Milk exosomes-mediated miR-31-5p delivery accelerates diabetic wound healing through promoting angiogenesis

Abstract

The refractory diabetic wound has remained a worldwide challenge as one of the major health problems. The impaired angiogenesis phase during diabetic wound healing partly contributes to the pathological process. MicroRNA (miRNA) is an essential regulator of gene expression in crucial biological processes and is a promising nucleic acid drug in therapeutic fields of the diabetic wound. However, miRNA therapies have limitations due to lacking an effective delivery system. In the present study, we found a significant reduction of miR-31-5p expression in the full-thickness wounds of diabetic mice compared to normal mice. Further, miR-31-5p has been proven to promote the proliferation, migration, and angiogenesis of endothelial cells. Thus, we conceived the idea of exogenously supplementing miR-31-5p mimics to treat the diabetic wound. We used milk-derived exosomes as a novel system for miR-31-5p delivery and successfully encapsulated miR-31-5p mimics into milk exosomes through electroporation. Then, we proved that the miR-31-5p loaded in exosomes achieved higher cell uptake and was able to resist degradation. Moreover, our miRNA-exosomal formulation demonstrated dramatically improved endothelial cell functions in vitro, together with the promotion of angiogenesis and enhanced diabetic wound healing in vivo. Collectively, our data showed the feasibility of milk exosomes as a scalable, biocompatible, and cost-effective delivery system to enhance the bioavailability and efficacy of miRNAs.

Keywords: Milk-derived exosomes; angiogenesis; diabetic wound; drug delivery; miR-31-5p.

Figure 1.

Schematic image. (A) Isolation of milk-derived exosomes (mEXO) and preparation of miR-31-5p-loaded exosomes (mEXO-31). (B) Action mechanism of mEXO-31 in vitro. (C) mEXO-31 treatment on mice model.

Figure 2.

Overexpression of miR-31-5p promoted endothelial cell proliferation, migration, and angiogenesis. (A) Relative expression levels of miRNAs in wound tissue compared between normal mice and diabetic mice. n = 5, **p < .01 vs. normal mice. (B,C) EdU assay analysis of the proliferation rate of HUVECs treated with mimic-NC/miR-31-5p mimics. The proliferative cells and cellular nuclei were stained with red and blue colors. n = 3, **p < .01 vs. NC. Scale bar, 50 μm. (D,E) Images of migrated HUVECs in each group. n = 3, *p < .05 vs. NC. Scale bar, 50 μm. (F,G) Images of tube formation of HUVECs in each group. n = 3, ***p < .001 vs. NC. Scar bar, 100 μm. Data were presented as mean ± SD. Unpaired Student’s t-test was used.

Figure 3.

HIF1AN was a direct target of miR-31-5p. (A) Venn diagram depicting the number of potential targets of miR-31-5p predicted by three algorithms. (B) RT-PCR analysis of HIF1AN mRNA expression in HUVECs transfected with mimic-NC/miR-31-5p mimics. n = 3, **p < .01 vs. NC (C,D) Western blotting analysis of HIF1AN protein expression in HUVECs in each group. n = 3, **p < .01 vs. NC. (E) Schematic drawing of the putative binding sites or mutations of miR-31-5p in HIF1AN mRNA 3′UTR. (F) Luciferase activity of each group was detected at 48 h post-transfection. n = 3, ns no significant vs. NC + MUT; **p < .01 vs. NC + WT. (G,H) Western blotting analysis of HIF1AN protein expression in wound tissue from normal and diabetic mice. n = 3, *p < .05 vs. normal mice. Data were presented as mean ± SD. Unpaired Student’s t-test was used in (B,D,H). One-way ANOVA with Tukey post-hoc test was used in (F).

