Lemon-derived nanoparticle-functionalized hydrogels regulate macrophage reprogramming to promote diabetic wound healing

Abstract

The orderly regulation of immune inflammation and promotion of the regeneration of skin vessels and fibers are key to the treatment of diabetic skin injury (DSI). Although various traditional polypeptide biological dressings continue to be developed, their efficacy is not satisfactory. In recent years, plant-to-mammal regulation has provided an effective approach for chronic wound management, but the development of effective plant-based treatments remains challenging. The development of exosomes from Chinese herbs is promising for wound healing. In this study, plant exosomes derived from lemons (Citrus limon) were extracted, and their biological efficacy was verified. Lemon exosomes regulated the polarization reprogramming of macrophages, promoted the proliferation and migration of vascular endothelial cells and fibroblasts, and thus promoted the healing of diabetic wounds. To solve the problems of continuous drug delivery and penetration depth, Lemon Exosomes were loaded into a hydrogel constructed of Gelatin Methacryloyl (GelMA) and Dialdehyde Starch (DAS) that closely fits to the skin, absorbs water, swells, and is moist and breathable, effectively promoting the sustained and slow release of exosomes and resulting in excellent performance for diabetic wound healing. Our GelMA-DAS-Lemon Exosomes hydrogel (GelMA/DAS/Exo hydrogel) patch represents a potentially valuable option for repairing diabetic wounds in clinical applications.

Keywords: Angiogenesis; Diabetic wound healing; Lemon exosomes; Macrophage reprogramming; Network pharmacology.

Fig. 1

GelMA/DAS/Exo hydrogel was used as a dressing for wound healing in DSI. (A) Preparation of the lemon exosome hydrogel. (B) The lemon exosome hydrogel promoted diabetic wound healing by regulating macrophage polarization and promoting fibroblast and vascular endothelial cell proliferation

Fig. 2

RNA sequencing revealed potential immune mechanisms of diabetic skin damage. (A) Three samples were diabetic skin lesions, and 3 samples were controls. The whole gene expression of each sample was normalized. (B) There were obvious differences between the two groups of samples, and there are no confounding factors identified through PCA. (C) Heatmap showing the expression of all differential genes between the two groups of samples. (D) Differentially expressed genes were screened using the following criteria: |log2 (FC) |>1 and FDR < 0.05. (E-G) GO and KEGG pathway enrichment analyses revealed that DSI was closely related to inflammation, oxidative stress, cell proliferation and apoptosis. (G) Intercellular communication of pathway molecules

Fig. 3

Non-targeted metabolomics of lemon juice and lemon exosomes. (A-B) Lemon juice non-targeted metabolomics pie and bar graphs; (C-D) Lemon exosomes non-targeted metabolomics pie and bar graphs; (E-F) Comparison of vitamin C and flavonoids in lemon juice and lemon exosomes

Fig. 4

Potential therapeutic relationship between lemon-related pharmaceutical active ingredients and DSI. (A) Potential target gene crossover between lemon and diabetic skin lesions. (B-C) Lemon and potential target gene networks in diabetic skin lesions. (D) KEGG pathway enrichment map of potential target genes

Fig. 5

Preparation and characterization of GelMA/DAS/Exo hydrogel. (A) Diagram of the synthesis of hydrogels. (B) Physical picture of Lemon Exosomes. (C) Lemon Exosome TEM. (D) Lemon Exosome NTA. (E-H) Lemon Exosomes could be targeted by macrophages, fibroblasts and HUVECs and could be uniformly loaded in a hydrogel. (I) ¹H NMR spectra of gelatin and GelMA. (J) IR spectra of the GelMA, DAS, and GelMA/DAS hydrogels. (K-L) SEM images of the GelMA/DAS and GelMA/DAS/Exo hydrogels

