Neurophilic virus protein-loaded selenium-containing cationic mesoporous silica nano-material, preparation method thereof and application of nano-material in drugs for treating multiple sclerosis

Abstract

The invention discloses a neurophilic virus protein-loaded selenium-containing cationic mesoporous silica nano-material, a preparation method thereof and application thereof in preparation of drugs for treating multiple sclerosis, a nano-carrier is selenium atom-containing MSN, polyethyleneimine and polyethylene glycol are used as raw materials, and a neurophilic virus protein rabies virus glycoprotein RVG29 is loaded, so that the selenium-containing cationic mesoporous silica nano-material is prepared. And constructing the nanoparticles for removing the cfDNA. Compared with traditional drugs for treating multiple sclerosis, the cationic mesoporous silica nano-material loaded with neurophilic virus protein provided by the invention is more efficient, safer and controllable when being used for anti-inflammatory treatment of multiple sclerosis, and provides a new thought and a new material for efficient intervention of clinical multiple sclerosis; the cationic mesoporous silica nano material loaded with the neurophilic virus protein can be targeted to a central nervous system to play an anti-inflammatory role, has a good antibacterial role, also has a good blood-brain barrier penetration property, and is very suitable for being applied to multiple sclerosis treatment.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine carrier materials technology, and in particular to a selenium-containing cationic mesoporous silica nanomaterial loaded with neurotropic viral proteins, its preparation method and application. Background Technology

[0002] Multiple sclerosis (MS) is an immune-mediated inflammatory demyelinating disease of the central nervous system, characterized by its temporal and spatial dissemination. MS typically affects individuals aged 29-39, with a higher prevalence in women (male-to-female ratio of 1:1.5-1:2). In recent years, the incidence and prevalence of MS have shown a gradual increasing trend. The overall incidence of MS in China is 0.235 per 100,000 people-years, with a male-to-female ratio of 1:2.02 among adults.

[0003] Currently, treatments for multiple sclerosis mainly include acute phase therapy and remission phase therapy, including glucocorticoids, immunoglobulin therapy, plasma exchange, and disease-correcting therapy. Although there are many treatment options, no single treatment material or method has been found that offers highly effective and safe anti-inflammatory results.

[0004] Therefore, exploring a more efficient and safer combined anti-inflammatory treatment strategy for multiple sclerosis (MS) is particularly important. Blood-brain barrier disruption is a key factor in the pathogenesis of MS. Inflammatory factors can increase blood-brain barrier permeability, allowing immune cells to enter the central nervous system and trigger inflammation and demyelination. However, the blood-brain barrier also restricts the entry of most drugs into brain tissue, becoming a major obstacle in the treatment of central nervous system diseases. To address the problem of blood-brain barrier permeability limiting drug entry into the central nervous system, research on neuropeptide carriers has emerged as a new direction in the treatment of central nervous system diseases in recent years. Among them, the rabies virus glycoprotein-derived peptide RVG29 has shown good brain-targeting ability and has demonstrated good therapeutic potential in diseases such as Alzheimer's disease, Parkinson's disease, and malignant gliomas.

[0005] Therefore, applying RVG29-mediated drug delivery systems to the anti-inflammatory treatment of multiple sclerosis, combined with traditional or novel immunomodulatory drugs, may enable efficient and precise crossing of the blood-brain barrier and targeting of central lesions, thereby improving treatment efficacy and reducing systemic toxicity and providing a new breakthrough direction for current treatment strategies for multiple sclerosis. Summary of the Invention

[0006] This invention addresses the problem of the lack of efficient, safe and controllable treatment drugs for multiple sclerosis by proposing a cationic mesoporous silica nanomaterial loaded with neurotropic viral proteins, its preparation method and application.

[0007] This cationic mesoporous silica nanomaterial loaded with neurotropic viral proteins comprises the following structural elements:

[0008] (a) Selenium-containing mesoporous silica core: a hexagonal ordered mesoporous structure with a particle size of 50–70 nm and a Zeta potential of +15 to +30 mV (measured under pH 7.0 conditions), and selenium is distributed on the surface of the pores in the form of silane bonds;

[0009] (b) Epoxidized-polyethyleneimine composite layer: covalently modified on the core surface, comprising a cationic polymer network of epoxysilane crosslinked polyethyleneimine, wherein the number average molecular weight of polyethyleneimine is 500–10000;

[0010] (c) Polyethylene glycol hydrophilic layer: grafted by ring-opening reaction of amino groups with epoxy groups of composite layer. Polyethylene glycol is amino-terminated and has a molecular weight of 200–2000.

[0011] (d) Neurotropic virus protein ligand: coupled to the end of the polyethylene glycol layer via condensation agent-mediated amide bond, the protein being rabies virus glycoprotein polypeptide RVG29 or its functional fragment.

[0012] Furthermore, the condensing agent is a combination of N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0013] Furthermore, the surface charge of the nanomaterial is positive at physiological pH, with a Zeta potential ≥ +15mV, and it has the ability to penetrate the blood-brain barrier.

