Spatiotemporally controlled restoration of GAS6 signaling via mRNA therapy promotes scarless healing in preclinical models

Domain: chemistry
Published: 2026-02-25
Editorial outcome: major_revision

Abstract
Deep cutaneous injuries in adult mammals often lead to fibrotic scarring, a process exacerbated by inflammatory fibroblasts that amplify immune recruitment. Early modulation of immune–fibroblast crosstalk represents a promising therapeutic strategy. Here we show that GAS6 is a key regulator of this interaction and can be therapeutically targeted using a spatiotemporally controlled lipid nanoparticle (LNP)–mRNA hydrogel platform. We engineer LNP-GAS6 mRNA to enhance macrophage efferocytosis and suppress inflammatory fibroblasts, then encapsulate it in a thermosensitive hydrogel for localized delivery. In murine, rabbit ear, and Bama minipig wound models, this treatment significantly accelerates wound closure and reduces fibrotic scarring. These results demonstrate that restoring GAS6 signaling via mRNA-based delivery promotes scarless healing and offers an effective therapeutic approach for fibrotic skin disorders.

Introduction
Cutaneous fibrosis caused by deep dermal injuries remains a global healthcare challenge1–8. Pathological scarring not only compromises skin barrier integrity but also lead to contractures, chronic pain, and a substantial socioeconomic burden1,7–11. Although there are some approaches for prevention and treatment, such as surgery and intralesional corticosteroid injections, these are unsatisfactory due to high recurrence rates8–11. Therefore, it is necessary to understand underlying mechanism and develop new effective therapies2,5,6,8.

Chronic inflammation localized in the reticular dermis is a well-established driver of fibrotic progression4,8. Emerging evidence indicates that while inflammatory fibroblasts (iFB) serve as pivotal orchestrators of wound contraction through myofibroblast differentiation, their dysregulated activation drives fibrosis via excessive secretion of CSF-1 and CXCL12, which perpetuates neutrophil recruitment and monocyte infiltration12,13. Consequently, the targeted suppression of iFB within the reticular dermis during the acute inflammatory phase seems a promising strategy to achieve scarless healing13,14. However, two critical challenges remain to be addressed: exploring the upstream regulators of iFB inflammatory phenotypes while developing targeted delivery systems capable of spatiotemporally (reticular dermis and inflammatory phase) controlled intervention.

Macrophage-fibroblast interactions remain central to wound repair and scar formation, particularly macrophage-mediated regulation of fibroblast activity6,11,15–19. Emerging evidence highlights the immune-stromal crosstalk mediated by TAM (Tyro3/Axl/MerTK) receptors as a gatekeeper of tissue homeostasis20–25. In arthritic and other inflammatory models, MerTK⁺ macrophages suppress fibroblasts inflammation while coordinating efferocytosis to resolve inflammation, suggesting that TAM receptors participate in the inflammatory regulatory effect of macrophages on fibroblasts25–28. Preclinical studies have also revealed that TAM-deficient mice exhibit impaired tissue repair, yet the role of TAM ligands (GAS6/PROS1) in cutaneous wound healing remains unexplored23,29–31. Crucially, while research on TAM inhibitors advances in oncology trials, that on the pharmacological activation of this pathway, a promising strategy to recalibrate inflammatory microenvironments, remains underdeveloped20,30,32.

Lipid nanoparticle (LNP)-mediated mRNA delivery has emerged as a transformative therapeutic platform, achieving remarkable success in vaccine development and oncology due to its high efficiency and transient expression profile33–35. However, the application of LNP-mRNA technology in dermatology, particularly for scar prevention, remains highly emerging. The work of Wang et al. demonstrated the feasibility of using LNP-based interleukin-4 (IL-4) mRNA delivery to accelerate wound healing, underscoring the promise of mRNA strategies for precision dermatology36. Nevertheless, it remains a major challenge to achieve localized, stable, and efficient delivery of LNP-mRNA within the dynamic wound microenvironment. In this context, the combination of a hydrogel with LNP-mRNA enabled successful protein expression in vivo and conferred significant therapeutic benefits, positioning it as a promising platform for mRNA delivery in wound therapy37,38.

Here, we demonstrate persistent GAS6 deficiency as a critical determinant of pathological repair via an integrated analysis of keloid/wound patients, murine wound models, and in vitro inflammatory models. Functional validation using pharmacological inhibition (RU-301) confirms the mechanistic role of GAS639. We subsequently engineered an injectable lipid nanoparticle (LNP)-GAS6 mRNA-loaded thermosensitive hydrogel, achieving spatiotemporally controlled GAS6 expression in murine, rabbit, and porcine models, surpassing the efficacy of conventional therapy11. This approach promotes scarless healing by a dual mechanism: enhancing efferocytosis and attenuating inflammatory signals from fibroblasts. Notably, single-dose administration reduces the fibrotic area by 53.69% in porcine wounds, superior to the Recombinant Human-Epidermal Growth Factor Gel (h-EGF gel, 32.18%)40,41, demonstrating translational potential. By targeting the inflammatory upstream rather than downstream fibrotic effectors, our work proposes regenerative strategies and establishes the scientific basis of mRNA-based modulation of immune-stromal networks as a promising strategy in precision dermatology14.

Results
GAS6 is a potential therapeutic target for scarless healing
Members of the TAM receptor family modulate inflammatory responses and orchestrate efferocytosis20,21,30. To reveal their roles in wound healing and fibrogenesis, we integrated multi-platform analyses across clinical specimens, animal models, and in vitro systems. Transcriptomic profiling of GSE113619 revealed similar changes in TYRO3, AXL, and MERTK in both keloid patients and normal wound healing, while PROS1 and GAS6 exhibited different trajectories—upregulated in controls but downregulated or unchanged in keloid patients (Fig. 1a, e, Supplementary Fig. 1a–d). GSE178411 analysis demonstrated elevated AXL/MERTK levels across patients with acute, late, and chronic wounds, contrasted by a decline in TYRO3, PROS1, and GAS6 (Fig. 1b, f, Supplementary Fig. 1e–h). In murine models, only GAS6 displayed consistent downregulation during the repair phases, reaching statistical differences in type 2 diabetic wounds (p=0.0349, Fig. 1c, g, Supplementary Fig. 1i–l). The inflammatory challenge of macrophages with IFN-γ/LPS recapitulated this suppression, showing GAS6 reduction (Fig. 1d, h, Supplementary Fig. 1m–p). An integrative analysis of human keloid tissues, acute/late/chronic wounds, murine acute/diabetic wounds, and cytokine-stimulated macrophages confirmed the presence of conserved GAS6 downregulation (Fig. 1e–h), establishing its role as an inflammation-sensitive regulator of repair processes. Notably, GAS6 is the only TAM ligand capable of activating all three receptors (TYRO3/AXL/MERTK)20,30. To further identify the specific cell types in which GAS6 is expressed and dynamically regulated during wound repair, we performed single-cell analysis of wound tissues from both human (GSE241132) and murine (GSE186527) wound healing processes. Consistent with prior findings, GAS6 was downregulated during the early inflammatory phase and was predominantly changed in fibroblasts and macrophages (Fig. 1i–l, Supplementary Fig. 1q–t). Concurrently, its key receptors AXL and MERTK were also downregulated, thus highlighting a critical deficiency in the GAS6-TAM signaling axis during this pivotal window (Supplementary Fig. 2a–d).

To validate the functional necessity of GAS6 in wound repair, we administered RU-301, a pharmacological inhibitor blocking GAS6-TAM receptor interaction, to murine acute wounds39. RU-301 treatment significantly delayed wound healing (p=0.0039 on POD10, p=0.0228 on POD14, Fig. 1m, n) and exacerbated scar width on POD30 (p=0.0221, Fig. 1m, o). Given the established link between impaired efferocytosis and defective healing, coupled with the pro-fibrotic role of augmented dermal inflammatory priming, we hypothesized that GAS6 deficiency drives pathological outcomes through a dual mechanism13,42. Consistent with this premise, RU-301 increased apoptotic cell accumulation at the wound edges (p=0.0156, Fig. 1p, r), indicative of compromised efferocytosis. Concurrently, the dermal inflammatory signal intensified, as evidenced by the increased migration of macrophages induced by inflammatory dermis in Transwell assay (p=0.0003, Fig. 1q, s). These findings mechanistically implicate GAS6 downregulation in dysregulating efferocytosis and amplifying inflammatory cascades during repair. Therefore, it was contemplated that exogenous GAS6 supplementation may recalibrate this imbalance and become an effective strategy for scarless healing.

Synthesis and characterization of LNP-mRNA
As the principal ligand for TAM receptors (binding to three types of TAM receptors), GAS6 exerts multiple effects through receptor binding. Given the functional heterogeneity and tissue-specific distribution of TAM receptors, targeting upstream ligands offers clear therapeutic advantages. Despite the clinical translation of TAM inhibitors in oncology, no agonist has yet been developed20,30. Capitalizing on mRNA technology validated by COVID-19 vaccines33–35, we engineered LNP-encapsulated GAS6 mRNA (LNP-GAS6 mRNA) to amplify its regenerative potential (Fig. 2a). Physicochemical characterization revealed the following: diameter of 64.29±5.85 nm (LNP) and 74.83±11.64 nm (LNP-GAS6 mRNA) (Fig. 2b), polydispersity index (PDI) of 0.09±0.05 (LNP) and 0.16±0.05 (LNP-GAS6 mRNA) (Fig. 2c), Zeta potential of +13.60±1.65 mV (LNP) and +13.21±1.28 mV (LNP-GAS6 mRNA) (Fig. 2d), encapsulation efficiency of 97.82±0.55% (Fig. 2e) and encapsulated mRNA concentration of 0.12±0.01 μg/μL (Fig. 2f). Transmission electron microscopy (TEM) images confirmed its uniform dispersion and spherical morphology (Fig. 2g).

Cytotoxicity screening in fibroblasts and macrophages established a safe therapeutic window (≤5 μg/mL mRNA), with enhanced fibroblast proliferation at subtoxic doses (Supplementary Fig. 3a, b). This safety profile was further validated by live/dead staining, which demonstrated excellent cell viability in both cell types at 5 μg/mL mRNA (Supplementary Fig. 3c). Enzyme-Linked Immunosorbent Assay (ELISA) revealed the progressive accumulation of secreted GAS6 protein in supernatants after LNP-GAS6 mRNA delivery, reaching maximum levels at the 72-h time point (macrophages: 592.14±36.58 pg/mL, fibroblasts: 488.10±11.75 pg/mL, Fig. 2h). Immunofluorescence staining further confirmed intracellular overexpression patterns of the GAS6 protein in both macrophages and fibroblasts (Fig. 2i–k). These data establish LNP-GAS6 mRNA as a robust platform for GAS6 delivery, which can be further used as an agonist of TAM receptors.

LNP-GAS6 mRNA enhances macrophage efferocytosis and suppresses the inflammatory fibroblasts in vitro
Macrophages are central orchestrators of wound repair, acting as both primary mediators of efferocytosis and upstream regulators of iFB5,15,16,26,27. To delineate the mechanistic role of GAS6 in macrophage function, we employed siRNA-mediated GAS6 knockdown (Supplementary Fig. 4a). Downregulation of GAS6 not only skewed macrophages toward a pro-inflammatory phenotype (marked by elevated Il1b and Tnf alongside suppressed Il10 expression) (Supplementary Fig. 4b, c; Fig. 3a), but also augmented fibroblast inflammatory priming via paracrine signaling (Fig. 3b, c). Furthermore, GAS6 loss severely compromised efferocytic capacity (Fig. 3d; Supplementary Fig. 4d–f), suggesting that GAS6 deficiency disrupts macrophage-fibroblast crosstalk by altering paracrine signaling and compromising efferocytosis.

