All studies were approved by the Medical Ethics Committee of Jiangsu University (IRB approval protocol number: 2020161).
Primary hucMSCs were isolated as previously described . Fresh umbilical cord tissues were obtained from the Fourth Affiliated Hospital of Jiangsu University after the informed consent of the puerpera. Briefly, the umbilical cord tissues were washed and cut into 2-cm pieces and then placed in phosphate buffer solution (PBS) containing penicillin–streptomycin for approximately 15 min. Subsequently, the arteries and veins of the umbilical cord tissue were removed and processed into tissue blocks of approximately 3 mm3. The small tissue blocks were pasted on the bottom of a petri dish and then placed upside-down in a cell culture incubator for approximately 30–60 min. Subsequently, the umbilical cord tissues were maintained in α-MEM medium (Invitrogen, USA) containing 20% FBS (Bovogen, Australia) containing penicillin–streptomycin (Leagene, China). The medium was changed every 3 days for approximately 10 days. The passaged cells were maintained with α-MEM containing 10% FBS and penicillin–streptomycin, and the P3 hucMSCs were used for subsequent experimental studies. Rat renal tubular epithelial cell NRK52E and mouse mononuclear macrophage RAW 264.7 cells were purchased from American Type Culture Collection. The NRK52E cells were cultured in high-glucose DMEM (Bioind, Israel) with 15% FBS, and the RAW 264.7 cells were cultured in RPMI 1640 medium (Invitrogen, USA) with 10% FBS. All cells were maintained in a humidified incubator with 5% CO2 at 37 °C.
Identification of hucMSCs
For the identification of multi-directional differentiation potential, the P3 hucMSCs were inoculated into 6-well plates at a density of 2 × 104 cells/cm2. According to the manufacturer’s instructions to perform adipogenic differentiation (Cyagen Biosciences, HUXUC-90031, USA). Briefly, when the degree of cell fusion reached 100%, 2 mL of adipogenic differentiation medium A was added into each well for 3 days, and then 2 mL of adipogenic differentiation medium B was replaced for 1 day. The above steps were repeated until the cells were cultured alternately for approximately 12–20 days. Oil red O staining was applied to evaluate the adipogenic differentiation of the hucMSCs. According to the manufacturer's instructions to perform osteogenic differentiation (Cyagen Biosciences, HUXUC-90021, USA). Briefly, when the degree of cell fusion reached 60–70%, 2 mL of osteogenic differentiation medium was added into each well, which was replaced with fresh complete medium every 3 days. The above steps were repeated until the cells were cultured for approximately 14 days. Alizarin Red S staining was performed to identify the osteogenic differentiation of the hucMSCs. According to the manufacturer’s instructions to detect the surface markers of hucMSCs (Cyagen Biosciences, HUXMX-09011, USA). The P3 hucMSCs suspension (3 × 106 cells/mL) was randomly divided into 1.5-mL sterile EP tubes. The primary antibodies, including isotype control, anti-human CD11b, anti-human CD14, anti-human CD45, anti-human CD29, anti-human CD73, and anti-human CD105, were added to each tube separately and incubated in the dark for 30 min. After the cells were washed twice by flow cytometry buffer, PE-labeled fluorescence secondary antibody was added to each tube, and incubated in the dark for 30 min. Ultimately, flow cytometry was performed to detect the surface markers of the hucMSCs.
Isolation and identification of hucMSC-sEVs
The hucMSC-sEVs were isolated and purified as previously described [24, 25]. The conditioned medium of P3-6 hucMSCs with good growth condition was collected. Cell supernatants were centrifuged to remove cell debris and organelles. Finally, the exosome pellets were resuspended in PBS and then passed through a 0.22-μm filter (Millipore, USA) and stored at − 80 °C. The protein content of the hucMSC-sEVs was determined by using a BCA protein assay kit (Vazyme, Nanjing, China). The morphology of the hucMSC-sEVs was observed using transmission electron microscopy (TEM; H-7800, Hitachi, Japan). The particle size, concentration, and zeta potential of the hucMSC-sEVs were analyzed by Nanoparticle tracking analyzer (NTA) (Germany, Particle Metrix, 220-Twin). The positive markers of hucMSC-sEVs, such as CD9, CD63, CD81, TSG101, Alix, and HSP70, as well as the negative control Calnexin, were determined by western blotting.
