Bionic microglia cell membrane coated ZnS-coated BSA nano-particles as well as preparation method and application thereof

Abstract

The invention provides a bionic microglial cell membrane coated ZnS-coated BSA (bovine serum albumin) nano-particle as well as a preparation method and application thereof, and belongs to the technical field of biological nano-materials. According to the invention, a self-assembly method is adopted, and zinc acetate and sodium hydrosulfide are mixed with bovine serum albumin (BSA) to synthesize zinc sulfide nanoparticles (ZnS-coated BSA) of bovine serum albumin. The BSA has the characteristics of affinity to metal ions and prolonged half-life period, so that the BSA can be widely applied to nano medicine. The ZnS-coated BSA nanoparticles (ZnS-coated BSA-coated MM) coated with bionic microglial cell membranes are prepared by using an extrusion method, the ZnS-coated BSA-coated MM enhances the accumulation of zinc sulfide nanoparticles of bovine serum albumin at an injured part, reduces the loss of hydrogen sulfide in blood circulation, promotes the regeneration of functional neurons, inhibits the formation of nervous scars, and improves the motor function recovery of SCI mice.

Description

Technical Field

[0001] The present invention belongs to the technical field of bio-nanomaterials, and particularly relates to a biomimetic microglial cell membrane-coated ZnS@BSA nanoparticle, a preparation method thereof, and an application thereof. Background Art

[0002] Due to the development of industries such as construction, transportation, and sports activities, the incidence of spinal cord injury (SCI) caused by trauma has been on the rise, especially having a greater impact on young men. Approximately 30 million people globally suffer from SCI, imposing a huge pressure on economic resources. Primary SCI results from direct impact, leading to secondary injury processes including ischemia, inflammation, and oxidative stress, which exacerbate the injury. After traumatic SCI, phagocytic immune cells (such as microglia, neutrophils, and macrophages) are activated and recruited to the injury site. These cells exacerbate neuronal death by releasing reactive oxygen species (ROS), which not only promote inflammation but also aggravate secondary injury. Therefore, treatment strategies focusing on alleviating oxidative stress and inflammatory responses are crucial for improving the prognosis after SCI. Existing SCI treatment methods, including drugs, surgery, and hyperbaric oxygen therapy, aim to promote axonal regeneration but have limited effects. Therefore, there is an urgent need to develop more effective protection strategies. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a biomimetic microglial cell membrane-coated ZnS@BSA nanoparticle, a preparation method thereof, and an application thereof. The present invention uses a biomimetic cell membrane-coated nanoparticle with low immunogenicity, long circulation time, and high drug delivery efficiency as a raw material to prepare a biomimetic microglial cell membrane-coated ZnS@BSA nanoparticle (ZnS@BSA@MM). The ZnS@BSA@MM prepared by the present invention promotes functional neuron regeneration, inhibits nerve scar formation, and improves the recovery of motor function in SCI mice.

[0004] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides a biomimetic microglial cell membrane-coated ZnS@BSA nanoparticle, which comprises a microglial cell membrane and a zinc sulfide nanoparticle of bovine serum albumin with a final concentration of 300 - 500 mM.

[0006] Preferably, the preparation method of the zinc sulfide nanoparticle of bovine serum albumin is as follows:

[0007] (2.1) Dissolve bovine serum albumin in water, and sequentially add Zn(CH 3 COO) 2A solution, a NaHS solution and a NaOH solution are used to obtain a mixture;

[0008] (2.2) A crosslinking agent is added to the mixture. After the crosslinking reaction, dialysis is carried out to obtain zinc sulfide nanoparticles of bovine serum albumin.

[0009] Preferably, the crosslinking agent is a glutaraldehyde solution, and the addition amount of the crosslinking agent to the volume ratio of the mixture is 1:400 - 600.

[0010] Preferably, the temperature of the crosslinking reaction is 20 - 30 °C, and the time of the crosslinking reaction is 3 - 5 h.