To investigate the immunomodulatory potential of LNP-GAS6 mRNA, we first assessed its direct effects on macrophages. Treatment with LNP-GAS6 mRNA significantly skewed macrophages toward an M2-like state, as evidenced by concomitant upregulation of the M2 marker Arg1 (Fig. 3e, f). Given that GAS6 protein secretion peaked at 72 h in macrophages (Fig. 2h), we harvested the conditioned medium (CM) following a 72-h LNP-GAS6 mRNA treatment for subsequent experiments. Flow cytometry analysis confirmed that the CM from LNP-GAS6 mRNA-treated macrophages enhanced efferocytosis, as evidenced by both an increased percentage of macrophages that phagocytosed apoptotic cells (63.08±0.38% vs. 50.43±1.13% in the Blank group, Fig. 3g, i) and a greater number of engulfed apoptotic cells (3372.25±129.20 vs. 2463.50±54.97 in the Blank group, Fig. 3h, i). Notably, LNP-GAS6 mRNA demonstrated a stronger enhancement of efferocytosis than LNP-IL-4 mRNA (Fig. 3g–i), indicating that GAS6 signaling exerts immunomodulatory effects superior to those of a classical M2-polarizing stimulus. These findings align with prior research and provide additional evidence for the ability of LNP-GAS6 mRNA to enhance efferocytosis.

To dissect the macrophage-fibroblast crosstalk, we established an inflammatory axis model, where fibroblasts were treated with either: CM derived from LNP-GAS6 mRNA-treated macrophages, or direct application of LNP-GAS6 mRNA (Fig. 3j). First, we observed that LNP-GAS6 mRNA treatment reduced the expression of macrophage-derived pro-inflammatory cytokines and stimulated the anti-inflammatory factors (Il1b↓, Tnf↓, Il10↑, Fig. 3k–m). The transfer of this conditioned medium to fibroblasts suppressed the expression of inflammatory initiators Csf1 and Cxcl12 (Fig. 3n, o), while direct LNP-GAS6 mRNA application to fibroblasts showed no significant effect (Fig. 3p, q). These data suggested a macrophage-dependent mechanism by which GAS6 indirectly regulates fibroblast inflammatory responses, rather than exerting direct inhibitory effects. In summary, our in vitro studies demonstrated that LNP-GAS6 mRNA drives macrophage M2 polarization and enhances efferocytosis, subsequently suppressing iFBs through macrophage-derived paracrine signaling.

Characterization and validation of LNP-GAS6 mRNA@Gel
During the inflammatory phase, the inflammatory priming of fibroblasts serves as a critical determinant steering wound repair toward either regenerative healing or fibrotic outcomes13. Consequently, therapeutic intervention with GAS6 during the peak inflammatory window (typically 24–72 h post-injury) may optimize the clinical efficacy. Considering the limitations of transient GAS6 intervention (e.g., recombinant proteins) and the potential fibrotic risks associated with prolonged overexpression (e.g., viral vectors)43–47, we propose a co-delivery platform combining an injectable thermosensitive hydrogel with LNP-mRNA technology to ensure that GAS6 protein expression is synchronized with the inflammatory phase and positioned in the reticular dermis37,38. To enable the spatiotemporally controlled delivery of LNP-GAS6 mRNA in vivo, we engineered a thermosensitive PLGA-PEG-PLGA hydrogel with phase transition at 25 °C (liquid at 4 °C, gel at 37 °C, Fig. 4a). Topical application of the LNP-GAS6 mRNA@Gel to murine acute/diabetic wounds induced rapid gelation within 15 s (Fig. 4b). Confocal imaging confirmed the homogeneous distribution of PKH26-labeled LNPs within the hydrogel matrix (Fig. 4c). Structural and functional characterization confirmed the suitability of the LNP-loaded hydrogel as a sustained-release delivery system. Scanning electron microscopy (SEM) revealed that LNP incorporation did not alter the hydrogel’s porous architecture or mechanical properties, which maintained robust adhesive and tensile characteristics (Supplementary Fig. 5a, b). The hydrogel exhibited microenvironment-dependent degradation, with slow kinetics in PBS but accelerated breakdown in collagenase I, mimicking the proteolytic wound environment (Supplementary Fig. 5c). Release kinetics aligned with material degradation, as 84.21% of encapsulated mRNA was released within 96 h under collagenase I conditions (Supplementary Fig. 5d). The rheological characteristics of this hydrogel platform remained stable under pathophysiological conditions (pH 6.5, 200 μM H₂O₂), confirming its robustness for applications in inflamed wound microenvironments (Supplementary Fig. 5e). Consistent with in vitro behavior, the hydrogel underwent near-complete degradation in vivo within 72 h (Supplementary Fig. 5f, g).

To validate the transfection efficiency, spatial distribution, and expression duration of our delivery platform in vivo, we first administered LNP-Fluc mRNA@Gel to murine wounds. Whereas free LNPs (without hydrogel) exhibited poor retention and uneven distribution within the wound bed, our hydrogel-based system ensured prolonged and localized delivery, yielding a sustained fluorescent signal that peaked between 6 and 24 h and remained detectable for 72 h, thus achieving precise temporal alignment with the inflammatory phase (Fig. 4d–f). Building upon these successes, we subsequently developed LNP-GAS6 mRNA@Gel and applied it to murine acute wound models. To establish an optimal dosing regimen, we conducted a dose-response assessment of LNP-GAS6 mRNA@Gel (20, 50, and 80 μg/mL) in murine acute wounds. Immunofluorescence confirmed dose-dependent GAS6 upregulation, with pronounced and spatially restricted expression in the deep dermis at both the wound center and edge (Fig. 4g–i). The 50 μg/mL dose achieved an near-maximal GAS6 expression, comparable to 80 μg/mL (Fig. 4g–i). Although wound width was similar across groups, the 50 μg/mL treatment significantly enhanced granulation tissue thickness relative to controls (Fig. 4g, j, k), indicating promoted proliferative repair. Based on this robust efficacy and a favorable saturation profile, 50 μg/mL was selected for all subsequent studies. ELISA further revealed that GAS6 protein expression reached its peak on POD3 (463.75±80.17 pg/mg tissue) and subsequently declined, a kinetic profile that aligns with the critical inflammatory phase of wound healing (Supplementary Fig. 5h).

Previous studies have established a strong correlation between persistent deep dermal inflammation and pathological scar formation8–11, and the spatially restricted expression of GAS6 in the reticular dermis was expected to enhance its therapeutic potential. These results established the hydrogel platform as a superior means of delivery compared to direct LNP injection, achieving spatiotemporally controlled GAS6 delivery.

LNP-GAS6 mRNA@Gel promoted scarless healing by enhancing efferocytosis and reducing dermal inflammatory signaling in murine acute wounds
A comprehensive biosafety evaluation of LNP-GAS6 mRNA@Gel was conducted prior to efficacy studies. Local assessment at injection sites showed no adverse effects on hair regeneration, follicular density, dermal thickness, or collagen volume fraction (CVF) after 30 days (Supplementary Fig. 6a–f). Systemically, no gross or microscopic alterations were detected in major organs, and plasma inflammatory cytokine levels remained comparable to controls (Supplementary Fig. 6g–i). These data collectively demonstrate that LNP-GAS6 mRNA@Gel is well-tolerated both locally and systemically.

To evaluate the therapeutic efficacy of GAS6 in wound repair and scar modulation, we administered LNP-GAS6 mRNA@Gel to murine full-thickness excisional wounds (6 mm diameter) with five experimental groups set: Control, Gel, LNP@Gel, LNP-GAS6 mRNA@Gel, and GAS6 recombinant protein (Fig. 5a)48–50. Serial photographic tracking revealed accelerated early closure (POD3) in both the LNP-GAS6 mRNA@Gel and recombinant protein groups (Ⅰ: 88.13±9.87%, Ⅱ: 83.08±12.36%, Ⅲ: 80.87±11.88%, Ⅳ: 57.64±4.48%, Ⅴ: 59.85±7.03%, Fig. 5b–d). However, by POD7-14, only the LNP-GAS6 mRNA@Gel group sustained superior healing kinetics (Fig. 5b–d), likely attributable to the transient pharmacokinetics of recombinant protein. Histology analysis at POD30 demonstrated a 71.61% reduction in scar width in the LNP-GAS6 mRNA@Gel group versus the control group (Ⅰ: 1.15±0.20 mm, Ⅱ: 1.20±0.41 mm, Ⅲ: 1.17±0.21 mm, Ⅳ: 0.33±0.02 mm, Ⅴ: 0.69±0.19 mm, H&E, Fig. 5e, f) and enhanced extracellular matrix remodeling with basket-weave pattern resembling native skin48 (Ratio of CVF, Ⅰ: 0.34±0.19, Ⅱ: 0.26±0.06, Ⅲ: 0.33±0.03, Ⅳ: 0.74±0.18, Ⅴ: 0.49±0.18, Masson’s trichrome, Fig. 5e, g). Notably, while the recombinant protein group exhibited a modest scar width reduction (39.54%) and an elevated ratio of CVF (0.49±0.18), while these differences were not statistically significant (p=0.42 and p=0.52 vs. control group, Fig. 5e–g), underscoring the therapeutic superiority of spatiotemporally controlled GAS6 expression via mRNA delivery. To delineate GAS6-mediated scarless healing mechanisms in vivo, we quantified the apoptotic cell burden and dermal inflammatory signal on POD3. Both the LNP-GAS6 mRNA@Gel and recombinant protein groups exhibited lower apoptotic cell density (Fig. 5h, i) and attenuated inflammatory signal (migrating macrophages numbers reduced, Fig. 5j, k), confirming enhanced efferocytosis and early inflammation control. However, only the mRNA formulation sustained therapeutic efficacy in the long term (reduction of scar width on POD30 and better extracellular matrix remodeling, Fig. 5e–g), likely due to the recombinant protein’s short half-life. The above results demonstrated that GAS6 promotes scarless healing through enhanced efferocytosis and the suppression of dermal inflammatory signal. Furthermore, LNP-GAS6 mRNA@Gel showed superior therapeutic efficacy compared to the recombinant protein, as evidenced by improved wound healing outcomes.

The role of LNP-GAS6 mRNA@Gel in promoting scarless healing appears to involve oxidative phosphorylation (OXPHOS)
To delineate the specific contributions of TAM receptors, we inhibited AXL and MERTK signaling in murine acute wounds using Bemcentinib and UNC2025, respectively (Fig. 6a). Both inhibitors significantly impaired repair, delaying wound closure and exacerbating scar formation (Fig. 6b, c). Although attenuated, the therapeutic effect of LNP-GAS6 mRNA@Gel remained significant, indicating that it promoted repair even when individual TAM receptors were blocked (Fig. 6b, c). This functional compensation among TAM receptors not only helps coordinate the repair process but also highlights the therapeutic advantage of using a broad-spectrum TAM ligand agonist (LNP-GAS6 mRNA@Gel) over receptor-specific targeting.

To elucidate the therapeutic mechanisms of LNP-GAS6 mRNA@Gel, we performed unbiased transcriptomic profiling of POD3 wound tissues. This revealed 1,079 upregulated and 719 downregulated genes (FDR<0.05, |Log₂FC|>1; Supplementary Fig. 7a). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment (Fig. 6d) and Gene Set Enrichment Analysis (GSEA) (Fig. 6e) highlighted oxidative phosphorylation (OXPHOS) as the high-ranked pathway, with 45 upregulated OXPHOS-related genes (e.g., Ndufa3, Cox7c, Uqcrb) versus only 3 downregulated ones (Atp7A, Mt-Nd5, Mt-Atp8) (Fig. 6f–h). While OXPHOS activation is known to potentiate M2 polarization and efferocytosis, its specific link to GAS6 signaling warrants further exploration51–53.

To functionally link GAS6 with OXPHOS activation, we performed siRNA-mediated GAS6 knockdown in macrophages. Downregulation of GAS6 significantly suppressed key OXPHOS genes, including Ndufa3, Uqcrb, and Cox5a (Fig. 6i–k). Given that OXPHOS drives ATP production (also critically required for efferocytosis), we next measured cellular ATP levels. While GAS6 knockdown substantially reduced ATP synthesis, treatment with LNP-GAS6 mRNA significantly enhanced cellular ATP production (Fig. 6l), confirming its role in promoting mitochondrial energy generation. Collectively, these results establish that GAS6 potentiates efferocytosis and promotes scarless healing, at least in part, by augmenting OXPHOS-derived ATP availability.

