointing out this oversight. We have now added the missing hydroxylamine oxygen atom in the first structure. The correction has been made in the revised manuscript.\nBefore revision:\nFig. 2a Schematic illustration of ROS-triggered CAPE/CA release from CAPE/CA-FPBA-DO.\nAfter revision (Results section, Page 6):\nFig. 2a Schematic illustration of ROS-triggered CAPE/CA release from CAPE/CA-FPBA-DO.\n11. Typo in Figure 2 Title: The word “Synthesis” is missing an “n.”\nResponse: Thank you very much for your detailed review and our sincerely apologies for the oversight. We have thoroughly checked the manuscript and correct the typos. In the revision, we have changed \" Syhthesis \" as “Synthesis”.\nBefore revision: “Syhthesis and characterization of CAPE-FPBA-DO and CA-FPBA-DO.”\nAfter revision (Results section, Page 6): “Synthesis and characterization of CAPE-FPBA-DO and CA-FPBA-DO.”\nReviewer #2 (Remarks to the Author):\nFan et al present an innovative nanotherapeutic platform based on macrophage–mitochondria hybrid membrane-derived artificial extracellular vesicles for mtROS regulation in diabetic wounds. The concept is attractive and the work demonstrates encouraging in vitro and in vivo results. However, the manuscript in its current form is not yet suitable for publication, as several critical issues need to be addressed.\nResponse: Thank you for your thoughtful review and positive recognition of our work. We sincerely appreciate your constructive feedback, which has helped us identify areas for improvement. We have carefully addressed the critical issues you raised through additional experiments, data analysis, and extensive revisions to the manuscript. Our point-by-point responses below detail the changes made for each specific comment. We hope that these revisions have adequately resolved your concerns and believe that the manuscript has been significantly strengthened as a result.\n1. “Notably, CAPE-FPBA-DO exhibited a higher release capacity than CA-FPBA-DO during the 480 min detection period, favoring faster ROS clearance (Supplementary Fig. 16)”. The authors should clarify the reason why CAPE-FPBA-DO exhibited a higher release capacity than CA-FPBA-DO. In addition, no experimental evidence was provided to substantiate the claim that CAPE-FPBA-DO achieves faster ROS clearance.\nResponse: We thank the reviewer for raising these important points. The observed difference in drug release, where CAPE-FPBA-DO exhibits a slightly higher release in solution compared to CA-FPBA-DO, may related to the distinct self-assembly behaviors of the two compounds in the aqueous buffer, which profoundly affect the accessibility of their boronic ester groups to H₂O₂1. CA-FPBA-DO (carboxylic acid): This molecule is amphiphilic, featuring a hydrophilic carboxylate/carboxylic acid head group and a large hydrophobic chain. Above a certain concentration, it is highly likely to form micelles or compact aggregates in the aqueous solution. In such structures, the reactive boronic ester moieties are likely shielded within the hydrophobic core, making them poorly accessible to the hydrophilic H₂O₂ molecules in the bulk solution. This physical encapsulation significantly hinders the reaction, leading to a lower apparent release capacity. CAPE-FPBA-DO (carboxylic ester): The ester derivative is much more hydrophobic and lacks a strong hydrophilic head group. Consequently, it is less prone to form well-defined, tightly packed micelles. It may form looser aggregates where the boronic esters remain more exposed and accessible to H₂O₂, resulting in a higher observed release rate. Notably, while drug release studies in standard buffers provide initial insight, they are of limited value for predicting in vivo performance due to the substantial compositional differences between these simplified media and biological fluids. Therefore, we apologize for the oversight about the statement of “CAPE-FPBA-DO achieves faster ROS clearance”. We have carefully revised the manuscript to present a more accurate interpretation of the data.\nBefore revision: “This result confirms the potential on-demand release capabilities of CAPE-FPBA-DO and CA-FPBA-DO in dysfunctional mitochondrial environments. Notably, CAPE-FPBA-DO exhibited a higher release capacity than CA-FPBA-DO during the 480 min detection period, favoring faster ROS clearance (Supplementary Fig. 16).”\nAfter revision (Results section, Page 5): “This result confirms the potential on-demand release capabilities of CAPE-FPBA-DO and CA-FPBA-DO in dysfunctional mitochondrial environments. Notably, CAPE-FPBA-DO exhibited a slightly higher release capacity than CA-FPBA-DO during the 480 min detection period. This divergence may be attributed to the greater propensity of CA-FPBA-DO to form micellar aggregates in aqueous environments, which sequester the reactive boronic ester groups within a hydrophobic core, thereby shielding them from H₂O₂ solution and attenuating the reaction-driven release (Supplementary Fig. 18).”\nReference\n1. Ghezzi, M., et al. Polymeric micelles in drug delivery: An insight of the techniques for their characterization and assessment in biorelevant conditions. J Control Release 332, 312-336 (2021).\n2. The authors demonstrated the excellent ROS scavenging ability of CAPE-FPBA-DO and CA-FPBA-DO using the ABTS·⁺ assay. However, a direct comparison with free CAPE and free CA is needed to evaluate how conjugation affects the intrinsic ROS scavenging capacity of these molecules. Moreover, since CAPE-FPBA-DO and CA-FPBA-DO already exhibit strong ROS scavenging ability prior to drug release, the authors should clarify the rationale and added benefit of incorporating a ROS-responsive drug release design. Since both processes involve ROS depletion, the authors should clarify the conceptual distinction between ROS responsive and ROS scavenging.\nResponse: We sincerely thank the reviewer for these insightful and constructive questions, which help us better clarify the innovative design of the prodrug. In response to your suggestion, we first included CAPE and CA group in the ABTS assay. The results confirm that both free drugs effectively scavenge free radicals (Fig. 2i). Regarding the introduction of the ROS-responsive drug, our objective was to provide precise mtROS scavenging. Due to the ROS-responsive nature of boronic ester bonds, they can cleave under the stimulation of elevated mtROS levels, triggering the controlled release of CAPE or CA to scavenge excess mtROS. The greatest advantage of this design is that it restores mtROS at a lower, safer baseline, which avoiding oxidative damage caused by excessive mtROS, while also preventing the impairment of M2 macrophage polarization induced by excessive mtROS depletion. However, intriguingly, in the ABTS•+ scavenging assay, we observed that CAPE-FPBA-DO and CA-FPBA-DO exhibit a certain degree of radical scavenging activity without exogenous ROS addition. We analyzed that this phenomenon may be attributed to the fact that K₂S₂O₈ (used to generate ABTS•+) can decompose in aqueous solution to produce H₂O₂, SO₄•-, and other radical species1,2, which also confirmed by HRP/TMB assay (Response Fig. 1). These endogenously generated ROS may induce partial cleavage of the boronic acid ester bonds in the prodrugs, leading to the premature release of a small amount of CAPE/CA, which then reacts with ABTS•+ and causes its decolorization.