Bridging mesoporous silica nanoparticles with diselenide linkage loaded with baicalin and applications thereof

Abstract

This invention relates to diselenylene-bridged mesoporous silica nanoparticles loaded with baicalin and their applications. The invention constructs a ROS-responsive, microglia-inspired Ba@Se-MSN&BV2 nanoplatform targeting the secondary phase of spinal cord injury. The BV2 cell membrane-mediated enrichment at the injury site, combined with the diselenylene-triggered, on-demand release of baicalin, enables targeted intervention in a highly oxidative and pro-inflammatory microenvironment. In vitro and in vivo studies show that Ba@Se-MSN&BV2 can alleviate oxidative stress and mitochondrial dysfunction, inhibit apoptosis, promote axonal and neuronal survival, and improve motor circuit function. This invention provides a new therapeutic target and research direction for neuroinflammatory injuries driven by redox homeostasis disruption, with significant clinical application prospects and social value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to mesoporous silica nanoparticles supported on baicalin and bridged by diselenyl bonds, their preparation method, and their application in the treatment of spinal cord injury. Background Technology

[0002] Spinal cord injury (SCI) causes severe damage to the central nervous system, often leading to lifelong disability, including persistent motor, sensory, and autonomic dysfunction, as well as systemic comorbidities such as chronic pain and metabolic disorders. Following primary SCI, SCI rapidly evolves into a secondary pathobiological cascade, in which the excessive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), mitochondrial dysfunction, and amplified neuroinflammation establish a self-reinforcing feedforward loop. Redox imbalance induces mitochondrial depolarization and bioenergy depletion, leading to excitotoxicity and disruption of ion homeostasis. These processes converge and amplify, ultimately resulting in axonal degeneration and reactive glial proliferation, followed by the formation and remodeling of glial scars, thereby disrupting the structural scaffold and creating an inhibitory microenvironment that impairs the reconstruction of neural circuits. Current clinical care focuses on early decompression and spinal stabilization, supplemented by pharmacological therapy, neurotrophic factor delivery, and cell-based therapies. However, clinical benefits remain limited, largely because drugs cannot reach therapeutic concentrations at the site of injury, delivery is rarely controlled with spatiotemporal precision and signal responsiveness, and long-term functional recovery is limited and often inconsistent. Therefore, integrating damage targeting, pathological signal response, and neural repair simultaneously in the complex SCI microenvironment remains a key, unmet challenge in treatment progress.

[0003] Nanoscale drug delivery offers a rational and engineerable strategy for improving drug exposure and therapeutic consistency at injury sites. Mesoporous silica nanoparticles (MSNs), with their tissue-penetrating size, tunable pore structure, and high specific surface area, enable high drug loading and controlled release kinetics. Abundant surface silanol groups facilitate the construction of pore-gated structures and anchored biomimetic shells, synergistically integrating pore regulation and surface functionalization for precise control of drug delivery and release. However, in the inflammatory and oxidative microenvironment of SCI, passive diffusion driven solely by concentration gradients cannot synchronize drug release with injury source signals; off-target leakage and premature drug loss remain prevalent, weakening durable bioavailability at injury sites. Therefore, integrating pathologically responsive "gated" structures into such carriers is crucial for matching drug release with local pathological signals. Increasing evidence suggests that diselenylene bonds are highly sensitive to oxidative stress. Elevated ROS triggers redox-mediated bond breaking, generating release kinetics proportional to local ROS load, which minimizes nonspecific premature leakage while enhancing drug exposure at injury sites. Furthermore, selenium can enhance the endogenous antioxidant system and synergize with the intrinsic redox network. Therefore, the diselenobond can serve as a ROS-responsive "gated" structure and an intrinsic antioxidant group, matching the significant oxidative imbalance in secondary SCI and supporting controlled in-situ release for sustained and reproducible therapeutic effects.

[0004] In addition to pathologically triggered release, therapeutic payloads should also target key nodes of secondary injury, particularly oxidative stress, neuroinflammation, and susceptibility to apoptosis. Baicalin is a structurally well-defined, bioactive flavonoid with antioxidant, anti-inflammatory, and anti-apoptotic properties. However, baicalin has poor water solubility, low membrane permeability, and is rapidly eliminated in vivo. Furthermore, it exhibits high non-specific protein binding, resulting in a low proportion of free drug and insufficient exposure to the site of injury, thus limiting its efficacy and reproducibility. Summary of the Invention

[0005] The purpose of this invention is to address the problems existing in the prior art, thereby providing a mesoporous silica nanoparticle, Ba@Se-MSN&BV2, loaded with baicalin and bridged by diselenyl bonds. The Ba@Se-MSN&BV2 provided by this invention can effectively reduce intracellular reactive oxygen species levels, achieve significant antioxidant capacity, and promote neuronal growth and recovery of motor function after spinal cord injury. Furthermore, the Ba@Se-MSN&BV2 of this invention exhibits good safety, with no significant toxic side effects on neurons or major organs and tissues, providing a new drug source for the treatment of spinal cord injury.

[0006] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution.

[0007] The first aspect of this invention provides mesoporous silica nanoparticles supported on baicalin and bridged by diselenyl bonds, which are prepared by the following method:

[0008] (1) In an ammonia solution containing CTAB (hexadecyltrimethylammonium bromide), add BTESePD (bis[3-(triethoxysilyl)propyl]diselenide) and TEOS (tetraethyl orthosilicate), heat and stir until the system gels;

[0009] (2) After the reaction was completed, the product was collected by centrifugation, washed and dried; then the dried product was refluxed to remove CTAB, and diselenylene bond-bridged mesoporous silica nanoparticles (Se-MSNs) were obtained.

[0010] (3) Disperse the diselenide-bridged mesoporous silica nanoparticles obtained in step (2) in baicalin solution, mix them evenly by ultrasonication, and then carry out a light-protected stirring reaction.

[0011] (4) After the reaction was completed, the precipitate was centrifuged and washed to remove unbound components, and then freeze-dried under vacuum to obtain Ba@Se-MSNs;

[0012] (5) Resuspend BV2 cells in hypotonic lysis buffer and perform repeated freeze-thaw cycles to separate the BV2 cell membrane; then collect the membrane components by differential centrifugation; extrude the obtained membrane components through 400nm and 200nm polycarbonate porous membranes 5-20 times to obtain BV2 cell membrane vesicles (BV2-CMVs).

[0013] (6) After mixing Ba@Se-MSNs with BV2 cell membrane vesicles, incubate on ice for 20-40 min; then extrude through 400 nm and 200 nm polycarbonate porous membranes 5-20 times at 4 °C; after centrifugation to remove free membranes, resuspend the precipitate in PBS buffer to obtain diselenyl disodium-bridged mesoporous silica nanoparticles (Ba@Se-MSN&BV2) loaded with baicalin.

[0014] Preferably, in step (1), the concentration of NH3 in the ammonia solution is 0.5-2.0 mol / L, the concentration of CTAB is 0.01-0.03 mol / L, and the pH of the ammonia solution is 10-12.

[0015] Preferably, in step (1), BTESePD and TEOS are used together as silicon sources, with a total concentration of 0.05-0.2 mol / L in the solution, and BTESePD accounts for 10-20% of the total molar amount of silicon source.

[0016] Preferably, the heating and stirring temperature in step (1) is 60-80℃.

[0017] Preferably, the centrifugation speed in step (2) is 5000-15000 rpm and the time is 5-20 min.

[0018] Preferably, the washing in step (2) specifically involves washing with deionized water and anhydrous ethanol in sequence, repeating 2-5 times.

[0019] Preferably, the drying temperature in step (2) is 50-70°C and the time is 8-24h.

