Reagents and antibodies
3-Methyladenine (cat. no. S2767), an autophagy inhibitor, was purchased from Sellect (Shanghai, China). Dimethyl sulfoxide (cat. no. 67-68-5) was supplied by Sigma (St. Louis, Missouri, USA). Erastin (cat. no. HY-15763), a ferroptosis inducer, was obtained from MedChemExpress (Shanghai, China). Rhodamine B isothiocyanate (RBITC; cat. no. GY418) was obtained from Goyoo Biotech (Nanjing, China). Collagenase type II (cat. no. A004174), N-hydroxysuccinimide (NSH) (cat. no. 6066-82-6), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (cat. no. 25952-53-8) were obtained from Sangon Biotech (Shanghai, China). PKH26 (cat. no. MINI26) and collagenase D (cat. no. 11088858001) were provided by Sigma (St. Louis, Missouri, USA). NGR peptide (cat. no. GC14926) was purchased from GlpBio Technology (Montclair, California, USA). HOOC-PEG-COOH (cat. no. R-1202-1 k) was purchased from RuiXi Biological Technology (Xi’An, China). Anti-glyceraldehyde 3-phosphate dehydrogenase (cat. no. 60004-1-Ig), anti-P53 (cat. no. 10442-1-AP), anti-LC3A/B (cat. no. 14600-1-AP), anti-P62 antibody (cat. no. 18420-1-AP), anti-GPX4 antibody (cat. no. 67763-1-Ig), anti-LC3B antibody (cat. no. 18725-1-AP), anti-CD13 antibody (cat. no. 14553-1-AP), anti-CD31 antibody (cat. no. 66065-2-Ig), anti-pink1 antibody (cat. no. 23274-1-AP), anti-parkin antibody (cat. no. 66674-1-Ig), anti-CD63 antibody (cat. no. 25682-1-AP), anti-CD14 antibody (cat. no. 17000-1-AP), and anti-α-SMA antibody (cat. no. 14395-1-AP) were obtained from Proteintech (Wuhan, China). Anti-TSG101 antibody (cat. no. A1692) was obtained from ABclonal Technology (Wuhan, China). Anti-ACSL4 antibody (cat. no. ab155282), goat anti-mouse IgG antibody (Alexa Fluor® 488) (cat. no. ab150113), and goat anti-rabbit IgG antibody (Alexa Fluor® 594) (cat. no. ab150080) were provided by Abcam (Cambridge, Massachusetts, USA). Immunol Fluorescence Staining Kit with Alexa Fluor 647-labeled goat anti-rabbit IgG (cat. no. A0468), DAPI nucleic acid dye (cat. no. C1006), and crystal violet staining solution (cat. no. C0121) were provided by Beyotime Biotechnology (Shanghai, China). Anti-MFGE8 (cat. no. sc-8029) was provided by Santa Cruz Biotechnology (Santa Cruz, USA).
Patients
Instead of necrotic tissues, mild and/or severe PU tissues were acquired from each patient who underwent skin debridement surgery. A total of thirteen tissues (mild ulcer tissues, n = 7; severe ulcer tissues, n = 6) were collected. Mild and severe PU tissues were used for western blotting as well as immunohistochemical and immunofluorescence staining. This study received official approval from the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University (approval number: 2022-KLSD-198). Written informed consent was obtained from all patients or their relatives prior to tissue collection.
Isolation of VECs
Two-week-old rats were anesthetized with 3% pentobarbital sodium (50 mg/kg) and sacrificed with a broken neck. The abdominal skin was cut open to expose the abdominal blood vessels. A section of the blood vessel was cut, the whole vessel was turned over to expose the vascular endothelium, and both ends of the vessel were ligated and digested with 2% collagenase D and 2% collagenase type II at 37 °C for 1–2 h. The supernatant was collected after digestion and was subsequently centrifuged at 1200 rpm/min for 5 min. Cell precipitates were collected, resuspended in a complete medium, and inoculated into culture dishes. Cell culture and identification of VECs were performed after cell fusion to 80–90%.
