A cyclic heptapeptide-based hydrogel boosts the healing of chronic skin wounds in diabetic mice and patients

The combined use of peptides, nanomaterials, and hydrogels is a promising strategy for chronic skin wound healing, which remains a huge clinical challenge. Here, we optimized the RL-QN15 peptide, which was shown to be a pro-healing drug candidate in our previous research, to obtain the cyclic heptapeptide (CyRL-QN15) with considerable therapeutic potency against skin wounds. Furthermore, a Zn2+-crosslinked sodium alginate (ZA) hydrogel containing hollow polydopamine (HPDA) nanoparticles loaded with CyRL-QN15 (HPDAlCyRL-QN15/ZA hydrogel) was prepared and characterized, which significantly enhanced the pro-healing potency of CyRL-QN15. At the cellular level, this nontoxic hydrogel accelerated the proliferation, migration, tube formation, and scratch healing of skin cells, regulated the secretion of cytokines from macrophages, directly scavenged free radicals, and decreased reactive oxygen species. Moreover, the HPDAlCyRL-QN15/ZA hydrogel significantly accelerated the healing of full-thickness skin wounds in type 2 diabetic mice by promoting the transition of macrophages to the M2 phenotype to reduce inflammation and cause re-epithelialization, formation of granulation tissue, deposition of collagen, and angiogenesis. Of note, the hydrogel also facilitated wound healing of diabetic patient skin cultured ex vivo. Overall, the HPDAlCyRL-QN15/ZA hydrogel presents a novel therapeutic strategy for clinical chronic skin wound (diabetic ulcer) healing. We optimized the RL-QN15 peptide to obtain the cyclic heptapeptide (CyRL-QN15) with excellent therapeutic potency against skin wounds. Then, a multifunctional HPDAlCyRL-QN15/ZA hydrogel was prepared and characterized, which significantly enhanced the pro-healing potency of CyRL-QN15. This non-toxic hydrogel accelerated the proliferation, migration, and tube formation of skin cells, regulated the secretion of cytokines, directly scavenged free radicals, and decreased reactive oxygen species. Moreover, the HPDAlCyRL-QN15/ZA hydrogel accelerated the healing of skin wounds in type 2 diabetic mice and diabetic patient skin cultured ex vivo by promoting the polarization of macrophages to reduce inflammation, re-epithelialization, deposition of collagen, and angiogenesis.


Introduction
Wound healing is a complex physiological process that maintains the structural integrity of the body and consists of hemostasis, inflammation, proliferation, and remodeling stages 1,2 . Wounds that fail to heal within a normal time frame are considered chronic wounds. Chronic wounds affect 0.2% to 1% of the population in developed countries, posing an increasing health and economic burden on society 3 . At present, chronic wound treatment lacks effective targeted therapies, focusing instead on optimizing controllable healing factors 4,5 . Therefore, exploring innovative intervention strategies to promote chronic skin wound healing remains essential.
Various novel interventions have been developed for chronic skin wounds, including the use of bioactive peptides, hydrogels, nanomaterials, and tissue engineering. In particular, bioactive peptides, hydrogel dressings, and nanomaterials have received considerable attention 6,7 . Several bioactive peptides derived from amphibian skin, such as OA-GL12, cathelicidin-OA1, cathelicidin-NV, and RL-QN15, have shown significant potential as novel prohealing agents in the treatment of skin wounds [8][9][10][11][12] . A variety of biomaterials (e.g., hydrogels, nanofibers, and films) have also been used in the treatment of chronic wounds 13 . Hydrogels are three-dimensional (3D) networks formed by cross-linking hydrophilic polymer chains, with properties similar to those of the extracellular matrix (ECM). They are considered ideal scaffolds for wound healing due to their ability to absorb wound exudates, maintain a moist environment, and promote fibroblast proliferation and keratinocyte migration 14,15 . Sodium alginate (SA) consists of different ratios of β-1,4-linked repeating units of D-mannuronic acid (M) and L-glutamine (G) 16 . The high L-glutamine (G) block content of alginate enables the formation of an insoluble gel network by building bridges in the polymer network with divalent cations such as Zn 2+17 . In addition, Zn 2+ is an essential element for cell proliferation and angiogenesis and has shown excellent results in chronic skin wound healing 18 . Nanomaterials have been widely used in wound repair due to their unique surface properties, physiological activities, adjustable porous structure, outstanding biocompatibility, and drug loading ability 19 . Hollow polydopamine (HPDA) nanoparticles exhibit excellent surface permeability, loadcarrying capacity, antioxidant activity, and controllable morphology, thus representing an ideal drug delivery system for chronic skin wound healing 20 . Therefore, incorporating peptides, nanomaterials, and hydrogels to create combination agents will lead to novel strategies for the treatment of chronic skin wounds. At present, however, relevant reports on skin wound healing remain scarce.
