Preparation of nanoformulation cemsn@vk and biomedical applications thereof

Abstract

This invention discloses the preparation of a nano-formulation CeMSN@VK and its biomedical applications. The CeMSN@VK nano-formulation uses cerium oxide-modified hollow mesoporous silica nanoparticles (CeMSN) as a carrier, internally loaded with vitamin K (VK), achieving responsive degradation and drug release in the acidic microenvironment of damaged tissue. Vitamin K synergistically with cerium oxide to scavenge reactive oxygen species, achieving a dual therapeutic effect of inhibiting ferroptosis, neuroprotection, and functional recovery after spinal cord injury, while exhibiting good biocompatibility.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of nervous system injury treatment and medical nanomaterials. Specifically, this invention relates to the preparation of a nano-formulation CeMSN@VK and its biomedical applications. Background Technology

[0002] Spinal cord injury is a serious central nervous system disorder characterized by rapid onset, rapid progression, and high disability rate. Besides the initial mechanical injury, the secondary injury process is a key factor leading to the continued deterioration of neurological function. This process can persist for hours to weeks after the injury and involves oxidative stress, abnormal iron accumulation, lipid peroxidation, and activation of multiple programmed cell death pathways.

[0003] Recent studies have shown that ferroptosis is an important form of cell death secondary to spinal cord injury, characterized by uncontrolled iron-dependent phospholipid peroxidation. Following spinal cord injury, glutamate excitotoxicity can inhibit systemic Xcase. - The transporter leads to reduced cysteine ​​uptake and depletion of glutathione (GSH), which in turn inactivates glutathione peroxidase 4 (GPX4), preventing timely repair of lipid peroxides. Simultaneously, heme release, mitochondrial dysfunction, and excessive generation of reactive oxygen species (ROS) further exacerbate ferroptosis, severely damaging the functional integrity of surviving neurons.

[0004] Currently, clinical treatment for spinal cord injury mainly includes drug therapy such as glucocorticoids, surgical decompression, and rehabilitation training. However, these treatments are mostly systemic or delayed interventions, making it difficult to precisely regulate the specific microenvironment of the injured area. Therefore, in the hyperacute phase after spinal cord injury, timely inhibition of ferroptosis and restoration of redox homeostasis are among the key treatment strategies for improving neurological function recovery.

[0005] Currently, nanomedicines or formulations for treating spinal cord injuries mainly focus on conventional drug delivery systems, such as liposomes, polymer nanoparticles, or traditional mesoporous silica nanoparticles. These systems are primarily used to deliver anti-inflammatory drugs, antioxidants, or neuroprotective agents, aiming to improve tissue damage and promote neurological function recovery to some extent. However, the design of these drug delivery systems has not been optimized for the key pathological mechanisms following spinal cord injury—iron ion accumulation and lipid peroxidation (ferroptosis).

[0006] Specifically, existing delivery systems suffer from several drawbacks: ① Insufficient targeting of pathological mechanisms: Current technologies are not systematically designed around the core aspects of ferroptosis in spinal cord injury (iron ion accumulation, lipid peroxidation, GPX4 inactivation, etc.), making it difficult to effectively block the secondary injury process; ② Limited material functionality: Most nanocarriers are merely "passive delivery tools," lacking the intrinsic function of scavenging ROS or participating in redox regulation, relying on single-drug action for treatment with insufficient synergistic effects; ③ Lack of environmental responsiveness in release mechanisms: Drug release largely depends on diffusion or slow material degradation, making it difficult to match with changes in the microenvironment such as acidity and increased oxidative stress in the injury area, resulting in insufficient precision in treatment timing and location; ④ Inadequate administration routes for hyperacute intervention: Current technologies mainly rely on intravenous or intrathecal administration, which is complex and invasive, unsuitable for the pre-hospital or transport phases after spinal cord injury, and prone to missing the critical time window for ferroptosis intervention.

