Application of ROS-responsive trifluoperazine nanomicelles combined with MSC exosomes in traumatic spinal cord injury

Abstract

The present application relates to the biomedical field, specifically is targeted to edema protein 4 (AQP4) ROS response type trifluoperazine (TFP) nanomicelles and MSC exosome synergistic combination in the preparation of the application of treating traumatic spinal cord injury drugs. The ROS (reactive oxygen) response type trifluoperazine (Trifluoperazine, TFP) nanomicelles combined with MSC exosome can treat spinal cord injury patients in all directions. By verifying its single drug efficacy, the combined treatment group is better in cell edema and animal level treatment effect, indicating that the combination strategy has significant advantages in improving the treatment effect. Further enriches the treatment of spinal cord injury, and provides a new theoretical basis and new idea for future clinical treatment scheme design.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of ROS-responsive trifluoperazine (TFP) nanomicelles combined with MSC exosomes in traumatic spinal cord injury. Background Technology

[0002] Spinal cord injury (SCI) is a particularly common trauma that often leads to devastating damage to neurological function, severely impacting patients' quality of life. With the development of modern society, economy, transportation, and construction, the incidence of SCI is rising annually. Statistics show that in the United States, the incidence of SCI is approximately 54 cases per million people, equivalent to about 17,900 SCI patients annually. After a spinal cord injury, patients' lifespan and quality of life can decline sharply. Statistics indicate that the average lifespan of SCI patients is only 3.7 years, and most patients require assistance from others or wheelchairs. Spinal cord injury imposes a heavy social and economic burden on patients, families, and society. Therefore, research on spinal cord injury treatment, and the protection and restoration of spinal cord function after injury, is of great significance for improving the quality of life of spinal cord injury patients and alleviating the burden on families and society.

[0003] The treatment principle for spinal cord injury is to salvage spinal cord function and avoid secondary spinal cord injury as much as possible. Early secondary spinal cord edema following primary spinal cord injury is extremely harmful, increasing intraspinal pressure, leading to microcirculatory disturbances, rapidly causing ischemia and necrosis of damaged local tissues, amplifying the inflammatory response, exacerbating nerve cell damage, reducing the number of functional residual nerve cells, and worsening spinal cord injury. Although surgical laminectomy within 24 hours of acute spinal cord injury is related to decompression and improved sensory and motor recovery, its function is limited by other structures of the spinal cord, such as the pia mater and dura mater. Aquaporin (AQP) is a protein located on the cell membrane, forming "pores" that control the movement of water in and out of the cell, maintaining the body's water balance. There are several subclasses of aquaporin, among which AQP4 is widely distributed in the brain and spinal cord and is an important protein involved in the formation of spinal cord edema. Within one hour of spinal cord injury, AQP4 is expressed in large quantities, leading to acute spinal cord edema and exacerbating spinal cord tissue damage. Inhibiting AQP4 protein expression can reduce spinal cord cell edema intoxication and the spinal cord edema index, thus alleviating spinal cord injury. In recent years, studies have shown that AQP4 can play a regulatory role in edema, glial scar formation, and improving the cellular inflammatory microenvironment after spinal cord injury (SCI). Summary of the Invention

[0004] The purpose of this invention is to provide the application of ROS-responsive trifluoperazine (TFP) nanomicelles combined with MSC exosomes in spinal cord injury, so as to enhance the therapeutic effect of spinal cord injury treatment through the synergy of the two.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] ① Design, synthesis and identification of ROS-responsive trifluoperazine (TFP) nanomicelles;

[0007] ②MSC culture, exosome extraction and identification;

[0008] ③The combined effects of ROS-responsive trifluoperazine (TFP) nanomicelles and MSC exosomes on astrocytes under hypoxic and hypoglycemic conditions, including cell edema, cell survival, and A1 and A2 differentiation of astrocytes;

[0009] ④ The combined effects of ROS-responsive trifluoperazine (TFP) nanomicelles and MSC exosomes on mice after spinal cord injury, including postoperative motor function recovery, spinal cord structure, scarring, and neuronal status.