Optimization of existing bioactive peptides is an effective approach for developing novel agents, such as ziconotide, exenatide, bivalirudin, and captopril 21,22 . We previously identified a novel pro-healing peptide RL-QN15 from frog skin secretions, which contains intramolecular disulfide bonds without posttranslational modifications 23 . At low concentrations, RL-QN15 showed remarkable therapeutic potential in the healing of acute wounds, chronic wounds, skin fibrosis, oral ulcers, and full-thickness skin wounds in pigs 10 . We also developed a novel strategy to promote dermal wound healing by loading RL-QN15 into HPDA nanoparticles, which increased the pro-healing ability of the peptide 19 . However, further refinement of the RL-QN15 structure is important to reduce costs and increase activity and thus facilitate the development of novel pro-healing drugs.
In the current study, we optimized the structure of RL-QN15 and obtained a shorter cyclic heptapeptide (Cy RL-QN15 ) with excellent skin wound healing activity. Furthermore, we successfully prepared and characterized a Zn 2+ cross-linked SA hydrogel containing HPDA nanoparticles loaded with Cy RL-QN15 (HPDAlCy RL-QN15 /ZA hydrogel) for chronic skin wound healing. At the cellular level, this nontoxic hydrogel accelerated the proliferation, migration, tube formation, and scratch healing of skin cells, regulated the secretion of cytokines from macrophages, directly scavenged free radicals, and decreased reactive oxygen species (ROS). The HPDAlCy RL-QN15 /ZA hydrogel also showed excellent therapeutic effects on full-thickness diabetic skin wounds in mice and full-thickness ex vivo foot skin wounds from diabetic patients. This study presents a prospective HPDAlCy RL-QN15 /ZA hydrogel for chronic skin wound healing and emphasizes the potential of combined therapy based on peptides, nanomaterials, and hydrogels for the clinical treatment of chronic skin trauma.

Experimental section
Animal ethics statement and informed consent Male Kunming and C57BL/6 mice (20-24 g, 6-8 weeks old) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China). All animal care and handling procedures were approved by and followed the requirements of the Ethics Committee of Kunming Medical University (kmmu20220069).
All human skin samples were obtained with informed consent from diabetic patients undergoing amputation surgeries at the Department of Endocrinology, Affiliated Hospital of Yunnan University (Kunming, Yunnan, China). Skin collection was approved by the Ethics Committee of the Affiliated Hospital of Yunnan University (2021103). Informed consent confirmed that the patients voluntarily donated their skin for wound healing research with no financial payment. This research abides by the Declaration of Helsinki principles.

Synthesis and stability of peptides
The RL-QN15 peptide, reduced linear peptide of RL-QN15 without disulfide bonds (Re RL-QN15 ), linear octapeptide in front of RL-QN15 disulfide bonds (Li RL-QN15 ), and cyclic heptapeptide composed of an RL-QN15 disulfide-bonded circular structure (Cy RL-QN15 ) (purity > 95%) were commercially synthesized by Bioyeargene Biotechnology Co., Ltd. (Wuhan, China). The structure of Cy RL-QN15 was predicted using PEP-FOLD3 online service 9 . The stability of RL-QN15 and Cy RL-QN15 was evaluated according to previous research 24 .

Keratinocyte scratch healing assay
The pro-healing effects of Cy RL-QN15 on HaCaT cells were evaluated according to a previous study 25 . Specific experimental methods are detailed in section S2.2 of the Supplementary Information.

Effects of RL-QN15-modified peptides on full-thickness skin wounds in mice
The healing effects of Cy RL-QN15 on full-thickness skin wounds in mice were examined according to a previous study 10  The loading efficiency of HPDA and hydrogel against Cy RL-QN15 and the release efficiency of the HPDAlCy RL-QN15 /ZA hydrogel against Cy RL-QN15 were determined according a previous study 19 .
Biocompatibility and degradation of the HPDAlCy RL-QN15 / ZA hydrogel The toxicity of the HPDAlCy RL-QN15 /ZA hydrogel was evaluated in C57BL/6 mice with full-thickness skin wounds and HaCaT cells using the live/dead cell viability assay according to a previous study 19 . The degradation of ZA, HPDA/ZA, Cy RL-QN15 /ZA, and HPDAlCy RL-QN15 /ZA in vitro and in vivo was determined according to previous studies 26,27 . Specific experimental methods are detailed in section S2.5 of the Supplementary Information.

