Preparation method of nanoparticles capable of adjusting crowding degree in cells

Abstract

The invention provides a preparation method of nano-particles capable of regulating the crowding degree in cells, which comprises the following steps: firstly preparing degradable silicon dioxide nano-particles BSN (at) Ficoll containing disulfide bonds entrapped with polysucrose, then modifying amino at the tail end to obtain BSN (at) Ficol-NH2 nano-particles, and then modifying with folic acid and hyaluronic acid to obtain FA-HA-BSN (at) Ficoll nano-particles, namely FHS (at) Ficoll for short. According to the FHS-Ficoll nano-particle disclosed by the invention, the polysucrose is used as a crowding agent, the silicon dioxide is used as a core skeleton, the disulfide bond is used as a polysucrose responsive release switch, and the folic acid and the hyaluronic acid are jointly used as targeting ligands, so that accurate targeted delivery of proinflammatory macrophages and glutathione responsive release crowding agents can be realized, and the crowding degree in cells can be dynamically regulated; and protein is induced to generate phase separation, so that the relieving of inflammatory response can be effectively promoted, and the repair and regeneration process of damaged tissues is accelerated.

Description

Technical Field

[0001] The invention belongs to the field of nanoparticles and biotechnology, and in particular relates to a method for preparing nanoparticles capable of regulating the degree of crowding in cells. Background Art

[0002] Tissue damage triggers a complex and coordinated inflammatory response, and an orderly inflammatory response is a necessary condition for tissue regeneration. Macrophages, as important effector cells in the body's innate immunity, clear pathogens and tissue debris by initiating inflammatory responses in the early stages of inflammation, and secrete anti-inflammatory cytokines during the inflammation resolution phase to promote stem cell recruitment and bone regeneration. However, in the inflammatory tissue microenvironment caused by diseases such as diabetes, the retention of pro-inflammatory macrophages hinders the resolution of inflammation, thereby damaging stem cell function and hindering the regeneration cascade process. Therefore, revealing the regulatory mechanism of post-injury inflammation and macrophage function is the key to regulating inflammatory responses and tissue regeneration under pathological conditions.

[0003] The cell is filled with a large number of biomacromolecules (proteins, nucleic acids, polysaccharides, etc.), and due to the excluded volume effect, the free space is extremely limited. This state is called macromolecular crowding. Macromolecular crowding affects key physiological functions such as protein structure, folding, enzyme activity, and protein interaction. Biomolecules are enriched by phase separation, and phase separation, as a self-organizing regulatory mechanism of complex biological processes in cells, widely affects physiological activities, including cell division, development, tumors, neurodegenerative diseases, immune responses, and plant photosynthesis. The degree of intracellular macromolecular crowding is closely related to the occurrence of phase separation. Studies have shown that intracellular crowding promotes the occurrence of phase separation by increasing the effective concentration of biomolecules. WNK kinase is a class of serine threonine protein kinases found in mammals. It can sense the intracellular crowding state, use disordered domains to drive the occurrence of phase separation, and then assemble into condensates to activate the WNK-SPAK / OSR1 pathway. The WNK-SPAK / OSR1 pathway can trigger the phosphorylation of downstream sodium-potassium-chloride cotransporter, drive ion influx, inhibit inflammasome activation, and play an important role in inhibiting inflammation. Therefore, WNK protein phase separation can be triggered by regulating the crowding state of intracellular macromolecules, providing a new way to regulate cell function.