\nIn conclusion, the prodrug delivery system in this study is capable of efficient ROS response and precise mtROS level regulation. This system not only effectively mitigates oxidative damage to cells caused by mtROS but also avoids the inhibition of macrophage M2 polarization due to excessive mtROS scavenging, thereby providing an ideal therapeutic strategy for diabetic wound healing. All these points have been thoroughly addressed in the revised manuscript.\nResponse Figure 1. Detection of H₂O₂ in the ABTS•+ solution by the HRP/TMB assay. The HRP-catalyzed oxidation of TMB in the presence of H₂O₂ was measured by detecting the absorbance of the colored product at 450 nm. The assay was conducted in a 96-well plate. Reagents were added sequentially: first the samples, followed by HRP, and finally TMB. A solution of 10 μmol/L H₂O₂ and PBS were used as the positive and negative controls, respectively. To validate specificity, samples were pre-incubated with CAT prior to the addition of HRP. Data are presented as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test was used. HRP, Horseradish peroxidase. TMB, 3,3’,5,5’- teramethylbencidine. CAT, Catalase.\nBefore revision:\n(1) “To assess the ROS scavenging ability of CAPE-FPBA-DO and CA-FPBA-DO, the stable radical cation ABTS·+ (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation) was utilized (Fig. 2h). The results demonstrated that CAPE-FPBA-DO and CA-FPBA-DO scavenge 73.30 % and 80.62 % (p>0.05) of ABTS•+ after incubation at 200 μg mL-1 for 30 min, respectively (Fig. 2i).”\n(2) “Targeting the clearance of mtROS in macrophages is an effective approach to promote wound healing. Although numerous mitochondrial-targeting strategies have been developed, their translational application value is significantly limited due to inefficiencies in targeted delivery systems, poor intracellular drug stability, and potential toxicity risks to healthy tissue. In this study, hybrid membrane vesicles formed by macrophage membrane and mitochondrial membrane offer a safer and more effective strategy. Native cellular membranes confer inherent biocompatibility and enhanced safety to the C@AH-EVs. Mitochondrial targeting is achieved by the mitochondrial membrane, which operates independently of mitochondrial membrane potential, offering significant advantages over membrane potential-dependent molecular targeting strategies, such as lipophilic cations.”\nFig. 2i ROS scavenging activities of CAPE-FPBA-DO and CA-FPBA-DO were assessed by ABTS assay (n = 3 independent samples).\nAfter revision\n(1) (Results section, Page 5): “Subsequently, ABTS•+ (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation) was incubated with the CAPE, CA, 2-FPBA, CAPE-FPBA-DO, or CAPE-FPBA-DO alone to assess the ROS scavenging ability (Fig. 2h). The results showed that CAPE and CA possessed comparable antioxidant capacity. Notably, CAPE/CA-FPBA-DO also decolorized ABTS•+ to some extent, which may be attributed to the production of ROS in the generation of ABTS•+1,2.”\nFig. 2i ROS scavenging activities of CAPE, CA, 2-FPBA, CAPE-FPBA-DO, and CA-FPBA-DO were assessed by ABTS assay (n = 3 independent samples).\n(2) (Discussion section, Page 22) “Targeting the clearance of mtROS in macrophages is an effective approach to promote wound healing. Although numerous mitochondrial-targeting strategies such as small-molecule compounds and synthetic nanoparticles have been developed, their translational application value is significantly limited due to inefficiencies in targeted delivery systems, poor intracellular drug stability, and potential toxicity risks to healthy tissue. Importantly, most existing systems focus solely on scavenging mtROS, which may lead to excessive depletion of physiological mtROS levels and thereby inhibit M2 macrophage polarization. This study effectively addresses these challenges by integrating macrophage-mitochondrial hybrid membrane vesicles with ROS-responsive prodrugs. The vesicles not only exhibit an improved safety profile but, more importantly, achieve specific delivery to macrophage mitochondria, independently of the mitochondrial membrane potential. Furthermore, its nano-encapsulation capability protects the prodrug from degradation, thereby ensuring the attainment of effective local drug concentrations. Upon internalization into the macrophage mitochondria, the prodrug CAPE-FPBA-DO were released and exhibits mtROS concentration-dependent antioxidant activity. This mechanism not only exerts anti-inflammatory effects but also maintains mtROS at a lower, homeostatic level, thereby preventing the impairment of M2 macrophage polarization caused by excessive mtROS scavenging.”\nReferences\n1. Wacławek, S., et al. Chemistry of persulfates in water and wastewater treatment: A review. Chem Eng J 330, 44-62 (2017).\n2. Wu, X.J., et al. A catalyst-free co-reaction system of long-lasting and intensive chemiluminescence applied to the detection of alkaline phosphatase. Mikrochim Acta 189, 181 (2022).\n3. “As shown in Fig. 4d, C@AH-EVs effectively targeted mitochondria of macrophages in both common and diabetic wounds”. In the arrow-indicated regions, no clear overlap between Dil red fluorescence and Tom20 green fluorescence is observed. Instead, the yellow signal appears to colocalize with F4/80 fluorescence. The authors should provide further clarification to substantiate mitochondrial targeting.\nResponse: We thank the reviewer for their careful observation regarding the evidence for mitochondrial targeting in Fig. 4d. We acknowledge that the original pseudocolor scheme, where the merged signal of Dil (red) and Tom20 (green) appeared yellow, was visually similar to the color used for the F4/80 marker, which could cause confusion and make specific colocalization difficult to discern. To address this point unequivocally and enhance the clarity of our data, we have undertaken the following revisions:\nRevised pseudo color scheme: We have changed the color of the F4/80 signal to a magenta hue in our revised figures. This change effectively eliminates the color ambiguity, allowing for a clear visual distinction between the yellow signal (indicating true colocalization of C@AH-EVs with mitochondria) and the magenta signal (identifying macrophages).\nHighlighted specific C@AH-EVs-mitochondria colocalization: In the revised figure panel, we feature the separate merge channel for Dil (C@AH-EVs) and Tom20 (mitochondria). This provides a direct view of their association, reinforcing the visual evidence for mitochondria targeting.\nProvided quantitative colocalization analysis: To provide robust numerical evidence, we have performed a quantitative colocalization analysis. The calculation of the Pearson's Correlation Coefficient between the Dil and Tom20 channels confirms a statistically significant correlation, substantiating the mitochondrial targeting capability of C@AH-EVs.\nBefore revision: “In the subsequent experiment, we observed the mitochondrial targeting properties of C@AH-EVs in macrophages within diabetic wounds using CLSM (Supplementary Fig. 23). As shown in Figure 4d, C@AH-EVs effectively targeted mitochondria of macrophages in both common and diabetic wounds.”