In vitro HUVEC migration and tube formation assays
Angiogenesis experiments were performed in vitro according to a previous study 28 . Specific experimental methods are detailed in section S2.7 of the Supplementary Information.
Antioxidant activity of the HPDAlCy RL-QN15 /ZA hydrogel The antioxidant activity of the HPDAlCy RL-QN15 /ZA hydrogel was evaluated based on free radical scavenging ability and reduction of intracellular reactive oxygen species (ROS). Specific experimental methods are detailed in Section S2.8 of the Supplementary Information.

Effects of samples on chronic diabetic skin wounds in mice
To evaluate the wound healing effects of the HPDAl-Cy RL-QN15 /ZA hydrogel, chronic full-thickness skin wounds in diabetic mice were established according to an earlier study 10 . Specific experimental methods are detailed in Section S2.9 of the Supplementary Information.

Effects of the HPDAlCy RL-QN15 /ZA hydrogel on cytokine secretion in skin wounds
Specimens were acquired from the central area of fullthickness skin wounds in diabetic mice on Days 3, 7, and 14 postoperation, homogenized in ice-cold saline (weight/ volume = 1:9) at 4°C and centrifuged at 12000 × g for 20 min at 4°C to collect the supernatant. The levels of TNF-α and TGF-β1 were detected using ELISA kits (NeoBioscience, Shanghai, China).

Human ex vivo diabetic skin wound model
The pro-tissue regenerative activity of the HPDAl-Cy RL-QN15 /ZA hydrogel was examined using a modified human skin wound healing assay [30][31][32] . Specific experimental methods are detailed in section S2.11 of the Supplementary Information.

Results and discussion
The RL-QN15-optimized cyclic heptapeptide Cy RL-QN15 promoted keratinocyte scratch repair and full-thickness skin wound healing in mice Structural optimization of existing bioactive peptides is important for the development of novel drugs. We previously revealed that the bioactive peptide RL-QN15 exhibits considerable therapeutic effects on skin wounds 10 . As shown in Fig. 1A, RL-QN15 consists of 15 amino acid residues and contains a pair of intramolecular disulfide bonds. Disulfide bonds are critical to the function of amphibian-derived bioactive peptides 10,33,34 . To optimize RL-QN15 and obtain peptides with shorter amino acid sequences and stronger activities, we synthesized Re RL-QN15 , Li RL-QN15 , and Cy RL-QN15 (Fig. 1A). As shown in Fig. 1B, Cy RL-QN15 contains seven amino acids and links two-terminal cysteine residues to form a closed-loop structure. The predicted structure of Cy RL-QN15 also demonstrated a closed-loop structure ( Fig. 1C).
The therapeutic effects of Cy RL-QN15 on full-thickness skin wounds in mice were explored (Fig. 1F). At a concentration of 1 nM, the wound healing rates of RL-QN15 and Cy RL-QN15 conditions were 91.8% and 94.0%, respectively, higher than those of the positive control rh-bFGF (87.7%) and the Re RL-QN15 (60.0%) and Li RL-QN15 (63.1%) conditions (Fig. 1G). Peptide stability is critical in skin wound treatment, and therefore, we examined the stability of Cy RL-QN15 in plasma. The halflife of Cy RL-QN15 in plasma was 8.07 h, longer than that Fig. 1 The cyclic heptapeptide Cy RL-QN15 showed pro-healing therapeutic potential. A Amino acid sequences of RL-QN15, Re RL-QN15 , Li RL-QN15 , and Cy RL-QN15 . B Chemical structure of Cy RL-QN15 . C Predicted structure of Cy RL-QN15 . D, E Representative and quantitative plots of the effects of RL-QN15, Re RL-QN15 , Li RL-QN15 , and Cy RL-QN15 on scratch repair of HaCaT cells. Data represent the mean ± standard deviation (SD), generated from three independent experiments performed in triplicate. ** indicates P < 0.01. F, G Representative and quantitative plots of the effects of RL-QN15, Re RL-QN15 , Li RL-QN15 , and Cy RL-QN15 on wound healing in Kunming mice with full-thickness skin wounds in vivo. Data represent the mean ± SD, n = 15. * and ** indicate P < 0.05 and P < 0.01, respectively. of RL-QN15 (7.79 h), indicating greater stability (Fig.  S1A, B). Thus, Cy RL-QN15 exhibited pro-trauma repair activity comparable to that of RL-QN15 but had better stability and lower synthesis costs than RL-QN15, suggesting that it may be a better pro-healing drug candidate.
