Hybrid nanovesicles promote diabetic wound healing via dual-targeted multimodal therapy

Abstract

Background: Diabetic wounds remain difficult to treat due to persistent oxidative stress, chronic inflammation, and vascular dysfunction. These factors reinforce each other, forming a vicious cycle that leads to delayed healing, poor angiogenesis, and high amputation risk. Existing therapies often fail because they are unable to address these challenges simultaneously. Therefore, this study aimed to develop a hybrid extracellular vesicle system that targets these multiple barriers concurrently to promote diabetic wound healing. Methods: A biohybrid nanovesicle system (DFO@HEVs) was built by fusing endothelial cell-derived extracellular vesicles with neutrophil-derived nanovesicles (forming hybrid extracellular vesicles, HEVs), which were loaded with deferoxamine (DFO). The vesicles were tested for their physicochemical properties, drug loading, and safety. Therapeutic effects were studied in vitro using HG/PA-stimulated endothelial cells and macrophages and in vivo in diabetic mouse wounds. The analyses included microscopy, flow cytometry, histology, transcriptomics, and database-based single-cell RNA sequencing. Results: DFO@HEVs showed dual targeting: homing to endothelial cells via CXCR4 and to inflamed sites via β2 integrin. They enhanced endothelial uptake, promoted angiogenesis through PI3K/AKT/HIF-1 α and VEGF signaling pathways, and reduced oxidative stress and ferroptosis by activating Nrf2 and upregulating antioxidant genes. They also shifted macrophages toward an anti-inflammatory M2 phenotype, boosted efferocytosis, and suppressed NF-κB/NLRP3-driven inflammation. In diabetic mice, treatment with DFO@HEVs accelerated wound closure, re-epithelialization, collagen deposition, and new vessel formation, while lowering neutrophil infiltration, reactive oxygen species levels, ferroptosis, and pro-inflammatory cytokines, creating a healing-supportive environment. Conclusions: DFO@HEVs provided a hybrid nanovesicle system for combined membrane and drug delivery. By promoting angiogenesis, limiting ferroptosis, and resolving inflammation, they disrupted the cycle that prevented diabetic wound repair. This approach shows a strong potential as a new treatment for chronic wounds.

Keywords

Diabetic wound / Engineering nanovesicles / Targeted delivery / Antioxidants / Macrophage polarization

References

[1] Lim JZ, Ng NS, Thomas C. 2 prevention and treatment of diabetic foot ulcers. J R Soc Med 2017; 110:104-9. https://doi.org/10.1177/0141076816688346.

[2] Lo ZJ, Surendra NK, Saxena A, Car J. Clinical and economic burden of diabetic foot ulcers: a 5-year longitudinal multi-ethnic cohort study from the tropics. Int Wound J 2021; 18:375-86. https://doi.org/10.1111/iwj.13540.

[3] Armstrong DG, Tan T-W, Boulton AJM, Bus SA. Diabetic foot ulcers: a review. JAMA 2023; 330:62-75. https://doi.org/10.1001/jama.2023.10578.

[4] Schaper NC, van Netten JJ, Apelqvist J, Bus SA, Hinchliffe RJ, Lipsky BA, et al. Practical guidelines on the prevention and management of diabetic foot disease (iwgdf 2019 update). Diabetes Metab Res Rev 2020; 36:e3266. https://doi.org/10.1002/dmrr.3266.

[5] Wang X, Tan X, Zhang T, Xu S, Zeng Y, Xu A, et al. Modeling diabetic cardiomyopathy using human cardiac organoids: effects of high glucose and lipid conditions. Chem Biol Interact 2025; 411:111421. https://doi.org/10.1016/j.cbi.2025.111421.

[6] Ceja-Galicia ZA, Cespedes-Acuña CLA, El-Hafidi M. Protection strategies against palmitic acid-induced lipotoxicity in metabolic syndrome and related diseases. Int J Mol Sci 2025; 26:788. https://doi.org/10.3390/ijms26020788.

[7] Deng L, Du C, Song P, Chen T, Rui S, Armstrong DG, et al. The role of oxidative stress and antioxidants in diabetic wound healing. Oxidative Med Cell Longev 2021; 2021:8852759. https://doi.org/10.1155/2021/8852759.