[0011] The present invention provides a preparation method of the ZnS@BSA nanoparticles coated with the biomimetic microglial cell membrane, comprising the following steps:

[0012] (5.1) Microglial cells are resuspended in a hypotonic solution and sonicated to obtain a mixture;

[0013] (5.2) The mixture is centrifuged, the supernatant is collected, and centrifuged again to obtain a precipitate;

[0014] (5.3) The precipitate is washed, centrifuged, and the precipitate is resuspended to obtain a cell membrane suspension;

[0015] (5.4) The cell membrane suspension is mixed with the zinc sulfide nanoparticles of bovine serum albumin, sonicated, filtered, and the filtered component is collected to obtain the ZnS@BSA nanoparticles coated with the biomimetic microglial cell membrane.

[0016] Preferably, the microglial cells are BV2 microglial cell line.

[0017] Preferably, the hypotonic solution uses water as a solvent and contains sucrose with a final concentration of 0.2 - 0.3 M, EDTA of 0.5 - 2 mM, HEPES-NaOH of 15 - 30 mM, and protease inhibitor of 0.1 - 1.0 mmol / L.

[0018] Preferably, the protease inhibitor is a serine protease inhibitor.

[0019] Preferably, in step (5.1), the power of the sonication is 100 - 300 W, the time of the sonication is 1 - 5 s, the number of sonication times is 20 - 30 times, and the time interval between two adjacent sonications is 5 - 10 s.

[0020] The present invention also provides an application of the ZnS@BSA nanoparticles coated with the biomimetic microglial cell membrane in the preparation of a preparation for treating spinal cord injury.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention detects the changes in the metal ion concentration in cerebrospinal fluid (CSF) by inductively coupled plasma mass spectrometry (ICP-MS). The results confirm that the increase in calcium and iron ion concentrations may lead to neuronal apoptosis and necrosis, while the zinc ion concentration decreases in the acute stage of SCI. To supplement the zinc ion concentration after SCI, the present invention uses a self-assembly method to synthesize zinc sulfide nanoparticles of bovine serum albumin (ZnS@BSA) by mixing zinc acetate and sodium hydrosulfide with bovine serum albumin (BSA). Due to the affinity of BSA for metal ions and its characteristic of extending the half-life, it is widely used in nanomedicine. The present invention prepares biomimetic microglial cell membrane-coated ZnS@BSA nanoparticles (ZnS@BSA@MM) by the extrusion method. ZnS@BSA@MM exhibits targeted delivery to the SCI site and can release Zn 2+ and H 2 S in an acidic microenvironment. Physical characterization confirms that ZnS@BSA releases H 2 S in the inflammatory microenvironment of spinal cord injury, showing an antioxidant effect. ZnS@BSA@MM inhibits the NF-κB signaling pathway in immune cells, reduces inflammation, and inhibits nerve scar formation. In vivo, intravenous injection of ZnS@BSA@MM induces axonal regeneration of neuronal cells in SCI mice, inhibits the inflammatory response, and thus promotes motor function recovery. The application of the biomimetic microglial cell membrane enhances the targeted effect of ZnS@BSA on the SCI site, while reducing the depletion of H 2 S in the circulation and its accumulation in other organ tissues. Finally, ZnS@BSA@MM can be excreted from the body through renal metabolism. The research results emphasize the potential of combining metal sulfides with biomimetic membranes to address various pathological complications and enhance SCI repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a schematic diagram of the synthesis of ZnS@BSA;

[0023] Figure 2 is a transmission electron micrograph of ZnS@BSA, where A is ZnS@BSA obtained after using a cross-linking agent, the scale bar is 500 nm, and B is ZnS@BSA obtained without using a cross-linking agent, the scale bar is 0.2 μm;

[0024] Figure 3 is an XRD pattern of ZnS@BSA;

[0025] Figure 4 is the particle size of ZnS@BSA in different solutions;

[0026] Figure 5 is the absorbance detection of hydrogen sulfide released by ZnS@BSA at different concentrations;