[0004] As an ideal drug delivery carrier, silica nanoparticles can efficiently and safely deliver crowding agents to the cytoplasm due to their controllable size and adjustable surface chemical properties. In addition, silica can also achieve responsive release to the intracellular environment by modifying its surface chemical properties, thereby more accurately controlling the release time and location of crowding agents in the cell. Therefore, there is an urgent need to develop functional silica nanoparticles that can selectively deliver crowding agents to the cytoplasm as an advanced tool to regulate the intracellular crowding state and trigger the phase separation process of specific proteins. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing nanoparticles that can regulate the degree of intracellular crowding in view of the deficiencies of the above-mentioned prior art. The nanoparticles use polysucrose as a crowding agent, silica as a core skeleton, disulfide bonds as polysucrose-responsive release switches, and folic acid and hyaluronic acid as targeting ligands. The nanoparticles have the ability to target pro-inflammatory macrophages, release crowding agents in a glutathione-responsive manner, and regulate the degree of intracellular crowding.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] A method for preparing nanoparticles capable of regulating the degree of intracellular crowding comprises the following steps:

[0008] S1. Preparation of BSN@Ficoll Nanoparticles

[0009] Dissolving polysucrose in water to obtain a polysucrose aqueous solution, then mixing the polysucrose aqueous solution, ethyl orthosilicate and bis[3-(triethoxysilyl)propyl] disulfide, and shaking to obtain a mixture; adding the mixture to a mixed organic solvent of TritonX-100, n-hexanol and cyclohexane, and then adding 30% by mass of ammonia water to obtain a water-oil emulsion, stirring at room temperature for 12-24 hours, adding acetone to precipitate solid particles, and then centrifuging and washing to obtain polysucrose-encapsulated disulfide-containing degradable silica nanoparticles BSN@Ficoll;

[0010] S2. Preparation of BSN@Ficoll-NH2 nanoparticles

[0011] The BSN@Ficoll nanoparticles obtained in S1 were dispersed in anhydrous ethanol, and 30% ammonia water was added. Under stirring conditions of 10000 rpm / min, 3-aminopropyltriethoxysilane was added, and the mixture was reacted at 25-45°C for 12-24 hours. The obtained product was washed and dried to obtain silica nanoparticles BSN@Ficoll-NH2 with amino terminal groups.

[0012] S3. Preparation of FHS@Ficoll Nanoparticles

[0013] Hyaluronic acid was mixed with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to activate the carboxyl group. After 30 minutes, the BSN@Ficoll-NH2 nanoparticles obtained by S2 were added and reacted at room temperature for 24-48 hours to obtain hyaluronic acid-modified nanoparticles HA-BSN@Ficoll-NH2.

[0014] Folic acid was mixed with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to activate the carboxyl group. After 30 minutes, HA-BSN@Ficoll-NH2 nanoparticles were added and reacted at room temperature for 24-48 hours to obtain folic acid and hyaluronic acid modified nanoparticles FA-HA-BSN@Ficoll, referred to as FHS@Ficoll.

[0015] Preferably, the polysucrose in S1 is Ficoll-400, with a relative molecular weight of 400,000 and a purity of ≥99%, and the concentration of the polysucrose aqueous solution is 20 mg / mL.

[0016] Preferably, the volume ratio of the polysucrose aqueous solution, tetraethyl orthosilicate, and bis[3-(triethoxysilyl)propyl] disulfide in the mixture of S1 is 10:(3-7):(5-9); the volume ratio of Triton X-100, n-hexanol, and cyclohexane in the mixed organic solvent is 1:(1-2):(2-5); the volume ratio of the polysucrose aqueous solution to 30% ammonia water is (2-3):1; the amount of acetone added is 1 to 3 times the total volume of the reaction system.

[0017] Preferably, the amount of BSN@Ficoll nanoparticles and anhydrous ethanol in S2 is 25 mg:25 mL, and the volume ratio of anhydrous ethanol, ammonia water and 3-aminopropyltriethoxysilane is (100-200):2:1.

[0018] Preferably, the drying temperature in S2 is 25-45° C., and the drying time is 12-24 h.

[0019] Preferably, the molecular weight of the hyaluronic acid in S3 is 11500Da, the mass ratio of the BSN@Ficoll-NH2 nanoparticles to the hyaluronic acid is 25mg:203mg; the molar ratio of the hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:(2-4):(1-2).

[0020] Preferably, the mass ratio of the HA-BSN@Ficoll-NH2 nanoparticles to folic acid in S3 is 25 mg:236 mg; the molar ratio of folic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:(2-4):(1-2).