[0007] Therefore, there is an urgent need in this field to develop a novel nanotherapy platform that combines rapid delivery capability, ferroptosis-targeting regulation function, and environmentally responsive release characteristics.

[0008] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of the present invention, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0009] The purpose of this invention is to provide an anti-ferrode death nanoparticle formulation suitable for intervention in the hyperacute phase of spinal cord injury, so as to overcome the shortcomings of existing technologies in terms of therapeutic timeliness, targeted mechanism of action, and synergistic therapeutic effect.

[0010] To achieve the above objectives, this invention constructs a cerium oxide-modified hollow mesoporous silica nanoplatform loaded with vitamin K (CeMSN@VK).

[0011] In a first aspect of the invention, a CeMSN@VK composite is provided, the composite being composed of mesoporous silica nanoparticles, cerium oxide, and vitamin K.

[0012] In another preferred embodiment, the CeMSN@VK complex is a nanoformulation.

[0013] In another preferred embodiment, the CeMSN@VK complex releases vitamin K in damaged tissue and can scavenge reactive oxygen species.

[0014] In a second aspect of the invention, a method for preparing the CeMSN@VK complex described in the first aspect of the invention is provided, the method comprising the steps of: (1) Provide cerium oxide modified hollow mesoporous silica nanoparticles (CeMSN); (2) Vitamin K load.

[0015] In another preferred embodiment, the cerium oxide-modified hollow mesoporous silica nanoparticles (CeMSN) are prepared by a modified Stöber method combined with homogeneous precipitation and alkaline etching.

[0016] In another preferred embodiment, the method for preparing the cerium oxide-modified hollow mesoporous silica nanoparticles (CeMSN) includes the following steps: (1a) Provide (preparation) silica precursor spheres; (1b) Homogeneous deposition of cerium oxide; (1c) Hollow structure formation (alkaline etching); (1d) Surface electrical property regulation.

[0017] In a third aspect of the invention, the use of the CeMSN@VK complex described in the first aspect of the invention in the preparation of a medicament for treating spinal cord injury is provided.

[0018] In another preferred embodiment, the drug for treating spinal cord injury is used for the prevention / treatment of hyperacute spinal cord injury.

[0019] In another preferred embodiment, the drug for treating spinal cord injury is used for the prevention / treatment of anti-ferrode death in the hyperacute phase of spinal cord injury.

[0020] In another preferred embodiment, the medication for treating spinal cord injury is administered via nasal delivery.

[0021] In a fourth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising: (i) the CeMSN@VK complex as described in the first aspect of the invention; and (ii) Pharmaceutically acceptable carriers.

[0022] In another preferred embodiment, the pharmaceutical composition is used for spinal cord injury repair.

[0023] In another preferred embodiment, the pharmaceutical composition is used to protect neural structures.

[0024] In another preferred embodiment, the pharmaceutical composition is used to regulate inflammation.

[0025] In another preferred embodiment, the pharmaceutical composition is used for the treatment of hyperacute spinal cord injury.

[0026] In another preferred embodiment, the pharmaceutical composition is used to inhibit ferroptosis following spinal cord injury.

[0027] In another preferred embodiment, the pharmaceutical composition is used for the prevention / treatment of antiferrodegeneration in the hyperacute phase of spinal cord injury.

[0028] In another preferred embodiment, the pharmaceutical composition is used to promote neuroprotection and neurological function recovery.

[0029] In another preferred embodiment, the pharmaceutical composition is used to promote axonal protection and / or remyelination.

[0030] In another preferred embodiment, the pharmaceutical composition is administered via nasal delivery.

[0031] In another preferred embodiment, the nasal delivery is selected from the group consisting of: nasal spray, nasal drops, and nasal nebulization.

[0032] In another preferred embodiment, the pharmaceutical composition is a pre-hospital medication or an early treatment medication.

[0033] In another preferred embodiment, the pharmaceutical composition is administered within the time interval from the injury to the surgery (e.g., within 0.5 hours after the injury).