Fig. 6

Rheological properties and shape adaptability of the GelMA/DAS/Exo hydrogel. (A) Rheological analysis of the GelMA/DAS and GelMA/DAS/Exo hydrogels before and after UV curing in frequency scan mode. (B) Mean storage modulus of the GelMA/DAS and GelMA/DAS/Exo hydrogels before and after UV curing at 1 Hz, with error bars indicating the standard deviation (SD) (*P < 0.05, **P < 0.01, ***P < 0.001, n = 3). (C) Scanning tests of strain amplitudes ranging from 0.001–300% at a fixed angular frequency (10 rad s-1). (D) Images showing shape changes in the GelMA/DAS hydrogel. (E) Sequential strain testing of the cured GelMA/DAS hydrogel ranging from a small strain (1%) to large strain (300%). (F) Image showing the self-healing ability of the cured GelMA/DAS hydrogel. (G) Shear-thinning experiment of the GelMA/DAS hydrogel. Statistical differences were determined via one-way ANOVA with Bonferroni’s multiple comparison test (*P < 0.05, **P < 0.01, and ***P < 0.001)

Fig. 7

Adhesive properties of the hydrogels. (A) Schematic diagram of lap-shear experiments involving hydrogels. (B) Lap-shear adhesion curves for the GelMA/DAS and GelMA/DAS/Exo hydrogels applied to pig skin. (C) Lap shear adhesion strengths of the GelMA/DAS and GelMA/DAS/Exo hydrogels applied to pig skin. (D) Lap-shear adhesion curves for UV-cured GelMA/DAS and GelMA/DAS/Exo hydrogels applied to pig skin. (E) Lap shear adhesion strengths of UV-cured GelMA/DAS and GelMA/DAS/Exo hydrogels applied to pig skin. (F) In situ bonding image of the hydrogel to pig skin. Statistical differences were determined via one-way ANOVA with Bonferroni’s multiple comparison test (*P < 0.05, **P < 0.01, and ***P < 0.001)

Fig. 8

Biocompatibility of the GelMA/DAS/Exo hydrogel. (A) Schematic diagram of the coculture of Lemon Exosome hydrogels with cells. (B) Live/dead staining of HUVECs cultured on each sample surface for 1 day. Live cells are stained green, and dead cells are stained red. The scale bar represents 200 μm. (C) Quantitative analysis of the live/dead staining results (n = 3). (D) The results of the CCK-8 assay demonstrated that the cell viability surpassed 80% for each sample for 48 h after seeding (n = 3). (E) Cytoskeleton images showing the adhesion of HUVECs cultured in each group after 3 days. The scale bar represents 100 μm. (F) Quantitative analysis of the cell spread area (n = 3). (G) Cytoskeleton images showing the adhesion of L929 cells cultured in each group after 3 days. The scale bar represents 100 μm. (H) Quantitative analysis of the cell spread area (n = 3). (I) Live/dead staining of L929 cells cultured on each sample surface for 1 day. Live cells are stained green, and dead cells are stained red. The scale bar represents 200 μm. (J) Quantitative analysis of the live/dead staining results (n = 3). (K) The results of the CCK-8 assay demonstrated that the cell viability surpassed 80% for each sample for 48 h after seeding (n = 3). (L) Hemolysis test of the materials. (M) Quantitative analysis of the hemolysis test results. Statistical differences were determined via one-way ANOVA with Bonferroni’s multiple comparison test (*P < 0.05, **P < 0.01, and ***P < 0.001)

Fig. 9

In vivo biocompatibility of lemon exosome hydrogels. (A) Pathological examinations of the heart, liver, kidney and lung of rats in the control and GelMA/DAS/Exo groups. (B) Serum levels of biomarkers reflecting liver function and kidney function and blood cell parameters in rats treated with GelMA/DAS/Exo hydrogels (n = 3, *p < 0.05, **p < 0.01, and ***p < 0.001)

Fig. 10

The GelMA/DAS/Exo hydrogel stimulated the polarization of RAW 264.7 cells from the M1 phenotype to the M2 phenotype through the NF-κB pathway. (A) An illustration of how GelMA/DAS/Exo modulates macrophage polarization from the M1 phenotype to the M2 phenotype. (B) Bar graph showing the RT‒qPCR results for the gene expression of anti-inflammatory cytokines (Arg-1 and IL-10) and proinflammatory cytokines (iNOS and TNF-α) in each group (n = 3). (C) Heatmap showing the RT‒qPCR results for the gene expression of anti-inflammatory cytokines (Arg-1 and IL-10) and proinflammatory cytokines (iNOS and TNF-α) in each group (n = 3). (D) IF images showing the number of Arg1 (red)-positive and iNOS (red)-positive RAW264.7 cells cultured on each hydrogel. The scale bar represents 50 μm. (E) Quantitative analysis of the fluorescence intensity of Arg-1 (n = 3). (F) Quantitative analysis of the fluorescence intensity of iNOS (n = 3). (G) WB analysis of Arg-1 and iNOS protein expression. (H) The protein band intensities of Arg-1 and iNOS were quantified using ImageJ (n = 3). (I) Flow cytometry analysis of the proportions of M1 macrophages (labeled with F4/80⁺CD86⁺) and M2 macrophages (labeled with F4/80 + CD206+); (F) percentages of F4/80⁺CD86⁺ and F4/80⁺CD206⁺ cells (n = 3); significant differences were determined via one-way ANOVA with Bonferroni’s multiple comparison test (*P < 0.05, **P < 0.01, and ***P < 0.001)

