Drug-loaded nano-microsphere for spinal cord injury recovery and application of drug-loaded nano-microsphere

Abstract

The invention provides a drug-loaded nano-microsphere for spinal cord injury recovery and application thereof, and relates to the technical field of biomedicine, the technical key point is that the drug-loaded nano-microsphere is provided, the drug-loaded nano-microsphere is characterized in that the drug-loaded nano-microsphere is prepared by an emulsified solvent evaporation method, a polymer and a drug are dissolved in an organic solvent, and the polymer and the drug are mixed uniformly to obtain a drug-loaded nano-microsphere solution; then emulsifying into a water phase, and finally, volatilizing an organic solvent to form microspheres; polyethylene glycol and a targeting ligand are added on the surface of a modified nano-microsphere, and the drug inhibits the SOCS3 / STAT pathway in a targeting manner. According to the research, a GM1-PLGA-PEG (at) FPR2 nano delivery system with a spinal cord targeting function is constructed, the system is good in slow release performance and high in biocompatibility, and neuronal apoptosis after H2O2 stimulation is effectively reduced. The drug-loaded nano-microspheres are used for simulating treatment of spinal cord injury, so that various movement functions and spinal cord health of rats with spinal cord injury can be improved, and the injured spinal cord can be targeted to promote neural restoration. The GM < 1 >-PLGA-PEG microspheres do not show strong liver retention, and show good biological safety in various tissues.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to drug-loaded nanospheres for spinal cord injury recovery and their applications. Background Technology

[0002] Spinal cord injury is a severe neurosurgical disease of the spine with a very poor prognosis. It is caused by direct or indirect external factors that damage the spinal cord tissue, leading to varying degrees of neurological dysfunction in the corresponding segment and below. The local microenvironment after spinal cord injury is complex, including disruption of the blood-spinal cord barrier integrity, cytokine storms, and persistent oxidative stress. This complex local microenvironment severely limits the therapeutic efficacy of traditional drug delivery systems. Although anti-inflammatory and neuroprotective drugs have shown reparative potential in animal models, their clinical application is often hampered by nonspecific distribution, short-acting nature, and systemic toxicity.

[0003] In recent years, nanoparticle-based drug delivery systems have become a key strategy for overcoming the aforementioned bottlenecks due to their tunable particle size, surface functionalization capabilities, and targeted delivery characteristics. Nanoparticle-based drug delivery technology has demonstrated unique advantages in the treatment of neurological diseases by precisely controlling the spatiotemporal characteristics of drug delivery. Among them, polymer nanospheres (such as PLGA and PEG-PLGA) have attracted much attention due to their excellent biocompatibility, modifiability, and sustained-release properties. Nanospheres are an ideal drug delivery system, capable of protecting drugs from degradation, prolonging drug release time, and improving drug bioavailability.

[0004] Therefore, designing nanospheres that can target and inhibit the SOCS3 / STAT3 signaling axis for spinal cord injury repair is a very promising research direction. Summary of the Invention

[0005] The purpose of this invention is to design a nanosphere capable of targeting and inhibiting the SOCS3 / STAT3 signaling axis for spinal cord injury repair, which is a very promising research direction.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A drug-loaded nanosphere for spinal cord injury recovery is characterized in that: the drug-loaded nanosphere is prepared by an emulsion solvent evaporation method, wherein a polymer and a drug are dissolved in an organic solvent, then emulsified into an aqueous phase, and finally the organic solvent is evaporated to form microspheres; and polyethylene glycol and a targeting ligand are added to modify the surface of the nanosphere, wherein the drug targets and inhibits the SOCS3 / STAT pathway.

[0008] Preferably, the drug is a small molecule inhibitor FPR2agonist 3 that targets and inhibits the SOCS3 / STAT3 axis, and its chemical formula is C. 25 H 20 F2N4O2.

[0009] Preferably, the polymer is PLGA, the organic solvent is an acetone-DCM mixed solvent, and the targeting ligand is ganglioside GM1.

[0010] Preferably, the method for preparing the drug-loaded nanospheres is as follows:

[0011] S1: Dissolve polymers PLGA and FPR2 agonist 3 together in 3 mL of acetone-DCM mixed solvent, vortex until completely dissolved, and use as the organic phase; prepare an appropriate amount of aqueous phase; inject the organic phase solution into 20 mL of aqueous phase containing 0.5% PVA at a rate of 0.5 mL / min, stir magnetically and sonicate simultaneously.

[0012] S2: Add ethanol to change the polarity of the solvent, causing the polymer to precipitate and form nanospheres; after the organic solvent evaporates, collect the microspheres by centrifugation at 10000×g for 15min, wash them three times with ultrapure water, and freeze-dry them for storage.

[0013] S3: Finally, the nanospheres were collected by centrifugation and washed with deionized water to remove unencapsulated drugs;

[0014] S4: Resuspend the lyophilized microspheres in PBS, add NH2-PEG2000-Mal, and react at room temperature; remove free PEG by ultrafiltration and centrifugation to obtain PEGylated microspheres.

[0015] S5: By modifying the surface of nanospheres and adding polyethylene glycol and GM1, their targeting and stability in spinal cord tissue are improved;

[0016] Preferably, in step S5, EDC / NHS chemical coupling is used: GM1 and PLGA-PEG microspheres are reacted in MES buffer containing 5mM EDC / 2mM NHS for 12h;

[0017] Unbound GM1 was removed by dialysis, and the final product was denoted as GM1-PLGA-PEG@FPR2.

[0018] Preferably, the quality control standards for the prepared GM1-PLGA-PEG@FPR2 are: particle size 150±20nm, PDI<0.2, Zeta potential -15 to -25mV, and encapsulation efficiency >80%.

[0019] Preferably, the preparation process of the drug-loaded nanospheres is carried out under sterile conditions, and all reagents are sterilized by filtration through a 0.22 μm filter membrane.

[0020] This application also provides the application of the drug-loaded nanospheres described above in the preparation of drugs for treating spinal cord injury.

[0021] Compared with the prior art, this application has the following beneficial effects:

[0022] This application utilizes the SOCS3 / STAT3 pathway inhibitor FRP2 as the drug, and prepares PLGA-based nanospheres via an emulsion solvent evaporation method. The surface of these nanospheres is then modified with ganglioside GM1 to achieve spinal cord-specific targeting. The particle size distribution, drug loading rate, in vitro drug release behavior, and blood-spinal cord barrier penetration ability of the nanospheres are systematically evaluated. Combined with in vitro neuronal apoptosis models and in vivo animal experiments on spinal cord injury, the neuroprotective effect is verified. This application aims to construct an efficient and safe nanodelivery platform, providing theoretical and technical support for the precision treatment of spinal cord injury. Attached Figure Description

[0023] Figure 1 The preparation and characterization of drug-loaded nanospheres for spinal cord injury recovery in this application include: (A) preparation method of drug-loaded nanospheres; (B) scanning electron microscope image of nanospheres; (C) particle size distribution of nanospheres; and (D) zeta potential distribution of nanospheres.