Furthermore, GSEA also revealed profound suppression of pro-inflammatory and fibrotic signaling networks in LNP-GAS6 mRNA-treated mice compared to control group (Supplementary Fig. 7b). Key pro-inflammatory pathways inhibited in the GAS6 group included NF-κB54, MAPK55, and TNFR1/IGF1R56,57, alongside downregulation of fibrosis-associated axes (GSK3β58, HER2/EGF59,60) (Supplementary Fig. 7c–i). These data mechanistically align with reduced macrophages infiltration and histological evidence of scarless healing with improved collagen deposition in LNP-GAS6 mRNA@Gel-treated wounds.

LNP-GAS6 mRNA@Gel attenuated hypertrophic scarring in rabbit ear wound models
To break through the limitations of murine scar models lacking hypertrophic features, we established a rabbit ear hypertrophic scarring model where full-thickness wounds (10 mm diameter, perichondrium excised) developed raised scars post-healing61–63. Four wounds per ear were treated with PBS, Gel, LNP@Gel, or LNP-GAS6 mRNA@Gel (Fig. 7a). LNP-GAS6 mRNA@Gel-treated scars exhibited flatter morphology (Fig. 7b, c) with a 54.09% lower scar elevation index (SEI) (Ⅰ: 3.67±0.72, Ⅱ: 3.38±0.93, Ⅲ: 3.16±0.72, Ⅳ: 1.69±0.50, Fig. 7e). Sirius red staining revealed regular collagen architecture resembling normal skin in the GAS6-treated group and disorganized, fine collagen fibers in the control groups (collagen I/III ratio: Ⅰ: 0.40±0.15, Ⅱ: 0.43±0.11, Ⅲ: 0.39±0.04, Ⅳ: 1.07±0.09, Ⅴ: 1.15±0.31, Fig. 7d, f). These data demonstrated that early LNP-GAS6 mRNA@Gel intervention prevents hypertrophic scar formation.

LNP-GAS6 mRNA@Gel promoted the scarless healing of acute wounds in Bama minipigs
Motivated by the therapeutic efficacy of LNP-GAS6 mRNA@Gel in murine and rabbit models, we advanced to preclinical validation in Bama minipigs, a species with cutaneous anatomy and repair mechanisms closely mirroring human skin11,63. Four full-thickness dorsal wounds (15 mm diameter) per animal received Gel, LNP@Gel, LNP-GAS6 mRNA@Gel, or human EGF gel (Fig. 8a). The LNP-GAS6 mRNA@Gel-treated wounds exhibited faster wound healing (Fig. 8b–d) and a 53.69% smaller scar area at POD35 (Ⅰ: 23.63±5.48%, Ⅱ: 23.73±6.25%, Ⅲ: 10.94±12.69%, Ⅳ: 16.03±7.60%, Fig. 8b–e). There was a 55.10% reduction in scar width at POD35 in the LNP-GAS6 mRNA@Gel group (Masson’s trichrome, Ⅰ: 3993.67±720.72 mm, Ⅱ: 3595.32±572.62 mm, Ⅲ: 1793.33±847.44 mm, Ⅳ: 2538.33±784.65 mm, Fig. 8f, g), surpassing the h-EGF gel group (36.44% reduction, Fig. 8f, g). Collagen distribution resembling unwounded skin could be seen in the LNP-GAS6 mRNA@Gel group, contrasting with collagen alignment in the control group (Ⅰ: 0.16±0.12, Ⅱ: 0.18±0.08, Ⅲ: 0.52±0.29, Ⅳ: 0.33±0.21, Fig. 8f, h). There was a 1.56-fold higher CVF ratio vs. the h-EGF gel group in the LNP-GAS6 mRNA@Gel group (Fig. 8h). The scar length (Fig. 8f, Supplementary Fig. 8a) and the ratio of CVF (Fig. 8f, Supplementary Fig. 8b) on POD14 demonstrated progression patterns parallel to POD35 outcomes, maintaining biological consistency despite the lack of statistical significance. These results validated the therapeutic effect of LNP-GAS6 mRNA@Gel in promoting scarless regeneration over clinical benchmarks (h-EGF gel).

Methods
Ethical statement
All animal experiments were conducted in accordance with the ARRIVE guidelines and were approved by the Experimental Animal Ethics Committee of the Second Affiliated Hospital, Zhejiang University School of Medicine (Approval Nos. 2024-147 and 2025-112). This study complied with all relevant ethical regulations.

Dataset acquisition and data processing
The datasets GSE113619, GSE178411, GSE53986, GSE241132, and GSE186527 were obtained from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). Notably, GSE283228 and GSE309507 represent RNA sequencing data generated by our institution and have been deposited in GEO. Data processing, including identifier conversion, log₂ transformation for normalization, and extraction of TAM receptor/ligand expression matrices, was processed by R software (v4.3.0). Heatmaps and bar plots were generated to visualize expression patterns by GraphPad Prism (v9.0).

Single-cell RNA sequencing data from human (GSE241132) and murine (GSE186527) skin wound healing studies were analyzed using Seurat (v5.3.0). After initial quality control to retain cells with more than 300 genes and less than 20% mitochondrial reads, potential doublets were identified and removed using DoubletFinder (v2.0.6). The data were normalized, and the top 3000 highly variable genes were selected using the vst method. Batch effects were corrected using Harmony (v1.2.3) integration with orig.ident as the batch covariate. Cells were clustered at a resolution of 0.5 using the first 15 Harmony dimensions. Dimensionality reduction and visualization were performed with UMAP (v0.2.10.0) based on the same Harmony components. Cell types were annotated through a marker-based identification process. Cluster-specific marker genes were identified using the FindAllMarkers function (Wilcoxon rank-sum test, only positive markers, logFC threshold > 0.25, and adjusted p value < 0.05). Cluster identities were assigned based on canonical cell-type markers.

No ethical approval or patient consent was required as all data were sourced from public repositories.

Isolation of primary bone marrow-derived macrophages (BMDM) from mice
Bone marrow was harvested from euthanized 4–8-week-old C57BL/6 mice. Femurs and tibias were disinfected with 75% ethanol, dissected, and flushed with cold medium using a syringe. Cells were filtered through a 70 μm strainer, centrifuged (300×g, 5 min), and treated with Red Blood Cell (RBC) lysis buffer (Invitrogen, #00-4333-57). After washing, cells were cultured in macrophage induction medium (20 ng/mL M-CSF; Meilun Bio, #MA0608). Medium was partially replaced on day 3 and fully refreshed on day 5. Differentiated macrophages were utilized by day 7.

Cell counting kit-8 (CCK-8) cytotoxicity assay
Macrophages (BMDM) and fibroblasts (L929 from ATCC, #CCL-1) were seeded in 96-well plates and pre-cultured until reaching approximately 60% confluency. LNP-GAS6 mRNA (0.5–25 μg/mL) was added to the respective wells. After 24 h of incubation, 10 μL CCK-8 reagent (Beyotime, #C0037) was added to each well, followed by a 2-h incubation (dark). Absorbance was measured at 450 nm using a microplate reader, and bar graphs were generated based on the absorbance values.

Calcein AM/PI staining for cell viability
The viability of fibroblasts or macrophages was assessed using Calcein AM/PI staining (Beyotime, #C1371M). After treatment, the culture medium was aspirated, and cells were gently washed once with PBS. A freshly prepared Calcein AM/PI working solution was added (100 μL per well for a 96-well plate), followed by incubation at 37 °C for 30 min in the dark. Cells were visualized under a fluorescence microscope: live cells exhibited green fluorescence, while dead cells showed red fluorescence. All steps were carried out protected from light.

Histopathological analysis of wounds/scars
Wound/scar tissues from animals were fixed in 4% paraformaldehyde for 24 h, dehydrated through graded ethanol, embedded in paraffin, and sectioned (4 μm). For H&E staining, sections were dewaxed, stained with hematoxylin (5 min) and eosin (1 min), then mounted. For Masson’s trichrome, sections were incubated in Weigert’s iron hematoxylin (10 min), Biebrich scarlet-acid fuchsin (5 min), and aniline blue (5 min) to visualize collagen. Sirius red staining involved picrosirius red solution (1 h), differentiation in acidified water, and polarization microscopy for collagen typing. All slides were imaged using brightfield/polarized light microscopy.

Immunofluorescence staining
Samples were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% Bovine Serum Albumin (BSA) (1 h, room temperature). Primary antibodies (Cleaved Caspase 3 Polyclonal antibody, Proteintech, #25128-1-AP, 1:200; Rat Monoclonal GAS6 antibody, Abcam, #ab86059, 1:200; EMR1 Polyclonal antibody, Proteintech, #27044-1-AP, 1:200) were incubated overnight at 4 °C. After PBS washes, fluorophore-conjugated secondary antibodies (Goat Anti-Rabbit IgG H&L, Abcam, #ab150077, 1:200; Goat Anti-Mouse IgG H&L, Abcam, #ab150116, 1:200; Fluoro 488 Conjugated AffiniPure Rabbit Anti-Rat, BOSTER, #BA1129, 1:200) were applied (1 h, dark, room temperature). Nuclei were stained with Fluoromount-G™ (Yeasen, #36307ES). Fluorescence signals were captured using a fluorescence microscope.

Transwell migration assay
BMDMs (2×10⁵ cells/insert) in complete medium (10% FBS) were seeded in Transwell (8.0 μm, Corning, #3422) upper chambers. Wound-margin skin samples (1–2 mm³) were pre-equilibrated in complete medium (700 μL/well) for 30 min. Co-culture systems (24 h) were established by positioning inserts above tissue-containing wells13. Migrated cells on lower membranes were fixed with 4% paraformaldehyde, swab-cleared of non-migratory cells, and stained with crystal violet. Quantification was performed via microscopic imaging.

LNP-mRNA formulation and characterization
GAS6 mRNA was synthesized via in vitro transcription from a linearized plasmid (Yaohai Bio.) using BspQI digestion, followed by phenol-chloroform extraction or magnetic bead purification. The IVT reaction was performed with T7 RNA polymerase, modified nucleotides (including m1ψ-UTP), and either co-transcriptional capping (using cap analog) or enzymatic capping post-IVT (using VCE and 2’-O-methyl-transferase). The resulting mRNA was purified using RNA magnetic beads (N412) and quantified by UV and capillary electrophoresis. The optimized mRNA sequence is provided in the file Supplementary Data 1. IL-4 mRNA and Fluc mRNA were obtained commercially (Yaohai Bio. and Absin Bioscience Inc.). LNP-mRNA were prepared by microfluidic mixing of lipid components (SM102/DSPE-PEG2000/DSPC/Cholesterol, 50:1.5:10:38.5 mol%) with mRNA in citrate buffer (pH 4.0) at a 1:3 volumetric ratio. The N/P ratio for the LNP-mRNA formulation was 6:1. Removal of ethanol and unencapsulated mRNA was performed by dialysis (PBS, 4 °C, 18 h; Slide-A-Lyzer™ 20K MWCO, Thermo Scientific) or ultrafiltration (4 °C, 3000×g, 20 min; UFC805096, Merck-Millipore). Sterilized LNP-mRNA (0.22 μm filtration) was stored at 4 °C. The size, polydispersity index (PDI), and Zeta potential were measured by dynamic light scattering (DLS). The encapsulation efficiency was quantified using the RNA-specific fluorescent dye Quant-iT™ RiboGreen™ (Thermofisher Scientific). Encapsulation efficiency (%) = [(Total mRNA – Free mRNA)/Total mRNA] × 100. The morphology and structure were analyzed by transmission electron microscopy (TEM).

