Abstract
Background:
Diabetic foot ulcers (DFUs) represent a severe global health challenge, often characterized by persistent oxidative stress and a stalled inflammatory phase that prevents normal wound healing. Chronic accumulation of reactive oxygen species (ROS) leads to an inflammatory lock where macrophages remain in a pro-inflammatory M1 phenotype. This study aims to develop kiwifruit-derived plant extracellular vesicles (K-PEVs) as a sustainable, cell-free therapeutic platform to remodel the diabetic wound microenvironment and accelerate regeneration.
Methods:
K-PEVs were isolated from fresh kiwifruit juice using differential centrifugation combined with ultracentrifugation. The vesicles were characterized via transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS). In vitro, the effects of K-PEVs on HaCaT cell migration, NIH3T3 cell viability, and ROS scavenging in Raw264.7 macrophages were evaluated. In vivo, a full-thickness foot wound model in STZ-induced diabetic rats was established to assess the healing efficacy of low-dose (5 μg/mL) and high-dose (20 μg/mL) K-PEV treatments. Histological (H&E, Masson’s trichrome) and immunofluorescence (CD86) analyses were performed to evaluate tissue remodeling and macrophage polarization.
Results:
K-PEVs exhibited a typical cup-shaped morphology with an average diameter of approximately 155 nm and high colloidal stability. In vitro assays demonstrated that K-PEVs dose-dependently promoted HaCaT migration and NIH3T3 viability while significantly scavenging intracellular ROS in Raw264.7 cells. In vivo, K-PEV treatment significantly accelerated wound closure, enhanced re-epithelialization, and increased collagen deposition. Notably, immunofluorescence staining revealed a marked reduction in CD86 expression in K-PEV-treated wounds, indicating a successful transition from the M1 pro-inflammatory phenotype to a pro-reparative microenvironment.
Conclusion:
Our findings demonstrate that K-PEVs effectively promote diabetic wound healing by mitigating oxidative stress, resolving chronic inflammation through the modulation of macrophage polarization, and stimulating essential cellular activities. K-PEVs represent a promising green nanomedicine strategy for the clinical management of chronic diabetic ulcers.
1 Introduction
Diabetes Mellitus (DM) has emerged as one of the most daunting global public health challenges of the twenty-first century, with its prevalence increasing at an explosive rate worldwide (Sun et al., 2022). According to the latest data from the International Diabetes Federation (IDF) 2025 report, approximately 589 million adults (aged 20–79) were living with diabetes in 2024, a figure projected to surge to 853 million by 2050 (Sun et al., 2022). Beyond systemic metabolic dysregulation, DM triggers a spectrum of severe chronic complications, among which Diabetic Foot Ulcers (DFU) represent a primary cause of disability, mortality, and staggering healthcare expenditures (Armstrong et al., 2017). Statistics indicate that 19%–34% of diabetic patients will experience a foot ulcer during their lifetime (Mcdermott et al., 2023; Zhang et al., 2024; Everett and Mathioudakis, 2018). Consequently, developing novel regenerative strategies that possess high biocompatibility and targeting capabilities, which can precisely modulate the wound microenvironment, has become a pressing research priority in clinical medicine and biomaterials science.
Healthy wound healing is a highly orchestrated, dynamically overlapping process typically divided into four stages: hemostasis, inflammation, proliferation, and remodeling (Yang et al., 2023; Shan et al., 2025; Peña and Martin, 2024; Velnar et al., 2009; Talbott et al., 2022). However, in diabetic individuals, this orderly sequence is severely disrupted by hyperglycemia, peripheral neuropathy, peripheral vascular disease, and immune dysfunction (Mao et al., 2024; Verdolino et al., 2021). As a result, wounds often become stalled in a chronic inflammatory phase, failing to transition effectively into the reparative stage (Zhao et al., 2016; Powers et al., 2016; Zhao et al., 2024). Chronic hyperglycemia is the fundamental driver of oxidative stress within the diabetic wound; the excessive accumulation of reactive oxygen species (ROS) leads to catastrophic biological consequences, including direct damage to proteins, lipids, and DNA, which compromises cell membrane integrity and impairs signal transduction (Wan et al., 2022; Li et al., 2024). Macrophages play a pivotal role in coordinating wound healing, as their remarkable plasticity allows them to dynamically switch between the pro-inflammatory M1 phenotype and the pro-reparative M2 phenotype in response to microenvironmental cues (Wan et al., 2022; Wu et al., 2024). Under persistent stimulation from high ROS levels, advanced glycation end products (AGEs), and the hyperglycemic milieu, macrophages enter an inflammatory lock state—remaining persistently in the M1 phenotype and failing to respond to M2-inducing signals (Hassanshahi et al., 2022). The over-accumulation of M1 macrophages in diabetic wound tissue results in a sustained high concentration of pro-inflammatory cytokines (Zhao et al., 2024). This activates the overexpression of matrix metalloproteinases (MMPs), which excessively degrade the extracellular matrix (ECM) and nascent granulation tissue, ultimately leading to stalled wound healing (Schuster et al., 2023; Bainbridge, 2013). Given that persistent oxidative stress sustains M1 macrophage accumulation and delays the transition to the proliferative phase, targeted intervention in redox homeostasis to facilitate M1-to-M2 phenotypic switching has emerged as a critical approach to restore healing trajectory in diabetic wounds.