\nFig. 4d CLSM images showing the co-localization of C@AH-EVs with mitochondria in wound tissue macrophages. Yellow: F4/80, Green: Tom20, Red: DiI, Blue: Hoechst. Scale bar, 40 μm. The CLSM images were representative data from three mice.\nAfter revision (Results section, Page 10): “In the subsequent experiment, we observed the mitochondrial targeting properties of C@AH-EVs in macrophages within diabetic wounds using CLSM (Supplementary Fig. 23). As shown in Figure 4d and Figure 4e, C@AH-EVs effectively targeted mitochondria of macrophages in both common and diabetic wounds.”\nFig. 4 CLSM images (d) and co-localization analysis (e) showing the co-localization of C@AH-EVs with mitochondria in wound tissue macrophages. Magenta: F4/80, Green: Tom20, Red: DiI, Blue: Hoechst. Scale bar, 40 μm. Rr: Pearson correlation coefficient. The CLSM images were representative data from three mice.\n4. “In addition, cells treated with CAPE-FPBA-DO showing no obvious mtROS reduction compared to the LPS group, further confirming that C@AH-EVs specifically target mtROS scavenging”. In Fig. 6a, the data appear to show that the CAPE-FPBA-DO group exhibits a noticeable reduction in mtROS relative to the LPS (PBS) group. The authors should clarify this discrepancy.\nResponse: We appreciate the reviewer's careful examination of our data. Figure 6a shows a trend of mtROS reduction in the CAPE-FPBA-DO group compared to the LPS group, potentially linked to CAPE-FPBA-DO's ability to scavenge cytosolic ROS. This activity may conceivably increase glutathione transport into mitochondria and boost the Nrf2 antioxidant pathway, mechanisms that would contribute to a partial reduction in mtROS1,2. However, upon rigorous statistical analysis, this reduction did not reach a level of statistical significance (P > 0.05). Therefore, we have now clarified this point in the revised manuscript by modifying the sentence. To ensure full transparency and to facilitate the review process, we have made the underlying raw data for the quantitative analysis available (Response Fig. 2).\nBefore revision: “In addition, cells treated with CAPE-FPBA-DO showing no obvious mtROS reduction compared to the LPS group, further confirming that C@AH-EVs specifically target mtROS scavenging.”\nAfter revision (Results section, Page 14): “In addition, although a slight visual decrease in mtROS was observed in cells treated with CAPE-FPBA-DO, this change was not statistically significant. This results further confirming that C@AH-EVs specifically target mtROS scavenging.”\nResponse Figure 2. Raw data for the quantitative analysis of MitoSOX fluorescence in RAW 264.7 cells treated with PBS, AH-EVs, CAPE-FPBA-DO, and C@AH-EVs for 24 h. Red: MitoSOX, Blue: Hoechst. Scale bar, 20 μm (n = 3 biologically independent samples).\nReferences\n1. Mari, M., et al. Mitochondrial glutathione: features, regulation and role in disease. Biochim Biophys Acta 1830, 3317-3328 (2013).\n2. Dinkova-Kostova, A.T. & Abramov, A.Y. The emerging role of Nrf2 in mitochondrial function. Free Radic Biol Med 88, 179-188 (2015).\n5. In both in vitro and in vivo assays on mtROS clearance and M1 repolarization, the authors did not demonstrate that loading CAPE-FPBA-DO into AH-EVs offers a clear advantage over loading free CAPE. It is therefore recommended to include a CAPE@AH-EVs group as an additional control in both cell-based and animal studies. Furthermore, in the animal experiments, a clinically used wound dressing for diabetic wound healing should be included as a reference control.\nResponse: We thank the reviewer for this insightful suggestion, which would indeed provide a direct comparison of the therapeutic efficacy between prodrug complex and the free drug. According to your suggestion, we have conducted experiments to include the CAPE@AH-EVs group in in vitro study. The results shown that while both CAPE@AH-EVs and C@AH-EVs reduced mtROS levels, a critical difference emerged in their functional outcomes: only C@AH-EVs robustly promoted M2 macrophage polarization. The CAPE@AH-EVs likely leads to a widespread and potentially excessive scavenging of mtROS. It is effective at reducing oxidative stress, but this global quenching may also ablate the physiological levels of mtROS that are essential for M2 polarization. In contrast, the mtROS responsive C@AH-EVs provide a more nuanced clearance of pathologically elevated mtROS. This precise intervention is sufficient to alleviate oxidative damage yet is preserved the requisite redox dynamics necessary to support the repolarization of macrophages.\nIn the in vivo study, we also evaluated the therapeutic efficacy of CAPE@AH-EVs and clinically used agent Beifuji (alongside free drug and single-membrane vesicle groups suggested by Reviewer #3) on wound healing. The results demonstrated that the C@AH-EVs group significantly promoted wound closure, enhanced granulation tissue formation, and boosted angiogenesis compared to other groups.\nBefore revision:\n(1) “Furthermore, these cells exhibited obvious upregulation of the regeneration genes including Arg1, Mrc1, Ym1, and Fizz1…Altogether, these results indicate that C@AH-EVs can effectively regulate the phenotype of macrophages.”\n(2) “Neovascularization is a key characteristic during the proliferative stage of wound healing…This suggests that C@AH-EVs hold great potential in facilitating wound healing by augmenting angiogenesis.”\nAfter revision:\n(1) (Results section, Page 16) “Furthermore, these cells exhibited obvious upregulation of the regeneration genes including Arg1, Mrc1, Ym1, and Fizz1…Altogether, these results indicate that C@AH-EVs can effectively regulate the phenotype of macrophages.\nNotably, we prepared hybrid membrane vesicles loaded with an equivalent mass of CAPE (CAPE@AH-EVs) as a control to evaluate the potential advantages of C@AH-EVs. The results demonstrated that whereas both AH-EVs effectively attenuated mtROS levels and reduced the proportion of M1 macrophages, only C@AH-EVs significantly promoted M2 polarization (Supplementary Fig. 30, 31). This discrepancy is likely attributable to the excessive scavenging of mtROS by CAPE@AH-EVs, Which may obliterate the physiological levels of mtROS essential for driving M2 polarization.”\nSupplementary Figure 30. C@AH-EVs inhibit M1 macrophage polarization. a Schematic illustration of the experimental procedure. RAW 264.7 cells were stimulated with LPS. After 12 h, cells were treated with AH-EVs for another 24h, or they remained untreated. Untreated macrophages served as control. Representative CLSM images (b) and quantitative analysis (c) of MitoSOX fluorescence in RAW 264.7 cells. Red: MitoSOX, Blue: Hoechst. Scale bar, 20 μm (n = 3 biologically independent samples). d Flow cytometry analysis of CD86+ macrophages (n = 3 biologically independent samples). e Relative mRNA expression of proinflammatory genes (Tnfα, Nos2, Il6, and Cxcl1) (n = 3 biologically independent samples). Data are presented as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test was used in (c), (d) and (e). Source data are provided as a Source data file.