[0024] Figure 2 To evaluate the biocompatibility of nanospheres, the following methods were used: (A) RT-QPCR detection of the expression of inflammatory factors in cells after co-culturing nanospheres with nerve cells; (B) cell counting after co-culturing nanospheres with nerve cells; (C) evaluation of the drug release capacity of nanospheres; and (D) staining of dead / live cells after co-culturing nanospheres with nerve cells.

[0025] Figure 3 The study simulated treatment for rat spinal cord injury, including (A) rat spinal cord injury modeling method; (B) BMS score after spinal cord injury; (C) gait analysis experiment; (D) stride length analysis experiment; (E) foot width analysis experiment; (F) rotarod test; (G) inclined plate test; and (H) heat-induced pain-induced foot retraction test.

[0026] Figure 4 Immunofluorescence staining of neural progenitor cells in the spinal cord was used to simulate treatment. (A) TUNEL fluorescence staining of the spinal cord in each experimental group; (B) ELISA analysis of changes in validation factors in the spinal cord tissue; (C) Western blotting analysis of the expression of neural repair proteins in the spinal cord tissue.

[0027] Figure 5 The nanospheres were used to target the damaged spinal cord. (A) In vivo imaging of the animal and (B) corresponding semi-quantitative fluorescence analysis; (C) fluorescence imaging of the microspheres' visceral retention.

[0028] Figure 6 H&E staining was performed on rat heart, liver, spleen, lung, and kidney samples 28 days after simulated treatment. The results show: (A) H&E staining of heart, liver, spleen, lung, and kidney after simulated treatment; (B) serum alanine aminotransferase (ALT) (U / L) level after simulated treatment; (C) serum aspartate aminotransferase (AST) (U / L) level after simulated treatment; (D) serum uric acid (UA) level after simulated treatment; and (E) serum blood urea nitrogen (BUN) (mg / dL) level after simulated treatment. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments.

[0030] This application provides the use of FRP2 agonist 3 in the preparation of drugs for treating spinal cord injury, wherein FRP2 agonist 3 promotes neuronal survival by inhibiting the SOCS3 / STAT3 pathway.

[0031] This application also provides the application of drug-loaded nanospheres targeting the spinal cord in the preparation of drugs for treating spinal cord injury, wherein the drug-loaded nanospheres are loaded with FPR2 agonist 3 (FPR2), and the molecular formula of FPR2 agonist 3 is: C 25 H 20 F2N4O2, its chemical formula is shown below:

[0032]

[0033] The method for preparing the drug-loaded nanospheres described above is as follows:

[0034] The entire preparation process was carried out under sterile conditions, and all reagents were sterilized by filtration through a 0.22μm filter membrane.

[0035] First, drug-loaded microspheres were prepared by an emulsion solvent evaporation method. Polymers PLGA and FPR2agonist 3 were dissolved in an organic solvent acetone-DCM mixed solvent, then emulsified into an aqueous phase, and finally the organic solvent was evaporated to form microspheres.

[0036] By modifying the surface of nanospheres, polyethylene glycol (PEG) and a targeting ligand (ganglioside GM1) were added.

[0037] The above content will be elaborated below with specific verification experiments:

[0038] All female SD rats (approximately 200g) used in this application were purchased from Zhaoyan New Drug Research Center Co., Ltd. (Production License No.: SCXK(Su)2018-0006). The animal research was reviewed by the Experimental Animal Ethics Committee of Soochow University (Review No.: 201902087). Animal handling and surgical procedures during the experiment followed the "Guiding Opinions on the Humane Treatment of Laboratory Animals" issued by the Ministry of Science and Technology.

[0039] Experimental materials and their sources:

[0040]

[0041]

[0042] Experimental apparatus:

[0043]

[0044]

[0045] Example 1: Preparation and characterization of drug-loaded nanospheres

[0046] 1.1 Preparation of drug-loaded nanospheres:

[0047] The preparation of the drug-loaded nanospheres was carried out under aseptic conditions, and all reagents were sterilized by filtration through a 0.22 μm filter membrane. The preparation steps are as follows: S1: Polymer PLGA (50 mg, Mw = 15 kDa, Sigma-Aldrich) and FPR2agonist 3 (5 mg, MedChemExpress) were dissolved together in 3 mL of acetone-DCM mixed solvent (v / v = 2:1), and vortexed until completely dissolved to form the organic phase. An appropriate amount of aqueous phase (an aqueous solution containing surfactant) was prepared. The organic phase solution was injected into 20 mL of the aqueous phase containing 0.5% PVA (stabilizer) at a rate of 0.5 mL / min, magnetically stirred (800 rpm) and simultaneously sonicated (20% amplitude, 5 s pulse).

[0048] S2: Ethanol is added to change the polarity of the solvent, causing the polymer to precipitate and form nanospheres. After the organic solvent evaporates, the microspheres are collected by centrifugation at 10000×g for 15 min, washed three times with ultrapure water, and then lyophilized for storage.

[0049] S3: Finally, the nanospheres were collected by centrifugation and washed with deionized water to remove unencapsulated drugs.

[0050] S4: The lyophilized microspheres were resuspended in PBS (pH 7.4), and NH2-PEG2000-Mal (molar ratio PLGA:PEG = 1:0.2) was added. The mixture was reacted at room temperature for 4 h. Free PEG was removed by ultrafiltration centrifugation (100 kDa molecular weight cutoff) to obtain PEGylated microspheres (PLGA-PEG).

[0051] S5: By modifying the surface of the nanospheres and adding polyethylene glycol (PEG, Thermo Fisher) and a targeting ligand (ganglioside GM1), the targeting and stability in spinal cord tissue were improved. EDC / NHS chemical coupling was employed: GM1 (1 mg / mL) was reacted with PLGA-PEG microspheres in MES buffer (pH 6.0) containing 5 mM EDC / 2 mM NHS for 12 h. Unbound GM1 was removed by dialysis (MWCO 300 kDa, 24 h), and the final product was designated GM1-PLGA-PEG@FPR2. Quality control standards were: particle size 150 ± 20 nm, PDI < 0.2, Zeta potential -15 to -25 mV, and encapsulation efficiency > 80%.

[0052] 1.2 Characterization of nanospheres

[0053] (1) After the preparation of the nanospheres was completed, characterization experiments were performed. The particle size distribution of the nanospheres was measured using dynamic light scattering technique.

[0054] (2) The morphology of the nanospheres was observed using a transmission electron microscope. The images were formed by 2% phosphotungstic acid negative staining and an accelerating voltage of 80 kV.

[0055] (3) Dynamic light scattering (DLS) was used to measure three batches of samples (n=3), which were dispersed in deionized water (1mg / mL) to ensure the particle size and zeta potential of the microspheres.

[0056] (4) Drug loading efficiency and release curve The drug loading efficiency was determined by high performance liquid chromatography (HPLC) (C18 column, mobile phase acetonitrile / water = 70:30, flow rate 1 mL / min, detection wavelength 254 nm).