Hypoxic culture of VECs
VECs that reached 90–100% confluence were digested with trypsin and then expanded three times for culture. Normal control cells were cultured with 21% O2 and 5% CO2 at 37 °C. The hypoxic culture of VECs was cultured in a hypoxic incubator (Whitley H35 Hypoxystation; Don Whitley, UK) under the following conditions: 1–3% O2, 5% CO2, and 37 °C. Then, the cells were collected for gene sequencing (Novogene, Beijing, China), flow cytometry, and western blotting.
Extraction, identification, and mass spectrometric detection of EVs
VECs that reached 90–100% confluence were digested with trypsin and then expanded for culture. When the cells reached 80–90% confluence, further culture was carried out for 3–4 d using serum-free DMEM/F12 medium, and the supernatant was collected after culture. The culture medium was centrifuged at 300 ×g for 10 min, 1000 ×g for 10 min, and 10,000 ×g for 30 min to collect the supernatant and finally at 100,000 ×g for 70 min to collect the EV precipitates. EVs were resuspended in 200 μL of phosphate-buffered saline (PBS). Subsequently, 100 μL of suspension EVs was taken out and mixed well with 5 μL of PKH26 dye in 300–500 μL of diluent in the dark for 5–10 min, and 500 μL of 5% BSA was then added to terminate the reaction. PKH26-labeled EVs (PKH26-EVs) were obtained by centrifugation at 100,000 ×g for 70 min. The particle size of EVs was detected using a zeta particle size analyzer. The characteristic protein of EVs was detected by western blotting, and the morphology of EVs was observed by transmission electron microscopy (TEM). EVs were examined by mass spectrometry at the Central Laboratory of the Army Medical University to detect the protein species.
Lentivirus transfection
To overexpress MFGE8, the lentivirus was obtained from HanBio (Shanghai, China). VECs were inoculated and cultured in six-well plates. Diluted Lenti-MFGE8 or Lenti-NC and 1 μL Polybrene (2 μg/mL) were added when the VECs reached 50% confluence. After shaking evenly, the VECs were cultured in an incubator at 37℃ and 5% CO2. After 24 h, the medium was changed, and the VECs continued to culture for 3–5 d. After the VECs were treated with puromycin (10 μg/mL) for 5–7 d, the transfection efficiency of the VECs was analyzed by western blotting.
Western blotting
After grinding and crushing, the VECs or skin tissues were lysed with RIPA (cat. no. P0013B) and PMSF (cat. no. ST506) from Beyotime Biotechnology (Shanghai, China). After being lysed for 30 min and centrifugated at 12,000 rpm/min for 10 min, the supernatant was collected and added with 5 × SDS-PAGE sample loading buffer (cat. no. P0015, Beyotime Biotechnology, Shanghai, China). When the samples were electrophoresed at the bottom of PAGE Gel at 120 V for 70 min, the protein samples were transferred to polyvinylidene fluoride (PVDF) at 300 mA for 70–120 min. PVDF was sealed with 5% degreased milk powder for 1 h. After washing, PVDF was incubated with the diluted first antibody overnight at 4 °C. On the second day, after washing, PVDF was incubated with the diluted second antibody (1:5000) at 20–30 °C for 1–2 h. After washing, the High Sensitivity ECL Kit was used to formulate the luminescent solution (A:B/1:1) and to image the PVDF in an imaging system.
Immunofluorescence and immunohistochemistry
Paraffin-embedded tissue sections were dewaxed to hydrate. The samples were then subjected to immunofluorescence or immunohistochemistry. Tissue samples were repaired with trypsin antigen repair solution and endogenous peroxidase blocker. After washing, the tissue samples were sealed with 5% BSA for 30 min and then incubated with dilute rat or rabbit first antibody at 4 °C overnight. After incubation, they were washed with PBS. In immunofluorescence experiments, the secondary antibodies of goat against rabbit or rat were diluted, and the samples were incubated at 37 °C for 1–2 h. Immunofluorescence staining was performed for 5 min using a DAPI staining solution, and the samples were then photographed under a fluorescence microscope or laser confocal microscope. In immunohistochemical tests, HRP-labeled secondary antibody was incubated with tissues at 37 °C for 30 min. After washing, the samples were stained with DAB solution and observed under a microscope; additionally, they were stained with hematoxylin for 5 min. After dehydration and fixation, the tissue sections were observed and photographed using the M8 digital scanning microscopic imaging system.
