Preparation and characterization of HCFD nanoparticles
To confirm that FAP1 and His were successfully attached to CTs, we performed infrared spectroscopy on CTs, HC (His-CTs), and HCF (His-CTs-FAP1 peptide). Compared to CTs, HC exhibits an enhanced absorption peak at 1085 cm−1 in its infrared spectrum. This peak may be associated with vibrations of glycosyl or His residues, indicating the formation of new bonds [26,27,28]. The characteristic peak shift at 1525 cm−1 and the enhanced absorption peak at 1630 cm−1 correspond to the amide I band (C = O stretching vibration), which typically indicates the formation or strengthening of amide bonds. This phenomenon is commonly observed during peptide-polymer conjugation and serves as a chemical signature for the binding of proteins or peptides to carriers such as CTs. The observed enhancement may originate from the covalent linkage between His peptides and CTs (Fig. 1A) [29]. Compared to HC, the infrared spectrum of HCF exhibits reduced intensity at the characteristic peak at 1064 cm⁻¹. This reduction may be attributed to the formation of an intermolecular hydrogen bond between the guanidino group (-NH-C(= NH)NH₂) of the arginine residue in the FAP1 peptide and the phenolic hydroxyl group (-OH) of tyrosine (Fig. 1A) [30]. We further validated this effect using nuclear magnetic resonance (NMR) spectroscopy. The results revealed that compared to the HC group, the NMR spectra of the HCF group exhibited a new peak at 2.6 ppm, likely originating from the introduction of methylene or methyl groups from the FAP1 peptide (Fig. 1B) [31]. These findings suggested that FAP1 and His were successfully conjugated to CTs.
We constructed the plasmid pGPU6/GFP/Neo MCP-1-shRNA to stably knock out MCP-1. To verify whether pDNA was successfully loaded onto HCF nanoparticles, we measured the zeta potential of CTs, CTs-pDNA, HC-pDNA, and HCF-pDNA (HCFD). Unlike CTs, CTs-pDNA, HC-pDNA, and HCFD exhibited a negative potential (Fig. 1C), consistent with the Phase Image (Fig. S2). We further measured the particle size changes of CTs, HC, and HCF, and found that that of HCFD nanoparticles primarily ranged from 50 to 100 nm (Fig. 1D). The morphological alterations of pDNA following binding to CTs, HC, and HCF were investigated using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In contrast to CTs, CTs–pDNA, and HC–pDNA, HCFD produced spherical particles that were mainly between 60 and 90 nm in diameter, which is in line with the particle size range that DLS detected (Fig. 1E, F). The above results indicated that compared to CTs and HC, HCF is more readily capable of forming spherical carriers with pDNA. The main particle size of these spherical carriers is less than 100 nanometers, which helps them enter cells more effectively and perform their functions.
Preparation and characterization of HCFD nanoparticles. (A) Infrared spectroscopy analysis of CTs, HC, and HCF. (B) Nuclear magnetic resonance hydrogen spectroscopy analysis of HC and HCF. (C) Zeta potential measurement of CTs, CTs–pDNA, HC–pDNA, and HCFD. (D) Particle size distribution of CTs, CTs–pDNA, HC–pDNA, and HCFD. (E) SEM analysis of CTs, C–pDNA, HC–pDNA, and HCFD. (F) TEM analysis of CTs, C–pDNA, HC–pDNA, and HCFD
Synthesis and physical and chemical characterization of the GPHCFD hydrogel
We performed infrared spectroscopy on Gelma (Gel), GP, GPHCF, and GPHCFD to verify the successful formation of the GPHCFD hydrogel. In the mid-infrared region, the characteristic peak at 1510 cm⁻¹ corresponded to the vibration of the benzene ring skeleton, indicating the successful incorporation of benzene-containing components into the hydrogel [32] (Fig. 2A). The infrared spectra of GPHCF and GPHCFD showed no changes in comparison with that of GP, which was attributable to the excessive number of groups attached to GP in GPHCF and GPHCFD (Fig. 2A) [33].