[8] Weigert C, Brodbeck K, Staiger H, Kausch C, Machicao F, Häring HU, et al. Palmitate, but not unsaturated fatty acids, induces the expression of interleukin-6 in human myotubes through proteasome-dependent activation of nuclear factor-kappab. J Biol Chem 2004; 279:23942-52. https://doi.org/10.1074/jbc.M312692200.

[9] Kusminski CM, Shetty S, Orci L, Unger RH, Scherer PE. Diabetes and apoptosis: lipotoxicity. Apoptosis 2009; 14:1484-95. https://doi.org/10.1007/s10495-009-0352-8.

[10] Xu Y, Li Y, Li J, Chen W. Ethyl carbamate triggers ferroptosis in liver through inhibiting gsh synthesis and suppressing nrf2 activation. Redox Biol 2022; 53:102349. https://doi.org/10.1016/j.redox.2022.102349.

[11] Forcina GC, Dixon SJ. Gpx4 at the crossroads of lipid homeostasis and ferroptosis. Proteomics 2019; 19:e1800311. https://doi.org/10.1002/pmic.201800311.

[12] Chun HJ, Kim ER, Lee M, Choi DH, Kim SH, Shin E, et al. Increased expression of sodium-glucose cotransporter 2 and o-glcnacylation in hepatocytes drives non-alcoholic steatohepatitis. Metabolism 2023; 145:155612. https://doi.org/10.1016/j.metabol.2023.155612.

[13] Rui S, Dai L, Zhang X, He M, Xu F, Wu W, et al. Exosomal mirna-26b-5p from prp suppresses nets by targeting mmp-8 to promote diabetic wound healing. J Control Release 2024; 372:221-33. https://doi.org/10.1016/j.jconrel.2024.06.050.

[14] Leventis PA, Grinstein S. The distribution and function of phosphatidylserine in cellular membranes. Annu Rev Biophys 2010; 39:407-27. https://doi.org/10.1146/annurev.biophys.093008.131234.

[15] Zhu H, Wang W, Zhu J, Chen X, Wang J, Wang J, et al. Methylglyoxal deteriorates macrophage efferocytosis in diabetic wounds through ros-induced ubiquitination degradation of klf4. Free Radic Biol Med 2025; 231:23-37. https://doi.org/10.1016/j.freeradbiomed.2025.02.030.

[16] Manoj H, Gomes SM, Thimmappa PY, Nagareddy PR, Jamora C, Joshi MB. Cytokine signalling in formation of neutrophil extracellular traps: implications for health and diseases. Cytokine Growth Factor Rev 2025; 81:27-39. https://doi.org/10.1016/j.cytogfr.2024.12.001.

[17] Gong H, Yang L, Li Y, Zhang X, Zheng C, Gan T, et al. Metal-polyphenol nanocomposite hybrid hydrogel: a multifunctional platform for treating diabetic foot ulcers through metabolic microenvironment reprogramming. Biomaterials 2025; 322:123414. https://doi.org/10.1016/j.biomaterials.2025.123414.

[18] Lei L, Yuan J, Dai Z, Xiang S, Tu Q, Cui X, et al. Targeting the labile iron pool with engineered dfo nanosheets to inhibit ferroptosis for parkinson’s disease therapy. Adv Mater 2024; 36:e2409329. https://doi.org/10.1002/adma.202409329.

[19] Zhu Y, Chang B, Pang Y, Wang H, Zhou Y. Advances in hypoxia-inducible factor-1α stabilizer deferoxamine in tissue engineering. Tissue Eng Part B Rev 2023; 29:347-57. https://doi.org/10.1089/ten.TEB.2022.0168.

[20] Duscher D, Neofytou E, Wong VW, Maan ZN, Rennert RC, Inayathullah M, et al. Transdermal deferoxamine prevents pressure-induced diabetic ulcers. Proc Natl Acad Sci USA 2015; 112:94-9. https://doi.org/10.1073/pnas.1413445112.