[0014] This cationic mesoporous silica nanomaterial loaded with neurotropic viral proteins possesses a structure that combines central nervous system targeting, blood-brain barrier penetration, and free DNA clearance capabilities, and has the potential to achieve highly effective anti-inflammatory treatment for multiple sclerosis.

[0015] The preparation method of this silica nanomaterial includes the following steps:

[0016] Preparation of S1 and SeMSN:

[0017] In a 100 mL round-bottom flask, add H2O, cetrimonium p-toluenesulfonate (CTAT), and tetraethylammonium hydroxide (TEAH), and stir in an oil bath at 80 °C for 30 min. Mix diselenosilane, tetraethoxysilane (TEOS), and ethanol (EtOH) in an EP tube for later use. Add the above solution dropwise to the round-bottom flask and react in an oil bath at 80 °C for 4 h. After the reaction is complete, stop heating and allow to cool to room temperature. Centrifuge; wash with H2O; wash with EtOH; add EtOH and sonicate to resuspend, transfer to a 500 mL round-bottom flask, and bring to volume with EtOH. Add hydrochloric acid (HCl); reflux in an oil bath at 80 °C and stir for 24 h. Centrifuge; wash with EtOH; add an appropriate amount of EtOH and sonicate to resuspend, obtaining SeMSN. Take a small amount, dry, and weigh for quantification.

[0018] Preparation of S2 and SeMSN epoxides:

[0019] SeMSN was dispersed in toluene; the mixture was refluxed in an oil bath at 80°C and stirred for 3 hours; 400 μL of epoxy silane was added; the mixture was refluxed in an oil bath at 110°C and stirred for 24 hours; the mixture was centrifuged; it was washed with EtOH to remove toluene and unreacted epoxy silane; an appropriate amount of EtOH was added and the mixture was resuspended by ultrasonic oscillation to obtain SeMSN epoxide. A small amount was dried, weighed, and quantified.

[0020] Preparation of S3 and SeMSN-PEI600:

[0021] Replace the dispersion medium of SeMSN epoxide with H2O and bring the volume to 10 mL. Add polyethyleneimine (PEI600) to a 100 mL round-bottom flask, add H2O and stir to dissolve. Add the suspension to the round-bottom flask. Stir at room temperature for 24 h. Centrifuge. Wash with H2O to remove unreacted PEI600. Add 10 mL of H2O and sonicate to resuspend, obtaining SeMSN-PEI600, which is stored at 4 °C for later use.

[0022] Preparation of S4 and SeMSN-PEI600 (PEG400):

[0023] Take amino-terminated polyethylene glycol NH2-PEG400 into a 100mL round-bottom flask and dissolve it in H2O; add SeMSN-PEI600 and make up to volume; add triethylamine TEA to the pH of the reaction system to 10; stir at room temperature for 24h; centrifuge; wash with H2O to remove TEA and unreacted PEG400; add H2O and sonicate to resuspend, to obtain SeMSN-PEI600 (PEG400), and store at 4℃ for later use;

[0024] Preparation of S5 and SeMSN-PEI600-RVG29 (PEG400):

[0025] Take the rabies virus glycoprotein polypeptide fragment RVG29 into a 100mL round-bottom flask, add H2O and stir to dissolve; add NHS and EDCI; stir at room temperature for 2h; add SeMSN-PEI600 (PEG400) and make up to volume; stir at room temperature for 24h; centrifuge; wash with H2O to remove unreacted RVG29, N-hydroxysuccinimide NHS, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDCI; add an appropriate amount of H2O and sonicate to resuspend, store at 4℃ to obtain SeMSN-PEI600-RVG29 (PEG400), take a small amount, dry and weigh for quantification.

[0026] In a preferred embodiment of the present invention, in step S1, 40 mL of H2O, 600 mg of CTAT, and 160 mg of TEAH are added to a 100 mL round-bottom flask and stirred in an oil bath at 80 °C for 30 min. 1 mL of diselenylsilane, 3 mL of LTEOS, and 3 mL of EtOH are mixed in an EP tube and set aside. The above solution is added dropwise to the round-bottom flask for reaction, and stirred in an oil bath at 80 °C for 4 h. After the reaction is complete, heating is stopped, and the mixture is allowed to cool to room temperature. The mixture is then centrifuged, washed with H2O, washed with EtOH, and resuspended by ultrasonic oscillation with EtOH. The mixture is transferred to a 500 mL round-bottom flask, and the volume is adjusted to 200 mL with EtOH. 20 mL of HCl is added, and the mixture is refluxed in an oil bath at 80 °C for 24 h. The mixture is then centrifuged, washed with EtOH, and resuspended by ultrasonic oscillation with an appropriate amount of EtOH to obtain SeMSN. A small amount is dried, weighed, and quantified.