In contrast to GelMA, GP showed additional peaks at 7.7 ppm and 7.5 ppm, which correspond to the ortho and para protons of the PBA benzene ring, respectively, according to a nuclear magnetic resonance hydrogen spectroscopy study [34]. The shift at 7.5 ppm indicates enhanced polarity of the B-O bond, leading to a conjugation effect, which is consistent with the expected formation of a borate ester bond (B-OR) with PBA (Fig. 2B). This reflects the formation process of dynamic covalent bonds. At the same time, the enhanced peaks at 3.1, 3.6, and 3.8 ppm in the H3-H6 proton region of the CTs sugar ring may indicate intermolecular interactions, such as hydrophobic effects, consistent with the mechanism of chitosan binding to FAP1 (Fig. 2B) [34]. Furthermore, the high-molecular-weight matrix background obscures the pDNA signal; therefore, there is no discernible difference between the GPHCFD and GPHCF spectra [35]. The aforementioned findings suggested the successful synthesis of GPHCFD.
We performed rheological characterization of Gel, GP, GPHCF, and GPHCFD hydrogels. Shear rate scanning tests showed that within the γ̇ = 1–10 s−1 range (corresponding to injection rates of 0.1-1 mL/min), GPHCF and GPHCFD exhibited significant shear thinning behavior, with viscosity (η) maintained at 7–20 mPa·s (Fig. 2C), confirming their injectability [36]. The development of a stable gel network was demonstrated by oscillation time scans. It showed that GPHCFD’s storage modulus (G’) remained higher than its loss modulus (G’’) (Fig. 2D). The curing time of GPHCFD hydrogel, influenced by the added CTs, ranged from 50 to 80 seconds (Fig. 2E, G). Frequency scanning revealed that the G’ value of GPHCFD hydrogel is below 1000 Pa (Fig. 2F), approaching the mechanical properties of soft tissues (e.g., adipose tissue ~ 300 Pa), which is advantageous for subsequent biomedical applications [37].
Fluorescence confocal microscopy was used to generate 3D images of fluorescently labeled HCFD nanoparticles in GPHCFD hydrogels, demonstrating that HCFD nanoparticles had successfully attached to the GPHCFD hydrogels (Fig. 2H). Atomic force microscopy (AFM) analysis of 2D and 3D images of the Gel, GP, GPHCF, and GPHCFD hydrogels revealed that only GPHCFD exhibited a distinct granular texture. GPHCFD contained a large number of HCFD nanoparticles (Fig. 2I, J), with particle sizes ranging from 50 to 90 nm (Fig. S3). SEM analysis was used to examine the morphology of the Gel, GP, GPHCF, and GPHCFD hydrogels. Adding a pore-forming agent during GP synthesis and linking HCF and HCFD resulted in GPHCF and GPHCFD hydrogels exhibiting larger pores (Fig. 2K). The synthesized GPHCFD hydrogel facilitated the effective release of HCFD nanoparticles. These results suggested the successful assembly of GPHCFD hydrogels (loaded HCFD nanoparticles). These results also indicated that the GPHCFD hydrogels were suitable for subsequent nanoparticle delivery and cell experiments.