\nSupplementary Figure 31 C@AH-EVs promote M2 macrophage polarization. a Schematic illustration of the experimental procedure. RAW 264.7 cells were first stimulated with LPS for 12 hours. Subsequently, the cells were received a subsequent 24 hours treatment with either IL-4 alone or IL-4 co-treated with AH-EVs. b Flow cytometry analysis of CD206+ macrophages (n = 3 biologically independent samples). c Relative mRNA expression of regeneration genes (Arg1, Mrc1, Ym1, and Fizz1) (n = 3 biologically independent samples). Data are presented as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test was used in (b) and (c). Source data are provided as a Source data file.\n(2) (Results section, Page 23) “Neovascularization is a key characteristic during the proliferative stage of wound healing…This suggests that C@AH-EVs hold great potential in facilitating wound healing by augmenting angiogenesis.\nBesides, macrophage membrane-derived artificial EVs (AMM-EVs), mitochondria membrane-derived artificial EVs (AMitoM-EVs), CAPE, CAPE@AH-EVs, and clinically used agent Beifuji were also served as controls to evaluate the therapeutic efficacy of C@AH-EVs. The results demonstrated that diabetic wounds treated with C@AH-EVs exhibited accelerated healing than other groups (Supplementary Fig. 44). Consistent with this, the C@AH-EVs group formed more mature granulation tissue, accompanied by accelerated re-epithelialization, skin appendage regeneration, and neovascularization relative to all controls (Supplementary Fig. 45).”\nSupplementary Figure 44. C@AH-EVs accelerate wound closure in diabetic mice. Digital photos of wounds (a) and traces of wounds closure (b) in different groups during the treatment period. Scale bar, 5 mm. The digital photos were representative data from six biologically independent wounds. c Quantitative analysis of wound area over time (n = 6 biologically independent wounds). d Quantitative analysis of wound closure rate over time (n = 6 biologically independent wounds). Data are presented as mean ± SD. Two-way ANOVA with Dunnett’s multiple comparisons was used in (d). Source data are provided as a Source data file\nSupplementary Figure 45. Histological evaluation of wound healing under different treatments. a Histological images of wound tissues stained with CD31, VEGF, H&E, and Masson's trichrome. Scale bar, 100 μm. The images were representative data from four biologically independent wounds. Quantitative analysis of VEGF expression (b), CD31 expression (c), granulation tissue thickness (d), gland count per HPF (e), hair follicle counts per HPF (f), and collagen content (g) (n = 4 biologically independent samples). Data are presented as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test was used in (b-g). Source data are provided as a Source data file.\nReviewer #3 (Remarks to the Author):\nThis manuscript presents a novel hybrid membrane-derived artificial extracellular vesicle (C@AH-EV) system, integrating macrophage and mitochondrial membranes with a ROS-responsive prodrug (CAPE-FPBA-DO), for the treatment of diabetic wounds. The study is well-designed, with a systematic evaluation that spans from material characterization to in vivo therapeutic effects. The manuscript is generally well-structured. However, several critical issues remain and must be properly addressed before the manuscript can be considered further.\nResponse: We sincerely thank you for your time and effort in reviewing our manuscript and for providing these insightful and constructive comments. We highly appreciate your positive assessment of our study's design and the novelty of the C@AH-EV system. We have carefully considered all the points you raised and have conducted additional experiments and analyses to address them thoroughly. The revisions have significantly improved the quality of our manuscript. Below, we provide a point-by-point response to your comments.\nComments:\n1. The authors should more clearly clarify the unique innovations of this study.\nResponse: We sincerely thank the reviewer for this valuable suggestion. The innovation is crucial. The innovation of this study lies in the development of a mitochondria-targeted and cell-specific nanotherapeutic that mimics endogenous communication mechanisms, offering a robust and precise mtROS scavenging strategy applicable to wound healing and other disorders. We have now more explicitly highlighted in the revised manuscript.\nBefore revision: “Herein, we report a macrophage-mitochondria hybrid membrane-derived artificial extracellular vesicle platform (C@AH-EV) that enables macrophage mitochondria-specific, on-demand mtROS scavenging. These biomimetic nanovesicles leverage membrane protein-mediated targeted of macrophage mitochondria to offer highly selective intracellular delivery”\n“This study presents a promising on-demand mtROS scavenging nanovesicle system, and offers the potential for broader therapeutic applications beyond wound healing.”\nAfter revision\n(Abstract section, Page 2) “Herein, we report a macrophage-mitochondria hybrid membrane-derived artificial extracellular vesicle platform (C@AH-EV) that enables macrophage mitochondria-specific, on-demand mtROS scavenging. Unlike small-molecule compounds or synthetic nanoparticles, C@AH-EV uniquely integrates biomimetic dual-targeting membranes with an mtROS activatable prodrug design to realize organelle-level precision and dynamic responsiveness. These biomimetic nanovesicles leverage membrane protein-mediated targeted of macrophage mitochondria to offer highly selective intracellular delivery.”\n(Introduction section, Page 4): “This study presents a mitochondria-targeted and cell-specific nanotherapeutic that mimics endogenous communication mechanisms, offering a robust and precise mtROS scavenging strategy applicable to wound healing and other disorders.”\n2. Please explain in greater detail why nanoscale therapeutics provide advantages over currently applied small-molecule antioxidants in diabetic wound healing.\nResponse: We thank the reviewer for raising this critical question. C@AH-EVs provide key advantages over small-molecule antioxidants by enabling targeted and on-demand action. Hybrid-membrane vesicle used in this system provides the precise macrophage mitochondrial delivery, independent of the impaired mitochondrial membrane potential, and protects the prodrug to ensure therapeutic concentrations at the site. Crucially, the system releases the antioxidant in a mtROS concentration dependent manner, which scavenges mtROS to a beneficial homeostatic level rather than quenching it entirely. This preserves the essential signaling role of mtROS in promoting M2 macrophage polarization, which play the important role in the process of wound healing.\nBefore revision: “Targeting the clearance of mtROS in macrophages is an effective approach to promote wound healing. Although numerous mitochondrial-targeting strategies have been developed, their translational application value is significantly limited due to inefficiencies in targeted delivery systems, poor intracellular drug stability, and potential toxicity risks to healthy tissue. In this study, hybrid membrane vesicles formed by macrophage membrane and mitochondrial membrane offer a safer and more effective strategy. Native cellular membranes confer inherent biocompatibility and enhanced safety to the C@AH-EVs. Mitochondrial targeting is achieved by the mitochondrial membrane, which operates independently of mitochondrial membrane potential, offering significant advantages over membrane potential-dependent molecular targeting strategies, such as lipophilic cations.”