As key mediators of paracrine signaling, exosomes have demonstrated immense potential as alternatives to whole-cell therapies (Bian et al., 2022; Yang et al., 2024; Ha et al., 2016). Exosomes are lipid bilayer vesicles (30–150 nm in diameter) enriched with proteins, lipids, DNA, and various non-coding RNAs (e.g., miRNAs) derived from their parent cells (Coughlan et al., 2020; Wu et al., 2019; Purushothaman, 2019). Compared to whole cells, mammalian cell-derived exosomes (MDEs) offer lower immunogenicity, higher stability, enhanced ability to cross biological barriers, and are more amenable to industrial scale-up and standardized quality control (Ju et al., 2023; Narauskaitė et al., 2021; Guillamat-Prats, 2021). Nevertheless, the preparation of MDEs still faces challenges such as high expansion costs, batch-to-batch inconsistency due to parental cell heterogeneity, and the risk of cross-pathogen contamination (Zhao et al., 2022; Woith et al., 2021). In this context, plant-derived extracellular vesicles (PEVs) have attracted widespread attention in the biomedical field as an emerging and sustainable cell-free therapeutic platform (Woith et al., 2021; Tan et al., 2024; Seo et al., 2023). PEVs are extracted from various edible plants, fruits, or herbal medicines via differential centrifugation and are structurally and functionally analogous to mammalian exosomes (Zhao et al., 2022; Liu et al., 2024; Jin et al., 2025; Zhu et al., 2023; Yan et al., 2024). PEVs not only effectively encapsulate and protect internal bioactive molecules (such as plant polyphenols, antioxidant enzymes, and regulatory sequences) but can also be internalized by mammalian cells through cross-kingdom communication mechanisms to regulate target cell gene expression (Seo et al., 2023). For instance, extracellular vesicles derived from ginger (Li Z. et al., 2018; Suresh et al., 2025; Yin et al., 2022), grapes (Savcı et al., 2021; Castelli et al., 2023), and lemons (Jin et al., 2025; Raimondo et al., 2022) have shown significant efficacy in anti-inflammatory, antioxidant, and cancer therapies.
Kiwifruit is renowned for its exceptionally high content of bioavailable Vitamin C (ascorbic acid). Vitamin C is not only a potent free radical scavenger but also an essential cofactor for prolyl hydroxylase in collagen synthesis, which is critical for maintaining skin barrier integrity and promoting healing (Li Hy. et al., 2018; Mohajeri et al., 2014). Kiwifruit also contains polyphenols, flavonoids, actinidin (a proteolytic enzyme), Vitamin E, carotenoids, and minerals, all of which play roles in antioxidant activity, inflammation regulation, and the debridement of necrotic tissue (Li Hy. et al., 2018; Moysidou et al., 2024; He et al., 2019; Wang et al., 2021). Studies have shown that the direct application of Kiwifruit Extract (KFE) can significantly accelerate wound healing in diabetic rats and clinical DFU cases (Mohajeri et al., 2014). However, raw extracts are limited by low bioavailability and the susceptibility of active ingredients to oxidative degradation. Utilizing K-PEVs as carriers can effectively overcome these bottlenecks.
About this study, we hypothesized that K-PEVs, as natural antioxidant nanocarriers, possess the intrinsic capacity to modulate the oxidative stress microenvironment and orchestrate macrophage phenotypic switching, thereby breaking the cycle of impaired healing in diabetic wounds. Aim of the study: To test this hypothesis, we isolated K-PEVs from fresh kiwifruit and systematically investigated their (i) antioxidant properties and cytoprotective effects against oxidative damage in vitro, and (ii) therapeutic efficacy and underlying mechanisms involving macrophage polarization in a diabetic rat foot wound model. Our findings demonstrate that K-PEVs represent a promising plant-derived therapeutic strategy for chronic diabetic wound management (Scheme 1).