[0021] The present invention has the following significant technical effects:

[0022] 1. The present invention provides a method for preparing nanoparticles capable of regulating the degree of intracellular crowding, using polysucrose as a crowding agent, silica as a core skeleton, disulfide bonds as a polysucrose responsive release switch, and folic acid and hyaluronic acid as targeting ligands. The FHS@Ficoll nanoparticles prepared by the preparation method of the present invention exhibit good performance, including precise targeted delivery, intracellular environment responsive release of crowding agents, dynamic regulation of intracellular crowding, and induction of protein phase separation.

[0023] 2. The highly efficient synergistic effect of folic acid and hyaluronic acid ensures that FHS@Ficoll nanoparticles can be accurately identified and delivered to pro-inflammatory macrophages, achieving precise intracellular delivery; after entering the cells, the disulfide bonds in the nanoparticles are specifically broken in the reducing environment of high-concentration glutathione in the cells. This process releases a large amount of polysucrose and triggers the phase separation of WNK protein, thereby activating a series of downstream signaling pathways and effectively inhibiting the excessive activation of inflammasomes. This mechanism not only promotes the relief of inflammatory responses, but also accelerates the repair and regeneration of damaged tissues, providing a novel, non-invasive autoregulatory strategy for disease treatment, which is expected to replace traditional drug treatments and significantly reduce the risk of immune rejection in vivo.

[0024] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The present invention is a process for synthesizing nanoparticles capable of regulating the degree of intracellular crowding;

[0026] Figure 2 is a graph showing the average particle size of BSN@Ficoll nanoparticles, BSN@Ficoll-NH2 nanoparticles, and FHS@Ficoll nanoparticles prepared in Example 1;

[0027] Figure 3 is a schematic diagram of the FHS@Ficoll nanoparticles of Example 4 being selectively engulfed by pro-inflammatory macrophages, releasing crowding agents in response to glutathione, and finally triggering WNK phase separation;

[0028] Figure 4 This is a fluorescence confocal microscopy result of the FHS@Ficoll nanoparticles of Example 4 being phagocytosed by pro-inflammatory macrophages;

[0029] Figure 5 This is a fluorescence confocal microscopy result of pro-inflammatory macrophages triggering WNK phase separation in Example 5;

[0030] Figure 6This is the fluorescence confocal microscopy result of iNOS expression in the pro-inflammatory macrophages of Example 5 after application of FHS@Ficoll. DETAILED DESCRIPTION

[0031] Example 1

[0032] This embodiment is a method for preparing nanoparticles capable of regulating the degree of intracellular crowding, the method comprising the following steps:

[0033] S1. Preparation of BSN@Ficoll Nanoparticles

[0034] 1.8 mL Triton X-100 and 1.8 mL n-hexanol were dissolved in 7.2 mL cyclohexane to obtain a mixed organic solvent; 100 μL of a 20 mg / mL crowding agent polysucrose (Ficoll) aqueous solution was mixed with 40 μL tetraethyl orthosilicate (TEOS) and 60 μL bis[3-(triethoxysilyl)propyl] disulfide, and the mixture was obtained after shaking. The mixture was added to the above mixed organic solvent, and then 50 μL of 30% ammonia water was added to obtain a water-oil emulsion. After stirring at room temperature for 12 hours, 22.1 mL acetone was added to precipitate Ficoll@BS-NP solid particles, and then centrifuged at 1000 rpm / min for 10 minutes, washed with ethanol 3 times, and then washed with deionized water 3 times, and the nanoparticles were recovered to obtain the degradable silica nanoparticles BSN@Ficoll containing disulfide bonds encapsulated with polysucrose.

[0035] S2. Preparation of BSN@Ficoll-NH2 nanoparticles

[0036] Take 25 mg of BSN@Ficoll nanoparticles obtained in S1 and disperse them in 25 mL of anhydrous ethanol, add 500 μL of ammonia water, stir at a speed of 10000 rpm / min and add 250 μL of 3-aminopropyltriethoxysilane (APTES), react at 40 °C for 12 h, wash the product with ethanol three times, and dry it at 40 °C for 24 h to obtain silica nanoparticles BSN@Ficoll-NH2 with amino terminal groups.