[0057] Please see Figure 1In this application, a GM1-PLGA-PEG@FPR2 nanodelivery system with spinal cord targeting function was successfully constructed. Through a nanoprecipitation method combined with a multi-step surface modification strategy, highly efficient encapsulation of FPR2 agonist 3 was achieved (encapsulation efficiency 82.3±3.5%, drug loading 9.8±0.6%). GM1 can specifically bind to surface receptors on spinal cord neurons (such as TrkA), enhancing the active targeting ability of microspheres in the damaged area. PEG modification can improve the colloidal stability of microspheres and reduce clearance by the reticuloendothelial system. Figure 1 A). Scanning electron microscopy images show that the GM1-PLGA-PEG@FPR2 microspheres are regularly spherical with smooth surfaces and no obvious depressions or pores. Figure 1 B). The microspheres were uniformly dispersed, and no significant aggregation was observed, indicating that the PEGylated surface modification effectively reduced the van der Waals interactions between particles. The median hydrated particle size measured by dynamic light scattering (DLS) was 152.5 (PDI = 0.15), slightly larger than the scanning electron microscopy result. This is attributed to the fact that the DLS measurement included the solvation layer and the hydration swelling effect of the PEG chains. Figure 1 C). The median zeta potential is -16.2 mV ( Figure 1 D).

[0058] 1.3 nanospheres co-cultured with nerve cells

[0059] (1) The neuronal cells were seeded in a 12-well culture plate pretreated with polylysine, and 1 mL of neuronal culture medium was added to each well. The cell density was 1×10^5 cells / mL.

[0060] (2) After the neurons adhere and grow to an appropriate density, the nanospheres are added to the culture medium, with a final concentration of 10 nM and 100 nM.

[0061] (3) Incubate at 37°C and 5% CO2 for 3 days. Change the culture medium daily to maintain cell growth. Perform cell counting after 3 days.

[0062] (4) The monolayer cells are digested with trypsin, or the suspended cells are directly aspirated. The cells are then added to a hemocytometer, and the number of cells is observed and counted under a microscope. Finally, the concentration, number, and density of the cells are calculated using a specific formula. The operation steps are as follows:

[0063] (5) After digestion, the nerve cells are fully resuspended and dispersed by blowing. Take 1 mL and transfer it into a clean EP tube.

[0064] (6) Gently wipe the surface of the counting plate and the cover glass with an alcohol swab to moisten the edge of the counting plate. Place the cover glass in the center of the counting plate and cover the groove of the counting plate with its edge.

[0065] (7) Repeatedly pipette the cell suspension transferred into the EP tube until it becomes a single-cell suspension, and aspirate 20uL of the suspension; place the pipette tip against the upper edge of the coverslip and slowly pipette the liquid, using the siphon effect to draw the liquid into the gap between the coverslip and the counting plate;

[0066] (8) Adjust the microscope and observe with a 10x objective lens. Adjust the focus to ensure that the grid lines of the counting plate are clear.

[0067] (9) Under normal circumstances, the field of view can accommodate 25 small squares, and the total area of ​​these 25 squares is 1 square millimeter. Next, change the objective lens to 40x and start counting each small square. To avoid double counting, if a cell is located on the edge, only count the top and left lateral lines; do not count those located on the bottom and right. After counting all 25 squares, multiply the total number by 10^4 to get the concentration of the cell suspension (cells / mL); multiply by the volume of the cell suspension to get the total number of cells.

[0068] 1.4 ELISA detection

[0069] In this application, an ELISA kit was used to quantitatively detect the content of inflammatory factors after co-culturing nanospheres with nerve cells.

[0070] (1) Neurons were treated with serum-free cell culture medium containing 300 μM H2O2, and samples were taken at 3 hours and 6 hours. After washing the cells three times with PBS, 1 mL of Trizol was added to each well and the cells were lysed for 20 min. Then, the lysate was aliquoted into 1.5 mL enzyme-free EP tubes, labeled and incubated at room temperature for 10 min.

[0071] (2) Prepare seven standards with concentrations of 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.50 pg / mL, and 31.25 pg / mL. Pipette 100 μL from each standard into a 96-well plate.

[0072] (3) Add 50 μL of buffer, 50 μL of the test sample, and 50 μL of diluted detection antibody to each well. Seal the plate and shake it at 300 r / min at room temperature for 90 min.

[0073] (4) Remove the supernatant, add 300 μL of washing buffer to each well, and repeat 3 times. After each washing, try to remove as much residual liquid as possible. Add 100 μL of diluted horseradish peroxidase-labeled streptavidin to each well.

[0074] (5) Seal the plate again, and shake it at 300 r / min at room temperature for 30 min. Remove the supernatant, add washing solution, and repeat 3 times.

[0075] (6) Add 100 μL of the chromogenic substrate and incubate at room temperature in the dark for 30 min. Add 100 μL of the stop solution to terminate the reaction.

[0076] (7) Microplate reader detection: Set the microplate reader wavelength to 450 nm and measure the OD value. Plot a standard curve based on the OD value and concentration of the standard, and calculate the concentration of each sample based on the curve.

[0077] Co-culturing primary spinal cord nerve cells with nanospheres showed that, compared to the negative control, the nanospheres did not significantly promote the secretion of inflammatory factors IL-6 and TNF-α by spinal cord nerve cells within 48 hours, while the positive control significantly promoted the secretion of inflammatory factors. Figure 2 A). In cell counts of primary spinal cord nerve cells co-cultured at different concentrations of GM1-PLGA-PEG@, no cell-killing effect of the nanospheres was observed. Figure 2 B). This indicates that GM1-PLGA-PEG@ does not cause significant inflammatory stimulation or cell killing of spinal cord nerve cells.

[0078] 1.5 Cumulative drug release curve

[0079] (1) Accurately weigh 5 mg of FRP2 raw material and 5 mg of GM1-PLGA-PEG@FPR2 sustained-release microspheres prepared from FRP2, and conduct drug release experiments under simulated physiological conditions (PBS, pH 7.4) to plot release curves. Place them separately in 100 mL of release solution containing 0.5% sodium dodecyl sulfate.

[0080] (2) Add 10 mL of release medium (same as the release solution) to the pretreated dialysis bag and immerse the dialysis bag in the release solution. Shake the bag in a constant temperature water bath at 37°C. Simultaneously replenish the same amount of release medium at the same temperature. Use 6 h, 12 h, 1 d, 2 d, 3 d, 6 d, 9 d, 12 d, 15 d, 18 d, 21 d, 24 d, 27 d, and 30 d as time monitoring points. Take 2 mL of release medium from the dialysis bag and determine the FRP2 content using HPLC. Calculate the cumulative release percentage Qn of the drug at different time points using the following formula.

[0081]

[0082] Where Qn is the cumulative release percentage (%) at point n, V is the volume of the release medium (mL), Cn is the drug concentration (mg·mL-1) measured at point n, 2 is the sample volume of 2 mL, and W is the total dose (mg).

[0083] (5) Use cytotoxicity assays (such as the MTT assay) to assess the biocompatibility of nanospheres with neurons and glial cells. Assess the in vivo distribution and safety of nanospheres through animal experiments.