Purified protein extraction
CHO cells were cultured in incubators under static conditions with 5% CO2 at 37 °C and then transfected with 3xFlag-labeled MFGE8-PURO lentivirus. After 3 d, the CHO cells were digested with trypsin after fusion to 80–100%. After treating with puromycin (10 μg/mL) for 5–7 days under static conditions, the CHO cells were digested and centrifuged at 1000 rpm/min for 5 min. Subsequently, the CHO cells were cultured in suspension in a shaker at a rotating speed of 80–100 rpm/min with the RAPID CHO 18 serum-free medium (cat. no. H180KJ, BasalMedia, Shanghai, China). After incubation for 48–72 h, the suspended cells were centrifuged, and CHO cell precipitates were collected. The CHO cells were lysed with RAPI and protease inhibitor PMSF for 30–60 min. Subsequently, ultrasound was performed under the following conditions: ultrasonic time, 20–30 s; amplitude, 20%; pause, 2 s; and ultrasound, 2 s. After centrifuging, the protein supernatant was collected and added to a 20-μL anti-Flag magnetic bead suspension according to the 500-μL protein supernatant. Magnetic beads were separated by a magnetic frame for 30 s following incubation for 2 h at 20–30 °C in a shaker. After being washed with TBS buffer two times, the 20 μL magnetic bead suspension was incubated with 100 μL of 3xFlag polypeptide for elution at 25 °C for 30–60 min. The magnetic beads were then separated by a magnetic frame for 30 s to collect the eluent containing purified MFGE8 protein.
Synthesis and characterization of NPs
Cocoons (2 g), provided by the State Key Laboratory of Resource Insects, Southwest University, Chongqing, were taken and placed into 1 L of water. Afterwards, 5–10 g of Na2CO3 was added so that the Na2CO3 concentration was 0.5–1%. These cocoons were then boiled in 90–100 °C water for 30–60 min. A dissolved solution (50 mL) was prepared with deionized water (30.43 mL), ethanol (19.57 mL), and calcium chloride (24 g). The cocoons were dissolved at 60 ± 5 °C for 1–2 h until complete dissolution of silk fibroin. Centrifugation was performed at 10,000 ×g for 10 min, and the supernatant was collected. The silk fibroin solution was filtered for 48 h in a dialysis bag with an interception molecular weight of 8000–14000 Da. After filtration, the solution was centrifuged at 10,000 ×g for 10 min. The obtained silk fibroin solution was diluted to 20 mg/mL (2%). Then, 5 mL of acetone solution was added to a 15-mL centrifuge tube, 1 mL of 1% silk fibroin solution was added to the acetone solution by drops, and vortexing was continued for 30–60 s. Then, ultrasound was conducted on ice under the following conditions: ultrasonic time, 2 min; pause, 2 s; ultrasound, 2 s; and amplitude, 30%. Then, the milky supernatant was obtained by centrifugation at 3840 × g for 5 min. The milky supernatant was centrifuged at 10,629 × g for 10 min to obtain no-load NPs. For obtaining the NPs@MFGE8, approximately 200 μl of purified MFGE8 protein (1 mg/ml) was added to 200 μl of silk fibroin solution (10 mg/ml or 5 mg/ml) with a mass ratio of 1:10 or 1:5. After swirling well, the mixed solution was added to the acetone solution (2–3 ml) by drops and then swirled in a vortex mixer for 30–60 s. Ultrasound was conducted on ice under the following conditions: ultrasonic time, 2 min; pause, 2 s; ultrasound, 2 s; and amplitude, 30%. Then the milky supernatant was obtained by centrifugation at 1699 ×g for 5 min. The milky supernatant was centrifuged at 10,629 ×g for 10 min to obtain MFGE8-coated NPs (NPs@MFGE8). Afterwards, 10 mL of deionized water was added for washing, centrifugation was performed at 10,629 ×g for 10 min, the supernatant was poured off to collect the precipitates, and the acetone residue was removed. NPs@MFGE8 was stored at 4℃ for short-term use within 3 days and at − 80 ℃ for long-term use within 6 months, and repeated freezing and thawing were avoided. The particle size and potential of NPs and NPs@MFGE8 were detected using a zeta potential and particle size analyzer. RBITC (10 mg) was dissolved in deionized water (1 mL), and 5 μL of 1% RBITC staining solution was added to 1 mL of resuspended NP solution. RBITC-labeled NPs (RBITC-NPs) were obtained after the reaction transpired for 30 min under a light shield at 20–30 °C.