[0037] S3. Preparation of FHS@Ficoll Nanoparticles

[0038] First, 203 mg of hyaluronic acid (HA) was mixed with 10.15 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and 4.06 mg of N-hydroxysuccinimide (NHS) to activate the carboxyl group. After 30 min, 25 mg of S2 was added to obtain BSN@Ficoll-NH2 nanoparticles. The mixture was reacted at room temperature for 24 h to obtain HA-BSN@Ficoll-NH2 nanoparticles.

[0039] Then, 236 mg of folic acid (FA) was mixed with 307.48 mg of EDC.HCl and 123.07 mg of NHS to activate the carboxyl group. After 30 min, 25 mg of HA-BSN@Ficoll-NH2 nanoparticles were added and reacted at room temperature for 24 h to finally obtain FA and HA modified nanoparticles FA-HA-BSN@Ficoll, referred to as FHS@Ficoll.

[0040] Figure 2 is the average particle size of the BSN@Ficoll nanoparticles, BSN@Ficoll-NH2 nanoparticles and FHS@Ficoll nanoparticles prepared in this example. It can be obtained that the average particle size of the BSN@Ficoll nanoparticles is 180nm, the average particle size of the BSN@Ficoll-NH2 nanoparticles is 180nm, and the average particle size of the FHS@Ficoll nanoparticles is 200nm.

[0041] Example 2

[0042] This embodiment is a method for preparing nanoparticles capable of regulating the degree of intracellular crowding, the method comprising the following steps:

[0043] S1. Preparation of BSN@Ficoll Nanoparticles

[0044] 1.8 mL Triton X-100 and 1.8 mL n-hexanol were dissolved in 3.6 mL cyclohexane to obtain a mixed organic solvent; 100 μL of a 20 mg / mL crowding agent polysucrose (Ficoll) aqueous solution was mixed with 30 μL tetraethyl orthosilicate (TEOS) and 50 μL bis[3-(triethoxysilyl)propyl] disulfide, and the mixture was obtained after shaking. The mixture was added to the above mixed organic solvent, and then 50 μL of 30% ammonia water was added to obtain a water-oil emulsion. After stirring at room temperature for 24 hours, 7.43 mL acetone was added to precipitate Ficoll@BS-NP solid particles, and then centrifuged at 1000 rpm / min for 10 minutes, washed with ethanol 3 times, and then washed with deionized water 3 times, and the nanoparticles were recovered to obtain the degradable silica nanoparticles BSN@Ficoll containing disulfide bonds encapsulated with polysucrose.

[0045] S2. Preparation of BSN@Ficoll-NH2 nanoparticles

[0046] Take 25 mg of BSN@Ficoll nanoparticles obtained in S1 and disperse them in 25 mL of anhydrous ethanol, add 500 μL of ammonia water, stir at a speed of 10000 rpm / min and add 250 μL of 3-aminopropyltriethoxysilane (APTES), react at 25 °C for 24 h, wash the product twice with ethanol, and dry it at 45 °C for 12 h to obtain silica nanoparticles BSN@Ficoll-NH2 with amino terminal groups.

[0047] S3. Preparation of FHS@Ficoll Nanoparticles

[0048] First, 203 mg of hyaluronic acid (HA) was mixed with 6.77 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and 2.03 mg of N-hydroxysuccinimide (NHS) to activate the carboxyl group. After 30 min, 25 mg of S2 was added to obtain BSN@Ficoll-NH2 nanoparticles. The mixture was reacted at room temperature for 48 h to obtain HA-BSN@Ficoll-NH2 nanoparticles.