[0027] Figure 6 Absorbance detection of the release of hydrogen sulfide by ZnS@BSA at different times;

[0028] Figure 7 Regression equation formula for the concentration and absorbance of ZnS@BSA;

[0029] Figure 8 Particle size change of ZnS@BSA in solutions with different pH values;

[0030] Figure 9 Potential change of ZnS@BSA in physiological saline and culture medium;

[0031] Figure 10 Detection of the release of hydrogen sulfide by ZnS@BSA in solutions with different pH values over time using lead acetate test paper;

[0032] Figure 11 Scavenging efficiency of ZnS@BSA on ABTS radicals;

[0033] Figure 12 Scavenging efficiency of ZnS@BSA on oxygen radicals;

[0034] Figure 13 Scavenging ability of ZnS@BSA and natural reactive oxygen species scavengers on ABTS radicals;

[0035] Figure 14 Inhibition of microglial polarization towards the M1 pro-inflammatory type by ZnS@BSA;

[0036] Figure 15 Western blot quantitative analysis chart of the inhibition of the activation of the microglial NF-κB signaling axis by ZnS@BSA;

[0037] Figure 16 Inhibition of the production of reactive oxygen species in microglia by ZnS@BSA;

[0038] Figure 17 Diagram of the promotion of axon growth of PC12 neuronal cells in the pathological microenvironment by ZnS@BSA;

[0039] Figure 18 Schematic diagram of the preparation of ZnS@BSA@MM by the extrusion method;

[0040] Figure 19 Transmission electron microscopy image of ZnS@BSA@MM;

[0041] Figure 20 Flow cytometry detection results of ZnS@BSA@MM;

[0042] Figure 21Figure showing the expression of CD9, CD36, Iba1, and β-actin membrane marker proteins by ZnS@BSA@MM;

[0043] Figure 22 Figure showing the endocytosis of different concentrations of ZnS@BSA@MM by microglia;

[0044] Figure 23 Figure showing ZnS@BSA@MM entering the cell by forming endosomes;

[0045] Figure 24 Figure showing the swimming experiment verifying the promotion of hindlimb motor function recovery in spinal cord injury mice by ZnS@BSA@MM;

[0046] Figure 25 Figure showing the footprint experiment verifying the promotion of hindlimb motor function recovery in spinal cord injury mice by ZnS@BSA@MM;

[0047] Figure 26 Figure showing the muscle electrophysiology experiment verifying the promotion of hindlimb motor function recovery in spinal cord injury mice by ZnS@BSA@MM;

[0048] Figure 27 Figure showing the fluorescence enrichment of ZnS@BSA@MM in various organs of spinal cord injury mice;

[0049] Figure 28 Figure showing the targeted action of ZnS@BSA@MM on the injured spinal cord;

[0050] Figure 29 Figure showing that ZnS@BSA@MM effectively promotes spinal cord tissue repair;

[0051] Figure 30 Figure showing that HE sections confirm the promotion of spinal cord tissue repair by ZnS@BSA@MM and the reduction of tissue cavitation;

[0052] Figure 31 Figure showing that ZnS@BSA@MM delays the atrophy of bladder wall muscles;

[0053] Figure 32 Figure showing that ZnS@BSA@MM promotes the regeneration of neuron cells and synaptic growth in the injury center. Detailed implementation method

[0054] The present invention provides a ZnS@BSA nanoparticle coated with a biomimetic microglial cell membrane, and the ZnS@BSA nanoparticle coated with the biomimetic microglial cell membrane comprises a microglial cell membrane and ZnS@BSA with a final concentration of 300 - 500 mM.

[0055] In the present invention, the preparation method of the ZnS@BSA is as follows:

[0056] (2.1) Dissolve bovine serum albumin in water, and successively add Zn(CH 3 COO) 2 solution, NaHS solution and NaOH solution to obtain a mixture;

[0057] (2.2) Add a crosslinking agent to the mixture, after crosslinking reaction, dialyze to obtain ZnS@BSA.