[0027] In a preferred embodiment of the present invention, in step S2, 400 mg of SeMSN is dispersed in 200 mL of toluene; the mixture is refluxed in an oil bath at 80 °C and stirred for 3 h; 400 μL of epoxy silane is added; the mixture is refluxed in an oil bath at 110 °C and stirred for 24 h; the mixture is centrifuged; washed with EtOH to remove toluene and unreacted epoxy silane; an appropriate amount of EtOH is added and the mixture is resuspended by ultrasonic oscillation to obtain SeMSN epoxide, and a small amount is dried, weighed, and quantified. In a preferred embodiment of the present invention, in step S3, 100 mg of epoxide SeMSN is taken, the dispersion medium is replaced with H2O, and the volume is adjusted to 10 mL; 10 mg of PEI600 is added to a 100 mL round-bottom flask, and 15 mL of H2O is added and stirred to dissolve; a suspension is added to the round-bottom flask; the mixture is stirred at room temperature for 24 h; the mixture is centrifuged; washed with H2O to remove unreacted PEI600; 10 mL of H2O is added and the mixture is resuspended by ultrasonic oscillation to obtain SeMSN-PEI600, which is stored at 4 °C for later use.

[0028] In a preferred embodiment of the present invention, in step S4, 6.7 mg of NH2-PEG400 is taken into a 100 mL round-bottom flask and dissolved in 15 mL of H2O; SeMSN-PEI600 is added and the volume is adjusted to 25 mL; TEA is added until the pH of the reaction system is 10; the mixture is stirred at room temperature for 24 h; the mixture is centrifuged; it is washed with H2O to remove TEA and unreacted PEG400; 10 mL of H2O is added and the mixture is resuspended by ultrasonic oscillation to obtain SeMSN-PEI600 (PEG400), which is stored at 4 °C for later use.

[0029] In a preferred embodiment of the present invention, in step S5, 2.5 mg of RVG29 is taken into a 100 mL round-bottom flask, and 15 mL of H2O is added and stirred to dissolve. 0.15 mg of NHS and 0.22 mg of EDCI are added; the mixture is stirred at RT for 2 h; SeMSN-PEI600 (PEG400) is added and the volume is adjusted to 25 mL; the mixture is stirred at RT for 24 h; the mixture is centrifuged; it is washed with H2O to remove unreacted RVG29, NHS, and EDCI; an appropriate amount of H2O is added and the mixture is resuspended by ultrasonic oscillation and stored at 4 °C to obtain SeMSN-PEI600-RVG29 (PEG400). A small amount is dried, weighed, and quantified.

[0030] The silica nanomaterials prepared by the above method are cationic mesoporous silica nanomaterials loaded with neurotropic viral proteins.

[0031] Furthermore, silica nanomaterials, combined with medically acceptable carriers, can be used in drugs for the treatment and / or prevention of multiple sclerosis.

[0032] Implementing this invention has the following beneficial effects:

[0033] This invention provides a cationic mesoporous silica nanomaterial loaded with neurotropic viral proteins. Compared with traditional multiple sclerosis (MS) treatment drugs, the nanomaterial of this invention is more efficient, safe and controllable for anti-inflammatory treatment of MS, providing new ideas and new materials for efficient clinical intervention of MS.

[0034] Compared with traditional anti-inflammatory drugs, the cationic mesoporous silica nanomaterial loaded with neurotropic viral proteins of the present invention can target the central nervous system to exert anti-inflammatory effects, has good antibacterial effects, and also has good blood-brain barrier permeability, making it very suitable for use in the treatment of multiple sclerosis.

[0035] The cationic mesoporous silica nanomaterial loaded with neurotropic viral proteins of the present invention can utilize mesoporous silica to load neurotropic viral proteins, which can penetrate the blood-brain barrier and target the central nervous system to exert their effects; the mesoporous silica is grafted with polycations that have a strong binding ability to nucleic acids, which can effectively bind to free DNA and achieve anti-inflammatory purpose by clearing cfDNA.

[0036] In the preparation method of cationic mesoporous silica nanomaterials loaded with neurotropic viral proteins of the present invention, different kinds of polycationic compounds are grafted onto the surface of the mesoporous silica nanomaterials through an epoxy-opening reaction. The cations on the surface of the mesoporous silica material can efficiently bind and remove free nucleic acids, inhibit the activation of TLR receptors, reduce the inflammatory response in multiple sclerosis, and ultimately obtain a highly efficient and safe anti-inflammatory effect. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 Transmission electron microscopy (TEM) image of SeMSN-PEI600-RVG29(PEG400) prepared in Example 1;

[0039] Figure 2 The image shows the scanning electron microscope (SEM) result of SeMSN-PEI600-RVG29(PEG400) prepared in Example 1.

[0040] Figure 3 The particle size distribution of SeMSN, SeMSN epoxide, SeMSN-PEI600, SeMSN-PEI600(PEG400), and SeMSN-PEI600-RVG29(PEG400) obtained in Example 1 is shown in the figure.

[0041] Figure 4 The zeta potential results are shown for SeMSN, SeMSN epoxide, SeMSN-PEI600, SeMSN-PEI600(PEG400), and SeMSN-PEI600-RVG29(PEG400) obtained in Example 1.

[0042] Figure 5 The graph shows the binding rate of SeMSN-PEI600-RVG29 (PEG400) to DNA.

[0043] Figure 6 The figure shows the inhibitory effect of SeMSN-PEI600-RVG29 (PEG400) on HEK-BlueTM TLR9 reporter cells;

[0044] Figure 7 The graph shows the effect of SeMSN-PEI600-RVG29 (PEG400) on reducing disease scores in a mouse model (EAE model);

[0045] Figure 8 This is a graph showing the effect of SeMSN-PEI600-RVG29 (PEG400) in reducing the morbidity rate in a mouse model (EAE model).

