Redox-Responsive Polymer Dot Nanozymes Coordinate Exosome-Mediated Cutaneous Regeneration via Laser-Modulated Microenvironment Remodeling

Redox-Responsive Polymer Dot Nanozymes Coordinate Exosome-Mediated Cutaneous Regeneration via Laser-Modulated Microenvironment Remodeling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Redox-Responsive Polymer Dot Nanozymes Coordinate Exosome-Mediated Cutaneous Regeneration via Laser-Modulated Microenvironment Remodeling Yen-Jen Wang, Chang-Cheng Chang, Tzong-Yuan Juang, Yi-Hsuan Tu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9132659/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cutaneous wound healing is orchestrated by tightly coordinated biochemical and biomechanical cues that regulate epithelial activation, angiogenesis, and extracellular matrix (ECM) remodeling. However, nanozyme-based biomaterial platforms capable of dynamically regulating the wound microenvironment remain limited. Here, we develop a nanozyme-enabled regenerative platform in which redox-responsive hyperbranched polymer dot nanozymes (PDNs) are integrated with porcine fallopian tube stem cell–derived exosomes and picosecond laser stimulation to modulate phase-specific microenvironmental responses. Unlike conventional carriers, PDNs act as catalytic nanozymes that regulate reactive oxygen species (ROS) dynamics within the wound microenvironment. Using a murine splinted excisional wound model that isolates epithelial-driven regeneration, treatment with exosomes and PDNs (Exo+PDN) significantly accelerated early wound closure and was associated with coordinated activation of the EGF–ERK1/2–AQP3 signaling axis. When combined with picosecond laser stimulation (Exo+Laser+PDN), the platform preferentially enhanced early angiogenic activation, followed by improved epidermal maturation and more organized collagen architecture. Analysis of epithelial plasticity markers revealed maintained E-cadherin expression with concurrent vimentin upregulation in the absence of SLUG induction, indicating a regulated partial epithelial plasticity state rather than a full epithelial–mesenchymal transition. Collectively, these findings demonstrate that PD nanozymes function as active microenvironment-modulating biomaterials that integrate biochemical and physical cues to guide phase-dependent wound regeneration. This work highlights a material-driven strategy for regulating the temporal dynamics of tissue repair beyond conventional delivery-based approaches. Nanozymes Exosome-Based Therapy Cutaneous Regeneration Microenvironment Modulation Nanomedicine Regenerative Biomaterials Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Cutaneous wound healing is orchestrated by tightly coordinated biochemical and biomechanical cues that regulate epithelial activation, angiogenesis, and extracellular matrix (ECM) remodeling. 1, 2 Although numerous biomaterial platforms have been developed to enhance repair through growth factor delivery, scaffold engineering, or smart wound dressings, most systems function primarily as passive carriers or structural supports. 3 , 4 Consequently, they lack the capacity to dynamically regulate the wound microenvironment in a temporally defined manner. Among critical microenvironmental regulators, redox balance plays a central role in controlling epithelial plasticity, angiogenic signaling, and matrix turnover. 5–8 However, material systems capable of actively tuning oxidative signaling within a permissive regenerative window remain limited. Polymer dot nanozymes (PDNs) constitute an ultrasmall (< 10 nm), redox-responsive carbon nanomaterial platform with intrinsic enzyme-mimetic catalytic functionality. 9 , 10 , 11 , 12 Unlike conventional nanoparticle carriers, PDNs are drug-free nanozymes that regulate reactive oxygen species (ROS) through intrinsic catalytic redox activity rather than acting as passive delivery carriers. 13 By maintaining ROS within a permissive signaling window, PDNs regulate redox signaling within the wound microenvironment, thereby facilitating epithelial activation and tissue repair. 11 , 14 , 15 , 16 Beyond amplifying exogenous biologics, PDNs function as bioactive material regulators that reshape the wound niche through microenvironmental modulation. 17,18, 19 This material-level control introduces the possibility of phase-programmable regeneration, in which the timing and directionality of repair processes can be engineered rather than passively supported. 20 , 21 Exosomes derived from porcine fallopian tube stem cells (PFTSC-Exo) provide a biologically defined source of growth factors and regulatory miRNAs capable of activating MAPK- and PI3K/AKT-associated pathways implicated in epithelial proliferation and tissue remodeling. 22 , 23 , 24 However, the efficacy of exosome-based therapies remains highly dependent on the surrounding microenvironment, which can either permit or constrain regenerative signaling. 25–28 Mechanical stimuli such as 755-nm picosecond laser–induced optical breakdown (LIOB) introduce localized microinjury that activates angiogenic and ECM remodeling pathways, including TGF-β/Smad signaling. 29–32 Yet without redox-level regulation, such mechanically triggered pathways may not be optimally synchronized with epithelial repair dynamics. Here, we introduce a redox-responsive PDN platform that functions as a gating nanozyme interface integrating exosome-derived biochemical cues with laser-triggered mechanical signaling. Using a murine splinted excisional wound model that isolates epithelial-driven repair, we investigated whether PDNs can selectively tune early epithelial activation while reprogramming laser-induced angiogenic and ECM remodeling responses in a phase-dependent manner. We hypothesized that PDNs act as catalytic material regulators capable of directing the temporal dynamics of wound healing, thereby coordinating epithelialization, angiogenesis, and tissue maturation. Accordingly, this study investigates whether nanozyme-mediated microenvironmental regulation can redistribute regenerative signaling during wound repair. Materials and Methods 1.1. Preparation and Isolation of porcine fallopian tube stem cell-derived exosomes Porcine fallopian tube stem cell (PFTSC)-derived exosomes, obtained from the National Pingtung University of Science and Technology and Chang Gung Memorial Hospital (Linkou, Taiwan), were used as the source of porcine oviduct–derived exosomes. Fresh porcine oviduct tissues were rinsed three times with sterile saline supplemented with 1% penicillin–streptomycin (P/S), minced into approximately 0.1–0.5 mm³ fragments, and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% P/S at 38.5°C in a humidified atmosphere with 5% CO₂. At passage five, when cells reached approximately 80–90% confluence, cultures were washed with Dulbecco’s phosphate-buffered saline (DPBS) and incubated for 3 h in TCM-199 medium supplemented with 10% FBS, 2.5 µg/mL follicle-stimulating hormone (FSH), 5 IU/µL human chorionic gonadotropin (hCG), 10 ng/mL epidermal growth factor (EGF), 1% antibiotic–antimycotic solution (ABAM), and 10% (v/v) porcine follicular fluid (PFF). Conditioned medium (CM) was subsequently collected, filtered through a 0.22-µm membrane to remove cellular debris, and stored at 4°C until further processing. For exosome isolation, PFTSC were cultured in CM supplemented with exosome-depleted FBS. Exosomes were isolated using the miRCURY™ Exosome Isolation Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Briefly, CM was mixed with the isolation reagent and centrifuged at 3,200 × g for 30 min. The resulting exosome-containing pellets were resuspended in the provided buffer and stored at − 80°C until use. For visualization, 10 µL of the exosome suspension was placed onto a glass slide and air-dried prior to imaging. For topical application experiments, exosome preparations were diluted in phosphate-buffered saline (PBS) to a final concentration of 2 mg per 100 µL. 1.2 Hyperbranched Polymer Dot (PD) Synthesis and Characterization Hyperbranched polymer dots (PDs) were synthesized via an A₂ + B₃ polycondensation strategy as previously reported. 15, 18,33 Briefly, wholly aliphatic monomers were subjected to a one-pot polymerization process to generate hyperbranched polymer dots with a heteroatom-enriched polymeric carbon framework. This synthetic route enables gram-scale production with yields of up to 44%, demonstrating good scalability and synthetic reproducibility. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) analyses revealed ultrasmall particle sizes in the range of approximately 3–5 nm. X-ray photoelectron spectroscopy (XPS) confirmed the presence of abundant nitrogen- and oxygen-containing functional groups within the polymer backbone, consistent with successful heteroatom incorporation. These surface functionalities contribute to the high aqueous dispersibility of the PDs without additional surface modification. The resulting PDs exhibit intrinsic fluorescence and low cytotoxicity, supporting their suitability for biological applications. In addition, the heteroatom-rich polymer framework confers redox-responsive catalytic activity, enabling reactive oxygen species (ROS) scavenging behavior characteristic of antioxidant nanozymes. For in vivo experiments, PDs were incorporated into a PEG1000-based hydrogel formulation prior to topical administration, as previously described. 15 , 18 1.3 Picosecond laser treatment A 755 nm picosecond laser with a diffractive lens array (PicoSure®, Cynosure, MA, USA) was used to induce LIOB. Laser parameters were selected based on previous studies demonstrating reliable induction of laser-induced optical breakdown (LIOB) in murine skin. 12 , 15 , 34 Optimized parameters included: 6 mm spot size, 0.71 J/cm² fluence, 750 ps pulse duration, and 5 Hz frequency. 12 , 15 , 34 A total of 500 pulses were applied immediately post-wounding in laser-treated groups. 1.4. Animal Model and Experimental Design Five-week-old female BALB/cAnN.Cg-Foxn1^nu/CrlNarl nude mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan). All animal procedures were approved by the Institutional Animal Care and Use Committee of China Medical University (IACUC approval no. CMUIACUC-2022-404) and conducted in accordance with institutional and national guidelines for animal welfare. A total of 45 nude mice were randomly assigned to five experimental groups using a computer-generated randomization sequence (3 mice per group per time point): Control, Exosome (Exo), Exosome plus laser (Exo + L), Exosome plus polymer dots (Exo + PD), and Exosome plus polymer dots combined with laser (Exo + L+PD). Each mouse carried two wounds, resulting in six wounds per group at each time point. On Day 0, mice were anesthetized with isoflurane, and two symmetric 6-mm full-thickness excisional wounds were created on the dorsal skin of each mouse. To minimize wound contraction and better mimic human wound healing, silicone splints (inner diameter, 10 mm; outer diameter, 18 mm; thickness, 0.5 mm) were secured around each wound using 6 − 0 nylon sutures (Ethicon, USA) and medical-grade tissue adhesive (3M Vetbond™, USA). On Day 0, laser-treated groups (Exo + L and Exo + L+PD) received picosecond laser irradiation designed to induce laser-induced optical breakdown (LIOB), delivered as 500 pulses. Immediately after laser treatment, topical treatments were applied according to group allocation. Mice in the exosome-treated groups (all groups except the control group) received topical application of exosomes (100 µL per wound). Mice assigned to the PD-treated groups (Exo + PD and Exo + L+PD) received 100 µL of PD hydrogel (5 mg/mL polymer dots formulated in 5% PEG1000), which was reapplied on Days 3, 6, 9, 12, and 15. Wound healing progression was documented by standardized digital photography every 3 days from Day 0 to Day 18, and wound areas were quantified using ImageJ software (National Institutes of Health, USA). For mechanistic and histological analyses, mice were sacrificed at predetermined time points as illustrated in Fig. 1 , and wound tissues were harvested for subsequent analyses. 