[21] Bao X, Luo X, Bai X, Lv Y, Weng X, Zhang S, et al. Cigarette tar mediates macrophage ferroptosis in atherosclerosis through the hepcidin/fpn/slc7a11 signaling pathway. Free Radic Biol Med 2023; 201:76-88. https://doi.org/10.1016/j.freeradbiomed.2023.03.006.

[22] Zhang AY, Su JB, Sun HT, Liu Q, Li R, Zhang Y, et al. Stachyose ameliorates myocardial ischemia-reperfusion injury by inhibiting cardiomyocyte ferroptosis and macrophage pyroptosis. Int Immunopharmacol 2024; 143:113334. https://doi.org/10.1016/j.intimp.2024.113334.

[23] Ding Z, Zhang Y, Guo P, Duan T, Cheng W, Guo Y, et al. Injectable desferrioxamine-laden silk nanofiber hydrogels for accelerating diabetic wound healing. ACS Biomater Sci Eng 2021; 7:1147-58. https://doi.org/10.1021/acsbiomaterials.0c01502.

[24] Li N, Zhan A, Jiang Y, Liu H. A novel matrix metalloproteinases-cleavable hydrogel loading deferoxamine accelerates diabetic wound healing. Int J Biol Macromol 2022; 222:1551-9. https://doi.org/10.1016/j.ijbiomac.2022.09.185.

[25] van Niel G, D’Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol 2018; 19:213-28. https://doi.org/10.1038/nrm.2017.125.

[26] Escudier B, Dorval T, Chaput N, André F, Caby MP, Novault S, et al. Vaccination of metastatic melanoma patients with autologous dendritic cell (dc) derived-exosomes: results of the first phase i clinical trial. J Transl Med 2005; 3:10. https://doi.org/10.1186/1479-5876-3-10.

[27] Yousefpour P, Chilkoti A. Co-opting biology to deliver drugs. Biotechnol Bioeng 2014; 111:1699-716.

[28] Aled C, Harris C, Jacquelyn C, Mason M, Paul M . Antigen-presenting cell exosomes are protected from complement-mediated lysis by expression of cd55 and cd59. Eur J Immunol 2003; 33:522-31.

[29] Kalra H, Adda CG, Liem M, Ang CS, Mechler A, Simpson RJ, et al. Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma. Proteomics 2013; 13:3354-64. https://doi.org/10.1002/pmic.201300282.

[30] Wang C, Chen J, Guo J, Li G, Yi K, Jiang T, et al. Photoresponsive multifunctional nanoplatform based on adsc-exos coated bpqds demonstrates effective antibacterial properties and induces macrophage polarization for infected wound healing. Adv Healthc Mater 2025; 14:e2501044. https://doi.org/10.1002/adhm.202501044.

[31] Adamo G, Fierli D, Romancino DP, Picciotto S, Barone ME, Aranyos A, et al. Nanoalgosomes: introducing extracellular vesicles produced by microalgae. J Extracell Vesicles 2021; 10:e12081. https://doi.org/10.1002/jev2.12081.

[32] Paterna A, Rao E, Adamo G, Raccosta S, Picciotto S, Romancino D, et al. Isolation of extracellular vesicles from microalgae: a renewable and scalable bioprocess. Front Bioeng Biotechnol 2022; 10:836747. https://doi.org/10.3389/fbioe.2022.836747.

[33] Jeyaram A, Jay SM. Preservation and storage stability of extracellular vesicles for therapeutic applications. AAPS J 2017; 20:1. https://doi.org/10.1208/s12248-017-0160-y.

[34] Manno M, Bongiovanni A, Margolis L, Bergese P, Arosio P. The physico-chemical landscape of extracellular vesicles. Nature Reviews Bioengineering 2025; 3:68-82. https://doi.org/10.1038/s44222-024-00255-5.

[35] Wen Y, Fu Q, Soliwoda A, Zhang S, Zheng M, Mao W, et al. Cell-derived nanovesicles prepared by membrane extrusion are good substitutes for natural extracellular vesicles. Extracellular Vesicle 2022; 1:100004. https://doi.org/10.1016/j.vesic.2022.100004.