[0010] 10-[3-(4-methylpiperazine-1-ylpropyl)]-2-trifluoromethyl-10H-phenothiazine (TFP) is an approved phenothiazine antipsychotic and anxiolytic drug. A single injection of TFP at the site of injury in a rat SCI model, equivalent to the human dose, reduced spinal cord edema by 56% within 72 hours and completely resolved it within 7 days. It also significantly improved the recovery of electrophysiological, sensory, and motor functions in the rat SCI model. Nanoparticle drug delivery systems can selectively target and deliver drugs to the lesion site of spinal cord injury, significantly improving drug concentration distribution and release efficiency. They allow for controlled release at different times and tissue sites, increasing drug loading efficiency and bioavailability, while also improving drug solubility and reducing side effects. Therefore, the development of a "smart" drug delivery system, specifically ROS-responsive targeted AQP4 nanomicelles for intravenous injection, is underway for early use in patients with spinal cord injury.

[0011] Exosomes are nanoscale vesicles secreted by cells, ranging in size from 30 to 150 nanometers. They can carry bioactive molecules such as proteins and RNA, playing a crucial role in intercellular communication. Due to their excellent biocompatibility, low immunogenicity, and ability to cross the blood-brain barrier, exosomes are considered ideal drug delivery systems and therapeutic tools. Mesenchymal stem cells (MSCs) were initially thought to function by differentiating and replacing damaged neurons, but later studies have found that their therapeutic effects mainly stem from the paracrine factors they secrete. Mesenchymal stem cell exosomes (MSC-Exos) are one of the most promising research directions in the treatment of spinal cord injury (SCI). Compared to direct transplantation of mesenchymal stem cells (MSCs), exosomes, as a "cell-free therapy," have higher safety and biological activity. Therefore, intravenous injection of nanomicelles + exosomes in the early stages of spinal cord injury can promote spinal cord recovery.

[0012] Based on the above technical solution, in a first aspect, the present invention provides a pharmaceutical composition for treating traumatic spinal cord injury, comprising: ROS (reactive oxygen species) responsive trifluoperazine (TFP) nanomicelles, and mesenchymal stem cell exosomes (MSC-Exos) derived from mesenchymal stem cells (MSC).

[0013] Furthermore, the nanomicelles are self-assembled from amphiphilic block copolymers, and the hydrophilic and hydrophobic segments of the copolymers are connected by ROS-sensitive chemical bonds; preferably, the chemical bonds are thioketal bonds (TK), arylboronic acid ester bonds, or thioether bonds.

[0014] Furthermore, the amphiphilic block copolymer is polyethylene glycol-thioketal-polycaprolactone (PEG-TK-PCL).

[0015] Furthermore, the trifluoperazine is a free trifluoperazine (TFP Base), which is encapsulated in the hydrophobic core of the nanomicelles.

[0016] Furthermore, the mesenchymal stem cell exosomes were extracted from the culture supernatant of human umbilical cord mesenchymal stem cells (hUC-MSCs) by differential ultracentrifugation.

[0017] A second aspect of the present invention provides the use of the pharmaceutical composition described above in the preparation of a medicament for treating traumatic spinal cord injury.

[0018] Furthermore, the pharmaceutical composition synergistically inhibits the translocation of edema protein 4 (AQP4), reduces spinal cord edema, and regulates the polarization of astrocytes towards the A2 type, thereby promoting the recovery of neurological function.

[0019] Furthermore, the drug is an intravenous injection preparation.

[0020] Furthermore, the pharmaceutical composition is used in the preparation of drugs that inhibit inflammatory responses following spinal cord injury, reduce glial scar formation, promote angiogenesis, and improve neuronal survival.

[0021] A third aspect of the present invention provides a formulation for treating traumatic spinal cord injury, comprising a therapeutically effective amount of reactive oxygen species (ROS)-responsive nanomicelles loaded with trifluoperazine (TFP) and mesenchymal stem cell exosomes (MSC-Exos) derived from mesenchymal stem cells (MSCs); wherein the nanomicelles are self-assembled from an amphiphilic block copolymer, wherein the hydrophilic and hydrophobic segments of the copolymer are linked by ROS-sensitive chemical bonds; wherein the trifluoperazine is in a free state and is encapsulated in the hydrophobic core of the nanomicelles.