Synthesis and physicochemical characterization of the GPHCFD hydrogel. (A) Infrared spectroscopy analysis of the Gel, GP, GPHCF, and GPHCFD hydrogels. (B) Nuclear magnetic resonance hydrogen spectroscopy analysis of the Gel, GP, GPHCF, and GPHCFD hydrogels. (C) Viscosity measurements of the Gel, GP, GPHCF, and GPHCFD hydrogels. (D) Frequency sweep analysis of the Gel, GP, GPHCF, and GPHCFD hydrogels. (E) Photocuring time analysis of the Gel, GP, GPHCF, and GPHCFD hydrogels. (F) Self-healing analysis of the Gel, GP, GPHCF, and GPHCFD hydrogels. (G) Typical images of the Gel, GP, GPHCF, and GPHCFD hydrogels before and after curing. (H) Fluorescence confocal microscopy of nanoparticles in GPHCFD hydrogel. (I, J) AFM analysis of the Gel, GP, GPHCF, and GPHCFD liquid hydrogels. (K) SEM images of the Gel, GP, GPHCF, and GPHCFD hydrogels
Biological function testing of ERS-responsive GPHCFD hydrogels
Fluorescence confocal imaging of fibroblasts cultured on GPHCFD hydrogels (2D/3D) showed multidirectional growth (green) and minimal cell death (red) (Fig. 3A), indicating good biocompatibility. The GPHCD hydrogel exhibited the strongest promotion of fibroblast cell proliferation when fibroblast cells were induced to undergo ERS in vitro using Tg. Only GPHCFD and GPHCF hydrogels demonstrated a significant promotional effect on fibroblast proliferation. Therefore, we selected GPHCD and GPHCF as the experimental groups and GP as the control group for the subsequent experiments (Fig. 3B). These results suggest that excessive ERS can activate GPHCFD hydrogel release plasmid, knock down the MCP-1 gene in fibroblasts, and inhibit the expression of MCP-1. Induce the ERS in fibroblasts using Tg concentrations of 0 mM, 10 mM, 50 mM, and 100 mM. Using flow cytometry, assess the fluorescence intensity of cells transfected with a GFP-labeled plasmid. The GFP fluorescence intensity was significantly increased in the 50 mM and 100 mM Tg induction groups, with the highest GFP fluorescence intensity observed in the 100 mM Tg induction group. This indicated that as ERS increased in fibroblasts, plasmid transfection efficiency was significantly enhanced (Fig. S4). Among the four groups of Tg-induced fibroblasts that underwent ERS–the non-hydrogel group, GP group, GPHCF group, and GPHCFD hydrogel group–the GPHCFD hydrogel group had the fastest migration speed of fibroblasts (Fig. 3C, D).
Fibroblast (F) and macrophage (M) cell lines expressing ESRE mCherry were established. When excessive ERS occurs, both fibroblasts and macrophages show positive expression of ESRE mCherry. In the coculture system, CD11b+ cells represented macrophages, and CD11b− cells represented fibroblasts. The following groups were analyzed: PBS without Tg induction, Tg-induced fibroblasts and macrophages, Tg-induced macrophages, GP hydrogel-treated Tg-induced fibroblasts and macrophages, GPHCF hydrogel-treated Tg-induced fibroblasts and macrophages, and GPHCFD hydrogel-treated Tg-induced fibroblasts and macrophages. The proportion of ESRE mCherry+CD11b+ cells was significantly higher in Tg-induced fibroblasts and macrophages and in Tg-induced macrophages, whereas the proportion of ERSE mCherry+CD11b+ cells was very low in the non-Tg-induced PBS group and in hydrogel-treated Tg-induced fibroblasts and macrophages. This finding indicated that the GPHCFD hydrogel was ERS-responsive and could only exert its ERS-inhibitory function in the presence of fibroblasts. The GPHCFD hydrogel targeted fibroblasts and inhibited ERS in both fibroblasts and macrophages (Fig. 3E, F). The proportion of ERSE mcherry+CD11b− cells was high in the Tg-induced fibroblast and macrophage groups, the GP hydrogel-treated Tg-induced fibroblast and macrophage groups, and the GPHCF hydrogel-treated Tg-induced fibroblast and macrophage