Exosome isolation
Extraction and induction of peritoneal macrophages: The abdominal skin of the mice was disinfected with ethanol, and 1 mL of 5% starch broth solution was injected into the abdominal cavity of each mouse. After 48 h, the mice were sacrificed by cervical dislocation. The peritoneum of the mice was exposed under aseptic conditions, and the peritoneal cavity was injected with 5 mL precooled Dulbecco's Modified Eagle’s Medium (DMEM). After gently massaging the mouse’s abdomen for 5 min, a 5-mL syringe was used to repeatedly flush the peritoneal cavity lavage fluid twice, and then the lavage fluid was recovered. The lavage fluid was centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and the peritoneal cell concentration was adjusted to 1×106/mL. One milliliter per well of the cell suspension was inoculated in a 12-well culture plate and placed in an incubator at 37°C with 5% carbon dioxide and saturated humidity to allow the macrophages to adhere to the wall. After incubating for 2 h to 4 h, the culture supernatant was discarded, and the cells were cultured in DMEM supplemented with exosome-free 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). After 24 h, interleukin-4 (IL-4) (20 µg/mL) was added to the medium to induce the differentiation of primary macrophages into M2 macrophages.
Exosome isolation: The cell supernatant of primary M2 macrophages was collected and placed in a centrifuge tube, which was sequentially centrifuged at 300 g for 10 min, 2,000 g for 10 min, 10,000 g for 30 min, and 100,000 g for 70 min at 4°C. The obtained precipitates were suspended in phosphate-buffered saline (PBS) and centrifuged at 100,000 g for 70 min to wash away impurities. The exosomes were resuspended in PBS for further characterization.
The preparation of EXS− cl−NGF and Cur@EXS− cl−NGF.
Modification of NGF with cleavable peptide substrate (NHS-Arg-Val-Gly-Leu-Pro-(6-Mal), RVGLP-(6-Mal)): The peptide (0.5 mg/mL) was added to the NGF solution (1 mg/mL) at a molar ratio of 50:1, keeping the linker in excess. The mixed solution was incubated at 4°C in PBS (pH 7.4) overnight. Then, the samples were filtered using a centrifugal filter device (Amicon Ultra-0.5, Millipore Co, Germany) at 7,000 g for 30 min to remove the excess linker. Next, the NGF was linked to EXs. The exosomes were pretreated with 1 mM TCEP at 37°C for 30 min to break the disulfide bonds on the surface of the exosomes and expose the sulfhydryl groups. Then, “-cl-NGF” was dissolved in PBS (pH 7.4) and reacted with the EXs at 25°C for 1 h. Finally, the EXS− cl−NGF was washed and collected by dialysis. Cur was dissolved in a 1:1 mixed solution of ethanol and acetonitrile. PBS was added to the mixed solution to a concentration of the organic solvent of 10%. EXs− cl−NGF was added at a concentration of 20% curcumin to the solution. Cur@EXS− cl−NGF was prepared by ultrasonic soaking using a 40- kHz and 100-W intermittent ultrasonic cleaner for 15 min and washing by centrifugation at 5000 rpm three times with a 100-kDa ultrafiltration tube. The loading capacity or percent drug load was calculated using the following formula.
Percent drug load = (amount of Cur in Cur@EXS− cl−NGF/amount of Cur@EXS− cl−NGF) *100
Characterization of EXS− cl−NGF and Cur@EXS− cl−NGF
Morphology of EXS− cl−NGF: Twenty milliliters of purified EXS/EXS− cl−NGF-linked AU nanoparticles (10 nm) were added onto a carbon-coated copper grid for 20 min, stained with 2% uranyl acetate for 1 min, and then washed with distilled water. Transmission electron microscopy (TEM) images were obtained on a JEM-1400 microscope (JEOL Japan) with an accelerating voltage of 120 kV. The size distribution and zeta potential of EXS/EXS− cl−NGF were analyzed on a Zetaview (Partical Metrix Germany).