[0022] The advantages of this invention are:

[0023] In the pathophysiology of spinal cord injury (SCI), edema is not only a symptom accompanying the injury but also a core driving force of "secondary injury," leading to further deterioration of neurological function. The spinal cord is encased within the rigid and inelastic vertebral canal and dura mater, making the impact of edema on the spinal cord far more severe than on other tissues. Current treatments are not ideal; therefore, this invention targets the regulatory proteins that cause edema, employing a targeted design to prepare ROS (reactive oxygen species)-responsive trifluoperazine (TFP) nanomicelles—an advanced drug delivery strategy. Besides its use as an antipsychotic, trifluoperazine has recently been found to inhibit calmodulin in SCI, thereby reducing edema and inhibiting glial scarring. The ROS-responsive design allows for targeted drug release at the injury site (where ROS concentration is high in the inflammatory area), improving efficacy and reducing systemic side effects. Simultaneously, intravenous injection allows for early administration. The pathophysiological mechanisms following spinal cord injury are complex. While edema is a significant mechanism, ischemia, hypoxia, and inflammation also play important roles. The preparation of ROS (reactive oxygen species)-responsive trifluoperazine (TFP) nanomicelles represents an advanced drug delivery strategy. The ROS-responsive design allows for targeted drug release at the injury site (where ROS concentrations are high in the inflammatory zone), thereby improving efficacy and reducing systemic side effects. MSC exosomes play a "microenvironment regulator" role in spinal cord injury treatment. They not only suppress harmful inflammatory storms but also provide essential signals and nutrients for nerve repair.

[0024] 1. The ROS-responsive trifluoperazine (TFP) nanomicelles developed in this invention inhibit the translocation of edema protein 4 (AQP4), and when combined with MSC exosomes, they can effectively improve the function of mice after traumatic spinal cord injury and promote the functional recovery after traumatic spinal cord injury, indicating that this combination strategy has significant advantages in improving the therapeutic effect.

[0025] 2. The ROS (reactive oxygen species) responsive trifluoperazine (TFP) nanomicelles combined with MSC exosomes of this invention can provide comprehensive treatment for patients with spinal cord injury. Verification of the combined treatment group showed better efficacy in cellular edema and animal studies compared to single-drug therapy, indicating that this combination strategy has significant advantages in improving treatment outcomes.

[0026] 3. This invention further enriches the treatment methods for spinal cord injury, and provides a new theoretical basis and new ideas for the design of future clinical treatment plans. Attached Figure Description

[0027] Figure 1 ROS (Reactive Oxygen Species) responsive trifluoperazine (TFP) nanomicelles were successfully constructed. (A) Schematic diagram of synthesis; (B) Morphology and hydrodynamic diameter of the micelles under a transmission electron microscope.

[0028] Figure 2 Morphological images of human mesenchymal stem cells (MSCs) at 5, 10, and 15 days during their primary culture.

[0029] Figure 3 Phenotypic identification of cultured MSCs. (A) Morphological images of MSCs, (B, C, and D) positive markers (≥95% expression): CD90, CD73, CD105, (E, F, G, H, I, and J) negative markers (≤2% expression): CD19, CD31, CD34, CD45, HLA-DR.

[0030] Figure 4 MSC three-differentiation function identification: the left image shows osteogenic differentiation, the middle image shows chondrogenic differentiation, and the right image shows adipogenic differentiation.

[0031] Figure 5 MSC exosome identification: Figure A shows the morphology of exosomes observed by transmission electron microscopy. B. Particle size distribution. C. Positive markers: transmembrane / membrane-associated proteins: CD81, CD9; endosome-derived proteins: TSG101; endoplasmic reticulum: Calnexin.

[0032] Figure 6Primary identification of astrocytes: immunofluorescence detection of GFAP.

[0033] Figure 7 Astrocyte exosome uptake: Exosomes were co-incubated with astrocytes, and PKH26 was expressed in the cytoplasm of astrocytes.

[0034] Figure 8 Effects of ROS (Reactive Oxygen Species) responsive trifluoperazine (TFP) nanomicelles combined with MSC exosomes on astrocytes under hypoxia and hypoglycemic conditions: (A) and (B) f are cell viability and apoptosis detection, respectively.

[0035] Figure 9 Effects of ROS (Reactive Oxygen Species) responsive trifluoperazine (TFP) nanomicelles combined with MSC exosomes on astrocyte edema under hypoxia and hypoglycemia.

[0036] Figure 10 Effects of ROS (reactive oxygen species) responsive trifluoperazine (TFP) nanomicelles combined with MSC exosomes on A1 and A2 differentiation of astrocytes under hypoxic and hypoglycemic conditions.