Characteristics of NPs clearance after the uptake by cells
After treatment with RBITC-NPs or RBITC-NPs@MFGE8, the mean fluorescence intensity (MFI) of VECs was statistically analyzed at 0, 1, 3, 5 and 7 days. MFI-0 was the mean fluorescence intensity at day 0. MFI-x was the mean fluorescence intensity at day 1, 3, 5 or 7. The clearance rate was calculated as follows: (%) = (MFI-0—MFI-x)/MFI-0 × 100%.
Synthesis and characterization of NGR-NPs@MFGE8
NPs or NPs@MFGE8 produced from 500 μL of 1% silk fibroin solution or/and 500 μL of purified MFGE8 protein (1 mg/mL) solution were dissolved in 1 mL of deionized water. EDC (5 mg), HOOC-PEG-COOH (10 mg), and NGR peptide (10 mg) were added to NPs solution (1 mL) and allowed to react at 20–30 °C for 0.5–1 h in a shaker. Subsequently, NSH (5 mg) was added and incubated overnight at 20–30 °C in a shaker. On the second day, the solution was centrifuged at 1699 ×g for 5 min, and the supernatant was discarded to obtain cross-linked NGR NPs (NGR-NPs) or cross-linked NGR NPs@MFGE8 (NGR-NPs@MFGE8) precipitate. The particle size and zeta potential of NGR-NPs and NGR-NPs@MFGE8 were detected using a zeta potential and particle size analyzer (Thermo Fisher Scientific, Massachusetts, USA). Fourier transform infrared spectrometry (FT-IR) was conducted for NPs, NPs@MFGE8, NGR-NPs, and NGR-NPs@MFGE8.
Envelopment efficiency assay
High-performance liquid chromatography (HPLC) was performed to measure the envelopment efficiency. First, the total amount of MFGE8 protein was detected using an ultra-fine ultraviolet spectrophotometer (NanoDrop OneC, Thermo Fisher Scientific, Massachusetts, USA) and divided into two equal fractions (namely, fractions A and B) before coating. Fraction A of the purified protein was used for NPs@MFGE8 according to the amount of silk fibroin protein and MFGE8 (10:1 or 5:1). After obtaining the precipitated NPs@MFGE8 by centrifugation, NPs@MFGE8 were lysed with lithium bromide (LiBr) (9.3 mol/L). Then, HPLC was conducted to detect the chromatogram and quantity of free MFGE8 in NPs@MFGE8 and fraction B of the purified MFGE8 protein. The envelopment efficiency was then calculated from the ratio of free MFGE8 to the total quantity of MFGE8 in fraction B.