[0049] Then, 236 mg of folic acid (FA) was mixed with 204.99 mg of EDC.HCl and 61.53 mg of NHS to activate the carboxyl group. After 30 minutes, 25 mg of HA-BSN@Ficoll-NH2 nanoparticles were added and reacted at room temperature for 48 hours to finally obtain FA and HA modified nanoparticles FA-HA-BSN@Ficoll, referred to as FHS@Ficoll.

[0050] Example 3

[0051] This embodiment is a method for preparing nanoparticles capable of regulating the degree of intracellular crowding, the method comprising the following steps:

[0052] S1. Preparation of BSN@Ficoll Nanoparticles

[0053] 1.8 mL Triton X-100 and 3.6 mL n-hexanol were dissolved in 9 mL cyclohexane to obtain a mixed organic solvent; 100 μL of a 20 mg / mL crowding agent polysucrose (Ficoll) aqueous solution was mixed with 70 μL tetraethyl orthosilicate (TEOS) and 90 μL bis[3-(triethoxysilyl)propyl] disulfide, and the mixture was obtained after shaking. The mixture was added to the above mixed organic solvent, and then 33.33 μL of 30% ammonia water was added to obtain a water-oil emulsion. After stirring at room temperature for 18 hours, 44.08 mL acetone was added to precipitate Ficoll@BS-NP solid particles, and then centrifuged at 1000 rpm / min for 10 minutes, washed with ethanol 3 times, and then washed with deionized water 2 times, and the nanoparticles were recovered to obtain the polysucrose-encapsulated disulfide-containing degradable silica nanoparticles BSN@Ficoll.

[0054] S2. Preparation of BSN@Ficoll-NH2 nanoparticles

[0055] Take 25 mg of BSN@Ficoll nanoparticles obtained in S1 and disperse them in 25 mL of anhydrous ethanol, add 250 μL of ammonia water, stir at a speed of 10000 rpm / min and add 125 μL of 3-aminopropyltriethoxysilane (APTES), react at 45 °C for 18 h, wash the product with ethanol three times, and dry it at 25 °C for 20 h to obtain silica nanoparticles BSN@Ficoll-NH2 with amino terminal groups.

[0056] S3. Preparation of FHS@Ficoll Nanoparticles

[0057] First, 203 mg of hyaluronic acid (HA) was mixed with 13.54 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and 4.06 mg of N-hydroxysuccinimide (NHS) to activate the carboxyl group. After 30 min, 25 mg of S2 was added to obtain BSN@Ficoll-NH2 nanoparticles. The mixture was reacted at room temperature for 36 h to obtain HA-BSN@Ficoll-NH2 nanoparticles.

[0058] Then, 236 mg of folic acid (FA) was mixed with 409.98 mg of EDC.HCl and 123.07 mg of NHS to activate the carboxyl group. After 30 min, 25 mg of HA-BSN@Ficoll-NH2 nanoparticles were added and reacted at room temperature for 36 h to finally obtain FA and HA modified nanoparticles FA-HA-BSN@Ficoll, referred to as FHS@Ficoll.

[0059] Example 4

[0060] This example is an application of the FHS@Ficoll nanoparticles prepared by the present invention. The FHS@Ficoll nanoparticles can accurately deliver the crowding agent Ficoll into pro-inflammatory macrophages. The process includes the following steps:

[0061] 1. Isolation and culture of mouse bone marrow-derived macrophages

[0062] Obtain femurs and tibias from 6-8 week old C57BL / 6J mice, rinse the bone marrow cavity with sterile PBS, add red blood cell lysis buffer to lyse at room temperature for 5 min, and centrifuge at 800 rpm for 5 min. Discard the supernatant, add 10% FBS high-glucose DMEM medium to resuspend, inoculate in a 24-well plate with cell slides, add mouse macrophage colony stimulating factor (M-CSF, 20 ng / mL) for induction. Place in a 37°C, 5% CO2 incubator for routine culture.