[0037] Figure 11 Effects of ROS (Reactive Oxygen Species) responsive trifluoperazine (TFP) nanomicelles combined with MSC exosomes on hind limb function in mice after spinal cord injury. (A) Schematic diagram, nanomicelles (immediately after model establishment) and exosomes (immediately after model establishment, 1W, 2W, 3W and 4W) were administered via tail vein injection; (B) Establishment of mouse spinal cord injury model, with post-spinal cord hemorrhage and edema; (C) Gross specimen of spinal cord after treatment; (D) Morphological changes of the right hind limb after treatment; (E) BMS score; (F) Imprint test; (G) Mine field test.

[0038] Figure 12 The effects of ROS (reactive oxygen species) responsive trifluoperazine (TFP) nanomicelles combined with MSC exosomes on spinal cord recovery after injury. CD31, GFAP, and NeuN fluorescent staining revealed angiogenesis, scarring, and neuronal survival.

[0039] Figure 13 The effects of ROS (reactive oxygen species) responsive trifluoperazine (TFP) nanomicelles combined with MSC exosomes on spinal cord recovery after injury. HE staining, Masson staining, and Nissan staining revealed spinal cord structure, scarring, and neuronal status.

[0040] Figure 14 Flowchart of the application of ROS-responsive trifluoperazine nanomicelles combined with MSC exosomes in traumatic spinal cord injury. Detailed Implementation

[0041] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0042] Example 1: Preparation of ROS (Reactive Oxygen Species) Responsive Trifluoperazine (TFP) Nanomicelles

[0043] I. Design Concept

[0044] 1. Drug (Cargo): Trifluoperazine (TFP). Since TFP usually exists in the form of hydrochloride (water-soluble), it needs to be liberated (made hydrophobic) before preparing micelles so that it can be encapsulated in the hydrophobic centers of the micelles.

[0045] 2. Carrier amphiphilic block copolymer. Must contain:

[0046] Hydrophilic segments, such as PEG (polyethylene glycol), provide long cycling capability.

[0047] Hydrophobic segments: such as PCL (polycaprolactone), PLA (polylactic acid), or PPS (polypropylene sulfide).

[0048] Responsive linkages: Introducing ROS-sensitive chemical bonds between hydrophilic and hydrophobic segments, such as thioethers (TK), aryl boronic esters, or thioethers.

[0049] II. Material Preparation (Taking PEG-TK-PCL as an example)

[0050] Polymer: PEG-TK-PCL (can be chemically synthesized by using a small molecule of thioacetate containing dihydroxyl groups as an initiator to ring-open polymerize caprolactone, and then couple it with PEG).

[0051] Solvents: Organic solvents (such as DMSO, DMF or dichloromethane), ultrapure water.

[0052] Drug: Free trifluoperazine (TFP Base).

[0053] III. Preparation steps (thin-film hydration method or dialysis method)

[0054] Method A: Thin-film hydration method (suitable for large-scale preparation)

[0055] 1. Dissolution: Dissolve the PEG-TK-PCL polymer and free TFP in an organic solvent (such as dichloromethane) at a certain mass ratio (e.g., 10:1).

[0056] 2. Membrane: Using a rotary evaporator, the solvent is removed under reduced pressure to form a uniform drug-polymer film on the inner wall of a round-bottom flask.

[0057] 3. Hydration: Add phosphate buffered saline (PBS) and hydrate by shaking or rotating at 40-50℃ to allow the film to detach and self-assemble into micelles.

[0058] 4. Refining: The particle size is uniformized by ultrasonic testing using a probe.

[0059] Method B: Solvent dialysis (prepares more uniform particle size)

[0060] 1. Mixing: Dissolve the polymer and TFP in a polar organic solvent (such as DMSO).

[0061] 2. Self-assembly: The mixed solution is loaded into a dialysis bag (the molecular weight cutoff is determined according to the polymer, usually 3500 Da).

[0062] 3. Dialysis: Place the dialysis bag in ultrapure water and change the water every few hours. As the organic solvent diffuses outward, the hydrophobic segments polymerize, and TFP is automatically encapsulated within the core.

[0063] IV. Post-processing and purification

[0064] 1. Impurity removal: Filter through a 0.22 μm microporous membrane to remove unencapsulated free drug precipitates.

[0065] 2. Concentration: Centrifugation is performed using ultrafiltration units.