Characteristics of MFGE8 released from NPs@MFGE8
The characteristics of MFGE8 released from NPs@MFGE8 were detected by HPLC. On day 0, we obtained 800 μl of MFGE8 solution (1 mg/ml), added the MFGE8 solution to 800 μl of silk fibroin solution (10 mg/ml), and prepared the NPs@MFGE8 solution after mixing evenly. The NPs@MFGE8 solution was divided equally into eight aliquots of 200 μl each. One aliquot was centrifuged at 10,629 ×g, and the NPs@MFGE8 precipitate was collected. NPs@MFGE8 was lysed with 9.3 mol/L LiBr, and the total content of MFGE8 (Mt) was determined by HPLC. The remaining 7 portions were rotated and shaken in a shaker (20 rpm/min) at 37 ℃. Then on days 1, 2, 3, 4, 5, 6, and 7, the NPs@MFGE8 solution was centrifuged at 10,629 ×g, the supernatant was collected, and the release content of MFGE8 (Mr) in the supernatant was detected by HPLC. The release rate was calculated as follows: (%) = Mr/Mt × 100%.
Synthesis and characterization of collagen, silk fibroin, and silk fibroin/collagen hydrogels
Collagen hydrogel was prepared from rat tail collagen. One rat was anesthetized with pentobarbital sodium (50 mg/kg) and subsequently sacrificed by breaking its neck. The tail was removed and washed with PBS. It was then cut through the skin, exposing the white collagen of the tail. The tail collagen was removed, cut to a size of 1–3 mm3, and washed with PBS two to three times. The collagen was centrifuged at 4000 rpm/min (3220 ×g) for 5 min, and the supernatant was poured away. The collagen precipitate was added to 100 mL of 0.5–1% (v/v) acetic acid solution. The collagen was dissolved by shaking at 4 °C for 48 h in a shaker and, after complete dissolution, was centrifuged at 12,000 ×g for 10 min. The supernatant was collected and then stirred and salted out for 5 min in 10% NaCl solution, after which a large amount of flocculent collagen was precipitated. The flocculent collagen was centrifuged at 12,000 ×g for 10 min at 4 °C to collect the collagen. To fully dissolve the collagen, 50–100 mL of 0.1 mmol/L HCl solution was added to the collagen. Additionally, 3 mL of 0.5% collagen solution was added to approximately 240 μL of 1 mol/L NaOH so that the pH value reached approximately 7.0. Collagen hydrogel was formed at 37 °C for 5–10 min, whereas silk fibroin hydrogel was prepared from the silk fibroin solution. Carbomer (0.24 g) was dissolved in 1 mL of deionized water and stirred well. The carbomer solution was mixed with 800 μL of 1 mol/L NaOH solution and stirred well. Afterwards, 800 μL of 20% polyvinyl alcohol was added. Subsequently, 4 mL of 6% silk fibroin solution was added to the carbomer solution, stirred well, and placed at 20–30 °C for 12–24 h until it cross-linked with the silk fibroin hydrogel. Silk fibroin/collagen hydrogel was prepared from rat tail collagen and silk fibroin solution. Carbomer (0.24 g) was dissolved in 1 mL of deionized water and stirred well. Furthermore, 800 μL of 1 mol/L NaOH solution was added to the carbomer solution and stirred well. Subsequently, 800 μL of 20% polyvinyl alcohol was added, and 4 mL of 6% silk fibroin solution was added to the carbomer solution and stirred well. Afterwards, 4 mL of 0.5–1% collagen solution was mixed evenly with about 320 μL of 1 mol/L NaOH solution, then added to the carbomer solution and stirred well, and placed at 20–30 °C for 12–24 h until the silk fibroin/collagen hydrogel formation. To prepare the silk fibroin/collagen hydrogel loaded with NPs, the prepared NPs were added to the uncrosslinked silk fibroin/collagen hydrogel. The silk fibroin/collagen hydrogel loaded with NPs was stirred thoroughly, filtered several times with a 70 μm filter by centrifugation at 3840 ×g to make its distribution uniform, and then placed at 20–30 °C for 12–24 h until the silk/collagen hydrogel loaded with NPs formation. Scanning electron microscopy (SEM) and rheometer were used to detect the void size and the energy storage modulus (G′) and loss modulus (G″) of hydrogels, respectively. The swelling rate was measured at different time points. The weight of the dried hydrogel was determined, and then the dried hydrogel was immersed in PBS solution at 37 ℃. The hydrogel soaked in PBS at the set time point was taken out, the water on the surface was quickly removed, and the hydrogel was weighed. Ws was the weight of the hydrogel after swelling, and Wd was the weight of the hydrogel after drying. The swelling ratio was calculated as follows: (%) = (Ws−Wd)/Wd × 100%.