\nAfter revision (Discussion section, Page 22): “Targeting the clearance of mtROS in macrophages is an effective approach to promote wound healing. Although numerous mitochondrial-targeting strategies such as small-molecule compounds and synthetic nanoparticles have been developed, their translational application value is significantly limited due to inefficiencies in targeted delivery systems, poor intracellular drug stability, and potential toxicity risks to healthy tissue. Importantly, most existing systems focus solely on scavenging mtROS, which may lead to excessive depletion of physiological mtROS levels and thereby inhibit M2 macrophage polarization. This study effectively addresses these challenges by integrating macrophage-mitochondrial hybrid membrane vesicles with ROS-responsive prodrugs. The vesicles not only exhibit an improved safety profile but, more importantly, achieve specific delivery to macrophage mitochondria, independently of the mitochondrial membrane potential. Furthermore, its nano-encapsulation capability protects the prodrug from degradation, thereby ensuring the attainment of effective local drug concentrations. Upon internalization into the macrophage mitochondria, the prodrug CAPE-FPBA-DO were released and exhibits mtROS concentration-dependent antioxidant activity. This mechanism not only exerts anti-inflammatory effects but also maintains mtROS at a lower, homeostatic level, thereby preventing the impairment of M2 macrophage polarization caused by excessive mtROS scavenging.”\n3. The authors claim that the mechanism of macrophage and mitochondrial targeting is attributed to membrane proteins, but the key proteins involved have not been validated.\nResponse: We thank the reviewer for this critical comment. Identifying the precise protein pairs would be very interesting. In our study, we primarily focused on providing functional evidence that membrane proteins as a whole are essential for the targeting process. Our results demonstrate that clathrin- and lipid raft-mediated endocytosis play crucial roles in macrophage targeting, suggesting that their associated proteins are likely key molecules involved in macrophage targeting. For mitochondrial targeting, MFN play the important role, though other proteins may also participate in this process. We have now revised the manuscript to more clearly articulate this point.\nBefore revision: “… This discrepancy may be attributed to the reduced surface area of macrophage membranes in the AH-EVs and suboptimal drug dosage.”\nAfter revision (Discussion section, Page 23): “... This discrepancy may be attributed to the reduced surface area of macrophage membranes in the AH-EVs and suboptimal drug dosage.\nBesides, our results indicate that the targeting capability of C@AH-EVs is closely associated with membrane proteins. Toward macrophages targeting, it is achieved through the CME and LrME, suggesting that proteins associated with these pathways may serve as key mediators in homologous targeting. Regarding mitochondrial targeting, we have verified a strong association with mitofusin (MFN), but it is unclear whether this involves synergy with other proteins. Consequently, the specific contributions and functional interplay of these proteins require further experimental validation.”\n4. Tissue penetration depth and mechanism should be addressed. As extracellular vesicles generally do not offer inherent advantages in tissue penetration, further clarification is needed.\nResponse: We thank the reviewer for raising this crucial point regarding tissue penetration. Indeed, extracellular vesicles often face limitations in deep tissue penetration due to their size and nonspecific interactions. In this study, we administered the formulation via subcutaneous injection to circumvent this issue of low tissue penetration. Furthermore, due to the macrophage membrane components in the hybrid membrane vesicles, C@AH-EVs are more readily recruited to inflammatory sites within the diabetic wound environment, which may consequently enhance their tissue penetration depth. To further evaluate the penetration depth, DiI-labeled C@AH-EVs were subcutaneously injected around the diabetic wound. The results demonstrated their effective distribution throughout the full thickness of the wound tissue (Supplementary Fig. 33). The corresponding results have been included in the revised manuscript.\nBefore revision: “First, the diabetic wound model was created by injecting streptozotocin at a dose of 50 mg/kg/day for 5 days, followed by surgical removal of 10-mm-diameter full-thickness skin after consistent blood glucose levels over 16.65 mmol/L for one week. Subsequently, PBS, AH-EVs, CAPE-FPBA-DO, and C@AH-EVs were subcutaneously administrated at the wound sites, respectively. Considering the prolonged inflammation associated with diabetic wounds, in vivo macrophage mtROS levels and macrophage phenotypes were determined on the day 3, 7, and 14.”\nAfter revision (Result section, Page 18): “First, the diabetic wound model was created by injecting streptozotocin at a dose of 50 mg/kg/day for 5 days, followed by surgical removal of 10-mm-diameter full-thickness skin after consistent blood glucose levels over 16.65 mmol/L for one week. Subsequently, PBS, AH-EVs, CAPE-FPBA-DO, and C@AH-EVs were subcutaneously administrated at the wound sites, respectively.\nTracking of DiI-labeled C@AH-EVs exhibited an efficient distribution throughout the full thickness of the wound tissue (Supplementary Fig. 33). Considering the prolonged inflammation associated with diabetic wounds, in vivo macrophage mtROS levels and macrophage phenotypes were determined on the day 3, 7, and 14.”\nSupplementary Figure 33. Penetration of C@AH-EVs in wound tissue. Representative CLSM images of wound tissue sections with DiI-labeled C@AH-EV (red). Nuclei are counterstained with DAPI (blue) (n = 3 biologically independent samples).\n5. The control groups could be improved. Free drug and single-membrane vesicle groups should be included, and comparisons with clinically used medicines would better demonstrate the specific advantage of C@AH-EVs.\nResponse: We sincerely thank the reviewer for this excellent suggestion. Accordingly, we have now included additional control groups comprising free drugs (CAPE), single-membrane vesicles (AMitoM-EVs, AMM-EVs), AH-EVs encapsulating CAPE (CAPE@AH-EVs), and a clinically used wound healing agent (Beifuji, a recombinant growth factor formulation). The results consistently demonstrate that C@AH-EVs were more effective than other groups in promoting wound repair. These findings underscore the unique advantage of C@AH-EVs. The corresponding data have been incorporated into the revised manuscript.\nBefore revision: “Neovascularization is a key characteristic during the proliferative stage of wound healing…This suggests that C@AH-EVs hold great potential in facilitating wound healing by augmenting angiogenesis.”\nAfter revision (Results section, Page 22): “Neovascularization is a key characteristic during the proliferative stage of wound healing…This suggests that C@AH-EVs hold great potential in facilitating wound healing by augmenting angiogenesis.