Characterization and properties of the HPDAlCy RL-QN15 /ZA hydrogel In the current study, we prepared HPDAlCy RL-QN15 /ZA hydrogel containing HPDA nanoparticles loaded with Cy RL-QN15 for skin wound healing. As shown in Fig. S2, Zn 2+ promoted the formation of ZA and HPDAlCy RL-QN15 /ZA hydrogels with greater cross-linking than the SA hydrogel. Transmission electron microscopy (TEM) revealed that the HPDA nanoparticles loaded with Cy RL-QN15 had an average grain size of approximately 50 nm, with a spherical morphology and hollow structure, indicating excellent loading capacity (Fig. S3A, B). The morphology and structure of ZA and HPDAlCy RL-QN15 / ZA hydrogels were characterized by scanning electron microscopy (SEM), showing a 3D network of microporous structures, which form the basis of the moisture retention and swelling of gels ( Fig. 2A, B). Due to the ability of HPDAlCy RL-QN15 to interact with the internal molecules of the ZA hydrogel, the HPDAlCy RL-QN15 /ZA hydrogel showed an increased cross-linking density and a denser surface, with a much lower porosity (48.52 ± 0.40%) compared to the ZA hydrogel (56.78 ± 0.10%) (Fig. 2C).
The four Fourier transform infrared (FTIR) spectral curves of the crystal structures of SA, ZA, HPDAlCy RL-QN15 , and HPDAlCy RL-QN15 /ZA were shown in Fig. 2D. The peak at 3384 cm −1 in the SA curve corresponded to the stretching vibration of O-H, which shifted to 3380 cm −1 and 3344 cm −1 with the formation of ZA and HPDAl-Cy RL-QN15 /ZA hydrogels, respectively. The shift in the HPDAlCy RL-QN15 /ZA hydrogel toward lower stretching energy may be related to the presence of imine, amine, catechol, and other groups in HPDA that cause stretching vibrations of the -OH group (Fig. 2D). In addition, the characteristic peak of the -COO stretching vibration at 1610 cm −1 in SA shifted to 1607 and 1599 cm −1 in the ZA and HPDAlCy RL-QN15 /ZA hydrogels, respectively (Fig. 2D). After HPDAlCy RL-QN15 was embedded in the HPDAl-Cy RL-QN15 /ZA hydrogel, the characteristic peak of 1220 cm −1 for HPDAlCy RL-QN15 shifted to 1226 cm −1 due to the intermolecular interaction between Cy RL-QN15 and alginate, while the SA and ZA hydrogels remained at 1215 cm −1 , indicating successful introduction of Cy RL-QN15 into the HPDAlCy RL-QN15 /ZA hydrogel (Fig. 2D). The X-ray photoelectron spectroscopy (XPS) spectra of SA, ZA, HPDAlCy RL-QN15 , and HPDAlCy RL-QN15 /ZA were shown in Fig. 2E. Notably, Na1s, Zn2p, O1s, N1s, C1s, and S2p signal peaks were detected in the HPPDAlCy RL-QN15 / ZA hydrogel, indicating the presence of SA, Zn 2+ , and cyclic peptides with disulfide bonds in the composite. Thus, based on these results, the multifunctional HPDAlCy RL-QN15 /ZA hydrogel containing HPDA, Cy RL-QN15 , and Zn 2+ was successfully prepared.
A decreased cross-linking density of the hydrogel results in higher swelling properties 35 . As seen in Fig. 2F, the ZA hydrogel showed excellent swelling properties, while the HPDAlCy RL-QN15 /ZA hydrogel showed a lower swelling ratio. The lower swelling ratio of the HPDAlCy RL-QN15 / ZA hydrogel may be related to HPDAlCy RL-QN15 interacting with the internal molecules of the ZA hydrogel and increasing its cross-linking density, consistent with the SEM and porosity results. The mechanical properties of hydrogels applied to wounds or tissue engineering are crucial 36 ; thus, we determined the rheological and compression properties of the hydrogels. The rheological properties of storage modulus (G') and loss modulus (G") of ZA, HPDA/ZA, Cy RL-QN15 /ZA, and HPDAlCy RL-QN15 / ZA hydrogels were measured in the frequency range of 0-10 Hz at 37°C. The results showed that G' was greater than G" for all groups, with the HPDAlCy RL-QN15 /ZA hydrogel showing the most significant difference, indicating greater flexibility (Fig. S4A). The G' and G" values of the HPDAlCy RL-QN15 /ZA hydrogel did not change with increasing oscillation frequency, indicating that the HPDAlCy RL-QN15 /ZA hydrogel has excellent stability (Fig.  S4A). The compression properties of the HPDAlCy RL-QN15 / ZA hydrogel were enhanced by the cross-linking of Zn 2+ and the internal molecular interactions between HPDAlCy RL-QN15 and ZA, demonstrating higher compressive strength (more than 250 kPa to 80% strain) than the ZA, HPDA/ZA, and Cy RL-QN15 /ZA hydrogels (Fig. S4B).