[0063] 2. FHS@Ficoll nanoparticles are phagocytosed by pro-inflammatory macrophages

[0064] Macrophages isolated and cultured in vitro were pre-stimulated with 500ng / mL LPS for 4h, and 5mM ATP was added for 30min to induce macrophage inflammasome activation. The culture medium was replaced with FHS@Ficoll nanoparticles (2μg / μL, 5μL) (RhB-labeled nanoparticles and FITC-labeled Ficoll), co-cultured with macrophages, washed twice with PBS after 3h, and 500μL 4% paraformaldehyde was added and fixed at room temperature for 15min. 500μL PBS was added to the 24-well plate and washed twice; far-infrared fluorescent labeled phalloidin was added for staining at room temperature for 30min, and the plate was sealed with anti-fluorescence quenching sealing agent containing DAPI after washing three times with PBS. The phagocytosis of FHS@Ficoll by macrophages was observed using a laser confocal microscope.

[0065] Figure 3 Schematic diagram of the selective engulfment of FHS@Ficoll nanoparticles by proinflammatory macrophages, the glutathione-responsive release of crowding agents, and the eventual triggering of WNK phase separation.

[0066] Figure 4 The fluorescence confocal microscopy results of FHS@Ficoll nanoparticles being phagocytosed by pro-inflammatory macrophages. Pro-inflammatory macrophages induced in vitro can phagocytize red fluorescently labeled nanoparticles, and the release of green fluorescently labeled crowding agent polysucrose can be seen inside the cells.

[0067] Example 5

[0068] This example is an application of the FHS@Ficoll nanoparticles prepared by the present invention, which is used to regulate the intracellular crowding state of pro-inflammatory macrophages and trigger specific protein phase separation to alleviate the inflammatory state of macrophages. The process includes the following steps:

[0069] 1. After macrophages induced inflammasome activation, FHS@Ficoll nanoparticles were co-cultured with macrophages. After 3 hours, the cells were washed twice with PBS, and 500 μL of 4% paraformaldehyde was added and fixed at room temperature for 15 minutes. Washed twice with PBS; permeabilized with 0.1% Trition for 10 minutes, washed twice with PBS, and blocked with 2.5% BSA at room temperature for 1 hour; anti-WNK antibody (1:200 dilution of antibody) was added and incubated overnight at 4°C; washed twice with PBS the next day; incubated with goat anti-rabbit red fluorescent secondary antibody (1:200 dilution of antibody) at room temperature for 2 hours; washed three times with PBS and sealed with anti-fluorescence quenching sealing agent containing DAPI. The phase separation of WNK in pro-inflammatory macrophages was observed using a laser confocal microscope.

[0070] Figure 5 This is a fluorescence confocal microscopy result of WNK phase separation triggered in pro-inflammatory macrophages. Immunofluorescence showed that WNK in activated macrophages was diffusely distributed in the cytoplasm, while WNK was observed to aggregate in bright spots in the FHS@Ficoll group, indicating that the delivery of polysucrose regulated the intracellular crowding and triggered WNK phase separation.

[0071] 2. After macrophages induced inflammasome activation, FHS@Ficoll nanoparticles were co-cultured with macrophages, washed twice with PBS after 3 hours, and 500μL 4% paraformaldehyde was added and fixed at room temperature for 15 minutes. Washed twice with PBS; added 0.1% Trition for permeabilization for 10 minutes, washed three times with PBS, blocked with 2.5% BSA at room temperature for 1 hour; added anti-iNOS antibody (1:200 diluted antibody) and incubated overnight at 4°C; washed twice with PBS the next day; incubated with goat anti-rabbit red fluorescent secondary antibody (1:200 diluted antibody) at room temperature for 2 hours; washed three times with PBS and sealed with anti-fluorescence quenching sealing agent containing DAPI. Laser confocal microscopy was used to observe the inflammatory changes in pro-inflammatory macrophages.

[0072] Figure 6 The fluorescence confocal microscopy results of iNOS (inducible nitric oxide synthase) expression in unactivated macrophages, proinflammatory macrophages, and proinflammatory macrophages after application of FHS@Ficoll. Immunofluorescence showed that iNOS was almost not expressed in unactivated macrophages, while iNOS expression in activated macrophages increased significantly. The application of FHS@Ficoll group showed that iNOS expression decreased, indicating that the delivery of polysucrose alleviated the inflammatory state of cells.

