Preparation of peptides
The peptides had been synthesized by Chinapeptide Co. Ltd. and saved as lyophilized powders at −80 °C. To arrange the exosomes, these peptides had been dissolved in PBS. The peptide sequences used had been as follows:
RVG-CP05, YTIWMPENPRPGTPCDIFTNSRGKRASNGGGGSCRHSQMTVTSRL
TAT-CP05, YGRKKRRQRRRGGGSCRHSQMTVTSRL
Isolation, purposeful modification, and characterization of MSC-EXOR&T-GA
The supernatants obtained from human umbilical wire MSC cultures underwent sequential centrifugation to get rid of cells and particles. The method adopted a stepwise protocol: preliminary centrifugation at 300 × g for 10 min, subsequent centrifugation at 2,000 × g for 20 min, and, in the end, centrifugation at 10,000 × g for 30 min. After passing by a 0.22-μm sterile membrane (Millipore), the ensuing supernatant was ultracentrifuged for 70 min at 130,000 × g (Optima L-100 XP, Beckman Coulter). A second ultracentrifugation step was carried out on the pellet on the identical parameters after the pellet was reconstituted in 1 mL of PBS. The BCA check was used to measure the quantity of protein.
MSC-EXOs had been incubated with RVG-CP05 and TAT-CP05, both individually or collectively, at 4 °C for six h. To take away unbound peptides, the mixtures had been handed by 100-Kd filter tubes (Millipore) and washed 5 instances with PBS. Lastly, PBS was used to resuspend the complexes, yielding MSC-EXOR and MSC-EXOR&T.
Exosomes had been conjugated to GA by a DIC/NHS-mediated amide response. First, MSC-EXOR&T had been handled with MES buffer (pH 6.0) and dispersed ultrasonically to activate carboxyl teams. DIC and NHS in DMSO activated these teams, forming NHS esters. After 30 min, an amino-containing GA answer was added, and the pH was adjusted to 7.4. NHS esters shaped amide bonds with GA amino teams throughout a 1-h incubation. Extra byproducts had been eliminated by ultrafiltration and ultracentrifugation.Profitable conjugation was validated utilizing UV absorption spectroscopy and fluorescence resonance power switch (FRET) assay. Ready contemporary for fast 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 measurement and distribution had been assessed utilizing NTA. EV-specific markers had been verified by Western blot evaluation.
Cell tradition
The HT22 and OLN93 cell traces had been cultured in Dulbecco’s modified Eagle medium/nutrient combination 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 tradition medium was modified each 2 days. To avoid the oxidative stress results of glutamate on the HT22 cell line, all medium utilized in vitro experiments had been devoid of glutamate.
Exosome uptake and endocytosis
Exosomes had been labeled with the lipophilic dye DiO (3,3’-dioctadecyloxacarbocyanine perchlorate, Invitrogen), whereas RVG-CP05 and TAT-CP05 had been tagged with FITC and Rhodamine B, respectively. MSC-EXOR and MSC-EXOR&T had been ready following customary protocols. Purified exosomes had been resuspended in PBS at 100 µg/mL. A ten µM DiO inventory answer in ethanol was diluted in PBS to a closing focus of 1 µM. Equal volumes of DiO working answer and the exosome suspension had been gently blended and incubated at 37 °C for 30 min, enabling the dye to be included into the exosome lipid bilayer. To take away any unbound dye, ultracentrifugation was carried out at 130,000 × g for 70 min at 4 °C (Beckman Coulter, USA). The ensuing pellet was washed with PBS and centrifuged once more to take away any residual dye, after which the labeled exosomes had been resuspended in PBS. Fluorescence microscopy confirmed profitable labeling.
For the endocytosis assay, 10 μg of DiO-labeled MSC-EXOR or MSC-EXOR&T had been added to the cell tradition medium. Following incubation, the cells had been mounted and stained with a rabbit anti-NeuN antibody (1:500, Abcam, ab104225), adopted by therapy with a secondary antibody. Exosome uptake was visualized utilizing an LSM900 inverted confocal microscope (Germany, ZEISS).