[0084] like Figure 2 As shown in Figure C, the FPR2 agonist 3 active pharmaceutical ingredient is released extremely rapidly, with a cumulative release rate exceeding 90% after 1 day of the experiment. In contrast, GM1-PLGA-PEG@FPR2 microspheres release FPR2 agonist 3 slowly in the release medium / external liquid. The release is slightly faster in the first 15 days of the experiment, and then gradually slows down. The cumulative release rate tends to stabilize at close to 97% from the 18th day onwards.

[0085] The GM1-PLGA-PEG@FPR2 microspheres exhibited stable release throughout the entire process, with no burst release effect observed, demonstrating good sustained-release performance. Figure 2 C).

[0086] 1.6 Dead Cell / Live Cell Staining Assay

[0087] (1) Collect sample cells. Aspirate and retain the original culture medium, digest the cells with trypsin-EDTA, stop the digestion with culture medium, collect the cells together with the original culture medium, centrifuge (1500 rpm, 5 min) to collect the cells, and discard the culture medium. Wash the cells with PBS, resuspend the collected cells in PBS, centrifuge (1200 rpm, 5 min) to wash thoroughly, and repeat 2-3 times;

[0088] (2) Cell counting. After washing, prepare a cell suspension with an appropriate amount of PBS to a density of 1 x 10⁻⁶. 5 ~3x10 5 cells / ml;

[0089] (3) Prepare the staining working solution.

[0090] 1. Stock solution concentration: Add 200ug calcein-AM to 200ul DMSO and use a pipette to dissolve it completely to prepare a stock solution with a concentration of 1mM. The PI solution is 1.5mM.

[0091] 2. Preparation of working solution: Take 2 μL of calcein-AM solution and 3 μL of PI solution, add them to 1 mL of PBS, and mix thoroughly. The concentration of calcein-AM is 2 μmol / L, and the concentration of PI is 4.5 μmol / L, which is the final concentration of the working solution. Mix 100 μL of staining working solution with 200 μL of cell suspension and incubate in the dark for 15 min.

[0092] (4) Detection results. Take 10 μL of the above mixed solution, pour it onto a manual cell counting plate, and observe it under a fluorescence microscope. Observe the green fluorescent live cells under fluorescence microscope channel 1, and observe the red fluorescent dead cells under fluorescence microscope channel 2. Remove the staining solution, observe and photograph under an inverted fluorescence microscope, and semi-quantitatively analyze the fluorescence density of live cells using ImageJ software.

[0093] Double staining of live and dead cells revealed that treatment with GM1-PLGA-PEG@FPR2 microspheres significantly protected nerve cells, reducing H2O2-induced cell death. However, GM1-PLGA-PEG@microspheres alone did not exhibit this effect. Figure 2 D).

[0094] Example 2: Construction and grouping of a rat spinal cord injury model

[0095] This application aims to systematically evaluate the biosafety of GM1-PLGA-PEG@FPR2 microspheres in the treatment of spinal cord injury. The experimental design strictly adheres to the ethical guidelines established by the Laboratory Animal Care and Use Committee. Healthy adult Sprague-Dawley rats (weighing 220±20g, half male and half female) were selected as research subjects and were acclimatized for one week in a standard SPF-grade animal laboratory. Environmental parameters were maintained at constant temperature (23±1℃), humidity (55±5%), and a 12-hour diurnal rhythm. They had free access to sterilized feed and purified water.

[0096] (1) In an animal model of spinal cord injury, the effects of intrathecal injection of nanospheres on neuronal survival, neuroinflammation, and functional recovery were evaluated. SD rats weighing 180 to 220 g were used. A rat model of spinal cord contusion / transection (Allen's method) was established, with sham-operated group and groups at 1d / 3d / 7d / 14d / 28d post-injury (n=6 / group). Drug-loaded nanospheres were injected locally 24 h post-injury, with blank nanosphere group, drug-loaded nanosphere group, free drug control group, and saline control group.

[0097] (2) After 7 days of acclimatization feeding, the rats were anesthetized.

[0098] (3) Palpate the boundary between the lowest rib and soft tissue on both sides of the spinal cord on the back of the rat as a bone landmark. Disinfect the surgical area, and make a 2.5cm midline incision centered on T10 to fully expose the T9 to T11 segments. Use bone forceps to remove the spinous process and lamina of T10, ensuring the dura mater remains intact. Use a 10g weight to drop vertically from a height of 25mm onto the exposed spinal cord surface to create a standard moderate contusion model. If rapid local congestion and edema occur after the injury, and both hind limbs twitch and the tail wags, the model is considered successfully established.

[0099] (4) After successful modeling, the sutures are used to suture the fascia and skin layer by layer;

[0100] (5) Postoperatively, each rat was injected intramuscularly with 200,000 units of antibiotics for 5 days, and manual bladder emptying was performed every 12 hours until the urination reflex was restored. The sham surgery group had only the spinal cord exposed without impact; the injury control group was injected with an equal volume of physiological saline; the blank microsphere group was given GM1-PLGA-PEG carrier; and the treatment group received GM1-PLGA-PEG@FPR2 microsphere suspension (dose 10 mg / kg).

[0101] The administration regimen was carried out via tail vein injection 6 hours after model establishment, with the injection rate controlled at 0.1 mL / min to avoid hemodynamic fluctuations.

[0102] Please see Figure 3 A. In this application, a rat spinal cord injury model is established by using the heavy object drop method, and the treatment effect is evaluated by injecting nanospheres into the tail vein to simulate treatment, and in vivo imaging and motor function detection are used to evaluate the treatment effect.

[0103] Example 3: GM1-PLGA-PEG@FPR2 microspheres promote motor function recovery in rats after spinal cord injury

[0104] In this embodiment, the efficacy of drug-loaded microspheres was evaluated using animal motor function scores.

[0105] 3.1 Basso Mouse Scale (BMS) Rating

[0106] The BMS score is a sensitive, reliable, and effective method for assessing motor function in mice after spinal cord injury, designed based on the characteristics of motor function changes. In this method, a higher score indicates better recovery; a score of 9 is considered normal in mice. The BMS score is used to assess the recovery of hindlimb motor function in mice following spinal cord injury.

[0107] The BMS score ranges from 0 to 9, and the specific scoring criteria are as follows:

[0108] 0 points: No ankle joint movement observed at all. 1 point: Mild ankle joint movement, range of motion ≤50%. 2 points: Significant ankle joint movement, range of motion >50%. 3 points: Paws actively placed on the ground, thumb and little toe touching the ground, weight-bearing or non-weight-bearing possible, or occasional / frequent / continuous dorsiflexion walking, no plantar weight-bearing walking. 4 points: Occasional (≤50%) plantar weight-bearing walking. 5 points: Frequent (51%–94%) to continuous (95%–100%) plantar weight-bearing walking, but no forelimb or hindlimb coordination. 6 points: Frequent to continuous plantar weight-bearing walking, with some forelimb and hindlimb coordination, and the hind paws are parallel upon initial contact with the ground. 7 points: Frequent to continuous plantar weight-bearing walking, with significant forelimb and hindlimb coordination, and the hind paws are parallel upon initial contact with the ground and upon lifting off, but with severe body instability. 8 points: Frequent to continuous weight-bearing walking on the soles of the feet, with extensive coordination between the forelimbs and hindlimbs, and the hind paws are parallel when they first touch the ground and when they lift off the ground, showing mild body instability. 9 points: Capable of weight-bearing walking on the soles of the feet, with coordination between the forelimbs and hindlimbs, and trunk stability.