[0073] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing nanoparticles capable of regulating the degree of intracellular crowding, characterized in that: The following steps are involved: S1. Preparation of BSN@Ficoll Nanoparticles Dissolving polysucrose in water to obtain a polysucrose aqueous solution, then mixing the polysucrose aqueous solution, ethyl orthosilicate and bis[3-(triethoxysilyl)propyl] disulfide, and shaking to obtain a mixture; adding the mixture to a mixed organic solvent of Triton X-100, n-hexanol and cyclohexane, and then adding 30% by mass of ammonia water to obtain a water-oil emulsion, stirring at room temperature for 12-24 hours, adding acetone to precipitate solid particles, and then centrifuging and washing to obtain polysucrose-encapsulated disulfide-containing degradable silica nanoparticles BSN@Ficoll; S2. Preparation of BSN@Ficoll-NH2 nanoparticles The BSN@Ficoll nanoparticles obtained in S1 were dispersed in anhydrous ethanol, and 30% ammonia water was added. Under stirring conditions of 10000 rpm / min, 3-aminopropyltriethoxysilane was added, and the mixture was reacted at 25-45°C for 12-24 hours. The obtained product was washed and dried to obtain silica nanoparticles BSN@Ficoll-NH2 with amino terminal groups. S3. Preparation of FHS@Ficoll Nanoparticles Hyaluronic acid was mixed with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to activate the carboxyl group. After 30 minutes, the BSN@Ficoll-NH2 nanoparticles obtained by S2 were added and reacted at room temperature for 24-48 hours to obtain hyaluronic acid-modified nanoparticles HA-BSN@Ficoll-NH2. Folic acid was mixed with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to activate the carboxyl group. After 30 minutes, HA-BSN@Ficoll-NH2 nanoparticles were added and reacted at room temperature for 24-48 hours to obtain folic acid and hyaluronic acid modified nanoparticles FA-HA-BSN@Ficoll, referred to as FHS@Ficoll.

2. The method according to claim 1, characterized in that The polysucrose in S1 is Ficoll-400, with a relative molecular weight of 400,000 and a purity of ≥99%. The concentration of the polysucrose aqueous solution is 20 mg / mL.

3. The method according to claim 1, characterized in that The volume ratio of polysucrose aqueous solution, ethyl orthosilicate and bis[3-(triethoxysilyl)propyl]disulfide in the mixture of S1 is 10:(3-7):(5-9); The volume ratio of Triton X-100, n-hexanol and cyclohexane in the mixed organic solvent is 1:(1-2):(2-5).

4. The method according to claim 1, characterized in that: The volume ratio of the polysucrose aqueous solution to 30% ammonia water in S1 is (2-3):1; the amount of acetone added is 1-3 times the total volume of the reaction system.

5. The method according to claim 1, characterized in that The amount of BSN@Ficoll nanoparticles and anhydrous ethanol used in S2 is 25 mg:25 mL, and the volume ratio of anhydrous ethanol, ammonia water and 3-aminopropyltriethoxysilane is (100-200):2:

1.

6. The method according to claim 1, characterized in that The drying temperature in S2 is 25-45° C., and the drying time is 12-24 hours.

7. The method according to claim 1, characterized in that The molecular weight of the hyaluronic acid in S3 is 11500 Da, and the mass ratio of the BSN@Ficoll-NH2 nanoparticles to the hyaluronic acid is 25 mg:203 mg.

8. The method according to claim 1, characterized in that The molar ratio of hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in S3 is 1:(2-4):(1-2).

9. The method according to claim 1, characterized in that: The mass ratio of HA-BSN@Ficoll-NH2 nanoparticles to folic acid described in S3 is 25 mg:236 mg.

10. The method according to claim 1, characterized in that The molar ratio of folic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in S3 is 1:(2-4):(1-2).

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