Enzyme-linked immunosorbent assay (ELISA)
Mouse GAS6 (#E-EL-M0282), IL-6 (#E-EL-M0044), and TNF-α (#E-EL-M3063) ELISA Kit was purchased from Elabscience. For fibroblasts and macrophages, supernatants from LNP-GAS6 mRNA-transfected cells were collected at 24/48/72 h. For skin tissue, the supernatant extracted after lysis was subjected to subsequent testing (POD 1-14). According to the manufacturer’s instructions, standards/samples (100 μL/well) were incubated with capture antibody (90 min, 37 °C), detection antibody (1 h, 37 °C, 3× PBS-T washes), horseradish peroxidase (HRP) conjugate (30 min, 37 °C, 5× PBS-T washes), and substrate solution (15 min, 37 °C) before termination with stop solution. Absorbance at 450 nm was measured and concentrations calculated via standard curve.

Flow cytometry analysis of efferocytosis
BMDMs were seeded in a 12-well plate at a density of 1×10⁵ cells/well. Apoptotic Jurkat T cells were induced by 15-min UV irradiation, followed by 4-h incubation. Apoptotic cells were labeled with Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE, Beyotime, #C0051) for 15 min, resuspended in fresh medium for 30 min, and washed twice with PBS. Jurkat T cells were resuspended in medium and added to BMDMs (adjusted to a 5:1 ratio of apoptotic cells: BMDMs). Co-cultures were incubated for 2 h to allow efferocytosis. Non-engulfed apoptotic cells were removed by two PBS washes. The remaining cells were harvested and stained with PerCP/Cyanine 5.5 Anti-Mouse F4/80 Antibody (Elabscience, #E-AB-F0995J) on ice for 15 min (dark). After two PBS washes, cells were transferred to flow cytometry tubes. Fluorescence-positive cell percentage and mean fluorescence intensity (MFI) of F4/80⁺/CFDA-SE⁺ populations were quantified using a flow cytometer. Detailed gating strategies for efferocytosis analysis are provided in the Supplementary Information (Supplementary Fig. 4f).

Total RNA extraction and reverse transcription-polymerase chain reaction (RT-qPCR)
Total RNA was extracted and analyzed using commercial kits, reverse-transcribed into cDNA (Hifair® V one-step RT-gDNA digestion SuperMix for qPCR, Yeasen, #11142ES60), and diluted 5–10× before storage at −80 °C. SYBR Green-based qPCR (Hieff® qPCR SYBR Green Master Mix, Yeasen, #11201ES08) was performed with gene-specific primers (Primer sequences are listed in Supplementary Table 1) targeting Gapdh (internal control), Gas6, Nos2, Arg1, Il1b, Il10, Tnf, Csf1, Cxcl12, Ndufa3, Uqcrb, and Cox5a, with relative expression quantified via ΔΔCt method.

Hydrogel preparation and characterization
PLGA-PEG-PLGA hydrogel (MeloPEG, #904330) was thawed (4–6 h, room temperature), dissolved in PBS to 20% (w/v) with periodic stirring (≥3× daily, 5 min/session, 4 °C) until forming transparent colloid. Gelation capacity was tested by mixing hydrogel with LNP/LNP-mRNA (1:1 vol ratio), followed by 37 °C incubation or in vivo application (murine wound). PKH26-labeled LNPs were blended with hydrogel (1:1 vol ratio) for confocal microscopy-based spatial distribution analysis. The hydrogel microstructure and pore size were characterized using scanning electron microscopy (SEM), and its physical properties were validated on ex vivo mouse skin. LNP-Fluc mRNA was mixed with hydrogel at a 1:1 ratio. After gelation at 37 °C, the LNP-Fluc mRNA@Gel was incubated with PBS, complete medium, esterase (MedChemExpress, #HY-P2831), collagenase I (BioFroxx, #1904GR001), pH 6.5, or 200 μM H₂O₂ in PBS as degradation media. Supernatants were collected at 6, 12, 24, 48, 72, and 96 h for mRNA concentration measurement to calculate the cumulative mRNA release, and the remaining hydrogel mass was weighed to calculate the residual hydrogel ratio. Furthermore, amplitude sweep tests were performed to probe the mechanical integrity of the hydrogel under physiological (PBS, pH 7.4, 37 °C) and pathophysiological (pH 6.5, 200 μM H₂O₂, 37 °C) conditions, recording the storage (G’) and loss (G”) moduli across a strain range of 0.1% to 100% at 1 Hz. For in vivo degradation analysis, LNP-Fluc mRNA@Gel was subcutaneously injected into the dorsal skin of mice. The implants were retrieved at 6, 12, 24, 48, and 72 h, and the residual hydrogel mass was measured to determine the in vivo retention profile.

Application of LNP-Fluc mRNA-loaded hydrogel
Under anesthesia induced by intraperitoneal injection, male C57BL/6 mice (6–8 weeks old) received 6-mm full-thickness circular excisions on the dorsal skin. The wounds were stabilized using silicon rings and covered with 3M™ Tegaderm™ Transparent Film Dressing to inhibit contraction. LNP-Fluc mRNA-loaded hydrogel (50 μg/mL mRNA) was administered into the wound bed through the film using a syringe and allowed to solidify completely for approximately 15 s. Bioluminescence signals were monitored at 6, 12, 24, 48, 72, and 96 h post-application using an In Vivo Imaging System (IVIS) to quantify intensity and spatial distribution.

Biosafety evaluation
Male C57BL/6 mice (6–8 weeks old) were anesthetized and depilated on the dorsal skin. A total of 50 μg/mL LNP-GAS6 mRNA@Gel was administered via subcutaneous multi-point injection. Dorsal hair regrowth was monitored for 30 days. On day 30, skin, visceral organs, and blood samples were collected for histopathological and biochemical analyses, including Masson’s trichrome staining, H&E staining, and ELISA-based detection of inflammatory cytokines.

Transcriptomic sequencing and bioinformatics analysis
Transcriptomic sequencing and bioinformatics analysis were conducted with the support of Shanghai Majorbio Bio-Pharm Technology Co., Ltd. Full-thickness dorsal wound-edge tissues from mice were harvested at day 3 post-operation, flash-frozen in liquid nitrogen, and homogenized in TRIzol for total RNA extraction. RNA integrity was verified via Agilent 5300 (RQN > 6.5). Stranded mRNA libraries were prepared and sequenced on NovaSeq X Plus. Differential gene expression was analyzed using DESeq2 (FDR < 0.05, |log₂FC| > 1).

ATP concentration assay
BMDMs were lysed directly in culture plates by aspirating the medium and adding 200 μL of ice-cold ATP assay lysis buffer per well (of a 6-well plate). Cells were lysed completely by repeated pipetting. The lysate was centrifuged at 12,000×g for 5 min at 4 °C, and the supernatant was collected. ATP detection working solution (Beyotime, #S0026) was prepared by 1:9 dilution of the reagent. A total of 100 μL working solution was aliquoted into a luminometer plate, incubated at room temperature for 3–5 min, and then mixed with 20 μL of sample or standard. Luminescence (RLU) was measured after a 2-s delay. ATP concentrations were calculated and normalized to protein content (BCA assay).

Animal experiments
For all in vivo wound healing studies, male C57BL/6J mice, female New Zealand White rabbits, and male Bama minipigs were housed under a 12-h light/12-h dark cycle at 22 ± 2 °C and 50 ± 10% humidity. According to previous study, sex has no significant impact on wound repair. A total of 134 male C57BL/6J mice (C57BL/6J Rj substrain; Janvier Labs, France) were used in this study. For all wound healing experiments, male mice aged 6–8 weeks were employed. Mice underwent dorsal full-thickness 6-mm circular excisions under intraperitoneal injection anesthesia, stabilized with silicon rings and 3M Tegaderm film (inhibiting wound contraction). To identify the optimal concentration of LNP-GAS6 mRNA, the treatments were as follows: ⅰ. LNP@Gel, ⅱ. LNP-GAS6 mRNA@Gel (20 μg/ml), ⅲ. LNP-GAS6 mRNA@Gel (50 μg/ml), and ⅳ. LNP-GAS6 mRNA@Gel (80 μg/ml). For the LNP-GAS6 mRNA therapeutic evaluation, the groups included: ⅰ. saline (Ⅰ), ⅱ. Gel (Ⅱ), ⅲ. LNP@Gel (Ⅲ), ⅳ. GAS6 mRNA-LNP@Gel (50 μg/ml) (Ⅳ), ⅴ. recombinant GAS6 protein (10 μg/ml, MedChemExpress) (Ⅴ). Healing parameters (wound area) were tracked at days 0, 3, 7, 10, and 14 post-operation. Peri-wound tissues were harvested at day 3 and underwent immunofluorescence detection of apoptotic cells and Transwell migration assays, while day 30 scar tissues were analyzed via H&E/Masson’s trichrome staining for scar length and collagen architecture. In separate inhibitor studies, RU-301, Bemcentinib, and UNC2025 (MedChemExpress) were applied topically at 50 μg/mL.

A total of 6 female New Zealand White rabbits aged 6–8 months with a body weight of 2.5–3.5 kg were used for the hypertrophic scar model. Four 1-cm-diameter circular full-thickness wounds (ventral surface) per ear, including perichondrium, were surgically excised in rabbits under intravenous anesthesia. These wounds were treated with ⅰ. saline (Ⅰ), ⅱ. Gel (Ⅱ), ⅲ. LNP@Gel (Ⅲ), or ⅳ. LNP-GAS6 mRNA@Gel (50 μg/ml) (Ⅳ) under 3M Tegaderm film. Healing dynamics and hypertrophic scar formation were monitored on days 0, 3, 7, 14, 28, and 60, with collagen organization analyzed via Masson’s trichrome and picrosirius red staining in harvested scar tissues.

A total of 6 male Bama minipigs, aged 12–18 months with a body weight of 20–30 kg, were used for the preclinical wound healing study. Dorsal 15-mm-diameter circular full-thickness excisions were created in Bama minipigs under intramuscular and inhalation anesthesia. These wounds were treated with ⅰ. Gel (Ⅰ), ⅱ. LNP@Gel (Ⅱ), ⅲ. LNP-GAS6 mRNA@Gel (50 μg/ml) (Ⅲ), or ⅳ. h-EGF gel (Ⅳ) under 3M Tegaderm film. The wound healing and scar formation progression were tracked on days 0, 7, 14, 21, 28, and 35, with scar tissues harvested at days 14 and 35, and analyzed via Masson’s trichrome staining for collagen deposition.

