Gallium-modified gelatin nanoparticles loaded with quercetin promote skin wound healing via the regulation of bacterial proliferation and macrophage polarization

Abstract

Background: Wound healing is a complicated process involving multiple cell components and can help the re-establishment of the skin's barrier function. Previous studies have pointed out that bacterial infection and sustained inflammatory reactions are the main causes of the delay of wound closure and scar formation during wound healing. The effect of current approaches for scar-free wound repair still faces many challenges, and alternative therapeutic methods are urgently needed to be established. Methods: The basic characteristics of the new-designed nanoparticles were clarified through the characterization of the material. The biocompatibility of the nanoparticles, as well as its effect on fibroblast function, anti-bacterial capacity, inflammation suppressive role, and the underlying mechanism were further verified by a panel of biochemical assays in vitro. Ultimately, pre-clinical rat model was employed to testify its role in wound healing and scar formation in vivo. Results: Firstly, gallium-modified gelatin nanoparticles loaded with quercetin was successfully established, displaying good biocompatibility and facilitative effect on fibroblast function. In addition, the nanoparticles showed prominent anti-bacterial and inflammation-suppressive effects. What's more important, the nanoparticles could also induce the polarization of macrophages from M1 to M2 phenotype to exert its inflammatory inhibitory role through TGF-β/Smad signaling pathway. Ultimately, in vivo experiment showed that the nanoparticles could effectively promote wound repair and inhibit scar formation during the process of wound healing. Conclusion: Taken together, the new nanoparticles have good anti-bacterial and anti-scar formation effects and great potential in the field of skin wound repair, which provides a promising therapeutic strategy for wound treatment.

Keywords: Gallium; Quercetin; macrophage; nanoparticles; wound healing.

FIGURE 1

Characterization of QCT@GNP-Ga. (A) A schematic view of the design of gallium-modified gelatin nanoparticles loaded with quercetin. (B) Representative image TEM images of QCT@GNP and QCT@GNP-Ga, Scale bar = 20 nm. (C) Particle size of QCT@GNP and QCT@GNP-Ga. (D) FTIR spectra of QCT, Ga, QCT@GNP, and QCT@GNP-Ga. (E) QCT release from QCT@GNP and QCT@GNP-Ga.

FIGURE 2

Effect of QCT@GNP-Ga on the cell function of primary fibroblasts. (A) Live/dead staining of fibroblasts treated with QCT@GNP or QCT@GNP-Ga. Scale bar = 100 μm. (B) Cell viability of fibroblasts treated with QCT@GNP or QCT@GNP-Ga for 24, 48, and 72 h. (C) The uncovered aera and (D) representative images of wound-healing assay of fibroblasts treated with GNP, QCT@GNP or QCT@GNP-Ga. (E) Western blot analysis and (E) statistical results of COL1, ELN protein expressions in fibroblasts treated with GNP, QCT@GNP or QCT@GNP-Ga. (G) qRT-PCR analysis of COL1, ELN mRNA expressions in fibroblasts treated with GNP, QCT@GNP or QCT@GNP-Ga for 48 h. Data are shown as mean ± SD, ^* p < 0.05 compared with GNP group.

FIGURE 3

Anti-bacterial activity of QCT@GNP-Ga. (A, B) Images and (C, D) statistical results of colony forming unit (CFU) of E. coli and S. aureus treated with QCT@GNP or QCT@GNP-Ga. Data are shown as mean ± SD, ^* p < 0.05 compared with QCT@GNP group.

FIGURE 4

Induction of macrophages from M1 to M2 phenotype by QCT@GNP-Ga. (A) Representative images of FCM analysis of LPS induced RAW264.7 cells treated with GNP, GNP-Ga, or QCT@GNP-Ga. (B) EILSA analysis of TNF-α, MCP-1, TGF-β3, and IL-4 protein expressions in LPS induced RAW264.7 cells treated with GNP, GNP-Ga, or QCT@GNP-Ga. (C) qPCR analysis of TNF-α, MCP-1, iNOS, TGF-β3, IL-4, Arg-1 mRNA expressions in LPS induced RAW264.7 cells treated with GNP, GNP-Ga, or QCT@GNP-Ga. (D) Western blot analysis and (E) statistical results of ARG1 protein expressions in LPS induced RAW264.7 cells treated with GNP, GNP-Ga, or QCT@GNP-Ga. Data are shown as mean ± SD, ^* p < 0.05 compared with LPS + GNP group.

FIGURE 5

Regulation of polarization of macrophages by QCT@GNP-Ga through TGFβ/Smad pathway. (A) Western blot analysis, (B) statistical results, and (C) immunofluorescent staining analysis of TGFβR2 protein expressions in RAW264.7 cells treated with GNP or QCT@GNP-Ga. (D) Western blot analysis and (E) statistical results of TGFβR2, p-Smad2, and Smad2 protein expressions in RAW264.7 cells treated with GNP or QCT@GNP-Ga. (F) Representative images of FCM analysis of RAW264.7 cells treated with GNP or QCT@GNP-Ga or LY2109761. (G, H) Relative expression and secretion of TNF-α, MCP-1, TGF-β3 and IL-4 after the treatment with GNP or QCT@GNP-Ga or with LY2109761 in LPS-stimulated macrophage. Data are shown as mean ± SD, ^* p < 0.05, Scale bar = 20 μm.

FIGURE 6

In vivo repair effect of QCT@GNP-Ga on rat skin wounds. (A) Representative images of wound healing covered by GNP, GNP-Ga or QCT@GNP-Ga from 0 to 14 days and (D) quantitative results. (B) Representative HE staining images of tissue from healing wounds of GNP, GNP-Ga, or QCT@GNP-Ga group, Scale bar = 100 μm. (C) Representative TNF-α immunohistochemical staining images and (E) quantitative results of tissue from healing wounds of GNP, GNP-Ga or QCT@GNP-Ga group, Scale bar = 20 μm. Data are shown as mean ± SD, ^* p < 0.05 compared with GNP group.