1.5 Wound Monitoring and Image Analysis Digital photographs of wounds were captured on Days 0, 3, 6, 9, and 12. Wound area was measured using ImageJ software, and percentage wound closure was calculated: % wound closure = [(A₀ – Aₜ)/A₀] × 100 where A₀ is initial wound area and At is area at time t. 1.6. Tissue Harvest and Processing On Days 3, 6, 12, and 18, three mice per group were euthanized at each predetermined time point. Full-thickness wound tissues, including the wound bed and adjacent periwound skin, were carefully excised. Harvested tissues were gently defatted, flattened on paperboard to maintain consistent orientation, and fixed in 10% neutral-buffered formalin for 24 h at room temperature. Following fixation, tissues were rinsed with deionized water, placed into tissue cassettes, and kept submerged in water to prevent desiccation prior to routine histological processing. For biochemical analyses, wound tissues were homogenized in ice-cold phosphate-buffered saline (PBS) supplemented with a protease inhibitor cocktail. Tissue lysates were clarified by centrifugation, and total protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. The resulting protein extracts were subsequently used for enzyme-linked immunosorbent assay (ELISA) analyses. 1.7 Time-Point–Specific Histological and Biochemical Analyses To capture the temporal dynamics of wound healing and tissue remodeling, histological and biochemical analyses were performed at predefined time points corresponding to distinct phases of wound repair. Day 3 (early inflammatory and activation phase). At Day 3, Masson’s trichrome staining was performed to assess early changes in collagen deposition and dermal structural integrity. ERK1/2 protein levels were quantified by ELISA to evaluate activation of MAPK signaling pathways associated with growth factor–mediated cellular activation. Immunohistochemical staining for CD31, VEGF, and EGF was conducted to examine angiogenic responses and growth factor signaling potentially mediated by exosome treatment. Hematoxylin and eosin (H&E) staining was used for general histopathological assessment and evaluation of early cellular changes within the wound bed. Day 6 (re-epithelialization and matrix synthesis phase). At Day 6, Masson’s trichrome staining was repeated to evaluate progressive collagen deposition and dermal remodeling. Procollagen type I and type III levels were quantified by ELISA to determine early extracellular matrix synthesis and to assess whether laser and/or polymer dot treatment enhanced collagen production. Immunohistochemical analyses of E-cadherin and vimentin were performed to assess epithelial–mesenchymal transition (EMT) dynamics, while filaggrin and aquaporin-3 were evaluated as markers of epidermal differentiation and keratinocyte functional activation, respectively. H&E staining was additionally used to assess overall tissue architecture and cellular morphology. Day 18 (late remodeling and maturation phase). At Day 18, ELISA was performed to quantify collagen type I and type III levels, reflecting cumulative collagen production and matrix maturation during the late remodeling phase of wound healing. Wound closure assessment. Standardized wound photographs were acquired every three days (Days 0, 3, 6, 9, 12, 15, and 18). Wound areas were measured using ImageJ software, and wound closure rates were calculated to compare healing kinetics among experimental groups. 1.8 Statistical Analysis All quantitative data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism (version 10.2; GraphPad Software, San Diego, CA, USA). For wound closure analysis, wound areas measured at multiple time points (Days 3, 6, 9, and 12) were analyzed using two-way repeated-measures analysis of variance (ANOVA), with treatment group and time as independent factors, followed by Tukey’s multiple comparisons test to assess differences between groups at individual time points. For biochemical and histological quantitative data obtained at single time points (including ELISA measurements of ERK1/2, collagen type I, procollagen type III, and immunohistochemical quantification of CD31, VEGF, E-cadherin, and vimentin), comparisons among multiple groups were performed using one-way ANOVA followed by Tukey’s post hoc test. When comparisons involved only two groups, an unpaired two-tailed Student’s t test was applied as appropriate. Qualitative macroscopic wound appearance was assessed descriptively based on standardized digital photographs acquired at predefined time points and was not subjected to statistical testing. A p value < 0.05 was considered statistically significant. Results 2.1 Exosome–PD treatment accelerates early wound closure Wound closure was quantitatively assessed by ImageJ analysis at Days 3, 6, 9, and 12 following wound creation (Fig. 2 ). All experimental groups demonstrated progressive wound contraction over time; however, distinct differences in healing kinetics were observed among treatment groups. The Exosome + PD (Exo + PD) group consistently exhibited the most rapid reduction in wound area during the early healing phase. By Day 6, wounds in the Exo + PD group had decreased to 42.0 ± 14.0% of the original wound size, compared with 59.0 ± 7.6% in the control group and 55.7 ± 7.6% in the exosome-only group. This accelerated closure became more pronounced by Day 9, at which time the Exo + PD group reached 19.5 ± 8.2% of the original wound area, representing the fastest wound closure among all treatment groups. These findings suggest that PD incorporation influences wound-healing kinetics, with Exo + PD accelerating early wound closure, whereas the Exo + L+PD group shows comparatively slower early closure but improved tissue appearance at later time points. 2.2 Macroscopic wound appearance reveals distinct healing trajectories Representative macroscopic images were acquired at Days 0, 3, 6, 9, 12, 15, and 18 to qualitatively evaluate wound closure dynamics and scar quality across treatment groups (Fig. 3 ). Immediately after injury (Day 0), all wounds presented as uniform 6-mm full-thickness defects stabilized by silicone splints. In the control group, wound closure progressed slowly, with limited size reduction by Day 6 and persistent granulation tissue and irregular wound beds. Although gradual contraction was observed between Days 9 and 12, complete closure was not achieved until Day 18, and residual scabbing with uneven epithelial surfaces remained apparent. Exosome-treated wounds exhibited moderate improvement, characterized by smoother wound margins and reduced inflammatory appearance by Day 6. Wound areas decreased steadily thereafter, with near-complete closure observed by Days 15–18. Notably, the Exo + PD group demonstrated the most rapid early macroscopic healing, with marked wound size reduction and flattened, dry wound beds evident by Day 6. Most wounds appeared fully closed by Day 9, and by Days 12–18 the regenerated skin surface was smooth and uniform, suggesting improved epithelial coverage and smoother surface morphology. In contrast, wounds treated with Exosome + Laser alone showed delayed early closure, with residual open areas persisting at Day 6. Nevertheless, wound contraction progressed after Day 9, and closure was achieved by Days 15–18, albeit with mild surface irregularity. Interestingly, the Exo+Laser + PD group displayed a distinct healing pattern, in which early wound closure was less pronounced than that observed with Exo + PD alone, yet substantial improvements in tissue appearance and surface regularity became evident by Days 9–12. By Day 18, wounds were fully closed with smooth, well-reconstructed skin, suggesting enhanced tissue remodeling and superior scar quality despite comparable final closure. 2.3 Early EGF–ERK1/2 Signaling and Subsequent AQP3 Upregulation Early molecular events associated with accelerated wound closure were evaluated by examining growth factor–related signaling pathways during the initial phase of repair. EGF expression assessed on Day 3 was significantly increased in the Exo + PD group compared with the control group (Tukey’s post hoc test, p = 0.0074), indicating enhanced early growth factor availability following combined exosome and polymer dot treatment. No other pairwise comparisons reached statistical significance (Fig. 4 A). Consistent with this upstream change, ERK1/2 expression on Day 3 exhibited a trend toward increased levels in exosome-treated groups, particularly in the Exosome and Exo + PD groups, whereas a modest attenuation was observed in laser-treated groups; however, inter-group differences did not reach statistical significance (Fig. 4 B). Aquaporin-3 (AQP3) expression assessed on Day 6 demonstrated an overall upregulation trend in treatment groups associated with accelerated wound closure (Fig. 4 C). Groups showing higher EGF availability and increased ERK1/2 signaling trends during the early phase tended to exhibit elevated AQP3 expression at later time points. Collectively, these findings suggest a temporal association between early growth factor–related signaling and subsequent epithelial functional activation during wound repair, particularly in PD-containing exosome-based treatments. 2.4 PD-Containing Treatments Enhance Early Angiogenic Signaling At Day 3 after full-thickness excisional wounding, angiogenesis-associated markers VEGF and CD31 were significantly modulated by treatment (Fig. 5 ). One-way ANOVA revealed a significant intergroup difference in CD31 expression (F = 3.22, p = 0.0298). Post hoc analysis demonstrated that CD31 expression was significantly higher in the Exosome + Laser + PD group compared with the Exosome-only group (p = 0.0379), suggesting enhanced early endothelial activation under combinatorial treatment. VEGF expression exhibited a more pronounced treatment-dependent effect (F = 9.64, p = 7.36 × 10⁻⁵). PD-containing regimens significantly increased VEGF levels compared with control, with both Exosome + PD (p = 0.0206) and Exosome + Laser + PD (p < 0.0001) showing marked elevation. In addition, Exosome + Laser + PD induced significantly higher VEGF expression than Exosome alone (p = 0.0048) and Exosome + Laser (p = 0.0063), indicating a synergistic enhancement of early pro-angiogenic signaling. 