Isolation and identification of human peripheral blood neutrophils
Fresh anticoagulant blood samples from healthy volunteers were collected, and 5 mL of fresh whole blood was slowly added along the tube wall into a 15-mL sterile centrifuge tube with 5 mL of PolymorphPrep separation solution at the bottom under sterile conditions. After centrifugation at 600g for 30 min at 23 ℃, a pipette was used to carefully remove the intermediate white film layer into serum-free 1640 medium for washing. After centrifugation at 800g for 5 min at 23 ℃, the supernatant was discarded, and freshly configured red blood cell lysis buffer was added to resuspend the cell precipitate. Then, the cells were gently blown and mixed, and they stood at room temperature for 5–10 min. The cells were terminated with serum-free 1640 medium. After centrifugation at 800g for 5 min at 23 ℃, the neutrophils precipitation was harvested, and precooled PBS was washed three times and centrifuged at 800g for 5 min at 4 ℃. Subsequently, the neutrophils cell precipitation was obtained. Phase contrast microscopy was applied to observe the morphological characteristics of the neutrophils. Wright–Giemsa staining was performed to detect the nuclear and cytoplasmic characteristics of the neutrophils. The surface specific antigen molecules CD11b and CD33 of the neutrophils were identified by laser scanning confocal microscopy and flow cytometry. The integrity of the plasma membrane of DIO and DIL stained neutrophils was determined by laser scanning confocal microscopy.
Cell membrane derivation
The plasma membranes of the neutrophils were extracted as previously described with added modifications [25,26,27]. Briefly, frozen cells were thawed and washed with precooled 1 × PBS three times before centrifuging at 800g for 10 min. The cells were suspended in isolation buffer 1 containing 225 mM mannitol, 75 mM sucrose, 0.5% (wt/vol) BSA, 0.5 mM EGTA, and 30 mM Tris–HCl at pH 7.4 (all reagents from Sigma), as well as a protease and phosphatase inhibitor cocktail (CST, USA, 5870S). The cells were broken using a tissue dissociator, and then the homogenized solution was centrifuged at 800g for 10 min at 4 °C. Subsequently, the pellet was discarded, and the supernatant was centrifuged at 10,000g for 30 min at 4 °C. Following the centrifugation, the pellet was discarded, and the supernatant was centrifuged at 100,000g for 2 h at 4 °C. Finally, the membranes were collected as a pellet and washed twice with 0.2 mM EDTA in water. The membrane protein content of the neutrophils was determined by using a BCA protein assay kit. Ultimately, the membranes were stored at − 80 °C for subsequent studies.
Preparation and identification of Neu-NVs and NEX
Nanovesicles derived from human neutrophil membrane (Neu-NVs) were synthesized using sonication combined with extrusion. Briefly, the plasma membrane solution of neutrophils was ultrasonicated in an ice bath for 2 min. Subsequently, in order to prepare the Neu-NVs, the membrane protein components were successively extruded through 400 nm, 200 nm, and 100 nm polycarbonate membranes of liposome extruder (Germany, Merck, Avanti) 33 times. In order to prepare a NEX hybrid of Neu-NVs and hucMSC-sEVs, Neu-NVs and hucMSC-sEVs were mixed at a ratio of 1:1, and the mixture was then sonicated in an ice bath for 2 min. Furthermore, the mixture was then successively extruded through 400 nm, 200 nm, and 100 nm polycarbonate membranes of liposome extruder for a total of 33 times to form NEX. TEM (Transmission electron microscope), AFM (Atomic force microscope), and NTA were performed to detect the size, morphology, appearance, height, particle size, concentration, and zeta potential of the Neu-NVs and NEX. Western blotting was employed to determine the specific protein markers of the neutrophils. Coomassie blue staining was used to evaluate the protein components of the hucMSC-sEVs and the neutrophil membranes.