SCHEME 1
2 Materials and methods
2.1 Materials and reagents
Fresh kiwifruits (Actinidia chinensis) were purchased from a local market. Streptozotocin (STZ), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging kits were obtained from MCE. Cell counting kit-8 (CCK-8) was purchased from Biosharp. Antibodies for CD86 were obtained from Proteintech. All other analytical grade reagents were used as received.
2.2 Isolation and purification of K-PEVs
Fresh kiwifruit (Actinidia chinensis, Hayward variety) was thoroughly washed with sterile deionized water, surface-sterilized with 75% ethanol for 5 min, and rinsed three times with sterile PBS. K-PEVs were isolated using a combination of differential centrifugation and ultracentrifugation. Briefly, fresh kiwifruit was juiced and filtered. The juice was sequentially centrifuged at 500 g for 10 min and 2,000×g for 20 min (4 °C) to remove debris and organelles. The supernatant was filtered (0.22 μm) and ultracentrifuged at 120,000×g for 90 min (4 °C). The resulting pellet was resuspended in phosphate-buffered saline (PBS) and further purified/condensed using an Amicon Ultra-15 centrifugal filter (100 kDa cutoff). The final K-PEVs were resuspended and stored in sterile PBS (Ca2+/Mg2+-free, pH 7.4) at −80 °C.
2.3 Characterization of K-PEVs
TEM: K-PEVs were diluted to approximately 1 × 108 particles/mL with sterile PBS. A 10 μL aliquot was deposited onto a formvar/carbon-coated copper grid (400-mesh, glow-discharged) for 5 min at room temperature. Excess liquid was blotted with filter paper, and the sample was negatively stained with 2% uranyl acetate aqueous solution (w/v) for 2 min. After air-drying for 10 min, samples were examined using a JEM-1400 transmission electron microscope (JEOL, Japan) operating at 80 kV. Images were captured at magnifications of 30,000–80,000× using a digital CCD camera.
NTA: K-PEV samples were diluted 1:100 to 1:500 with particle-free PBS (0.01 μm filtered) to achieve a concentration of 1–10 × 108 particles/mL (within the linear detection range). Measurements were performed using NanoSight NS300 (Malvern Panalytical) equipped with a 405 nm laser and a CMOS camera. Three videos of 30 s each were recorded per sample (camera level = 16, detection threshold = 5). Data were analyzed using NTA 3.4 software, capturing motion under Brownian motion principles at 25 °C. Results represent the mean ± SD of three independent measurements.
DLS: Hydrodynamic diameter and zeta potential were measured using a Zetasizer Nano ZS (Malvern Panalytical). For size measurements, K-PEVs were diluted in PBS to a final concentration of 50–100 μg/mL (protein equivalent) and analyzed at 25 °C with a backscatter angle of 173° (NIBS optics). For zeta potential, samples were diluted in 10 mM HEPES buffer (pH 7.4) with conductivity ∼0.1 mS/cm and analyzed using folded capillary zeta cells (DTS1070). Three measurements of 10–15 runs each were performed per sample. Data are presented as intensity-weighted mean diameter (Z-average) and electrophoretic mobility-converted zeta potential.
2.4 Cell culture and viability assay
RAW264.7 cells and human keratinocyte HaCaT cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, ExCell Bio), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Cells at 80%–90% confluence were harvested using 0.25% trypsin-EDTA and subcultured at a 1:3 ratio every 2–3 days (passages 3–10 were used for experiments). Cell viability was assessed using the CCK-8 assay. Cells were seeded in 96-well plates and treated with various concentrations of K-PEVs (0, 5, 10, and 20 μg/mL) for 24 h before adding the CCK-8 reagent, Cell viability was calculated as: Viability (%) = (A treated–A blank)/(A control–A blank) × 100%, where A blank represents wells containing medium without cells, and A control represents untreated cells (0 μg/mL K-PEVs).
2.5 Cell scratch assay
To evaluate cell migration, a scratch assay was performed with HaCaT cell. A sterile 200 μL pipette tip was used to create a linear wound on the confluent cell monolayer. After washing with PBS, cells were treated with different concentrations of K-PEVs (5, 10, 20 μg/mL) in serum-free medium. Images were captured at 0 and 24 h using an inverted microscope, and the migration rate was calculated using ImageJ software.