Figure 4.

miR-31-5p/HIF1AN axis regulated endothelial cell function. (A,B) Western blotting analysis of HIF1AN protein expression in HUVECs in different treated groups. n = 3, ****p < .0001, ^##p < .01 vs. NC + empty; ^$$p < .01 vs. miR-31-5p + empty. (C,D) EdU assay analysis of the proliferation rate of HUVECs in each group. The proliferative cells and cellular nuclei were stained with red and blue colors. Scale bar, 50 μm. n = 3, ***p < .001, ^####p < .0001 vs. NC + empty; p < .0001 vs. miR-31-5p + empty. (E,F) Images of migrated HUVECs in each group. Scar bar, 50 μm. n = 3, **p < .01, ^####p < .0001 vs. NC + empty; p < .0001 vs. miR-31-5p + empty. (G,H) Images of tube formation of HUVECs in each group. Scar bar, 100 μm. n = 3, **p < .01, ^####p < .0001 vs. NC + empty; p < .0001 vs. miR-31-5p + empty. Data were presented as mean ± SD. One-way ANOVA with Tukey post-hoc test was used.

Figure 5.

Preparation and characterization of mEXO-31. (A) RT-PCR analysis of relative miR-31-5p levels in mEXO, mEXO-NC, and mEXO-31. n = 3, ****p < .0001 vs. mEXO-NC. (B) Confocal images showed successful loading of miR-31-5p into mEXO. Red and green fluorescence represented mEXO and miR-31-5p mimics, respectively. Scale bar, 10 µm. (C) RT-PCR analysis of remaining miR-31-5p in each group. n = 3, ns no significant, ****p < .0001 vs. free mimics mixed with mEXO. (D) NTA identified the size distribution of mEXO and mEXO-31. (E) TEM identified the morphology of mEXO and mEXO-31. Scale bar, 50 µm. (F) Western blotting analysis of exosome specific markers including CD9, CD81, HSP70, and TSG101 of mEXO and mEXO-31. Data were presented as mean ± SD. One-way ANOVA with Tukey post-hoc test was used in (A). Unpaired Student’s t-test was used in (C).

Figure 6.

mEXO-31 delivered miR-31-5p into cells more efficiently. (A) Confocal images showed the delivery of miR-31-5p into HUVECs in each group. Blue, red, and green fluorescence represented the cellular nuclei, mEXO, and miR-31-5p, respectively. Scale bar, 50 µm. (B) RT-PCR analysis of relative miR-31-5p levels in HUVECs in different treated groups. (C,D) Western blotting analysis of HIF1AN protein expression in HUVECs in different treated groups. n = 3, ns: no significant, **p < .01, ****p < .0001. Data were presented as mean ± SD. One-way ANOVA with Tukey post-hoc test was used.

Figure 7.

mEXO-31 promoted endothelial cell proliferation, migration, and angiogenesis in vitro. (A,B) EdU assay analysis of the proliferation rate of HUVECs in different treated groups The proliferative cells and cellular nuclei were stained with red and blue colors. Scale bar, 50 μm. (C,D) Images of migrated HUVECs in each group. Scar bar, 50 μm. (E,F) Images of tube formation of HUVECs in each group. Scar bar, 100 μm. n = 3, ns: no significant, ***p < .001, ****p < .0001. Data were presented as mean ± SD. One-way ANOVA with Tukey post-hoc test was used.

Figure 8.

mEXO-31 accelerated diabetic wound healing in vivo. (A,B) Representative images of wound closure in a wound model at the dorsum of diabetic mice at day 0, 5, 10, and 15 post-wounding. n = 5, ns: no significant, *p < .05 vs. mimic-NC; ^###p < .001 vs. mEXO-NC; ^$p < .05 vs. miR-31-5p. (C,D) H&E staining analysis of wound sections at day 15 post-wounding. The single-headed arrows indicate the un-epithelialized areas. Scar bar, 500 μm. (E,F) Masson staining evaluated the collagen deposition at day 15 post-wounding. Scar bar, 100 μm. (G–J) Immunofluorescence staining of cellular nuclei (DAPI, blue), CD31 (red), and α-SMA (green) of wound bed at day 15 post-wounding. Scar bar, 50 μm. n = 3, ns: no significant, *p < .05, **p < .01, ***p < .001, ****p < .0001. Data were presented as mean ± SD. One-way ANOVA with Tukey post-hoc test was used.