\nBesides, macrophage membrane-derived artificial EVs (AMM-EVs), mitochondria membrane-derived artificial EVs (AMitoM-EVs), CAPE, CAPE@AH-EVs, and clinically used agent Beifuji were also served as controls to evaluate the therapeutic efficacy of C@AH-EVs. The results demonstrated that diabetic wounds treated with C@AH-EVs exhibited accelerated healing than other groups (Supplementary Fig. 44). Consistent with this, the C@AH-EVs group formed more mature granulation tissue, accompanied by accelerated re-epithelialization, skin appendage regeneration, and neovascularization relative to all controls (Supplementary Fig. 45).”\nSupplementary Figure 44. C@AH-EVs accelerate wound closure in diabetic mice. Digital photos of wounds (a) and traces of wounds closure (b) in different groups during the treatment period. Scale bar, 5 mm. The digital photos were representative data from six biologically independent wounds. c Quantitative analysis of wound area over time (n = 6 biologically independent wounds). d Quantitative analysis of wound closure rate over time (n = 6 biologically independent wounds). Data are presented as mean ± SD. Two-way ANOVA with Dunnett’s multiple comparisons was used in (d). Source data are provided as a Source data file\nSupplementary Figure 45. Histological evaluation of wound healing under different treatments. a Histological images of wound tissues stained with CD31, VEGF, H&E, and Masson's trichrome. Scale bar, 100 μm. The images were representative data from four biologically independent wounds. Quantitative analysis of VEGF expression (b), CD31 expression (c), granulation tissue thickness (d), gland count per HPF (e), hair follicle counts per HPF (f), and collagen content (g) (n = 4 biologically independent samples). Data are presented as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test was used in (b-g). Source data are provided as a Source data file.\n6. The authors should clarify whether blood glucose levels remained within the diabetic range throughout the wound healing study, to ensure that the model reflects a persistent diabetic state.\nResponse: We thank the reviewer for this critical point. We systematically monitored blood glucose levels and body weight throughout the entire wound healing study to confirm the stability of diabetic model. The data demonstrate that all animals in the diabetic groups sustained hyperglycemia (Blood glucose levels > 16.65 mmol/L) and exhibited characteristic slow body weight gain typical of progressive diabetes (Supplementary Fig. 32). These findings confirm a persistent diabetic state throughout the experiment. We have showed these data in the Results section.\nBefore revision: “First, the diabetic wound model was created by injecting streptozotocin at a dose of 50 mg/kg/day for 5 days, followed by surgical removal of 10-mm-diameter full-thickness skin after consistent blood glucose levels over 16.65 mmol/L for one week. Subsequently, PBS, AH-EVs, CAPE-FPBA-DO, and C@AH-EVs were subcutaneously administrated at the wound sites, respectively.”\nAfter revision (Results section, Page 18): “First, the diabetic wound model was created by injecting streptozotocin at a dose of 50 mg/kg/day for 5 days, followed by surgical removal of 10-mm-diameter full-thickness skin after consistent blood glucose levels over 16.65 mmol/L for one week. The persistent hyperglycemia and impaired weight gain collectively demonstrate that the mice maintained a sustained diabetic state throughout the experimental period (Supplementary Fig. 32). Subsequently, PBS, AH-EVs, CAPE-FPBA-DO, and C@AH-EVs were subcutaneously administrated at the wound sites, respectively.”\nSupplementary Figure 32. Records of blood glucose (a) and body weight (b) in mouse models within two weeks (n = 6 biologically independent samples). Data are presented as mean ± SD.\n7. The therapeutic administration in this study was performed via subcutaneous injection at the wound site. While this approach ensures local delivery in the animal model, the authors should provide an evaluation of the overall safety of the system under this administration route. In addition, it would be valuable for the authors to clarify whether alternative delivery methods, such as topical application or wound dressings, could be feasible, as these are more commonly used in clinical diabetic wound management.\nResponse: We sincerely thank the reviewer for raising these important points regarding safety and clinical translation. To evaluate the safety of C@AH-EVs system administered via subcutaneous injection, we have conducted comprehensive biosafety assessments including histological examination and serum biochemical analysis of major organs, all of which revealed no indications of significant systemic toxicity (Supplementary Fig. 22, 23).\nRegarding alternative delivery methods, while the subcutaneous injection approach has yielded promising results in our study, non-invasive therapeutic strategies (such as hydrogels and wound dressings) hold greater potential for clinical application due to their improved feasibility and patient compliance. Therefore, in our subsequent research, we will focus on enhancing the tissue penetration depth of these nanomaterials by employing more efficient delivery systems to accelerating their translation into clinical practice. These additional safety and applicability considerations have been incorporated into the revised manuscript.\nBefore revision:\n(1) “…achieving a 69% encapsulation efficiency for CAPE-FPBA-DO and a loading capacity of 403 ng/1010 particles (Fig. 3h, Supplementary Fig. 20). Furthermore, C@AH-EVs maintained size stability for at least 72 h in both PBS and RPMI medium (Fig. 3i).”\n(2) “In summary, we developed a novel drug delivery system capable of on-demand and precise clearance of mtROS in macrophages.”\nAfter revision:\n(1) (Results section, Page 7): “…achieving a 69% encapsulation efficiency for CAPE-FPBA-DO and a loading capacity of 403 ng/1010 particles (Fig. 3h, Supplementary Fig. 20). Subsequent CCK-8 assays and histological analysis further confirmed the biosafety of the prepared C@AH-EVs (Supplementary Fig. 21-23). Furthermore, C@AH-EVs maintained size stability for at least 72 h in both PBS and RPMI medium (Fig. 3i).”\nSupplementary Figure 23. Potential toxicity of the C@AH-EVs on major organs. a. H&E images of major organs harvested on day 14 after receiving daily injections of PBS or C@AH-EVs for 14 consecutive days. Scale bar, 200 μm. The H&E images were representative data from three biologically independent wounds. b. Plasma levels of ALT, AST, Cr, and CK in different groups on day 14 (n = 3 biologically independent samples). Data are presented as mean ± SD. Two-tailed Student’s t test was used. Source data are provided as a Source data file.\n(2) (Discussion Page 23): “As mimics of EVs, C@AH-EVs inherently lack sufficient tissue penetration capability. While the subcutaneous injection approach has yielded promising results in our study, non-invasive therapeutic strategies (such as hydrogels and wound dressings) hold greater potential for clinical application due to their improved feasibility and patient compliance. Therefore, in our subsequent research, we will focus on enhancing the tissue penetration depth of these nanomaterials by employing more efficient delivery vehicles to accelerating their translation into clinical practice… Hence, elucidating their in vivo behavior will enable a more efficient path toward clinical translation.