groups. In contrast, the proportion of ERSE mcherry+CD11b− cells was low in the non-Tg-induced PBS group and the GPHCDFD hydrogel-treated Tg-induced fibroblast and macrophage group (Fig. 3E, G). While the non-Tg-induced PBS group and the GPHCFD hydrogel-treated Tg-induced fibroblast and macrophage groups showed a low proportion of mcherry+CD11b+ cells, the Tg-induced fibroblast and macrophage group, the GP hydrogel-treated Tg-induced fibroblast and macrophage group, and the GPHCF hydrogel-treated Tg-induced fibroblast and macrophage group showed a high proportion of ERSE mcherry+CD11b+cells (Fig. 3E, H), suggesting that the ERS-responsive GPHCFD can simultaneously inhibit ERS in fibroblasts and macrophages. Tg-induced fibroblasts and macrophages underwent ERS. A coculture system was established with two cell types divided into the no hydrogel, GP, GPHCF, and GPHCFD hydrogel-treated groups. Western blotting analysis of fibroblasts in the coculture system revealed that the GPHCFD group exhibited the lowest expression levels of MCP-1 and the ERS-specific molecules p-IRE1 and CHOP among the four groups, confirming that the GPHCFD hydrogel can inhibit MCP-1 expression in fibroblasts and reduce fibroblast ERS levels (Fig. 3I, J, S5). The GPHCFD group showed the greatest abundance of the M2-type characteristic molecule Arg1 and the lowest expression of the M1-type characteristic molecules TNF-α, iNOS, and IL-1β, according to fluorescent quantitative PCR analysis of macrophages in the coculture system. This suggested that the GPHCFD hydrogel can prevent the promotion of M1 to M2 macrophage transformation (Fig. 3K). The above results demonstrated that the GPHCFD hydrogel is an ERS-responsive hydrogel. By suppressing MCP-1 expression, the GPHCFD hydrogel could decrease excessive ERS in fibroblasts and encourage fibroblast migration and proliferation. It could also decrease ERS in macrophages and encourage the polarization of M1-type macrophages toward the M2 phenotype.
Biological function testing of ERS-responsive GPHCFD hydrogels. (A) Fluorescence confocal detection of fibroblast proliferation in the GPHCFD hydrogels. Red: dead fibroblast; green: live fibroblast. (B) CCK8 detection of fibroblast proliferation in the Gel GP, GPHCF, and GPHCFD hydrogels induced by Tg on days 1, 3, 5, and 7, in that order. n = 3 per group. (C, D) Tg-induced fibroblasts produce excessive ERS; scratch assay of fibroblast migration in the no hydrogel, GP, GPHCF, and GPHCFD hydrogel-treated teams. n = 3 per team. (E) Flow cytometry assessment of the PBS group without Tg induction, the Tg-induced fibroblast (F) and macrophage (M) group, the GP hydrogel-treated Tg-induced fibroblast and macrophage group, the GPHCF hydrogel-treated Tg-induced fibroblast and macrophage group, the GPHCFD hydrogel-treated Tg-induced fibroblast and macrophage group, and the GPHCFD hydrogel-treated Tg-induced macrophage group. (F) The proportion of mcherry+CD11b+ cells within CD11b+ cells is statistically analyzed. n = 5 per team. NS: non-significant, *P < 0.05. (G) The proportion of mcherry+CD11b− cells within CD11b− cells is statistically analyzed. (H) The proportion of mcherry+CD11b+ cells within CD11b+ cells is statistically analyzed. n = 5 per group. (I) Tg-induced four groups without hydrogels; GP, GPHCF, and GPHCFD hydrogel-treated fibroblasts and macrophages underwent ERS. Western blotting was used to assess the expression levels of MCP-1, p-IRE1, and CHOP in fibroblasts from the four groups. (J) The statistical analysis was performed via a heatmap. n = 3 per group. (K) Fluorescent quantitative PCR was used to measure the expression levels of TNF-α, iNOS, IL-1β, and Arg1 in macrophages from the four groups. n = 5 per team. NS: non-significant, *P < 0.05. The data is displayed as mean ± SD.
