Glycyrrhizic Acid Hydrogel Microparticles Encapsulated with Mesenchymal Stem Cell Exosomes for Wound Healing

Abstract

Hydrogel microparticles have been proved to be curative to diabetic wounds. Current trends focus on the integration of bioactive matrix and their smart stimulus-responsive release to meet the complex demand of regeneration in diabetic wound. In this paper, we present novel stem cell exosome-encapsulated Chinese herb glycyrrhizic acid (GA) hydrogel microparticles for wound healing. The integrated GA endows the hydrogel microparticles with antibacterial properties, while the encapsulated exosomes impart them with pro-angiogenesis ability. In addition, as the black phosphorus is incorporated into these hybrid hydrogel microparticles, the release profile of GA and exosomes could be controllable under near-infrared irradiation due to the excellent photothermal effect of black phosphorus and the reversible phase transformation properties of GA. Based on these features, we have demonstrated that these microparticles can effectively kill bacteria, scavenge free radical, and promote angiogenesis from in vitro experiments. Besides, they could also markedly accelerate the wound healing process by down-regulating inflammation and promoting collagen deposition and angiogenesis in bacteria-infected in vivo diabetic wound. These results indicate that the proposed exosome-integrated GA hydrogel microparticles present great potential for clinical diabetic wound treatment.

Fig. 1.

(A) Schematic diagram of microfluidic preparation of exosome-encapsulated Chinese herb hydrogel microparticles. (B) Healing process of the diabetic wound accelerated by hydrogel microparticles with exosomes and Chinese herb.

Fig. 2.

Preparation and characterization of BMSCs-exo-encapsulated GA hydrogel microparticles. (A) TEM image of BMSCs-exo. (B and C) Concentration and size distribution of BMSCs-exo. (D) Real-time generation of droplets in the microfluidic channel at the outer phase flow rate of (i) 1 ml/h, (ii) 2 ml/h, (iii) 3 ml/h, (iv) 4 ml/h, and (v) 5 ml/h. Inner phase flow rate: 0.3 ml/h. (E) Optical image of spherical droplets. Scale bar, 600 μm. (F) Optical image of photopolymerized microparticles. (G) Relationship of droplet size with the change of flow rate. (H) Histogram of size distribution of microparticles. (I) SEM image of the microparticles. Scale bars, 100 nm (A), 1 mm (D), 600 μm (E), 300 μm (F), and 50 μm (I).

Fig. 3.

Multifunction evaluation of GA microparticles encapsulated with BMSCs-exo. (A) Plot of the temperature variations of microparticles containing different concentrations of BP under NIR irradiation. (B) Plot of the temperature variations of microparticles during 5 cycles. (C) Characterization of DPPH clearance activity of GA microparticles containing BMSCs-exo. (D) Fluorescent images of cells treated with different microparticles during 3 d. (E) Quantified viability of cells of different groups. n = 3 for each group. (F) Hemolysis assessment of microparticles. Scale bar, 500 μm (D).

Fig. 4.

Characterization of scratch assay and in vitro antibacterial experiment. (A) Representative images of the scratch wound. (B) Images of bacterial colonies on culture plates of different groups. (C and D) Fluorescent images of E. coli and S. aureus stained by SYTO (green) and propidium iodide (PI) (red). (E) Quantification of scratch test (n = 3). (F) Reduction rates of E. coli and S. aureus in different groups. Scale bars, 400 μm (A), 2 cm (B), and 50 μm (C). **P < 0.01; ns, not significant.

Fig. 5.

Effects of different treatments on the infected diabetic wounds. (A) Representative images of the wounds in different groups. (B) H&E staining images of skin tissues. i, ii, iii, iv, and v in (B) represent control group, BM group, Drug group, BM-Drug group, and BM-Drug-NIR group,respectively. (C) Data analysis of wound closure in different groups. (D) Quantitative analysis of the granulation tissue width on day 11. Scale bars, 1 cm (A) and 1 mm (C). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.

Study of biological mechanisms of GA microparticles with BMSCs-exo to promote wound healing. (A) Masson staining of the regenerated tissues in different groups at day 11. (B) IL-6 immunohistochemistry staining images of the wounds in 5 groups at day 11. (C) Double immunofluorescent staining of CD31 (green) and α-SMA (red) at day 11. i, ii, iii, iv, and v in (A) to (C) represent control group, BM group, Drug group, BM-Drug group, and BM-Drug-NIR group,respectively. (D) Analysis of collagen deposition of different groups. (E) Analysis of relative expression of IL-6 of different groups. (F) Analysis of CD31/α-SMA of different groups. Scale bars, 50 μm (A to C). *P < 0.05, **P < 0.01.