[0109] BMS score analysis showed motor function in the Sham group, SCI+FPR2 group, SCI group, and SCI+GM1-PLGA-PEG@FPR2 group over 28 days. The SCI group treated with GM1-PLGA-PEG@FPR2 microspheres had higher scores and better recovery than those treated directly with FPR2.

[0110] like Figure 3 As shown in Figure B, all rats exhibited complete hind limb paralysis (BMS score = 0) one day after SCI, indicating successful and uniform modeling. Starting on day 3, the animals showed gradual, time-dependent recovery, with varying degrees of improvement observed across groups. Compared to other groups, the GM1-PLGA-PEG@FPR2 microsphere group showed the fastest recovery of motor function in SCI rats, significantly exceeding the group with a score of 1.8 and the spinal cord injury group (0.9) starting in week 2. The BMS score of the spinal cord injury group was below 4, indicating very limited self-recovery capacity of the spinal cord in spinal cord injury rats. Notably, the motor function of rats in the FPR2 group also showed relative improvement compared to the SCI group. These results demonstrate that while FPR2 can partially improve motor function in SCI rats, constructing GM1-PLGA-PEG@FPR2 microspheres can significantly enhance motor function in rats with spinal cord injury.

[0111] 3.2 Footprint Analysis Experiment

[0112] Gait, stride length, and stride width were assessed by analyzing the footprints produced by the animals. Twenty-eight days post-surgery, the hind limbs of rats were immersed in non-toxic ink and guided through a 1m × 10cm narrow path. Stride length (distance between the front and hind paw prints on the same side) and stride width (distance between the midlines of the paw prints on both hind limbs) were recorded. Each rat was tested three times, and the average was taken. Three days prior to the experiment, the rats underwent acclimatization training to familiarize them with the experimental environment and procedures. The rats were trained to walk on a narrow board, with a dark box placed on the other side. Utilizing the rats' preference for darkness, they were guided to run from one side to the other side of the dark box. This training was conducted three times daily, with 5-10 minute intervals between each session.

[0113] Experimental Procedure: Record the animal's number and test date on a piece of white paper. Apply red ink to the instep of the animal's hind leg and black ink to the sole. Immediately place the animal on one side of the white paper and guide it to walk towards the dark box. After the animal enters the dark box, collect the white paper and allow it to dry. Repeat the above steps until all animals have been analyzed.

[0114] Please see Figure 3 C. Footprint analysis showed that the SCI group had obvious dragging of the hind limbs, while the SCI+FPR2 group had obvious footprints on the hind limbs and the dragging was weakened. The GM1-PLGA-PEG@FPR2 group had the best recovery effect and the weakest dragging.

[0115] 3.3 Step length and step width analysis experiment

[0116] Step length analysis: Footprints of rats in each group were scanned and imaged, then processed and analyzed using image analysis software. The length of the hind limb footprints (distance from heel to toe) was measured in the footprint images. At least 4-6 clear footprints were selected from each rat for measurement, and the average value was taken as the rat's step length. Statistical software was used to analyze the step length data of different groups, comparing differences between groups, and the changes in step length at different time points in the spinal cord injury group. Methods such as t-tests or analysis of variance could be used to assess changes in step length among different groups of rats.

[0117] Step width analysis: In footprint images, the width of the rat's hind limb footprints was measured, i.e., the maximum distance from the outer edge to the inner edge of the footprint. At least 4-6 clear footprints were selected from each rat for measurement, and the average value was taken as the rat's step width. Statistical software was used to analyze the step width data of different groups, comparing the differences between groups, and the changes in step width at different time points in the spinal cord injury group. Methods such as t-tests or analysis of variance can be used to assess the influence of different groups on step width.

[0118] In a spinal cord injury model, stride length and stride width reflect the motor coordination and gait stability of rats. Changes in stride length directly reflect the impact of spinal cord injury on motor function and the progress of recovery after injury. The SCI group, compared to the Sham group, showed a wider stride width and a shorter stride length, suggesting impaired motor function or gait instability. In contrast, the GM1-PLGA-PEG@FPR2 group showed a narrower stride width and a longer stride length compared to the SCI group, indicating some recovery of motor function. The GM1-PLGA-PEG@FPR2 group showed better recovery in motor coordination and gait stability than the FPR2 group. Figure 3 D and 3E).

[0119] 3.4 Rotating Rod Experiment

[0120] The experiment began once the rats were firmly standing on the rotating bar. The experimental environment was kept quiet during testing. A stopwatch was used to record the time the rats remained on the bar. Each animal was tested five times, and the three longest durations were recorded as the experimental results. The acclimatization phase consisted of three stages with no time intervals between them.

[0121] (1) In stage T1, the rotor accelerates at 5 r / min 2 The vehicle accelerates uniformly from rest to a speed of 5 r / min.

[0122] (2) In stage T2, the acceleration is changed to 10 r / min 2 Accelerate to 15 r / min and then rotate at a constant speed for 2 minutes;

[0123] (3) In the T3 stage, continue with an acceleration of 10 r / min 2 Accelerate to 20 r / min and continue at a constant speed for 2 minutes. If the mouse falls during the test, record the fall time. After 20 minutes, restart the experiment on the fallen rat and record the duration the rat remains on the rotundus. During the adaptation experiment, the rats' physical strength and coordination abilities are improved.

[0124] (4) During the formal experimental phase, the acceleration remained constant in each of the T1, T2, and T3 phases. At the end of the phase, the uniform velocity reached 5 r / min, 15 r / min, and 18 r / min, respectively. The duration of the rat on the rotator was recorded. Rats that failed the experiment more than three times in a row were discarded.

[0125] (5) The maximum time a rat can stay on the stick is 180 seconds. Any time exceeding this limit is recorded as 180 seconds. During the test, the experimental environment should be kept quiet. Use a stopwatch to record the time the rat stays on the stick. Each animal is tested 5 times, and the 3 longest times are taken as the experimental results.

[0126] The fall delay (in seconds) test was used to assess the recovery of motor function in rats after spinal cord injury. The average time was 32±2 s in the sham-operated group, 19±4 s in the spinal cord injury group, 27±2 s in the GM1-PLGA-PEG@FPR2 group, and 23±3 s in the FPR2 group. The GM1-PLGA-PEG@FPR2 group showed the best recovery of motor function. Figure 3 F).