2.5 Re-epithelialization occurs without full EMT activation To further characterize the epithelial phenotype during the re-epithelialization phase, the expression of epithelial and mesenchymal markers was evaluated on Day 6. Quantitative analyses revealed no significant differences in E-cadherin expression among treatment groups (one-way ANOVA, p = 0.18), indicating that epithelial cell–cell adhesion was not completely lost during re-epithelialization across conditions (Fig. 6 A, B). Similarly, Vimentin expression did not differ significantly between groups (one-way ANOVA, p = 0.22), suggesting the absence of a full mesenchymal transition despite enhanced wound closure observed in the combination treatment groups (Fig. 6 C). Consistent with these findings, the expression of the EMT-associated transcription factor SLUG remained comparable across all groups on Day 6 (one-way ANOVA, p = 0.31; Fig. 6 D). Collectively, these results indicate that the accelerated wound healing induced by exosome-based combination therapies is accompanied by partial EMT–like features, characterized by maintained E-cadherin expression without overt induction of mesenchymal markers, thereby supporting efficient keratinocyte migration while preserving epithelial integrity. 2.6 Collagen remodeling during wound maturation Collagen deposition and remodeling were evaluated by quantifying Collagen I and Pro-collagen III expression at Days 6 and 18 (Fig. 7 A–D). At Day 6, exosome-based treatments exhibited numerically higher collagen levels compared with control, although these differences did not reach statistical significance by one-way ANOVA. By Day 18, sustained collagen remodeling was observed across treatment groups, with exosome-based treatments demonstrating higher median Collagen I and Pro-collagen III expression than control, although not all comparisons reached statistical significance. Representative Masson’s trichrome–stained sections at Day 6 revealed increased collagen fiber density and more organized extracellular matrix architecture in treated wounds compared with control (Fig. 7 E–I). Collagen fibers were more uniformly distributed within the dermal compartment, indicating ongoing matrix maturation during the remodeling phase. 2.7 Filaggrin expression during epidermal differentiation Filaggrin expression was assessed at Day 6 to evaluate epidermal differentiation during re-epithelialization (Fig. 8 ). Quantitative analysis demonstrated increased filaggrin levels in treated groups compared with control, although intergroup differences did not reach statistical significance. Immunohistochemical staining revealed filaggrin localization predominantly within the suprabasal layers of the regenerating epidermis at the wound edge, consistent with active epidermal differentiation during wound closure. DISCUSSION This study presents a phase-programmable regenerative platform in which exosomes, polymer dot nanozymes (PDNs), and picosecond laser stimulation function as temporally integrated system components rather than additive therapeutics. Within this engineered framework, exosomes provide biochemical activation cues, PDNs operate as catalytic microenvironment regulators, and laser exposure introduces controlled mechanical perturbation. The coordinated interaction among these modules enables phase-dependent redistribution of reparative signaling. During the early epithelialization phase, the Exo + PD configuration was associated with accelerated wound closure and coordinated trends in EGF, ERK1/2, and AQP3 expression. Although not all molecular differences reached statistical significance, the kinetic alignment between AQP3 upregulation and wound area reduction suggests that PDNs enhance epithelial responsiveness within a permissive signaling range rather than amplifying a single pathway. 35, 36 In the splinted wound model, where contraction is minimized, this effect highlights PDN-mediated optimization of epithelial-driven repair dynamics. Mechanistically, the biological initiation signal originates from exosome-derived miRNAs regulating MAPK- and PI3K/AKT-associated pathways. 22 However, PDNs introduce an additional material-level layer of regulation. As hyperbranched nanozymes with intrinsic catalytic properties, PDNs have been reported to modulate reactive oxygen species within physiologically relevant ranges. 15, 16 The antioxidant nanozyme activity of PDNs, including hydroxyl and superoxide radical scavenging, has been experimentally demonstrated in our previous study. 15 Although redox parameters were not directly measured in the present study, the phase-selective response patterns observed here are consistent with previous reports describing the redox-responsive nanozyme activity of PDNs and their capacity to regulate oxidative signaling dynamics. 13, 15, 20 Collectively, these findings support a model in which PDNs function as microenvironment-conditioning materials that shape system-level repair responses rather than acting as passive delivery carriers. Integration of picosecond laser treatment introduced a controlled mechanical perturbation that reshaped the wound microenvironment and redistributed regenerative outputs across healing phases. Laser-induced optical breakdown (LIOB) generates localized microinjury that has been reported to activate remodeling-associated signaling pathways, including TGF-β/Smad-mediated matrix turnover and angiogenic activation. 30, 32 Consistent with this mechanism, the Exo + L + PD group demonstrated enhanced early angiogenic activation and improved collagen organization during later stages of repair. Notably, immediate laser exposure did not accelerate early wound closure. Instead, wound closure kinetics and ERK1/2 signaling patterns suggest that laser-induced microinjury may transiently attenuate epithelial activation during the earliest phase of healing. Such transient suppression of epithelial signaling is consistent with the known response to fractional laser–induced microinjury, in which initial epithelial disruption precedes subsequent tissue remodeling. Within this context, PD nanozymes likely function as microenvironmental regulators that stabilize redox signaling following laser-induced perturbation, enabling the subsequent transition toward coordinated angiogenesis and extracellular matrix organization. 23, 37-39 In addition to modulating epithelial signaling, PD-containing treatments also influenced early angiogenic responses. VEGF expression was significantly elevated in the Exo + PD group and further increased in the Exo + L + PD group, while CD31 expression showed significant upregulation only under the combined Exo + L + PD condition. This pattern suggests that PD nanozymes may prime the angiogenic microenvironment, establishing a permissive signaling state that can be further amplified by laser-induced mechanical stimulation. Within this framework, PD-mediated redox regulation may create a microenvironment favorable for pro-angiogenic signaling, whereas laser-induced optical breakdown provides a secondary stimulus that enhances endothelial activation and vascular remodeling. The sequential elevation of VEGF followed by CD31 expression observed in the present study is consistent with this two-stage model, in which PDs prepare the regenerative niche and laser exposure amplifies angiogenic responses. Such cooperative regulation highlights the complementary roles of catalytic biomaterials and mechanically induced stimuli in directing tissue repair. Rather than acting independently, PD nanozymes and laser stimulation appear to function synergistically to coordinate vascular activation and extracellular matrix remodeling during wound healing. Coordinated but incomplete epithelial–mesenchymal marker modulation, particularly in the Exo + L + PD group, further supports a controlled partial EMT state consistent with regulated tissue regeneration rather than fibrotic transition. 40, 41 The absence of SLUG upregulation suggests that SLUG may be required for EMT initiation but is not essential for maintaining partial EMT during wound repair. 41, 42 These findings imply that PDNs may influence epithelial–mesenchymal plasticity under mechanically perturbed conditions, reinforcing their role as dynamic regulators of repair-state transitions. 16, 19, 21 Collectively, these findings demonstrate that PD nanozymes may function as catalytic regulators of the wound microenvironment, coordinating epithelial activation, angiogenesis, and matrix remodeling during tissue repair. Rather than uniformly accelerating repair, the platform enables redistribution and tuning of phase-specific regenerative outputs, providing a materials-driven strategy for programmable tissue regeneration. Several limitations should be acknowledged. Mechanistic confirmation using pathway-specific inhibition was not performed, and validation in large-animal or chronic wound models will be required to assess translational scalability. In addition, although the antioxidant nanozyme activity of PDNs has been previously demonstrated, including reactive oxygen species (ROS) scavenging capability reported in earlier studies, 15 ROS levels were not directly quantified in the present work. Future investigations incorporating direct redox measurements will help further clarify the relationship between PD-mediated redox modulation and downstream regenerative signaling. Despite these limitations, the present work establishes a material-centric framework for phase-specific modulation of wound repair, positioning PD nanozymes as regulatory interfaces that direct the temporal distribution of regenerative signaling. This systems-level design principle highlights how catalytic biomaterials can be used to program the temporal trajectory of tissue repair. Conclusion This study establishes a nanozyme-enabled biomaterial framework for phase-specific regulation of cutaneous wound healing. Redox-responsive polymer dot nanozymes act as catalytic microenvironmental regulators that coordinate epithelial activation, angiogenesis, and extracellular matrix remodeling when integrated with exosome signaling and picosecond laser stimulation. By conditioning biochemical and biomechanical cues into temporally organized regenerative responses, this platform demonstrates how catalytic biomaterials can modulate healing trajectories rather than merely support tissue repair. Declarations Funding sources: This study was funded by the research grant from China Medical University (CMU111-S-08). Conflicts of interest: The authors declare no competing interests. Data Availability Statement The data supporting the findings of this study are available in this article and the supplementary material. AUTHOR CONTRIBUTIONS Yen-Jen Wang: Methodology, Conceptualization, Visualization, Formal analysis, Writing (original draft) Chang-Cheng Chang: Conceptualization, Investigation, Project administration, Methodology, Writing (review & editing) Tzong-Yuan Juang: Investigation, Conceptualization, Methodology, Funding acquisition, Project administration, Writing (review & editing) Yi-Hsuan Tu: Conceptualization, Methodology, Data curation, Formal analysis, Visualization Jia-Chee Siew: Conceptualization, Methodology, Data curation, Formal analysis, Visualization Shao-Yu Peng: Investigation Steven W. 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Wang Z, Liu L, Bu W, Zheng M, Jin N, Zhang K, et al. Carbon Dots Induce Epithelial-Mesenchymal Transition for Promoting Cutaneous Wound Healing via Activation of TGF-β/p38/Snail Pathway. Adv Funct Mater. 