[36] Neupane YR, Handral HK, Alkaff SA, Chng WH, Venkatesan G, Huang C, et al. Cell-derived nanovesicles from mesenchymal stem cells as extracellular vesicle-mimetics in wound healing. Acta Pharm Sin B 2023; 13:1887-902. https://doi.org/10.1016/j.apsb.2022.10.022.

[37] Cui Y, Li Z, Guo Y, Qi X, Yang Y, Jia X, et al. Bioinspired nanovesicles convert the skeletal endothelium-associated secretory phenotype to treat osteoporosis. ACS Nano 2022; 16:11076-91. https://doi.org/10.1021/acsnano.2c03781.

[38] Poch CM, Foo KS, DeAngelis MT, Jennbacken K, Santamaria G, Bähr A, et al. Migratory and anti-fibrotic programmes define the regenerative potential of human cardiac progenitors. Nat Cell Biol 2022; 24:659-71. https://doi.org/10.1038/s41556-022-00899-8.

[39] Jiang G, Guo J, Yan C, He Y, Chen J, Zhang M, et al. Biomimetic hybrid nanovesicles improve infected diabetic wound via enhanced targeted delivery. J Control Release 2024; 365:193-207. https://doi.org/10.1016/j.jconrel.2023.11.019.

[40] Sun H, Hu L, Fan Z. B2 integrin activation and signal transduction in leukocyte recruitment. Am J Phys Cell Phys 2021; 321:C308-16. https://doi.org/10.1152/ajpcell.00560.2020.

[41] Wen L, Moser M, Ley K. Molecular mechanisms of leukocyte β2 integrin activation. Blood 2022; 139:3480-92. https://doi.org/10.1182/blood.2021013500.

[42] Yang L, Froio RM, Sciuto TE, Dvorak AM, Alon R, Luscinskas FW. Icam-1 regulates neutrophil adhesion and transcellular migration of tnf-α-activated vascular endothelium under flow. Blood 2005; 106:584-92. https://doi.org/10.1182/blood-2004-12-4942.

[43] Zhang Q, Hu C, Feng J, Long H, Wang Y, Wang P, et al. Anti-inflammatory mechanisms of neutrophil membrane-coated nanoparticles without drug loading. J Control Release 2024; 369:12-24. https://doi.org/10.1016/j.jconrel.2024.03.030.

[44] Sun M, Yang J, Fan Y, Zhang Y, Sun J, Hu M, et al. Beyond extracellular vesicles: hybrid membrane nanovesicles as emerging advanced tools for biomedical applications. Adv Sci (Weinh) 2023; 10:e2303617. https://doi.org/10.1002/advs.202303617.

[45] Li Y, Zhu J, Zhang X, Li Y, Zhang S, Yang L, et al. Drug-delivery nanoplatform with synergistic regulation of angiogenesis-osteogenesis coupling for promoting vascularized bone regeneration. ACS Appl Mater Interfaces 2023; 15:17543-61. https://doi.org/10.1021/acsami.2c23107.

[46] Chen C, Li Y, Wang Q, Cai N, Wu L, Yan X. Single-particle assessment of six different drug-loading strategies for incorporating doxorubicin into small extracellular vesicles. Anal Bioanal Chem 2023; 415:1287-98. https://doi.org/10.1007/s00216-022-04248-4.

[47] Qian S, Mao J, Zhao Q, Zhao B, Liu Z, Lu B, et al. “find-eat” strategy targeting endothelial cells via receptor functionalized apoptotic body nanovesicle. Sci Bull (Beijing) 2023; 68:826-37. https://doi.org/10.1016/j.scib.2023.03.030.

[48] Cui S, Liu X, Liu Y, Hu W, Ma K, Huang Q, et al. Autophagosomes defeat ferroptosis by decreasing generation and increasing discharge of free fe(2+) in skin repair cells to accelerate diabetic wound healing. Adv Sci (Weinh) 2023; 10:e2300414. https://doi.org/10.1002/advs.202300414.

[49] Lin C, Hu Y, Lin Z, Du L, Hu Y, Ouyang L, et al. Mmp-9 responsive hydrogel promotes diabetic wound healing by suppressing ferroptosis of endothelial cells. Bioact Mater 2025; 43:240-54. https://doi.org/10.1016/j.bioactmat.2024.09.006.