2.6 Antioxidant activity and ROS scavenging
The DPPH radical scavenging activity of K-PEVs was measured according to the manufacturer’s instructions, Briefly, K-PEVs were serially diluted in methanol to final concentrations ranging from 5 to 20 μg/mL (5, 10, 20 μg/mL). An equal volume (200 μL) of 0.1 mM DPPH solution (freshly prepared in methanol) was added to each sample and vortexed thoroughly. The mixture was incubated in the dark at room temperature for 30 min, followed by centrifugation at 12,000×g for 5 min to pellet any precipitated material. The absorbance of the supernatant was measured at 517 nm using a microplate reader. The scavenging activity was calculated using the formula: Scavenging rate (%) = [1−(As − Ab)/Ac] × 100%, where Ac is the absorbance of DPPH without K-PEVs.
Intracellular ROS levels were detected using the DCFH-DA fluorescent probe. RAW264.7 macrophages (1 × 105 cells/well in 24-well plates) were pre-treated with H2O2 (200 μM, freshly diluted from 30% stock) for 2 h at 37 °C in serum-free DMEM to induce oxidative stress. Control cells received serum-free medium only. Raw264.7 cell were pre-treated with H2O2 to induce oxidative stress, followed by incubation with K-PEVs (5, 10, 20 μg/mL). Fluorescence intensity was observed via confocal microscopy and quantified.
2.7 In Vitro macrophage polarization assay (flow cytometry)
To evaluate the immunomodulatory effect of K-PEVs on macrophage polarization in vitro, RAW264.7 macrophages were seeded into 6-well plates at a density of 2 times 10^5 cells per well and incubated overnight to allow for cell attachment. The cells were then treated with various concentrations of K-PEVs (0, 5, 10, and 20 μg/mL) in fresh culture medium for 24 h. After the designated treatment period, the cells were gently harvested using a cell scraper to preserve surface antigens, washed twice with cold PBS, and resuspended in 100 μL of cell staining buffer. For phenotypic analysis, the cell suspensions were incubated with PE-conjugated anti-CD86 antibody (an M1 macrophage marker) and FITC-conjugated anti-CD206 antibody (an M2 macrophage marker) in the dark at 4 °C for 30 min. Following incubation, the cells were washed twice with cold PBS to remove unbound antibodies and finally resuspended in 300 μL of PBS. The fluorescence signals were acquired using a flow cytometer, and the data were subsequently analyzed using FlowJo software. Proper compensation controls and unstained cells were used to set the gates and quadrants.
2.8 In Vivo diabetic wound healing model
Male Sprague-Dawley (SD) rats (8 weeks old) were used. Diabetes was induced by a single intraperitoneal injection of STZ (60 mg/kg). Rats with fasting blood glucose levels >16.7 mmol/L for two consecutive weeks were considered diabetic. After anesthesia, a full-thickness excisional wound (diameter = 0.8 cm) was created on the dorsal surface of the rat foot. The rats were randomly assigned into three groups using a computer-generated random number sequence, with no less than 3 biological replicates per group (n ≥ 3): (1) Control (PBS), (2) K-PEVs-L (low dose), and (3) K-PEVs-H (high dose). The selection of these specific doses was based on our in vitro assays, where 5 μg/mL represented the minimal effective concentration for promoting cellular activities, and 20 μg/mL showed maximal efficacy without cytotoxicity. To minimize bias, investigators performing downstream measurements and histological analyses were blinded to the treatment allocations. Treatments were applied topically every 2 days.
2.9 Wound closure analysis
Wound healing was monitored by photography on days 0, 2, 4, 8, and 16. The wound area and wound length were measured using ImageJ, and the wound healing rate was calculated as follows:
2.10 Histological and immunofluorescence analysis
Wound tissues collected on day 8 were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Sections were stained with Hematoxylin and Eosin (H&E) to observe tissue morphology and Masson’s Trichrome to evaluate collagen deposition (collagen volume fraction). For M1 macrophage detection, wound tissue sections (5 μm thick) were deparaffinized in xylene (3 × 10 min) and rehydrated through a graded ethanol series (100%, 95%, 70%, 50%, distilled water, 5 min each). Antigen retrieval was performed by microwave heating in citrate buffer (10 mM sodium citrate, pH 6.0) at 95 °C–100 °C for 15 min, followed by natural cooling to room temperature. Sections were washed three times with PBS (5 min each) and permeabilized with 0.3% Triton X-100 in PBS for 15 min at room temperature. Non-specific binding was blocked with 5% normal goat serum (Beyotime, P0096) in PBS for 1 h at room temperature. Sections were incubated overnight at 4 °C with rabbit polyclonal anti-CD86 antibody (1:200 dilution; Proteintech, Cat# 13395-1-AP), which specifically recognizes rat CD86 (validated for rat macrophage staining). After three PBS washes (5 min each), sections were incubated with Alexa Fluor® 488-conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:500; Thermo Fisher, A-11008) in the dark for 1 h at room temperature. Sections were then washed three times with PBS (5 min each). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/mL; Beyotime, C1002) for 5 min at room temperature in the dark, followed by three PBS washes. ImageJ software (version 1.53c, National Institutes of Health) was used for semi-quantitative analysis. To ensure the validity and reproducibility of all image-based quantifications (including scratch assays, macroscopic wound areas, and fluorescence intensities), analyses were performed independently by two blind investigators using ImageJ software. Standardized processing parameters, such as consistent background subtraction and fixed intensity thresholds, were applied uniformly across all samples within each experiment to eliminate subjective bias.