Membrane fusion verification
The fusion of Neu-NVs and hucMSC-sEVs was first investigated by a FRET(Fluorescence resonance energy transfer) method [28, 29]. Briefly, the hucMSC-sEVs were simultaneously labeled with DIO (excitation/emission = 480/510 nm) and DIL (excitation/emission = 549/565 nm) membrane dyes. The DIO-DIL-labeled hucMSC-sEVs and Neu-NVs were mixed by sonication combined extrusion to facilitate membrane fusion at a ratio of 1:1. The fluorescence spectrum of each sample was recorded between 490 and 650 nm using a Cytation 5 automatic microplate reader (BioTek, USA) with an excitation wavelength of 470 nm. For fluorescence colocalization experiments, DIO-labeled Neu-NVs and DIL-labeled hucMSC-sEVs were mixed at a ratio of 1:1 by simply stirring or the ultrasound combined extrusion method to facilitate membrane fusion. The fusion of Neu-NVs and hucMSC-sEVs was detected by super-resolution microscopy.
Mouse model of AKI and therapeutic experiments
Male ICR mice (8 to 10 weeks old, weighing 20 g ± 2 g) were purchased from the Laboratory Animal Center of Jiangsu University (Zhenjiang, China) and randomly divided into five groups (n = 6). All animal experiments were compliant with standard guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committees of Jiangsu University. A mouse model of cisplatin-induced AKI was established as described previously. After 10 mg/kg cisplatin (Sigma-Aldrich, USA, P4394) intraperitoneal injection for 72 h, Neu-NVs, hucMSC-sEVs, and NEX with the same particle number were administered to the AKI mice by tail vein for 2 consecutive days. AKI mice injected with PBS were used as a positive control. A normal group without any treatment served as a negative control. All animals were sacrificed at day 6 after cisplatin injection. Serum and kidney tissues were removed for further analysis of the renal function, histological changes, and tubular apoptosis.
Small animal in vivo imaging
One milliliter volumes of Neu-NVs, hucMSC-sEVs, and NEX were incubated with 5 μL of the membrane dye DIL (Thermo, USA, D3911) for 30 min at 37 °C. The labeled three types of vesicle suspension were transferred to a 100-kDa MWCO ultrafiltration centrifugal tube (Millipore, USA). In order to remove the unconjugated DIL, the samples were washed with PBS 3 times and centrifuged at 1500×g for 30 min. DIL-labeled Neu-NVs, hucMSC-sEVs, and NEX with the same particle number were administered to the AKI mice by tail vein, and small animals in vivo imaging (PerkinElmer, USA) was used to detect the major tissue organs distribution of the three types of transplanted vesicles at 24 h.
HE, IHC, and TUNEL staining
Bilateral renal tissues were obtained from the sacrificed mouse. The samples were fixed in 4% paraformaldehyde and gradually dehydrated, embedded in paraffin, and then cut into 4-μm sections. The slides were dewaxed to water and prepared via Hematoxylin and Eosin (HE) staining in accordance with standard protocols. Immunohistochemistry (IHC) staining was performed in accordance with the manufacturer’s instructions (Boster, USA, SA1020). PCNA primary antibody (CST, USA, 4711S) was diluted with 5% BSA at the ratio of 1:50, and then was incubated with sections for IHC. The stained slides were sealed and dried for observation. The apoptotic renal cells in tissue slides were measured by using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining according to the manufacturer’s protocol (Vazyme, USA, A113-02).
Quantification of serum cytokines
AKI mouse serum samples were collected on day 6, and the concentrations of IFN-α, IFN-γ, TNF-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-17, IL-10, and IL-12P70 were quantified with a multifunctional flow cytometer. Specifically, the whole blood of the AKI mice was collected and allowed to clot at room temperature for 30 min. The samples were then centrifuged at 2000g for 10 min to collect serum from the supernatant. The serum samples were immediately frozen at − 80 °C until they were analyzed via multiparameter flow cytometry within 3 days of collection.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Trizol reagent (Invitrogen, MA, USA) was used for total RNA extraction from cells, followed by cDNA synthesis with a commercial reverse transcription kit (Vazyme, Nanjing, China) according to the manufacturer’s protocol. On a QuantStudio™ 3 Real-Time PCR detection system (ABI, USA), qRT-PCR was run with SYBR Green PCR kit (CWBIO). The changes in the mRNA levels of the samples were evaluated using the 2−ΔΔ Ct method and were relative to the β-actin levels. The PCR primer sequences of the genes are listed in Additional file 1: Table S1.