The loading of Cy RL-QN15 and its slow-release from Cy RL-QN15 /ZA, HPDAlCy RL-QN15 , and HPDAlCy RL-QN15 / ZA were evaluated. As shown in Fig. 2G, the loading efficiencies of HPDAlCy RL-QN15 /ZA, HPDAlCy RL-QN15 , and Cy RL-QN15 /ZA against Cy RL-QN15 were 44.61, 50.62, and 87.86%, respectively. When dispersed in PBS, HPDAlCy RL-QN15 , Cy RL-QN15 /ZA, and HPDAlCy RL-QN15 / ZA released Cy RL-QN15 into the solvent in a sustained manner (Fig. 2H). As shown in Fig. 2H, the efficiency of Cy RL-QN15 release from Cy RL-QN15 /ZA, HPDAlCy RL-QN15 , and HPDAlCy RL-QN15 /ZA sequentially decreased, with release rates of >50% at 24 h, reaching 93.51, 79.89, and 73.48% at 48 h, respectively. The sustained-release of Cy RL-QN15 by the HPDAlCy RL-QN15 /ZA hydrogel prolonged the effects of the peptide, and the release rates of Cy RL-QN15 peaked at 48 h, thus exerting effects on the inflammatory and proliferative phases of wound repair. In summary, we successfully prepared the HPDAlCy RL-QN15 / ZA hydrogel with excellent mechanical properties and Cy RL-QN15 loading and slow-release properties.
Biocompatibility and degradation of the HPDAlCy RL-QN15 / ZA hydrogel Biocompatibility is an important property regarding the potential safety of novel biomaterials 37 . As shown in Fig.  S5A, nearly all HaCaT cells were stained with calcein-AM ester (green fluorescence), with very few dead cells stained with PI (red fluorescence), indicating that ZA, HPDA, Cy RL-QN15 , HPDA/ZA, Cy RL-QN15 /ZA, HPDAlCy RL-QN15 , and HPDAlCy RL-QN15 /ZA exhibited no toxicity toward HaCaT cells. The C57BL/6 mouse dorsal skin wound toxicity assay also revealed that ZA, HPDA, Cy RL-QN15 , HPDA/ZA, Cy RL-QN15 /ZA, HPDAlCy RL-QN15 , and HPDAlCy RL-QN15 /ZA caused no mortality in mice, and histological sections of major organs (heart, liver, spleen, lung, and kidney) from treated mice showed no abnormalities (Fig. S5B). The degradation of hydrogels in vivo is also crucial for their application in skin wound healing 38 . Thus, we evaluated the degradation of hydrogels in vitro and in vivo (Fig. S6). The results showed that the hydrogels gradually decreased in size with increasing incubation time under hyaluronidase and collagenase treatment; after 120 h of treatment, the remaining amounts of ZA, HPDA/ZA, Cy RL-QN15 /ZA, and HPDAl-Cy RL-QN15 /ZA hydrogels were 19.8, 23.2, 23.4, and 29.8%, respectively (Fig. S6A). The ZA, HPDA/ZA, Cy RL-QN15 / ZA, and HPDAlCy RL-QN15 /ZA hydrogels also showed great degradation efficacy when injected subcutaneously into the abdomens of mice, with all hydrogels fully degrading two weeks after injection (Fig. S6B). In conclusion, the HPDAlCy RL-QN15 /ZA hydrogel showed negligible cytotoxicity, excellent biocompatibility, and excellent degradation properties, providing a foundation for its pro-healing therapeutic potential in skin wounds.