[0020] Preferably, step (2) involves reflux in a mixed solution of NH4NO3 and ethanol; more preferably, the mass-to-volume ratio of NH4NO3 to ethanol in the NH4NO3-containing ethanol solution is 1% W / V.

[0021] Preferably, the reflux temperature in step (2) is 80-90℃ and the time is 8-24h.

[0022] Preferably, in step (3), the mass ratio of baicalin to diselenylene-bridged mesoporous silica nanoparticles is 1:2-10; more preferably, the mass ratio of baicalin to diselenylene-bridged mesoporous silica nanoparticles is 1:3-7.

[0023] Preferably, the temperature of the stirring reaction in step (3) is 20-30°C and the time is 6-24h; more preferably, the temperature of the stirring reaction is 23-28°C and the time is 12-18h.

[0024] The vacuum freeze-drying temperature in step (4) is -40 to -70°C; more preferably, the vacuum freeze-drying temperature is -45 to -65°C.

[0025] Preferably, the washing in step (4) is specifically performed using PBS.

[0026] Preferably, the repeated freeze-thaw cycle in step (5) is as follows: after standing at 4°C for 20-40 minutes, the freeze-thaw cycle is repeated 2-5 times, consisting of freezing at -80°C for 5-20 minutes and thawing in a water bath at 37°C for 5-20 minutes.

[0027] Preferably, in step (5), differential centrifugation specifically involves centrifuging at 500-1000g for 5-20 minutes to remove cell nuclei and unlysed cells; followed by further centrifugation at 5000-20000g for 10-30 minutes to collect membrane components.

[0028] Preferably, the mass ratio of Ba@Se-MSNs to BV2 cell membrane vesicles in step (6) is 1:0.5-2; more preferably, the mass ratio of Ba@Se-MSNs to BV2 cell membrane vesicles is 1:0.8-1.2.

[0029] A second aspect of this invention provides a method for preparing diselenylene-bridged mesoporous silica nanoparticles loaded with baicalin, comprising the following steps:

[0030] (1) In an ammonia solution containing CTAB (hexadecyltrimethylammonium bromide), add BTESePD (bis[3-(triethoxysilyl)propyl]diselenide) and TEOS (tetraethyl orthosilicate), heat and stir until the system gels;

[0031] (2) After the reaction was completed, the product was collected by centrifugation, washed and dried; then the dried product was refluxed to remove CTAB, and diselenylene bond-bridged mesoporous silica nanoparticles (Se-MSNs) were obtained.

[0032] (3) Disperse the diselenide-bridged mesoporous silica nanoparticles obtained in step (2) in baicalin solution, mix them evenly by ultrasonication, and then carry out a light-protected stirring reaction.

[0033] (4) After the reaction was completed, the precipitate was centrifuged and washed to remove unbound components, and then freeze-dried under vacuum to obtain Ba@Se-MSNs;

[0034] (5) Resuspend BV2 cells in hypotonic lysis buffer and perform repeated freeze-thaw cycles to separate the BV2 cell membrane; then collect the membrane components by differential centrifugation; extrude the obtained membrane components through 400nm and 200nm polycarbonate porous membranes 5-20 times to obtain BV2 cell membrane vesicles (BV2-CMVs).

[0035] (6) After mixing Ba@Se-MSNs with BV2 cell membrane vesicles, incubate on ice for 20-40 min; then extrude through 400 nm and 200 nm polycarbonate porous membranes 5-20 times at 4 °C; after centrifugation to remove free membranes, resuspend the precipitate in PBS buffer to obtain diselenyl disodium-bridged mesoporous silica nanoparticles (Ba@Se-MSN&BV2) loaded with baicalin.

[0036] Preferably, in step (1), the concentration of NH3 in the ammonia solution is 0.5-2.0 mol / L, the concentration of CTAB is 0.01-0.03 mol / L, and the pH of the ammonia solution is 10-12.

[0037] Preferably, in step (1), BTESePD and TEOS are used together as silicon sources, with a total concentration of 0.05-0.2 mol / L in the solution, and BTESePD accounts for 10-20% of the total molar amount of silicon source.

[0038] Preferably, the heating and stirring temperature in step (1) is 60-80℃.

[0039] Preferably, the centrifugation speed in step (2) is 5000-15000 rpm and the time is 5-20 min.

[0040] Preferably, the washing in step (2) specifically involves washing with deionized water and anhydrous ethanol in sequence, repeating 2-5 times.

[0041] Preferably, the drying temperature in step (2) is 50-70°C and the time is 8-24h.

[0042] Preferably, step (2) involves reflux in a mixed solution of NH4NO3 and ethanol; more preferably, the mass-to-volume ratio of NH4NO3 to ethanol in the NH4NO3-containing ethanol solution is 1% W / V.

[0043] Preferably, the reflux temperature in step (2) is 80-90℃ and the time is 8-24h.

[0044] Preferably, in step (3), the mass ratio of baicalin to diselenylene-bridged mesoporous silica nanoparticles is 1:2-10; more preferably, the mass ratio of baicalin to diselenylene-bridged mesoporous silica nanoparticles is 1:3-7.

[0045] Preferably, the temperature of the stirring reaction in step (3) is 20-30°C and the time is 6-24h; more preferably, the temperature of the stirring reaction is 23-28°C and the time is 12-18h.

[0046] Preferably, the temperature of the vacuum freeze-drying in step (4) is -40 to -70°C; more preferably, the temperature of the vacuum freeze-drying is -45 to -65°C.

[0047] Preferably, the washing in step (4) is specifically performed using PBS.

[0048] Preferably, the repeated freeze-thaw cycle in step (5) is as follows: after standing at 4°C for 20-40 minutes, the freeze-thaw cycle is repeated 2-5 times, consisting of freezing at -80°C for 5-20 minutes and thawing in a water bath at 37°C for 5-20 minutes.

[0049] Preferably, in step (5), differential centrifugation specifically involves centrifuging at 500-1000g for 5-20 minutes to remove cell nuclei and unlysed cells; followed by further centrifugation at 5000-20000g for 10-30 minutes to collect membrane components.

[0050] Preferably, the mass ratio of Ba@Se-MSNs to BV2 cell membrane vesicles in step (6) is 1:0.5-2; more preferably, the mass ratio of Ba@Se-MSNs to BV2 cell membrane vesicles is 1:0.8-1.2.

[0051] The third aspect of the present invention provides the application of the above-mentioned diselenylene-bridged mesoporous silica nanoparticles loaded with baicalin in the preparation of medicaments for treating diseases related to oxidative stress.

[0052] Preferably, the oxidative stress-related disease is selected from spinal cord injury.

[0053] A fourth aspect of the present invention provides a pharmaceutical composition for treating diseases related to oxidative stress, comprising the above-described diselenylene-bridged mesoporous silica nanoparticles loaded with baicalin, and a pharmaceutically acceptable carrier.

[0054] Preferably, the oxidative stress-related disease is selected from spinal cord injury.

[0055] Preferably, the pharmaceutically acceptable carrier is selected from one or more of fillers, disintegrants, lubricants, binders, antioxidants, antibacterial agents, flavoring agents, fragrances, and chelating agents.

[0056] Encapsulating baicalin in diselenylene bond-gated microspheres (MSNs) enables spatiotemporally controlled intralesional release at high ROS-loaded injury sites. To translate this programmable release into consistent in vivo efficacy, further enhancement of retention and tissue penetration at injury sites is necessary through nano-biointerface engineering. The cell membrane encapsulation retains transmembrane receptors and adhesion ligands on the nanoparticles. This self-mimicking immune signature attenuates the clearance effect of mononuclear phagocytes and enhances interactions with the inflamed endothelium and extracellular matrix, thereby promoting accumulation at targeted injury sites. Given the microglial-dominated inflammatory environment after SCI, BV2-derived microglial cell membrane encapsulation is more likely to bind to injury-associated receptors and ligand-receptor interactions, thereby enhancing selective accumulation and deep tissue penetration. This stable intralesional exposure, in turn, supports diselenylene bond-driven controlled release.