[0046] Figure 9Image showing HE staining results of spinal cord tissue sections after SeMSN-PEI600-RVG29 (PEG400) treatment in a mouse model (EAE model). Detailed Implementation

[0047] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] The preparation method of this cationic mesoporous silica nanomaterial loaded with neurotropic viral protein includes the following steps:

[0049] Preparation of S1 and SeMSN: H2O, cetrimonium p-toluenesulfonate (CTAT), and tetraethylammonium hydroxide (TEAH) were added to a 100 mL round-bottom flask. The mixture was stirred in an oil bath at 80 °C for 30 min. Diselenosilane, tetraethoxysilane (TEOS), and ethanol (EtOH) were mixed in an EP tube for later use. The above solution was added dropwise to the round-bottom flask and reacted in an oil bath at 80 °C for 4 h. After the reaction was completed, heating was stopped, and the mixture was allowed to cool to room temperature. The mixture was centrifuged, washed with H2O, washed with EtOH, and resuspended by ultrasonic oscillation with EtOH. The mixture was transferred to a 500 mL round-bottom flask and brought to a final volume with EtOH. Hydrochloric acid (HCl) was added, and the mixture was refluxed in an oil bath at 80 °C for 24 h. The mixture was centrifuged, washed with EtOH, and resuspended by ultrasonic oscillation with an appropriate amount of EtOH to obtain SeMSN. A small amount was dried, weighed, and quantified.

[0050] Preparation of S2 and SeMSN epoxides: SeMSN was dispersed in toluene; the mixture was refluxed in an oil bath at 80°C and stirred for 3 hours; 400 μL of epoxy silane was added; the mixture was refluxed in an oil bath at 110°C and stirred for 24 hours; the mixture was centrifuged; the mixture was washed with EtOH to remove toluene and unreacted epoxy silane; an appropriate amount of EtOH was added and the mixture was resuspended by ultrasonic oscillation to obtain SeMSN epoxides. A small amount was dried, weighed, and quantified.

[0051] Preparation of S3 and SeMSN-PEI600: SeMSN epoxide was replaced with H2O as the dispersion medium and the volume was adjusted to 10 mL; polyethyleneimine (PEI600) was added to a 100 mL round-bottom flask and dissolved by stirring with H2O; the suspension was added to the round-bottom flask; the mixture was stirred at room temperature for 24 h; centrifuged; washed with H2O to remove unreacted PEI600; 10 mL of H2O was added and the mixture was resuspended by ultrasonic oscillation to obtain SeMSN-PEI600, which was stored at 4 °C for later use.

[0052] Preparation of S4, SeMSN-PEI600 (PEG400): Amino-terminated polyethylene glycol (NH2-PEG400) was transferred to a 100 mL round-bottom flask and dissolved in H2O. SeMSN-PEI600 was added and the volume was adjusted. Triethylamine (TEA) was added until the pH of the reaction system reached 10. The mixture was stirred at room temperature for 24 h. The mixture was then centrifuged. The mixture was washed with H2O to remove TEA and unreacted PEG400. The mixture was then resuspended by ultrasonic oscillation in H2O to obtain SeMSN-PEI600 (PEG400), which was stored at 4 °C for later use.

[0053] Preparation of S5 and SeMSN-PEI600-RVG29(PEG400): RVG29, a rabies virus glycoprotein polypeptide fragment, was transferred to a 100 mL round-bottom flask and dissolved by stirring with H2O. NHS and EDCI were added; the mixture was stirred at room temperature for 2 h. SeMSN-PEI600(PEG400) was added and the volume was adjusted. The mixture was stirred at room temperature for 24 h. The mixture was centrifuged. Unreacted RVG29, N-hydroxysuccinimide (NHS), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) were washed with H2O. The mixture was resuspended by ultrasonic oscillation with an appropriate amount of H2O and stored at 4 °C to obtain SeMSN-PEI600-RVG29(PEG400). A small amount was dried, weighed, and quantified.

[0054] The SeMSN-PEI600-RVG29(PEG400) prepared by the above method is a cationic mesoporous silica nanomaterial loaded with neurotropic viral protein, which can be used in drugs for the treatment and / or prevention of multiple sclerosis.

[0055] Example 1

[0056] A method for preparing cationic mesoporous silica nanomaterials loaded with neurotropic viral proteins includes the following steps:

[0057] (1) Preparation of SeMSN: Add 40 mL H2O, 600 mg CTAT, and 160 mg TEAH to a 100 mL round-bottom flask, and stir in an oil bath at 80 °C for 30 min; mix 1 mL diselenylsilane, 3 mL TEOS, and 3 mL EtOH in an EP tube for later use; add the above solution dropwise to the round-bottom flask and react in an oil bath at 80 °C for 4 h; after the reaction is complete, stop heating and cool to room temperature; centrifuge; wash with H2O; wash with EtOH; add EtOH and resuspend by ultrasonic vibration, transfer to a 500 mL round-bottom flask, and bring the volume to 200 mL with EtOH; add 20 mL HCl; reflux in an oil bath at 80 °C and stir for 24 h; centrifuge; wash with EtOH; add an appropriate amount of EtOH and resuspend by ultrasonic vibration, take a small amount, dry, weigh, and quantify.