The HPDAlCy RL-QN15 /ZA hydrogel regulated the release of cytokines from macrophages, promoted angiogenesis, and exerted antioxidant activities Macrophages play a pivotal role in wound healing, particularly in the secretion of cytokines and inflammatory factors that regulate the wound repair process, thereby facilitating skin wound healing 39 . Cy RL-QN15 significantly decreased the expression of TNF-α induced by LPS but promoted the expression of TGF-β1. More importantly, the HPDAlCy RL-QN15 and HPDAlCy RL-QN15 / ZA enhanced the regulatory activity of Cy RL-QN15 on cytokine release from macrophages (Fig. 4A, B). The HPDAlCy RL-QN15 /ZA hydrogel exerted the best effects, reducing the expression of TNF-α from 850.89 ± 75.25 pg/ mL (LPS) to 674.83 ± 34.48 pg/mL while promoting the expression of TGF-β1 from 99.45 ± 20.96 pg/mL (vehicle) to 315.50 ± 63.50 pg/mL (Fig. 4A, B).
The HPDAlCy RL-QN15 /ZA hydrogel promoted full-thickness wound healing, epidermal regeneration, and collagen deposition in diabetic mice The application of multifunctional hydrogel dressings with anti-inflammatory, antioxidant, and angiogenic activities provides a new strategy for skin wound treatment, especially chronic wound healing 42 . With its superior biocompatibility, the multifunctional HPDAlCy RL-QN15 /ZA hydrogel showed excellent potential for the treatment of chronic skin wounds (Figs. 3, 4, S5, and S7). Thus, we further investigated its prohealing activity on diabetic skin wounds in mice. Wounds in type 2 diabetic mice were treated with the different compounds and then monitored at different times to determine the change in wound area (Fig. 5A). The results showed that the repairing effect of Cy RL-QN15 on wounds was significantly better than that of the vehicle or commercial dressing (Tegaderm), and the HPDAlCy RL-QN15 /ZA hydrogel significantly enhanced the skin regenerative effects of Cy RL-QN15 . Notably, after 14 days of treatment, the HPDAl-Cy RL-QN15 /ZA hydrogel-treated wounds were almost completely healed without obvious scarring, while the Cy RL-QN15and HPDAlCy RL-QN15 -treated wounds were mostly healed with a small amount of scabbing. In contrast, the Tegadermand vehicle-treated wounds were not healed and showed considerable scarring. On Day 14, compared with the vehicle (66.38 ± 3.41%), the skin tissue repair rates of the Cy RL-QN15 , HPDAlCy RL-QN15 , and HPDAlCy RL-QN15 /ZA were 88.74 ± 2.14, 92.64 ± 2.72, and 95.23 ± 4.49%, respectively, demonstrating that the HPDAlCy RL-QN15 /ZA hydrogel exhibited the highest pro-regeneration effects on diabetic skin wounds in mice (Fig. 5B).
H&E staining was performed to evaluate skin wound healing and regeneration on Days 3, 7, and 14 ( Fig. 5C-F). From Days 3 to 14, wounds treated with the HPDAlCy RL-QN15 /ZA hydrogel exhibited the best regeneration of epidermal integrity and thickness. On Day 14, Cy RL-QN15 treatment showed superior neo-epidermal (173 ± 6.23 μm) and granulation tissue thickness (576.33 ± 9.80 μm) in the wound compared to the vehicle and Tegaderm groups. Notably, compared to treatment with Cy RL-QN15 , wounds treated with the HPDAlCy RL-QN15 / ZA hydrogel exhibited better therapeutic efficacy, in which neo-epidermal and granulation tissue thicknesses reached 198 ± 5.09 μm and 669 ± 8.64 μm, respectively (Fig. 5D, E). As shown in Fig. S8A, C, compared with the vehicle group (99.95 ± 26.97), immunofluorescence staining of Ki67 in injured tissue on postoperative Day 14 also indicated that the HPDAlCy RL-QN15 /ZA hydrogel (279.52 ± 25.60) significantly enhanced the expression of the epidermal cell proliferation marker Ki67.
Masson trichrome and PAS staining showed significantly greater collagen deposition and basement membrane completion in the regenerated tissue with HPDAlCy RL-QN15 /ZA hydrogel treatment (Fig. 5C). As seen in Fig. S8B, D, E, the HPDAlCy RL-QN15 /ZA hydrogeltreated wounds showed significantly higher positive staining intensity for collagen types I and III (COL I and COL III, respectively) compared with the other treatment groups, consistent with its promotion of fibroblast proliferation and collagen deposition. These findings indicated that the HPDAlCy RL-QN15 /ZA hydrogel enhanced the pro-healing effects of Cy RL-QN15 in chronic skin wounds, resulting in shortened healing time, thicker neoepidermis, superior granulation tissue formation, and greater collagen deposition.