[0057] Maintaining mitochondrial homeostasis is a key determinant of neuronal survival and axonal regeneration during the repair phase following spinal cord injury (SCI). CHCHD2, a highly conserved protein located in the mitochondrial intermembrane space, supports cristae integrity and mitochondrial bioenergetics. It is involved in energy metabolism, oxidative stress response, and apoptosis susceptibility, and plays a role in neuroprotection in various neurological disorders. Increasing evidence suggests that CHCHD2 deficiency or pathogenic mutations disrupt cristae structure, reduce mitochondrial membrane potential, and induce persistent reactive oxygen species (ROS) production in mitochondria. These changes exacerbate neuronal apoptosis, axonal degeneration, and neurological dysfunction. Conversely, restoring or enhancing CHCHD2 can protect mitochondrial function and neuronal survival. These findings collectively establish CHCHD2 as a key regulator of mitochondrial homeostasis in the central nervous system. However, the dynamic changes of CHCHD2 in the secondary phase of SCI, its interactions with the oxidative and inflammatory microenvironment, and its role in spinal cord neuronal survival and axonal regeneration remain unclear. Therefore, it is necessary to investigate CHCHD2 as a mechanistic node in ROS-responsive nanotherapy. Elucidating its contribution to mitochondrial homeostasis restoration and post-SCI neural repair will help link nanomaterial-mediated effects with endogenous neuroprotective pathways.

[0058] To address this, this invention designs a diselenylene-bridged MSN nanocarrier, Ba@Se-MSN&BV2, loaded with baicalin and coated with a microglia membrane derived from BV2. The BV2 membrane provides an inflammatory interface, enhancing accumulation at the injury site and local bioavailability. Simultaneously, the diselenylene bond endows it with oxidative stress response and pathologically coupled release properties, thereby promoting the simultaneous inhibition of secondary oxidative stress and neuroinflammatory amplification. Comprehensive multi-scale in vitro and in vivo evaluations confirmed its ability to restore mitochondrial homeostasis, alleviate neuronal damage, and promote axonal regeneration and motor function recovery. Proteomics analysis and perturbation experiments targeting CHCHD2 indicate that its therapeutic benefits are largely attributed to CHCHD2-mediated restoration of mitochondrial homeostasis. This mechanistic link provides a basis for combining ROS-responsive nanotherapy with endogenous neuroprotective circuits, offering a new therapeutic target and research direction for neuroinflammatory injuries driven by redox homeostasis disruption, with significant clinical application prospects and social value. Attached Figure Description

[0059] Figure 1 Scanning electron microscopy and transmission electron microscopy images of Ba@Se-MSN&BV2.

[0060] Figure 2 This is a schematic diagram of the hydrodynamic size distribution and polydispersity index results of Ba@Se-MSN&BV2 under dynamic light scattering analysis.

[0061] Figure 3This is a schematic diagram of the zeta potential measurement results.

[0062] Figure 4 This is a schematic diagram of the ultraviolet-visible absorption spectrum results.

[0063] Figure 5 This is a schematic diagram showing the in vitro cumulative release curve of Ba@Se-MSN&BV2 in PBS.

[0064] Figure 6 This is a schematic diagram showing the results of the ABTS•⁺ free radical scavenging experiment, the DPPH• free radical scavenging experiment, and the ABTS•⁺ kinetic experiment.

[0065] Figure 7 This is a schematic diagram of fluorescence imaging results in mice.

[0066] Figure 8 This is a schematic diagram of the in vitro fluorescence imaging results of the spinal cord and major organs 6 hours after drug administration.

[0067] Figure 9 This diagram illustrates the effects of different treatments on the viability of HT22 cells.

[0068] Figure 10 This is a schematic diagram illustrating the results of flow cytometry analysis of total reactive oxygen species (ROS) and total ROS levels in HT22 cells after different treatments.

[0069] Figure 11 This is a schematic diagram showing the representative DCFH-DA fluorescence analysis and DHE fluorescence analysis results of HT22 cells in each group.

[0070] Figure 12 This is a schematic diagram illustrating the apoptosis results of HT22 cells after different treatments using flow cytometry.

[0071] Figure 13 This is a schematic diagram of representative immunofluorescence images of primary hippocampal neurons treated with various methods after glutamate-induced injury.

[0072] Figure 14 This is a schematic diagram showing the results of quantitative analysis of the total neurite length, total number of branches, primary neurite length, and primary neurite number of primary hippocampal neurons.

[0073] Figure 15 This is a schematic diagram of L-012 chemiluminescence imaging results of reactive oxygen species in the spinal cord injury site after different treatment methods.

[0074] Figure 16 This is a schematic diagram showing the time change curve of BMS score from surgery to 2 months after injury, as well as representative results of hindlimb weight-bearing and standing behavior in mice.

[0075] Figure 17This is a schematic diagram showing representative results of non-weight-bearing swimming behavior in mice two months after surgery.

[0076] Figure 18 This is a schematic diagram showing the curves of joint trajectories and the changes in the angles of major hind limb joints over time during movement.

[0077] Figure 19 This is a schematic diagram showing the results of analysis on the footprints, quantitative step length, and quantitative step width of the movement trajectory of mice after spinal cord injury.

[0078] Figure 20 This is a schematic diagram illustrating the results of the step sequence analysis of the claw print contact sequence and the phase relationship between the left and right limbs.

[0079] Figure 21 This is a schematic diagram of the three-dimensional claw print pressure analysis results for load distribution and propulsion mode.

[0080] Figure 22 A schematic diagram showing the quantitative analysis results of MEP amplitude in the motor cortex, the cephalic side of the injury, and the caudal side of the injury.

[0081] Figure 23 The diagram shows representative MEP waveforms for each part.

[0082] Figure 24 This diagram illustrates the gross specimen of the brain and spinal cord, the location of the injured segment, and the HE staining results of a cross-section of the spinal cord.

[0083] Figure 25 This is a schematic diagram of the immunofluorescence staining results of NFH and GFAP in a cross-section of the spinal cord.

[0084] Figure 26 This is a schematic diagram of the immunofluorescence staining results of NeuN, a neuronal marker, in a cross-section of the spinal cord.

[0085] Figure 27 This is a schematic diagram showing the results of the analysis of CHCHD2 expression in HT22 cells under different treatment conditions.

[0086] Figure 28 This is a schematic diagram showing the results of CHCHD2 expression analysis under damaged background after transfection with si-CHCHD2 and treatment with Ba@Se-MSN&BV2.

[0087] Figure 29 This is a schematic diagram of the flow cytometry analysis results for the distribution of mitochondrial superoxide fluorescence intensity and the proportion of MitoSOX-positive cells.

[0088] Figure 30 A schematic diagram of the results of flow cytometry analysis of JC-1 fluorescence distribution to reflect mitochondrial membrane potential status.

[0089] Figure 31Confocal JC-1 images for subcellular localization of polymer and monomer signals.

[0090] Figure 32 This diagram illustrates the results of Annexin V-FITC / PI flow cytometry analysis of apoptosis distribution, Western blot analysis of Bcl-2 and Bax expression under damage background after transfection with si-CHCHD2 and Ba@Se-MSN&BV2, and quantitative analysis of the Bcl-2 / Bax ratio.

