DOI: 10.6084/m9.figshare.26646673.v1
title: Additional file 1 of Extracellular vesicle-derived silk fibroin nanoparticles loaded with MFGE8 accelerate skin ulcer healing by targeting the vascular endothelial cells

description: Additional file 1: Figure S1. Identification of vascular endothelial cells (VECs). A Representative immunofluorescence staining of CD31 in VECs. B The proportion of CD31 + cells in extracted primary cells was determined by flow cytometry. Figure S2. Gene sequencing analysis of VECs treated with normoxia or hypoxia. A, B The volcano and Venn diagram of VECs treated with normoxia or hypoxia. C The differential expression of genes related to the ferroptosis and autophagy of VECs treated with normoxia or hypoxia was analyzed by heat map. D KEGG enrichment analysis of upregulated genes in hypoxic VECs compared with normoxic VECs. E GO enrichment analysis of upregulated genes in normoxic VECs compared with hypoxic VECs. Figure S3. Extracellular vesicles (EVs) inhibit the hypoxia-induced ferroptosis of VECs. A Propidium iodide (PI) staining of VECs treated with normoxia, hypoxia, EVs, or hypoxia + EVs was detected by flow cytometry. B, C The iron, MDA, and GSH levels and mitochondrial changes related to the ferroptosis of VECs treated as above. D, E Representative western blotting of GPX4 and mean fluorescence intensity (MFI) associated with reactive oxygen species (ROS) levels analyzed in VECs treated with normoxia, hypoxia, EVs, or hypoxia + EVs. ns: p &gt; 0.05; *p &lt; 0.05; **p &lt; 0.01; ***p &lt; 0.001. Figure S4. Ferroptosis was enhanced by autophagy in VECs. A KEGG enrichment analysis of all differential genes in VECs treated with or without hypoxia. B, C Representative western blot analysis of LC3A/B, ACSL4, GPX4, P53, P62, and lipid peroxidation in VECs treated with dimethyl sulfoxide (DMSO), erastin, or erastin + 3-methyladenine. D, E Iron, MDA, and GSH levels and mitochondrial changes associated with the ferroptosis of VECs treated as above. Figure S5. MFGE8 inhibited ferroptosis by diminishing autophagy in VECs. A Correlation analysis of MFGE8, ferroptosis-related proteins, and autophagy-related proteins. B–D Representative western blot analysis of P53, ACSL4, GPX4, LC3A/B, and MFGE8, reactive oxygen species (ROS) detected by flow cytometry, and invasive capability detected by transwell assay of VECs treated with dimethyl sulfoxide (DMSO), erastin, Lenti-MFGE8, or erastin + Lenti-MFGE8. J, K Representative immunofluorescence staining of MFGE8/LC3B and GPX4/ACSL4 in VECs treated as above. NC: normal control. ns: p &gt; 0.05; *p &lt; 0.05; **p &lt; 0.01; ***p &lt; 0.001. Figure S6. Characteristics of MFGE8 released from NPs@MFGE8. The rate of MFGE8 released from NPs@MFGE8 was examined by HPLC of the NPs@MFGE8 on days 1, 2, 3, 4, 5, 6, and 7. Figure S7. Detection and analysis of the NPs clearance uptake by VECs. A The preservation of RBITC-NPs in VECs was observed by fluorescence microscopy at days 0, 1, 3, 5, and 7. B The mean fluorescence intensity (MFI) of VECs after uptake of RBITC-NPs was statistically analyzed at days 0 1, 3, 5, and 7. C The clearance rate of NPs and NPs@MFGE8 in VECs was statistically analyzed at days 1, 3, 5, and 7. ns: p &gt; 0.05. Figure S8. The critical concentration of NPs@MFGE8 in enhancing VEC viability. The CCK-8 assay was used to further illustrate the critical concentration of NPs@MFGE8. Approximately 1 ml of purified MFGE8 protein (1 mg/ml) was added to 1 ml of silk fibroin solution (10 mg/ml) with a mass ratio of 1:10 to prepare the NPs@MFGE8. The VECs were cultured under hypoxic conditions and subsequently treated with different concentrations of NPs@MFGE8 (100, 250, 500, 750, or 1000 μg/ml). The VECs viability was detected by the CCK-8 assay, and the critical concentration of NPs@MFGE8 in enhancing VEC viability might be 500 μg/ml in vitro. ns: p &gt; 0.05; *p &lt; 0.05. Figure S9. The concentration of MFGE8 in NPs@MFGE8 (500 μg/ml) was determined. A The detection of MFGE8 standard solution (0.2 mg/ml) by HPLC. B The detection of MFGE8 in NPs@MFGE8 by HPLC after lysis with LiBr. The peaks of the MFGE8 protein were 2.053 min and 2.127 min, respectively. C The concentration of MFGE8 in NPs@MFGE8 (500 μg/ml) was calculated according to the concentration and the area of the waveform of MFGE8 (0.2 mg/ml). Figure S10. The degradation rate of the Silk fibroin/collagen hydrogel. After treated with collagenase I and II, trypsin, and neutral proteinase for 10 days, the hydrogel carriers were gradually degraded. Figure S11. The effect of hydrogel sustained-release carrier on promoting PU healing. A, B Wound size and its statistical analysis at days 0, 1, 3, 6, 9, and 12. ns (green): PU + Gels-NPs vs. PU + Gels; ns (yellow) and *(yellow): PU + Gels-NPs vs. PU + Gels-NPs@MFGE8. C Masson staining was conducted to detect wound healing in the PU + Gels, PU + Gels-NPs, and PU + Gels-NPs@MFGE8 groups. Figure S12. Immunohistochemistry of parkin, pink1, MFGE8, and GPX4 in VECs in rats' skin tissues, which were divided into NC group, PU group, PU + silk fibroin/collagen hydrogel (Gels) group, PU + silk fibroin/collagen hydrogel-NPs@MFGE8 (Gels-NPs@MFGE8) group, and PU + silk fibroin/collagen hydrogel-NGR-NPs@MFGE8 (Gels-NGR-NPs@MFGE8) group. Figure S13. Analysis of the expression levels of macrophage marker protein CD14 and fibroblast marker protein α-SMA in skin tissues. A, B Immunohistochemical staining of macrophage marker protein CD14 and statistical analysis of macrophage numbers in the NC group, PU group, PU + Gels group, PU + Gels-NPs@MFGE8 group, and PU + Gels-NGR-NPs@MFGE8 group. C, D Immunohistochemical staining of fibroblast marker protein α-SMA and statistical analysis of fibroblast numbers in the five groups above. ns: p &gt; 0.05; *p &lt; 0.05; **p &lt; 0.01; ***p &lt; 0.001.