[0127] 3.5 Inclined Plate Experiment

[0128] All rats were placed in the testing apparatus (i.e., covered with a rubber pad containing horizontal ridges spaced 3 mm apart). The rats' body longitudinal axis was parallel to the longitudinal axis of the inclined plane, with their heads facing the raised side of the inclined plane. Starting from 0°, the maximum angle at which the rat could remain on the inclined plane for 5 seconds was observed in increments of 5°. Five measurements were taken for each animal, and the average value was recorded. This assesses the animal's ability to maintain its position on a board raised in 5° increments, thus serving as an indicator of hind limb strength. For each position, the maximum angle at which the rat could maintain its position for 5 seconds without falling was recorded and averaged to obtain an individual score for each rat. The maximum angle at which a rat could maintain its position for at least 5 seconds constituted its inclined plane score.

[0129] In the inclined plane test, the GM1-PLGA-PEG@FPR2 microsphere group had the highest angle, indicating better balance in this group and further improving hind limb function and coordination in SCI mice. Figure 3 G).

[0130] 3.6 Thermal pain stimulation foot withdrawal test

[0131] The pain perception of rats was assessed using their escape behavior from heat stimuli. When the soles of the rats' hind limbs were heated, the increased pain sensation prompted them to quickly lift their legs to avoid the heat. A hot plate temperature was set (50-55℃), and rats were placed on it. The time from contact with the hot plate to leg lifting was recorded, i.e., the heat-pain latency period. After model establishment, experiments were conducted separately for each group.

[0132] Sensory function in the hind limbs of rats was assessed by evaluating paw withdrawal time in response to thermal pain stimuli. No significant abnormalities were observed in the sensory function of the hind limbs in all rats one day before surgery. The hot plate test time decreased rapidly within the first week post-surgery, slowing down from the second to the third week, and gradually stabilizing. The GM1-PLGA-PEG@FPR2 group showed the most significant improvement in hot plate withdrawal time and demonstrated better sensory function recovery. The SCI group exhibited significant sensory dysfunction in both hind limbs post-surgery, with sluggish responses to thermal pain stimuli and a significantly longer hot plate test time compared to one day before surgery or the sham-operated group at the same time point. Although sensory function in both hind limbs of the SCI group gradually recovered from 7 days post-surgery, the recovery was very limited. These functional assessments confirmed that GM1-PLGA-PEG@FPR2 microspheres can improve various motor functions and spinal cord health in SCI rats (Figure H).

[0133] Example 4: Verifying that nanospheres promote nerve repair at the site of spinal cord injury

[0134] 4.1 TUNEL in situ apoptosis detection

[0135] Rats were euthanized on days 7 and 28, and 1 cm spinal cord specimens were obtained. These specimens were fixed in 10% formalin for 24 hours and then embedded in paraffin for sectioning. After section preparation, TUNEL staining was performed.

[0136] (1) On days 7 and 28, rats were euthanized to obtain 1 cm long spinal cord tissue, which was then fixed in 10% formalin solution for 24 hours and subsequently embedded in paraffin for sectioning. After sectioning, TUNEL staining was performed.

[0137] (2) Reagent preparation: Prepare 1× Proteinase K working solution fresh for each use. Add 1 μL of Proteinase K (100×) to 99 μL of PBS and mix well. Prepare 1× DNase I Buffer working solution fresh for each use. Dilute DNase I Buffer (10×) with ddH2O at a ratio of 9:1. Prepare 200 U / mL DNase I working solution fresh for each use. Dilute 20 U / μL DNase I with 1× DNase I Buffer working solution at a ratio of 99:1. Prepare 2× DAPI working solution fresh for each use. Add 4 μL of DAPI Reagent (25 μg / mL) to 96 μL of PBS and mix well.

[0138] (3) Dewaxing and hydration of sections: Immerse the sections in xylene twice for 10 minutes each time; soak them in anhydrous ethanol twice for 5 minutes each time; and treat them with 90%, 80%, and 70% ethanol aqueous solutions once each for 3 minutes each time.

[0139] (4) Sample preparation: After dewaxing, the samples were rinsed three times with PBS for 5 minutes each time. The moisture around the slides was blotted dry with filter paper, and 100 μL of 1× proteinase K working solution was added to each sample. The samples were then reacted at 37°C for 20 minutes.

[0140] (5) Sample incubation: Immerse the treated samples in PBS for 5 minutes each time, rinsing three times. For the negative control, add 100 μL of 1×DNase I Buffer working solution to the sample and equilibrate at room temperature for 5 minutes; incubate the negative sample with DNase I Buffer at 37°C for 10 to 30 minutes. Then immerse the sample in PBS for 5 minutes each time. The experimental group samples are left to stand in PBS until they are processed and then labeled and stained together with the other control groups. Prepare the required reagents according to the sample volume, and mix each sample thoroughly according to the requirements in the table, preparing the reagents fresh for each use.

[0141] Table 1. TUNEL Reagent Formulation

[0142]

[0143] 4.2 Western Blot

[0144] (1) Protein supernatant extraction: Preparation: Prepare protein lysis buffer and add protease inhibitor (PMSF, at a volume ratio of protein lysis buffer to PMSF of 100:1), phosphorylated protease inhibitor (at a volume ratio of protein lysis buffer to phosphorylated protease inhibitor of 100:2), and aprotinin protease inhibitor (at a volume ratio of protein lysis buffer to aprotinin (500×) of 100:0.2) in sequence. Wash the cells twice with PBS buffer, then add protein lysis buffer, transfer the cells and protein lysis buffer to centrifuge tubes with a cell scraper, sonicate the cells, centrifuge at 4℃, 12000rpm for 10min, collect the supernatant, place the protein on ice, and freeze some samples at -80℃;

[0145] (2) BCA protein concentration measurement: BSA protein standard is used to create a standard curve and the protein concentration is measured; after incubation at 37℃, the next step is to measure the OD value of the target protein using an enzyme-linked immunosorbent assay (ELISA) reader.

[0146] (3) Protein denaturation: The protein supernatant was mixed with 5× loading buffer at a ratio of 4:1 and heated in a boiling water bath for 5 minutes. After the water bath, the mixture was centrifuged at room temperature, and then a gel was prepared for sample loading.

[0147] (4) Gel Preparation: First, prepare the stacking gel (upper layer, 5%), then determine the concentration of the separating gel (lower layer) based on the molecular weight of the target protein. Before preparing the gel, check for leaks using pure water, add pure water, and let stand for 2-3 minutes. When preparing the lower layer gel, slowly pour the prepared gel solution from the kit along the edge of the glass plate, avoiding air bubbles as much as possible. Add anhydrous ethanol or double-distilled water to flatten the gel surface and let it stand until the gel solidifies (usually more than 30 minutes depending on room temperature). After the lower layer gel has completely solidified, pour off the anhydrous ethanol or double-distilled water and blot away any remaining liquid with filter paper. Next, prepare the upper layer gel, slowly pour it between the glass plates, insert a comb, and wait for it to solidify before electrophoresis.

[0148] (5) Electrophoresis: Prepare the loading system by mixing protein solution, 5× loading buffer, and PBS in the specified proportions, ensuring that the protein loading volume for each sample is 30 μg. During electrophoresis, set the initial voltage to 80 V and hold for 30 min to concentrate the sample in the stacking gel. Then, adjust the voltage to 120 V to run the separating gel. Stop electrophoresis when bromophenol blue in the loading buffer is observed to migrate to 1 cm from the bottom of the gel.