[0091] Figure 33 This is a schematic diagram showing the analysis results of representative immunofluorescence images of primary hippocampal neurons with and without Ba@Se-MSN&BV2 treatment, under conditions of injury combined with si-CHCHD2 treatment, as well as the total number of branches and the length of primary axons in primary hippocampal neurons. Detailed Implementation

[0092] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0093] Unless otherwise specified, all reagents used in this invention context are commercially available. For animal experiments, the procedures and methods complied with medical ethics requirements; C57BL / 6 mice were used throughout the invention, and all mice were housed under SPF conditions (25°C, 50–60% relative humidity) with free access to food and water. To reduce the incidence of urinary retention after spinal cord injury and minimize postoperative care variability, only female mice were included. At the experimental endpoint, mice were deeply anesthetized until pain reflexes disappeared, euthanized via cervical dislocation, and spinal cord tissue was immediately collected for subsequent analysis. The experimental methods used in this invention, such as cell biology experiments, molecular biology experiments, animal experiments, and immunohistochemistry, are all conventional methods and techniques in the art.

[0094] Representative results from biological experiments were selected from replicates and presented in the contextual figures. Data were displayed as mean ± SD and mean ± SEM as specified in the figures. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 9.0 or SPSS 22.0 software. Standard medical statistical methods such as t-tests, chi-square tests, and ANOVA were used to compare differences in means between two or more groups. p < 0.05 was considered statistically significant.

[0095] Example 1

[0096] First, mesoporous silica nanoparticles (Ba@Se-MSN&BV2) loaded with baicalin and bridged by diselenyl bonds were prepared, specifically including the following steps:

[0097] (1) Add BTESePD and TEOS to a solution with CTAB as a template agent and ammonia as an alkaline catalyst, and heat and stir at 60°C until gelation occurs; wherein the effective concentration of NH3 in the ammonia solution is 1.0 mol / L, the concentration of CTAB is 0.02 mol / L, and the pH is 11.0; the total concentration of BTESePD and TEOS as silicon sources is 0.10 mol / L, and BTESePD accounts for 15% of the total molar amount of silicon sources.

[0098] (2) After the reaction was completed, the product was collected by centrifugation at 10,000 rpm for 10 min. The product was then washed with deionized water and anhydrous ethanol three times each. The washed product was dried at 60 °C for 12 h. The dried product was then placed in an ethanol solution containing 1% W / V NH4NO3 and refluxed at 80 °C for 12 h to remove CTAB, thus obtaining diselenylene bond-bridged mesoporous silica nanoparticles (Se-MSNs).

[0099] (3) The Se-MSNs obtained in step (2) are dispersed in the baicalin solution, and after being mixed evenly by ultrasound for a short time, they are stirred in the dark at 25°C for 15 h; wherein the mass ratio of Se-MSNs to baicalin is 5:1.

[0100] (4) After the reaction was completed, the precipitate was collected by centrifugation at 10,000 rpm for 10 min. The precipitate was then washed three times with PBS buffer (pH 7.4) to remove unbound components. The washed product was then freeze-dried under vacuum at −60℃ and vacuum degree ≤10Pa for 24 h to obtain Ba@Se-MSNs.

[0101] (5) The BV2 cell membrane was separated by hypotonic lysis and repeated freeze-thaw cycles. Specifically, the collected BV2 cells were resuspended in hypotonic lysis buffer and allowed to stand at 4°C for 30 min. Then, the cells were frozen at −80°C for 10 min and thawed in a water bath at 37°C for 10 min three times. The membrane components were then collected by differential centrifugation. The cell nuclei and unlyzed cells were removed by centrifugation at 800g for 10 min, and the cell membrane components were collected by centrifugation at 10000g for 20 min. The obtained membrane components were extruded through 400nm and 200nm polycarbonate porous membranes 10 times to obtain BV2 cell membrane vesicles (BV2-CMVs).

[0102] (6) Ba@Se-MSNs and BV2-CMVs were mixed at a mass ratio of 1:1 and incubated on ice for 30 min. Then, they were extruded 10 times through 400 nm and 200 nm polycarbonate porous membranes at 4 °C. The free membrane fragments were removed by centrifugation, and the precipitate was resuspended in PBS to obtain diselenylene-bridged mesoporous silica nanoparticles (Ba@Se-MSN&BV2) loaded with baicalin.

[0103] The morphology and mesostructure of Ba@Se-MSN&BV2 were subsequently detected by SEM and TEM. Dynamic light scattering was used to determine the hydrodynamic diameter and polydispersity index of Ba@Se-MSN&BV2, and the Zeta potentials of Se-MSNs, Ba@Se-MSNs, and Ba@Se-MSN&BV2 were measured using a Zeta potential analyzer. A matrix-matched baicalin calibration curve was established using UV-Vis spectrophotometry, and the drug loading and encapsulation efficiency were calculated accordingly. In vitro release of baicalin was evaluated in PBS at 37°C with gentle shaking. The supernatant was collected at predetermined time points and replaced with an equal volume of fresh PBS. The concentration of baicalin was determined by UV-Vis spectroscopy, and a cumulative release curve was plotted. Oxidation-responsive release mediated by the diselenyl bond backbone was evaluated in PBS with and without 100 μM H2O2 to simulate near-physiological and oxidative conditions, respectively.

[0104] SEM and TEM results showed that Ba@Se-MSN particles were well dispersed, approximately spherical, and possessed regularly arranged mesopores and intact pore walls (see [link to TEM]). Figure 1 Its average diameter is approximately 60 nm, which is advantageous for nanodelivery while maintaining mesoscopic structural integrity and compatibility with tissue permeability in the central nervous system after local administration. Under oxidative stimulation (100 μM H₂O₂, 2 h), its backbone undergoes diselenide bond breakage, accompanied by pore wall collapse and fragmentation. These changes are consistent with the oxidative remodeling of the diselenide bond-bridged backbone and ROS-responsive release behavior, indicating that this carrier can couple drug release with the characteristic oxidative signaling of the secondary phase of SCI. After membrane coating, Ba@Se-MSN&BV₂ exhibits a unimodal DLS distribution with a hydrodynamic diameter of 72.0 ± 0.7 nm and a PDI of 0.16, indicating that it is a monodisperse colloid (see [link to relevant documentation]). Figure 2 Compared to Ba@Se-MSN, a slight increase in particle size was observed, but no peak broadening or aggregation was observed, indicating that the membrane coating maintained colloidal stability. Its Zeta potential shifted towards the potential direction of the native BV2 membrane, and the absolute value was larger (see [link to relevant documentation]). Figure 3 This indicates that its surface has transformed from a silica surface rich in silanol groups to a biomimetic interface dominated by membrane proteins and phospholipids, which helps reduce nonspecific protein adsorption and facilitates damage-related interactions in vivo.

[0105] UV-Vis quantitative analysis using matrix-matched calibration curves showed that the encapsulation efficiency (EE) of Ba@Se-MSN was 93.47%, and the drug loading (LC) was 11.12%. After membrane coating, the EE remained at a high level (91.82%), while the LC decreased slightly to 8.85%, and no baicalin leakage was detected. Coomassie brilliant blue stained SDS-PAGE results showed that Ba@Se-MSN&BV2 had a highly similar protein spectrum to the natural BV2 membrane and retained key membrane proteins, indicating the successful construction of the biomimetic interface, consistent with the observed Zeta potential shift. UV-Vis spectral analysis showed that Ba@Se-MSN&BV2 retained the characteristic peaks of baicalin at 280 nm and 316 nm, without shift or the appearance of new bands (see [link to relevant documentation]). Figure 4 This indicates that encapsulation and membrane coating did not interfere with the spectral characteristics of the drug. Under simulated physiological conditions, in vitro release was slow, but release was significantly accelerated in the presence of 100 μM H2O2, resulting in a higher cumulative release (see [link to relevant documentation]). Figure 5 The curve showing accelerated ROS release corresponds to the oxidative fragmentation of the carrier, indicating that diselenyl bond breaking can loosen the mesostructure, promote pore opening, and thus enhance baicalin diffusion. Compared to simple passive diffusion-driven release, this ROS-triggered release holds promise for better synchronization between local drug availability and the pathological ROS surge in ROS-rich SCI lesions.