\nIn summary, we developed a novel drug delivery system capable of on-demand and precise clearance of mtROS in macrophages.”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript is much improved, and I appreciate the authors' thoughtful responses and the changes made throughout the text and figures. The work is now significantly clearer, and I would be happy to recommend its publication.\nHowever, there is still one issue that remains unclear to me and that should be addressed.\nThe new legend for Supplementary Figures 11 and 12 has greatly enhanced the readability and interpretation of the NMR data. However, while the CA spectrum shows all expected peaks with good definition, the CAPE spectrum displays poor resolution, with small and unclear peaks, being difficult to confirm the purity of the product.\nMore critically, in the CA/CAPE-FPBA-DO spectra in both figures and all timepoints, only a few peaks are visible (around 1.25, 3.5, and 4.75 ppm) which are not theoretically attributed to any of the signals of both molecules. None of the expected aromatic signals from either CA or FPBA is visible. This suggests that there may still be an issue with data quality or sample preparation, and I would recommend a careful re-evaluation of these spectra.", "reviewer_2": "The authors have adequately addressed my comments, and the revised manuscript shows clear improvement. Therefore, I recommend acceptance.", "reviewer_3": "The authors have addressed my comments completely, I suggest accepting the paper at its current version."}, "author_response": "We are extremely grateful for your positive feedback on the revisions we made to the previous version, as well as the critical observations regarding the NMR spectra, which provide an opportunity to clarify the experimental design and interpretation. Due to the poor aqueous solubility of the conjugates, direct NMR analysis in deuterated solvents with D O was challenging to get well-resolved spectra. 2 As a result, we conducted the experiment in deuterated PBS with surfactant Brij 58 which emulsified the conjugates (CAPE/CA-FPBA-DO) to form micelles while give simple peaks in the NMR spectra. For consistency in experimental conditions, the free drug control group (CAPE/CA) was treated in the same manner. In micelles, the molecular components present on the solvated surface with sufficient mobility give rise to detectable or well resolved resonance peaks (CAPE/CA), while the internal core of particles is tightly packed with much lower mobility that is “invisible” in the NMR analysis (emulsified CAPE/CA-FPBA-DO)1. Notably, the free drug CAPE, being more hydrophobic than CA, exhibited NMR signals with lower resolution in the presence of the surfactant. Nevertheless, these signals remained valuable for observing the hydrolysis behavior. We apologize for the lack of clarity in the presentation of the Figure 11 and 12. To validate the results, we repeated the experiments again and performed detailed peak assignments to obtain clearer spectral data. The resulting spectra and corresponding explanations have been included in the supplementary file.\nBefore revision:\nSupplementary Figure 11. Hydrolytic Stability of CAPE-FPBA-DO in PBS by 1H NMR. Spectra of CAPE-FPBA-DO (5 mg in 1mL PBS-D/DMSO-d6, 8:2 v/v, with 0.5 wt% Brij-58) were acquired at 0, 24, and 72 h. Reference spectrum of CAPE (5 mg) prepared under identical conditions. Preparation of PBS-D (12.5 mM): A 20 mg PBS tablet was dissolved in 1.6 mL D₂O.\nSupplementary Figure 12. Hydrolysis stability of CA-FPBA-DO in PBS by 1H NMR. Spectra of CA-FPBA-DO (5 mg in 1mL PBS-D/DMSO-d6, 8:2 v/v, with 0.5 wt% Brij-58) were acquired at 0, 24, and 72 h. Reference spectrum of CA (5 mg) prepared under identical conditions. Preparation of PBS-D (12.5 mM): A 20 mg PBS tablet was dissolved in 1.6 mL D₂O.\nAfter revision: (Supplementary information, Page 41, 42)\nSupplementary Figure 11. Hydrolytic Stability of CAPE-FPBA-DO in PBS by 1H NMR. Spectra of CAPE-FPBA-DO (5 mg in 1mL PBS-D/DMSO-d6, 8:2 v/v, with 0.5 wt% Brij-58) were acquired at 0, 24, and 72 h. Reference spectrum of CAPE (5 mg) prepared under identical conditions. Preparation of PBS-D (12.5 mM): A 20 mg PBS tablet was dissolved in 1.6 mL D₂O. Brij 58 was used to emulsify the conjugates/prodrugs to overcome poor aqueous solubility, which can also provide a mimicry of the EV environment. Under this condition, free CAPE was readily detectable due to its surface-accessible mobility, whereas the CAPE-FPBA-DO conjugates, sequestered within the micelle core, remained largely undetectable1.\nSupplementary Figure 12. Hydrolysis stability of CA-FPBA-DO in PBS by 1H NMR. Spectra of CA-FPBA-DO (5 mg in 1mL PBS-D/DMSO-d6, 8:2 v/v, with 0.5 wt% Brij-58) were acquired at 0, 24, and 72 h. Reference spectrum of CA (5 mg) prepared under identical conditions. Preparation of PBS-D (12.5 mM): A 20 mg PBS tablet was dissolved in 1.6 mL D₂O. Brij 58 was used to emulsify the conjugates/prodrugs to overcome poor aqueous solubility, which can also provide a mimicry of the EV environment. Under this condition, free CA was readily detectable due to its surface-accessible mobility, whereas the CA-FPBA-DO conjugates, sequestered within the micelle core, remained largely undetectable1.\nReferences\n1. Wang, M.M., et al. Elucidation of lipid nanoparticle surface structure in mRNA vaccines. Sci Rep 13, 16744 (2023).\nReviewer #2 (Remarks to the Author):\nThank you very much for your positive feedback and for recommending our manuscript for acceptance. We are deeply grateful for the time and thoughtful consideration you have dedicated to reviewing our work. Your constructive comments throughout the review process have been invaluable in strengthening the manuscript. We sincerely appreciate your support.\nReviewer #3 (Remarks to the Author):\nWe sincerely thank you for the valuable time and for acknowledging our revisions. We are very pleased that the manuscript in its current form meets with your approval, and we are grateful for your constructive input throughout the review process, which has been instrumental in enhancing the quality of our work."}, {"round": 3, "reviewer_comments": {"reviewer_1": "I thank the authors for their detailed response and for clarifying the use of Brij 58 to address solubility issues in the NMR experiments. While this explanation helps rationalize the poor resolution and partial signal suppression observed in micellar systems, it does not resolve the central problem with this experiment.\nThe stated goal of Supplementary Figures 11 and 12 is to assess the stability of CA/CAPE FPBA DO over time. However, in the 1H NMR spectra of CA FPBA DO at 0, 24, and 72 h, the only visible resonances correspond to Brij 58 (≈1.2, 3.5, and 4.9 ppm), as indicated by the authors’ assignments. Importantly, none of the resonances attributable to CA FPBA DO are visible at any time point, nor are signals corresponding to free CA observed. Under these conditions, it is therefore unclear how any conclusions regarding conjugate stability can be drawn from the NMR data, since the species of interest is not spectroscopically observable.