Preparation of peptides
The peptides were synthesized by Chinapeptide Co. Ltd. and stored as lyophilized powders at −80 °C. To prepare the exosomes, these peptides were dissolved in PBS. The peptide sequences used were as follows:
RVG-CP05, YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGSCRHSQMTVTSRL
TAT-CP05, YGRKKRRQRRRGGGSCRHSQMTVTSRL
Isolation, functional modification, and characterization of MSC-EXOR&T-GA
The supernatants obtained from human umbilical cord MSC cultures underwent sequential centrifugation to eliminate cells and debris. The process followed a stepwise protocol: initial centrifugation at 300 × g for 10 min, subsequent centrifugation at 2,000 × g for 20 min, and, ultimately, centrifugation at 10,000 × g for 30 min. After passing through a 0.22-μm sterile membrane (Millipore), the resulting supernatant was ultracentrifuged for 70 min at 130,000 × g (Optima L-100 XP, Beckman Coulter). A second ultracentrifugation step was performed on the pellet at the same parameters after the pellet was reconstituted in 1 mL of PBS. The BCA test was used to measure the amount of protein.
MSC-EXOs were incubated with RVG-CP05 and TAT-CP05, either individually or together, at 4 °C for 6 h. To remove unbound peptides, the mixtures were passed through 100-Kd filter tubes (Millipore) and washed five times with PBS. Finally, PBS was used to resuspend the complexes, yielding MSC-EXOR and MSC-EXOR&T.
Exosomes were conjugated to GA through a DIC/NHS-mediated amide reaction. First, MSC-EXOR&T were treated with MES buffer (pH 6.0) and dispersed ultrasonically to activate carboxyl groups. DIC and NHS in DMSO activated these groups, forming NHS esters. After 30 min, an amino-containing GA solution was added, and the pH was adjusted to 7.4. NHS esters formed amide bonds with GA amino groups during a 1-h incubation. Excess byproducts were removed by ultrafiltration and ultracentrifugation.Successful conjugation was validated using UV absorption spectroscopy and fluorescence resonance energy transfer (FRET) assay. Prepared fresh for immediate use, avoiding repeated freeze–thaw cycles.
The morphology of the MSC-EXOs, MSC-EXOR&T, and MSC-EXOR&T-GA was examined by TEM, and their size and distribution were assessed using NTA. EV-specific markers were verified by Western blot analysis.
Cell culture
The HT22 and OLN93 cell lines were cultured in Dulbecco’s modified Eagle medium/nutrient mixture F-12 (Cat# 11,330,057; Gibco, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Cat# 16,000,044; Gibco) and 1% penicillin–streptomycin (Cat# 15,140,122; Gibco) at 37 °C and 5% CO2. The culture medium was changed every 2 days. To circumvent the oxidative stress effects of glutamate on the HT22 cell line, all medium used in vitro experiments were devoid of glutamate.
Exosome uptake and endocytosis
Exosomes were labeled with the lipophilic dye DiO (3,3’-dioctadecyloxacarbocyanine perchlorate, Invitrogen), while RVG-CP05 and TAT-CP05 were tagged with FITC and Rhodamine B, respectively. MSC-EXOR and MSC-EXOR&T were prepared following standard protocols. Purified exosomes were resuspended in PBS at 100 µg/mL. A 10 µM DiO stock solution in ethanol was diluted in PBS to a final concentration of 1 µM. Equal volumes of DiO working solution and the exosome suspension were gently mixed and incubated at 37 °C for 30 min, enabling the dye to be incorporated into the exosome lipid bilayer. To remove any unbound dye, ultracentrifugation was performed at 130,000 × g for 70 min at 4 °C (Beckman Coulter, USA). The resulting pellet was washed with PBS and centrifuged again to remove any residual dye, after which the labeled exosomes were resuspended in PBS. Fluorescence microscopy confirmed successful labeling.