Fig. 11

Status of angiogenesis. (A) Vessel formation analysis of HUVECs cultured on different materials for 12 h. (B) Quantitative analysis of angiogenesis (n = 3). (C) IF images showing the number of VEGFA (red)-positive HUVECs cultured on each hydrogel. The scale bar represents 50 μm. (D) Quantitative analysis of the fluorescence intensity of VEGFA (n = 3). (E) IF images showing the number of CD31 (red)-positive HUVECs cultured on each hydrogel. The scale bar represents 50 μm. (F) Quantitative analysis of the fluorescence intensity of CD31 (n = 3). (G) WB analysis of CD31 and VEGFA protein expression. (H) The protein band intensities of CD31 and VEGFA were quantified using ImageJ (n = 3) Statistical differences were determined via one-way ANOVA with Bonferroni’s multiple comparison test (*P < 0.05, **P < 0.01, and ***P < 0.001)

Fig. 12

HUVECs and L929 cell migration and the fibrillogenesis were studied in vitro. (A) Wound-healing migration assay of HUVECs on each hydrogel at different time points. The scale bar represents 200 μm. (B) Quantitative analysis of the scratch area in the bare region at 0 and 12 h (n = 3). (C) Wound-healing migration assay of L929 cells on each hydrogel at different time points. The scale bar represents 200 μm. (D) Quantitative analysis of the scratch area in the bare region at 0 and 12 h (n = 3). (E) IF displaying the number of α-SMA (green)-positive L929 cells cultured in each group. The scale bar represents 50 μm. (F) Quantitative analysis of the fluorescence intensity of α-SMA (n = 3). (G) WB analysis of α-SMA protein expression. (H) The protein band intensity of α-SMA was quantified using ImageJ (n = 3). (I) Transwell experiment. The scale bar represents 200 μm. (J) Quantitative analysis of transwell experiment (n = 3). Statistical differences were determined via one-way ANOVA with Bonferroni’s multiple comparison test (*P < 0.05, **P < 0.01, and ***P < 0.001)

Fig. 13

Hemostatic properties of the GelMA/DAS/Exo hydrogel. (A) Schematic diagram of rat tail hemostasis. (B-C) Gross hemostatic map of the rat tail and quantitative analysis. (D) Schematic diagram of liver hemostasis. (E-F) Liver hemostasis and quantitative analysis. Statistical differences were determined via one-way ANOVA with Bonferroni’s multiple comparison test (*P < 0.05, **P < 0.01, and ***P < 0.001)

Fig. 14

The GelMA/DAS/Exo hydrogel promote diabetic wound healing. (A) Diabetic skin damage modeling diagram. (B) Visual observation of DSI. (C) H&E images of the skin of diabetic model rats after different treatments. Scale bar, 2 mm at 2 x magnification and 400 μm at 10 x magnification. (D) Representative image of Masson’s trichrome staining. Scale bar, 2 mm at 2 x magnification and 400 μm at 10 x magnification

Fig. 15

The GelMA/DAS/Exo hydrogel can reprogram macrophage polarization from the M1 to M2 phenotype and angiogenesis. (A) Immunohistochemistry of Arg-1 and iNOS in different groups. (B) Immunohistochemistry of CD31 and VEGFA in different groups. Scale bar, 2 mm at 2 x magnification, 100 μm at 40 x magnification, and 25 μm at the highest magnification

Fig. 16

The GelMA/DAS/Exo hydrogel can collagen fiber formation. (A) Immunohistochemistry of α-SMA in different groups. Scale bar, 2 mm at 2 x magnification, 100 μm at 40 x magnification, and 25 μm at the highest magnification