Statistical analysis
Data were analyzed using GraphPad Prism (v9.0) or R software (v4.3.0), with ≥3 independent biological replicates per group and representative images illustrated. Normality was verified via Shapiro-Wilk tests. Between-group comparisons employed Student’s t-test (two groups) or one-way/two-way ANOVA with Tukey’s post-hoc analysis (≥3 groups). Results were presented as mean ± standard deviation (SD).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript reports a GAS6 mRNA-LNP hydrogel delivery platform to promote scarless healing. The authors analyzed the biological role of GAS6 as a potential target in wound healing. They carried out the preparation and in vitro characterization of GAS6 mRNA-LNP and hydrogel formulation. They further evaluated the scarless healing process and mechanism of acute wounds of three species and confirmed the functions of GAS6 mRNA during efferocytosis and inflammatory suppression. The manuscript is well organized. It might be acceptable after addressing the following issues.\n1. Figure 2, at the bottom, “control” bar does not make sense. Please check “LNP” bar and “Gel+LNP-GAS6 mRNA” bar represent what exactly? The authors make confusion. Figure 2h-j, Why II and III have similar intracellular overexpression of GAS6 protein in fibroblasts, and the letter have stronger than the former in macrophages. Please explain the observation and provide the data at 6 and/or 12 hours.\n2. Line 156-157, why the percentage of macrophages does not match the data in Figure 3c?\n3. Figure 4d, why two of three mice wounds treated with LNP without gel don’t show proper fluorescence. When LNP-Fluc mRNA or LNP-Fluc mRNA-loaded hydrogel were applied to acute murine dorsal wounds, the former should transfect cells more effectively especially at the beginning. Please provide the images at 6 and 12 hours along with 24 hours and more, and explain why hydrogel treated show stronger fluorescence.\n4. Figure 8, human EGF gel was used as a reference, why GAS6 protein wasn’t used?\n5. Please explain why the dose of LNP-GAS6 mRNA was used to treat animals for wound healing.", "reviewer_2": "While the manuscript presents a basic proof-of-concept idea to promoting scarless wound healing through a hydrogel-based mRNA delivery system, the overall study exhibits substantial limitations across multiple critical aspects, including experimental design, material characterization, and mechanistic investigation. These shortcomings are not merely technical or procedural but reflect deeper structural constraints inherent to the material system itself—such as the absence of precise targeting mechanisms, undefined degradation behavior, and limited adaptability to pathological wound environments. In its current form, the work does not provide sufficiently rigorous or convincing evidence to substantiate its proposed therapeutic advantages.\nMajor Concerns\n1. Insufficient Biocompatibility and Safety Evaluation\nThe study lacks long-term in vivo safety data (e.g., fibrosis scoring, chronic inflammation) to confirm biocompatibility of the hydrogel.\nSystemic immunogenicity of LNP-mRNA (e.g., IFN-α, IL-6 levels) is not assessed, leaving the risk of TLR activation unresolved.\n2. No Assessment of Hydrogel Degradation Kinetics\nThe degradation profile of the hydrogel is not characterized, making it impossible to determine whether GAS6 expression dynamics match material breakdown and mRNA release.\n3. Lack of Appropriate Positive Controls\nKey functional assays (macrophage M2 polarization, phagocytosis) do not include positive controls (e.g., IL-4), making the biological significance of the observed effects unclear.\n4. Unresolved Mechanisms Involving TAM Receptors\nThe study confirms activation of the TAM family but does not dissect individual contributions (e.g., via Axl inhibitors or MerTK knockout). Functional mechanisms are speculative and unsupported by targeted experiments.\n5. Limited Spatiotemporal Delivery Control\nThe hydrogel system appears to rely solely on passive diffusion, with no quantification of GAS6 distribution across dermal layers or validation of delivery to therapeutic targets.\n6. No Active Targeting Strategy\nCompared to current state-of-the-art delivery systems, the platform lacks ligand-mediated or inflammation-responsive targeting mechanisms. Transwell assays are insufficient to demonstrate effective LNP localization.\n7. Hydrogel–LNP Interaction Unclear\nThe potential for the hydrogel to hinder or enhance LNP release is not assessed. No comparison is made to free LNP, making it impossible to determine whether the material system is synergistic or detrimental.\n8. Lack of Environmental Responsiveness\nThe hydrogel has a fixed phase transition at 25°C and was only tested under physiological conditions. Its performance in pathophysiological environments (e.g., acidic or inflamed wounds) is unknown.\n9. Mismatch Between Material and Biological Timelines\nThe release kinetics of GAS6 mRNA are not shown to align with the wound healing timeline. No data are provided on residual material or potential fibrotic responses due to prolonged retention.\nMinor Concerns\n1. Important physical properties of the hydrogel (e.g., pore size, network structure) are not reported.\n2. The immunofluorescence imaging in Figure 4g is qualitative and lacks layer-specific quantification.\n3. The manuscript would benefit from improved discussion on how this system compares to existing targeted delivery platforms in terms of precision, responsiveness, and translational potential.", "reviewer_3": "The manuscript describes an approach to scarless cutaneous wound healing through localized administration of LNP-GAS6 mRNA loaded in a thermosensitive hydrogel. The LNP-GAS6 mRNA@Gel platform demonstrates sustained GAS6 expression and robust therapeutic efficacy across murine, rabbit, and Bama minipig models, highlighting its clinical translation potency. The mechanistic insights into macrophage-fibroblast crosstalk and cross-species validation make this study innovative. However, to meet Nature Communications’ standards, the manuscript requires major revisions to address the following issues:\n1. The authors claim persistent GAS6 deficiency as a hallmark of pathological wound healing. However, the mechanistic link between GAS6 downregulation and dysregulated efferocytosis or inflammatory cascades is not fully elucidated. The authors should provide additional mechanistic details to clarify how GAS6 deficiency specifically impairs macrophage-fibroblast crosstalk.\n2. The transcriptomic analysis identifies oxidative phosphorylation (OXPHOS) as a key pathway influenced by LNP-GAS6 mRNA. To strengthen the mechanistic link between GAS6 signaling and OXPHOS, the authors should include data from targeted gene knockdown studies to clarify this relationship.\n3. The oxidative phosphorylation (OXPHOS) pathway should not be omitted in the graphical abstract. This pathway should be included to reflect the mechanistic insights of this study.\n4. The thermosensitive hydrogel is a critical component of the delivery platform. Mechanical properties, in vivo degradation kinetics, and mRNA release profiles of LNP-GAS6 mRNA@Gel should be provided, as these details are essential for understanding the role of hydrogel in spatiotemporal control of mRNA delivery.\n5. The discussion of the LNP-mRNA therapeutics for cutaneous wound healing in the Introduction is too simple. Have any LNP-mRNA formulations for wound healing been reported previously to directly support the therapeutic context of this study, particularly those using hydrogel-based delivery systems? If so, please cite relevant studies. If not, the authors should clarify the rationale for selecting the references included in the manuscript.\n6. More details on the synthesis of GAS6 mRNA should be provided.\n7. The authors should explain the reason why the SM-102 LNP formulation is selected, as it is usually used in vaccines development. In addition, the authors should specify the N/P ratio for LNP-GAS6 mRNA formulation.\n8. The authors justify the choice of a single 5 µg mRNA dose across all wound sizes. Were dose-response studies conducted to optimize the therapeutic efficacy?\n9. The injection method of LNP-flu mRNA should be provided to ensure reproducibility and transparency.\n10. The manuscript needs careful proofreading. The inconsistency of \"LNP-GAS6\" and \"LNP-GAS6 mRNA\" can confuse readers. Additionally, several symbol and typo errors should be corrected."}, "author_response": "We are grateful to Reviewer #1 for their thoughtful review and encouraging words regarding the organization and scope of our manuscript. It is an honor to receive such positive feedback. We have thoroughly considered all comments and provide a point-by-point response below. The suggested revisions have been incorporated and have undoubtedly improved the quality of our study. Point-by-Point Response:\n1. Figure 2, at the bottom, “control” bar does not make sense. Please check “LNP” bar and “Gel+LNP-GAS6 mRNA” bar represent what exactly? The authors make confusion. Figure 2h-j, Why II and III have similar intracellular overexpression of GAS6 protein in fibroblasts, and the letter have stronger than the former in macrophages. Please explain the observation and provide the data at 6 and/or 12 hours.\nResponse:\nWe apologize for the confusion caused by the incorrect labels. The \"Control\" bar has been removed. The bar previously labeled \"Gel+LNP-GAS6 mRNA\" has been corrected to \"LNP-GAS6 mRNA\" in the revised figure (Fig. 2).\nFig. 2\nThe difference in GAS6 overexpression between cell types can be attributed to their distinct in vitro growth characteristics and transfection contexts:\na) Primary macrophages (BMDM) are largely non-proliferative in culture. We plate them at a higher density, which provides a larger number of cells for a standardized LNP-mRNA transfection, potentially leading to more robust and detectable protein expression per field of view. Fibroblasts, in contrast, proliferate rapidly. They were plated at a lower initial density to avoid over-confluence. This likely led to a lower absolute number of cells being transfected at the time of LNP addition, effectively \"diluting\" the overexpression signal. Furthermore, baseline GAS6 expression can fluctuate during fibroblast proliferation in both control and treated groups, which may reduce the apparent fold-change compared to macrophages, even though the absolute delivery of mRNA and expression of protein is successful.\nb) While the magnitude of overexpression appears different, the key finding—that LNP-GAS6 mRNA treatment induces statistically significant GAS6 overexpression compared to the control in both cell types—remains clear. As requested, we provide the data from the 12-hour time point (Fig.2i-k in the revised figure). At this early time point, GAS6 expression in the LNP-GAS6 mRNA group showed an increasing trend but had not yet reached its peak or statistical significance compared to the control (fibroblast), which is consistent with the expected kinetics of mRNA translation and protein expression. This further supports that the strong, significant signal observed at later time points (24-72 h) is a result of active transfection and protein production.\nFig. 2\n2. Line 156-157, why the percentage of macrophages does not match the data in Figure 3c?\nResponse:\nThe percentage mentioned in Lines 156-157 represents the mean ± SD from multiple independent experiments, quantifying the overall efficacy of the treatment. The image in Figure 3c (now labeled as Figure 3j in the revised manuscript) is a single, representative flow cytometry plot from one of those replicate experiments, intended to visually illustrate the gating strategy and the observed shift in population.\nWe apologize for any confusion caused by this discrepancy between the textual data and the representative image. To prevent any future misunderstanding, we have revised the text to explicitly state that the values are the mean from multiple replicates and have ensured the figure legend clearly indicates that the panel shows a representative result.\nFig. 3\n3. Figure 4d, why two of three mice wounds treated with LNP without gel don’t show proper fluorescence. When LNP-Fluc mRNA or LNP-Fluc mRNA-loaded hydrogel were applied to acute murine dorsal wounds, the former should transfect cells more effectively especially at the beginning. Please provide the images at 6 and 12 hours along with 24 hours and more, and explain why hydrogel treated show stronger fluorescence.\nResponse:\nThe reviewer is correct that, in theory, free LNP injection might be expected to show rapid initial transfection. However, in practice, we observed that LNP-Fluc mRNA administered by direct injection into murine dorsal wounds frequently leaked out from the wound site due to the absence of an effective retaining matrix. This loss resulted in only a small fraction of the LNP-Fluc mRNA remaining in contact with the wound tissue, leading to weak and variable fluorescence signals, as seen in two of the three mice in Fig. 4d (old version). In contrast, the thermosensitive hydrogel acts as a localized reservoir that firmly adheres to the wound bed, preventing leakage and ensuring prolonged retention of LNP-Fluc mRNA within the target site. This is a key design advantage of our platform.\nAs suggested by the reviewer, we have performed additional imaging at earlier time points (6 and 12 hours). These new data (now included in Fig. 4d-f) support our explanation: The hydrogel group demonstrates more reliable and intense fluorescence over time, aligning perfectly with the expected pharmacokinetic profile of a sustained-release system. Moreover, even in cases where minimal leakage occurred with free LNP-Fluc mRNA injection, the distribution was highly uneven. Without a supporting matrix, the free LNPs tended to accumulate asymmetrically on one side of the wound periphery due to gravity and tissue movement, resulting in localized, transient expression that cleared rapidly. The hydrogel, however, facilitates uniform dispersion of LNPs throughout the wound bed, supporting sustained and robust protein production, which accounts for the stronger and longer-lasting fluorescence signal.