For the endocytosis assay, 10 μg of DiO-labeled MSC-EXOR or MSC-EXOR&T were added to the cell culture medium. Following incubation, the cells were fixed and stained with a rabbit anti-NeuN antibody (1:500, Abcam, ab104225), followed by treatment with a secondary antibody. Exosome uptake was visualized using an LSM900 inverted confocal microscope (Germany, ZEISS).
Spinal cord injury
This research utilized female C57BL/6 mice (8 weeks old, 20 g body weight) from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Guangdong, China). To induce the spinal contusion model, the mice were anesthetized with isoflurane (RWD, R510-22), and a midline incision was made at the T10 vertebra. The surrounding muscles and connective tissues of the thoracic spine were carefully dissected with minimal disruption to adjacent structures, exposing the T9–T11 spinous processes. Laminectomy was performed at T10 to expose the spinal cord, and precise incisions were made along the vertebral arch to remove the lamina. To stabilize the spine, a fixation device was used, and moderate spinal cord contusion was induced via the NYU Impact-III device (WM Keck, USA) by applying a free-fall force of 5 g from a 12.5 mm height. Postinjury, the wound was sutured in layers. The bladder was manually expressed for two weeks following the injury. All the animal experiments were approved by the Medical Ethics Committee and the Animal Welfare Ethics Committee of Tianjin Medical University General Hospital (IRB2021-KY-317, IRB2021-DWFL-354).
Tissue distribution
To assess the biodistribution of MSC-EXOR&T-GA in an SCI mouse model, DiR dye (Life Technologies) was used for exosome labeling. DiR-labeled MSC-EXOs (30 μg), MSC-EXOR&T (DiR-labeled MSC-EXOs incubated with RVG-CP05 and TAT-CP05 (30 μg each), MSC-EXOR&T-GA, or MSC-EXOR were injected intravenously into SCI mice. Two hours after injection, 50 mL of cold PBS was used to perfuse the mice to clear the circulating exosomes. Tissue samples, including the spinal cord, quadriceps, triceps, liver, spleen, heart, lungs, kidneys, and brain, were collected. Imaging was performed using an IVIS Spectrum system (PerkinElmer), and signal intensity was quantified using Living Image software. Regions of interest (ROIs) were analyzed to determine the signal distribution.
For spinal cord tissue analysis, MSC-EXOs were labeled with DiO, while RVG-CP05 and TAT-CP05 were labeled with FITC and rhodamine B. Multifunctional targeted exosomes (MSC-EXOs, MSC-EXOR&T, and MSC-EXOR&T-GA, 30 μg each) were injected into SCI mice or C57BL/6 control mice. Spinal cord tissues were fixed with 4% paraformaldehyde, dehydrated with graded sucrose solutions, and embedded for sectioning. Sections (10 μm thick) were visualized using a Zeiss 900 inverted confocal microscope (Germany) for analysis of MSC-EXO colocalization.
In vivo experimental design
To assess the therapeutic efficacy of these multifunctional exosomes, groups of mice were established for intravenous injection. These included the sham, equal-volume Injury, MSC-EXO, MSC-EXOR&T, and MSC-EXOR&T-GA groups. Exosomes were administered at a dose of 1 mg/kg (dissolved in 200 μL of PBS) via tail vein injection on Days 1, 3, 7, and 14 post SCI.
BMS score
To evaluate hindlimb motor function recovery, open field BMS scores were recorded for the mice prior to SCI [57], as well as on Day 1, Day 7, and weekly until Week 8. Scoring was conducted independently by two observers. The BMS ranges from 0 to 9, reflecting the degree of hindlimb functional recovery. A score of 0 indicates no voluntary hindlimb movement, whereas a score of 9 represents fully normal hindlimb function.
CatWalk analysis
The CatWalk XT system (version 10.6, Noldus, Wageningen, Netherlands) and its software were used to objectively assess gait, locomotion, and body coordination in the mice [58]. The system consists of a transparent glass runway, with a camera positioned beneath the glass runway to capture and record the mouse paw print data in real time. A red LED background above the platform enhances the visibility of the mouse’s body outlines and movement paths. All the experiments were conducted in a quiet, dimly illuminated environment. Prior to testing, the mice received training, which required at least three consecutive spontaneous crossings of the illuminated walkway. During the experiment, the camera-acquired paw print data were analyzed automatically using CatWalk XT software to evaluate gait and coordination.