Spinal wire damage
This analysis utilized feminine C57BL/6 mice (8 weeks previous, 20 g physique weight) from Beijing Important River Laboratory Animal Expertise Co., Ltd. (Guangdong, China). To induce the spinal contusion mannequin, the mice had been anesthetized with isoflurane (RWD, R510-22), and a midline incision was made on the T10 vertebra. The encompassing muscle mass and connective tissues of the thoracic backbone had been fastidiously dissected with minimal disruption to adjoining buildings, exposing the T9–T11 spinous processes. Laminectomy was carried out at T10 to reveal the spinal wire, and exact incisions had been made alongside the vertebral arch to take away the lamina. To stabilize the backbone, a fixation gadget was used, and average spinal wire contusion was induced by way of the NYU Impression-III gadget (WM Keck, USA) by making use of a free-fall pressure of 5 g from a 12.5 mm top. Postinjury, the wound was sutured in layers. The bladder was manually expressed for 2 weeks following the damage. All of the animal experiments had been authorised by the Medical Ethics Committee and the Animal Welfare Ethics Committee of Tianjin Medical College Normal Hospital (IRB2021-KY-317, IRB2021-DWFL-354).
Tissue distribution
To evaluate the biodistribution of MSC-EXOR&T-GA in an SCI mouse mannequin, DiR dye (Life Applied sciences) 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 every), MSC-EXOR&T-GA, or MSC-EXOR had been injected intravenously into SCI mice. Two hours after injection, 50 mL of chilly PBS was used to perfuse the mice to clear the circulating exosomes. Tissue samples, together with the spinal wire, quadriceps, triceps, liver, spleen, coronary heart, lungs, kidneys, and mind, had been collected. Imaging was carried out utilizing an IVIS Spectrum system (PerkinElmer), and sign depth was quantified utilizing Dwelling Picture software program. Areas of curiosity (ROIs) had been analyzed to find out the sign distribution.
For spinal wire tissue evaluation, MSC-EXOs had been labeled with DiO, whereas RVG-CP05 and TAT-CP05 had been labeled with FITC and rhodamine B. Multifunctional focused exosomes (MSC-EXOs, MSC-EXOR&T, and MSC-EXOR&T-GA, 30 μg every) had been injected into SCI mice or C57BL/6 management mice. Spinal wire tissues had been mounted with 4% paraformaldehyde, dehydrated with graded sucrose options, and embedded for sectioning. Sections (10 μm thick) had been visualized utilizing a Zeiss 900 inverted confocal microscope (Germany) for evaluation of MSC-EXO colocalization.
In vivo experimental design
To evaluate the therapeutic efficacy of those multifunctional exosomes, teams of mice had been established for intravenous injection. These included the sham, equal-volume Harm, MSC-EXO, MSC-EXOR&T, and MSC-EXOR&T-GA teams. Exosomes had been administered at a dose of 1 mg/kg (dissolved in 200 μL of PBS) by way of tail vein injection on Days 1, 3, 7, and 14 publish SCI.
BMS rating
To judge hindlimb motor operate restoration, open subject BMS scores had been recorded for the mice previous to SCI [57], in addition to on Day 1, Day 7, and weekly till Week 8. Scoring was performed independently by two observers. The BMS ranges from 0 to 9, reflecting the diploma of hindlimb purposeful restoration. A rating of 0 signifies no voluntary hindlimb motion, whereas a rating of 9 represents absolutely regular hindlimb operate.
CatWalk evaluation
The CatWalk XT system (model 10.6, Noldus, Wageningen, Netherlands) and its software program had been used to objectively assess gait, locomotion, and physique coordination within the mice [58]. The system consists of a clear glass runway, with a digital camera positioned beneath the glass runway to seize and report the mouse paw print information in actual time. A pink LED background above the platform enhances the visibility of the mouse’s physique outlines and motion paths. All of the experiments had been performed in a quiet, dimly illuminated atmosphere. Previous to testing, the mice acquired coaching, which required at the very least three consecutive spontaneous crossings of the illuminated walkway. In the course of the experiment, the camera-acquired paw print information had been analyzed mechanically utilizing CatWalk XT software program to guage gait and coordination.
