Additional file 1 of Extracellular vesicle-derived silk fibroin nanoparticles loaded with MFGE8 accelerate skin ulcer healing by targeting the vascular endothelial cells

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 > 0.05; *p < 0.05; **p < 0.01; ***p < 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 > 0.05; *p < 0.05; **p < 0.01; ***p < 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 > 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 > 0.05; *p < 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 > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

附加文件1：图S1 血管内皮细胞（vascular endothelial cells, VECs）的鉴定。A 血管内皮细胞中CD31的代表性免疫荧光染色结果。B 采用流式细胞术（flow cytometry）测定提取的原代细胞中CD31阳性细胞的占比。图S2 常氧（normoxia）或低氧（hypoxia）处理的VECs的基因测序分析。A、B 常氧或低氧处理的VECs的火山图与韦恩图。C 通过热图（heat map）分析常氧与低氧处理的VECs中与铁死亡（ferroptosis）、自噬（autophagy）相关的差异基因表达情况。D 对低氧VECs相较于常氧VECs的上调基因进行KEGG富集分析（KEGG enrichment analysis）。E 对常氧VECs相较于低氧VECs的上调基因进行GO富集分析（GO enrichment analysis）。图S3 细胞外囊泡（extracellular vesicles, EVs）抑制低氧诱导的VECs铁死亡。A 采用流式细胞术检测常氧、低氧、EVs或低氧+EVs处理的VECs的碘化丙啶（propidium iodide, PI）染色情况。B、C 上述各组处理的VECs中与铁死亡相关的铁离子、丙二醛（MDA）、谷胱甘肽（GSH）水平及线粒体形态变化。D、E 对上述各组处理的VECs进行GPX4的代表性蛋白质免疫印迹（western blotting）检测，以及活性氧（reactive oxygen species, ROS）水平的平均荧光强度（mean fluorescence intensity, MFI）分析。显著性标注：ns: p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001。图S4 自噬增强VECs的铁死亡。A 对经低氧处理与未处理的VECs的全部差异基因进行KEGG富集分析。B、C 分别采用二甲基亚砜（dimethyl sulfoxide, DMSO）、erastin、erastin+3-甲基腺嘌呤（3-methyladenine）处理的VECs中LC3A/B、ACSL4、GPX4、P53、P62的代表性蛋白质免疫印迹分析，以及脂质过氧化情况检测。D、E 上述各组处理的VECs中与铁死亡相关的铁离子、MDA、GSH水平及线粒体形态变化。图S5 MFGE8通过减弱VECs的自噬抑制铁死亡。A MFGE8与铁死亡相关蛋白、自噬相关蛋白的相关性分析。B–D 分别采用DMSO、erastin、Lenti-MFGE8、erastin+Lenti-MFGE8处理的VECs中P53、ACSL4、GPX4、LC3A/B及MFGE8的代表性蛋白质免疫印迹分析，流式细胞术检测的活性氧（ROS）水平，以及Transwell实验检测的侵袭能力。J、K 上述各组处理的VECs中MFGE8/LC3B与GPX4/ACSL4的代表性免疫荧光染色结果。NC: 正常对照组。显著性标注：ns: p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001。图S6 NPs@MFGE8释放的MFGE8的特性。采用高效液相色谱（HPLC）分别于第1、2、3、4、5、6、7天检测NPs@MFGE8中MFGE8的释放率。图S7 VECs对NPs摄取与清除的检测分析。A 于第0、1、3、5、7天通过荧光显微镜观察RBITC标记的NPs在VECs中的留存情况。B 统计分析第0、1、3、5、7天VECs摄取RBITC-NPs后的平均荧光强度（MFI）。C 统计分析第1、3、5、7天VECs对NPs与NPs@MFGE8的清除率。显著性标注：ns: p > 0.05。图S8 NPs@MFGE8增强VEC活力的临界浓度。采用CCK-8实验进一步明确NPs@MFGE8的临界作用浓度。将约1mL浓度为1mg/mL的纯化MFGE8蛋白与1mL浓度为10mg/mL的丝素蛋白溶液按1:10的质量比混合，制备得到NPs@MFGE8。将VECs置于低氧环境中培养，随后分别用不同浓度（100、250、500、750或1000μg/mL）的NPs@MFGE8处理。通过CCK-8实验检测VEC活力，体外实验中NPs@MFGE8增强VEC活力的临界浓度约为500μg/mL。显著性标注：ns: p > 0.05; *p < 0.05。图S9 NPs@MFGE8（500μg/mL）中MFGE8的浓度测定。A 采用高效液相色谱（HPLC）检测MFGE8标准溶液（0.2mg/mL）。B 采用LiBr裂解NPs@MFGE8后，通过HPLC检测其中的MFGE8。MFGE8蛋白的峰保留时间分别为2.053min与2.127min。C 根据MFGE8标准溶液（0.2mg/mL）的浓度与峰面积，计算得到500μg/mL NPs@MFGE8中MFGE8的浓度。图S10 丝素蛋白/胶原蛋白水凝胶的降解率。经Ⅰ型、Ⅱ型胶原酶、胰蛋白酶及中性蛋白酶处理10天后，该水凝胶载体可逐渐被降解。图S11 水凝胶缓释载体促进压疮（PU, pressure ulcer）愈合的效果。A、B 第0、1、3、6、9、12天的创面面积及其统计分析结果。显著性标注：ns（绿色）: PU+Gels组 vs PU+Gels组；ns（黄色）与*（黄色）: PU+Gels-NPs组 vs PU+Gels-NPs@MFGE8组。C 采用Masson染色检测PU+Gels、PU+Gels-NPs及PU+Gels-NPs@MFGE8三组的创面愈合情况。图S12 大鼠皮肤组织中VECs的parkin、pink1、MFGE8及GPX4的免疫组化检测，实验分组为正常对照组（NC组）、压疮组（PU组）、PU+丝素蛋白/胶原蛋白水凝胶（Gels）组、PU+丝素蛋白/胶原蛋白水凝胶-NPs@MFGE8（Gels-NPs@MFGE8）组及PU+丝素蛋白/胶原蛋白水凝胶-NGR-NPs@MFGE8（Gels-NGR-NPs@MFGE8）组。图S13 皮肤组织中巨噬细胞标志物CD14与成纤维细胞标志物α-SMA的表达水平分析。A、B 对上述五组（NC组、PU组、PU+Gels组、PU+Gels-NPs@MFGE8组、PU+Gels-NGR-NPs@MFGE8组）的巨噬细胞标志物CD14进行免疫组化染色，并统计巨噬细胞数量。C、D 对上述五组的成纤维细胞标志物α-SMA进行免疫组化染色，并统计成纤维细胞数量。显著性标注：ns: p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001。