[0066] 3. Storage: Add a freeze-drying protectant (such as 5% sucrose or trehalose), freeze-dry to obtain a powder for long-term storage.

[0067] V. Characterization and Detection

[0068] To ensure successful preparation, the following tests are required:

[0069] 1. Particle size and potential (DLS): Measures the hydrodynamic diameter (typically 50-150 nm) and surface charge of micelles.

[0070] 2. Morphology (TEM): Observe whether the micelles are regular spherical shapes using transmission electron microscopy.

[0071] 3. Encapsulation efficiency and drug loading (HPLC): The amount of TFP was determined by high performance liquid chromatography after demulsification.

[0072] Drug loading (DLC) = (Material mass in micelles / Total mass of micelles) × 100%

[0073] 4. ROS responsive release experiment:

[0074] Micelles were placed in release media containing different concentrations of H2O2 (simulating a ROS environment). Drug release was monitored at different time points. Due to the breaking of TK bonds and the disintegration of the micelle structure, the higher the ROS concentration, the faster the release.

[0075] VI. Application in Spinal Cord Injury (SCI)

[0076] Targeting: Specific ligands (such as RGD peptides targeting damaged vascular endothelium or ligands targeting neurons) can be coupled to the PEG terminus to enhance the ability to target the spinal cord injury area.

[0077] Injection method: Considering the pathological environment of SCI, the prepared nanomicelles can be administered via intravenous injection (relying on the EPR effect or inflammation targeting).

[0078] VII. Precautions

[0079] Hydrophobication of TFP: Trifluoperazine hydrochloride must first be treated with an alkali (such as NaOH) to extract the free alkali form, otherwise it cannot be effectively loaded into the micelle core.

[0080] Stability: Although the TK bond is sensitive to ROS, it is stable under light-protected and low-temperature conditions. Strong light exposure should be avoided during the preparation process.

[0081] VIII. Experimental Results

[0082] ROS (reactive oxygen species) responsive trifluoperazine (TFP) nanomicelles were successfully constructed. Figure 1 Schematic diagram A: Under ROS response, the TK disulfide bond opens, releasing trifluoperazine; Figure 1 B. Observation using transmission electron microscopy revealed that the micelles were regular spherical shapes. Figure 1 C shows that the hydrodynamic diameter of the micelles is between 50 and 150 nm.

[0083] This ROS-responsive TFP nanomicelle can precisely release drugs during the peak of inflammation in spinal cord injury, not only inhibiting edema through TFP but also reducing secondary damage to nerve cells caused by oxidative stress.

[0084] Example 2: Preparation of mesenchymal stem cell exosomes (MSC-Exos)

[0085] The preparation of mesenchymal stem cell exosomes (MSC-Exos) includes four main stages: MSC cell culture, supernatant collection, separation and purification, and identification.

[0086] I. Cell Origin and Culture

[0087] 1. Source Selection: Human Umbilical Cord (hUC-MSCs)

[0088] 2. Cell expansion: MSCs are cultured to the 3rd to 6th generation (P3-P6), at which time the cell activity is strongest and the secretory capacity is most stable.

[0089] 3. Key Step: Replace with "Exosome-Free Culture Medium"

[0090] When the cells reach 70%-80% confluence, discard the culture medium containing conventional fetal bovine serum (FBS).

[0091] Wash 2-3 times with PBS to remove residual FBS (regular FBS contains a large number of bovine exosomes, which can cause contamination).

[0092] Replace with exosome-free serum culture medium (use serum that has been de-exosome-free by ultracentrifugation).

[0093] 4. Collection time: After culturing for another 24-48 hours, collect the cell culture supernatant (Conditioned Medium, CM). At this time, ensure that the cell viability is >90%.

[0094] II. Isolation and Purification of Exosomes

[0095] Differential Ultracentrifugation

[0096] Cells, debris, and large vesicles are removed by gradually increasing the rotation speed.

[0097] 300 g × 10 min: Remove residual cells.

[0098] 2,000 g × 20 min: Remove cell debris.

[0099] 10,000 g × 30 min: Remove large vesicles (such as microvesicles).

[0100] 100,000 - 120,000 g × 70 min (or longer): precipitate exosomes.

[0101] Washing: The resulting precipitate was resuspended in a large amount of PBS and centrifuged again at 100,000 g to remove excess protein.