[0034] In a fifth aspect of the invention, a pharmaceutical combination is provided, comprising: (i) A first active ingredient, comprising the CeMSN@VK complex of the first aspect of the present invention or the pharmaceutical composition of the fourth aspect of the present invention; (ii) A second active ingredient, wherein the second active ingredient is a central nervous system therapeutic drug (e.g., a small molecule drug).

[0035] In another preferred embodiment, the small molecule drug is selected from the group consisting of curcumin and rapamycin.

[0036] In a sixth aspect of the invention, a method for treating spinal cord injury is provided, using the pharmaceutical composition described in the fourth aspect of the invention or the pharmaceutical composition described in the fifth aspect of the invention.

[0037] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit the invention. Attached Figure Description

[0038] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention. It is obvious that the drawings described below are merely some embodiments of the invention, and those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0039] Figure 1The preparation and characterization of CeMSN (cerium oxide-modified hollow mesoporous silica) are shown. (a) Scanning electron microscope image of CeMSN. (b) Transmission electron microscope image of CeMSN. (c) Line scan elemental distribution of a single CeMSN. (d) Elemental distribution map of CeMSN. (e) CeL3 edge normalized X-ray absorption near-edge structure spectrum and extended X-ray absorption fine structure spectrum of CeMSN. (f) CeL3 edge Fourier transform extended X-ray absorption fine structure spectrum of CeMSN. (g) Wavelet transform image of extended X-ray absorption fine structure signal of CeMSN.

[0040] Figure 2 The results show the dual staining of iNOS (inducible nitric oxide synthase, which labels activated inflammatory cells) and Iba1 (labeling all microglia / macrophages) in the spinal cord tissues of mice in the SCI (spinal cord injury) group, CeMSN treatment group, and CeMSN@VK treatment group.

[0041] Figure 3 The triple staining results of NF200 (red, neuronal axons), GFAP (green, astrocytes), and HLP (yellow, myelin markers) in the spinal cord tissue of mice in the SCI (spinal cord injury) group and the CeMSN@VK treatment group are shown.

[0042] Figure 4 The diagram shows the reconstruction results of the Sham (sham surgery) group, SCI (spinal cord injury) group, CeMSN treatment group, and CeMSN@VK treatment group after markerless motion tracking. Gray represents the stance phase and red represents the swing phase. Detailed Implementation

[0043] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, they are provided so that the invention will be thorough and complete, and the concept of the exemplary embodiments will be fully conveyed to those skilled in the art.

[0044] Unless otherwise defined, all terms and phrases used herein include their meanings as they have in the art, unless explicitly stated otherwise or clearly indicated from the context of their use. While any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the invention, specific methods and materials are now described.

[0045] Through extensive and in-depth research, the inventors have designed and prepared for the first time a CeMSN@VK nanoparticle formulation that can be delivered nasally. This formulation releases vitamin K in response to an acidic environment and works synergistically with cerium oxide to scavenge reactive oxygen species, achieving a dual therapeutic effect of inhibiting ferroptosis, protecting the nerves, and restoring function after spinal cord injury, while also exhibiting good biocompatibility.

[0046] Specifically, this invention relates to a nasally delivered anti-ferroptosis nanoparticle formulation for the treatment of spinal cord injury (SCI). The formulation uses cerium oxide-modified hollow mesoporous silica nanoparticles (CeMSN) as a carrier, internally loaded with vitamin K (VK), achieving responsive degradation and drug release in the acidic microenvironment of damaged tissue. Its technical advantages are reflected in: (1) Nasal delivery and non-invasive drug administration: Taking advantage of the nanoparticle scale, the drug can bypass the first-pass effect of the liver and quickly enter the central nervous system through nasal delivery, so as to achieve effective intervention within hours after the injury occurs and meet the needs of pre-hospital or early treatment. (2) Response release in acidic microenvironment: In the acidic microenvironment of the spinal cord injury area, the carrier degrades and precisely releases vitamin K, improving the effectiveness of the drug in the lesion area; (3) Synergistic inhibition of ferroptosis and oxidative stress: Released vitamin K inhibits lipid peroxidation by activating the FSP1-VK antiferroptosis pathway; simultaneously, cerium oxide in Ce³ + / Ce 4+ During the reversible process, it exhibits SOD-like and CAT-like activities, effectively scavenging ROS and restoring redox homeostasis; (4) Promote neuroprotection and functional recovery: By synergistically inhibiting secondary damage through multiple mechanisms, the survival rate of neurons is improved, creating a favorable microenvironment for the reconstruction of neurological function after spinal cord injury.