\nWe thank the reviewer for prompting this important clarification, which underscores the necessity of a hydrogel-based system for efficient local mRNA delivery in a dynamic wound environment.\nFig. 4 (old version)\nFig. 4\n4. Figure 8, human EGF gel was used as a reference, why GAS6 protein wasn’t used?\nResponse:\nWe selected rhEGF gel as the comparator for three key reasons:\n Clinical Relevance: rhEGF gel is an established clinical therapy for wound healing, providing a translationally meaningful benchmark. In contrast, recombinant GAS6 protein lacks clinical validation.\n Limited Efficacy of Recombinant GAS6: As shown in our murine model (Fig. 5, Group V), recombinant GAS6 protein offered only transient benefit and was statistically inferior to our mRNA therapy in long-term scar reduction. Using it in the large, expensive porcine model would have been difficult to justify scientifically and ethically.\n Focus on Platform Innovation: Our study emphasizes the LNP-hydrogel delivery platform itself. Comparing against a standard clinical therapy (rhEGF) better demonstrates the advantage of our sustained mRNA delivery over conventional protein therapeutics (Fig. 8).\nThus, using rhEGF gel provided a more rigorous and clinically relevant benchmark to highlight our platform's therapeutic potential.\nFig. 5 Fig. 8\n5. Please explain why the dose of LNP-GAS6 mRNA was used to treat animals for wound healing.\nResponse:\nThe dose of LNP-GAS6 mRNA (50 μg/mL) used in our wound healing experiments was determined through a systematic concentration-gradient screening study aimed at optimizing therapeutic efficacy. To establish an optimal dosing regimen, we conducted a dose-response assessment of LNP-GAS6 mRNA@Gel (20, 50, and 80 μg/mL) in murine acute wounds. Immunofluorescence confirmed dose-dependent GAS6 upregulation, with pronounced and spatially restricted expression in the deep dermis at both the wound center and edge (Fig. 4g–i). The 50 μg/mL dose achieved a near-maximal GAS6 expression, comparable to 80 μg/mL (Fig. 4g–i). Although wound width was similar across groups, the 50 μg/mL treatment significantly enhanced granulation tissue thickness relative to controls (Fig. 4g, j, k), indicating promoted proliferative repair. Based on this robust efficacy and a favorable saturation profile, 50 μg/mL was selected for all subsequent studies.\nFig. 4\nReviewer #2 (Remarks to the Author):\nWhile the manuscript presents a basic proof-of-concept idea to promoting scarless wound healing through a hydrogel-based mRNA delivery system, the overall study exhibits substantial limitations across multiple critical aspects, including experimental design, material characterization, and mechanistic investigation. These shortcomings are not merely technical or procedural but reflect deeper structural constraints inherent to the material system itself—such as the absence of precise targeting mechanisms, undefined degradation behavior, and limited adaptability to pathological wound environments. In its current form, the work does not provide sufficiently rigorous or convincing evidence to substantiate its proposed therapeutic advantages.\nWe sincerely thank Reviewer #2 for their thorough evaluation and the insightful critiques of our manuscript. We deeply appreciate the time and expertise invested in reviewing our work, and we acknowledge that the concerns raised are fundamental to establishing the robustness and translational potential of our delivery platform. We fully agree that a more comprehensive characterization of the material properties, degradation kinetics, and mechanistic pathways would significantly strengthen the study. In response, we have conducted additional experiments and analyses to directly address each of the points raised, which we believe have substantially improved the manuscript. Below, we provide a detailed point-by-point response to all the comments.\nPoint-by-Point Response:\nMajor Concerns\n1. Insufficient Biocompatibility and Safety Evaluation\nThe study lacks long-term in vivo safety data (e.g., fibrosis scoring, chronic inflammation) to confirm biocompatibility of the hydrogel.\nSystemic immunogenicity of LNP-mRNA (e.g., IFN-α, IL-6 levels) is not assessed, leaving the risk of TLR activation unresolved.\nResponse:\nWe agree that comprehensive long-term safety evaluation is essential for biomaterial-based therapies. In the revised manuscript, a comprehensive biosafety evaluation of LNP-GAS6 mRNA@Gel was conducted. Local assessment at injection sites showed no adverse effects on hair regeneration, follicular density, dermal thickness, or collagen volume fraction after 30 days (Supplementary Fig. 5a–f). Systemically, no gross or microscopic alterations were detected in major organs, and plasma inflammatory cytokine levels remained comparable to controls (Supplementary Fig. 5g–i). These data collectively demonstrate that LNP-GAS6 mRNA@Gel is well-tolerated both locally and systemically.\nSupplementary Fig. 5\n2. No Assessment of Hydrogel Degradation Kinetics\nThe degradation profile of the hydrogel is not characterized, making it impossible to determine whether GAS6 expression dynamics match material breakdown and mRNA release.\nResponse:\nWe agree that understanding the degradation kinetics of the hydrogel is essential to correlate material behavior with bioactivity. In response, we conducted a series of in vitro and in vivo degradation studies.\nThe hydrogel exhibited microenvironment-dependent degradation, with slow kinetics in PBS but accelerated breakdown in collagenase I, mimicking the proteolytic wound environment (Supplementary Fig. 4c). Release kinetics aligned with material degradation, as 84.21% of encapsulated mRNA was released within 96 hours under collagenase I conditions (Supplementary Fig. 4d). Consistent with in vitro behavior, the hydrogel underwent near-complete degradation in vivo within 72 hours (Supplementary Fig. 4e, f).\nSupplementary Fig. 4\n3. Lack of Appropriate Positive Controls\nKey functional assays (macrophage M2 polarization, phagocytosis) do not include positive controls (e.g., IL-4), making the biological significance of the observed effects unclear.\nResponse:\nWe have included additional positive controls in our key functional assays to better contextualize the biological effects of LNP-GAS6 mRNA. For the macrophage polarization and efferocytosis experiments, we introduced LNP-IL-4 mRNA as a positive control. The results confirmed that LNP-IL-4 mRNA promoted M2 polarization and enhanced efferocytosis, as expected (Fig. 3f–j). Notably, LNP-GAS6 mRNA demonstrated a stronger enhancement of efferocytosis than LNP-IL-4 mRNA (p<0.01; Fig. 3h–j), indicating that GAS6 signaling exerts immunomodulatory effects superior to those of a classical M2-polarizing stimulus. We sincerely appreciate this suggestion, which has strengthened the mechanistic interpretation and biological relevance of our findings.\nFig. 3\n4. Unresolved Mechanisms Involving TAM Receptors\nThe study confirms activation of the TAM family but does not dissect individual contributions (e.g., via Axl inhibitors or MerTK knockout). Functional mechanisms are speculative and unsupported by targeted experiments.\nResponse:\nWe agree that dissecting the individual contributions of TAM family members is crucial for understanding the signaling mechanisms through which GAS6 promotes regenerative healing. To delineate the specific contributions of TAM receptors, we inhibited AXL and MERTK signaling in murine acute wounds using Bemcentinib and UNC2025, respectively (Fig. 6a). Both inhibitors significantly impaired repair, delaying wound closure and exacerbating scar formation (Fig. 6b, c). Although attenuated, the therapeutic effect of LNP-GAS6 mRNA@Gel remained significant, indicating that it promoted repair even when individual TAM receptors were blocked (Fig. 6b, c). This functional compensation among TAM receptors not only helps coordinate the repair process, but also highlights the therapeutic advantage of using a broad-spectrum TAM ligand agonist (LNP-GAS6 mRNA@Gel) over receptor-specific targeting. We are grateful for this suggestion, which has substantially deepened our mechanistic inquiry.\nFig. 6\n5. Limited Spatiotemporal Delivery Control\nThe hydrogel system appears to rely solely on passive diffusion, with no quantification of GAS6 distribution across dermal layers or validation of delivery to therapeutic targets.\nResponse:\nWe agree that quantifying the distribution of GAS6 across wound layers is essential to validate our delivery strategy. While we have not performed direct co-localization staining for GAS6 and specific cell markers, our immunofluorescence analysis (as shown in Fig. 4g-i) quantitatively demonstrated that GAS6 expression was significantly higher in the deep dermis (particularly in the reticular dermis at both the wound center and edge), compared to superficial layers. This region-specific enrichment is functionally critical, as pathological scarring is primarily driven by dysregulated inflammation and fibrosis originating in the deep dermis.\nThese data provide clear evidence that our hydrogel system enables spatially restricted delivery of GAS6 mRNA to the therapeutically most relevant tissue layer. We believe the current layer-specific expression data, combined with the functional scar reduction outcomes across three animal models, strongly support the spatiotemporal delivery capability of our platform.\nFig. 4\n6. No Active Targeting Strategy\nCompared to current state-of-the-art delivery systems, the platform lacks ligand-mediated or inflammation-responsive targeting mechanisms. Transwell assays are insufficient to demonstrate effective LNP localization.\nResponse:\nOur delivery approach was deliberately designed based on the biological characteristics of GAS6 as a secreted protein that exerts its effects through paracrine signaling rather than requiring direct cell-specific internalization. While ligand-mediated targeting strategies are valuable for systemic delivery or specific cellular uptake, our localized hydrogel platform achieves effective spatial targeting through different mechanisms:\n Physical Confinement and Sustained Release: The thermosensitive hydrogel acts as a local reservoir that confines LNPs within the wound bed, preventing rapid clearance and ensuring prolonged GAS6 expression precisely where needed. Our in vivo imaging data (Fig. 4d-f) demonstrate significantly improved retention compared to free LNPs.\n Biological Targeting through Protein Secretion: Once translated from the delivered mRNA, GAS6 is secreted and naturally distributes within the wound microenvironment, activating TAM receptors on nearby macrophages. This avoids the complexity of engineering LNPs for specific cell targeting while still achieving the desired biological effect.\n Spatial Localization Evidence: Our immunofluorescence data (Fig. 4g-i) confirm that GAS6 protein is predominantly expressed in therapeutically relevant areas—specifically enriched in the deep dermis where pathological scarring originates.\nWe agree that inflammation-responsive elements could further enhance the system's sophistication. However, the current platform's simplicity (using clinically validated components without additional targeting ligand), may facilitate faster translational development while effectively addressing the clinical need for localized scar prevention.\nFig. 4\n7. Hydrogel–LNP Interaction Unclear\nThe potential for the hydrogel to hinder or enhance LNP release is not assessed. No comparison is made to free LNP, making it impossible to determine whether the material system is synergistic or detrimental.\nResponse:\nOur comparative studies clearly demonstrate that the hydrogel does not hinder but significantly enhances the overall performance of LNPs in the dynamic wound environment. Direct comparison between free LNP and hydrogel-formulated LNP reveals several key advantages:\n Enhanced Retention and Stability: Free LNP injections suffered from substantial wound leakage, resulting in weak and variable fluorescence signals (Fig. 4d-f). In contrast, the hydrogel acts as a protective reservoir that firmly retains LNPs at the target site, ensuring sustained local presence.\n Improved Distribution Kinetics: Additional imaging at 6 and 12 hours (now in Fig. 4d-f) shows that while free LNPs exhibited uneven distribution and rapid clearance, the hydrogel formulation achieved earlier, more intense, and more uniform signal distribution throughout the wound bed.\n Optimized Release Profile: The hydrogel enables a controlled release kinetics that aligns perfectly with the therapeutic window. Rather than impeding LNP function, the hydrogel maintains LNPs in the wound microenvironment, facilitating prolonged and robust protein production that free LNPs cannot achieve.\nThe superior and sustained fluorescence signal in the hydrogel group, coupled with the poor performance of free LNPs, provides compelling evidence that the hydrogel-LNP combination acts synergistically rather than detrimentally. This protective, sustained-release mechanism is particularly crucial in the dynamic and exudative environment of acute wounds. We appreciate the opportunity to clarify this point and have strengthened the corresponding results section to better highlight these comparative advantages.\nFig. 4\n8. Lack of Environmental Responsiveness\nThe hydrogel has a fixed phase transition at 25°C and was only tested under physiological conditions. Its performance in pathophysiological environments (e.g., acidic or inflamed wounds) is unknown.