Electrophysiological assessment
Electrophysiological testing was performed to assess the recovery of descending motor conduction pathways in 8 weeks post SCI. The mice were deeply anesthetized using pentobarbital, and a single electrical stimulus of 5 mA was applied to the motor cortex to elicit motor-evoked potentials (MEPs). The amplitude of the recorded MEPs were measured for further analysis using an electrophysiological system (YRKJ-G2008, Zhuhai Yirui Technology Co., Ltd.).
Louisville swim scale
The swim test was conducted to evaluate motor function recovery in mice after SCI [59, 60]. Each mouse was placed individually in a water tank maintained at 25 °C for 1 min, and their swimming behavior was observed. Hindlimb coordination, movement, and posture during swimming were evaluated on a scale from 0 to 3 by two independent, blinded observers. Each mouse performed three trials, and the average score was used for further analysis.
Hot-plate test
The hot-plate test was performed to assess sensory function recovery in the mice following SCI. Each mouse was placed on a heated metal plate set at 52 ± 0.5 °C. The time required for the mice to exhibit a nociceptive response, such as shaking, licking, or withdrawal of the hindlimbs, was recorded as the reaction time. A maximum time limit of 30 s was applied to avoid tissue damage. Each mouse experienced three trials with a 10 min interval between each trial. The average reaction time was calculated and analyzed.
Iron content detection
The tissue iron content was determined by tissue iron content assay Kit(Cat# BC4355; Beijing Solarbio Science & Technology Co., Ltd., China), with the iron concentration measured by a spectrophotometer. At the beginning of the experiment, approximately 0.1 g of tissue sample was weighed, and 1 mL of extraction solution was added for homogenization in an ice bath. The mixture was then centrifuged at 4,000 × g for 10 min at 4 °C, and the supernatant was collected for analysis. A visible spectrophotometer was preheated for 30 min, with the wavelength set to 520 nm, and calibrated to zero with distilled water. During sample preparation, the blank tube, standard tube, and sample tube were filled with distilled water, standard solution (0.125 mM Fe3+), and sample, respectively. Reagents one and two were added, followed by mixing and heating in a boiling water bath for 5 min. Chloroform was then added, and the mixture was shaken thoroughly. After centrifugation, the upper inorganic phase was collected, and its absorbance was measured. The tissue iron content was calculated by determining the change in absorbance (ΔA) on the basis of the difference in absorbance between the standard solution and the sample, along with the sample weight and protein concentration.
MDA detection
The MDA detection kit was used to perform the assay.(Cat# BC0025; Beijing Solarbio Science & Technology Co., Ltd., China). First, tissue samples of approximately 0.1 g were homogenized with 1 mL of extraction solution in an ice bath. For the cell or bacterial samples, ultrasound disruption was followed by centrifugation. The supernatant from the processed samples was used for subsequent analysis. The MDA detection working solution was prepared by dissolving reagent two in reagent one, thoroughly mixing, and then the mixture was stored. Next, the visible spectrophotometer or microplate reader was preheated, with the wavelength set to 532 nm, and calibrated to zero using distilled water. In accordance with the instructions of the kit, the sample, MDA detection working solution, and reagent three were added to the measurement tube, mixed thoroughly, and then incubated at 100 °C in a water bath for 60 min. After incubation, the samples were cooled to room temperature and centrifuged to remove impurities, and the supernatant was collected for absorbance measurement in a cuvette. Finally, the MDA content was calculated on the basis of the difference in absorbance (ΔA), specifically the difference between ΔA532 and ΔA600. The MDA content was then calculated according to the tissue weight, sample protein concentration, or cell count to ensure the accuracy of the experiment.
Perfusion, tissue processing, and sectioning
Fully anesthetized mice underwent a midline incision, to fully expose the heart by opening the chest and abdominal cavity. Cardiac perfusion was first performed with precooled PBS and continued until the drainage fluid was clear, indicating that any residual blood had been effectively washed out. Concurrently, the liver tissue gradually turned pale. Perfusion was then continued with precooled 4% paraformaldehyde.
