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

Wound healing is a complex physiological process that maintains the structural integrity of the body and consists of hemostasis, inflammation, proliferation, and remodeling stages. 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. At present, chronic wound treatment lacks effective targeted therapies, focusing instead on optimizing controllable healing factors. 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. Several bioactive peptides derived from amphibian skin, such as AO-GL12, cathelicidin-OA1, cathelicidin-NV, and RL-QN15, have shown significant potential as novel pro-healing agents in the treatment of skin wounds. A variety of biomaterials (e.g., hydrogels, nanofibers, and films) have also been used in the treatment of chronic wounds. Hydrogels are three-dimensional networks formed by cross-linking hydrophilic polymer chains, with properties similar to those of the extracellular matrix. 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. Sodium alginate consists of different ratios of β-1,4-linked repeating units of D-mannuronic acid (M) and L-glutamine (G). 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 Zn²⁺. In addition, Zn²⁺ is an essential element for cell proliferation and angiogenesis and has shown excellent results in chronic skin wound healing. 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. Hollow polydopamine nanoparticles exhibit excellent surface permeability, load-carrying capacity, antioxidant activity, and controllable morphology, thus representing an ideal drug delivery system for chronic skin wounds. Therefore, incorporating HPDA nanoparticles into hydrogels can improve the treatment of chronic skin wounds. At present, relevant reports on skin wound healing are scarce. Optimization of existing bioactive agents previously identified a novel pro-healing peptide RL-QN15 from skin secretions, which contains intramolecular disulfide bonds without posttranslational modifications and shows therapeutic potential in multiple wound models. Loading RL-QN15 into HPDA nanoparticles increased its pro-healing ability. However, further refining of the RL-QN15 structure is important to reduce costs and increase activity. In the current study, we optimized the structure of RL-QN15 and obtained a shorter cyclic heptapeptide (Cyl-QN15/CYRL-QN15) with excellent skin wound healing activity. We prepared and characterized a Zn²⁺ cross-linked sodium alginate hydrogel containing HPDA nanoparticles loaded with Cyl-QN15 (HPDA/Cyl-QN15/ZA hydrogel) for chronic skin wound healing. At the cellular level, this nontoxic hydrogel accelerated proliferation, migration, tube formation, and scratch healing of skin cells, regulated cytokine secretion from macrophages, directly scavenged free radicals, and decreased reactive oxygen species. The hydrogel 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 emphasizes the potential of combining peptides, nanomaterials, and hydrogels for clinical treatment of chronic skin trauma.

The study situates its approach within prior work demonstrating that bioactive peptides (e.g., AO-GL12, cathelicidin-OA1, cathelicidin-NV, RL-QN15) from amphibian skin possess strong pro-healing properties for skin wounds. Hydrogels are highlighted as effective wound dressings due to ECM-like properties, moisture retention, exudate absorption, and support for fibroblast and keratinocyte activity. Sodium alginate can be crosslinked by divalent cations (notably Zn²⁺), where Zn²⁺ is also biologically beneficial for cell proliferation and angiogenesis and has shown efficacy in chronic wound healing. Nanomaterials, particularly hollow polydopamine (HPDA) nanoparticles, offer high drug-loading capacity, antioxidant activity, and controlled morphology, making them suitable for wound repair applications. Prior optimization and nanocarrier loading of RL-QN15 improved its pro-healing performance, motivating further structural refinement and integration into a hydrogel platform.