[0102] Resuspension: Finally, resuspend the precipitate with a small amount of sterile PBS.

[0103] III. Characterization of Exosomes

[0104] Identification was conducted according to the standards of the International Society for Extracellular Vesicles (ISEV), based on three dimensions:

[0105] 1. Morphological observation (TEM):

[0106] Use a transmission electron microscope.

[0107] Typical characteristics: It exhibits a typical "cup-shaped" or "cup-shaped" double-layer membrane structure with a diameter between 30 and 150 nm.

[0108] 2. Particle size distribution (NTA):

[0109] The concentration (particles / mL) and peak particle size distribution (typically around 100 nm) of exosomes were detected using a nanoparticle tracking analysis instrument.

[0110] V. Storage and Quality Control

[0111] Store at -80°C. Minimize the number of freeze-thaw cycles, as repeated freeze-thaw cycles can cause exosome membrane rupture and loss of function.

[0112] Concentration: Protein concentration needs to be determined by BCA method or particle concentration by NTA method before the experiment to achieve quantitative drug administration.

[0113] VI. Experimental Results

[0114] 1. MSC extraction, culture and identification

[0115] Figure 2 Images of continuously cultured human mesenchymal stem cells (cells isolated from tissues) extracted from primary cells; Figure 3 The marker was identified as a positive marker for MSC cell identification. Figure 3 A, B, and C show MSC markers CD90, CD73, and CD105 expression ≥95%, while negative markers CD19, CD31, CD34, CD45, and HLA-DR expression ≤2%, thus excluding contamination from hematopoietic stem cells, endothelial cells, leukocytes, etc. Figure 4 Functional identification: It can differentiate into tissues derived from three different germ layers: bone, fat, and cartilage, directly demonstrating the pluripotency of MSCs and their plasticity in induced differentiation.

[0116] 2. Exosome identification

[0117] Figure 5 A. Transmission electron microscopy: the "gold standard" for observing exosome morphology, clearly showing its double membrane structure and cup-shaped morphology; B. The particle size distribution shows a single peak in the range of 30-150 nm, indicating good exosome enrichment; C. Positive markers: transmembrane / membrane-associated proteins: CD81, CD9 (tetratransmembrane protein family); endosome-derived protein: TSG101; negative marker (not expressed): Calnexin (endoplasmic reticulum), proving that there is no obvious organelle debris contamination in the sample.

[0118] 3. Primary identification of astrocytes and exosome uptake

[0119] Figure 6 Cellular GFAP immunofluorescence showed persistent expression in astrocytes; Figure 7 When PKH26-labeled exosomes were co-incubated with cells, clear, discrete red fluorescent spots were observed in the cytoplasm of astrocytes in the experimental group. This directly demonstrates that astrocytes can actively take up exosomes.

[0120] Example 3: Effects of ROS (Reactive Oxygen Species) Responsive Trifluoperazine (TFP) Nanomicelles Combined with MSC Exosomes on Cellular Level

[0121] The extraction and culture of primary astrocytes is a fundamental technique in neuroscience research. The cerebral cortex of young mice (rats or mice) aged 0-3 days (P0-P3) is typically used as the source because astrocytes at this age have strong proliferative capacity and relatively few neurons. Extraction is performed using the McCarthy and de Vellis differential adhesion method.

[0122] I. Experimental Materials

[0123] Animals: Newborn C57BL / 6 mice aged P0-P3 days.

[0124] Main reagents:

[0125] Culture medium: DMEM / F12 (1:1) or DMEM high glucose.

[0126] Serum: 10%-15% high-quality fetal bovine serum (FBS).

[0127] Double antibody: 1% Penicillin-Streptomycin.

[0128] Digestive fluid: 0.25% Trypsin-EDTA (pancreatic enzyme).

[0129] Dissection solution: ice-cold HBSS (calcium and magnesium-free) or PBS.

[0130] Consumables: 75cm 2 Culture flask (T75), 70μm cell sieve, ophthalmic scissors, fine forceps.

[0131] II. Extraction Steps

[0132] (1) Brain harvesting and cortical separation

[0133] 1. Disinfect the young mouse's head by immersing it in 75% alcohol for 30 seconds.

[0134] 2. Decapitation, rapid separation of the scalp and skull, removal of intact brain tissue and placement in a cold HBSS.