2020;30:2004886. Touqeer M, Siddiqui A, Haider MA, Ullah N, Senanu-James Ocloo O, Ahmed A et al. Breaking the Vicious Cycle: Nanozyme-Driven Multimodal Therapeutics for Diabetic Wound Regeneration. Adv Healthc Mater 2025:e04482. Fu TY, Wang SH, Lin TY, Shen PC, Chang SC, Lin YH, et al. The Exploration of miRNAs From Porcine Fallopian Tube Stem Cells on Porcine Oocytes. Front Vet Sci. 2022;9:869217. Asl ER, Amini M, Najafi S, Mansoori B, Mokhtarzadeh A, Mohammadi A, et al. Interplay between MAPK/ERK signaling pathway and MicroRNAs: A crucial mechanism regulating cancer cell metabolism and tumor progression. Life Sci. 2021;278:119499. González-Brusi L, Algarra B, Moros-Nicolás C, Izquierdo-Rico MJ, Avilés M, Jiménez-Movilla M. A Comparative View on the Oviductal Environment during the Periconception Period. Biomolecules 2020;10. Bian D, Wu Y, Song G, Azizi R, Zamani A. The application of mesenchymal stromal cells (MSCs) and their derivative exosome in skin wound healing: a comprehensive review. Stem Cell Res Ther. 2022;13:24. Zhang Y, Pan Y, Liu Y, Li X, Tang L, Duan M, et al. Exosomes derived from human umbilical cord blood mesenchymal stem cells stimulate regenerative wound healing via transforming growth factor-β receptor inhibition. Stem Cell Res Ther. 2021;12:434. Zhou C, Zhang B, Yang Y, Jiang Q, Li T, Gong J, et al. Stem cell-derived exosomes: emerging therapeutic opportunities for wound healing. Stem Cell Res Ther. 2023;14:107. Zhou Y, Zhang XL, Lu ST, Zhang NY, Zhang HJ, Zhang J, et al. Human adipose-derived mesenchymal stem cells-derived exosomes encapsulated in pluronic F127 hydrogel promote wound healing and regeneration. Stem Cell Res Ther. 2022;13:407. Lee WR, Hsiao CY, Chang ZY, Wang PW, Aljuffali IA, Lin JY et al. Cutaneous Delivery of Cosmeceutical Peptides Enhanced by Picosecond- and Nanosecond-Domain Nd:YAG Lasers with Quick Recovery of the Skin Barrier Function: Comparison with Microsecond-Domain Ablative Lasers. Pharmaceutics 2022;14. Liu C, Wu PJ, Chia SH, Sun CK, Liao YH. Characterization of picosecond laser-induced optical breakdown using harmonic generation microscopy. Lasers Surg Med. 2023;55:561–7. Hwang CY, Chen CC. Serial change in laser-induced optical breakdown by 1064-nm Nd:YAG picosecond laser. Photodermatol Photoimmunol Photomed. 2020;36:63–4. Zhou Y, Hamblin MR, Wen X. An update on fractional picosecond laser treatment: histology and clinical applications. Lasers Med Sci. 2023;38:45. Chang C-C, Chen Y-Y, Chiang H-M, Shen Y-F, Wang J-C, Ma K-C, et al. Nonconventional Fluorescent Hyperbranched Polymer Dots as Skin Nanocarriers Constructed from an Olefinic Aliphatic AB2-Type Monomer. ACS Appl Polym Mater. 2022;4:7790–800. Wang YJ, Chang CC, Lu ME, Wu YH, Shen JW, Chiang HM et al. Photoaging and Sequential Function Reversal with Cellular-Resolution Optical Coherence Tomography in a Nude Mice Model. Int J Mol Sci 2022;23. Sebastian R, Chau E, Fillmore P, Matthews J, Price LA, Sidhaye V, et al. Epidermal aquaporin-3 is increased in the cutaneous burn wound. Burns. 2015;41:843–7. Sugimoto T, Huang L, Minematsu T, Yamamoto Y, Asada M, Nakagami G, et al. Impaired aquaporin 3 expression in reepithelialization of cutaneous wound healing in the diabetic rat. Biol Res Nurs. 2013;15:347–55. Sun Y, Liu WZ, Liu T, Feng X, Yang N, Zhou HF. Signaling pathway of MAPK/ERK in cell proliferation, differentiation, migration, senescence and apoptosis. J Recept Signal Transduct Res. 2015;35:600–4. Sharma GD, He J, Bazan HE. p38 and ERK1/2 coordinate cellular migration and proliferation in epithelial wound healing: evidence of cross-talk activation between MAP kinase cascades. J Biol Chem. 2003;278:21989–97. Park JI. MAPK-ERK Pathway. Int J Mol Sci 2023;24. Koirala R, Priest AV, Yen CF, Cheah JS, Pannekoek WJ, Gloerich M et al. Inside-out regulation of E-cadherin conformation and adhesion. Proc Natl Acad Sci U S A 2021;118. Yao W, Wang Z, Ma H, Lin Y, Liu X, Li P, et al. Epithelial-mesenchymal plasticity (EMP) in wound healing: Exploring EMT mechanisms, regulatory network, and therapeutic opportunities. Heliyon. 2024;10:e34269. Subbalakshmi AR, Sahoo S, Biswas K, Jolly MK. A Computational Systems Biology Approach Identifies SLUG as a Mediator of Partial Epithelial-Mesenchymal Transition (EMT). Cells Tissues Organs. 2022;211:689–702. Additional Declarations No competing interests reported. Supplementary Files sFig1.jpg GraphicalAbstract.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9132659","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":609835647,"identity":"cd78e99d-1d91-4f49-86d2-193ccf77ddd0","order_by":0,"name":"Yen-Jen Wang","email":"","orcid":"","institution":"MacKay Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yen-Jen","middleName":"","lastName":"Wang","suffix":""},{"id":609835648,"identity":"e0accfb2-edfe-4a8d-abaf-e09e888b84f6","order_by":1,"name":"Chang-Cheng Chang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYDACCQjFwy///uEDEIOPWC0ykg05zAYgLWzEarExOJDDBmYT1MI/u/nZwy81djwGB84eq/yaYyfDxsD88NENfJbcOWZuLHMsmUfyYF/abdltyUCHsRkb5+DRYiCRYCYt2XCAh+8wg9ltyW3MQC08bNL4taR/A2thOMZgViy5rZ4YLTlmkh+BWgTO8Jgxftx2mLAWiRs5ZdIMIL/MYEuWZtx2nIeNmYBf+Gekb5P8UWNnzy/BfPDjz23V9vzszQ8f49MCAsw8KAxmAspBgPEHOmMUjIJRMApGATIAAOVMQiUSY0qaAAAAAElFTkSuQmCC","orcid":"","institution":"China Medical University Hospital","correspondingAuthor":true,"prefix":"","firstName":"Chang-Cheng","middleName":"","lastName":"Chang","suffix":""},{"id":609835649,"identity":"b22339c1-ddc9-4653-9524-3d84c80cb3fc","order_by":2,"name":"Tzong-Yuan Juang","email":"","orcid":"","institution":"China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tzong-Yuan","middleName":"","lastName":"Juang","suffix":""},{"id":609835650,"identity":"beeb942e-28b1-44cd-a72f-dbd93cd6c79a","order_by":3,"name":"Yi-Hsuan Tu","email":"","orcid":"","institution":"China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yi-Hsuan","middleName":"","lastName":"Tu","suffix":""},{"id":609835651,"identity":"228a4752-fab3-4e57-b94e-8f518e1fbc4c","order_by":4,"name":"Jia-Chee Siew","email":"","orcid":"","institution":"China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jia-Chee","middleName":"","lastName":"Siew","suffix":""},{"id":609835652,"identity":"f86a835b-ea2b-47b5-b2b8-9ece20ac7482","order_by":5,"name":"Shao-Yu Peng","email":"","orcid":"","institution":"National Pingtung University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Shao-Yu","middleName":"","lastName":"Peng","suffix":""},{"id":609835653,"identity":"aebc96a7-3695-4252-8143-652afb63a971","order_by":6,"name":"Steven W. Shaw","email":"","orcid":"","institution":"Chang Gung University","correspondingAuthor":false,"prefix":"","firstName":"Steven","middleName":"W.","lastName":"Shaw","suffix":""},{"id":609835654,"identity":"18c15f7d-c71a-4073-8091-526849cad127","order_by":7,"name":"Hoi-Man Iao","email":"","orcid":"","institution":"China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hoi-Man","middleName":"","lastName":"Iao","suffix":""},{"id":609835655,"identity":"31dbdf5c-cbae-4088-9f91-8283fa3747db","order_by":8,"name":"Siao-Cian Fan","email":"","orcid":"","institution":"China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Siao-Cian","middleName":"","lastName":"Fan","suffix":""},{"id":609835656,"identity":"9937ba8f-acc0-4844-8ecd-82a504b76530","order_by":9,"name":"Hsiu-Mei Chiang","email":"","orcid":"","institution":"China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hsiu-Mei","middleName":"","lastName":"Chiang","suffix":""},{"id":609835657,"identity":"bdf13cce-b97e-40c2-b767-6973f89a1f17","order_by":10,"name":"Bor-Shyh Lin","email":"","orcid":"","institution":"University College London","correspondingAuthor":false,"prefix":"","firstName":"Bor-Shyh","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2026-03-16 03:53:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9132659/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9132659/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105564887,"identity":"3fef2d0a-43b2-42e1-a05e-f5c17bf8d014","added_by":"auto","created_at":"2026-03-27 12:51:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":214709,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental design and treatment schedule of the murine splinted excisional wound model.\u003c/strong\u003e\u003cbr\u003e\nBALB/c nude mice were randomly assigned to five treatment groups: Control, Exosome, Exosome + Laser, Exosome + PDs, and Exosome + Laser + PDs. Two full-thickness excisional wounds were created on the dorsum of each mouse (n = 6 wounds per group per time point). Early epithelial and angiogenic markers (EGF, ERK1/2, VEGF, and CD31) were analyzed on Day 3. Markers associated with epithelial plasticity and matrix remodeling were evaluated on Day 6, and late collagen remodeling was assessed on Day 18.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9132659/v1/68509ebcccf038de713bc07a.png"},{"id":105300908,"identity":"66288220-d6ee-45c2-b6d7-92b8bbd042fa","added_by":"auto","created_at":"2026-03-24 13:47:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":87719,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantitative analysis of wound closure kinetics following full-thickness excisional wounding.\u003c/strong\u003e\u003cbr\u003e\nWound areas were measured at Days 0, 3, 6, 9, and 12 and expressed as the percentage of the original wound area. Data are presented as mean wound area percentages for each treatment group. The Exosome + PD group exhibited the most rapid reduction in wound size during the early phase of repair.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9132659/v1/efa0e0c7d271cbd093e5f631.png"},{"id":105564976,"identity":"56e6553d-ab97-46fb-8ead-f05bdd18f98c","added_by":"auto","created_at":"2026-03-27 12:51:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":751688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative macroscopic images illustrating wound healing progression.\u003c/strong\u003e\u003cbr\u003e\nRepresentative photographs of wounds from each treatment group at Days 0, 3, 6, 9, 12, 15, and 18 after injury. The Exosome + PD group showed faster early wound closure, whereas the Exosome + Laser + PD group exhibited improved tissue appearance at later stages.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9132659/v1/4287af96e9b2d59f3554fb29.png"},{"id":105564855,"identity":"0b8ef0d8-c119-4298-b783-1f152433b9a7","added_by":"auto","created_at":"2026-03-27 12:51:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":68898,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEarly EGF–ERK1/2 signaling and subsequent AQP3 expression during wound healing.\u003c/strong\u003e\u003cbr\u003e\n(A) EGF expression in wound tissue on Day 3. PD-containing treatments, particularly the Exo+PD group, showed significantly higher EGF levels compared with control (**p \u0026lt; 0.01).\u003cbr\u003e\n(B) ERK1/2 expression on Day 3 quantified by ELISA. ERK1/2 levels showed a modest increase in exosome-treated groups, although intergroup differences were not statistically significant.\u003cbr\u003e\n(C) Aquaporin-3 (AQP3) expression on Day 6 assessed by immunohistochemistry. Increased AQP3 expression was observed in Exo+PD and Exo+L+PD groups compared with control. Box plots represent the median and interquartile range with whiskers indicating minimum and maximum values. Individual points represent biological replicates (n = 6 wounds per group).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9132659/v1/ff74f5405c22db5d172fc218.png"},{"id":105564740,"identity":"dd572d9e-6722-4d87-8d4e-da5002dacc00","added_by":"auto","created_at":"2026-03-27 12:50:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":61526,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAngiogenesis-related marker expression during the early phase of wound healing.