2.11 Statistical analysis
All data are presented as mean ± standard deviation. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).
3 Results
3.1 Characterization of K-PEVs
To verify the successful extraction and ensure the quality of the isolated vesicles, a comprehensive characterization of the K-PEVs was performed. K-PEVs were isolated from fresh kiwi fruit juice using a standardized protocol combining differential centrifugation and ultracentrifugation (Figure 1a). TEM imaging confirmed the presence of vesicles with a typical cup-shaped or spherical morphology, displaying the characteristic intact lipid bilayer structure associated with extracellular vesicles (Figure 1b). To further assess the yield and size distribution, NTA was employed, demonstrating a high concentration of 1.96 × 1013 particles/mL with a primary peak at approximately 155 nm (Figure 1c). Additionally, DLS measurements showed a hydrodynamic diameter of 151.6 nm and a zeta potential of −17.9 mV (Figure 1d). The negative surface charge and narrow size distribution indicate that the K-PEVs possess excellent colloidal stability and mono-dispersity.
FIGURE 1
3.2 K-PEVs enhance cell viability and migration in vitro
Having confirmed the physical integrity of K-PEVs, we next investigated their biological impact on skin-related cells, as the migration of keratinocytes and the proliferation of fibroblasts are fundamental to the re-epithelialization of wounds. We first evaluated the influence of K-PEVs on the dynamic process of wound closure using HaCaT cells in a scratch assay. The results showed that K-PEVs significantly accelerated HaCaT cell migration in a dose-dependent manner (Figure 2a). Quantitatively, while the negative control (NC) group exhibited a migration rate of only 35.2% after 24 h, the groups treated with 5, 10, and 20 μg/mL K-PEVs showed significantly higher closure rates of approximately 50%, 61%, and 78%, respectively (P < 0.001 for the 20 μg/mL group, Figure 2b). To ensure that this promotive effect was not accompanied by cytotoxicity and to assess the impact on fibroblast growth, a CCK-8 assay was conducted on NIH3T3 cells. The assay revealed that K-PEVs were highly biocompatible at all tested concentrations and even significantly enhanced the viability of NIH3T3 cells at higher doses (10 and 20 μg/mL) (Figure 2c).
FIGURE 2
3.3 K-PEVs scavenge intracellular ROS and exhibit potent antioxidant activity
Given that persistent oxidative stress is a hallmark of the diabetic wound microenvironment and a major barrier to healing, we sought to determine whether K-PEVs could mitigate this stress. We first examined their intrinsic antioxidant capacity using a DPPH radical scavenging assay. The scavenging rate increased markedly with higher K-PEV concentrations, exceeding 80% in the 20 μg/mL group (Figure 3c). To further validate this effect at the cellular level, intracellular ROS levels in Raw264.7 macrophages were visualized using the DCFH-DA fluorescent probe under oxidative stress conditions. While the positive control group exhibited intense green fluorescence indicating high ROS accumulation, and treatment with K-PEVs resulted in a dramatic, dose-dependent reduction in fluorescence intensity in Raw264.7 cells (Figures 3a,b). This potent ROS-scavenging ability suggests that K-PEVs may break the oxidative stress impasse in diabetic wounds, thereby creating a more favorable environment for regeneration.
FIGURE 3
3.4 K-PEVs modulate macrophage polarization from M1 to M2 phenotype in vitro
Macrophages play a crucial role in the inflammatory phase of wound healing, and their timely transition from a pro-inflammatory M1 phenotype to a pro-reparative M2 phenotype is essential for tissue regeneration. To investigate the immunomodulatory effects of K-PEVs, we evaluated the polarization of macrophages in vitro following treatment with varying concentrations of K-PEVs (0, 5, 10, and 20 μg/mL) using flow cytometry.