[50] Yu X, Liu Z, Yu Y, Qian C, Lin Y, Jin S, et al. Hesperetin promotes diabetic wound healing by inhibiting ferroptosis through the activation of sirt3. Phytother Res 2024; 38:1478-93. https://doi.org/10.1002/ptr.8121.

[51] Birge RB, Boeltz S, Kumar S, Carlson J, Wanderley J, Calianese D, et al. Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer. Cell Death Differ 2016; 23:962-78. https://doi.org/10.1038/cdd.2016.11.

[52] Xie J, Shen Q, Huang K, Zheng T, Cheng L, Zhang Z, et al. Oriented assembly of cell-mimicking nanoparticles via a molecular affinity strategy for targeted drug delivery. ACS Nano 2019; 13:5268-77. https://doi.org/10.1021/acsnano.8b09681.

[53] Zhang W, Liu W, Long L, He S, Wang Z, Liu Y, et al. Responsive multifunctional hydrogels emulating the chronic wounds healing cascade for skin repair. J Control Release 2023; 354:821-34. https://doi.org/10.1016/j.jconrel.2023.01.049.

[54] Rankin-Turner S, Vader P, O’Driscoll L, Giebel B, Heaney LM, Davies OG. A call for the standardised reporting of factors affecting the exogenous loading of extracellular vesicles with therapeutic cargos. Adv Drug Deliv Rev 2021; 173:479-91. https://doi.org/10.1016/j.addr.2021.04.012.

[55] Han X, Shen S, Fan Q, Chen G, Archibong E, Dotti G, et al. Red blood cell-derived nanoerythrosome for antigen delivery with enhanced cancer immunotherapy. Sci Adv 2019; 5:eaaw6870. https://doi.org/10.1126/sciadv.aaw6870.

[56] Zhou SH, Lei YJ, Wen Y, Ding D, Luo MQ, Cui HY, et al. Engineered hybrid membrane vesicles combined with autologous and synthetic antigens as therapeutic vaccines to efficiently suppress tumor recurrence. J Control Release 2025; 385:113979. https://doi.org/10.1016/j.jconrel.2025.113979.

[57] Zhang W, Wang L, Guo H, Chen L, Huang X. Dapagliflozin-loaded exosome mimetics facilitate diabetic wound healing by hif-1 α-mediated enhancement of angiogenesis. Advanced Healthcare Materials 2023; 12:2202751. https://doi.org/10.1002/adhm.202202751.

[58] Chen L, Wu F, Xia W-H, Zhang Y-Y, Xu S-Y, Cheng F, et al. Cxcr4 gene transfer contributes to in vivo reendothelialization capacity of endothelial progenitor cells. Cardiovasc Res 2010; 88:462-70. https://doi.org/10.1093/cvr/cvq207.

[59] Luo Y, Feng J, Xu Q, Wang W, Wang X. Nsun2 deficiency protects endothelium from inflammation via mrna methylation of icam-1. Circ Res 2016; 118:944-56. https://doi.org/10.1161/circresaha.115.307674.

[60] Regal-McDonald K, Somarathna M, Lee T, Litovsky SH, Barnes J, Peretik JM, et al. Assessment of icam-1 n-glycoforms in mouse and human models of endothelial dysfunction. PLoS One 2020; 15:e0230358. https://doi.org/10.1371/journal.pone.0230358.

[61] Liu X, Bi Y, Wei C, Zhang Y, Liu X, Guo X, et al. Engineered neutrophil nanovesicles for inhibiting corneal neovascularization by synergistic anti-inflammatory, anti-vegf, and chemoexcited photodynamic therapy. Adv Mater 2025; 37:e2411030. https://doi.org/10.1002/adma.202411030.

[62] Jia M, Miao W, Li Y, Guo Y, Zeng J, Gao Y, et al. A polymerized probucol nanoformulation with neutrophil extracellular vesicle camouflage for cerebral ischemia-reperfusion injury therapy. Innovation (Camb) 2024; 6:100761. https://doi.org/10.1016/j.xinn.2024.100761.