The GPHCFD hydrogel promoted diabetic wound healing
We further investigated the therapeutic effects of the GPHCFD hydrogel on diabetic wounds. Three hydrogels, namely, GP, GPHCF, and GPHCFD, were used to treat diabetic wounds, and the wound areas of the untreated, GP, GPHCF, and GPHCFD groups were assessed. GPHCFD hydrogel and GPHCF hydrogel significantly promoted wound contraction in comparison with that observed in the untreated group, with the GPHCFD hydrogel showing the best therapeutic effect (Fig. 4A, B). HE staining was used to assess changes in the dermis of the untreated, GP, GPHCF, and GPHCFD hydrogel groups. The GPHCFD and GPHCF hydrogels promoted dermal formation, with the most significant dermal thickening observed in the GPHCFD hydrogel group on day 8 (Fig. 4C, D). By day 15, the dermis thickness in the GPHCFD hydrogel group had already begun to reduce (Fig. 4C, E), indicating that the dermis in the GPHCFD hydrogel group matured earliest. Additionally, on day 15, the GPHCFD hydrogel group exhibited more dermal hair follicles than the other three groups (Fig. 4C).
To explore the mechanism of action of the GPHCFD hydrogel on wounds, we performed single-cell sequencing on diabetic wound tissue without treatment on day 3 and diabetic wound tissue treated with GPHCFD hydrogel. Wound cells were classified into 10 cell groups: fibroblasts, macrophages, neutrophils, NK cells, keratinocytes, monocytes, endothelial cells, pericytes, DCs, and smooth muscle cells (Fig. 4F). The percentage of fibroblasts was much higher in GPHCFD hydrogel-treated diabetic wound tissue than in untreated diabetic wound tissue, but the percentage of NK cells was significantly lower (Fig. 4G). GO enrichment analysis related to wound healing showed that gene sets related to wound healing, collagen fibril organization, and angiogenesis were significantly enriched in the GPHCFD hydrogel treatment group (Fig. 4H). The results indicated that GPHCFD hydrogel promoted the healing of diabetic wounds, potentially by enhancing fibroblast proliferation and suppressing abnormal natural killer cell proliferation.
The GPHCFD hydrogel facilitated diabetic wound healing. (A) Typical images of diabetic wounds treated with no hydrogel, GP, GPHCF, and GPHCFD hydrogel at days 5, 10, and 15. n = 6 per group. (B) Statistical analysis of the wound areas. (C) H&E stained images showing wound healing in the no hydrogel, GP, GPHCF, and GPHCFD hydrogel treatment groups for diabetic wounds. Scale bar, 1000 μm and 200 μm. (D, E) Statistical analysis of dermal thickness in the four groups at days 8 and 15. n = 6 in each group. (F) UMAP plot analysis of 10 cell clusters in the GPHCFD hydrogel-treated diabetic wound group. (G) Bar chart analysis of the proportion of 10 cell clusters in the non-hydrogel-treated diabetic wound group and the GPHCFD hydrogel-treated diabetic wound group. (H) GO enrichment analysis of wound healing-related gene sets in the GPHCFD hydrogel-treated diabetic wound group. n = 3 per team. The data is displayed as mean ± SD.
The GPHCFD hydrogel inhibited excessive ERS in fibroblasts by suppressing MCP-1 expression, thereby promoting collagen formation
We analyzed the functional roles of local fibroblasts in diabetic wound tissue treated with the GPHCFD hydrogels using single-cell sequencing data. GO enrichment analysis showed that gene sets related to positive regulation of cell proliferation and migration were significantly enriched in fibroblasts treated with the GPHCFD hydrogels (Fig. 5A). GO enrichment circle diagram analysis showed that in diabetic wound tissue, local fibroblasts treated with the GPHCFD hydrogels exhibited significant downregulation of genes related to ERS functions, including unfolded protein binding (GO:0051082), endoplasmic reticulum unfolded protein response (GO:0030968), and protein folding chaperone (GO:0044183) (Fig. 5B). These findings confirmed that the GPHCFD hydrogel might inhibit excessive ERS in the local fibroblasts of diabetic wounds, thereby promoting fibroblast proliferation and migration.