Ethics: Animal procedures were approved by the Ethics Committee of Kunming Medical University (kmmu202200269). Human diabetic skin samples were collected with informed consent and institutional approval in accordance with the Declaration of Helsinki. Peptide synthesis and stability: RL-QN15, a reduced linear form (ReL-QN15), a linear octapeptide segment (LrL-QN15), and a cyclic heptapeptide (Cyl/CYRL-QN15) were synthesized at 95% purity. The CYRL-QN15 structure was predicted using PEP-FOLD3. Stability of RL-QN15 and CYRL-QN15 was assessed per prior protocols. Cell culture: HaCaT keratinocytes, human skin fibroblasts (HSFs), HUVECs, and RAW 264.7 macrophages were cultured in DMEM with glucose, 10% FBS, and antibiotics at 37 °C, 5% CO₂. In vitro assays: Keratinocyte scratch healing assay evaluated pro-healing effects (methods in Supplementary S2.2). HUVEC migration and tube formation assays modeled angiogenesis (S2.7). Cell proliferation was measured by MTS assay (S2.6). Cytokine secretion (TNF-α, TGF-β1) from LPS-stimulated RAW 264.7 cells treated with vehicle, CYRL-QN15, HPDA/CYRL-QN15, or HPDA/CYRL-QN15/ZA was quantified by ELISA. Antioxidant activity was tested via free radical scavenging and intracellular ROS reduction (S2.8). Live/dead assays assessed cytotoxicity in HaCaT cells and in vivo; hydrogel degradation was measured in vitro and in vivo (S2.5). Hydrogel preparation and characterization: A Zn²⁺-crosslinked sodium alginate (ZA) hydrogel containing HPDA nanoparticles loaded with CYRL-QN15 (HPDA/CYRL-QN15/ZA) was prepared and characterized (S2.4). Loading efficiency of HPDA and hydrogel for CYRL-QN15 and release profiles were determined per previous methods. In vivo wound models: Full-thickness skin wounds in mice were created to assess peptide efficacy and hydrogel performance. Chronic full-thickness wounds were induced in type 2 diabetic mice to evaluate healing with HPDA/CYRL-QN15/ZA (S2.9). Cytokine levels (TNF-α, TGF-β1) in wound tissue were measured on days 3, 7, and 14 by ELISA. Histology and immunostaining: Wound tissues underwent H&E, Masson trichrome, PAS staining, and immunohistochemistry/immunofluorescence to assess macrophage polarization (F4/80, iNOS, ARG), keratinocyte proliferation (Ki67), collagen deposition (COL I, COL III), angiogenesis (VEGF, α-SMA, CD31), and inflammatory cytokines (IL-1β, IL-10) (S2.10). Human model: A modified human ex vivo diabetic skin wound healing assay was used to evaluate pro-regenerative activity of the hydrogel (S2.11).

- Structural optimization of RL-QN15 yielded a cyclic heptapeptide (CYR-L/CYRL-QN15) maintaining a closed-loop structure and strong pro-healing activity. - In HaCaT scratch assays at 1 nM, cell scratch repair rates were: RE-RL-QN15 55.1%, LIRL-QN15 60.8%, RL-QN15 81.8%, and CYR-L-QN15 90.5%. - In a mouse full-thickness wound model at 1 nM, wound healing rates were: RL-QN15 91.8%, CYR-L-QN15 94.0%, rh-bFGF (positive control) 87.6%, RE-RL-QN15 60.0%, LIRL-QN15 63.1%. - CYR-L-QN15 demonstrated improved plasma stability with a half-life of 0.87 h. - A Zn²⁺-crosslinked sodium alginate hydrogel incorporating HPDA nanoparticles loaded with CYRL-QN15 (HPDA/CYRL-QN15/ZA) was successfully prepared; it significantly enhanced the peptide’s pro-healing potency. - Cellular effects of the hydrogel included accelerated proliferation, migration, tube formation, and scratch healing of skin cells; modulation of macrophage cytokine secretion (elevated TGF-β1, reduced TNF-α as implied by ELISA assays); direct free radical scavenging and reduction of intracellular ROS. - In type 2 diabetic mice with chronic full-thickness wounds, the hydrogel accelerated healing by promoting macrophage polarization toward the reparative M2 phenotype, facilitating resolution of inflammation, enhancing re-epithelialization, granulation tissue formation, collagen deposition, and angiogenesis. - The hydrogel also facilitated healing in a diabetic skin ulcer context (including a human ex vivo diabetic skin wound model).

The study addresses the need for effective targeted therapies for chronic skin wounds by optimizing a known bioactive peptide (RL-QN15) into a shorter cyclic heptapeptide (CYR-L/CYRL-QN15) that retains or improves pro-healing activity and shows better stability. Embedding this peptide within an HPDA nanoparticle-loaded, Zn²⁺-crosslinked alginate hydrogel provided a multifunctional wound dressing capable of sustained peptide delivery, antioxidation, and immunomodulation. The hydrogel’s biological effects—enhanced keratinocyte and endothelial activities, reduced oxidative stress, and a shift of macrophages toward an M2 phenotype—translate into accelerated wound closure and improved tissue regeneration in diabetic mouse wounds and pro-regenerative effects in human diabetic skin ex vivo. These findings support the therapeutic relevance of combining peptides, nanomaterials, and hydrogels to overcome impaired healing in chronic diabetic ulcers.

Optimizing RL-QN15 to a cyclic heptapeptide (CYRL-QN15) and integrating it into an HPDA nanoparticle-loaded Zn²⁺-alginate hydrogel created a nontoxic, multifunctional dressing that promotes cellular activities essential for repair, modulates inflammation, reduces oxidative stress, and accelerates healing in diabetic wound models. Overall, the HPDA/CYRL-QN15/ZA hydrogel represents a promising therapeutic strategy for chronic skin wound (diabetic ulcer) healing and highlights the potential of combining peptides, nanomaterials, and hydrogels for clinical treatment of chronic skin trauma.