Release assay of NPs in silk fibroin/collagen hydrogel
NPs were labeled with 1% RBITC (RBITC-NPs), with an absorption wavelength of approximately 550 nm. The NPs release capacity of silk fibroin/collagen hydrogel was characterized by analyzing the in vitro release of RBITC-NPs from the hydrogel. Briefly, the absorbance of gradient dilutions of RBITC-NPs with PBS at 550 nm was used to draw a standard curve for RBITC-NPs. RBITC-NPs loaded silk fibroin/collagen hydrogel was suspended in 4 mL of PBS in a centrifuge tube that was rotated at 37 °C. At various time points, supernatants were collected, and tubes were replenished with the same volume of PBS. The integrated absorbance of RBITC-NPs released from the silk fibroin/collagen hydrogel was determined using the iMark™ Microplate Reader (Bio-Rad, USA). The cumulative release of RBITC-NPs was calculated with the help of a standard curve.
Biocompatibility assay
VECs were inoculated and cultured in six-well plates. Diluted Lenti-NC (1:50) and 1 μL Polybrene (2 μg/mL) were added when the VECs reached 50% confluence. Then puromycin (10 μg/mL) was added and continued to culture for 3–5 d until 80–90% or more cells appeared green. The silk fibroin/collagen hydrogels were synthesized and soaked in complete medium for 24 h. The medium was changed every 6 h until the color of the medium did not change. The silk fibroin/collagen hydrogel was cut into disks with a thickness of 1–2 mm and a diameter of 1 cm and stained with RBITC for 2 h. 100 μM of 1 × 105 VECs transfected with Lenti-NC were inoculated and cultured on silk fibroin/collagen hydrogels. Two hours later, 1 mL of complete medium was added. On the second day, the hydrogel was removed and imaged using a microscope (Olympus, Japan).
Degradation of the hydrogel carrier assay
Collagenase I and II, trypsin and neutral proteinase were used to degrade hydrogel carriers for 10 days. On day 0, the mass of the dry hydrogel carrier was weighed as H0, and then on days 1, 3, 5, 7, and 10, the mass of the dry hydrogel was weighed as Hm. The degradation rate of the hydrogel was calculated as follows: (%) = (H0−Hm) /H0 × 100%.
Flow cytometry
The mitochondrial membrane potential of VECs was detected using the Mitochondrial Membrane Potential Detection Kit (cat. no. E-CK-A301) from Elabscience Biotechnology (Wuhan, China). Cells were digested with trypsin for 1–2 min, and a complete medium was added to terminate the digestion. The cell suspension was collected and centrifuged at 300 ×g for 5 min. The cells were resuspended in 500 μL of JC-1 working solution and incubated at 37 °C for 20 min. After incubation, the cells were centrifuged at 300 ×g for 5 min. The cell precipitates were resuspended using 1xJC-1 assay buffer and then assayed by flow cytometry (Beckman, USA). Hypoxia-induced cells were collected and stained using an apoptosis kit (cat. no. PI0100, Invigentect, USA). Propidium iodide staining was performed for 5 min. The death rate was then determined by flow cytometry.