As shown in Figure 4a the control group (0 μg/mL) exhibited a predominantly M1 pro-inflammatory phenotype, characterized by high expression of the M1 marker CD86. However, treatment with K-PEVs induced a remarkable shift in macrophage polarization. The flow cytometry dot plots visually demonstrated a dose-dependent decrease in the CD86+ cell population and a concurrent increase in the CD206+ cell population (an M2 marker).
FIGURE 4
Quantitative analysis further confirmed these significant transitions. The percentage of M1 macrophages steadily decreased from approximately 71.0% in the control group to 15.0% in the high-dose K-PEVs (20 μg/mL) group (**P < 0.001, Figure 4b). Conversely, the proportion of M2 macrophages exhibited a dramatic, dose-dependent upregulation, soaring from about 11.0% in the untreated control to over 80.0% following 20 μg/mL K-PEVs treatment (***P < 0.001, Figure 4c). Collectively, these in vitro findings robustly demonstrate that K-PEVs can effectively reprogram macrophages, break the pro-inflammatory lock and promote a regenerative M2 phenotype, which provides a strong cellular basis for their therapeutic application in chronic diabetic wounds.
3.5 K-PEVs accelerate diabetic wound healing in vivo
Encouraged by the promising vitro results, we progressed to an in vivo study using a full-thickness foot wound model in STZ-induced diabetic rats. The rats were treated with two doses of K-PEVs: a low-dose group (K-PEVs-L, 5 μg/mL) and a high-dose group (K-PEVs-H, 20 μg/mL). Macroscopic observation over 16 days revealed that wounds treated with K-PEVs closed significantly faster than those in the Control group (Figure 5a). Notably, the K-PEVs-H (20 μg/mL) group achieved near-complete closure on day 16, whereas the control wounds remained relatively large. Statistical analysis confirmed that the wound healing rate was significantly higher in the K-PEV-treated groups across all recorded time points (Figure 5b). Furthermore, histological evaluation via H&E staining on day 8 showed that K-PEV treatment, especially at the high dose, led to superior re-epithelialization and the formation of well-organized granulation tissue, in contrast to the control group which exhibited disorganized tissue and persistent inflammatory cell infiltration (Figures 5c,d).
FIGURE 5
3.6 K-PEVs promote collagen deposition and modulate macrophage polarization
To further elucidate the microscopic mechanisms by which K-PEVs accelerate healing, we analyzed the structural integrity of the extracellular matrix (ECM) and the status of the inflammatory response. Masson’s Trichrome staining on day 8 revealed a dense and orderly arrangement of blue-stained collagen fibers in wounds treated with K-PEVs (Figure 6a), with the collagen volume fraction in the K-PEVs-H group reaching approximately 36% compared to only 11% in the control (Figure 6c).
FIGURE 6
Since the transition from a pro-inflammatory to a pro-reparative phase is often stalled in diabetic wounds, we examined the polarization state of macrophages using immunofluorescence for CD86 (an M1-type pro-inflammatory marker) and CD206 (an M2-type pro-reparative marker). The control group showed a high density of CD86-positive cells alongside scarce CD206-positive cells, reflecting a severe state of inflammatory lock (Figure 6b). Remarkably, K-PEV treatment significantly reduced the fluorescence intensity of CD86 in a dose-dependent manner (Figure 6d), with the K-PEVs-H (20 μg/mL) group showing the most pronounced inhibitory effect. Concurrently, K-PEV treatment substantially upregulated the expression of CD206 in the wound tissues. As shown in Figures 6b,e, the fluorescence intensity of CD206 was significantly elevated in a dose-dependent manner, increasing by nearly 2-fold in the K-PEVs-L group (*P < 0.05) and nearly 5-fold in the K-PEVs-H group (*P < 0.001) compared to the control. These findings compellingly demonstrate that K-PEVs effectively drive the macrophage phenotypic switch from the pro-inflammatory M1 state to the pro-reparative M2 state in vivo, thereby alleviating chronic inflammation and paving the way for optimal tissue regeneration.
4 Discussion
The physiological process of wound healing is frequently compromised in diabetic patients, leading to the development of chronic, non-healing ulcers. The pathophysiology of DFUs is characterized by a vicious cycle of persistent oxidative stress and chronic inflammation. In this study, we successfully isolated K-PEVs and demonstrated their superior therapeutic potential in accelerating diabetic wound closure. Our findings highlight that K-PEVs can effectively remodel the impaired wound microenvironment by scavenging ROS and modulating macrophage polarization from a pro-inflammatory M1 phenotype to a pro-reparative state.
