A multifunctional injectable ε-poly-L-lysine-loaded sodium-alginate/gelatin hydrogel promotes the healing of infected wounds by regulating macrophage polarization and the skin microbiota

Abstract

Background: Infected wounds caused by bacteria such as Escherichia coli and Staphylococcus aureus pose significant challenges during the healing process. Hydrogels have emerged as promising materials for the treatment of such infections, as they have the potential to deliver therapeutic agents while supporting tissue repair. This study aimed to develop ε-PLL@SA/Gel (PSG) hydrogels by incorporating varying concentrations of ε-poly-L-lysine (ε-PLL) into sodium alginate/gelatin (SA/Gel) using calcium chloride as a crosslinking agent, and to evaluate their antibacterial efficacy. Methods: The mechanical properties, biocompatibility, antibacterial activity of hydrogels were evaluated. Biocompatibility was examined by measuring cell viability and proliferation of human skin fibroblasts in vitro. Antibacterial efficacy against Escherichia coli and Staphylococcus aureus was quantified using bacterial inhibition assays. The wound healing efficacy of the hydrogels were evaluated in mouse models of infected wounds. Results: PSG hydrogels exhibited excellent mechanical strength, injectability, and self-adhesive properties. In vitro, hydrogel treatment resulted in high cell viability and promoted human skin fibroblast proliferation. PSG15 exhibited the highest antibacterial activity and inhibited E. coli and S. aureus by 89.53% and 92.21%, respectively. In vivo, PSG15 significantly accelerated wound healing, enhanced angiogenesis, and regulated macrophage polarization by increasing CD206 expression and decreasing CD80 expression. Additionally, PSG15 modulated the skin microbiota, reduced pathogenic bacterial abundance and maintained microbiota diversity. Conclusions: The PSG15 hydrogel is a promising candidate for the treatment of infected wounds because it inhibits bacterial growth, promotes tissue repair, and modulates the wound microbiota.

Keywords

ε-PLL@SA/gel / PSG / Infected wounds / Skin healing / Skin microbiota

References

[1] Liu W, Gao R, Yang C, Feng Z, Ou-Yang W, Pan X, et al. ECM-mimetic immunomodulatory hydrogel for methicillin-resistant Staphylococcus aureus-infected chronic skin wound healing. Sci Adv 2022; 8: eabn7006. https://doi.org/10.1126/sciadv.abn7006.

[2] Liang Y, He J, Guo B. Functional hydrogels as wound dressing to enhance wound healing. ACS Nano 2021; 15: 12687-722. https://doi.org/10.1021/acsnano.1c04206.

[3] Ming Z, Han L, Bao M, Zhu H, Qiang S, Xue S, et al. Living bacterial hydrogels for accelerated infected wound healing. Adv Sci (Weinh) 2021; 8: e2102545. https://doi.org/10.1002/advs.202102545.

[4] Wang X, Song R, Johnson M, Sigen A, Shen P, Zhang N, et al. Chitosan-based hydrogels for infected wound treatment. Macromol Biosci 2023; 23: e2300094. https://doi.org/10.1002/mabi.202300094.

[5] Rodrigues B, Morais TP, Zaini PA, Campos CS, Almeida-Souza H, Dandekar AM, et al. Antimicrobial activity of epsilon-poly-L-lysine against phytopathogenic bacteria. Sci Rep 2020; 10: 11324. https://doi.org/10.1038/s41598-020-68262-1.

[6] Jiang X, Radko Y, Gren T, Palazzotto E, Jørgensen TS, Cheng T, et al. Distribution of epsilon-poly-l-lysine Synthetases in Coryneform bacteria isolated from cheese and human skin. Appl Environ Microbiol 2021; 87: e01841-20. https://doi.org/10.1128/AEM.01841-20.

[7] Shu C, Cui K, Li Q, Cao J, Jiang W. Epsilon-poly-l-lysine (epsilon-PL) exhibits multifaceted antifungal mechanisms of action that control postharvest Alternaria rot. Int J Food Microbiol 2021; 348: 109224. https://doi.org/10.1016/j.ijfoodmicro.2021.109224.

[8] Padilla-Garfias F, Rios-Cifuentes L, Sanchez NS, Calahorra M, Pena A. Study of the mechanism of epsilon-poly-l-lysine as an antifungal on Candida albicans and Saccharomyces cerevisiae. Biochim Biophys Acta Gen Subj 2022; 1866: 130197. https://doi.org/10.1016/j.bbagen.2022.130197.