[0047] This invention not only overcomes the shortcomings of existing nanomedicine delivery systems in terms of ferroptosis regulation and drug delivery timeliness, but also provides a new technical path for developing rapidly deliverable nanotherapeutic strategies suitable for acute central nervous system injuries, demonstrating promising application prospects and clinical translational potential. Based on this, this invention was completed.

[0048] the term

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0050] As used in this article, when referring to a specific listed value, the term “about” means that the value can vary by no more than 1% from the listed value.

[0051] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0052] Spinal cord injury (SCI)

[0053] Spinal cord injury can be classified according to its etiology into traumatic spinal cord injury (such as traffic accidents, falls from heights, violent impacts, etc.) and non-traumatic spinal cord injury (such as tumor compression, infection, vascular malformation, intervertebral disc degeneration, etc.); according to the injury mechanism into primary injury (tissue structure destruction directly caused by mechanical external force) and secondary injury (including a series of secondary pathological processes such as blood-spinal cord barrier disruption, ion homeostasis imbalance, neuroexcitotoxicity, oxidative stress, inflammatory cascade reaction and cell apoptosis); according to pathological and functional status into complete injury and incomplete injury; and according to the stage of the disease into acute phase, subacute phase and chronic phase. Currently, the comprehensive treatment strategies for spinal cord injury mainly include (1) acute intervention—such as early surgical decompression, hemodynamic support, and the use of anti-inflammatory drugs such as methylprednisolone to limit the spread of secondary injury; (2) promoting regeneration and repair—including neurotrophic factor delivery, stem cell transplantation, biomaterial bridging, neutralization of axon regeneration inhibitory factors (such as Nogo, MAG, and OMGp), and targeted drug delivery systems based on nanomaterials; (3) symptom management—covering neuropathic pain control, spasticity management, bladder / intestinal function rehabilitation, respiratory function support, and rehabilitation training.

[0054] CeMSN@VK complex

[0055] In this invention, the CeMSN@VK complex is composed of mesoporous silica nanoparticles, cerium oxide, and vitamin K.

[0056] CeMSN@VK complex is a vitamin K controlled-release cerium oxide-modified mesoporous silica nanoparticle formulation that can be delivered nasally for the prevention / treatment of anti-ferrode death in the hyperacute phase of spinal cord injury.

[0057] The CeMSN@VK complex in this invention can be used to treat spinal cord injury.

[0058] In one specific example of the present invention, the CeMSN@VK complex targets acute spinal cord injury, acts on nerve cells, and improves motor function.

[0059] Pharmaceutical Composition

[0060] The present invention also provides a pharmaceutical composition comprising the CeMSN@VK complex described above, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substances and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): oral, inhalation, intravenous, or local administration (e.g., nasal delivery).

[0061] The pharmaceutical composition of this invention can be directly and precisely targeted to spinal cord injury tissue, releasing vitamin K and scavenging reactive oxygen species, regulating inflammation, and promoting axonal protection and / or remyelination, thereby promoting neuroprotection and neurological function recovery through multiple actions. Furthermore, other therapeutic agents (such as central nervous system therapeutics like curcumin and rapamycin) can be used simultaneously.