[0058] In the present invention, dissolve bovine serum albumin in water, and successively add Zn(CH 3 COO) 2 solution, NaHS solution and NaOH solution to obtain a mixture. Dissolve bovine serum albumin in water, and the mass-volume ratio of the bovine serum albumin to water is 15 - 30 mg: 2 - 4 mL, preferably 17 - 25 mg: 2.5 - 3.5 mL, and further preferably 20 mg: 3 mL; successively add Zn(CH 3 COO) 2 solution, NaHS solution and NaOH solution, wherein after adding Zn(CH 3 COO) 2 solution, carry out stirring treatment, and the stirring time is 3 - 5 h, preferably 3.5 - 4.5 h, and further preferably 4 h. The concentration of the added Zn(CH 3 COO) 2 solution is 23 - 30 mg / mL, preferably 25 - 28 mg / mL, and further preferably 26 mg / mL; the mass-volume ratio of bovine serum albumin to Zn(CH 3 COO) 2 solution is 30 - 50 mg: 1 mL, preferably 35 - 45 mg: 1 mL, and further preferably 40 mg: 1 mL; the concentration of the NaHS solution is 40 - 60 mg / mL, preferably 45 - 55 mg / mL, and further preferably 50 mg / mL, and the mass-volume ratio of bovine serum albumin to NaHS solution is 30 - 50 mg: 1 - 5 mL, preferably 35 - 45 mg: 2 - 4 mL, and further preferably 40 mg: 3 mL; the concentration of the NaOH solution is 0.05 - 0.2 M, preferably 0.07 M - 0.15 M, and further preferably 0.1 M, and the mass-volume ratio of bovine serum albumin to NaOH solution is 30 - 50 mg: 1 mL, preferably 35 - 45 mg: 1 mL, and further preferably 40 mg: 1 mL to obtain a mixture.

[0059] In the present invention, a crosslinking agent is added to a mixture. After the crosslinking reaction, dialysis is performed to obtain ZnS@BSA. A crosslinking agent with a volume ratio to the mixture of 1:400 to 600, preferably 1:450 to 550, and more preferably 1:500 is added to the mixture. The crosslinking agent is a glutaraldehyde solution, and a crosslinking reaction is carried out. The temperature of the crosslinking reaction is 20 to 30 °C, preferably 23 to 28 °C, and more preferably 25 °C. The time of the crosslinking reaction is 3 to 5 h, preferably 3.5 to 4.5 h, and more preferably 4 h. After the crosslinking reaction, dialysis is performed. The dialysis uses a dialysis bag with an MW of 8000 to 14000, and the dialysis time is 3 to 5 h, preferably 3.5 to 4.5 h, and more preferably 4 h, thereby obtaining ZnS@BSA. The diameter of the ZnS@BSA is 80 to 120 nm, preferably 90 to 110 nm, and more preferably 100 nm.

[0060] The present invention provides a method for preparing the ZnS@BSA nanoparticles coated with the biomimetic microglial cell membrane, comprising the following steps:

[0061] (5.1) Resuspend microglial cells in a hypotonic solution and perform sonication to obtain a mixture;

[0062] (5.2) Centrifuge the mixture, collect the supernatant, and centrifuge again to obtain a precipitate;

[0063] (5.3) Wash the precipitate, centrifuge, and resuspend the precipitate to obtain a cell membrane suspension;

[0064] (5.4) Mix the cell membrane suspension with ZnS@BSA, perform sonication, filter, and collect the filtered fraction to obtain the ZnS@BSA nanoparticles coated with the biomimetic microglial cell membrane.

