Development of multi-functional diabetic wound healing therapeutics via interleukin-4 surface display on fibroblast-derived extracellular vesicles

다국어 초록 (Multilingual Abstract)

Diabetes mellitus is a rapidly escalating global health burden characterized by chronic hyperglycemia, which disrupts immune homeostasis, impairs fibroblast and endothelial cell function, and results in persistent inflammation that prevents normal wound repair. Diabetic wounds become pathologically arrested in the inflammatory phase due to sustained M1 macrophage activation, reduced growth factor production, defective extracellular matrix (ECM) deposition, and impaired angiogenesis. These abnormalities create a chronic, non-healing wound environment that remains refractory to current therapeutic strategies, underscoring the need for multifunctional approaches capable of simultaneously restoring fibroblast activity, enhancing endothelial repair, and reprogramming dysregulated immune responses. Extracellular vesicles (EVs) have emerged as promising biomimetic therapeutic agents owing to their ability to deliver bioactive cargos and modulate cellular communication. Fibroblast-derived EV (FEV), in particular, contain regenerative factors that promote fibroblast proliferation, collagen synthesis, and angiogenesis. However, their therapeutic potential in diabetic wounds remains limited by the persistent pro-inflammatory microenvironment. To address this challenge, we engineered IL-4–displaying fibroblast-derived EVs (FEV-IL4) using a DSPE-PEG– based surface conjugation strategy, enabling stable presentation of IL-4 on the vesicle membrane. This design integrates the intrinsic regenerative functions of FEV with the potent immunomodulatory activity of IL-4, which drives macrophage repolarization from the pro-inflammatory M1 phenotype toward a reparative M2 state. In vitro, FEV-IL4 effectively restored hyperglycemia-impaired fibroblast proliferation, migration, and ECM-related gene expression, while significantly enhancing endothelial tube formation. Furthermore, FEV-IL4 induced robust M1-to- M2 repolarization in macrophages, confirming preservation of IL-4 bioactivity on the EV surface. In a streptozotocin-induced diabetic mouse model, topical administration of FEV-IL4 markedly accelerated wound closure, improved granulation tissue formation, and enhanced angiogenesis compared to unmodified Collectively, this study demonstrates that FEV-IL4 functions as a multifunctional therapeutic platform that concurrently corrects inflammatory dysregulation, reinstates fibroblast and endothelial cell function, and promotes effective tissue regeneration in diabetic wounds. These findings highlight the potential of cytokine-engineered EVs as an advanced and clinically translatable strategy for treating chronic, non-healing diabetic ulcers. Keyword: Extracellular vesicles, Diabetic wound, Interleukin-4, Surface modification, Anti-inflammation

목차 (Table of Contents)

• Abstact i
• Table of Contents ii
• List of Tables iii
• List of Figures iv
• Chapter 1. Introduction
• 1.1. Diabetes mellitus: an escalating global health crisis
• 1.2. Wound healing: normal versus diabetic cutaneous repair
• 1.2.1. Normal skin wound healing
• 1.2.2. Diabetic wound healing under hyperglycemic conditions
• 1.3. Extracellular vesicles: emerging therapeutics for impaired diabetic wound healing
• 1.4. Interleukin-4: restoring immune balance in hyperglycemia-impaired wound healing
• 1.5. Surface engineering strategies of extracellular vesicles: integrating cytokines for enhanced therapeutic activity
• Chapter 2. Materials and Methods
• 2.1. Cell culture
• 2.2. Fibroblast-derived extracellular vesicles isolation
• 2.3. Small RNA sequencing and bioinformatic analysis
• 2.4. Construction of FEV-IL4
• 2.5. Quantification of surface IL-4 on FEV
• 2.6. Characterization of FEV-IL4
• 2.6.1. Quantification of particles
• 2.6.2. Transmission electron microscopy (TEM) analysis
• 2.6.3. Dynamic light scattering (DLS) analysis
• 2.6.4. EV maker expression analysis
• 2.7. Effects of FEV-IL4 on cell proliferation
• 2.8. Stability and cytotoxicity assessment of FEV-IL4
• 2.9. Effects of FEV-IL4 on cell migration in fibroblasts and endothelial cells
• 2.10. Effects of FEV-IL4 on tube formation in endothelial cells
• 2.11. Effects of FEV-IL4 on repolarization in macrophages
• 2.12. qRT-PCR analysis
• 2.13. Immunofluorescence (IF) analysis
• 2.14. Animals
• 2.15. Establishment of a streptozotocin-Induced diabetic wound model and topical administration of FEV-IL4
• 2.16. Statistical analysis
• Chapter 3. Results and Discussion
• 3.1. Isolation of fibroblast-derived extracellular vesicles
• 3.2. miRNA profiling and functional enrichment analysis of FEV
• 3.3. Surface modification of FEV with IL-4
• 3.4. Characterization of FEV and FEV-IL4
• 3.5. Stability and cytotoxicity assessment of FEV-IL4
• 3.6. FEV-IL4 restores fibroblast proliferation and migration in HG conditions
• 3.7. Restoration of ECM synthesis and myofibroblast differentiation by FEV-IL4 in HG conditions
• 3.8. Restoration of endothelial function and angiogenesis by FEV in HG conditions
• 3.9. FEV-IL4–mediated repolarization of M1 macrophages toward the M2 phenotype
• 3.10. Optimization of STZ dosage and establishment of a diabetic mouse model
• 3.11. FEV-IL4 accelerates wound closure in STZ-induced diabetic mouse model
• 3.12. Dose-dependent therapeutic efficacy of FEV-IL4 in STZ-induced diabetic wounds
• Chapter 4. Conclusion
• Reference
• 국문초록