We further investigated the effect of the GPHCFD hydrogel on fibroblast collagen secretion. Masson’s trichrome was used to assess collagen formation on the diabetic wound surfaces of the four groups (no hydrogel treatment, GP, GPHCF, and GPHCFD hydrogel treatment) on days 8 and 15, and collagen thickness was statistically analyzed. In comparison to the collagen layers in the other three groups, the GPHCFD hydrogel treatment group’s layer was the thickest on day 8 (Fig. 5C, D). By day 15, because collagen in the GPHCFD hydrogel-treated group had begun to mature, the collagen layer in the GPHCF hydrogel-treated group was the thickest (Fig. 5C, S6). This finding suggested that the GPHCFD hydrogel significantly promoted collagen formation and maturation in fibroblasts. DCN is a characteristic marker of fibroblasts. We used immunofluorescence to assess the expression of MCP-1 and the characteristic ERS protein CHOP in diabetic wounds on day 3 in the untreated, GP, GPHCF, and GPHCFD hydrogel groups. In contrast to the remaining three groups, the GPHCFD group exhibited the lowest local MCP-1 expression level and the lowest proportion of DCN+CHOP+ cells in diabetic wounds (Fig. 5E, G). Western blot analysis was performed to detect MCP-1 expression levels at the wound sites across all groups. We similarly found that the GPHCFD hydrogel group exhibited the lowest MCP-1 expression levels compared to the other three groups (Fig. S7). This finding indicated that the GPHCFD hydrogel could reduce the expression level of MCP-1 and inhibit excessive ERS in fibroblasts at the wound site. The above results indicated that GPHCFD hydrogel promoted tissue regeneration in wounds from diabetes by preventing the expression of MCP-1 in fibroblasts, suppressing ERS in fibroblasts, and promoting collagen formation.
The GPHCFD hydrogel inhibits excessive ERS in diabetic wound fibroblasts by suppressing MCP-1 expression and promotes collagen formation. (A) GO enrichment analysis bubble chart analysis of wound healing-related functions in diabetic wound fibroblasts treated with the GPHCFD hydrogel. n = 3 per group. (B) GO enrichment circle plot analysis of ERS-related functions in local fibroblasts from diabetic wound tissue treated with the GPHCFD hydrogel. n = 3 per group. (C) Masson’s trichrome to detect collagen formation in four groups of diabetic wounds: no hydrogel treatment and GP, GPHCF, and GPHCFD hydrogel treatment on days 8 and 15. 500 μm and 200 μm Scale bar. (D) Statistical evaluation of collagen thickness on day 8. n = 6 per team. (E) Immunofluorescence detection of MCP-1 expression levels in diabetic wounds treated with the GPHCFD hydrogel (Red: DCN; Blue: nuclei; Green: MCP-1). Scale bar, 20 μm. (F) Immunofluorescence detection of DCN+CHOP+ cells in the local area of diabetic wounds treated with no hydrogel and GP, GPHCF, and GPHCFD hydrogels. Scale bar, 20 μm. (G) Statistical evaluation of MCP-1’s mean fluorescence intensity. n = 6 in each group. (H) The percentage of DCN+CHOP+ cells among DCN+ cells is statistically analyzed in the wound area. n = 6 per team. NS: non-significant, *P < 0.05. The data is displayed as mean ± SD.