[0106] Example 2

[0107] Based on the structure and release behavior of Ba@Se-MSN&BV2 clarified in Example 1, the in vitro antioxidant capacity and in vivo accumulation at the spinal cord injury site of Ba@Se-MSN&BV2 were systematically evaluated. Specifically, under the same solvent system and baicalin dosage, the free radical scavenging activity at a series of mass concentrations was evaluated to directly compare the in vitro scavenging abilities of baicalin, Ba@Se-MSNs, and Ba@Se-MSN&BV2. The sample was mixed with the free radical working solution at a 1:1 volume ratio and incubated at room temperature in the dark. The absorbance was recorded at preset time points within 0-120 min to monitor the scavenging kinetics. For the ABTS experiment, 7 mM ABTS was mixed with 2.45 mM potassium persulfate and reacted for 12-16 h to generate ABTS⁺· to prepare a stock solution. The stock solution was diluted with the appropriate solvent to obtain the working solution, which was mixed with an equal volume of sample, and the absorbance at 734 nm was measured. For the DPPH experiment, a 0.10 mM DPPH ethanol solution was mixed with an equal volume of sample, incubated at room temperature in the dark, and the absorbance at 517 nm was measured.

[0108] The results showed that baicalin, Ba@Se-MSN, and Ba@Se-MSN&BV2 all exhibited dose-dependent free radical scavenging activity in ABTS and DPPH free radical scavenging experiments. At the same concentration and incubation time, Ba@Se-MSN&BV2 showed higher scavenging efficiency and a steeper dose-response curve (see [link to relevant documentation]). Figure 6 This is consistent with the preservation of antioxidant function and the effects of nanoscale carriers and interface engineering. This enhancement was observed at equivalent doses, suggesting that packaging baicalin in a ROS-responsive, high-surface-area carrier may improve the effective utilization of its free radical scavenging ability.

[0109] To translate the in vitro antioxidant benefits into in vivo accessibility to injury sites, coumarin-6 (C6) was used as a fluorescent tracer to assess its in vivo distribution and accumulation at spinal cord injury sites. Specifically, under the same baicalin equivalent dosage conditions as in the treatment experiments, C6 was loaded into the formulation at 1% of the baicalin mass. After 12 hours of gentle stirring in the dark, the particles were collected by centrifugation, repeatedly washed with PBS until no fluorescence was detected in the supernatant to remove free dye, and finally resuspended in PBS to prepare C6-Ba@Se-MSNs and C6-Ba@Se-MSN&BV2. SCI mice were randomly assigned to the treatment groups and injected with the corresponding formulation via the tail vein under isoflurane anesthesia. Six hours after injection, the spinal cord and major organs were removed, and in vitro fluorescence imaging was performed using an AniView 600 system.

[0110] Experimental results showed that after tail vein injection of an equivalent dose of the C6-labeled formulation, in vivo imaging revealed preferential accumulation of fluorescence at the site of the damaged spinal cord. Although Ba@Se-MSN-C6 showed significant enrichment, Ba@Se-MSN&BV2-C6 produced a stronger and more spatially confined signal (see [link to study]). Figure 7 This indicates that BV2 membrane encapsulation enhances the ability to target and persist at the injury site. This pattern is consistent with the inflammatory homing phenomenon reported in other biomimetic carriers derived from immune cells, suggesting that microglia can be used to guide spinal cord injury localization. Ex vivo imaging further confirmed the strong accumulation of Ba@Se-MSN & BV2-C6 at the injury site, showing predominantly liver and kidney signals (consistent with metabolic clearance pathways), with no significant off-target accumulation observed in other organs (see [link to relevant documentation]). Figure 8 These results collectively demonstrate that BV2 membrane coating enhances accumulation and retention at the injury site. Combined with a ROS-responsive diselenyl bond-gated carrier, this design preserves and enhances the antioxidant properties of baicalin, ensuring that the payload is not only highly effective in vitro but also preferentially delivered and retained at ROS-rich SCI injury sites.

[0111] Subsequently, glutamate-induced mouse hippocampal HT22 cells were used as an excitotoxicity model to evaluate the antioxidant properties of Ba@Se-MSN&BV2 under neuronal oxidative stress. Specifically, HT22 cells or primary neurons were seeded in 96-well plates and, after stable adhesion, were exposed to different concentrations of baicalin, Ba@Se-MSNs, or Ba@Se-MSN&BV2 for 24 h. The medium was then replaced with fresh medium containing 10% CCK-8 working solution and incubated for another 1-2 h. Absorbance was measured at 450 nm using a microplate reader, and relative cell viability was calculated by comparing with the untreated control group. The results showed that at concentrations up to 25 μg·mL⁻¹, baicalin, Ba@Se-MSN, or Ba@Se-MSN&BV2 did not exhibit significant cytotoxicity. Therefore, a concentration of 25 μg·mL⁻¹ was selected for subsequent experiments (see [link to relevant documentation]). Figure 9 ).

[0112] At this dose, intracellular total ROS and superoxide levels were assessed using DCFH-DA and DHE fluorescent probes, respectively. The antioxidant effects of baicalin, Ba@Se-MSNs, and Ba@Se-MSN&BV2 were evaluated using flow cytometry and confocal microscopy. Specifically, after HT22 cells adhered, they were treated with glutamate (120 mM) for 12 h to induce oxidative stress, followed by treatment with equal doses of baicalin, Ba@Se-MSNs, or Ba@Se-MSN&BV2 for 24 h. After treatment, cells were incubated at 37°C in the dark for 20-30 min with working solutions of DCFH-DA (10 μM) or DHE (5 μM) prepared in serum-free, phenol red-free medium, washed three times with PBS, and then resuspended or covered with phenol red-free complete medium. Mean fluorescence intensity was quantified by flow cytometry, and representative images were acquired by confocal microscopy to provide spatial localization and qualitative visualization of ROS and superoxide signals. Flow cytometry results showed a rightward shift in fluorescence signal and an increase in mean fluorescence intensity (MFI) in the damaged group, consistent with significant ROS accumulation. All treatments attenuated ROS signals, with Ba@Se-MSN & BV2 most significantly normalizing them, approaching control levels (see [link to treatment]). Figure 10 Confocal microscopy confirmed the results of flow cytometry. DCFH-DA fluorescence significantly increased after injury; baicalin or Ba@Se-MSN only partially reduced fluorescence intensity, while Ba@Se-MSN & BV2 produced the greatest reduction. DHE fluorescence showed the same trend (see [link to relevant documentation]). Figure 11 This further demonstrates that the Ba@Se-MSN&BV2 biomimetic nanoplatform has a superior inhibitory effect on superoxide.