[0062] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the CeMSN@VK complex described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated as sprays or drops, for example, prepared by conventional methods using physiological saline or aqueous solutions containing glucose and other excipients. Pharmaceutical compositions such as sprays or drops, solutions, are preferably manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 10 mg / kg body weight per day. Furthermore, the CeMSN@VK complex of the present invention can also be used with other therapeutic agents.

[0063] When using the pharmaceutical composition, a safe and effective amount of the CeMSN@VK complex is administered to mammals, where the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 8 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0064] The main beneficial effects of this invention include: (1) Synergistic inhibition of ferroptosis and oxidative stress: CeMSN@VK can significantly reduce RSL3-induced Fe²⁺ +It accumulates and inhibits ROS generation, and alleviates lipid peroxidation and mitochondrial membrane depolarization, thereby significantly improving the survival rate of nerve cells (ROS decreased by 48.8±3.5%, and cell survival was close to the control level). (2) Targeting the injury site and achieving high efficacy: Nanoparticles are delivered rapidly to the center of spinal cord injury via nasal delivery and release vitamin K in an acidic microenvironment to achieve lesion localization and long-term local action, significantly reducing MDA content and enhancing GPX4 expression; (3) Regulation of lipid metabolism and glutamate levels: CeMSN@VK reduces PE-AA oxidation, increases α-tocopherol levels, and inhibits lipid peroxidation; at the same time, it reduces glutamate levels, which helps to restore red oxygen balance without relying on GSH or GPX4 activity to be directly enhanced. (3) Remodeling the neuroinflammatory microenvironment: CeMSN@VK significantly inhibited microglial activation and IBA1 / iNOS expression, and reduced the levels of IL-6, IL-1β, and TNF-α, thereby alleviating secondary inflammatory damage and protecting surviving neurons; (5) Promotes tissue repair and functional recovery: In the SCI model, CeMSN@VK reduced the lesion area (31.8±3.4%) and restored axonal NF200. + Area (increased by 224% ± 28.9%), enhanced myelin sheath PLP + Region, while reducing GFAP + Astral glial coverage improves neural networks and remyelination; (6) Good biocompatibility: No significant damage was observed in major organs in vivo, the material is highly safe and suitable for clinical translation.

[0065] The present invention will be further explained below with reference to specific embodiments.

[0066] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in *Molecular Cloning: A Laboratory Guide* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the manufacturer's recommendations (e.g., product instructions). Unless otherwise stated, percentages and parts are by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available or can be prepared according to literature methods.

[0067] Preparation method

[0068] I. Preparation of Cerium Oxide Modified Hollow Mesoporous Silica Nanoparticles (CeMSN)

[0069] The CeMSN described in this invention is prepared using a modified Stöber method combined with homogeneous precipitation and alkaline etching. The specific steps are as follows.

[0070] (1) Preparation of silica precursor spheres

[0071] 14.4 mL of ammonia solution was slowly added dropwise to 516 mL of a mixed solution of TEOS / deionized water / anhydrous ethanol, with a volume ratio of 1:7:35. The reaction was carried out under magnetic stirring at 25 °C for 24 h. After the reaction was completed, the sample was centrifuged (10000 rpm). Collect the precipitate (10 min) and wash it three times each with deionized water and anhydrous ethanol to obtain silica precursor particles.

[0072] (2) Homogeneous deposition of cerium oxide

[0073] Weigh 0.1 g of the above silica precursor and ultrasonically disperse it in a mixed solution of 25 mL deionized water and 25 mL anhydrous ethanol. Then, add 0.15 g Ce(NO3)3·6H2O and 0.1 g hexamethylenetetramine (HMT) sequentially. Heat the reaction system to 70 °C and reflux for 4 h. After cooling to room temperature, centrifuge at 12000 rpm. The product was collected after 10 min and washed three times with deionized water, then dried at 60°C.