The GPHCFD hydrogel inhibited excessive ERS in NK cells, thereby suppressing abnormal proliferation and inflammatory factor secretion in NK cells and promoting NK cell maturation
The GO enrichment analysis bubble chart shows that genes significantly downregulated in local NK cells treated with the GPHCFD hydrogel in diabetic wound tissue are enriched in the following functional pathways: negative regulation of natural killer cell differentiation, ERS-related functions (PERK complex, response to unfolded proteins, and endoplasmic reticulum membrane) (Fig. 6A). Volcano plot analysis revealed that the expression of NK cell CCR2, ERS-related genes XBP1, ATF6B, IRE1, PERK, ATF6, ATF4, and inflammatory factors TNF-α and IFN-γ was significantly downregulated (Fig. 6B). To further validate the GPHCFD hydrogel’s impact on NK cells in diabetic wounds, we used flow cytometry to detect NK cells in diabetic wounds on day 3 in the untreated, GP, GPHCF, and GPHCFD groups—specifically CD45+CD3−NK1.1+ cells. The GPHCFD group had the lowest percentage of CD3−NK1.1+cells among the CD45 + cells when compared to the other three groups. This suggests that while the NK cells in the other three groups displayed significant abnormal proliferation, the GPHCFD group showed only mild abnormal NK cell proliferation (Fig. 6C, E). The proportion of CD11b+CD27+ NK cells among CD3−NK1.1+cells in diabetic wound sites increased most significantly among the four groups in the GPHCFD group (Fig. 6C, F), but the percentage of CD11b+CD27−NK cells among CD3−NK1.1+ cells did not significantly alter (Fig. 6C, G). The GPHCFD hydrogel treatment significantly enhanced NK cell maturation toward the CD11b+CD27+ NK cells. The NK cells from diabetic wound sites in the untreated, GP, GPHCF, and GPHCFD groups were then subjected to Western blotting analysis. The expression levels of CCR2 and ERS-related proteins p-IRE1 and XBP1 were lowest in the wound-site NK cells of the GPHCFD hydrogel treatment group (Fig. 6H, I, S8). These results demonstrated that the GPHCFD hydrogel might inhibit NK cell CCR2 expression, reduce excessive ERS levels, suppress abnormal NK cell proliferation and inflammatory factor secretion, and promote NK cell maturation. Mature NK cells can regulate wound inflammation homeostasis, increase re-epithelialization speed, and improve collagen deposition [38].
The GPHCFD hydrogel inhibits abnormal proliferation and inflammatory factor secretion of NK cells by suppressing excessive ERS, thereby promoting NK cell maturation. (A) GO enrichment analysis bubble chart analysis of ERS-related functions and NK cell differentiation functions in local NK cells of diabetic wound tissue treated with the GPHCFD hydrogel. n = 3 per group. (B) Volcano plot analysis of ERS-related gene expression and inflammatory factor expression in local NK cells of diabetic wound tissue treated with the GPHCFD hydrogel. n = 3 per team. (C) Flow cytometry was used to detect CD45+CD3−NK1.1+ NK cells from diabetic wounds in the untreated, GP, GPHCF, and GPHCFD groups. (D) Flow cytometry was used to detect CD11b+CD27+ NK cells and CD11b+CD27− NK cells in diabetic wounds from the untreated group, GP group, GPHCF group, and GPHCFD group, respectively. (E) The percentage of CD45+CD3−NK1.1+ cells within CD45+ cells is statistically analyzed. n = 5 in each group. (F) Statistical analysis of the percentage of CD11b+CD27+ NK cells among CD3−NK1.1+ cells. n = 5 per group. (G) The percentage of CD11b+CD27− NK cells among CD3−NK1.1+ cells. n = 5 per group. (H) Flow cytometry-sorted NK cells from diabetic wounds in the untreated, GP, GPHCF, and GPHCFD hydrogel treatment groups were subjected to Western blotting. (I) A heatmap was used to statistically assess the levels of CCR2, p-IRE1, and XBP1 protein expression according to Western blotting data. n = 5 per team. *P < 0.05, NS: non-significant. The data is displayed as mean ± SD.