[0113] To evaluate the anti-apoptotic effect of Ba@Se-MSN&BV2 under oxidative damage, Annexin V / PI double staining and flow cytometry analysis were performed. Specifically, after HT22 cells adhered, they were treated with glutamate (120 mM) for 12 h to induce oxidative stress, and then treated with baicalin, Ba@Se-MSN, or Ba@Se-MSN&BV2 at the same dose as baicalin for 24 h. After treatment, the culture supernatant and trypsin-digested adherent cells were collected, washed with PBS, centrifuged, and resuspended in binding buffer. Annexin V-FITC and PI were added, and the cells were incubated at room temperature in the dark for 10-15 min. Binding buffer was added to the total volume, and flow cytometry analysis was performed immediately; the apoptosis rate was calculated using Annexin V / PI quadrant gating. The results showed that the proportion of early and late apoptotic cells was significantly increased in the damage group, while all treatments reduced the total apoptosis rate. Among them, Ba@Se-MSN&BV2 produced the lowest proportion of apoptotic cells in the treatment group (see [link to relevant documentation]). Figure 12 These results are consistent with the view that reduced ROS at the same dosage contributes to improved cell viability. In conclusion, in HT22 cells, Ba@Se-MSN & BV2 were more effective than free baicalin and Ba@Se-MSN in reducing total ROS and superoxide levels and inhibiting glutamate-induced apoptosis.

[0114] To assess the effects of Ba@Se-MSN & BV2 on neurite remodeling in primary hippocampal neurons, given the reduction of oxidative stress and the shift of microglia to a repair phenotype, we examined its influence. Specifically, primary hippocampal neurons were obtained from 1-day-old SD rats, mildly digested with papain, and seeded in poly-L-lysine-coated culture dishes. Cells were cultured for 48 hours to achieve stable adhesion and neurite growth. Oxidative damage was induced with glutamate, followed by co-culturing with equal doses of baicalin, Ba@Se-MSN, or Ba@Se-MSN & BV2 for 24 hours. Neurons were then subjected to Tuj1 immunofluorescence staining. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 5% BSA, incubated overnight at 4°C with anti-β-III-tubulin primary antibody (1:1000), and then incubated at room temperature in the dark with fluorescent secondary antibody (1:1000). After DAPI counterstaining of cell nuclei, images were acquired under the same parameters. ImageJ software was used to quantify neurite length, total number of branches, and related morphological parameters to compare the neuroprotective effects of different treatments. Results showed that at a concentration of 12.5 μg·mL⁻¹, all groups (Ba@Se-MSN&BV2, Ba@Se-MSN, and baicalin) showed no significant cytotoxicity; therefore, this concentration was used for subsequent experiments. Representative images showed that neurites were significantly shortened and branches were reduced after injury. All treatments partially salvaged these defects, with Ba@Se-MSN&BV2 showing the most significant recovery of neurite structure in representative fields (see [link to image]). Figure 13 ) Morphometric quantitative analysis results showed that, compared with the lesion group, Ba@Se-MSN&BV2 significantly increased total neurite length, branching complexity, and the length and number of primary neurites (see Figure 14 Scratch-based neurite growth experiments yielded consistent results, indicating significantly impaired gap coverage after injury. All treatments improved both coverage and closure, with Ba@Se-MSN & BV2 achieving the greatest recovery in neurite growth across the gap. Combining morphometric and scratch-based experimental results, at the same dosage, Ba@Se-MSN & BV2 was significantly more effective than free baicalin or Ba@Se-MSN in promoting neurite regeneration and network reconstruction after injury, thus linking redox regulation to the structural repair of damaged neurons.

[0115] Example 3

[0116] Based on in vitro antioxidant and neuroprotective studies, a mouse spinal cord injury model was used to assess in vivo ROS control. The mouse spinal cord injury model was constructed as follows: Female C57BL / 6 mice were randomly divided into a normal group, a sham-operated group, an injury group, a baicalin group, a Ba@Se-MSN group, and a Ba@Se-MSN&BV2 group. After deep anesthesia with 3% tribromoethanol via intraperitoneal injection, laminectomy was performed at the T8-T10 segments to expose the spinal cord. Standardized injuries were created at the T9 segment using a spinal cord impactor. The sham-operated group underwent only laminectomy without impaction. Postoperatively, animals received routine warming, analgesia, and prophylactic antibiotics, and daily assisted urination was provided until spontaneous urination function recovered. In vivo imaging using L-012 chemiluminescence immunoassay was then performed to assess oxidative stress levels at the spinal cord injury site; specifically, mice were administered the drug via tail vein for 3 consecutive days. On day 3, freshly prepared L-012 solution was injected intraperitoneally. After incubation for 30 minutes under isoflurane anesthesia and normal body temperature, chemiluminescent signals of the damaged segments were acquired using an animal in vivo imaging system in bioluminescent mode.

[0117] The results showed that normal and sham-operated mice exhibited only basal levels of luminescence, while damaged mice showed a significant increase in luminescence signal, indicating the presence of an acute oxidative burst. All treatments reduced L-012 signal intensity, with Ba@Se-MSN&BV2 showing the most significant attenuation effect, demonstrating the strongest in vivo ROS inhibition at the same dose (see [link to treatment]). Figure 15 This result is consistent with BV2 membrane-mediated damage targeting and ROS-responsive baicalin release via diselenyl bond gating, both of which may amplify the local antioxidant effect during the acute oxidative-neuroinflammatory phase of SCI.

[0118] Given the early in vivo inhibitory activity of Ba@Se-MSN & BV2 against ROS, we next evaluated long-term motor function recovery in the same spinal cord injury model. Specifically, mice were randomly divided into six groups and assessed at specific time points after spinal cord injury. Overall hind limb function was assessed using the Bass Mouse Score and the Louisville Swimming Scale. For the BMS score, mice were observed in an open field for 4 minutes, scored independently by two trained researchers unaware of the group assignments, and the average was reported. For the LSS score, mice swam freely in a transparent tank, and scoring was also done blinded. Two months post-injury, a detailed gait kinematic analysis was performed. Mice walked at a constant speed on a treadmill while being videotaped from the side by a high-speed camera, with markers placed at the hip, knee, and ankle joints. Gait phases, left-right coordination, and dynamic parameters were extracted from the joint trajectory and angle time series. In the inclined plane test, the inclination angle was gradually increased, and the maximum angle at which the mouse could maintain its position without slipping was determined; the average of repeated trials was calculated. In the grid walking task, each mouse completed at least three effective walks, and the hind limb and overall foot error rates were calculated. In footprint analysis, the hind paws of mice were dipped in a non-toxic dye, and the mice were allowed to walk along a closed straight path. Stride length, stride width, and left-right symmetry were quantified from the footprints. Under constant illumination and a uniform detection threshold, CatWalk automated gait analysis was performed to extract parameters such as paw print contact area, stance phase duration, weight-bearing intensity, and gait regularity, comprehensively evaluating coordination and gait recovery.

[0119] Two-way repeated measures ANOVA of Basso mouse scores during the 2-month observation period showed significant main effects in both time and treatment groups, with scores gradually improving over time and stabilizing in the later stages. Throughout the observation period, the Ba@Se-MSN&BV2 group showed the most significant improvement in BMS scores, the Ba@Se-MSN group showed moderate improvement, while the injury group and the free baicalin group showed only limited recovery (see [link to relevant documentation]). Figure 16 Under non-weight-bearing conditions, the Louisville Swimming Scales further showed that the Ba@Se-MSN&BV2 group scored highest in hindlimb range of motion, alternating rhythm, and postural balance (see [link to data]). Figure 17 This indicates that its hindlimb drive and posture control capabilities are enhanced.

[0120] To perform higher-resolution kinematic analysis, gait parameters were analyzed using high-speed lateral imaging and joint tracking. Results showed that both the injury group and the free baicalin group exhibited significantly reduced range of motion in the hip, knee, and ankle joints, prolonged gait cycles, and disordered inter-joint phase relationships. Ba@Se-MSN and Ba@Se-MSN&BV2 increased joint range of motion and partially restored gait timing and coordination, with Ba@Se-MSN&BV2 showing the closest recovery to the control group (see [link to relevant documentation]). Figure 18An analysis integrating multiple kinematic indicators, including ankle, knee, and hip joint range of motion, toe and sacral height, and mean speed, revealed that Ba@Se-MSN&BV2 scored highest across all key movement dimensions, consistent with the results of the behavioral scales.