\u003c/strong\u003e\u003cbr\u003e\nVEGF (A) and CD31 (B) expression levels were quantified on Day 3 after injury. PD-containing treatments increased VEGF expression, while the Exosome + Laser + PD group exhibited the highest CD31 levels.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9132659/v1/e5439a6ca28db00303f51481.png"},{"id":105300910,"identity":"8164e7f8-0121-4b98-bff8-74752b177c93","added_by":"auto","created_at":"2026-03-24 13:47:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":401791,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of epithelial and mesenchymal markers indicating partial epithelial plasticity during wound repair.\u003c/strong\u003e\u003cbr\u003e\n(A) Quantitative analysis of E-cadherin expression in wound tissue on Day 6.\u003cbr\u003e\n(B) Representative immunohistochemical staining of E-cadherin at the wound edge on Day 6 (arrows).\u003cbr\u003e\n(C) Quantification of vimentin expression on Day 6.\u003cbr\u003e\n(D) Quantitative analysis of SLUG expression on Day 6.\u003cbr\u003e\nData are presented as box plots showing the median, interquartile range, and individual data points (n = 6 wounds per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Original magnification, 200×.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9132659/v1/c26fe366f4ae595f1e7846cc.png"},{"id":105300913,"identity":"bd5b2b6c-88bb-4d0f-80c5-d146c75d292e","added_by":"auto","created_at":"2026-03-24 13:47:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1232019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCollagen deposition and extracellular matrix remodeling during wound healing.\u003c/strong\u003e\u003cbr\u003e\n(A–D) Quantitative analysis of Collagen I and Pro-collagen III expression in wound tissues at Days 6 and 18. Box plots represent the median, interquartile range, and individual data points for each treatment group (n = 6 wounds per group).\u003cbr\u003e\n(E–I) Representative Masson’s trichrome–stained sections of wound tissue on Day 6 showing collagen deposition and dermal organization. Original magnification, 200×. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9132659/v1/7d23a53b051aaedb6655d876.png"},{"id":105564774,"identity":"04ba4586-3847-4752-9424-6ccf02540fa9","added_by":"auto","created_at":"2026-03-27 12:50:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":261540,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFilaggrin expression during re-epithelialization at Day 6.\u003c/strong\u003e\u003cbr\u003e\nLeft: Quantitative analysis of filaggrin expression in wound tissue on Day 6. Data are presented as box plots showing the median, interquartile range, and individual data points (n = 6 wounds per group).\u003cbr\u003e\nRight: Representative immunohistochemical staining of filaggrin in regenerating epidermis at the wound edge. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Original magnification, 200×.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9132659/v1/e11667d619ca202cbf530920.png"},{"id":106414278,"identity":"d86d0d8f-899d-4251-b4e1-69602142c540","added_by":"auto","created_at":"2026-04-08 10:07:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4140318,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9132659/v1/a4c31ae0-86dd-4bfc-89fe-40562f4427b9.pdf"},{"id":105564888,"identity":"d3a7b244-ae1a-42d3-811c-fb928106062c","added_by":"auto","created_at":"2026-03-27 12:51:13","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":414037,"visible":true,"origin":"","legend":"","description":"","filename":"sFig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9132659/v1/11b50b085facde2d8cd513cd.jpg"},{"id":105300917,"identity":"aa56a9d1-7ef5-49b3-82f5-e14df946cd89","added_by":"auto","created_at":"2026-03-24 13:47:32","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":337721,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-9132659/v1/e78a026bfeff5280adcaa4c1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Redox-Responsive Polymer Dot Nanozymes Coordinate Exosome-Mediated Cutaneous Regeneration via Laser-Modulated Microenvironment Remodeling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCutaneous wound healing is orchestrated by tightly coordinated biochemical and biomechanical cues that regulate epithelial activation, angiogenesis, and extracellular matrix (ECM) remodeling. \u003csup\u003e1, 2\u003c/sup\u003e Although numerous biomaterial platforms have been developed to enhance repair through growth factor delivery, scaffold engineering, or smart wound dressings, most systems function primarily as passive carriers or structural supports.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Consequently, they lack the capacity to dynamically regulate the wound microenvironment in a temporally defined manner. Among critical microenvironmental regulators, redox balance plays a central role in controlling epithelial plasticity, angiogenic signaling, and matrix turnover. \u003csup\u003e5\u0026ndash;8\u003c/sup\u003e However, material systems capable of actively tuning oxidative signaling within a permissive regenerative window remain limited.\u003c/p\u003e \u003cp\u003ePolymer dot nanozymes (PDNs) constitute an ultrasmall (\u0026lt;\u0026thinsp;10 nm), redox-responsive carbon nanomaterial platform with intrinsic enzyme-mimetic catalytic functionality.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Unlike conventional nanoparticle carriers, PDNs are drug-free nanozymes that regulate reactive oxygen species (ROS) through intrinsic catalytic redox activity rather than acting as passive delivery carriers. \u003csup\u003e13\u003c/sup\u003e By maintaining ROS within a permissive signaling window, PDNs regulate redox signaling within the wound microenvironment, thereby facilitating epithelial activation and tissue repair.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Beyond amplifying exogenous biologics, PDNs function as bioactive material regulators that reshape the wound niche through microenvironmental modulation. \u003csup\u003e17,18, 19\u003c/sup\u003e This material-level control introduces the possibility of phase-programmable regeneration, in which the timing and directionality of repair processes can be engineered rather than passively supported.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eExosomes derived from porcine fallopian tube stem cells (PFTSC-Exo) provide a biologically defined source of growth factors and regulatory miRNAs capable of activating MAPK- and PI3K/AKT-associated pathways implicated in epithelial proliferation and tissue remodeling.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e However, the efficacy of exosome-based therapies remains highly dependent on the surrounding microenvironment, which can either permit or constrain regenerative signaling. \u003csup\u003e25\u0026ndash;28\u003c/sup\u003e Mechanical stimuli such as 755-nm picosecond laser\u0026ndash;induced optical breakdown (LIOB) introduce localized microinjury that activates angiogenic and ECM remodeling pathways, including TGF-β/Smad signaling. \u003csup\u003e29\u0026ndash;32\u003c/sup\u003e Yet without redox-level regulation, such mechanically triggered pathways may not be optimally synchronized with epithelial repair dynamics.\u003c/p\u003e \u003cp\u003eHere, we introduce a redox-responsive PDN platform that functions as a gating nanozyme interface integrating exosome-derived biochemical cues with laser-triggered mechanical signaling. Using a murine splinted excisional wound model that isolates epithelial-driven repair, we investigated whether PDNs can selectively tune early epithelial activation while reprogramming laser-induced angiogenic and ECM remodeling responses in a phase-dependent manner. We hypothesized that PDNs act as catalytic material regulators capable of directing the temporal dynamics of wound healing, thereby coordinating epithelialization, angiogenesis, and tissue maturation. Accordingly, this study investigates whether nanozyme-mediated microenvironmental regulation can redistribute regenerative signaling during wound repair.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv\u003e\n \u003ch2\u003e1.1. Preparation and Isolation of porcine fallopian tube stem cell-derived exosomes\u003c/h2\u003e\n \u003cp\u003ePorcine fallopian tube stem cell (PFTSC)-derived exosomes, obtained from the National Pingtung University of Science and Technology and Chang Gung Memorial Hospital (Linkou, Taiwan), were used as the source of porcine oviduct\u0026ndash;derived exosomes. Fresh porcine oviduct tissues were rinsed three times with sterile saline supplemented with 1% penicillin\u0026ndash;streptomycin (P/S), minced into approximately 0.1\u0026ndash;0.5 mm\u0026sup3; fragments, and cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% P/S at 38.5\u0026deg;C in a humidified atmosphere with 5% CO₂.\u003c/p\u003e\n \u003cp\u003eAt passage five, when cells reached approximately 80\u0026ndash;90% confluence, cultures were washed with Dulbecco\u0026rsquo;s phosphate-buffered saline (DPBS) and incubated for 3 h in TCM-199 medium supplemented with 10% FBS, 2.5 \u0026micro;g/mL follicle-stimulating hormone (FSH), 5 IU/\u0026micro;L human chorionic gonadotropin (hCG), 10 ng/mL epidermal growth factor (EGF), 1% antibiotic\u0026ndash;antimycotic solution (ABAM), and 10% (v/v) porcine follicular fluid (PFF). Conditioned medium (CM) was subsequently collected, filtered through a 0.22-\u0026micro;m membrane to remove cellular debris, and stored at 4\u0026deg;C until further processing.\u003c/p\u003e\n \u003cp\u003eFor exosome isolation, PFTSC were cultured in CM supplemented with exosome-depleted FBS. Exosomes were isolated using the miRCURY\u0026trade; Exosome Isolation Kit (Qiagen, Hilden, Germany) according to the manufacturer\u0026rsquo;s instructions. Briefly, CM was mixed with the isolation reagent and centrifuged at 3,200 \u0026times; g for 30 min. The resulting exosome-containing pellets were resuspended in the provided buffer and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use.\u003c/p\u003e\n \u003cp\u003eFor visualization, 10 \u0026micro;L of the exosome suspension was placed onto a glass slide and air-dried prior to imaging. For topical application experiments, exosome preparations were diluted in phosphate-buffered saline (PBS) to a final concentration of 2 mg per 100 \u0026micro;L.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003e\u003cstrong\u003e1.2 Hyperbranched Polymer Dot (PD) Synthesis and Characterization\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eHyperbranched polymer dots (PDs) were synthesized via an A₂ + B₃ polycondensation strategy as previously reported. \u003csup\u003e15, 18,33\u003c/sup\u003e Briefly, wholly aliphatic monomers were subjected to a one-pot polymerization process to generate hyperbranched polymer dots with a heteroatom-enriched polymeric carbon framework. This synthetic route enables gram-scale production with yields of up to 44%, demonstrating good scalability and synthetic reproducibility.\u003c/p\u003e\n \u003cp\u003eDynamic light scattering (DLS) and transmission electron microscopy (TEM) analyses revealed ultrasmall particle sizes in the range of approximately 3\u0026ndash;5 nm. X-ray photoelectron spectroscopy (XPS) confirmed the presence of abundant nitrogen- and oxygen-containing functional groups within the polymer backbone, consistent with successful heteroatom incorporation. These surface functionalities contribute to the high aqueous dispersibility of the PDs without additional surface modification.