[0149] (6) Transfer: Transfer the membrane using the fast transfer method for 15 minutes;

[0150] (7) Sealing: Use Biyuntian rapid sealing solution to seal for 15 minutes;

[0151] (8) Incubate with primary antibody. The antibody dilution ratio is shown in the table below. Incubate overnight at 4°C.

[0152] (9) Wash the membrane 3 times with 1×TBST, every 5 min. Incubate with secondary antibody, 1:2000. After incubation for half an hour, wash the membrane 3 times with 1×TBST, every 10 min.

[0153] (10) Development and exposure: The film is placed in the machine, the imprint is selected, automatic exposure is performed, and the imaging results are saved.

[0154] Table 2. Antibody Usage Ratio

[0155]

[0156] TUNEL (TdT-mediated dUTP nick end labeling) apoptosis staining determined that spinal cord nerves in the SCI group showed the highest apoptosis rate after injury, and GM1-PLGA-PEG@FPR2 treatment significantly reduced nerve apoptosis by day 7. Figure 4 A). At day 7, severe neuronal apoptosis occurred in the SCI group, which subsided after day 28, but recovery was limited. Analysis of inflammatory factor expression in the spinal cord at days 7 and 14 revealed decreased expression of pro-inflammatory factors IL-6 and TNF-α in the GM1-PLGA-PEG@FPR2 group, while increased expression of anti-inflammatory factors IL-10 and TGF-β. Figure 4 B). More importantly, in the GM1-PLGA-PEG@FPR2 group, the levels of neuronal repair-related proteins MAP-2 and Tuj1 were increased, while the level of neuronal damage-related protein GFAP was decreased. Although FPR2 can also help with nerve repair, the therapeutic effect is relatively poor. Figure 4 C).

[0157] 4.3 In vivo imaging in animals

[0158] DiR fluorescent dye-labeled GM1-PLGA-PEG@FPR2 microspheres were injected via the tail vein into rats with spinal cord injury. Whole-body fluorescence signals were collected using a small animal in vivo imaging system (IVIS Spectrum, excitation / emission wavelengths 745 / 800 nm) at days 1, 3, 7, 14, and 28. The fluorescence intensity ratio of the spinal cord injury site to the liver within the region of interest (ROI) was quantitatively analyzed to assess the microsphere targeting and metabolic clearance. At the experimental endpoint, animals were sacrificed, and frozen sections of spinal cord tissue were harvested for confocal microscopy to observe the distribution of microspheres in and around the injury core.

[0159] In vivo imaging was performed on rats in different treatment groups on days 1, 3, 7, 14, and 28 using fluorescently labeled nanospheres. The GM1-PLGA-PEG@FPR2 group showed the best recovery after 28 days and could stably accumulate at the damaged spinal cord, with faster recovery than other groups. The GM1-PLGA-PEG group could also locate the damaged spinal cord, but the luminescent area of ​​the spinal cord did not decrease or weaken, indicating limited self-repair of the spinal cord after injury. Spinal cord injury persisted in the SCI group. No material accumulation was observed in the PLGA group, which served as a negative control during the detection process. Figure 5 (A and 5B). PLGA microspheres did not show strong hepatic retention, indicating that non-GM1 / PEG modified nanospheres were rapidly recognized and cleared by the autoimmune system. GM1-PLGA-PEG microspheres did not show strong hepatic retention; the weak hepatic retention is mainly attributed to circulating metabolism after tail vein injection. Figure 5 C).

[0160] 4.4 Hematoxylin-eosin (H&E) staining

[0161] Tissue samples were collected following systemic anatomy guidelines, with vital organs such as the heart, liver, spleen, lungs, and kidneys being completely removed. After rinsing with pre-cooled PBS, the samples were immediately fixed in 4% paraformaldehyde for 24 hours. Paraffin-embedded sections were prepared into 5μm serial sections using a Leica RM2235 microtome. Hematoxylin-eosin (H&E) staining followed Bancroft's theoretical pathological technique: dewaxing to water followed by hematoxylin staining for 5 minutes, differentiation with 1% hydrochloric acid alcohol for 30 seconds, counterstaining with eosin for 2 minutes, and mounting with neutral resin. Histopathological observation was then performed using an Olympus BX53 optical microscope.

[0162] (1) Preparation of paraffin sections: ① Dehydration: The fixed tissue sample is dehydrated by passing it through a series of ethanol solutions of increasing concentration to remove water from the tissue. ② Clearing: The dehydrated tissue sample is immersed in a clearing agent such as xylene to make it transparent, facilitating subsequent observation and mounting. ③ Embedding: The cleared tissue sample is embedded in paraffin to form a paraffin block. ④ Slicing: The paraffin block is sliced ​​into thin sections of 4-6 micrometers thickness using a microtome, i.e., paraffin sections.

[0163] (2) Staining: ① Dewaxing: Place the paraffin sections in a dewaxing agent such as xylene to dissolve and remove the wax from the sections. ② Rehydration: Use ethanol solutions of decreasing concentration for gradient dehydration to gradually restore the sections to a hydrated state. ③ Staining: Hematoxylin staining: Place the sections in diluted hematoxylin staining solution for 2-10 minutes to stain the cell nuclei. The staining time can be adjusted as needed to avoid over-staining. Eosin staining: Place the sections in eosin dye solution for 1-5 minutes to stain the cytoplasm. The staining time can also be adjusted according to experimental needs. ④ Washing: Rinse the sections with distilled water to remove excess hematoxylin dye. ⑤ Differentiation: Place the sections in acidic alcohol or 1% hydrochloric acid ethanol for a few seconds to 30 seconds to remove excess hematoxylin dye and make the staining of the cell nuclei clearer. ⑥ Blueing: Place the sections in acidic alcohol or a blue agent, such as 0.6% ammonia water, for blueing treatment, usually for 1-5 minutes, to make the cell nuclei appear a brighter blue. ⑦ Dehydration: Gradual dehydration is performed again using ethanol solutions of increasing concentration to prepare for subsequent clearing and mounting.

[0164] (3) Mounting and observation: ① Clearing: Immerse the dehydrated sections in a clearing agent such as xylene to make them transparent. ② Mounting: Remove the sections from the xylene, let them dry slightly, and then mount them with a suitable mounting medium (such as neutral resin). ③ Observation: Observe the stained sections under a microscope. The cell nuclei should appear blue, and the cytoplasm should appear red or pink.

[0165] 4.5 Serum marker detection

[0166] Before euthanizing animals on day 28 post-injury, animals were deeply anesthetized via intraperitoneal injection of sodium pentobarbital (50 mg / kg). 5 mL of blood was collected via the abdominal aorta and placed in a heparin anticoagulant tube. Plasma was immediately separated by centrifugation at 3000 rpm for 15 minutes and stored at -80°C for later testing. Hepatorenal toxicity was assessed using a Hitachi 7180 fully automated biochemical analyzer to detect alanine aminotransferase (ALT, catalog number A052-1-1), aspartate aminotransferase (AST, catalog number C010-2-1), uric acid (UA, catalog number C012-1-1), and blood urea nitrogen (BUN, catalog number C013-2-1). The procedure was strictly followed according to the instructions, and standard quality control was established.