Consistent with our findings, previous studies have demonstrated the therapeutic potential of PEVs in wound healing. Grapefruit-derived extracellular vesicles have been shown to promote cutaneous wound healing through M2 macrophage polarization and ROS reduction (Savcı et al., 2021; Castelli et al., 2023). Similarly, lemon-derived extracellular vesicles achieved antioxidant and anti-inflammatory effects by activating the AhR/Nrf2 signaling pathway (Jin et al., 2025; Raimondo et al., 2022). Our in vitro results using Raw264.7 cells showed that K-PEVs possess potent antioxidant activity, significantly reducing intracellular ROS levels. This antioxidant effect is likely attributed to the rich bioactive cargo within K-PEVs, such as Vitamin C and polyphenols, which are preserved within the protective lipid bilayer of the vesicles. By neutralizing excessive ROS, K-PEVs alleviate the primary stimulus for sustained M1 polarization. This was further confirmed by our in vivo immunofluorescence data, where a significant downregulation of the M1 marker CD86 was observed in the K-PEV-treated groups. The core difficulty in treating diabetic wounds lies in the inflammatory lock state, where macrophages remain stuck in the M1 phenotype due to the high-ROS and hyperglycemic milieu. The suppression of M1 macrophages subsequently reduces the secretion of pro-inflammatory cytokines and matrix metalloproteinases (MMPs), thereby preventing the excessive degradation of the extracellular matrix (ECM) and allowing the wound to transition into the proliferative phase.
Furthermore, we observed that K-PEVs directly promote the biological functions of essential skin cells. The enhanced migration of HaCaT keratinocytes and the increased viability of NIH3T3 fibroblasts in vitro provided a cellular basis for the improved re-epithelialization and granulation tissue formation seen in the diabetic rat model. Masson’s Trichrome staining corroborated these findings, showing significantly higher collagen deposition and a more organized ECM structure in the high-dose K-PEV group. This suggests that K-PEVs do not just passive clear barriers (like ROS), but also actively stimulate the regenerative machinery of the host tissue.
Compared to mammalian cell-derived exosomes (MDEs), K-PEVs offer several distinct advantages as a cell-free therapeutic platform (Bian et al., 2022). Their production is more cost-effective, sustainable, and avoids the ethical concerns and pathogen contamination risks associated with mammalian cell culture. The high yield (1.96 × 1013 particles/mL) and robust stability (−17.9 mV zeta potential) demonstrated in our characterization section further support their potential for large-scale pharmaceutical preparation. Moreover, the ability of PEVs to participate in cross-kingdom communication allows them to deliver plant-derived regulatory molecules directly into mammalian cells with high efficiency and low immunogenicity.
This study possesses several strengths. First, comprehensive characterization (TEM, NTA, DLS) ensured the quality of K-PEV preparations. Second, in vitro and in vivo models were employed to systematically evaluate therapeutic efficacy. Third, we elucidated multi-faceted mechanisms including ROS scavenging, macrophage polarization modulation, and direct stimulation of skin cell functions, providing a strong foundation for clinical translation. Despite these promising results, several limitations of the current study should be acknowledged. First, while we have demonstrated the overall therapeutic effect, the specific bioactive components within K-PEVs (such as specific miRNAs or proteins) that drive these processes remain to be fully elucidated through omics-based analysis. Second, the STZ-induced rodent model, while widely used, does not fully replicate the complexity of human diabetic ulcers, which often involve pressure-related ischemia and complex bacterial biofilms. Third, the current study lacks direct in vivo comparative controls utilizing crude kiwifruit extract (KFE) or free antioxidants. While the lipid bilayer of K-PEVs theoretically provides essential protection against rapid degradation in the ROS-rich diabetic wound bed and facilitates cellular uptake via endocytosis, future studies incorporating KFE and vehicle-antioxidant control groups are necessary to definitively quantify the specific therapeutic advantage conferred by the vesicular structure. Future studies should focus on identifying the key molecular drivers within K-PEVs and validating these findings in larger animal models or clinical pilot studies. Future studies should focus on: (1) comprehensive omics analysis to identify key molecular drivers within K-PEVs; (2) validation in larger animal models (e.g., diabetic pigs); that better recapitulate human wound healing; (3) development of optimized delivery systems (e.g., hydrogels); for sustained therapeutic effects; (4) evaluation of long-term safety and immunogenicity; and (5) clinical pilot studies to assess efficacy in human diabetic patients.