[9] Moon AY, Bailey EJ, Polanco JA, Kurata WE, Pierce LM. Antibacterial efficacy of a chitosan-based hydrogel modified with epsilon-poly-l-lysine against Pseudomonas aeruginosa in a murine-infected burn wound model. Mil Med 2023; 188: 52-60. https://doi.org/10.1093/milmed/usad013.

[10] Zhou C, Li P, Qi X, Mohamed AR, Poon YF, Cao Y, et al. A photopolymerized antimicrobial hydrogel coating derived from epsilon-poly-L-lysine. Biomaterials 2011; 32: 2704-12. https://doi.org/10.1016/j.biomaterials.2010.12.040.

[11] Rastogi P, Kandasubramanian B. Review of alginate-based hydrogel bioprinting for application in tissue engineering. Biofabrication 2019; 11: 042001. https://doi.org/10.1088/1758-5090/ab331e.

[12] Du J, Hu X, Su Y, Tuo W, Jiao Z, Liu T, et al. Gelatin/sodium alginate hydrogel-coated decellularized porcine coronary artery to construct bilayer tissue engineered blood vessels. Int J Biol Macromol 2022; 209: 2070-83. https://doi.org/10.1016/j.ijbiomac.2022.04.188.

[13] Yan Y, You A, Chen X, Huang W, Lu X, Gu C, et al. (+)4-cholesten-3-one/sodium alginate/gelatin hydrogel for full-thickness wound repair and skin regeneration. Biomed Mater 2024; 19: 055026. https://doi.org/10.1088/1748-605X/ad6966.

[14] Liu C, Qin W, Wang Y, Ma J, Liu J, Wu S, et al. 3D printed Gelatin/sodium alginate hydrogel scaffolds doped with Nano-Attapulgite for bone tissue repair. Int J Nanomedicine 2021; 16: 8417-32. https://doi.org/10.2147/IJN.S339500.

[15] Sun D. Sacrificial gelatin of PAM-alginate-BC hydrogel tube with tunable diameter as nerve conduit. J Biomater Sci Polym Ed 2023; 34: 1398-407. https://doi.org/10.1080/09205063.2023.2167047.

[16] Han Z, Deng L, Chen S, Wang H, Huang Y. Zn2+-Loaded adhesive bacterial cellulose hydrogel with angiogenic and antibacterial abilities for accelerating wound healing. Burns Trauma 2023; 11: tkac048. https://doi.org/10.1093/burnst/tkac048.

[17] Ding X, Yang C, Li Y, He T, Xu Y, Cheng X, et al. Reshaped commensal wound microbiome via topical application of Calvatia gigantea extract contributes to faster diabetic wound healing. Burns Trauma 2024; 12: tkae037. https://doi.org/10.1093/burnst/tkae037.

[18] Hu JQ, Wang J, Shen ZH, Lai YX, You JY, Yan Q, et al. Mechanical enhancement of the Gelatin/poly(zinc acrylate) hydrogel stent in bile. ACS Appl Bio Mater 2023; 6: 5621-9. https://doi.org/10.1021/acsabm.3c00786.

[19] Ranamalla SR, Tavakoli S, Porfire AS, Tafas LR, Banciu M, Tomuta L, et al. A quality by design approach to optimise disulfide-linked hyaluronic acid hydrogels. Carbohydr Polym 2024; 339: 122251. https://doi.org/10.1016/j.carbpol.2024.122251.

[20] Wang Y, Gao X, Wu J, Jiang M, Zhang H, Yan C. Antifreezing/Antiswelling hydrogels: synthesis strategies and applications as flexible motion sensors. ACS Appl Mater Interfaces 2024; 16: 58100-20. https://doi.org/10.1021/acsami.4c13621.

[21] Wang Y, Frascella F, Gaglio CG, Pirri CF, Wei Q, Roppolo I. Vat Photopolymerization 3D printing of hydrogels embedding metal-organic frameworks for photodynamic antimicrobial therapy. ACS Appl Mater Interfaces 2024; 16: 57778-57791. https://doi.org/10.1021/acsami.4c15168.

[22] Zhou W, Zhu L, Zu X, Ma YL, Shen P, Hu YY, et al. A novel antioxidant and anti-inflammatory carboxymethylcellulose/chitosan hydrogel loaded with cannabidiol promotes the healing of radiation-combined wound skin injury in the (60)Co gamma-irradiated mice. Phytomedicine 2025; 142: 156790. https://doi.org/10.1016/j.phymed.2025.156790.

