Bioengineered Extracellular Vesicle Hydrogel Modulating Inflammatory Microenvironment for Wound Management

Abstract

Chronic wounds, frequently arising from conditions like diabetes, trauma, or chronic inflammation, represent a significant medical challenge due to persistent inflammation, heightened infection risk, and limited treatment solutions. This study presents a novel bioengineered approach to promote tissue repair and improve the healing environment. We developed a bioactive hydrogel patch, encapsulated zeolitic imidazolate framework-8 (ZIF-8) into extracellular vesicles (EVs) derived from anti-inflammatory M2 macrophages, and synthesized ZIF@EV, then embedded it in the sodium alginate matrix. This hydrogel structure enables the controlled release of therapeutic agents directly into the wound site, where it stimulates endothelial cell proliferation and promotes new blood vessel formation. These processes are key components of effective tissue regeneration. Crucially, the EV-infused patch influences the immune response by polarizing macrophages towards an M2 phenotype, shifting the wound environment from inflammation toward regenerative healing. When applied in a murine model of chronic wounds, the EV hydrogel patch demonstrated notable improvements in healing speed, quality, and tissue integration compared to traditional approaches such as growth factor therapies and foam dressings. These promising findings suggest that this bioactive hydrogel patch could serve as a versatile, practical solution for chronic wound management, providing an adaptable platform that addresses both the biological and logistical needs of wound care in clinical settings.

Keywords: bioactive hydrogel; extracellular vesicles; tissue healing.

Figure 1

Synthesis of ZIF@EV–Gel ink. (A) Schematic illustration of the synthetic process of ZIF@EV nanoparticles and corresponding TEM images of the prepared ZIF@EV. Scale bar: 100 nm. (B) NTA of the prepared ZIF@EVs demonstrating the average size of around 180 nm. (C) Zeta potential of EV and ZIF@EV. (D) The gelation of ZIF@EV–Gel ink. (E) SEM image of the macrostructure of ZIF@EV–Gel ink. Scale bar: 3 μm. (F) The degradation curve of ZIF@EV forms the Gel. (G) The continuous release profile of ZIF@EV from Gel. ns denotes non-significant difference.

Figure 2

Angiogenesis ability of ZIF@EV. (A) Representative images of the transwell migration assay of HUVECs under the treatment of ZIF@EV. Scale bar, 200 μm. (B) Quantification of the migratory capacity of HUVECs. (C) Images of HUVEC migration under ZIF@EV treatment at different time points. Scale bar, 200 μm. (D) Corresponding quantification of HUVEC migration at different time points. (E) Formation of tubes by HUVECs with various treatments. (F) percentage area of vessels as a representation of tube formation capability in various groups (n = 3). ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 3

ZIF@EV induced macrophage polarization into M2 phenotype. (A) Representative image of macrophages under different treatments. Scale bar, 50 μm. (B) Flow cytometry analysis of CD206+ macrophages. (C) Flow cytometry analysis of CD86+ macrophages. (D) Quantification of CD206+ macrophages (n = 3). (E) Histogram analysis of CD206+ macrophages. (F) Quantification of the ratio of M1 and M2 macrophages (n = 3). (G) Relative protein expression of M1 and M2 macrophage markers after macrophages incubated with various treatments (n = 3). The color contour from blue to red denotes the intensified signal. **** p < 0.0001.

Figure 4

In vivo wound healing efficacy of ZIF@EV-Gel. (A) Representative image of wound size on different days. (B) Fluorescence images of cutaneous wounds extracted at 6 and 12 h after different treatments. The color contour from blue to red denotes the intensified signal. (C) Hematoxylin and eosin (HE) and Masson stain of the wounded skin on day 12. Scale bar = 100 μm. (D) Quantification of wound size in different groups. (E) Quantitative analysis of epidermal thickness. (F) Quantitative analysis of collagen deposition (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 5

Therapeutic mechanism of ZIF@EV-Gel ink. ZIF@EV-Gel accelerated wound healing in vivo. (A) Image of immunofluorescence staining to assess the formation of new blood vessels and the inflammatory status of the wounds. The blue color indicate nucleus and red color indicate the presence of CD31 and CD206 separately. (B) bactericidal effects of ZIF@EV-Gel. (C) Quantification of blood vessels with various treatments. (D) Quantitative analysis of activated M2 macrophages in epidermal tissues. **** p < 0.0001.