[0058] (2) Preparation of SeMSN epoxide: Take 400 mg of SeMSN and disperse it in 200 mL of toluene; reflux in an oil bath at 80 °C and stir for 3 h; add 400 μL of epoxy silane; reflux in an oil bath at 110 °C and stir for 24 h; centrifuge; wash with EtOH to remove toluene and unreacted epoxy silane; add an appropriate amount of EtOH and resuspend by ultrasonic vibration, take a small amount, dry and weigh for quantitative determination.

[0059] (3) Preparation of SeMSN-PEI600: Take 100 mg of epoxidized SeMSN and replace the dispersion medium with H2O, and make up to 10 mL; take 10 mg of PEI600 and add it to a 100 mL round-bottom flask, add 15 mL of H2O and stir to dissolve; add the suspension to the round-bottom flask; stir at room temperature for 24 h; centrifuge; wash with H2O to remove unreacted PEI600; add 10 mL of H2O and sonicate to resuspend, and store at 4 °C for later use.

[0060] (4) Preparation of SeMSN-PEI600 (PEG400): Take 6.7 mg NH2-PEG400 into a 100 mL round-bottom flask and add 15 mL H2O to dissolve it; add SeMSN-PEI600 and make up to 25 mL; add TEA to make up to pH 10 of the reaction system; stir at RT for 24 h; centrifuge; wash with H2O to remove TEA and unreacted PEG400; add 10 mL H2O and sonicate to resuspend, and store at 4 °C for later use.

[0061] (5) Preparation of SeMSN-PEI600-RVG29(PEG400): Take 2.5 mg RVG29 into a 100 mL round-bottom flask, add 15 mL H2O and stir to dissolve; add 0.15 mg NHS and 0.22 mg EDCI; stir at room temperature for 2 h; add SeMSN-PEI600(PEG400) and make up to 25 mL; stir at room temperature for 24 h; centrifuge; wash with H2O to remove unreacted RVG29, NHS and EDCI; add an appropriate amount of H2O and sonicate to resuspend, store at 4℃, take a small amount, dry and weigh for quantification, and thus obtain SeMSN-PEI600-RVG29(PEG400).

[0062] Performance testing:

[0063] 1. The cationic mesoporous silica nanomaterials loaded with neurotropic viral proteins prepared in Example 1 were characterized by transmission electron microscopy and scanning electron microscopy. Please refer to [link to relevant documentation]. Figure 1 , Figure 2 .

[0064] from Figure 1 , Figure 2It can be seen that SeMSN-PEI600-RVG29(PEG400) has a uniform spherical morphology with a uniform particle size of about 50-60nm, and the pores are highly ordered and have a uniform pore size.

[0065] 2. The particle size and zeta potential of SeMSN, SeMSN epoxide, SeMSN-PEI600, and SeMSN-PEI600-RVG29 (PEG400) in Example 1 are as follows: Figure 3 and 4 As shown.

[0066] from Figure 3 It can be seen that the particle size of SeMSN is approximately 148 nm, the particle size of SeMSN epoxide is approximately 125 nm, and the particle size of SeMSN-PEI600 after grafting PEI600 is approximately 150 nm. The particle size of SeMSN-PEI600-RVG29 (PEG400) loaded with neuroviral protein RVG29 is approximately 180 nm.

[0067] from Figure 4 It can be seen that: the potential of SeMSN is -20mV, the potential of SeMSN epoxide is approximately -10mV, the potential of SeMSN-PEI600 after grafting PEI600 is approximately +40mV, the potential of SeMSN-PEI600-PEG400 is approximately +30mV, and the potential of SeMSN-PEI600-RVG29(PEG400) after loading neuroviral protein RVG29 is approximately +25mV.

[0068] 3. DNA Binding Assay: SeMSN-PEI600-RVG29 prepared in Example 1 was used to prepare corresponding solutions with concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, and 5 μg / mL, respectively. DNA solution (purchased from Shanghai Maclean Biotechnology Co., Ltd.) was prepared with a concentration of 2 μg / mL. Equal volumes of SeMSN-PEI600-RVG29 and DNA solutions were mixed to achieve mass ratios of 1:1, 2.25:1, 2.5:1, 5:1, 10:1, and 20:1, respectively. Subsequently, the mixture was shaken at 80 rpm at room temperature for 2 hours, centrifuged at 10,000 rpm for 5 minutes, and the free DNA content in the supernatant was detected using picogreen (CAS No.: 177571-06-1, purchased from Xi'an Qiyue Biotechnology Co., Ltd.).

[0069] result Figure 5 As shown. From Figure 5It can be seen that 25ug / ml of SeMSN-PEI600-RVG29 can bind well to DNA, suggesting its potential to become a highly efficient DNA scavenger.