The spinal cord was immersed overnight in a 4% paraformaldehyde solution for fixation. Following fixation, the spinal cord samples were subjected to dehydration using 30% sucrose solution. The dehydrated samples were embedded in optimal cutting temperature (OCT) compound and sectioned into 10-μm-thick slices via a cryostat (Leica CM3050S, Germany).
Immunofluorescence
Following TBST washes, the tissue sections were incubated in QuickBlock™ solution (P0260, Beyotime) for blocking. The sections were subsequently incubated with primary antibodies diluted to their working concentrations. The primary antibodies used included goat anti-GFAP (1:500, Abcam, ab53554), rabbit anti-NeuN (1:500, Abcam, ab104225), rabbit anti-Oligo2 (1:500, Abcam, ab254043), mouse anti-NF200 (1:400, Servicebio, GB12144-100), and rabbit anti-MBP (1:400, Servicebio, GB11226-100) antibodies.
After thorough washing with TBST, the sections were incubated for 2 h with appropriate secondary antibodies conjugated to Alexa Fluor 488 or Cy3. Additional TBST washes were performed to eliminate any unbound reagents, and the tissue sections were mounted with DAPI-stained glass slides. Imaging was conducted using a panoramic imaging system (Vectra Polaris, USA). Areas positive for GFAP, NeuN, Oligo2, NF200, and MBP were quantified via ImageJ software, and threshold analysis was applied to exclude background signals.
RNA-Seq analysis
TRIzol reagent (Invitrogen, USA) was used to extract total RNA from spinal cord tissues or cultured cells in accordance with the manufacturer’s instructions. An Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) were used to assess the quality and amount of RNA. For library development, only samples with RNA integrity numbers (RINs) greater than 7.0 were utilized.
The NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, USA) was used to create RNA-seq libraries. Oligo(dT) beads were used to enrich the mRNAs, which were then broken up and converted into cDNA. To produce 150 bp paired-end reads, the libraries were indexed, amplified by PCR, and sequenced using the Illumina NovaSeq 6000 platform.
The raw sequences were processed using FastQC and Trimmomatic for adapter trimming to guarantee data quality. Gene expression levels were measured using featureCounts after the cleaned reads were matched to the mouse reference genome (GRCm39) using STAR. DESeq2 was used to identify differentially expressed genes (DEGs) with an adjusted P value < 0.05 and a |log2(fold change)| threshold > 1.2. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to perform functional enrichment analysis of the DEGs, and ClusterProfiler in R was used for visualization.
UV–Vis calibration of GA in MSC-EXOR&T-GA
MSC-EXOR&T-GA, MSC-EXOR&T and GA were dissolved in PBS and analyzed by ultraviolet–visible (UV–Vis) spectroscopy between 200 and 400 nm, revealing an absorption maximum for GA at 300 nm.
Serial dilutions of MSC-EXOR&T-GA (1, 2, 4, 6, 8, and 10 μg/mL) were analyzed by UV–Vis spectrophotometry at 300 nm. A linear calibration curve (Y vs X) was generated, with the regression equation Y = 0.003926X + 0.7629 (R2 = 0.9882).
Statistical analysis
The data are expressed as the mean ± SD. For comparisons among three or more groups, one-way analysis of variance (one-way ANOVA) was performed, followed by Tukey’s post-hoc test; while statistical comparisons between two groups were conducted using Student’s t-test. Details of the specific statistical analyses are offered in the figure legends. Significance thresholds were defined as follows: P > 0.05 (not significant, ns); P ≤ 0.05 (significant, *); P ≤ 0.01 (highly significant, **); P ≤ 0.001 (very significant, ***); and P ≤ 0.0001 (extremely significant, ****).
Sample sizes were determined on the basis of prior power analyses to ensure sufficient statistical power. All experimental procedures and analyses adhered to ethical guidelines to ensure reproducibility and consistency throughout the study.
