| Objective:Non-healing chronic diabetic skin wound,a major complication of diabetes,have caused serious physical problems to patients and imposed an economic burden on the social healthcare system.Active peptide RL-QN15 showed remarkable therapeutic potential in the healing of acute wounds,chronic wounds,skin fibrosis,and oral ulcers,as well as full-thickness skin wounds in porcine.In addition,an external modification strategy was used to combine RL-QN15 with materials to prepare novel wound healing agents,which showed excellent therapeutic efficacy and significantly enhanced the skin pro-healing activity of RL-QN15.Importantly,excavating the active region and optimizing the structure of RL-QN15 can not only increase its biological activity and reduce synthesis costs,but also provide new insights and strategies for exploring the functional region of RL-QN15 and developing peptide drugs.Our study is devoted to explore the active sites of the pro-healing peptide RL-QN15 and optimize its structure.Exploring the functions of optimized peptide and preparing novel multifunctional wound formulations by external modification will provide new insights into the development of peptide-derived drugs and therapeutic strategies for chronic skin wounds.Methods:Here,the pro-healing peptide RL-QN15 was optimized,and the modified peptides with different sequence structures of RL-QN15(Li RL-QN15,Re RL-QN15and Cy RL-QN15)were chemically synthesized to explore the active regions of RL-QN15.The pro-wound repair activities of RL-QN15 and its optimized peptides were investigated by cell scratch assay in vitro and the full-thickness skin wound assay in mice.Next,the Cy RL-QN15,the optimizing peptide of RL-QN15,was loaded into the hollow polydopamine（HPDA）nanoparticles to form HPDAlCy RL-QN15,and then HPDAlCy RL-QN15 was mixed with alginate solution（SA）to prepare pro-healing hydrogel dressing(HPDAlCy RL-QN15/ZA hydrogel)via Zn2+crosslinking.The HPDAlCy RL-QN15/ZA hydrogel was characterized by Scanning electron microscopy（SEM）,Fourier transform infrared spectroscopy（FTIR）,Transmission electron microscopy（TEM）,and X-ray photoelectron spectroscopy（XPS）.And the biocompatibility of the HPDAlCy RL-QN15/ZA hydrogel was explored through in vitro and in vivo toxicity experiments.In addition,the physicochemical properties of HPDAlCy RL-QN15/ZA hydrogel were explored by the loading and slow-releasing rates of Cy RL-QN15,porosity,swelling,rheological and compressive assays.Cell scratch,cell proliferation,cell migration assays,tube formation,and cytokine release assays were performed to explore the pro-healing activities of the HPDAlCy RL-QN15/ZA hydrogel.Meanwhile,the ABTS+and DPPH radical scavenging,and intracellular scavenging of reactive oxygen species（ROS）assays were performed to detect the antioxidant activity of HPDAlCy RL-QN15/ZA hydrogel.Full thickness skin wounds of type II diabetic mice and ex vivo diabetic patient skin wound model were established to detect the effect of the HPDAlCy RL-QN15/ZA hydrogel in wound healing.hematoxylin and eosin（H&E）staining,Masson’s trichrome staining,periodic acid schiff（PAS）staining,immunohistochemistry,immunofluorescence,and enzyme-linked immunosorbent assay（ELISA）were performed to investigate the molecular mechanism of HPDAlCy RL-QN15/ZA hydrogel in chronic wound healing.Results:The results of cell scratch assay and full-thickness skin wounds healing showed that the cyclic heptapeptide Cy RL-QN15 had a comparable pro-healing ability to RL-QN15,indicating that the active region of RL-QN15 is the cyclic structure formed by seven amino acid sequences at its C-terminus through disulfide bonds.TEM results showed that HPDA nanoparticles were spherical hollow structures with a particle size of about 50 nm.The SEM results revealed that HPDAlCy RL-QN15/ZA hydrogel had a 3D network structure with microporous morphology.The HPDAlCy RL-QN15/ZA hydrogel structure became compact and the deeper color.Furthermore,XPS spectra analysis revealed that the presence of SA,Zn2+and cyclic peptide Cy RL-QN15with disulfide bond in the