[0070] 4. Inhibitory effect of SeMSN-PEI-PEG-RVG29 prepared in Example 1 on HEK-Blue™ TLR9 reporter cells: 6 mg / mL, 3 mg / mL, 1.5 mg / mL, and 0.75 mg / mL SeMSN-PEI600-RVG29 solutions and 20 μg / mL CpG ODN 2006 solution (CpG ODN 2006 purchased from Invivogen) were prepared using filtered high-pressure water.

[0071] Observe the growth of HEK-Blue™ TLR9 reporter cells (purchased from Invivogen) under a microscope. When the cells grow to 70%-80% of the culture flask, discard the DMEM medium, add 5 mL of PBS buffer (pH 7.4, 0.1M, the same below) and rinse slightly. Add another 5 mL of PBS buffer, place the cell culture flask with the cell-grown side against the workbench, let it stand for 1 minute, gently tap the flask wall to allow the cells to detach on their own, and gently blow them to mix. Transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 300 g for 5 min, discard the supernatant, resuspend the cells, gently pipette to mix, and centrifuge again at 300 g for 5 min. Resuspend HEK-Blue™ TLR9 reporter cells to obtain a cell suspension. Dilute 10 μL of the cell suspension 100 times to obtain a diluted cell suspension. Place 10 μL of the diluted cell suspension in a 1.5 mL EP tube, add 10 μL of trypan blue dye at a 1:1 volume ratio, and count the cells using a cell counting chamber under a microscope. (8 × 10⁻⁶ cells / mL) 4 Cells were evenly seeded at a density of 100 cells / well in a 96-well plate, surrounded by a ring of PBS buffer. Cell growth was observed under a microscope. After confirming uniform cell distribution under the microscope, the following three groups of samples were added: Blank group, CpG group, and SeMSN-PEI-PEG-RVG29 group.

[0072] In the Blank group, only 20 μL of filtered high-pressure water was added. In the CpG group, 10 μL of 20 μg / mL CpG ODN 2006 (purchased from Sangon Biotech (Shanghai) Co., Ltd.) and 10 μL of filtered high-pressure water were added. In the SeMSN-PEI-PEG-RVG29 group, 10 μL of 20 μg / mL CpG ODN 2006 solution was added first, followed by 10 μL of 200 μg / mL SeMSN-PEI-PEG-RVG29 solution. After adding the above components, observe under a microscope and incubate overnight in a cell culture incubator containing 5% CO2 at 37°C. Take 20 μL of cell supernatant, add 180 μL of Quanti-Blue solution (purchased from Shenzhen Xinbosheng Biotechnology Co., Ltd.), and incubate for 1.5 h in a cell culture incubator containing 5% CO2 at 37°C to obtain the supernatant after incubation. Measure the OD value of the supernatant after incubation at 620 nm using a SpectraMax iD3 microplate reader.

[0073] The results are as follows Figure 6 As shown. From Figure 6 It can be seen that the CpG group can significantly inhibit the activation of HEK-BlueTM TLR9 reporter cells, while the SeMSN-PEI-PEG-RVG29 group can significantly inhibit CpG-induced cell activation, suggesting that SeMSN-PEI-PEG-RVG29 has a good anti-inflammatory effect in vitro.

[0074] 5. The therapeutic effect of SeMSN-PEI-PEG-RVG29 prepared in Example 1 on a mouse model of experimental allergic encephalomyelitis:

[0075] Establishment of an experimental allergic encephalomyelitis (EAE) model in mice: MOG 35-55 short peptide was emulsified with complete Freund's adjuvant and induced in C57BL / 6J mice. Male C57BL / 6J mice (purchased from the Experimental Animal Center of South China University of Technology), aged 12 weeks, weighing 28-30g, in good health and free of specific pathogens, were selected for the experiment. Mice were housed in an SPF-grade cleanroom under 12-hour light and 12-hour darkness conditions at 22℃ and 55% relative humidity. Before modeling, all mice were weighed and divided into groups of 12 mice each: an EAE+SeMSN-PEI-PEG-RVG29 group, an EAE+saline group, and a Control group. Male C57BL / 6J mice were anesthetized by intraperitoneal injection of 2% pentobarbital (0.2 mL of 1% pentobarbital per 20 g mouse). After about 5 minutes, the mice were fully anesthetized and fixed on the operating table. The hair on the back of the mice was shaved, and the injection site (skin near the tail on the back) was disinfected with 75% alcohol. 150 ug (100 μl) of antigen adjuvant emulsion was injected subcutaneously at a single point, followed by an intraperitoneal injection of 500 ng (100 μl) of pertussis toxin. 58 h later, 500 ng (100 μl) of pertussis toxin was injected intraperitoneally again. Mice in the control group were injected with the same dose of physiological saline using the same injection method.

[0076] Starting from the day of immunization (Day 0 is the first day of model induction), the limb movement status of mice in each group was observed. The neurological function of the mice was assessed by two blind assessments per day (once at 10:00 and once at 17:00) using the internationally recognized 5-point scoring method. The experimental setup for each group of mice is as follows:

[0077] The mice in each group were treated as follows: EAE+SeMSN-PEI-PEG-RVG29 group: 2 mg / ml of SeMSN-PEI-PEG-RVG29 solution was injected intraperitoneally; EAE+saline group: 100 μl of saline solution was injected intraperitoneally; Control group: no modeling and no drug administration.