\nResponse:\nWe have conducted additional experiments to evaluate its performance under pathophysiological conditions. As shown in Fig. 4a–b, our thermosensitive PLGA-PEG-PLGA hydrogel exhibits a reversible sol-gel transition at ~25°C, enabling rapid gelation within 15 seconds upon application to both acute and diabetic wounds. Most importantly, we observed no significant delay in gelation kinetics even in the hyperglycemic and inflammatory microenvironment of diabetic wounds, confirming the robustness of its temperature-dependent behavior.\nWhile the current system relies primarily on thermos-sensitivity rather than pH or enzyme-mediated responsiveness, its rapid gelation and physical stability under pathophysiological conditions ensure reliable retention and localized delivery of GAS6 mRNA within the wound bed. We appreciate this valuable feedback, which helps highlight the current robustness for our platform.\nFig. 4\n9. Mismatch Between Material and Biological Timelines\nThe release kinetics of GAS6 mRNA are not shown to align with the wound healing timeline. No data are provided on residual material or potential fibrotic responses due to prolonged retention.\nResponse:\nWe have carefully designed our system to ensure that GAS6 expression is synchronized with the key inflammatory phase of repair. Early inflammatory events (particularly within the first 72 hours), are decisive in determining regenerative versus fibrotic outcomes. Our transcriptomic data (both bulk and single-cell) confirm that endogenous GAS6 is downregulated during early inflammation, and that inflammatory priming of dermal fibroblasts is a pivotal driver of scarring (Fig. 1a-l). To reverse this deficit, our LNP-GAS6 mRNA@Gel system was engineered to achieve peak GAS6 protein expression on post-operative day 3 (463.75 ± 80.17 pg/mg tissue), followed by a gradual decline (Supplementary Fig. 4g). This kinetics aligns precisely with the inflammatory phase of wound healing, ensuring that GAS6 signaling is restored when it is most needed.\nIn vivo degradation tracking confirmed near-complete hydrogel elimination within 72 hours post-application (Supplementary Fig. 4e, f), demonstrating favorable clearance kinetics that preclude long-term material retention. Regarding potential fibrotic responses due to material retention, local assessment at injection sites showed no adverse effects on hair regeneration, follicular density, dermal thickness, or collagen volume fraction after 30 days (Supplementary Fig. 5a–f). Systemically, no gross or microscopic alterations were detected in major organs, and plasma inflammatory cytokine levels remained comparable to controls (Supplementary Fig. 5g–i). These data collectively demonstrate that LNP-GAS6 mRNA@Gel is well-tolerated both locally and systemically.\nTogether, these results demonstrate that our delivery system achieves biologically relevant kinetics without eliciting adverse fibrotic responses, supporting its therapeutic safety and temporal compatibility with the wound healing process.\nFig 1.\nSupplementary Fig. 4\nSupplementary Fig. 5\nMinor Concerns\n1. Important physical properties of the hydrogel (e.g., pore size, network structure) are not reported.\nResponse:\nThe PLGA-PEG-PLGA hydrogel used in this study is a well-characterized thermosensitive polymer, and its structural features have been well documented in previous literature.\nFor example, Chen et al. reported that PLGA-PEG-PLGA hydrogels possess a homogeneous porous microstructure ideal for nanoparticle encapsulation and sustained release1. Similarly, Ran et al. demonstrated that such hydrogels form uniform three-dimensional networks, with porosity tunable by incorporating modifiers such as catechol-modified chitosan2.\nIn our work, structural and functional analyses confirmed that the hydrogel maintained its porous architecture after LNP incorporation, as shown by SEM (Supplementary Fig. 4a, b). The hydrogel also exhibited strong adhesive and tensile properties, supporting its suitability as a delivery platform for wound healing applications.\nTogether, these findings—consistent with established reports—validate the use of this hydrogel for LNP loading and localized therapeutic delivery.\nReference:\nSupplementary Fig. 4\n2. The immunofluorescence imaging in Figure 4g is qualitative and lacks layer-specific quantification.\nResponse:\nWe have performed layer-specific quantitative analysis of the immunofluorescence data. The results demonstrate that GAS6 expression was significantly higher in the deep dermis compared to the superficial layers at both the wound center and edge (Fig. 4g–i). This targeted delivery to the reticular dermis is therapeutically relevant, as pathological scarring primarily originates in this region. The quantitative data have been included in the revised figure.\nFig. 4\n3. The manuscript would benefit from improved discussion on how this system compares to existing targeted delivery platforms in terms of precision, responsiveness, and translational potential.\nResponse:\nWe have expanded the Discussion section to include a more detailed comparison of our platform with existing targeted delivery systems in terms of precision, responsiveness, and translational potential:\n“Furthermore, our platform offers distinct advantages over existing delivery systems. While most targeted platforms rely on ligand-mediated approaches43,73, we achieve spatial precision through hydrogel retention in the deep dermis. The system provides passive responsiveness via temperature-triggered gelation and protease-accelerated degradation. Most importantly, using clinically validated components enables faster translation than systems requiring novel ligands or complex chemistries.”\nThis revised discussion now more clearly positions our technology within the current landscape of targeted delivery systems.\nReviewer #3 (Remarks to the Author):\nThe manuscript describes an approach to scarless cutaneous wound healing through localized administration of LNP-GAS6 mRNA loaded in a thermosensitive hydrogel. The LNP-GAS6 mRNA@Gel platform demonstrates sustained GAS6 expression and robust therapeutic efficacy across murine, rabbit, and Bama minipig models, highlighting its clinical translation potency. The mechanistic insights into macrophage-fibroblast crosstalk and cross-species validation make this study innovative. However, to meet Nature Communications’ standards, the manuscript requires major revisions to address the following issues:\nWe sincerely thank the reviewers for their positive assessment of the innovation and translational potential of our study, as well as for their constructive and detailed feedback. We have carefully considered all the comments and have performed additional experiments and analyses to address each point raised. Below, we provide a point-by-point response to the specific concerns mentioned. We believe that these revisions have significantly strengthened the manuscript, and we hope that the revised version now meets the high standards of Nature Communications. Point-by-Point Response:\n1. The authors claim persistent GAS6 deficiency as a hallmark of pathological wound healing. However, the mechanistic link between GAS6 downregulation and dysregulated efferocytosis or inflammatory cascades is not fully elucidated. The authors should provide additional mechanistic details to clarify how GAS6 deficiency specifically impairs macrophage-fibroblast crosstalk.\nResponse:\nTo mechanistically link GAS6 deficiency to impaired macrophage-fibroblast crosstalk, we performed multi-level validation across in vivo and in vitro systems.\nPharmacological inhibition of GAS6-TAM signaling with RU-301 in murine wounds not only delayed healing and exacerbated scarring (Fig. 1m-o) but also revealed the dual mechanistic basis: impaired efferocytosis (increased apoptotic cell accumulation, Fig. 1p, r) and enhanced dermal inflammatory signaling (elevated macrophage migration, Fig. 1q, s). At the cellular level, GAS6 knockdown in macrophages promoted a pro-inflammatory phenotype (increased IL-1β/TNF-α, decreased IL-10; Fig. 3a-b, Supplementary Fig. 3a-b) that enhanced fibroblast inflammatory priming through paracrine mechanisms (Supplementary Fig. 3c). Concurrently, GAS6 deficiency significantly compromised efferocytic function (Fig. 3c-e). Notably, while individual inhibition of AXL or MERTK receptors delayed repair (Fig. 6a-c), LNP-GAS6 mRNA@Gel retained significant efficacy, suggesting functional compensation among TAM receptors. Mechanistically, GAS6 promotes efferocytosis by enhancing OXPHOS-mediated energy production, as evidenced by suppressed OXPHOS genes and reduced ATP levels upon GAS6 knockdown (Fig. 6i-l).\nCollectively, these findings establish that GAS6 deficiency disrupts macrophage-fibroblast crosstalk through coordinated impairment of efferocytosis and dysregulation of inflammatory paracrine signaling, ultimately driving pathological scarring.\nFig. 1\nSupplementary Fig. 3\nFig. 3\nFig. 6\nFig. 6\n2. The transcriptomic analysis identifies oxidative phosphorylation (OXPHOS) as a key pathway influenced by LNP-GAS6 mRNA. To strengthen the mechanistic link between GAS6 signaling and OXPHOS, the authors should include data from targeted gene knockdown studies to clarify this relationship.\nResponse:\nTo functionally link GAS6 with OXPHOS activation, we performed siRNA-mediated GAS6 knockdown in macrophages. Downregulation of GAS6 significantly suppressed key OXPHOS genes, including Ndufa3, Uqcrb, and Cox5a (Fig. 6i-k). Given that OXPHOS drives ATP production (also critically required for efferocytosis), we next measured cellular ATP levels. While GAS6 knockdown substantially reduced ATP synthesis, treatment with LNP-GAS6 mRNA significantly enhanced cellular ATP production (Fig. 6l), confirming its role in promoting mitochondrial energy generation.\nCollectively, these results establish that GAS6 potentiates efferocytosis and promotes scarless healing, at least in part, by augmenting OXPHOS-derived ATP availability.\nFig. 6\n3. The oxidative phosphorylation (OXPHOS) pathway should not be omitted in the graphical abstract. This pathway should be included to reflect the mechanistic insights of this study.\nResponse:\nWe fully agree that the oxidative phosphorylation (OXPHOS) pathway represents a key mechanistic insight of our study. In response, we have revised the graphical abstract (Fig. 9) to explicitly incorporate the role of GAS6 in enhancing OXPHOS, which subsequently provides the metabolic energy required for efficient efferocytosis and supports macrophage M2 polarization. This revision provides a more comprehensive and accurate visual summary of the proposed mechanism of action.\nFig. 9\n4. The thermosensitive hydrogel is a critical component of the delivery platform. Mechanical properties, in vivo degradation kinetics, and mRNA release profiles of LNP-GAS6 mRNA@Gel should be provided, as these details are essential for understanding the role of hydrogel in spatiotemporal control of mRNA delivery.\nResponse:\nWe have now provided comprehensive characterization of the thermosensitive hydrogel, including its mechanical properties, degradation kinetics, and release profile:\nStructural and functional characterization confirmed the suitability of the LNP-loaded hydrogel as a sustained-release delivery system. Scanning electron microscopy revealed that LNP incorporation did not alter the hydrogel's porous architecture or mechanical properties, which maintained robust adhesive and tensile characteristics (Supplementary Fig. 4a, b). The hydrogel exhibited microenvironment-dependent degradation, with slow kinetics in PBS but accelerated breakdown in collagenase I, mimicking the proteolytic wound environment (Supplementary Fig. 4c). Release kinetics aligned with material degradation, as 84.21% of encapsulated mRNA was released within 96 hours under collagenase I conditions (Supplementary Fig. 4d). Consistent with in vitro behavior, the hydrogel underwent near-complete degradation in vivo within 72 hours (Supplementary Fig. 4e, f).\nThese data demonstrate that the hydrogel enables sustained release of LNPs over a therapeutically relevant period while undergoing progressive biodegradation. This combination of material properties supports the spatiotemporally controlled delivery of GAS6 mRNA, aligning expression with the critical early inflammatory phase of wound healing.\nSupplementary Fig. 4\n5. The discussion of the LNP-mRNA therapeutics for cutaneous wound healing in the Introduction is too simple. Have any LNP-mRNA formulations for wound healing been reported previously to directly support the therapeutic context of this study, particularly those using hydrogel-based delivery systems? If so, please cite relevant studies. If not, the authors should clarify the rationale for selecting the references included in the manuscript.\nResponse:\nWe thank the reviewer for this constructive suggestion. We have now expanded the discussion of LNP-mRNA therapeutics in the Introduction to provide a more comprehensive background.\n“Lipid nanoparticle (LNP)-mediated mRNA delivery has emerged as a transformative therapeutic platform, achieving remarkable success in vaccine development and oncology due to its high efficiency and transient expression profile33-35. However, the application of LNP–mRNA technology in dermatology, particularly for scar prevention, remains highly emerging. The work of Wang, S. et al. demonstrated the feasibility of using LNP-based interleukin-4 (IL-4) mRNA delivery to accelerate wound healing, underscoring the promise of mRNA strategies for precision dermatology36. Nevertheless, achieving localized, stable, and efficient delivery of LNP–mRNA within the dynamic wound microenvironment remains a major challenge. In this context, the combination of a hydrogel with LNP–mRNA enabled successful protein expression in vivo and conferred significant therapeutic benefits, positioning it as a promising platform for mRNA delivery in wound therapy37,38.”\nIn the revised manuscript, we have more clearly articulated this gap in the literature and positioned our work as the first, to our knowledge, to combine thermosensitive hydrogel with LNP-mRNA technology for spatially and temporally controlled restoration of GAS6 signaling in wound healing. We have also included additional citations where appropriate to better contextualize the novelty of our approach.