[0135] 3. Under an anatomical microscope, separate the left and right cerebral hemispheres and remove the olfactory bulb and midbrain.

[0136] 4. Key step: Meninges removal. Carefully remove the transparent meninges and their blood vessels covering the cortical surface using fine forceps.

[0137] (2) Mechanical dispersion and enzymatic digestion

[0138] 1. Cut the dermal tissue into pieces approximately 1mm thick. 3 Fragments.

[0139] 2. Add 0.25% trypsin and digest in a 37°C water bath for 10-15 minutes. Gently shake every few minutes during digestion.

[0140] 3. Add serum-containing complete culture medium to stop digestion.

[0141] 4. Gently pipette 15-20 times with a 1ml pipette to make the tissue suspension a homogeneous suspension (avoid generating a large number of air bubbles).

[0142] (3) Filtration and inoculation

[0143] 1. Pass the suspension through a 70μm cell sieve to remove undigested tissue residue.

[0144] 2. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant.

[0145] 3. Resuspend the cells in complete culture medium, count them, and then seed them into T75 culture flasks. Typically, the cortex of 2-3 rats is seeded into one T75 flask.

[0146] III. Purification Steps (Differential Centrifugation)

[0147] After inoculation, the cells form a mixed glial cell culture containing astrocytes, microglia, and oligodendrocyte precursor cells.

[0148] 1. Initial fluid change: Change the fluid once after 24 hours to remove neurons and debris that have not adhered to the wall.

[0149] 2. Maintenance culture: Change the medium every 2-3 days until the cells reach confluence (approximately 7-10 days). At this point, the cell layer will exhibit stratification.

[0150] The basal layer consists of converging astrocytes.

[0151] Upper layer: Translucent, highly refractive granular cells (microglia and oligodendrocytes).

[0152] 3. Purification by isothermal shaking:

[0153] Fix the culture flask on a constant temperature shaker (37°C).

[0154] Step 1: Shake at 200 rpm for 2 hours, then aspirate the supernatant (mainly to remove microglia).

[0155] Step 2: Add fresh culture medium and shake at 250 rpm for 18-24 hours.

[0156] Collection: After shaking, aspirate the supernatant containing oligodendrocytes. The cells remaining firmly attached to the bottom of the flask are high-purity astrocytes.

[0157] IV. Identification and Propagation

[0158] 1. Passaging: After purification, the cells are digested with trypsin and passaged at a ratio of 1:2 or 1:3. It is recommended to use P2-P4 generation cells for experiments; excessively high passage numbers may cause cell hypertrophy or changes in reactivity.

[0159] 2. Identification:

[0160] GFAP (glial fibrillary acidic protein) immunofluorescence staining: Astrocyte-specific marker, purity should reach above 95%.

[0161] V. Effects of ROS (Reactive Oxygen Species) Responsive Trifluoperazine (TFP) Nanomicelles Combined with MSC Exosomes on Astrocytes Under Hypoxia and Glucose-Free (FBS-Free) Simulated Spinal Cord Injury Conditions

[0162] 1) Grouping: control group, nanomicelle group, MSC exosome group, and nanomicelle combined with MSC exosome group;

[0163] 2) Establishment of a hypoxic and glucose-free astrocyte culture model;

[0164] 3) After cell treatment, the activity, anti-apoptotic ability, cell edema, and differentiation of A1 and A2 cells of astrocytes were identified.

[0165] VI. Experimental Results

[0166] Figure 8 A and B show that the combined treatment strategy of MSC exosomes and TFP smart drug delivery (nanomicelles) can significantly improve the cell activity and anti-apoptotic ability of ischemic hypoxic astrocytes; Figure 9 The combined treatment showed that it could reduce cellular edema; Figure 10 The combined therapy showed that it could modulate astrocyte polarization, causing it to transform into neuroprotective type A2.

[0167] Example 4: Effects of ROS (Reactive Oxygen Species) Responsive Trifluoperazine (TFP) Nanomicelles Combined with MSC Exosomes on Spinal Cord Injury in Animals

[0168] 1. Construction of a mouse spinal cord injury model

[0169] Experimental preparation:

[0170] Animal selection: Healthy adult mice, C57BL / 6 strain, weighing about 25 to 30g, are usually selected;

[0171] Anesthesia: Chloral hydrate anesthesia was used;

[0172] Surgical instruments: Prepare spinal injury impactor, microsurgical instruments, etc.