Western blot analysis: The expression of special marker proteins of EXS/EXS− cl−NGF was detected using a western blot analyzer with BeyoECL Plus (Beyotime, Beijing, China). The primary antibodies used were as follows: rabbit anti-CD9 (1:1000, Abcam, Cambridge, UK), rabbit anti-TSG101 (1:1000, Abcam, Cambridge, UK), rabbit anti-CD206 (1:1000, Cell Signaling Technology, MA, USA), rabbit anti-IL10 (1:1000, Cell Signaling Technology, MA, USA), rabbit anti-CCR2 (1:1000, Cell Signaling Technology, MA, USA), rabbit anti-ARG-1 (1:1000, Cell Signaling Technology, MA, USA), rabbit anti-NGF (1:1000, Cell Signaling Technology, MA, USA), and mouse anti-β-actin (1:1000, Abcam, Cambridge, UK).
Colocalization analysis of EXS− cl−NGF and Cur@EXS− cl−NGF: First, EXs were dyed with DID, and NGF was labeled with CY3. Subsequently, the double fluorescently labeled nanomaterial EXS− cl−NGF was prepared using the above method. EXS− cl−NGF was imaged by confocal laser scanning microscopy (CLSM) (Nikon A1 Japan) and visualized with stimulated emission depletion (STED) (Leica SP8, Germany).
Release and biological activity of NGF from EXS− cl−NGF: MMP-9 was added to a PBS solution of EXS− cl−NGF with/without inhibitor, and the supernatant was removed at different time points. After ultrafiltration, the content of NGF in the filtrate was detected with an NGF ELISA kit. The biological activity of NGF was detected by the MTT test in PC12 cells, in which different concentrations of NGF from EXS− cl−NGF/free NGF were added to the culture medium of PC12 cells.
Construction of the Transwell™ coculture model
The Transwell™ coculture model was built with Transwell™ plates (pore size of 8 µm, Corning, USA), and 0.5-1×105 PC12 cells were loaded in each upper chamber. Primary macrophages were removed from the mouse abdominal cavity and pretreated in the lower chamber with lipopolysaccharide (LPS) (0.1 µg/mL) for 24 h before building the Transwell™ coculture model. All the cells were cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with fetal bovine serum (10%), penicillin (100 units/mL), and streptomycin (100 µg/mL) (Gibco, Grand Island, NY, USA) at 37°C in a humidified atmosphere containing CO2 (5%).
Apoptosis test
For the apoptosis experiment of PC12 cells, PC12 cells were loaded in each upper chamber and stimulated with 200 µM hydrogen peroxide (H2O2) for 12 h to simulate damage to cells caused by oxidative stress. After changing to normal medium, primary M1 macrophages were placed in the lower layer of the Transwell™ coculture model for 12 h. Different nanoparticles (PBS, EXS, NGF, EXS− NGF, EXS− cl−NGF) were added to the different chambers, and after 12 h, the apoptosis rate of PC12 cells and the macrophage polarization level in each chamber were evaluated by flow cytometry and CLSM.
Flow Cytometry
For the cell uptake experiment, medium containing NGF, EXS− NGF, and EXS− cl−NGF (FITC-labeled NGF, 5 µg/mL) was added to the Transwell™ coculture model. At the different time points, cells were removed and resuspended in standing buffer to assess the fluorescence of different cells in different groups (Beckman Coulter, USA).
For the apoptosis rate of PC12 cells, the Transwell™ coculture model was removed after different stimulations, and the upper PC12 cells were resuspended. A total of 105 PC12 cells were mixed in 100 µL of binding buffer. Cells were stained with Annexin V-Alexa Fluor 488/PI for 15 min at room temperature to assess cell apoptosis.
For macrophage polarization, macrophage cells were removed, and flow cytometry antibodies were added for 30 min at 4°C. M1 macrophages were labeled with F4/80 (Biolegend, USA) and CD86 (Biolegend, USA), and M2 macrophages were labeled with F4/80 (Biolegend, USA) and CD206 (Biolegend, USA) to evaluate the differentiation of macrophage phenotypes.