Cell viability and proliferation assay
VECs that reached 90–100% confluence were digested and then inoculated into 96-well plates. The cell viability was tested using the Cell Counting Kit-8 (cat. no. BG0025, BG Biotech Beijing, China). After processing the different groups, 10 μl of CCK-8 reagent was added, and the cells were incubated at 37 ℃ in the dark for 2–5 h. The absorbance of the medium for each culture well was measured at a wavelength of 450 nm, and the cell viability level was statistically analyzed. Moreover, the proliferation of VECs was also measured using 5-ethynyl-2′-deoxyuridine (EdU) (cat. no. C10310-3, RiboBio, Guangzhou, China). After 12 h of incubation with Edu solution, Apollo staining solution was added to the VECs for incubation for 30 to 60 min. The VECs were then washed with PBS and subjected to DNA staining. Fluorescence images were obtained by fluorescence microscopy (Olympus, Japan).
TEM
VECs or EVs were collected and fixed overnight with 3% glutaraldehyde. After washing, they were fixed with 1% osmium acid for 2 h. Subsequently, the samples were subjected to gradient dehydration with different acetone concentrations. After the samples were embedded, ultrathin sections (70–90 nm) were prepared and stained with 5% uranium and lead citrate. Finally, the samples were observed using TEM (Philips, Amsterdam, The Netherlands).
SEM
Silk fibroin or collagen was used in preparing the hydrogel or NPs. Droplets of NPs were added to silicon wafers and naturally dried at 25 °C. The hydrogel was freeze-dried overnight. On the second day, the dried hydrogel was taken out, soaked in liquid nitrogen for 1–2 min, and cut into 1–2-mm thin slices. The NPs and hydrogel were treated with gold spray, and their structure and morphology were then observed via SEM.
Lipid peroxidation and reactive oxygen species (ROS) assay
Lipid peroxidation and ROS levels were analyzed using C11-BODIPY™ 581/591 (cat. no. D3861, Thermo Fisher Scientific, Massachusetts, USA) or CellROX™ Deep Red Flow Cytometry Assay Kit (cat. no. C10491, Thermo Fisher Scientific, Massachusetts, USA). Adherent cells were taken and washed with PBS. The cells were digested with trypsin for 1–2 min. After 30–50% of cells were digested, a complete medium was added to terminate the digestion. The cell suspension was collected and centrifuged at 300 ×g for 5 min. The cells were resuspended with C11-BODIPY™ 581/591 or CellROX™ Deep Red reagent at an appropriate concentration and were then incubated at 37 °C for 30–60 min away from light. Afterwards, the cells were washed with PBS and analyzed using flow cytometry.
Glutathione (GSH) assay
GSH levels were detected using the GSH Colorimetric Assay Kit (cat. no. E-BC-K030-M) from Elabscience Biotechnology (Wuhan, China). The VECs were harvested by cell scraping and washed once or twice with PBS, after which ultrasound was performed on ice. After centrifuging at 1500 ×g for 10 min, the cell supernatant was incubated with 100 μL of acid reagent at 20–30 °C and then centrifuged at 4500 ×g for 10 min. The protein concentration of the supernatant was determined using a BCA kit (cat. no. P0012S, Beyotime Biotechnology, Shanghai, China). Subsequently, 25 μL of 5,5′-dithiobis(2-nitrobenzoic acid) solution was added to a 96-well plate, along with 100 μL of the above mentioned supernatant, which was incubated for 5 min at 20–30 °C. A microplate reader was then used to measure the absorbance values of each well at 405–414 nm. The amount of GSH in the cells was calculated based on the GSH standard curve and normalized to the protein concentration.
Malondialdehyde (MDA) assay
MDA levels of VECs were detected by the MDA Colorimetric Assay Kit (cat. no. E-BC-K028-M) from Elabscience Biotechnology (Wuhan, China). The VECs were harvested by cell scraping and washed once or twice with PBS. For ultrasound on ice, an extracting solution (0.5 mL) was added to the cell suspension. A 0.1 mL sample was mixed with 1 mL working solution and incubated in 100 °C water for 40 min. After cooling to 20–30 °C, the samples were centrifuged at 1078 ×g for 10 min. The protein concentration was measured using a BCA kit (cat. no. P0012S, Beyotime Biotechnology, Shanghai, China). The supernatant (250 μL) was added to a 96-well plate, and a microplate reader was then used to measure the absorbance values at 532 nm. The MDA level in each sample was normalized to the protein concentration.