[0167] (1) The rats were perfused with 0.9% saline and 5 mL of fresh blood was collected simultaneously using blood collection tubes containing a coagulant. The blood was left to stand at room temperature for 30 minutes.

[0168] (2) Centrifuge the collected blood samples at 3000 rpm for 5 minutes at room temperature.

[0169] (3) After centrifugation, the upper serum layer of each tube was collected. The corresponding indicators in the serum of rats in each group were quantitatively detected by a kit for ALT (alanine aminotransferase), AST (aspartate aminotransferase), UA (uric acid), and BUN (blood urea nitrogen) to assess the damage of the nanospheres to the organs.

[0170] Twenty-eight days after spinal cord injury, heart, liver, spleen, lung, and kidney organs were collected from rats in different treatment groups for histological analysis to assess biocompatibility. No observable damage or lesions were found under an optical microscope. Figure 6 A).

[0171] Serum alanine aminotransferase ( Figure 6 B), aspartate aminotransferase ( Figure 6 C), uric acid Figure 6 D) and urea nitrogen ( Figure 6 No obvious abnormalities were found during the examination of E).

[0172] In summary, GM1-PLGA-PEG@FPR2 microspheres demonstrated good biocompatibility in the treatment of spinal cord injury.

[0173] All experimental data in this application are presented as mean ± standard deviation. Data analysis and graph creation were performed using Origin 9.1 or GraphPadPrism 10.0 software. One-way or two-way ANOVA was used, combined with Turkey's test, with a p-value less than 0.05 as the criterion for statistical significance.

[0174] In this application, the inflammatory stimulation and cytotoxicity of primary spinal cord nerve cells and nanospheres were systematically evaluated through co-culture experiments. The results showed that GM1-PLGA-PEG@microspheres did not significantly promote the secretion of IL-6 and TNF-α within 48 hours, and cell survival rates at different concentrations were not statistically different from the negative control group. Compared with positive controls (such as lipopolysaccharide), the nanosystem of this application did not activate inflammatory pathways, indicating its suitability for the microenvironment of high inflammation following spinal cord injury. GM1-PLGA-PEG@FPR2 microspheres achieved approximately 80% cumulative release of FPR2 agonist 3 within 21 days without a burst release effect, which is closely related to the degradation kinetics of PLGA. This sustained-release mode can maintain drug concentration during the therapeutic window and avoid the side effects of frequent dosing. Live / dead cell double staining experiments further confirmed that drug-loaded microspheres (GM1-PLGA-PEG@FPR2) significantly reduced H2O2-induced neuronal death, while blank microspheres showed no such effect, indicating that the neuroprotective effect originates from the sustained release of FPR2 agonist 3 rather than the carrier itself. Combined with previous studies, FPR2 agonist 3 restores STAT3 activity by inhibiting the SOCS3 / STAT3 axis, thereby regulating the Bax / Bcl-2 balance and inhibiting caspase-3 activation, ultimately blocking the apoptosis pathway. The sustained-release characteristics of the nanodelivery system allow this molecular mechanism to persist over time, thus enhancing the therapeutic effect.

[0175] In summary, this application constructed a GM1-PLGA-PEG@FPR2 nanodelivery system with spinal cord targeting function. This system exhibits good sustained-release performance, high biocompatibility, and effectively reduces neuronal apoptosis after H2O2 stimulation. Using drug-loaded nanospheres to simulate spinal cord injury treatment can improve various motor functions and spinal cord health in rats with spinal cord injury, and can target the damaged spinal cord to promote nerve repair. GM1-PLGA-PEG microspheres did not show strong liver retention and demonstrated good biocompatibility in various tissues.

Claims

1. A drug-loaded nanosphere for spinal cord injury recovery, characterized in that: The drug-loaded nanospheres are prepared by an emulsion solvent evaporation method, in which polymers and drugs are dissolved in an organic solvent, then emulsified into an aqueous phase, and finally the organic solvent is evaporated to form microspheres. Polyethylene glycol and a targeting ligand are added to modify the surface of the nanospheres, wherein the drug targets and inhibits the SOCS3 / STAT pathway.

2. The drug-loaded nanospheres for spinal cord injury recovery according to claim 1, characterized in that: The drug is a small molecule inhibitor, FPR2 agonist 3, that targets and inhibits the SOCS3 / STAT3 axis. Its chemical formula is C2. 25 H 20 F2N4O2.

3. The drug-loaded nanospheres for spinal cord injury recovery according to claim 2, characterized in that: The polymer is PLGA, the organic solvent is an acetone-DCM mixed solvent, and the targeting ligand is ganglioside GM1.

4. The drug-loaded nanospheres for spinal cord injury recovery according to claim 3, characterized in that: The method for preparing the drug-loaded nanospheres is as follows: S1: Dissolve polymers PLGA and FPR2 agonist 3 together in 3 mL of acetone-DCM mixed solvent, vortex until completely dissolved, and use as the organic phase; prepare an appropriate amount of aqueous phase; inject the organic phase solution into 20 mL of aqueous phase containing 0.5% PVA at a rate of 0.5 mL / min, stir magnetically and sonicate simultaneously. S2: Add ethanol to change the polarity of the solvent, causing the polymer to precipitate and form nanospheres; after the organic solvent evaporates, collect the microspheres by centrifugation at 10000×g for 15min, wash them three times with ultrapure water, and freeze-dry them for storage. S3: Finally, the nanospheres were collected by centrifugation and washed with deionized water to remove unencapsulated drugs; S4: Resuspend the lyophilized microspheres in PBS, add NH2-PEG2000-Mal, and react at room temperature; remove free PEG by ultrafiltration and centrifugation to obtain PEGylated microspheres. S5: By modifying the surface of nanospheres and adding polyethylene glycol and targeting ligands, their targeting and stability in spinal cord tissue are improved.

5. The drug-loaded nanospheres for spinal cord injury recovery according to claim 4, characterized in that: In S5, EDC / NHS chemical coupling is used: GM1 and PLGA-PEG microspheres are reacted in MES buffer containing 5mM EDC / 2mM NHS for 12h; Unbound GM1 was removed by dialysis, and the final product was denoted as GM1-PLGA-PEG@FPR2.

6. The drug-loaded nanospheres for spinal cord injury recovery according to claim 5, characterized in that: The quality control standards for the prepared GM1-PLGA-PEG@FPR2 were: particle size 150±20nm, PDI<0.2, Zeta potential -15~-25mV, and encapsulation efficiency >80%.

7. The drug-loaded nanospheres for spinal cord injury recovery according to claim 6, characterized in that: The preparation process of the drug-loaded nanospheres was carried out under sterile conditions, and all reagents were sterilized by filtration through a 0.22 μm filter membrane.

8. The use of the drug-loaded nanospheres for spinal cord injury recovery as described in any one of claims 1-7 in the preparation of drugs for treating spinal cord injury.

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