\u003c/p\u003e\n \u003cp\u003eThe resulting PDs exhibit intrinsic fluorescence and low cytotoxicity, supporting their suitability for biological applications. In addition, the heteroatom-rich polymer framework confers redox-responsive catalytic activity, enabling reactive oxygen species (ROS) scavenging behavior characteristic of antioxidant nanozymes.\u003c/p\u003e\n \u003cp\u003eFor in vivo experiments, PDs were incorporated into a PEG1000-based hydrogel formulation prior to topical administration, as previously described.\u003csup\u003e\u003cspan citationid=\"CR15\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\"\u003e18\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\"\u003e\n \u003ch2\u003e1.3 Picosecond laser treatment\u003c/h2\u003e\n \u003cp\u003eA 755 nm picosecond laser with a diffractive lens array (PicoSure\u0026reg;, Cynosure, MA, USA) was used to induce LIOB. Laser parameters were selected based on previous studies demonstrating reliable induction of laser-induced optical breakdown (LIOB) in murine skin.\u003csup\u003e\u003cspan citationid=\"CR12\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR34\"\u003e34\u003c/span\u003e\u003c/sup\u003e Optimized parameters included: 6 mm spot size, 0.71 J/cm\u0026sup2; fluence, 750 ps pulse duration, and 5 Hz frequency.\u003csup\u003e\u003cspan citationid=\"CR12\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR34\"\u003e34\u003c/span\u003e\u003c/sup\u003e A total of 500 pulses were\u003c/p\u003e\n \u003cp\u003eapplied immediately post-wounding in laser-treated groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\"\u003e\n \u003ch2\u003e1.4. Animal Model and Experimental Design\u003c/h2\u003e\n \u003cp\u003eFive-week-old female BALB/cAnN.Cg-Foxn1^nu/CrlNarl nude mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan). All animal procedures were approved by the Institutional Animal Care and Use Committee of China Medical University (IACUC approval no. CMUIACUC-2022-404) and conducted in accordance with institutional and national guidelines for animal welfare.\u003c/p\u003e\n \u003cp\u003eA total of 45 nude mice were randomly assigned to five experimental groups using a computer-generated randomization sequence (3 mice per group per time point): Control, Exosome (Exo), Exosome plus laser (Exo\u0026thinsp;+\u0026thinsp;L), Exosome plus polymer dots (Exo\u0026thinsp;+\u0026thinsp;PD), and Exosome plus polymer dots combined with laser (Exo\u0026thinsp;+\u0026thinsp;L+PD). Each mouse carried two wounds, resulting in six wounds per group at each time point.\u003c/p\u003e\n \u003cp\u003eOn Day 0, mice were anesthetized with isoflurane, and two symmetric 6-mm full-thickness excisional wounds were created on the dorsal skin of each mouse. To minimize wound contraction and better mimic human wound healing, silicone splints (inner diameter, 10 mm; outer diameter, 18 mm; thickness, 0.5 mm) were secured around each wound using 6\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon sutures (Ethicon, USA) and medical-grade tissue adhesive (3M Vetbond\u0026trade;, USA).\u003c/p\u003e\n \u003cp\u003eOn Day 0, laser-treated groups (Exo\u0026thinsp;+\u0026thinsp;L and Exo\u0026thinsp;+\u0026thinsp;L+PD) received picosecond laser irradiation designed to induce laser-induced optical breakdown (LIOB), delivered as 500 pulses. Immediately after laser treatment, topical treatments were applied according to group allocation. Mice in the exosome-treated groups (all groups except the control group) received topical application of exosomes (100 \u0026micro;L per wound). Mice assigned to the PD-treated groups (Exo\u0026thinsp;+\u0026thinsp;PD and Exo\u0026thinsp;+\u0026thinsp;L+PD) received 100 \u0026micro;L of PD hydrogel (5 mg/mL polymer dots formulated in 5% PEG1000), which was reapplied on Days 3, 6, 9, 12, and 15.\u003c/p\u003e\n \u003cp\u003eWound healing progression was documented by standardized digital photography every 3 days from Day 0 to Day 18, and wound areas were quantified using ImageJ software (National Institutes of Health, USA). For mechanistic and histological analyses, mice were sacrificed at predetermined time points as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\"\u003e1\u003c/span\u003e, and wound tissues were harvested for subsequent analyses.\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003e1.5 Wound Monitoring and Image Analysis\u003c/h2\u003e\n \u003cp\u003eDigital photographs of wounds were captured on Days 0, 3, 6, 9, and 12. Wound area was measured using ImageJ software, and percentage wound closure was calculated:\u003c/p\u003e\n \u003cp\u003e% wound closure = [(A₀ \u0026ndash; Aₜ)/A₀] \u0026times; 100 where A₀ is initial wound area and At is area at time t.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003e1.6. Tissue Harvest and Processing\u003c/h2\u003e\n \u003cp\u003eOn Days 3, 6, 12, and 18, three mice per group were euthanized at each predetermined time point. Full-thickness wound tissues, including the wound bed and adjacent periwound skin, were carefully excised. Harvested tissues were gently defatted, flattened on paperboard to maintain consistent orientation, and fixed in 10% neutral-buffered formalin for 24 h at room temperature. Following fixation, tissues were rinsed with deionized water, placed into tissue cassettes, and kept submerged in water to prevent desiccation prior to routine histological processing.\u003c/p\u003e\n \u003cp\u003eFor biochemical analyses, wound tissues were homogenized in ice-cold phosphate-buffered saline (PBS) supplemented with a protease inhibitor cocktail. Tissue lysates were clarified by centrifugation, and total protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, USA) according to the manufacturer\u0026rsquo;s instructions. The resulting protein extracts were subsequently used for enzyme-linked immunosorbent assay (ELISA) analyses.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003e1.7 Time-Point\u0026ndash;Specific Histological and Biochemical Analyses\u003c/h2\u003e\n \u003cp\u003eTo capture the temporal dynamics of wound healing and tissue remodeling, histological and biochemical analyses were performed at predefined time points corresponding to distinct phases of wound repair.\u003c/p\u003e\n \u003cp\u003eDay 3 (early inflammatory and activation phase).\u003c/p\u003e\n \u003cp\u003eAt Day 3, Masson\u0026rsquo;s trichrome staining was performed to assess early changes in collagen deposition and dermal structural integrity. ERK1/2 protein levels were quantified by ELISA to evaluate activation of MAPK signaling pathways associated with growth factor\u0026ndash;mediated cellular activation. Immunohistochemical staining for CD31, VEGF, and EGF was conducted to examine angiogenic responses and growth factor signaling potentially mediated by exosome treatment. Hematoxylin and eosin (H\u0026amp;E) staining was used for general histopathological assessment and evaluation of early cellular changes within the wound bed.\u003c/p\u003e\n \u003cp\u003eDay 6 (re-epithelialization and matrix synthesis phase).\u003c/p\u003e\n \u003cp\u003eAt Day 6, Masson\u0026rsquo;s trichrome staining was repeated to evaluate progressive collagen deposition and dermal remodeling. Procollagen type I and type III levels were quantified by ELISA to determine early extracellular matrix synthesis and to assess whether laser and/or polymer dot treatment enhanced collagen production. Immunohistochemical analyses of E-cadherin and vimentin were performed to assess epithelial\u0026ndash;mesenchymal transition (EMT) dynamics, while filaggrin and aquaporin-3 were evaluated as markers of epidermal differentiation and keratinocyte functional activation, respectively. H\u0026amp;E staining was additionally used to assess overall tissue architecture and cellular morphology.\u003c/p\u003e\n \u003cp\u003eDay 18 (late remodeling and maturation phase).\u003c/p\u003e\n \u003cp\u003eAt Day 18, ELISA was performed to quantify collagen type I and type III levels, reflecting cumulative collagen production and matrix maturation during the late remodeling phase of wound healing.\u003c/p\u003e\n \u003cp\u003eWound closure assessment.\u003c/p\u003e\n \u003cp\u003eStandardized wound photographs were acquired every three days (Days 0, 3, 6, 9, 12, 15, and 18). Wound areas were measured using ImageJ software, and wound closure rates were calculated to compare healing kinetics among experimental groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003e1.8 Statistical Analysis\u003c/h2\u003e\n \u003cp\u003eAll quantitative data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were performed using GraphPad Prism (version 10.2; GraphPad Software, San Diego, CA, USA).\u003c/p\u003e\n \u003cp\u003eFor wound closure analysis, wound areas measured at multiple time points (Days 3, 6, 9, and 12) were analyzed using two-way repeated-measures analysis of variance (ANOVA), with treatment group and time as independent factors, followed by Tukey\u0026rsquo;s multiple comparisons test to assess differences between groups at individual time points.\u003c/p\u003e\n \u003cp\u003eFor biochemical and histological quantitative data obtained at single time points (including ELISA measurements of ERK1/2, collagen type I, procollagen type III, and immunohistochemical quantification of CD31, VEGF, E-cadherin, and vimentin), comparisons among multiple groups were performed using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. When comparisons involved only two groups, an unpaired two-tailed Student\u0026rsquo;s t test was applied as appropriate.\u003c/p\u003e\n \u003cp\u003eQualitative macroscopic wound appearance was assessed descriptively based on standardized digital photographs acquired at predefined time points and was not subjected to statistical testing. A p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.1 Exosome\u0026ndash;PD treatment accelerates early wound closure\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003cp\u003eWound closure was quantitatively assessed by ImageJ analysis at Days 3, 6, 9, and 12 following wound creation (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All experimental groups demonstrated progressive wound contraction over time; however, distinct differences in healing kinetics were observed among treatment groups.\u003c/p\u003e\n \u003cp\u003eThe Exosome\u0026thinsp;+\u0026thinsp;PD (Exo\u0026thinsp;+\u0026thinsp;PD) group consistently exhibited the most rapid reduction in wound area during the early healing phase. By Day 6, wounds in the Exo\u0026thinsp;+\u0026thinsp;PD group had decreased to 42.0\u0026thinsp;\u0026plusmn;\u0026thinsp;14.0% of the original wound size, compared with 59.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6% in the control group and 55.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6% in the exosome-only group. This accelerated closure became more pronounced by Day 9, at which time the Exo\u0026thinsp;+\u0026thinsp;PD group reached 19.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.2% of the original wound area, representing the fastest wound closure among all treatment groups.