The HPDAlCy RL-QN15 /ZA hydrogel promoted macrophage polarization (M1 to M2) to reduce inflammation and angiogenesis Markers of the macrophage M1 (F4/80/INOS) and M2 phenotypes (F4/80/ARG) were stained with immunofluorescence to evaluate the effects of the HPDAlCy RL-QN15 / ZA hydrogel on macrophage polarization (Fig. 6A, B). Immunofluorescence showed that the positive rate of F4/ 80/INOS (INOS + /F4/80 + ) in the vehicle, Tegaderm, Fig. 5 The HPDAlCy RL-QN15 /ZA hydrogel promoted the repair and tissue regeneration of full-thickness skin wounds in type 2 diabetic mice. A Representative images of wounds in each group during the healing process and schematic of the wound healing process in the five groups. B Quantitative data of wound repair rates at different time points in the five groups. C Representative H&E staining images of wound specimens from different groups on Days 3, 7, and 14. Masson trichrome and PAS staining on Day 14. Es eschar, NE newborn epithelium, GT granulation tissue. Scale bar: 500 µm. D-F Quantification of regenerated epidermis thickness, granulation tissue thickness, and collagen deposition in wounds. All data represent the mean ± SD generated from three independent experiments performed in triplicate. *, **, and *** indicate P < 0.05, P < 0.01, and P < 0.001, respectively.  Cy RL-QN15 group and 27.81 ± 6.22 in the HPDAlCy RL-QN15 / ZA hydrogel group (Fig. 6D). On Days 3 and 7, the positive rate of ARG/F4/80 (ARG + /F4/80 + ) showed the opposite trend to INOS + /F4/80 + in Cy RL-QN15 , HPDAlCy RL-QN15 , and HPDAlCy RL-QN15 /ZA, with the greatest increase being seen in the HPDAlCy RL-QN15 /ZA hydrogel (from 24.97 ± 8.02 to 308.26 ± 40.72) (Fig. 6E), which indicated the greatest increase in M2 phenotype macrophages in the wound tissue (Fig. 6E, F). These results suggested that Cy RL-QN15 regulated macrophage polarization and the HPDAl-Cy RL-QN15 /ZA hydrogel significantly enhanced Cy RL-QN15 activity.
As shown in Fig. S9A, B, the expression level of TNF-α decreased after Cy RL-QN15 , HPDAlCy RL-QN15 , and HPDAlCy RL-QN15 /ZA treatment, whereas the expression level of TGF-β1 showed the opposite pattern, consistent with the in vitro results. After 7 days of treatment with Cy RL-QN15 , HPDAlCy RL-QN15 , and HPDAlCy RL-QN15 /ZA, the expression levels of IL-1β decreased, while the expression levels of IL-10 increased (Fig. S9C-E). Thus, Cy RL-QN15 promoted macrophage polarization to the M2 phenotype to reduce inflammation, while the HPDAl-Cy RL-QN15 /ZA hydrogel enhanced the regulatory activity of Cy RL-QN15 .
As M2 macrophages and Zn 2+ can promote angiogenesis 18,43 and both Cy RL-QN15 and HPDAlCy RL-QN15 / ZA promoted the transformation of macrophages to M2, their effects on angiogenesis were explored by immunofluorescence analysis of VEGF, CD31, and α-SMA in skin wounds. VEGF can promote endothelial cell migration and angiogenesis, CD31 and α-SMA are common markers of angiogenesis, and α-SMA also promotes wound contraction 44 . Compared with the vehicle, the positive staining intensities of VEGF, α-SMA, and CD31 were enhanced after treatment with Cy RL-QN15 , HPDAlCy RL-QN15 , and HPDAlCy RL-QN15 /ZA, indicating promotion of angiogenic activity (Figs. 7A-D, S10A, B). Cy RL-QN15 enhanced the expression of CD31, and HPDAlCy RL-QN15 markedly enhanced the expression levels of VEGF, α-SMA, and CD31 (Figs. 7C, D, S10B). Notably, the HPDAlCy RL-QN15 /ZA hydrogel showed the greatest promotion of angiogenesis, increasing the expression levels of VEGF, α-SMA, and CD31 by 3.1-, 2.6-, and 3.3-fold, respectively, compared to the vehicle (Figs. 7C, D, S10B). Cy RL-QN15 significantly promoted angiogenesis in diabetic mouse skin wounds. The HPDAlCy RL-QN15 and HPDAlCy RL-QN15 /ZA enhanced Cy RL-QN15 activity, with the HPDAlCy RL-QN15 / ZA hydrogel exhibiting the strongest angiogenic activity due to the combination of Zn 2+ and Cy RL-QN15 . In summary, the HPDAlCy RL-QN15 /ZA hydrogel markedly enhanced Cy RL-QN15 peptide activity, suppressed the inflammatory response by stimulating macrophage polarization from the M1 to M2 phenotype, and promoted Ki67, VEGF, CD31, α-SMA, COL I, and COL III expression to accelerate epithelialization and facilitate blood vessel regeneration, collagen deposition, and granulation tissue regeneration. Thus, this hydrogel exhibits excellent therapeutic potential in the healing of chronic diabetic skin wounds.