[0074] (3) Formation of hollow structure (alkaline etching)

[0075] The above product was dispersed in 50 mL of 0.5 M NaOH solution and stirred overnight at 60 °C. Fresh NaOH solution was then added and mechanical stirring continued for 24 h to thoroughly remove the internal silicon core structure. After the reaction was complete, the solution was washed repeatedly with deionized water until the pH was close to neutral, and finally dried at 60 °C to obtain cerium oxide-modified hollow mesoporous silica nanoparticles (CeMSN).

[0076] (4) Surface electrical property regulation

[0077] To regulate the surface charge properties of CeMSN, its surface can be modified with 3-aminopropyltriethoxysilane (APTES) to improve its biocompatibility and drug loading performance.

[0078] II. Vitamin K loading (Preparation of CeMSN@VK)

[0079] Vitamin K was loaded into the internal channels of CeMSN using a solvent impregnation method. The specific steps are as follows: Vitamin K was dissolved in dimethyl sulfoxide (DMSO) to prepare a solution of a certain concentration (10 mg / mL). - ¹) A vitamin K solution was prepared; then 20 mg of CeMSN nanoparticles were added to this solution, and the mixture was stirred and incubated at room temperature in the dark for 8 h, allowing vitamin K to be passively adsorbed into the mesoporous structure of CeMSN through hydrophobic interactions. After the reaction was completed, the mixture was centrifuged (12000 rpm). (10 min) Remove unloaded free vitamin K, redisperse the precipitate in deionized water to obtain an aqueous dispersion of CeMSN@VK. The vitamin K loading concentration was determined to be approximately 3 μmol·mL⁻¹. - ¹.

[0080] Example 1: Structural and physicochemical characterization of CeMSN@VK

[0081] (1) Morphological and structural characterization

[0082] The morphology of CeMSN was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results showed that the obtained CeMSN nanoparticles possessed good monodispersity and a regular spherical structure, with a distinct hollow internal morphology (Figure 1, A, B). Figure 1 (B)

[0083] TEM elemental distribution maps and line scan analysis show that silicon (Si) and cerium (Ce) are uniformly distributed in the nanoparticles, indicating that cerium oxide has been successfully modified into the mesoporous silica structure (Figure 1C and Figure 1D).

[0084] (2) Analysis of valence state and coordination environment of cerium

[0085] The chemical state of cerium in CeMSN was analyzed using synchrotron X-ray absorption fine structure spectroscopy (XAFS). Ce L3 edge XANES spectra showed that cerium coexists with Ce³. + and Ce 4+ The average valence state of the two valence states is approximately +3.52 (E in Figure 1) after fitting calculation.

[0086] EXAFS analysis and wavelet transform results show that Ce–O coordination structures mainly exist in CeMSN, with the first coordination shell distance being approximately 2.04 Å (F in Figure 1). Figure 1 The presence of cerium (G) indicates that cerium exists in the nanostructure as a stable oxide.

[0087] Example 2 Animal Experiment

[0088] Animal experiments were conducted, applying the nano-formulation to spinal cord-injured mice (8-week-old male C57BL / 6 mice, purchased from the Experimental Animal Center of Fudan University). Immunohistochemical analysis of the spinal cord tissue was performed on day 7 post-injury. Figure 2 As shown, in the simple spinal cord injury group, a large number of IBA1-positive microglia were observed to aggregate in the injury area, accompanied by significant iNOS co-expression, indicating a significant pro-inflammatory response. In contrast, iNOS expression was reduced in the CeMSN treatment group, while the density of IBA1-positive cells and the level of iNOS expression were significantly reduced in the CeMSN@VK treatment group, and were effectively inhibited in the injury center and surrounding areas.

[0089] Immunofluorescence staining of axonal and myelin-related markers was performed on spinal cord tissue 4 weeks after injury. Figure 3 As shown, compared with the spinal cord injury group, the CeMSN@VK treatment group exhibited more continuous NF200-positive axonal structures, fewer GFAP-positive astrocyte aggregates, and enhanced PLP-positive myelin signal. Quantitative analysis of these indicators showed that the CeMSN@VK treatment group had a significantly reduced GFAP-positive coverage area, an NF200-positive axonal area that was approximately 224% ± 28.9% higher than that of the spinal cord injury group, and a significantly increased PLP-positive remyelination area (e.g., ...). Figure 3 As shown in the figure, this nano-formulation can effectively promote axonal protection and remyelination.