[0121] Two months post-injury, footprint analysis showed shortened stride and increased stride width in both the injury group and the free baicalin group, indicating disordered walking rhythm and impaired weight-bearing stability. Ba@Se-MSN partially improved these two parameters but did not reach the levels of the control group, while Ba@Se-MSN & BV2 showed the best performance in stride length recovery and essentially restored stride width to normal (see [link to relevant documentation]). Figure 19 Walking on a metal grid and on an inclined plane showed similar improvement. The injured group and the free baicalin group had higher foot error rates and lower maximum grip angles. Ba@Se-MSN could partially recover, while the Ba@Se-MSN&BV2 group had the lowest foot error rate and recovered the grip angle to near normal values, indicating improvements in both coordination and balance.

[0122] CatWalk analysis was performed at the end of the 2-month trial to quantitatively assess walking quality and load-bearing capacity. Gait sequencing showed that the gait in the injury group was irregular and discontinuous, the Ba@Se-MSN group showed partial reorganization, while the Ba@Se-MSN&BV2 group exhibited a near-normal, symmetrical, alternating left-right gait (see [link to study]). Figure 20 Three-dimensional paw print contact thermograms and quantitative parameter analysis showed that Ba@Se-MSN&BV2 reconstructed a typical biphasic contact pattern, achieved the highest proportion of normal gait, and showed the most significant recovery in ipsilateral contact intensity and walking speed, while the free baicalin group showed little effect (see [link to study]). Figure 21 In summary, among the various behavioral and gait parameters, Ba@Se-MSN&BV2 was most effective at correcting gait geometry, restoring weight-bearing capacity, and reconstructing rhythmic coordination at the same dose. These functional benefits are consistent with sustained intralesional effects achieved through accumulation at the lesion site and ROS-responsive baicalin release, providing a basis for further histological and mechanistic investigations.

[0123] To examine whether behavioral recovery was accompanied by recovery of cortical-spinal conduction, motor evoked potentials (MEPs) were performed at three sites at the 2-month endpoint of the experiment to assess cortical output and translesion conduction. The results are as follows: Figure 22 As shown. The results showed that transcranial stimulation induced cortical MEP, and spinal cord MEP was recorded simultaneously on both the cephalic and caudal sides of the lesion segment. The cortical output MEP amplitude was significantly reduced in the lesion group and the free baicalin group, partially recovered in the Ba@Se-MSN group, and showed the most significant recovery in the Ba@Se-MSN&BV2 group (see...). Figure 22(a) indicates that Ba@Se-MSN&BV2 can more significantly restore cortical spinal cord output function. MEP recorded on the cephalic side of the injury showed no difference between groups (see [link]). Figure 22 (b) indicates that conduction was preserved proximal to the lesion. A key difference occurred approximately 5 mm caudally to the lesion. Responses were almost undetectable in the lesion group and the free baicalin group, moderate in the Ba@Se-MSN group, and significantly enhanced in the Ba@Se-MSN&BV2 group, approaching the level of the sham-operated group (see [link to relevant documentation]). Figure 22 (c) Representative waveforms show that the Ba@Se-MSN&BV2 group showed the greatest amplitude recovery and a trend towards shorter latency, while the Ba@Se-MSN group showed less improvement, and the damaged group and the free baicalin group showed weak or absent signals (see [link]). Figure 23 Electrophysiological recovery patterns in the cortex, cephalic, and caudal regions were consistent with improvements in footprinting, CatWalk, and balance tests, further confirming the association between Ba@Se-MSN&BV2-mediated behavioral benefits and partial corticospinal pathway reconnection and enhanced translesion conduction.

[0124] To corroborate the tissue-level repair underlying the sustained behavioral and electrophysiological improvements, histological analysis was performed at the 2-month endpoint of the experiment. Gross observation and H&E staining revealed that the injury control group exhibited significant spinal cord thinning, discontinuity, and large cavity formation, accompanied by parenchymal collapse and thickened lesion margins; while the Ba@Se-MSN&BV2 group produced the smallest cavities, better preserved spinal cord continuity, and had smoother, more continuous margins, suggesting more complete parenchymal filling (see [link to study]). Figure 24 Co-staining with GFAP / NFH revealed that the injury control group formed a dense GFAP⁺ stellate glial scar ring, accompanied by fragmented and disordered NFH⁺ axons; the free baicalin group showed minimal changes; the Ba@Se-MSN group partially reduced glial scar formation and improved NFH⁺ alignment; most notably, the Ba@Se-MSN&BV2 group reduced GFAP⁺ scarring and formed continuous NFH⁺ axonal bundles traversing the injury area, aligned with the surrounding white matter bundles (see [link to relevant documentation]). Figure 25 This is consistent with the recovery of the caudal MEP and suggests enhanced translesion connectivity. NeuN staining revealed parallel gradient changes in neuronal preservation: NeuN⁺ neurons around the lesion were sparse and discontinuous in the lesion control group, the rescue effect of free baicalin was minimal, the density and continuity were increased in the Ba@Se-MSN group, and the distribution density of NeuN⁺ neurons around the lesion was the highest and most continuous in the Ba@Se-MSN&BV2 group (see [link to relevant documentation]). Figure 26Systemic histopathological examination revealed no significant lesions in major organs (including the heart, liver, spleen, lungs, kidneys, and brain), indicating that Ba@Se-MSN&BV2 has good biocompatibility. These results collectively suggest that Ba@Se-MSN&BV2 can reduce cavity formation and stellate scarring, promote trans-injury axonal continuity, and protect neurons, thus providing an anatomical basis for sustained pathway integrity and long-term functional recovery.

[0125] Example 4

[0126] The aforementioned experiments demonstrated that Ba@Se-MSN&BV2 can alleviate oxidative stress and promote the recovery of neuronal structure and function. To elucidate the underlying molecular basis of these effects, particularly focusing on oxidative stress-related signaling pathways influencing the pathogenesis of secondary SCI, label-free quantitative proteomics analysis was performed in glutamate-damaged HT22 cells. After normalization and batch correction, the sample distribution was highly consistent, allowing for reliable differential analysis. A treatment reversal strategy was employed to screen differentially expressed proteins, selecting those downregulated due to injury but rescued by Ba@Se-MSN&BV2, or those upregulated due to injury but downregulated by treatment. This resulted in two sets of reversed proteins: those reversed upward under the action of Ba@Se-MSN&BV2 (364 proteins) and those reversed downward (297 proteins). Given the central role of redox imbalance in SCI, further investigation was conducted on proteins related to oxidative stress and metabolism. The results showed that most redox and metabolic-related proteins disturbed by glutamate tended to return to baseline levels after Ba@Se-MSN&BV2 treatment, suggesting a partial restoration of the redox-metabolic program. Enrichment analysis revealed CHCHD2 as a key target, linked to oxidative stress, hypoxia signaling, and mitochondrial homeostasis pathways. Considering the important role of mitochondrial dysfunction in secondary SCI, these results establish CHCHD2 as a mitochondrial-related candidate mediator downstream of Ba@Se-MSN&BV2.