Iron assay
Intracellular iron in VECs was measured by the Iron Colorimetric Assay Kit (cat. no. E1042) from Applygen (Beijing, China). The VECs cultured in 24-well plates were washed with cold PBS, and 200 μL of lysis buffer was then added to each well and lysed for 2 h in a shaker. Working solution A was prepared by mixing the buffer with 4.5% potassium permanganate solution at a 1:1 ratio. Next, 100 μL of working solution A and 100 μL of the sample were mixed well and incubated at 60 °C for 1 h. After cooling to 20–30 °C, 30 μL of iron ion agent was added to the sample, mixed well, and incubated at 20–30 °C for 30 min. The supernatant (200 μL) was added to a 96-well plate, and a microplate reader was used to detect the absorbance at 550 nm. The iron levels were normalized to the protein concentration.
Animal experiments
The 3-week-old rats (about 60–80 g) were provided by the Animal Center, Army Medical University, and anesthetized with 3% pentobarbital sodium (50 mg/kg). After hair removal, pressure was applied using cylindrical magnets with a diameter of 8 mm and height of 3 mm, which was continued for 6–8 h daily until ulcers formed. 10 rats were used to observe the diffusion of RBITC-NPs in vivo. Silk fibroin/collagen hydrogel loaded with RBITC-NPs was applied to prepare hydrogel sustained-release carrier. Necrotic tissues were removed, and the hydrogel sustained-release carrier was fixed on the ulcer surface with a thin film. The treatment was continued by changing the hydrogel daily for 5 d. In vivo imaging and fluorescence microscopy were performed to observe the diffusion of RBITC-NPs in PU skin tissues at days 0, 1, 3, 5, and 7.
Six rats were divided into the normal control (NC) and PU groups to evaluate whether RBITC-labeled NGR-NPs@MFGE8 (RBITC-NGR-NPs@MFGE8) could target VECs in PU tissues. The NC group was not damaged with PU; however, the surface skin structure was partially removed using a blade to ensure that RBITC-NGR-NPs@MFGE8 had crossed the barrier between the skin and subcutaneous tissues. Subsequently, the skin in both the NC group and the PU group was treated with RBITC-NGR-NPs@MFGE8-loaded hydrogel and observed under fluorescence.
To assess the healing effect of hydrogel sustained-release carrier on the ulcer surface, 24 rats were divided into six groups—namely, the NC (n = 3), PU (n = 3), PU + silk fibroin/collagen hydrogel (Gels) (n = 6), PU + silk fibroin/collagen hydrogel-NPs (Gels-NPs) (n = 3), PU + silk fibroin/collagen hydrogel-NPs@MFGE8 (Gels-NPs@MFGE8) (n = 6), and PU + silk fibroin/collagen hydrogel-NGR-NPs@MFGE8 (Gels-NGR-NPs@MFGE8) (n = 3) groups. Necrotic tissues were removed, and the Gels, Gels-NPs, Gels-NPs@MFGE8 or Gels-NGR-NPs@MFGE8 were fixed on the ulcer surface with a thin film, respectively. The ulcer surface was then photographed at days 0, 1, 3, 5, and 10. After treatment, the blood perfusion of PU tissues was statistically analyzed, and PU tissues were used for Masson staining, immunohistochemistry staining, immunofluorescence staining, and electron microscopy observation of mitochondrial structure changes in VECs. Animal use and experimental procedures were given official approval by the Animal Ethics Committee of the Army Medical University (no. AMUWEC20230372).
Statistical analyses
The data are presented as means ± standard deviations on at least three measurements and were analyzed by Student’s t-test or one-way analysis of variance using SPSS version 22.0 (IBM Corp., NY, USA) or GraphPad Prism version 7.0 (GraphPad Software Inc., CA, USA). Graph analysis was performed using GraphPad Prism version 7.0. Statistical significance was set at p < 0.05.
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