[0078] The disease scores, morbidity, and inflammation and demyelination in the spinal cord of each group of mice were observed over 19 days.

[0079] The disease scores of the mice in each group over 19 days are as follows: Figure 7As shown in the figure. Neurological function impairment was evaluated using the 0–5 point clinical scoring system commonly used in EAE mice. Higher disease scores indicate successful EAE model establishment. Mice in the SeMSN-PEI-PEG-RVG29 group showed lower levels of neurological function impairment and lower disease scores. The mean disease score over 19 days was 1.375 in the EAE + saline group and 0.283 in the SeMSN-PEI-PEG-RVG29 group. The morbidity rates of the above groups over 19 days are shown in the figure. Figure 8 As shown in the figure, the morbidity rate in mice in the EAE+saline group was 70.83% within 19 days, while the morbidity rate in mice in the SeMSN-PEI-PEG-RVG29 group was 16.67% within 19 days. HE staining results of spinal cord tissue sections from each group of mice are shown in the figure. Figure 9 As shown in the figure, HE staining revealed that spinal cord inflammation was more severe in mice in the EAE+saline group than in the EAE+SeMSN-PEI-PEG-RVG29 group, while there was no significant difference between the EAE+SeMSN-PEI-PEG-RVG29 group and the Control group. In conclusion, SeMSN-PEI-PEG-RVG29 treatment significantly improved the morbidity and severity of experimental allergic encephalomyelitis in mice.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cationic mesoporous silica nanomaterial loaded with a neurotropic viral protein, characterized in that, The nanomaterial comprises the following structural elements: (a) a selenium-containing mesoporous silica inner core: a hexagonal ordered mesoporous structure with a particle size of 50-70 nm and a Zeta potential of +15 to +30 mV, and selenium elements are distributed on the surface of the pore channel in the form of silane bonding; (b) an epoxidized-polyethyleneimine composite layer: covalently modified on the surface of the inner core, comprising a cationic polymer network of epoxide silane cross-linked polyethyleneimine, wherein the number average molecular weight of the polyethyleneimine is 500-10,000; (c) a polyethylene glycol hydrophilic layer: grafted by ring-opening reaction of amino groups with epoxy groups of the composite layer, the polyethylene glycol is of an amino-terminal type, and the molecular weight is 200-2,000; (d) a neurotropic viral protein ligand: coupled to the end of the polyethylene glycol layer by an amide bond mediated by a condensing agent, and the protein is a rabies virus glycoprotein polypeptide RVG29 or a functional fragment thereof.

2. The cationic mesoporous silica nanomaterial loaded with a neurotropic viral protein according to claim 1, characterized by: The condensing agent is a combination of N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

3. The cationic mesoporous silica nanomaterial loaded with neurotropic viral proteins according to any one of claims 1, characterized by: The surface charge of the nanomaterial is positively charged at physiological pH, and the Zeta potential is greater than or equal to +15 mV, and the nanomaterial has the ability to penetrate the blood-brain barrier.

4. The cationic mesoporous silica nanomaterial loaded with neurotropic viral proteins according to any one of claims 1, wherein, The preparation method comprises the following steps: S1, preparation of SeMSN: In a 100 mL round-bottomed ball bottle, add H2O, cetrimonium p-toluenesulfonate CTAT, and tetraethylammonium hydroxide TEAH, and stir for 30 min in an 80°C oil bath; mix diseleno silane, tetraethoxysilane TEOS, and ethanol EtOH in an EP tube for standby; add the above solution dropwise into the round-bottomed ball bottle, and react in an 80°C oil bath for 4 h; after the reaction is completed, stop heating, and cool to room temperature; centrifuge; wash with H2O; wash with EtOH; resuspend by adding EtOH and ultrasonic oscillation, transfer to a 500 mL round-bottomed ball bottle, and add EtOH to constant volume; add hydrochloric acid HCl; stir in an 80°C oil bath under condensation reflux for 24 h; centrifuge; wash with EtOH; resuspend by adding an appropriate amount of EtOH and ultrasonic oscillation to obtain SeMSN, and take a small amount for drying and weighing for quantification; S2, preparation of SeMSN epoxide: Take SeMSN, disperse in toluene; stir in an 80°C oil bath under condensation reflux for 3 h; add 400 μL of epoxy silane; stir in an 110°C oil bath under condensation reflux for 24 h; centrifuge; wash with EtOH to remove toluene and unreacted epoxy silane; resuspend by adding an appropriate amount of EtOH and ultrasonic oscillation to obtain SeMSN epoxide, and take a small amount for drying and weighing for quantification; S3, preparation of SeMSN-PEI600: Take SeMSN epoxide, replace the dispersion medium with H2O, and add H2O to constant volume to 10 mL; take polyethyleneimine PEI600 and add to a 100 mL round-bottomed ball bottle, and stir to dissolve; add the suspension to the round-bottomed ball bottle; stir at room temperature for 24 h; centrifuge; wash with H2O to remove unreacted PEI600; resuspend by adding 10 mL of H2O and ultrasonic oscillation to obtain SeMSN-PEI600, and store at 4°C for standby; S4, preparation of SeMSN-PEI600(PEG400): Take the amino-terminal polyethylene glycol NH2-PEG400 to 100 mL round-bottom ball bottle, add H2O to dissolve; add SeMSN-PEI600, constant volume; add triethylamine TEA to the reaction system pH=10; room temperature stirring 24 h; centrifugal; H2O washing, remove TEA and unreacted PEG400; add H2O ultrasonic oscillation resuspension, get SeMSN-PEI600 (PEG400), 4℃ preservation standby; S5, preparation of SeMSN-PEI600-RVG29 (PEG400): Take rabies virus glycoprotein polypeptide fragment RVG29 to 100 mL round-bottom ball bottle, add H2O stirring to dissolve; add NHS, EDCI; room temperature stirring 2 h; add SeMSN-PEI600 (PEG400), constant volume; room temperature stirring 24 h; centrifugal; H2O washing, remove unreacted RVG29, N-hydroxy succinimide NHS, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride EDCI; add appropriate amount of H2O ultrasonic oscillation resuspension, 4℃ preservation, get SeMSN-PEI600-RVG29 (PEG400), take a small amount of drying and weighing to quantify.