Cell proliferation assay
The NRK52E cells (5 × 103 cells/well) were seeded in 96-well plates. After the cells adhered for 12–24 h, different concentrations of cisplatin were added to each well for treatment for 12 h and 24 h. The Cell Counting Kit-8 (CCK-8) assay was performed to evaluate cell proliferation activity according to the manufacturer’s procedures. One hundred µL of medium containing 10% CCK-8 reagent (Vazyme, Nanjing, China) was added to each well and incubated in the dark for 2 h at 37 °C. The absorbance values of each well at 450 nm were measured by the Cytation 5 automatic microplate reader (BioTek, USA). The NRK52E cells were seeded in 6-well plates at a density of 2 × 104 cells/cm2. When the cell density reached 80–90%, Neu-NVs, hucMSC-sEVs, and NEX with the same particle number were added to the cells and treated with cisplatin at a concentration of 10 ng/mL for 12 h. The cell proliferation activity was evaluated according to the manufacturer’s protocol by using an EDU (5-Ethynyl-2′-deoxyuridine) staining proliferation detection kit (Beyotime, Shanghai, China). After staining, the cells were immediately detected for green fluorescence intensity at 519 nm using the Cytation 5 automatic microplate reader (BioTek, USA).
Cells and exosomes were lysed in radio-immunoprecipitation assay (RIPA) buffer containing proteinase inhibitors (Pierce). The total protein concentration was detected using a BCA protein assay kit. An equal amount of extracted protein was separated by 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, MA, USA) followed by blocking with 5% nonfat milk for 1 h. Afterward, the membranes were incubated with primary antibodies at 4 °C overnight. The membranes were washed with 1xTBST and then incubated with an HRP-conjugated goat anti-rabbit/mouse IgG secondary antibody (USA, Invitrogen, 31460/31430) for 1 h at room temperature. The primary antibodies used in this study were: CD9 (Proteintech, USA, 60232-1-Ig), CD63 (Abcam, USA, ab59479), CD81 (Proteintech, USA, 18250-1-AP), HSP70 (CST, USA 2679S), Alix (CST, USA, 2171S), TSG101 (Abcam, ab30871), Calnexin (CST, USA, 4872S), CCL2 (Abclonal, USA, A7277), CXCR4 (Abcam, USA, ab124824), Fas (Absin, China, abs115046), ICAM-1 (Abcam, ab179707), integrin αV (CST, 4711S), integrin β3 (CST, 4702S), and β-actin (Bioworld, USA, AP0060).
Cellular uptake experiment
One-milliliter volumes of hucMSC-sEVs and NEX were incubated with 5 μL of the membrane dye DIO (Thermo, V22886) for 30 min at 37 °C. The stained hucMSC-Ex and NEX were then transferred to 100-kDa MWCO ultrafiltration centrifugal tubes (Millipore, USA). In order to remove the unconjugated DIO, the samples were washed three times with PBS and centrifuged at 1500g for 20 min. DIO-labeled Neu-NVs, hucMSC-sEVs, and NEX with the same particle number were administered to the NRK52E cells and RAW264.7 cells, and small animals in vivo imaging (PerkinElmer, USA) was used to detect the major tissue organ distribution of the three types of transplanted vesicles at 24 h. Laser scanning confocal microscopy and flow cytometry were applied to identify the internalization of the DIO-labeled hucMSC-sEVs and NEX by the NRK52E and RAW264.7 cells at different time points.
Cell ROS detection
The NRK52E cells were seeded in six-well plates at a density of 2 × 104 cells/cm2. When the cell density reached 80–90%, Neu-NVs, hucMSC-sEVs and NEX with the same particle number were added to cells and treated with cisplatin at a concentration of 10 ng/mL for 12 h. The intracellular ROS expression levels in each group were evaluated according to the manufacturer’s procedures. One milliliter of serum-free medium containing 10 μmoL DCFH-DA working solution (Beyotime) was added to each well and incubated in the dark for 20 min at 37 °C. The cells were washed in serum-free medium three times and were immediately detected for green fluorescence intensity at 525 nm using the Cytation 5 automatic microplate reader (BioTek, USA).
All data are presented as mean ± standard deviation (SD). Statistical analyses were performed with GraphPad Prism software (Version 5.01). The statistically significant differences between any two groups were determined using the two-tailed unpaired Student’s t-test. Comparisons between more than two groups were analyzed using one-way analysis of variance (ANOVA). P value < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, and ***P < 0.001.