Electrophysiological evaluation
Electrophysiological testing was carried out to evaluate the restoration of descending motor conduction pathways in 8 weeks publish SCI. The mice had been deeply anesthetized utilizing pentobarbital, and a single electrical stimulus of 5 mA was utilized to the motor cortex to elicit motor-evoked potentials (MEPs). The amplitude of the recorded MEPs had been measured for additional evaluation utilizing an electrophysiological system (YRKJ-G2008, Zhuhai Yirui Expertise Co., Ltd.).
Louisville swim scale
The swim check was performed to guage motor operate restoration in mice after SCI [59, 60]. Every mouse was positioned individually in a water tank maintained at 25 °C for 1 min, and their swimming habits was noticed. Hindlimb coordination, motion, and posture throughout swimming had been evaluated on a scale from 0 to three by two unbiased, blinded observers. Every mouse carried out three trials, and the common rating was used for additional evaluation.
Sizzling-plate check
The recent-plate check was carried out to evaluate sensory operate restoration within the mice following SCI. Every mouse was positioned on a heated steel plate set at 52 ± 0.5 °C. The time required for the mice to exhibit a nociceptive response, reminiscent of shaking, licking, or withdrawal of the hindlimbs, was recorded because the response time. A most time restrict of 30 s was utilized to keep away from tissue injury. Every mouse skilled three trials with a ten min interval between every trial. The typical response time was calculated and analyzed.
Iron content material detection
The tissue iron content material was decided by tissue iron content material assay Equipment(Cat# BC4355; Beijing Solarbio Science & Expertise Co., Ltd., China), with the iron focus measured by a spectrophotometer. At the start of the experiment, roughly 0.1 g of tissue pattern was weighed, and 1 mL of extraction answer was added for homogenization in an ice bathtub. The combination was then centrifuged at 4,000 × g for 10 min at 4 °C, and the supernatant was collected for evaluation. A visual spectrophotometer was preheated for 30 min, with the wavelength set to 520 nm, and calibrated to zero with distilled water. Throughout pattern preparation, the clean tube, customary tube, and pattern tube had been full of distilled water, customary answer (0.125 mM Fe3+), and pattern, respectively. Reagents one and two had been added, adopted by mixing and heating in a boiling water bathtub for five min. Chloroform was then added, and the combination was shaken totally. After centrifugation, the higher inorganic section was collected, and its absorbance was measured. The tissue iron content material was calculated by figuring out the change in absorbance (ΔA) on the idea of the distinction in absorbance between the usual answer and the pattern, together with the pattern weight and protein focus.
MDA detection
The MDA detection package was used to carry out the assay.(Cat# BC0025; Beijing Solarbio Science & Expertise Co., Ltd., China). First, tissue samples of roughly 0.1 g had been homogenized with 1 mL of extraction answer in an ice bathtub. For the cell or bacterial samples, ultrasound disruption was adopted by centrifugation. The supernatant from the processed samples was used for subsequent evaluation. The MDA detection working answer was ready by dissolving reagent two in reagent one, totally mixing, after which the combination was saved. Subsequent, the seen spectrophotometer or microplate reader was preheated, with the wavelength set to 532 nm, and calibrated to zero utilizing distilled water. In accordance with the directions of the package, the pattern, MDA detection working answer, and reagent three had been added to the measurement tube, blended totally, after which incubated at 100 °C in a water bathtub for 60 min. After incubation, the samples had been cooled to room temperature and centrifuged to take away impurities, and the supernatant was collected for absorbance measurement in a cuvette. Lastly, the MDA content material was calculated on the idea of the distinction in absorbance (ΔA), particularly the distinction between ΔA532 and ΔA600. The MDA content material was then calculated in response to the tissue weight, pattern protein focus, or cell rely to make sure the accuracy of the experiment.