hydrogel.The FTIR curves showed the characteristic peaks of the SA,ZA,HPDAlCy RL-QN15,and HPDAlCy RL-QN15/ZA hydrogels,respectively.Compared with SA,ZA,and HPDAlCy RL-QN15 the characteristic peak of the HPDAlCy RL-QN15/ZA hydrogel was significantly shifted due to the cross-linking of alginate carboxylic acid and Zn2+.In summary,the TEM,SEM,FTIR and XPS results demonstrated that the HPDAlCy RL-QN15/ZA hydrogel was successfully prepared.HaCaT live/dead staining and wound toxicity tests on the dorsal skin of C57BL/6mice confirmed that the HPDAlCy RL-QN15/ZA hydrogel has a great biocompatibility.The hydrogels exhibited excellent degradability in hyaluronidase and collagenase solutions,and in the abdominal subcutaneously of mice.Moreover,the HPDAlCy RL-QN15/ZA hydrogel exhibited an excellent loading and slow-releasing effect against Cy RL-QN15,and the loading and release rates reached 53.39%and 73.48%,respectively.Notably,compared to sodium alginate（SA）,zinc ions significantly promoted the cross-linking of the HPDAlCy RL-QN15/ZA hydrogel.Compared to ZA hydrogel,HPDAlCy RL-QN15/ZA hydrogel had more compact porosity and more stable structure.The HPDAlCy RL-QN15/ZA hydrogel also exhibited excellent swelling properties,and its swelling ratio reached 48.92%at 5 hours.Rheological properties and compressive strength examination revealed that Zn2+cross-linking enhanced the mechanical properties of HPDAlCy RL-QN15/ZA hydrogel.Cell proliferation and cell scratch assay results indicated that the HPDAlCyRL-QN15/ZA hydrogel significantly enhanced the pro-proliferative and pro-scratch healing effects of Cy RL-QN15 at the cellular level.In addition,the HPDAlCy RL-QN15/ZA hydrogel also significantly enhanced HUVECs cells migration and tube formation.The HPDAlCy RL-QN15/ZA hydrogel significant modulated cytokines and inflammatory factors secretion from macrophages,indicating its potential for regulating the wound inflammatory response.Notably,the HPDAlCy RL-QN15/ZA hydrogel exhibited significant activity in scavenging ABTS+and DPPH radicals and reducing excessive reactive oxygen species（ROS）in HUVECs induced by hydrogen peroxide（H2O2）stimulation.HPDAlCy RL-QN15/ZA hydrogel exhibited great therapeutic effects on diabetic mice skin wounds.The wound repair rate reached 95.23±4.49%after 14 days treatment of HPDAlCy RL-QN15/ZA hydrogel,which was significantly higher than vehicle（66.38±3.41%）and Cy RL-QN15（88.74±2.14%）.More importantly,the HPDAlCy RL-QN15/ZA hydrogel also facilitated wound healing of diabetic patient skin cultured ex vivo.Exploring the mechanism of HPDAlCy RL-QN15/ZA hydrogel significantly promoted wound healing,which revealed that the HPDAlCy RL-QN15/ZA hydrogel accelerated the wound repair process by regulating the polarization of macrophages from M1phenotype to M2 phenotype and suppressing the excessive inflammatory response to promote the wound repair to a proliferative phase.In addition,HPDAlCy RL-QN15/ZA hydrogel promoted the expression of epidermal Ki67,VEGF,CD31,α-SMA,COL I,and COL III expression,thus accelerating re-epithelialization and promoting angiogenesis,collagen deposition,and granulation tissue regeneration,which exhibited excellent therapeutic effects on diabetic chronic skin wounds.Conclusion:In this study,the multifunctional HPDAlCy RL-QN15/ZA hydrogel dressing with great biocompatibility and physicochemical properties was successfully prepared through the internal structure optimization of RL-QN15 and external modification strategies.The HPDAlCy RL-QN15/ZA hydrogel accelerated the proliferation,migration,and tube formation of skin cells,regulated the secretion of cytokines,and directly scavenged free radicals and ROS.Furthermore,HPDAlCy RL-QN15/ZA hydrogel markedly accelerated the healing of diabetic skin wounds by promoting re-epithelialization,granulation tissue formation,collagen deposition,and angiogenesis,and by reducing inflammation.Thus,the HPDAlCy RL-QN15/ZA hydrogel provides a novel therapeutic strategy for the clinical treatment of chronic skin wounds. |
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