\u003c/p\u003e\n \u003cp\u003eThese findings suggest that PD incorporation influences wound-healing kinetics, with Exo\u0026thinsp;+\u0026thinsp;PD accelerating early wound closure, whereas the Exo\u0026thinsp;+\u0026thinsp;L+PD group shows comparatively slower early closure but improved tissue appearance at later time points.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Macroscopic wound appearance reveals distinct healing trajectories\u003c/h2\u003e\n \u003cp\u003eRepresentative macroscopic images were acquired at Days 0, 3, 6, 9, 12, 15, and 18 to qualitatively evaluate wound closure dynamics and scar quality across treatment groups (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Immediately after injury (Day 0), all wounds presented as uniform 6-mm full-thickness defects stabilized by silicone splints.\u003c/p\u003e\n \u003cp\u003eIn the control group, wound closure progressed slowly, with limited size reduction by Day 6 and persistent granulation tissue and irregular wound beds. Although gradual contraction was observed between Days 9 and 12, complete closure was not achieved until Day 18, and residual scabbing with uneven epithelial surfaces remained apparent.\u003c/p\u003e\n \u003cp\u003eExosome-treated wounds exhibited moderate improvement, characterized by smoother wound margins and reduced inflammatory appearance by Day 6. Wound areas decreased steadily thereafter, with near-complete closure observed by Days 15\u0026ndash;18.\u003c/p\u003e\n \u003cp\u003eNotably, the Exo\u0026thinsp;+\u0026thinsp;PD group demonstrated the most rapid early macroscopic healing, with marked wound size reduction and flattened, dry wound beds evident by Day 6. Most wounds appeared fully closed by Day 9, and by Days 12\u0026ndash;18 the regenerated skin surface was smooth and uniform, suggesting improved epithelial coverage and smoother surface morphology.\u003c/p\u003e\n \u003cp\u003eIn contrast, wounds treated with Exosome\u0026thinsp;+\u0026thinsp;Laser alone showed delayed early closure, with residual open areas persisting at Day 6. Nevertheless, wound contraction progressed after Day 9, and closure was achieved by Days 15\u0026ndash;18, albeit with mild surface irregularity.\u003c/p\u003e\n \u003cp\u003eInterestingly, the Exo+Laser\u0026thinsp;+\u0026thinsp;PD group displayed a distinct healing pattern, in which early wound closure was less pronounced than that observed with Exo\u0026thinsp;+\u0026thinsp;PD alone, yet substantial improvements in tissue appearance and surface regularity became evident by Days 9\u0026ndash;12. By Day 18, wounds were fully closed with smooth, well-reconstructed skin, suggesting enhanced tissue remodeling and superior scar quality despite comparable final closure.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Early EGF\u0026ndash;ERK1/2 Signaling and Subsequent AQP3 Upregulation\u003c/h2\u003e\n \u003cp\u003eEarly molecular events associated with accelerated wound closure were evaluated by examining growth factor\u0026ndash;related signaling pathways during the initial phase of repair. EGF expression assessed on Day 3 was significantly increased in the Exo\u0026thinsp;+\u0026thinsp;PD group compared with the control group (Tukey\u0026rsquo;s post hoc test, p\u0026thinsp;=\u0026thinsp;0.0074), indicating enhanced early growth factor availability following combined exosome and polymer dot treatment. No other pairwise comparisons reached statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eConsistent with this upstream change, ERK1/2 expression on Day 3 exhibited a trend toward increased levels in exosome-treated groups, particularly in the Exosome and Exo\u0026thinsp;+\u0026thinsp;PD groups, whereas a modest attenuation was observed in laser-treated groups; however, inter-group differences did not reach statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eAquaporin-3 (AQP3) expression assessed on Day 6 demonstrated an overall upregulation trend in treatment groups associated with accelerated wound closure (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Groups showing higher EGF availability and increased ERK1/2 signaling trends during the early phase tended to exhibit elevated AQP3 expression at later time points.\u003c/p\u003e\n \u003cp\u003eCollectively, these findings suggest a temporal association between early growth factor\u0026ndash;related signaling and subsequent epithelial functional activation during wound repair, particularly in PD-containing exosome-based treatments.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 PD-Containing Treatments Enhance Early Angiogenic Signaling\u003c/h2\u003e\n \u003cp\u003eAt Day 3 after full-thickness excisional wounding, angiogenesis-associated markers VEGF and CD31 were significantly modulated by treatment (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eOne-way ANOVA revealed a significant intergroup difference in CD31 expression (F\u0026thinsp;=\u0026thinsp;3.22, p\u0026thinsp;=\u0026thinsp;0.0298). Post hoc analysis demonstrated that CD31 expression was significantly higher in the Exosome\u0026thinsp;+\u0026thinsp;Laser\u0026thinsp;+\u0026thinsp;PD group compared with the Exosome-only group (p\u0026thinsp;=\u0026thinsp;0.0379), suggesting enhanced early endothelial activation under combinatorial treatment.\u003c/p\u003e\n \u003cp\u003eVEGF expression exhibited a more pronounced treatment-dependent effect (F\u0026thinsp;=\u0026thinsp;9.64, p\u0026thinsp;=\u0026thinsp;7.36 \u0026times; 10⁻⁵). PD-containing regimens significantly increased VEGF levels compared with control, with both Exosome\u0026thinsp;+\u0026thinsp;PD (p\u0026thinsp;=\u0026thinsp;0.0206) and Exosome\u0026thinsp;+\u0026thinsp;Laser\u0026thinsp;+\u0026thinsp;PD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) showing marked elevation. In addition, Exosome\u0026thinsp;+\u0026thinsp;Laser\u0026thinsp;+\u0026thinsp;PD induced significantly higher VEGF expression than Exosome alone (p\u0026thinsp;=\u0026thinsp;0.0048) and Exosome\u0026thinsp;+\u0026thinsp;Laser (p\u0026thinsp;=\u0026thinsp;0.0063), indicating a synergistic enhancement of early pro-angiogenic signaling.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Re-epithelialization occurs without full EMT activation\u003c/h2\u003e\n \u003cp\u003eTo further characterize the epithelial phenotype during the re-epithelialization phase, the expression of epithelial and mesenchymal markers was evaluated on Day 6. Quantitative analyses revealed no significant differences in E-cadherin expression among treatment groups (one-way ANOVA, p\u0026thinsp;=\u0026thinsp;0.18), indicating that epithelial cell\u0026ndash;cell adhesion was not completely lost during re-epithelialization across conditions (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B).\u003c/p\u003e\n \u003cp\u003eSimilarly, Vimentin expression did not differ significantly between groups (one-way ANOVA, p\u0026thinsp;=\u0026thinsp;0.22), suggesting the absence of a full mesenchymal transition despite enhanced wound closure observed in the combination treatment groups (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\n \u003cp\u003eConsistent with these findings, the expression of the EMT-associated transcription factor SLUG remained comparable across all groups on Day 6 (one-way ANOVA, p\u0026thinsp;=\u0026thinsp;0.31; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e\n \u003cp\u003eCollectively, these results indicate that the accelerated wound healing induced by exosome-based combination therapies is accompanied by partial EMT\u0026ndash;like features, characterized by maintained E-cadherin expression without overt induction of mesenchymal markers, thereby supporting efficient keratinocyte migration while preserving epithelial integrity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Collagen remodeling during wound maturation\u003c/h2\u003e\n \u003cp\u003eCollagen deposition and remodeling were evaluated by quantifying Collagen I and Pro-collagen III expression at Days 6 and 18 (Fig. \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;D). At Day 6, exosome-based treatments exhibited numerically higher collagen levels compared with control, although these differences did not reach statistical significance by one-way ANOVA. By Day 18, sustained collagen remodeling was observed across treatment groups, with exosome-based treatments demonstrating higher median Collagen I and Pro-collagen III expression than control, although not all comparisons reached statistical significance.\u003c/p\u003e\n \u003cp\u003eRepresentative Masson\u0026rsquo;s trichrome\u0026ndash;stained sections at Day 6 revealed increased collagen fiber density and more organized extracellular matrix architecture in treated wounds compared with control (Fig. \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE\u0026ndash;I). Collagen fibers were more uniformly distributed within the dermal compartment, indicating ongoing matrix maturation during the remodeling phase.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Filaggrin expression during epidermal differentiation\u003c/h2\u003e\n \u003cp\u003eFilaggrin expression was assessed at Day 6 to evaluate epidermal differentiation during re-epithelialization (Fig. \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Quantitative analysis demonstrated increased filaggrin levels in treated groups compared with control, although intergroup differences did not reach statistical significance. Immunohistochemical staining revealed filaggrin localization predominantly within the suprabasal layers of the regenerating epidermis at the wound edge, consistent with active epidermal differentiation during wound closure.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study presents a phase-programmable regenerative platform in which exosomes, polymer dot nanozymes (PDNs), and picosecond laser stimulation function as temporally integrated system components rather than additive therapeutics. Within this engineered framework, exosomes provide biochemical activation cues, PDNs operate as catalytic microenvironment regulators, and laser exposure introduces controlled mechanical perturbation. The coordinated interaction among these modules enables phase-dependent redistribution of reparative signaling.\u003c/p\u003e\n\u003cp\u003eDuring the early epithelialization phase, the Exo + PD configuration was associated with accelerated wound closure and coordinated trends in EGF, ERK1/2, and AQP3 expression. Although not all molecular differences reached statistical significance, the kinetic alignment between AQP3 upregulation and wound area reduction suggests that PDNs enhance epithelial responsiveness within a permissive signaling range rather than amplifying a single pathway.\u003csup\u003e35, 36\u003c/sup\u003e In the splinted wound model, where contraction is minimized, this effect highlights PDN-mediated optimization of epithelial-driven repair dynamics.\u003c/p\u003e\n\u003cp\u003eMechanistically, the biological initiation signal originates from exosome-derived miRNAs regulating MAPK- and PI3K/AKT-associated pathways. \u003csup\u003e22\u003c/sup\u003e However, PDNs introduce an additional material-level layer of regulation. As hyperbranched nanozymes with intrinsic catalytic properties, PDNs have been reported to modulate reactive oxygen species within physiologically relevant ranges. \u003csup\u003e15, 16\u003c/sup\u003e The antioxidant nanozyme activity of PDNs, including hydroxyl and superoxide radical scavenging, has been experimentally demonstrated in our previous study.