The HPDAlCy RL-QN15 /ZA hydrogel boosted skin wound repair in cultured ex vivo diabetic patient skin The HPDAlCy RL-QN15 /ZA hydrogel exhibited similar antiinflammatory and angiogenesis-promoting properties as the multifunctional GelMA/AA/Cu and PC/GO/Met hydrogels, showing excellent therapeutic effects on chronic diabetic wounds at the animal level 45,46 . In addition, the HPDAlCy RL-QN15 /ZA hydrogel also exhibited free radical scavenging and oxidative stress reducing activities, which are essential for the recovery of cellular function in diabetic wound areas (Fig. S7). To better explore the therapeutic effects of the HPDAlCy RL-QN15 /ZA hydrogel on skin wounds in diabetic patients, an ex vivo diabetic skin wound healing model was established (Fig. 8A, B). PAS staining showed that the skin in all treatment groups maintained a normal structure, with no epidermal detachment and no significant changes at the dermal-epidermal junction, indicating successful construction of the model (Fig. 8C). Compared with the vehicle (0.56 ± 0.07 mm and 0.29 ± 0.05 mm), the HPDAlCy RL-QN15 / ZA hydrogel significantly stimulated re-epithelialization, with a markedly thicker epidermis (0.77 ± 0.07 mm) and epidermal migration (0.5 ± 0.05 mm) from the edge to the center of the wound (Fig. 8D, E). The level of collagen deposition in granulation tissue is a principal marker of wound healing 47 . As shown in Fig. 8F, collagen deposition in the HPDAlCy RL-QN15 /ZA hydrogel group (81.62 ± 7.90%) was significantly higher than that in the HPDAlCy RL-QN15 (70.21 ± 5.71%), Cy RL-QN15 (65.86 ± 4.38%), and vehicle groups (58.15 ± 5.00%).
The ELISA results showed that the expression level of TNF-α in the in vitro diabetic skin wounds was significantly lower in the Cy RL-QN15 , HPDAlCy RL-QN15 , and HPDAlCy RL-QN15 /ZA groups than in the vehicle group, while the expression level of TGF-β1 was significantly higher after HPDAlCy RL-QN15 and especially HPDAl-Cy RL-QN15 /ZA treatment (Fig. 8G, H). Immunofluorescence staining of the vascular regenerationrelated markers CD31 and α-SMA was performed to detect angiogenesis in ex vivo foot skin wounds of diabetic patients (Fig. S11). As shown in Fig. S11B, D, α-SMA expression in the Cy RL-QN15 , HPDAlCy RL-QN15 , and HPDAlCy RL-QN15 /ZA groups differed insignificantly from that in the vehicle (PBS) group. In contrast, the expression levels of CD31 in the HPDAlCy RL-QN15 and HPDAlCy RL-QN15 /ZA groups were 169.60 ± 9.63 and 170.10 ± 10.92%, respectively, significantly higher than that of Cy RL-QN15 (150.32 ± 12.80%) (Fig. S11A, C).
These results demonstrated that the HPDAlCy RL-QN15 / ZA hydrogel inhibited inflammation, promoted re-epithelialization, and accelerated angiogenesis, thus exhibiting excellent therapeutic effects on ex vivo diabetic patient skin wounds and providing a new strategy and candidate for the treatment of chronic wounds. Fig. 7 Effect of the HPDAlCy RL-QN15 /ZA hydrogel on angiogenesis. A, B Representative images of immunofluorescence staining of VEGF and α-SMA in diabetic mice on Day 14 after treatment. Scale bar: 50 µm. C, D Quantitative data of VEGF and α-SMA in wounds, respectively. Data represent the mean ± SD, generated from three independent experiments performed in triplicate. * and ** indicate P < 0.05 and P < 0.01, respectively.