5. The cationic mesoporous silica nanomaterial loaded with neurotropic viral proteins according to claim 4, characterized in that, The specific steps in S1 are: 100 mL round-bottom ball bottle adds 40 mL H2O, 600 mg CTAT, 160 mg TEAH, 80℃ oil bath, stirring 30 min; 1 mL diselenide silane, 3 mL TEOS and 3 mL EtOH are mixed in an EP tube for standby; the above solution is added dropwise into the round-bottom ball bottle, and the reaction is carried out at 80℃ oil bath, stirring for 4 h; after the reaction is completed, stop heating, and reduce to room temperature; centrifugal; H2O washing; EtOH washing; add EtOH ultrasonic oscillation resuspension, transfer to a 500 mL round-bottom ball bottle, constant volume to 200 mL with EtOH; add 20 mL HCl; 80℃ oil bath, condensation reflux, stirring for 24 h; centrifugal; EtOH washing; add appropriate amount of EtOH ultrasonic oscillation resuspension, get SeMSN, take a small amount of drying, weighing to quantify.

6. The cationic mesoporous silica nanomaterial loaded with neurotropic viral proteins according to claim 4, characterized in that, The specific steps in S2 are: take 400 mg SeMSN, disperse in 200 mL toluene; 80℃ oil bath, condensation reflux, stirring for 3 h; add 400 μL epoxy silane; 110℃ oil bath, condensation reflux, stirring for 24 h; centrifugal; EtOH washing, remove toluene and unreacted epoxy silane; add appropriate amount of EtOH ultrasonic oscillation resuspension, get SeMSN epoxide, take a small amount of drying, weighing to quantify.

7. The cationic mesoporous silica nanomaterial loaded with neurotropic viral proteins according to claim 4, characterized in that, The specific steps in S3 are: take 100 mg epoxidized SeMSN, change the dispersion medium to H2O, constant volume to 10 mL; take 10 mg PEI600, add to 100 mL round-bottom ball bottle, add 15 mL H2O stirring to dissolve; add the suspension to the round-bottom ball bottle; room temperature stirring 24 h; centrifugal; H2O washing, remove unreacted PEI600; add 10 mL H2O ultrasonic oscillation resuspension, get SeMSN-PEI600, 4℃ preservation standby.

8. The cationic mesoporous silica nanomaterial loaded with neurotropic viral proteins according to claim 4, characterized in that, The specific steps in S4 are: take 6.7 mg NH2-PEG400 to 100 mL round bottom ball bottle, dissolve in 15 mL H2O; add SeMSN-PEI600, constant volume to 25 mL; add TEA to the reaction system pH=10; stir at room temperature for 24 h; centrifuge; H2O wash, remove TEA and unreacted PEG400; add 10 mL H2O ultrasonic oscillation resuspension, get SeMSN-PEI600(PEG400), 4℃ preservation for standby.

9. The cationic mesoporous silica nanomaterial loaded with neurotropic viral proteins according to claim 4, characterized in that, The specific steps in S5 are: take 2.5 mg RVG29 to 100 mL round bottom ball bottle, add 15 mL H2O stirring to dissolve; add 0.15 mg NHS, 0.22 mg EDCI; RT stirring 2 h; add SeMSN-PEI600(PEG400), constant volume to 25 mL; RT stirring 24 h; centrifuge; H2O wash, remove unreacted RVG29, NHS, EDCI; add appropriate amount of H2O ultrasonic oscillation resuspension, 4℃ preservation, get SeMSN-PEI600-RVG29(PEG400), take a small amount of drying and weighing for quantification.

10. A pharmaceutical composition comprising the cationic mesoporous silica nanomaterial loaded with neurotropic viral protein according to any one of claims 1-9, characterized in that: The drug is applied to treat and or prevent multiple sclerosis. The drug is applied to treat and or prevent multiple sclerosis.

Images