\nMoreover, the authors state that free CA is “readily detectable due to its surface accessible mobility,” while the conjugate remains sequestered within the micelle core. However, this behavior is not reflected in the presented spectra: in all three time points, no signals corresponding to either conjugated or released CA are observed. As such, the NMR data does not support the interpretation provided in the manuscript and, in fact, appear inconsistent with the conclusions drawn from them.\nI fully understand the experimental challenges associated with the poor aqueous solubility of these systems. Nevertheless, if the compound under investigation is not detectable under the chosen NMR conditions, the authors should therefore provide alternative NMR conditions where the conjugate (or released CA/CAPE) is directly observable. One possibility would be to use a larger percentage of a co-solvent such as d6-DMSO/D2O.\nIn the end, if this is still not successful, then the presented UV-Vis data may be sufficient to support stability claims. But the NMR data in its current form must be removed from the manuscript, as well as the conclusions supported on this data.\nI apologize for my persistence, as it is not my intent to delay publication, but I must ensure that any conclusions presented in the final version are rigorously supported by the data."}, "author_response": "Thank you once again for reviewing our response and providing this crucial and insightful feedback. Our initial assumption was that CAPE/CA could only be detected by NMR after being hydrolyzed and released. However, since neither the resonances attributable to conjugates (CAPE/CA-FPBA-DO) nor the signals corresponding to free drug (CAPE/CA) observed in the spectra, this explanation indeed appears to be insufficiently rigorous. We apologize for this oversight.\nTo address this issue, we conducted the following experiments: We reduced the Brij 58 concentration to 0.25% (from the previous 0.5%) to minimize its interference with the NMR, and simultaneously lowered the conjugates concentration to 2.0 mM (from the previous 10 mM) to improve solubility. Under this condition, slight hydrolysis of CA-FPBA-DO was observed (Response Fig. 1). However, CAPE-FPBA-DO remained undetectable, which is indeed unfavorable for monitoring its hydrolysis (Response Fig. 2). Subsequently, we removed Brij 58 and increased the proportion of DMSO/D₂O as you suggested. In 60% DMSO-d6/D2O (v/v), signals attributed to CA and CA-FPBA-DO could be observed. However, hydrolysis percentage from the integration remained difficult (Response Fig. 3). The NMR signals for CAPE-FPBA-DO were still difficult to observe clearly under this adjusted condition (Response Fig. 4). As a result, we removed these NMR data (Supplementary Figures 11 and 12), as well as the conclusions supported on this data.\nWe are once again deeply grateful for your rigorous review and constructive comments, which have significantly enhanced the scientific rigor of our study. The aforementioned revisions have been incorporated into the manuscript.\nBefore revision:\n(Results section): “NMR results indicate that both CAPE-FPBA-DO and CA-FPBA-DO exhibit good hydrolytic stability at high concentrations. However, significant hydrolysis of CA-FPBA-DO is observed when the concentration is closer to the non-cytotoxic concentration (25 μM) (Fig. 2e, Supplementary Fig. 11-13).”\n(Supplementary Information):\nSupplementary Figure 11. Hydrolytic Stability of CAPE-FPBA-DO in PBS by 1H NMR. Spectra of CAPE-FPBA-DO (5 mg in 1mL PBS-D/DMSO-d6, 8:2 v/v, with 0.5 wt% Brij-58) were acquired at 0, 24, and 72 h. Reference spectrum of CAPE (5 mg) prepared under identical conditions. Preparation of PBS-D (12.5 mM): A 20 mg PBS tablet was dissolved in 1.6 mL D₂O. Brij 58 was used to emulsify the drugs due to poor aqueous solubility. Under these conditions, free CAPE (surface-mobile) was detectable, whereas the CAPE-FPBA-DO conjugates (micelle core) remained largely invisible.\nSupplementary Figure 12. Hydrolysis stability of CA-FPBA-DO in PBS by 1H NMR. Spectra of CA-FPBA-DO (5 mg in 1mL PBS-D/DMSO-d6, 8:2 v/v, with 0.5 wt% Brij-58) were acquired at 0, 24, and 72 h. Reference spectrum of CA (5 mg) prepared under identical conditions. Preparation of PBS-D (12.5 mM): A 20 mg PBS tablet was dissolved in 1.6 mL D₂O. Brij 58 was used to emulsify the drugs due to poor aqueous solubility.\nAfter revision\n(Results section, Page 5): “UV-Vis analysis indicated that CA-FPBA-DO undergoes significant hydrolysis at concentrations near the non-cytotoxic level (25 μM), whereas CAPE-FPBA-DO exhibits relatively good hydrolytic stability at this concentration (Fig. 2e, Supplementary Fig. 11).”\nResponse Figure:\nResponse Figure 1. Hydrolysis of CA-FPBA-DO in PBS-D monitored by 1H NMR (2.0 mM in PBS-D). The hydrolysis degree was estimated by the integrations of peak d using peak a as the internal standard. Preparation of PBS-D with 0.25 wt% brij 58: PBS tablet 50 mg was dissolved in 5 mL D O to make a PBS-D solution. Then 12.5 mg of brij 58 was added to the 2 PBS-D solution to make a 0.25 wt% solution for the surfactant. Preparation of CA-FPBA-DO solution for NMR: 200 µL of CA-FPBA-DO in DMSO-d6 (10 mM) was added to 800 µL of the above PBS-D solution with 0.25 wt% surfactant. The mixture was vortexed for 30 s before NMR analysis.\nResponse Figure 2. Hydrolytic stability of CAPE-FPBA-DO in PBS emulsified by brij 58 (2.0 mM in PBS-D). Preparation of PBS-D with 0.25 wt% brij 58: PBS tablet 50 mg was dissolved in 5 mL D2O to make a PBS-D solution. Then 12.5 mg of brij 58 was added to the PBS-D solution to make a 0.25 wt% solution for the surfactant. Preparation of CAPE-FPBA-DO solution for NMR: 200 µL of CA-FPBA-DO in DMSO-d6 (10 mM) was added to 800 µL of the above PBS-D solution with 0.25 wt% surfactant. The mixture was vortexed for 30 s before NMR analysis.\nResponse Figure 3. ¹H NMR spectra of CA-FPBA-DO (2.0 mM) in PBS-D/DMSO-d₆ mixed solvents with varying ratios. The spectra were acquired in four solvent conditions: pure DMSO-d₆ (a), PBS-D/DMSO-d₆ 8:2 v/v (b), 6:4 v/v (c), and 4:6 v/v (d). All spectra did not exhibit well-resolved peaks.\nResponse Figure 4. ¹H NMR spectra of CAPE-FPBA-DO (2.0 mM) in PBS-D/DMSO-d₆ mixed solvents with varying ratios. The spectra were acquired in four solvent conditions: pure DMSO-d₆ (a), PBS-D/DMSO-d₆ 8:2 v/v (b), 6:4 v/v (c), and 4:6 v/v (d). All spectra did not exhibit well-resolved peaks."}, {"round": 4, "reviewer_comments": {"reviewer_1": "I would like to thank the authors for the additional experiments performed during the revision process. It is now clear that solubility is the limiting factor in observing the hydrolysis of the conjugate by NMR. I am satisfied with the decision to remove the NMR data and the corresponding discussion in the manuscript. The remaining UV-Vis and HPLC results should suffice to support the conclusions regarding conjugate stability. I have no further concerns and recommend the publication of this work."}, "author_response": "We sincerely appreciate your meticulous review and thoughtful feedback on our manuscript. Your insights and constructive suggestions have been invaluable in enhancing the clarity and rigor of our work. We are pleased that our revisions have addressed your concerns and are grateful for your support for the publication of this study. Thank you once again for dedicating your time and expertise."}]

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