[0173] Surgical steps:

[0174] Positioning: The mouse was fixed in a prone position on the operating table with its back facing up to ensure the stability of the surgical area.

[0175] Disinfection and incision: The back of the mouse is disinfected, and then a midline incision is made in the predetermined surgical area (usually the thoracic or cervical spine) to expose the spine.

[0176] Laminectomy: Carefully remove the lamina (T10) of the target segment to expose the dura mater, taking care to avoid spinal cord injury.

[0177] Positioning and fixation: The spinal cord is positioned and fixed using the fixation device of the striker to ensure accurate strike location.

[0178] Implementation of the strike: According to the experimental design, adjust the parameters of the striker (such as strike height, weight, etc.) and then carry out the strike to cause spinal cord injury.

[0179] Closing the incision: After the operation, rinse the wound with saline and suture the back incision layer by layer.

[0180] 2. The medication is administered via tail vein injection;

[0181] 3. Perform functional evaluation on mice (BMS score, mineral field test and imprint test).

[0182] 4. Collect spinal cord injury specimens for testing: including HE staining, Masson staining and Nissan staining to show spinal cord structure, scars and neuronal status, and CD31, GFAP, and NeuN fluorescent staining to show angiogenesis, scars and neuronal survival.

[0183] 5. Experimental Results

[0184] Figure 11 A is a schematic diagram of an animal experiment; Figure 11 B, C, D, E, F, G, and H show that combined treatment significantly improves lower limb function in mice after spinal cord injury; Figure 12 CD31, GFAP, and NeuN fluorescence staining showed that combined treatment promoted angiogenesis, reduced scarring, and promoted neuronal recovery after spinal cord injury. Figure 13 HE staining, Masson staining, and Nissan staining showed that the combined treatment promoted spinal cord structural recovery, reduced scarring, and promoted neuronal recovery. Figure 14 Flowchart for the application of ROS-responsive trifluoperazine nanomicelles combined with MSC exosomes in traumatic spinal cord injury.

[0185] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A pharmaceutical composition for treating traumatic spinal cord injury, characterized in that, include: The invention comprises ROS-responsive trifluoperazine TFP nanomicelles and mesenchymal stem cell exosomes (MSC-Exos) derived from mesenchymal stem cells. The nanomicelles are self-assembled from amphiphilic block copolymers, wherein the hydrophilic and hydrophobic segments of the copolymers are connected by ROS-sensitive chemical bonds. The trifluoperazine is in a free state and is encapsulated in the hydrophobic core of the nanomicelles.

2. The pharmaceutical composition according to claim 1, characterized in that, The chemical bonds are thioketal bonds (TK), arylboronic acid ester bonds, or thioether bonds.

3. The pharmaceutical composition according to claim 1, characterized in that, The amphiphilic block copolymer is polyethylene glycol-thioketal-polycaprolactone PEG-TK-PCL.

4. The pharmaceutical composition according to claim 1, characterized in that, The mesenchymal stem cell exosomes were extracted from the culture supernatant of human umbilical cord mesenchymal stem cells by differential ultracentrifugation.

5. The use of a pharmaceutical composition according to any one of claims 1-4 in the preparation of a medicament for treating traumatic spinal cord injury.

6. The application according to claim 5, characterized in that, The pharmaceutical composition inhibits the translocation of edema protein 4, reduces spinal cord edema, and regulates the polarization of astrocytes towards type A2, thereby promoting the recovery of nerve function.

7. The application according to claim 5, characterized in that, The drug is an intravenous injection preparation.

8. The application according to claim 5, characterized in that, The pharmaceutical composition is used in the preparation of drugs that inhibit inflammatory responses after spinal cord injury, reduce glial scar formation, promote angiogenesis, and improve neuronal survival.

9. A preparation for treating traumatic spinal cord injury, characterized in that, It contains a therapeutically effective amount of ROS-responsive trifluoperazine TFP nanomicelles and mesenchymal stem cell exosomes (MSC-Exos) derived from mesenchymal stem cells; the nanomicelles are self-assembled from amphiphilic block copolymers, and the hydrophilic and hydrophobic segments of the copolymers are connected by ROS-sensitive chemical bonds; the trifluoperazine is in a free state and is encapsulated in the hydrophobic core of the nanomicelles.

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