[0127] Western blot experiments in HT22 cells validated the proteomics findings. CHCHD2 expression was abundant under normal conditions, significantly decreased after glutamate injury, and Ba@Se-MSN&BV2 restored it to near baseline levels (see [link to study]). Figure 27This aligns with trends in proteomics. Subsequently, a si-CHCHD2 knockdown model was established (sense strand sequence as shown in SEQ ID NO: 1, 5'-AATGTGGACCCTTATATT-3', antisense strand sequence as shown in SEQ ID NO: 2, 5'-AATATAAGGGTCCACACTT-3') to significantly reduce CHCHD2 expression. Under CHCHD2 silencing conditions, Ba@Se-MSN&BV2 partially restored CHCHD2 levels (see...). Figure 28 Immunofluorescence further confirmed that CHCHD2 signaling was weakened under damage and the influence of si-CHCHD2, but was significantly restored by Ba@Se-MSN&BV2. These results are consistent with the conclusion that Ba@Se-MSN&BV2 upregulates CHCHD2, thereby helping to restore mitochondrial homeostasis under glutamate-induced stress and CHCHD2 inhibition.

[0128] To investigate how CHCHD2 deficiency and its regulation by Ba@Se-MSN&BV2 affect mitochondrial homeostasis and neuronal integrity, flow cytometry was used to quantify mitochondrial ROS and total ROS using MitoSOX (mitochondrial superoxide) and DCFH-DA (total ROS). CHCHD2 silencing significantly increased mitochondrial superoxide levels, while Ba@Se-MSN&BV2 mitigated this increase (see [link to relevant documentation]). Figure 29 DCFH-DA fluorescence showed similar results; CHCHD2 knockdown increased total ROS, while Ba@Se-MSN&BV2 partially reversed this increase. Dual-channel confocal imaging confirmed that Ba@Se-MSN&BV2 treatment simultaneously reduced mitochondrial ROS and total ROS under CHCHD2 silencing conditions. Mitochondrial functional status was assessed using JC-1. CHCHD2 knockdown further reduced mitochondrial membrane potential, while Ba@Se-MSN&BV2 partially restored JC-1 aggregate formation, consistent with improved ΔΨm (see [link to study]). Figure 30 Confocal JC-1 imaging showed the same trend: CHCHD2 silencing exacerbated the change in the red / green fluorescence ratio, while Ba@Se-MSN&BV2 mitigated this phenomenon (see [link to image]). Figure 31 Apoptosis assays showed that CHCHD2 knockdown increased the total apoptosis rate, while Ba@Se-MSN&BV2 significantly reduced apoptotic cells and decreased the Bax / Bcl-2 ratio (see [link to study]). Figure 32 In primary hippocampal neurons, CHCHD2 silencing further impairs neurite growth, while Ba@Se-MSN&BV2 salvages neurite length and branching complexity under CHCHD2-deficient conditions (see [link to original text]). Figure 33Overall, CHCHD2 knockdown exacerbated damage-related mitochondrial ROS accumulation, mitochondrial depolarization, apoptosis susceptibility, and neurite degeneration, while Ba@Se-MSN&BV2 restored these abnormalities to normal and improved neuronal structural integrity, indicating that the neuroprotective effect of Ba@Se-MSN&BV2 depends at least in part on CHCHD2-mediated mitochondrial homeostasis regulation.

[0129] This invention constructs a ROS-responsive, microglia-inspired Ba@Se-MSN&BV2 nanoplatform for the secondary spinal cord injury period. The platform combines BV2 cell membrane-mediated enrichment at the injury site with diselenyl bond-triggered, on-demand release of baicalin, enabling targeted intervention in a highly oxidative and pro-inflammatory microenvironment. In vitro and in vivo studies show that Ba@Se-MSN&BV2 can alleviate oxidative stress and mitochondrial dysfunction, inhibit apoptosis, promote axonal and neuronal survival, and improve motor circuit function. Label-free quantitative proteomics combined with si-CHCHD2 knockdown and rescue experiments further demonstrate that the neuroprotective efficacy of Ba@Se-MSN&BV2 is largely related to the restoration of mitochondrial homeostasis mediated by CHCHD2, thereby helping to translate the reduction of oxidative load into improved neuronal structure and function. In summary, these findings highlight a microenvironment-responsive, mitochondrial-targeting nanotherapy strategy for spinal cord injury, and suggest that mitochondrial stabilization centered on CHCHD2 provides a new therapeutic target and research direction for neuroinflammatory injuries driven by redox homeostasis disruption, with significant clinical application prospects and social value.

[0130] The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. A mesoporous silica nanoparticle supported on baicalin and bridged by diselenyl bonds, characterized in that, It is prepared by the following method: (1) Add BTESePD and TEOS to an ammonia solution containing CTAB, and heat and stir the reaction until the system gels; (2) After the reaction was completed, the product was collected by centrifugation, and then washed and dried. The dried product was then refluxed to remove CTAB, and diselenylene bond-bridged mesoporous silica nanoparticles were obtained. (3) Disperse the diselenide-bridged mesoporous silica nanoparticles obtained in step (2) in baicalin solution, mix them evenly by ultrasonication, and then carry out a light-protected stirring reaction. (4) After the reaction was completed, the precipitate was centrifuged and washed to remove unbound components, and then freeze-dried under vacuum to obtain Ba@Se-MSNs; (5) Resuspend BV2 cells in hypotonic lysis buffer and perform repeated freeze-thaw cycles to separate the BV2 cell membrane; then collect the membrane components by differential centrifugation; extrude the obtained membrane components through 400nm and 200nm polycarbonate porous membranes 5-20 times to obtain BV2 cell membrane vesicles. (6) After mixing Ba@Se-MSNs with BV2 cell membrane vesicles, incubate on ice for 20-40 min; then extrude through 400 nm and 200 nm polycarbonate porous membranes 5-20 times at 4 °C; remove free membrane fragments by centrifugation, and resuspend the precipitate in PBS to obtain diselenylene bond-bridged mesoporous silica nanoparticles loaded with baicalin.

2. The mesoporous silica nanoparticles supported on baicalin and bridged by diselenyl bonds according to claim 1, characterized in that, The heating and stirring temperature in step (1) is 60-80℃.

3. The mesoporous silica nanoparticles supported on baicalin and bridged by diselenyl bonds according to claim 1, characterized in that, Step (2) involves refluxing the mixture in an ethanol solution containing NH4NO3.

4. The mesoporous silica nanoparticles supported on baicalin and bridged by diselenyl bonds according to claim 1, characterized in that, In step (3), the mass ratio of baicalin to diselenylene-bridged mesoporous silica nanoparticles is 1:2-10.

5. The mesoporous silica nanoparticles supported on baicalin and bridged by diselenyl bonds according to claim 1, characterized in that, The repeated freeze-thaw cycle in step (5) is as follows: after standing at 4℃ for 20-40 minutes, repeat the freeze-thaw cycle of freezing at -80℃ for 5-20 minutes and thawing in a water bath at 37℃ for 5-20 minutes 2-5 times.

6. The diselenylene-bridged mesoporous silica nanoparticles loaded with baicalin according to claim 1, characterized in that, In step (5), differential centrifugation specifically involves centrifuging at 500-1000g for 5-20 minutes to remove cell nuclei and unlysed cells; followed by further centrifugation at 5000-20000g for 10-30 minutes to collect membrane components.

7. The diselenylene-bridged mesoporous silica nanoparticles loaded with baicalin according to claim 1, characterized in that, The mass ratio of Ba@Se-MSNs to BV2 cell membrane vesicles in step (6) is 1:0.5-2.

8. The use of diselenylene-bridged mesoporous silica nanoparticles loaded with baicalin according to any one of claims 1-7 in the preparation of medicaments for treating diseases related to oxidative stress.

9. A pharmaceutical composition for treating diseases related to oxidative stress, characterized in that, This includes diselenylene-bridged mesoporous silica nanoparticles loaded with baicalin according to any one of claims 1-7, and pharmaceutically acceptable carriers.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutically acceptable carrier is selected from one or more of the following: fillers, disintegrants, lubricants, binders, antioxidants, antibacterial agents, flavoring agents, fragrances, and chelating agents.

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