[63] Liu Y, He M, Yuan Y, Nie C, Wei K, Zhang T, et al. Neutrophil-membrane-coated biomineralized metal-organic framework nanoparticles for atherosclerosis treatment by targeting gene silencing. ACS Nano 2023; 17:7721-32. https://doi.org/10.1021/acsnano.3c00288.

[64] Liu Y, Chen W, Li C, Li L, Yang M, Jiang N, et al. Dsba-l interacting with catalase in peroxisome improves tubular oxidative damage in diabetic nephropathy. Redox Biol 2023; 66:102855. https://doi.org/10.1016/j.redox.2023.102855.

[65] Huang S, Chen G, Sun J, Chen Y, Wang N, Dong Y, et al. Histone deacetylase 3 inhibition alleviates type 2 diabetes mellitus-induced endothelial dysfunction via nrf2. Cell Commun Signal 2021; 19:35. https://doi.org/10.1186/s12964-020-00681-z.

[66] Wu X, Zhang C, Li X, Rong Y, Du Z, Gao F, et al. Ezh2-mediated phlda1 governs mitochondrial dysfunction and oxidative damage in traumatic brain injury via the akt/nrf2/sirt3 pathway. Neuroreport 2025; 36:822-832. https://doi.org/10.1097/wnr.0000000000002203.

[67] Sun D, Chang Q, Lu F. Immunomodulation in diabetic wounds healing: the intersection of macrophage reprogramming and immunotherapeutic hydrogels. J Tissue Eng 2024; 15:20417314241265202. https://doi.org/10.1177/20417314241265202.

[68] Wu X, He W, Mu X, Liu Y, Deng J, Liu Y, et al. Macrophage polarization in diabetic wound healing. Burns Trauma 2022; 10:tkac051. https://doi.org/10.1093/burnst/tkac051.

[69] Boada-Romero E, Martinez J, Heckmann BL, Green DR . The clearance of dead cells by efferocytosis. Nat Rev Mol Cell Biol 2020; 21:398-414. https://doi.org/10.1038/s41580-020-0232-1.

[70] Ngai D, Schilperoort M, Tabas I. Efferocytosis-induced lactate enables the proliferation of pro-resolving macrophages to mediate tissue repair. Nat Metab 2023; 5:2206-19. https://doi.org/10.1038/s42255-023-00921-9.

[71] Huang Y, Wang B, Ma Z, Chen T, Zou H, Chen Y, et al. Sulforaphane promotes diabetic wound healing by regulating macrophage efferocytosis and polarization. Int Immunopharmacol 2025; 150:114243. https://doi.org/10.1016/j.intimp.2025.114243.

[72] Zhu Y, Qiao S, Pang Y, Wang H, Zhou Y. Deferoxamine treatment effectively prevents periodontitis progression by reducing inflammation and osteoclastogenesis in experimental periodontitis rats. J Inflamm Res 2024; 17:9637-50. https://doi.org/10.2147/jir.S490823.

[73] Wang S, Liu C, Pan S, Miao Q, Xue J, Xun J, et al. Deferoxamine attenuates lipopolysaccharide-induced inflammatory responses and protects against endotoxic shock in mice. Biochem Biophys Res Commun 2015; 465:305-11. https://doi.org/10.1016/j.bbrc.2015.08.032.

[74] Singh G, Singh A, Mishra S, Singh D, Kumar A. Intracellular iron accumulation induces inflammatory and oxidative status of the host after japanese encephalitis viral infection. Mol Neurobiol 2024; 61:175-87. https://doi.org/10.1007/s12035-023-03538-x.

[75] Nambi N, Radhakrishnan L, Prasad MK, Ramkumar KM. Neutrophil migration is a crucial factor in wound healing and the pathogenesis of diabetic foot ulcers: insights into pharmacological interventions. Curr Vasc Pharmacol 2024; 23:98-112. https://doi.org/10.2174/0115701611308960241014155413.

[76] Long LY, Hu C, Liu W, Wu C, Lu L, Yang L, et al. Microfibrillated cellulose-enhanced carboxymethyl chitosan/oxidized starch sponge for chronic diabetic wound repair. Mater Sci Eng C Mater Biol Appl 2022; 135:112669. https://doi.org/10.1016/j.msec.2022.112669.