\u003csup\u003e15\u003c/sup\u003e Although redox parameters were not directly measured in the present study, the phase-selective response patterns observed here are consistent with previous reports describing the redox-responsive nanozyme activity of PDNs and their capacity to regulate oxidative signaling dynamics.\u003csup\u003e13, 15, 20\u003c/sup\u003e Collectively, these findings support a model in which PDNs function as microenvironment-conditioning materials that shape system-level repair responses rather than acting as passive delivery carriers.\u003c/p\u003e\n\u003cp\u003eIntegration of picosecond laser treatment introduced a controlled mechanical perturbation that reshaped the wound microenvironment and redistributed regenerative outputs across healing phases. Laser-induced optical breakdown (LIOB) generates localized microinjury that has been reported to activate remodeling-associated signaling pathways, including TGF-β/Smad-mediated matrix turnover and angiogenic activation.\u003csup\u003e30, 32\u003c/sup\u003e Consistent with this mechanism, the Exo + L + PD group demonstrated enhanced early angiogenic activation and improved collagen organization during later stages of repair.\u003c/p\u003e\n\u003cp\u003eNotably, immediate laser exposure did not accelerate early wound closure. Instead, wound closure kinetics and ERK1/2 signaling patterns suggest that laser-induced microinjury may transiently attenuate epithelial activation during the earliest phase of healing. Such transient suppression of epithelial signaling is consistent with the known response to fractional laser–induced microinjury, in which initial epithelial disruption precedes subsequent tissue remodeling. Within this context, PD nanozymes likely function as microenvironmental regulators that stabilize redox signaling following laser-induced perturbation, enabling the subsequent transition toward coordinated angiogenesis and extracellular matrix organization.\u003csup\u003e23, 37-39\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition to modulating epithelial signaling, PD-containing treatments also influenced early angiogenic responses. VEGF expression was significantly elevated in the Exo + PD group and further increased in the Exo + L + PD group, while CD31 expression showed significant upregulation only under the combined Exo + L + PD condition. This pattern suggests that PD nanozymes may prime the angiogenic microenvironment, establishing a permissive signaling state that can be further amplified by laser-induced mechanical stimulation.\u003c/p\u003e\n\u003cp\u003eWithin this framework, PD-mediated redox regulation may create a microenvironment favorable for pro-angiogenic signaling, whereas laser-induced optical breakdown provides a secondary stimulus that enhances endothelial activation and vascular remodeling. The sequential elevation of VEGF followed by CD31 expression observed in the present study is consistent with this two-stage model, in which PDs prepare the regenerative niche and laser exposure amplifies angiogenic responses.\u003c/p\u003e\n\u003cp\u003eSuch cooperative regulation highlights the complementary roles of catalytic biomaterials and mechanically induced stimuli in directing tissue repair. Rather than acting independently, PD nanozymes and laser stimulation appear to function synergistically to coordinate vascular activation and extracellular matrix remodeling during wound healing.\u003c/p\u003e\n\u003cp\u003eCoordinated but incomplete epithelial–mesenchymal marker modulation, particularly in the Exo + L + PD group, further supports a controlled partial EMT state consistent with regulated tissue regeneration rather than fibrotic transition.\u003csup\u003e40, 41\u003c/sup\u003e\u0026nbsp; The absence of SLUG upregulation suggests that SLUG may be required for EMT initiation but is not essential for maintaining partial EMT during wound repair.\u003csup\u003e41, 42\u003c/sup\u003e These findings imply that PDNs may influence epithelial–mesenchymal plasticity under mechanically perturbed conditions, reinforcing their role as dynamic regulators of repair-state transitions. \u003csup\u003e16, 19, 21\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eCollectively, these findings demonstrate that PD nanozymes may function as catalytic regulators of the wound microenvironment, coordinating epithelial activation, angiogenesis, and matrix remodeling during tissue repair. Rather than uniformly accelerating repair, the platform enables redistribution and tuning of phase-specific regenerative outputs, providing a materials-driven strategy for programmable tissue regeneration.\u003c/p\u003e\n\u003cp\u003eSeveral limitations should be acknowledged. Mechanistic confirmation using pathway-specific inhibition was not performed, and validation in large-animal or chronic wound models will be required to assess translational scalability. In addition, although the antioxidant nanozyme activity of PDNs has been previously demonstrated, including reactive oxygen species (ROS) scavenging capability reported in earlier studies,\u003csup\u003e15\u003c/sup\u003e ROS levels were not directly quantified in the present work. Future investigations incorporating direct redox measurements will help further clarify the relationship between PD-mediated redox modulation and downstream regenerative signaling.\u003c/p\u003e\n\u003cp\u003eDespite these limitations, the present work establishes a material-centric framework for phase-specific modulation of wound repair, positioning PD nanozymes as regulatory interfaces that direct the temporal distribution of regenerative signaling. This systems-level design principle highlights how catalytic biomaterials can be used to program the temporal trajectory of tissue repair.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study establishes a nanozyme-enabled biomaterial framework for phase-specific regulation of cutaneous wound healing. Redox-responsive polymer dot nanozymes act as catalytic microenvironmental regulators that coordinate epithelial activation, angiogenesis, and extracellular matrix remodeling when integrated with exosome signaling and picosecond laser stimulation. By conditioning biochemical and biomechanical cues into temporally organized regenerative responses, this platform demonstrates how catalytic biomaterials can modulate healing trajectories rather than merely support tissue repair.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding sources:\u003c/strong\u003e This study was funded by the research grant from China Medical University (CMU111-S-08).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available in this article and the supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYen-Jen Wang: Methodology, Conceptualization, Visualization, Formal analysis, Writing (original draft)\u003c/p\u003e\n\u003cp\u003eChang-Cheng Chang: Conceptualization, Investigation, Project administration, Methodology, Writing (review \u0026amp; editing) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTzong-Yuan Juang: Investigation, Conceptualization, Methodology, Funding acquisition, Project administration, Writing (review \u0026amp; editing) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYi-Hsuan Tu:\u0026nbsp;Conceptualization, Methodology, Data curation, Formal analysis, Visualization\u003c/p\u003e\n\u003cp\u003eJia-Chee Siew: Conceptualization, Methodology, Data curation, Formal analysis, Visualization\u003c/p\u003e\n\u003cp\u003eShao-Yu Peng: Investigation\u003c/p\u003e\n\u003cp\u003eSteven W. Shaw: Investigation\u003c/p\u003e\n\u003cp\u003eHoi-Man Iao: Investigation, Methodology, Data curation\u003c/p\u003e\n\u003cp\u003eSiao-Cian Fan: Investigation, Methodology, Data curation\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration. Nature. 2008;453:314\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEming SA, Martin P, Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation. Sci Transl Med. 2014;6:265sr6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDowner M, Berry CE, Parker JB, Kameni L, Griffin M. Current Biomaterials for Wound Healing. Bioeng (Basel) 2023;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarik S, Keswani K, Ray P, Chakraborty R, Mohini S, Banoth E, et al. 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Cells Tissues Organs. 2022;211:689\u0026ndash;702.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nanozymes, Exosome-Based Therapy, Cutaneous Regeneration, Microenvironment Modulation, Nanomedicine, Regenerative Biomaterials","lastPublishedDoi":"10.21203/rs.3.rs-9132659/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9132659/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCutaneous wound healing is orchestrated by tightly coordinated biochemical and biomechanical cues that regulate epithelial activation, angiogenesis, and extracellular matrix (ECM) remodeling. However, nanozyme-based biomaterial platforms capable of dynamically regulating the wound microenvironment remain limited.\u003c/p\u003e\n\u003cp\u003eHere, we develop a nanozyme-enabled regenerative platform in which redox-responsive hyperbranched polymer dot nanozymes (PDNs) are integrated with porcine fallopian tube stem cell–derived exosomes and picosecond laser stimulation to modulate phase-specific microenvironmental responses. Unlike conventional carriers, PDNs act as catalytic nanozymes that regulate reactive oxygen species (ROS) dynamics within the wound microenvironment.\u003c/p\u003e\n\u003cp\u003eUsing a murine splinted excisional wound model that isolates epithelial-driven regeneration, treatment with exosomes and PDNs (Exo+PDN) significantly accelerated early wound closure and was associated with coordinated activation of the EGF–ERK1/2–AQP3 signaling axis. When combined with picosecond laser stimulation (Exo+Laser+PDN), the platform preferentially enhanced early angiogenic activation, followed by improved epidermal maturation and more organized collagen architecture. Analysis of epithelial plasticity markers revealed maintained E-cadherin expression with concurrent vimentin upregulation in the absence of SLUG induction, indicating a regulated partial epithelial plasticity state rather than a full epithelial–mesenchymal transition.\u003c/p\u003e\n\u003cp\u003eCollectively, these findings demonstrate that PD nanozymes function as active microenvironment-modulating biomaterials that integrate biochemical and physical cues to guide phase-dependent wound regeneration. This work highlights a material-driven strategy for regulating the temporal dynamics of tissue repair beyond conventional delivery-based approaches.\u003c/p\u003e","manuscriptTitle":"Redox-Responsive Polymer Dot Nanozymes Coordinate Exosome-Mediated Cutaneous Regeneration via Laser-Modulated Microenvironment Remodeling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-24 13:47:27","doi":"10.21203/rs.3.rs-9132659/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"da68d0d5-4fc9-43c5-8cac-434e295f2ebe","owner":[],"postedDate":"March 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-08T09:59:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-24 13:47:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9132659","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9132659","identity":"rs-9132659","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}