[23] Tu C, Lu H, Zhou T, Zhang W, Deng L, Cao W, et al. Promoting the healing of infected diabetic wound by an anti-bacterial and nano-enzyme-containing hydrogel with inflammation-suppressing, ROS-scavenging, oxygen and nitric oxide-generating properties. Biomaterials 2022; 286: 121597. https://doi.org/10.1016/j.biomaterials.2022.121597.

[24] Zhao C, Wang X, Wu L, Wu W, Zheng Y, Lin L, et al. Nitrogen-doped carbon quantum dots as an antimicrobial agent against staphylococcus for the treatment of infected wounds. Colloids Surf B Biointerfaces 2019; 179: 17-27. https://doi.org/10.1016/j.colsurfb.2019.03.042.

[25] Devi VKA, Shyam R, Palaniappan A, Jaiswal AK, Oh TH, Nathanael AJ. Self-healing hydrogels: preparation, mechanism and advancement in biomedical applications. Polymers (Basel) 2021; 13: 3782. https://doi.org/10.3390/polym13213782.

[26] Lou J, Wang X, Zhang H, Yu G, Ding J, Zhu X, et al. Inhibition of PLA2G4E/cPLA2 promotes survival of random skin flaps by alleviating lysosomal membrane permeabilization-induced necroptosis. Autophagy 2022; 18: 1841-63. https://doi.org/10.1080/15548627.2021.2002109.

[27] Suh JW, Lee KM, Ko EA, Yoon DS, Park KH, Kim HS, et al. Promoting angiogenesis and diabetic wound healing through delivery of protein transduction domain-BMP2 formulated nanoparticles with hydrogel. J Tissue Eng 2023; 14: 20417314231190641. https://doi.org/10.1177/20417314231190641.

[28] Genin M, Clement F, Fattaccioli A, Raes M, Michiels C. M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide. BMC Cancer 2015; 15: 577. https://doi.org/10.1186/s12885-015-1546-9.

[29] Xu GY, Xu S, Zhang YX, Yu ZY, Zou F, Ma XS, et al. Cell-free extracts from human fat tissue with a Hyaluronan-based hydrogel attenuate inflammation in a spinal cord injury model through M2 microglia/microphage polarization. Small 2022; 18: e2107838. https://doi.org/10.1002/smll.202107838.

[30] White EK, Grice EA.The wound microbiome. Cold Spring Harb Perspect Biol 2023; 15: a041218. https://doi.org/10.1101/cshperspect.a041218.

[31] Li Y, Fu R, Duan Z, Zhu C, Fan D. Construction of multifunctional hydrogel based on the tannic acid-metal coating decorated MoS(2) dual nanozyme for bacteria-infected wound healing. Bioact Mater 2022; 9: 461-74. https://doi.org/10.1016/j.bioactmat.2021.07.023.

[32] Wang Y, Wang L, Hu Y, Qin J, Yu B. Design and optimization of epsilon-poly-l-lysine with specific functions for diverse applications. Int J Biol Macromol 2024; 262: 129513. https://doi.org/10.1016/j.ijbiomac.2024.129513.

[33] Liu JN, Chang SL, Xu PW, Tan MH, Zhao B, Wang XD, et al. Structural changes and antibacterial activity of epsilon-poly-l-lysine in response to pH and phase transition and their mechanisms. J Agric Food Chem 2020; 68: 1101-9. https://doi.org/10.1021/acs.jafc.9b07524.

[34] Zielińska M, Pawłowska A, Orzeł A, Sulej L, Placzyńska KM, Baran A, et al. Wound microbiota and its impact on wound healing. Int J Mol Sci 2023; 24: 17318. https://doi.org/10.3390/ijms242417318.

[35] Hyldgaard M, Mygind T, Vad BS, Stenvang M, Otzen DE, Meyer RL. The antimicrobial mechanism of action of epsilon-poly-l-lysine. Appl Environ Microbiol 2014; 80: 7758-70. https://doi.org/10.1128/AEM.02204-14.

[36] Yi X, He J, Wei X, Li H, Liu X, Cheng F. A polyphenol and epsilon-polylysine functionalized bacterial cellulose/PVA multifunctional hydrogel for wound healing. Int J Biol Macromol 2023; 247: 125663. https://doi.org/10.1016/j.ijbiomac.2023.125663.

[37] Ferdous Z, Nemmar A. Health impact of silver nanoparticles: a review of the biodistribution and toxicity following various routes of exposure. Int J Mol Sci 2020; 21: 2375. https://doi.org/10.3390/ijms21072375.