Antimicrobial dual-crosslinked hydrogel synergizes bioengineered extracellular vesicles for enhanced diabetic wound healing

Abstract

Diabetic wound healing remains a major clinical challenge owing to impaired angiogenesis, prolonged inflammation, and bacterial infection. Stem cell-derived extracellular vesicles (EVs) offer a promising solution for improving diabetic wound healing. The biological activity of EVs can be increased by engineering modifications. Antimicrobial hydrogel dressings combined with bioengineered EVs, will provide a good solution to the problem of difficult healing of diabetic wounds. Therefore, this study aims to investigate the potential of BCL-2-engineered EVs to enhance wound healing in a diabetic mouse model. BCL-2 engineered adipose mesenchymal stem cells were constructed using the lentiviral embedding method, and analyzed their transcriptional changes through transcriptome sequencing. Their secreted EVs were isolated and characterized by proteomic sequencing. Integrating bioinformatics analysis, we found that BCL-2 engineered EVs may play a powerful role in angiogenesis and tissue repair. Furthermore, we developed an antimicrobial hydrogel based on epsilon-poly-lysine and hyaluronic acid to encapsulate them. The hydrogel-EVs system demonstrated a comprehensive promotion of wound healing, including increased angiogenesis, enhanced cell proliferation, reduced inflammation, and improved tissue architecture. These findings highlighted the potential of BCL-2-engineered EV-loaded antimicrobial hydrogels as a novel strategy for managing diabetic wounds, providing a promising alternative to overcome the limitations of current therapeutic approaches.

Keywords: Angiogenesis; Antimicrobial hydrogel; BCL-2; Diabetic wound; Extracellular vesicles.

Graphical abstract

Fig. 1

Schematic diagram of BCL-2 engineered extracellular vesicle-loaded antimicrobial hydrogel for comprehensive promotion of diabetic wound healing a.BCL-2 overexpressing ADSCs were constructed using lentiviral vectors, EVs were collected using differential ultracentrifugation, cells were analyzed by transcriptome sequencing technology, and EVs were analyzed by proteome sequencing. b. Hybrid hydrogel composed of glycidyl methacrylate-modified epsilon-poly-lysine (EPLMA), epsilon-poly-lysine (EPL), and oxidized hyaluronic acid (OHA), creating a dynamic and biocompatible matrix. c. The presence of EPL makes the hydrogel antimicrobial and anti-inflammatory, while providing barrier protection. The sustained release of BCL-2 engineered EVs has a powerful pro-angiogenic effect, promotes cell proliferation and migration, and comprehensively promotes the healing of diabetic wounds.

Fig. 2

Construction and transcriptome sequencing analysis of BCL-2-overexpressing ADSCs a. Fluorescence pictures of hADSCs after transfection of blank vector and BCL-2 vector, the green fluorescence represents that the gene has been successfully transfected into the cell. The scale is 200 μm. b. The fluorescence intensity of the cells was quantified by flow cytometry to determine the transfection efficiency. c. BCL-2 protein expression level using WB. d. Relative transcript levels of the BCL-2 gene compared to the ACTB gene were detected using qRT-PCR. e. Consistency test for samples. f. Gene enrichment analysis heat map. g. Venn diagram showing co-expressed genes. h. The volcano diagram shows the differentially expressed genes. i. From the results of GO enrichment analysis, the most significant 30 Terms were selected to be plotted in a scatter plot for presentation. j. The correlation of our signaling pathways of interest with BCL-2 was analyzed using GSEA enrichment analysis. ∗∗∗:P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Isolation and characterization of EVs and proteomics sequencing a. Transmission electron micrographs of two groups of EVs and results of NTA particle size analysis. The scale is 100 nm. b. WB detection of positive markers CD63, CD81 and negative marker Calnexin in EVs. c. Cluster analysis heat map. d. Heatmap of differential protein expression between the two groups of samples. e. Scatterplot of GO functional enrichment analysis of differential proteins showing significantly different Top30 terms. f. Co-expression Venn diagram of the proteome and transcriptome. g. Clustered heatmap analysis was performed on the FC values (log2FC) of the shared differential genes obtained in the above Venn analysis in both histologies. h. We visualize the shared GO features in a bar chart. By default, in the shared the top 10 with the smallest sum of GO function. i. Cluster analysis of Up-Up, Down-Down, Up-Down, and Down-Up in the shared differential genes is presented as a heat map. j. Cellular uptake of EVs and EVs^BCL−2. The scale is 50 μm.

Fig. 4

Synthesis and characterization of EPHA hydrogels a. Macroscopic photograph of EPHA hydrogels formation. b. Dynamic rheological studies. c, d. Rheological behavior of hydrogels at different angular frequencies and under different shear strains. e. Shear thinning characterization. f, g. Characterization of the adhesion capacity of hydrogels. h, i. Characterization of mechanical properties of hydrogels. j. Rheological properties of hydrogels under alternating strains. k. Scanning electron micrograph of the hydrogels. The scale is 100 μm. l, m. The antibacterial activity against S. aureus and E. coli was determined using the agar diffusion method. n, o. The electron microscopies of S. aureus (the scale is 1 μm) and E. coli (the scale is 2 μm). ∗∗∗:P < 0.001.

Fig. 5

Hydrogel system has potent angiogenesis-promoting effects in vitro a, e. Cell scratch assay for HUVEC. The scale is 200 μm. b, f. Transwell migration assay for HUVEC. The scale is 100 μm. c, g. Tubule formation assay. The scale is 200 μm. d, h. EdU cell proliferation assay. The scale is 100 μm ∗:P < 0.05, ∗∗:P < 0.01, ∗∗∗:P < 0.001.

Fig. 6

BCL-2 engineered EVs loaded EPHA hydrogels can comprehensive enhancement of diabetic wound healing a. Photographs of healing wounds in each group of diabetic mice were taken on days 0, 3, 6, 9, 12, 15. b. Statistical analysis of wound healing. c. H&E staining of mouse wounds on day 9 and day 15. The scale is 1000 μm. d. Statistical results of wound width in mice. e. Masson staining of mouse wounds on day 15. The scale is 1000 μm. f. Collagen deposition result statistics. g. Immunofluorescence staining of CD31 in mouse wounds on day 9 and day 15. The scale is 20 μm. h. Statistics of trauma vessel density. i. Immunochemical staining of TNF-α, the scale is 50 μm. j. Immunochemical staining of IL-6, the scale is 50 μm∗:P < 0.05, ∗∗:P < 0.01, ∗∗∗:P < 0.001.