[0090] Example 3: CeMSN@VK helps restore motor function in mice with spinal cord injury.

[0091] To evaluate the effect of the CeMSN@VK nanoformulation described in this invention on functional recovery after spinal cord injury, marker-free hindlimb motor function was tracked in mice of different treatment groups. The sham-operated group showed regular and periodic oscillation characteristics at various joint angles; the spinal cord injury group showed significant loss of the above-mentioned periodic movements; the CeMSN treatment group only partially recovered joint angle oscillations, while the CeMSN@VK treatment group, especially at the ankle and hip joints, recovered clear and repetitive gait-like movement trajectories, suggesting a significant improvement in walking rhythm and coordination.

[0092] Experimental Data and Comparison: To verify the technical efficacy of the CeMSN@VK composite material of this invention in spinal cord injury repair, a comparative analysis was conducted on different treatment groups from multiple aspects, including neural structure protection, inflammation regulation, tissue repair, and neurological function recovery. Regarding neurological function recovery, behavioral assessment results showed that the control group animals exhibited significant long-term motor dysfunction. CeMSN treatment improved motor function to some extent, raising their BMS scores to approximately 6 points. In contrast, the CeMSN@VK treatment group showed a further increase in BMS scores, approaching 7 points, demonstrating more stable plantar gait and a certain degree of coordination recovery, with an overall functional recovery level significantly superior to the CeMSN group. Results are shown below. Figure 4 .

[0093] Regarding tissue repair, histological analysis showed that the control group exhibited significant tissue defects and cavity formation in the damaged area. CeMSN treatment could partially alleviate tissue damage, while CeMSN@VK treatment significantly reduced the damaged area, with the damaged area decreasing by approximately 31.8% ± 3.4% compared to the control group, indicating that this material can effectively improve the tissue integrity of the damaged area.

[0094] Regarding nerve axons and remyelination-related indicators, CeMSN@VK treatment significantly promoted axonal structural recovery compared to the control group, with NF200... + The axonal area increased by approximately 224% ± 28.9%, significantly higher than that in the CeMSN treatment group. Simultaneously, remyelination-related signals also showed an enhancing trend, indicating effective reconstruction of the neural conduction structure.

[0095] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the appended claims.

Claims

1. A CeMSN@VK complex, characterized in that, The complex consists of mesoporous silica nanoparticles, cerium oxide, and vitamin K.

2. The method for preparing the CeMSN@VK complex according to claim 1, characterized in that, The method includes the following steps: (1) Provide cerium oxide modified hollow mesoporous silica nanoparticles; (2) Vitamin K load.

3. The preparation method according to claim 2, characterized in that, The preparation method of the cerium oxide modified hollow mesoporous silica nanoparticles includes the following steps: (1a) Provide silica precursor spheres; (1b) Homogeneous deposition of cerium oxide; (1c) Hollow structure formation; (1d) Surface electrical property regulation.

4. The use of the CeMSN@VK complex according to claim 1 in the preparation of a medicament for treating spinal cord injury.

5. The application as described in claim 4, characterized in that, The medication for treating spinal cord injury is used for the prevention / treatment of hyperacute spinal cord injury.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) the CeMSN@VK complex as claimed in claim 1; and (ii) Pharmaceutically acceptable carriers.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition is used for spinal cord injury repair.

8. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition is used to promote neuroprotection and the recovery of neurological function.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition is administered via nasal delivery.

10. A drug combination, characterized in that, include: (i) A first active ingredient, comprising the CeMSN@VK complex of claim 1 or the pharmaceutical composition of claim 6; (ii) A second active ingredient, wherein the second active ingredient is a central nervous system therapeutic drug.

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