Perfusion, tissue processing, and sectioning
Totally anesthetized mice underwent a midline incision, to totally expose the guts by opening the chest and stomach cavity. Cardiac perfusion was first carried out with precooled PBS and continued till the drainage fluid was clear, indicating that any residual blood had been successfully washed out. Concurrently, the liver tissue progressively turned pale. Perfusion was then continued with precooled 4% paraformaldehyde.
The spinal wire was immersed in a single day in a 4% paraformaldehyde answer for fixation. Following fixation, the spinal wire samples had been subjected to dehydration utilizing 30% sucrose answer. The dehydrated samples had been embedded in optimum reducing temperature (OCT) compound and sectioned into 10-μm-thick slices by way of a cryostat (Leica CM3050S, Germany).
Immunofluorescence
Following TBST washes, the tissue sections had been incubated in QuickBlock™ answer (P0260, Beyotime) for blocking. The sections had been subsequently incubated with major antibodies diluted to their working concentrations. The first 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 had been incubated for two h with acceptable secondary antibodies conjugated to Alexa Fluor 488 or Cy3. Extra TBST washes had been carried out to get rid of any unbound reagents, and the tissue sections had been mounted with DAPI-stained glass slides. Imaging was performed utilizing a panoramic imaging system (Vectra Polaris, USA). Areas optimistic for GFAP, NeuN, Oligo2, NF200, and MBP had been quantified by way of ImageJ software program, and threshold evaluation was utilized to exclude background alerts.
RNA-Seq evaluation
TRIzol reagent (Invitrogen, USA) was used to extract whole RNA from spinal wire tissues or cultured cells in accordance with the producer’s directions. An Agilent 2100 Bioanalyzer (Agilent Applied sciences, USA) and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) had been used to evaluate the standard and quantity of RNA. For library growth, solely samples with RNA integrity numbers (RINs) larger than 7.0 had been utilized.
The NEBNext Extremely RNA Library Prep Equipment for Illumina (New England Biolabs, USA) was used to create RNA-seq libraries. Oligo(dT) beads had been used to complement the mRNAs, which had been then damaged up and transformed into cDNA. To provide 150 bp paired-end reads, the libraries had been listed, amplified by PCR, and sequenced utilizing the Illumina NovaSeq 6000 platform.
The uncooked sequences had been processed utilizing FastQC and Trimmomatic for adapter trimming to ensure information high quality. Gene expression ranges had been measured utilizing featureCounts after the cleaned reads had been matched to the mouse reference genome (GRCm39) utilizing STAR. DESeq2 was used to establish differentially expressed genes (DEGs) with an adjusted P worth < 0.05 and a |log2(fold change)| threshold > 1.2. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses had been used to carry out purposeful enrichment evaluation 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 had been dissolved in PBS and analyzed by ultraviolet–seen (UV–Vis) spectroscopy between 200 and 400 nm, revealing an absorption most for GA at 300 nm.
Serial dilutions of MSC-EXOR&T-GA (1, 2, 4, 6, 8, and 10 μg/mL) had been 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 evaluation
The info are expressed because the imply ± SD. For comparisons amongst three or extra teams, one-way evaluation of variance (one-way ANOVA) was carried out, adopted by Tukey’s post-hoc check; whereas statistical comparisons between two teams had been performed utilizing Pupil’s t-test. Particulars of the precise statistical analyses are supplied within the determine legends. Significance thresholds had been outlined as follows: P > 0.05 (not vital, ns); P ≤ 0.05 (vital, *); P ≤ 0.01 (extremely vital, **); P ≤ 0.001 (very vital, ***); and P ≤ 0.0001 (extraordinarily vital, ****).
Pattern sizes had been decided on the idea of prior energy analyses to make sure adequate statistical energy. All experimental procedures and analyses adhered to moral tips to make sure reproducibility and consistency all through the examine.