The GPHCFD hydrogel promoted M1–M2 macrophage polarization by inhibiting ERS in macrophages
To further explore the effects of the GPHCFD hydrogel on local macrophages in diabetic wounds, we analyzed macrophage data from the untreated and GPHCFD groups using single-cell sequencing. GSEA analysis showed that in the diabetic wound tissue of the GPHCFD group, the negatively regulated genes related to tumor necrosis factor production (inhibiting macrophage polarization to M1) were significantly upregulated in local macrophages (Fig. 7A). The response-related genes of ERS were significantly downregulated (Fig. 7B), while the KEGG pathway that promotes macrophage polarization towards the M2 type, namely the TGF-β signaling pathway-related genes, was significantly upregulated (Fig. 7C). The KEGG PPAR signal pathway-related genes that interact with ERS and macrophage polarization to M2 type were significantly up-regulated, further indicating that the GPHCFD hydrogel can promote macrophage polarization to M2 type by regulating ERS (Fig. 7D). The heatmap analysis revealed that compared with those in untreated diabetic wound tissue, the levels of inflammatory factors CCL1, CCL3, CCL3L1, CCL3L3, CSF1, CSF2, CXCL1, CXCL15, CXCL6, TNF-α, TNFSF15, and TNFSF18 were significantly decreased in macrophages in the GPHCFD group (Fig. 7E). CD68 is a characteristic marker of macrophages; thus, we used immunofluorescence to assess the level of CCR2, the ERS biomarker XBP1, the M1 signature iNOS, and the M2 biomarker CD206 in CD68+ cells in diabetic wound tissues from the untreated, GP, GPHCF, and GPHCFD groups. The proportions of CD68+CCR2+ and CD68+XBP1+ cells were significantly reduced (Fig. 7F-I), indicating that the GPHCFD hydrogel can inhibit macrophage CCR2 expression and reduce macrophage ERS. The proportion of CD68+iNOS+ cells significantly decreased, while that of CD68+CD206+ cells significantly increased, indicating that the GPHCFD hydrogel promotes the M1–M2 conversion of diabetic wound macrophages (Fig. 7J-M). The above results demonstrated that the GPHCFD hydrogel might inhibit the expression of CCR2 in local macrophages at diabetic wound sites, reduce excessive ERS in macrophages, and promote macrophage M1–M2 conversion.
The GPHCFD hydrogel promotes macrophage polarization toward the M2 type by inhibiting ERS in macrophages. (A) GSEA plot analysis of gene enrichment related to the negative regulation of tumor necrosis factor production function in local macrophages in diabetic wound tissue in the GPHCFD group. (B) The TGF-β signaling pathway in macrophages in the GPHCFD group was analyzed using a GSEA plot. (C) GSEA plot analysis of gene enrichment related to the response to ERS function in macrophages in the GPHCFD group. n = 3 per group. (D) GSEA plot analysis of gene enrichment related to the KEGG PPAR signaling pathway in macrophages in the GPHCFD group. n = 3 per group. (E) Heatmap analysis of inflammatory factor expression in local macrophages from the untreated diabetic wound group and the GPHCFD hydrogel-treated group. n = 3 per group. (F) Immunofluorescence detection of CD68+CCR2+ cells in diabetic wounds in the untreated, GP, GPHCF, and GPHCFD hydrogel-treated groups. Scale bar, 20 μm. (G) Identification of CD68+XBP1+ cells in the diabetic wounds in the four groups by immunofluorescence. 20 μm Scale bar. (H) The percentage of CD68+CCR2+ cells among CD68+ cells is statistically analyzed. n = 6 each group. *P < 0.05, NS: non-significant. (I) Statistical analysis of the proportion of CD68+XBP1+ cells among CD68+ cells. n = 6 in each group. (J) Immunofluorescence detection of CD68+ iNOS+ cells in the diabetic wounds in the four groups. Scale bar, 20 μm. (K) Immunofluorescence detection of CD68+CD206+ cells in the diabetic wounds in the four groups. 20 μm Scale bar. (L) The percentage of CD68+iNOS+ cells among CD68+ cells is statistically analyzed. (M) Statistical analysis of the proportion of CD68+CD206+ cells among CD68+ cells. n = 6 per team. *P < 0.05, NS: non-significant. The data is displayed as mean ± SD.