5 Conclusion
In summary, our study provides strong evidence that K-PEVs serve as a potent, multi-target regenerative agent for the treatment of diabetic wounds. We successfully isolated stable, high-yield K-PEVs that exhibit remarkable biocompatibility and biological activity. Through a series of in vitro and in vivo experiments, we demonstrated that K-PEVs can synergistically suppress oxidative stress, resolve chronic inflammation by modulating macrophage polarization, and actively promote cellular migration and extracellular matrix deposition. These findings suggest that K-PEVs can effectively break the pathological deadlock of chronic diabetic ulcers and restore the orderly progression of the healing process.
Overall, this research identifies K-PEVs as a promising green nanomedicine platform that overcomes the limitations of traditional cell-based therapies. Given their natural origin, cost-effectiveness, and potent bioactivity, K-PEVs hold significant potential for clinical translation in the management of diabetic foot ulcers and other chronic inflammatory conditions. Future exploration into their specific molecular cargo will further pave the way for the development of standardized, plant-based cell-free regenerative strategies.
Statements
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Ethics statement
The animal study was approved by Animal Experiments and Experimental Animal Welfare Committee of Zhejiang University. The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
ZX: Writing – review and editing, Methodology, Writing – original draft, Data curation, Funding acquisition. Y-LW: Writing – review and editing. CF: Writing – review and editing. YW: Writing – review and editing, Investigation. W-XL: Supervision, Writing – review and editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This study was supported by Jinhua City Public Welfare Technology Application Research Project (No. 2024-4-227). Zhejiang Provinical Science Foundation of China (NO.LQN26H150014).
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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Glossary
- DM
Diabetes Mellitus
- DFU
Diabetic Foot Ulcers
- OS
Oxidative Stress
- ROS
Reactive Oxygen Species
- AGEs
Advanced Glycation End products
- MMPs
Matrix Metalloproteinases
- ECM
Extracellular Matrix
- EVs
Extracellular Vesicles
- PEVs
Plant-derived Extracellular Vesicles
- K-PEVs
Kiwifruit-derived Plant Extracellular Vesicles
- MDEs
Mammalian cell-derived Exosomes
- PELNs
Plant-derived Exosome-like Nanovesicles
- KFE
Kiwifruit Extract
- TEM
Transmission Electron Microscopy
- NTA
Nanoparticle Tracking Analysis
- DLS
Dynamic Light Scattering
- CCK-8
Cell Counting Kit-8
- DCFH-DA
2,7-dichlorodihydrofluorescein diacetate
- DPPH
2,2-diphenyl-1-picrylhydrazyl
- STZ
Streptozotocin
- H&E
Hematoxylin and Eosin
- PBS
Phosphate-Buffered Saline
- DAPI
4',6-diamidino-2-phenylindole
- HaCaT
Human adult low calcium high temperature
- NIH3T3
NIH 3T3 mouse embryonic fibroblasts
- Raw264.7
RAW 264.7 mouse macrophages
- NC
Negative Control
- K-PEVs-L
Low-dose Kiwifruit-derived Plant Extracellular Vesicles (5 μg/mL)
- K-PEVs-H
High-dose Kiwifruit-derived Plant Extracellular Vesicles (20 μg/mL)
- M1
Pro-inflammatory macrophage phenotype
- M2
Pro-reparative macrophage phenotype
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Summary
Keywords
diabetic foot ulcers, kiwifruit, macrophage polarization, oxidative stress, plant-derived extracellular vesicles
Citation
Xu Z, Wang Y-L, Fang C, Wang Y and Li W-X (2026) Remodeling the oxidative and inflammatory microenvironment with kiwifruit-derived plant extracellular vesicles for enhanced diabetic foot ulcer repair. Front. Mater. 13:1825814. doi: 10.3389/fmats.2026.1825814
Received
08 March 2026
Revised
03 June 2026
Accepted
12 June 2026
Published
13 July 2026
Volume
13 - 2026
Edited by
Luiz Fernando Romanholo Ferreira, Catholic University of Brasilia (UCB), Brazil
Reviewed by
Yifan Li, Zhejiang University, China
Thilaga D., Saveetha Dental College and Hospitals, India
Updates
Copyright
© 2026 Xu, Wang, Fang, Wang and Li.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Yong Wang, wongyong@zju.edu.cn; Wen-Xin Li, 451457570@qq.com
Disclaimer
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