Animals and the SCI model
Adult male C56BL/6 mice (8–10 weeks old, 22–25 g) were used. All animal experiments were approved by the Animal Protection and Use Committee of Jinzhou Medical University. A contusion-induced SCI model was developed by an improved weightlessness method. In brief, after intraperitoneal anesthesia with 1% sodium pentobarbital (50 mg/kg), the mice were fixed on a sterile operating table, fully exposing the T9 spinal cord. Using a 12.5-g impactor device (diameter: 2 mm), the uniform height was 5 cm down to the spinal cord, resulting in a moderate spinal cord contusion. After successful modeling, the surgical incision was sutured layer by layer, and antibiotics were given to prevent infection. From 2 d after surgery, the mouse bladder was massaged twice a day to help them urinate until the mice could urinate spontaneously. Sham-operated mice underwent the same procedures, except for contusion of the spinal cord. To investigate the treatment effect in vivo, 100 µL of 2 mg/mL Cur, EXS− cl−NGF or Cur@EXs− cl−NGF was injected through the tail vein at 2 h, 2 d, 4 d, and 6 d after injury.
In vivo imaging
For in vivo imaging of nanoparticles, 100 µL of NGF, red vesicles (RVs− cl−NGF), or EXs− cl−NGF (2 mg/mL, Alexa Fluor® 647-labeled NGF) was injected via the tail vein. At different time points, mice were imaged using a Kodak Imaging System FX Pro to evaluate the fluorescence signal distribution in vivo. Twelve hours after tail vein injection, the mice were sacrificed by cervical dislocation, and the mouse heart, liver, spleen, lung, kidney, and spinal cord were removed for fluorescence imaging.
Immunofluorescence staining
For the cell uptake experiment, medium containing NGF, EXS− NGF, and EXS− cl−NGF (FITC-labeled NGF, 5 µg/mL) was added to the Transwell™ coculture model for 6 h. Cells were removed from the incubator, and the cells were fixed with immune tissue fixative. Finally, the cell membrane was labeled with phalloidin 561, and the cell nucleus was labeled with 4’,6-diamidino-2-phenylindole (DAPI).
For immunofluorescent dual-labeling staining, cells or frozen sections were fixed with immune tissue fixative and incubated with Triton X-100 (0.3%) followed by goat serum (10%) for 2 h. The cells were incubated with the first antibody overnight at 4°C, washed, and then incubated with Alexa Fluor 488 goat anti-rabbit IgG or Alexa Fluor 594 goat anti-mouse IgG (A-11034/A-11005, 1:250, Thermo Fisher Science) for 2 h at room temperature. Next, the cell nucleus was stained with DAPI. The primary antibodies were as follows: rabbit anti-F4/80 (1:200, Abcam, Cambridge, UK), rabbit anti- cleaved-Caspase3 (c-caspase3) (1:1000, Abcam, Cambridge, UK), mouse anti-ARG-1 (1:1000, Cell Signaling Technology, MA, USA), rabbit anti-iNOS (1:1000, Cell Signaling Technology, MA, USA), rabbit anti-NeuN (1:50, Cell Signaling Technology, MA, USA), rabbit anti-GFAP (1:200, Cell Signaling Technology, MA, USA), and rabbit anti-β3-Tubulin (1:200, Cell Signaling Technology, MA, USA).
Behavioral analysis
The basso mouse scale (BMS) exercise rating scale was used to evaluate the functional recovery of injured animals on days 1, 3, 5, 7, 14, 21 and 28 according to scores ranging from 0 to 9 (9 for complete normality and 0 for complete paralysis). The animals were placed in the open field and observed for 4 min. Three examiners who did not know the mouse grouping observed and scored the mouse's ankle joints, the touch degree of the sole and dorsum of the foot, trunk stability and tail position. The experiment was repeated three times.
Statistical analysis
All data are expressed as the average value of the data distribution evaluated by the Shapiro-Wilk test. The Mann-Whitney U test was used for two-group comparisons. One-way ANOVA and Bonferroni post hoc tests were used to compare more than two groups. All data were graphed and statistically analyzed using GraphPad Prism 9. P < 0.05 was indicated with “*”, P < 0.01 was indicated with “**”, and P < 0.001 was indicated with “***”.