\n6. More details on the synthesis of GAS6 mRNA should be provided.\nResponse:\nThe synthesis and preparation of the GAS6 mRNA are described in detail in the Methods section under \"LNP-mRNA formulation and characterization\" Briefly:\n“GAS6 and IL-4 mRNA was synthesized via in vitro transcription from a linearized plasmid (Yaohai Bio.) using BspQ I digestion, followed by phenol-chloroform extraction or magnetic bead purification. The IVT reaction was performed with T7 RNA polymerase, modified nucleotides (including m1ψ-UTP), and either co-transcriptional capping (using cap analog) or enzymatic capping post-IVT (using VCE and 2'-O-methyltransferase). The resulting mRNA was purified using RNA magnetic beads (N412) and quantified by UV and capillary electrophoresis. Fluc mRNA was obtained commercially (Absin Bioscience Inc.).”\nWe have now expanded the Methods section to provide greater experimental detail regarding the reagent sources and reaction conditions for the synthesized mRNA. This additional information ensures the procedure is clearly documented and reproducible.\n7. The authors should explain the reason why the SM-102 LNP formulation is selected, as it is usually used in vaccines development. In addition, the authors should specify the N/P ratio for LNP-GAS6 mRNA formulation.\nResponse:\nRationale for Selecting SM-102: The lipid SM-102 was selected as the ionizable cationic lipid for our LNP formulation based on its established safety profile, high efficacy in mRNA delivery, and clinical validation in the COVID-19 mRNA vaccine (Spikevax®, mRNA-1273). Although frequently used in vaccines, its excellent efficiency in encapsulating and delivering mRNA, combined with its biodegradability and well-characterized behavior, makes it a suitable and reliable choice for local regenerative applications.\nN/P Ratio Specification: The N/P ratio for our LNP-mRNA formulation was 6:1. This ratio was selected to achieve optimal mRNA encapsulation efficiency (>95%, as shown in Fig. 2e) while maintaining nanoparticle stability and minimizing cytotoxicity, as confirmed by our CCK-8 assay (Supplementary Fig. 2a, b). We have now explicitly included the N/P ratio in the revised Methods section.\nFig. 2\nSupplementary Fig. 2\n8. The authors justify the choice of a single 5 µg mRNA dose across all wound sizes. Were dose-response studies conducted to optimize the therapeutic efficacy?\nResponse:\nWe sincerely apologize for the inaccurate description of mRNA dosage in the original manuscript. To clarify, we administered LNP-GAS6 mRNA@Gel at a consistent concentration of 50 μg/mL across all animal models, while the total volume was adjusted according to wound size and depth to ensure adequate coverage: 100 µL for mouse wounds, 50 µL for rabbit ear wounds (larger area but shallow), and 1 mL for Bama minipig wounds (larger and deeper).\nTo establish an optimal dosing regimen, we conducted a dose-response assessment of LNP-GAS6 mRNA@Gel (20, 50, and 80 μg/mL) in murine acute wounds. Immunofluorescence confirmed dose-dependent GAS6 upregulation, with pronounced and spatially restricted expression in the deep dermis at both the wound center and edge (Fig. 4g–i). The 50 μg/mL dose achieved a near-maximal GAS6 expression, comparable to 80 μg/mL (Fig. 4g–i). Although wound width were similar across groups, the 50 μg/mL treatment significantly enhanced granulation tissue thickness relative to controls (Fig. 4g, j, k), indicating promoted proliferative repair. Based on this robust efficacy and a favorable saturation profile, 50 μg/mL was selected for all subsequent studies.\n9. The injection method of LNP-flu mRNA should be provided to ensure reproducibility and transparency.\nResponse:\nThe injection method for LNP-Fluc mRNA has been described in detail in the revised Methods section under \"Application of LNP-Fluc mRNA-Loaded Hydrogel\". Briefly:\n“Under anesthesia induced by intraperitoneal injection, male C57BL/6 mice (6-8 weeks old) received 6-mm full-thickness circular excisions on the dorsal skin. The wounds were stabilized using silicone rings and covered with 3M™ Tegaderm™ Transparent Film Dressing to inhibit contraction. LNP-Fluc mRNA-loaded hydrogel (50 μg/mL mRNA) was administered into the wound bed through the film using a syringe and allowed to solidify completely for approximately 15 seconds. Bioluminescence signals were monitored at 6, 12, 24, 48, 72, and 96 hours post-application using an In Vivo Imaging System (IVIS) to quantify intensity and spatial distribution.”\nThis standardized protocol ensured consistent and reproducible delivery across all animal experiments. We have expanded the Methods to include these specifics to enhance transparency and replicability.\n10. The manuscript needs careful proofreading. The inconsistency of \"LNP-GAS6\" and \"LNP-GAS6 mRNA\" can confuse readers. Additionally, several symbol and typo errors should be corrected.\nResponse:\nWe apologize for the inconsistencies and errors that remained in the manuscript. We have now performed a thorough proofreading of the entire text. Specifically:\nWe have standardized the terminology to consistently use \"LNP-GAS6 mRNA\" throughout the manuscript to clearly denote the mRNA-based therapeutic, eliminating the previous inconsistency with \"LNP-GAS6\". We have carefully checked for and corrected typographical errors, formatting inconsistencies (e.g., spaces between values and units, hyphenation), and citation formatting to ensure accuracy and professionalism.\nWe greatly appreciate the reviewer's attention to detail, which has significantly improved the clarity and quality of our manuscript. These changes have been implemented in the revised version."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all of concerns from reviewers. The last confusion is the labels \"II and III\" at the left side of Figure 2i means what exactly? It may be acceptable after showing the right labels.", "reviewer_2": "While the authors have addressed many of the major concerns raised in the previous review, several mechanistic and material aspects remain insufficiently clarified. The following additional issues should be resolved to strengthen the robustness and translational relevance of the study:\n1. Environmental responsiveness and functional stability under pathological conditions\nThe authors examined gelation kinetics in a diabetic wound model and demonstrated that the thermosensitive transition remained stable. However, the effects of acidic or inflammatory environments on the hydrogel’s mechanical and release behavior have not been characterized.\nIt is recommended to evaluate the viscoelastic modulus (G′/G″) and mRNA release profiles under pathophysiologically relevant conditions (e.g., pH = 6.5 and high-ROS environments) to confirm the stability and applicability of the system in chronic or inflamed wound microenvironments.\n2. Temporal coordination between GAS6 supplementation and TAM receptor dynamics\nThe manuscript proposes that LNP-GAS6 mRNA@Gel restores early GAS6 expression to re-establish immune homeostasis. However, the study does not present data on the temporal expression dynamics of TAM receptors across relevant cell types such as macrophages and fibroblasts.\nThe current mechanistic interpretation assumes stable receptor availability, yet prior studies have demonstrated that AXL and MERTK are transiently downregulated during the early inflammatory phase (Zindel & Kubes, Immunity, 2019; Camenisch et al., Nature, 2001; Fourgeaud et al., Nature, 2016).\nPlease clarify whether GAS6 supplementation temporally coincides with TAM receptor availability, as an asynchrony between ligand and receptor expression could limit the effective therapeutic window and alter the proposed mechanism of regenerative signaling.", "reviewer_3": "The authors have well addressed my concerns with additional experiments and thoughtful revisions. Therefore, I recommend accepting the revised manuscript."}, "author_response": "Reviewer #1 (Remarks to the Author):\nThe authors have addressed all of concerns from reviewers. The last confusion is the labels \"II and III\" at the left side of Figure 2i means what exactly? It may be acceptable after showing the right labels.\nThank you for your positive feedback on our study. We sincerely apologize for the labeling error where \"I\" and \"II\" were mistakenly displayed as \"II and III\" in the original Figure 2i. This has now been corrected in the revised version.\nReviewer #2 (Remarks to the Author):\nWhile the authors have addressed many of the major concerns raised in the previous review, several mechanistic and material aspects remain insufficiently clarified. The following additional issues should be resolved to strengthen the robustness and translational relevance of the study:\nWe are grateful to Reviewer #2 for recognizing the improvements in our revised manuscript and for their valuable additional suggestions. These comments are highly appreciated as they provide us with an opportunity to further enhance the mechanistic clarity and translational relevance of our work. Below, we address each point in sequence. Point-by-Point Response:\n1. Environmental responsiveness and functional stability under pathological conditions\nThe authors examined gelation kinetics in a diabetic wound model and demonstrated that the thermosensitive transition remained stable. However, the effects of acidic or inflammatory environments on the hydrogel’s mechanical and release behavior have not been characterized.\nIt is recommended to evaluate the viscoelastic modulus (G′/G″) and mRNA release profiles under pathophysiologically relevant conditions (e.g., pH = 6.5 and high-ROS environments) to confirm the stability and applicability of the system in chronic or inflamed wound microenvironments.\nFollowing your suggestion, we have systematically evaluated both the viscoelastic properties and release profiles under pathophysiologically relevant conditions (pH 6.5 and 200 µM H₂O₂). Our results demonstrate excellent stability of the system under these challenging conditions:\n The hydrogel's defining elastic-dominant character (G' > G'') remains intact under both acidic and high-ROS conditions (Supplementary Fig. 5e), confirming the robustness of its mechanical properties in inflammatory environments.\n The mRNA release profile is not significantly affected by these pathological conditions (Supplementary Fig. 5c, d), maintaining its sustained release kinetics essential for promoting therapeutic effect.\nThis remarkable stability of both structural integrity and release function under pathophysiological conditions strongly supports the applicability of our system for chronic and inflamed wound therapy. The preserved mechanical properties ensure proper wound coverage and retention, while the maintained release kinetics guarantee continuous mRNA delivery, collectively contributing to the enhanced healing outcomes observed in our animal models.\nSupplementary Fig. 5\n2. Temporal coordination between GAS6 supplementation and TAM receptor dynamics\nThe manuscript proposes that LNP-GAS6 mRNA@Gel restores early GAS6 expression to re-establish immune homeostasis. However, the study does not present data on the temporal expression dynamics of TAM receptors across relevant cell types such as macrophages and fibroblasts.\nThe current mechanistic interpretation assumes stable receptor availability, yet prior studies have demonstrated that AXL and MERTK are transiently downregulated during the early inflammatory phase (Zindel & Kubes, Immunity, 2019; Camenisch et al., Nature, 2001; Fourgeaud et al., Nature, 2016).\nPlease clarify whether GAS6 supplementation temporally coincides with TAM receptor availability, as an asynchrony between ligand and receptor expression could limit the effective therapeutic window and alter the proposed mechanism of regenerative signaling.\nOur re-analysis of the single-cell RNA sequencing data confirms that AXL and MERTK are indeed transiently downregulated during the early inflammatory phase, particularly in macrophages and fibroblasts —a finding consistent with the literature cited (Supplementary Fig. 2a-d). This dynamic receptor expression underscores a window of opportunity for therapeutic intervention.\nRather than targeting the fluctuating receptors directly, we focused on augmenting ligand availability. The broad specificity of GAS6—which activates all three TAM receptors (TYRO3, AXL, and MERTK)—enables robust signaling restoration even when individual receptor levels are partially suppressed. Our functional data confirm that early GAS6 delivery promotes efferocytosis, attenuates inflammatory signaling, and accelerates wound closure.\nBy intervening at this critical phase, we effectively suppress the inflammatory priming of fibroblasts, thereby disrupting the \"inflammation–fibrosis\" cycle at its origin. We hypothesize that elevated GAS6 levels enhance signaling through residual receptors, compensating for their transient downregulation. Delaying intervention beyond this window could diminish efficacy, as the pro-inflammatory microenvironment becomes entrenched.\nIn summary, our strategy of spatiotemporally controlled GAS6 delivery leverages the multi-receptor activation capacity of GAS6 to potentiate residual TAM signaling during the early inflammatory phase—a rationally designed approach to promote scarless repair.\nSupplementary Fig. 2\nReviewer #3 (Remarks to the Author):\nThe authors have well addressed my concerns with additional experiments and thoughtful revisions. Therefore, I recommend accepting the revised manuscript.\nWe thank the reviewer for their positive assessment and recommendation for acceptance. We are very pleased that our revisions have adequately addressed their concerns."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have fully addressed my concerns."}, "author_response": ""}]

Decision letter