[0065] In the present invention, microglial cells are resuspended in a hypotonic solution and sonicated to obtain a mixture. The microglial cells are the BV2 microglial cell line, and the final concentration of the microglial cells is 1×10 7 cells / mL. The hypotonic solution uses water as a solvent and contains sucrose with a final concentration of 0.2 to 0.3 M, EDTA with a concentration of 0.5 to 2 mM, HEPES-OH with a concentration of 15 to 30 mM, and a protease inhibitor with a concentration of 0.1 to 1.0 mmol / L. The water is preferably ultrapure water, and the protease inhibitor is a serine protease inhibitor. The power of the sonication in step (5.1) is 100 to 300 W, preferably 150 to 250 W, and more preferably 200 W. The time of the sonication is 1 to 5 s, preferably 2 to 4 s, and more preferably 3 s. The number of sonication times is 20 to 30 times, preferably 23 to 28 times, and more preferably 25 times. The time interval between two adjacent sonications is 5 to 10 s, preferably 6 to 8 s, and more preferably 7 s, thereby obtaining a mixture.

[0066] In the present invention, the mixture is centrifuged, and the supernatant is collected and centrifuged again to obtain a precipitate. The mixture is centrifuged at 3000 - 5000 rpm for 5 - 15 min. The centrifugation speed is preferably 3500 - 4500 rpm, more preferably 4000 rpm, and the centrifugation time is preferably 8 - 12 min, more preferably 10 min, to remove large cell debris. The supernatant is collected and centrifuged again at 14000 - 16000 rpm for 15 - 30 min. The speed of the second centrifugation is preferably 14500 - 15000 rpm, more preferably 14800 rpm, and the time of the second centrifugation is preferably 18 - 25 min, more preferably 20 min, to obtain the precipitate.

[0067] In the present invention, the precipitate is washed, centrifuged, and resuspended to obtain a cell membrane suspension. The precipitate is washed 1 - 3 times, preferably 2 times, with physiological saline and centrifuged at 14000 - 16000 rpm for 15 - 30 min. The centrifugation speed is preferably 14500 - 15000 rpm, more preferably 14800 rpm, and the centrifugation time is preferably 18 - 25 min, more preferably 20 min. The supernatant is removed, and 200 μL of resuspension solution, which is a hypotonic solution, is added to obtain the cell membrane suspension.

[0068] In the present invention, the cell membrane suspension is mixed with ZnS@BSA, sonicated, filtered, and the filtered fraction is collected to obtain ZnS@BSA nanoparticles coated with biomimetic microglial cell membranes. ZnS@BSA with a final concentration of 300 - 500 mM, preferably 350 - 450 mM, more preferably 400 mM, is added to the cell membrane suspension and sonicated for 1 - 3 h, preferably 1.5 - 2.5 h, more preferably 2 h. The product passing through a 200 nm polycarbonate porous membrane for 15 times is collected to obtain ZnS@BSA nanoparticles coated with biomimetic microglial cell membranes.

[0069] The present invention also provides the application of the ZnS@BSA nanoparticles coated with biomimetic microglial cell membranes in the preparation of a preparation for treating spinal cord injury.

[0070] The technical solutions provided by the present invention are described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present invention.

[0071] Example 1 Synthesis and Verification of ZnS@BSA

[0072] 1. Synthesis of ZnS@BSA

[0073] Dissolve 40.0 mg of bovine serum albumin (BSA, B2064-10G, SIGMA) in 6.0 mL of deionized water, and then add 1.0 mL of a Zn(CH 3 COO) 2 solution with a concentration of 26 mg / mL. After stirring for 4 h, add 3.0 mL of a NaHS solution with a concentration of 50 mg / mL and 1.0 mL of a NaOH solution with a concentration of 0.1 M. Then add 20 μL of glutaraldehyde solution and carry out a cross-linking reaction at room temperature (25 °C) for 4 h. After the reaction, dialyze the mixture in deionized water (MW: 8000-14000) for 4 h to obtain ZnS@BSA.

[0074] 2. Verification of ZnS@BSA

[0075] 2.1 Structural characterization

[0076] Characterize the morphology of ZnS@BSA using a transmission electron microscope (TEM, tecnai F20). Measure the crystal structure and surface chemical composition of ZnS@BSA by X-ray diffraction (XRD, Panalytical Empyrean). Detect the particle size and potential of ZnS@BSA by Zetasizer Nano-ZS (Malvern Instruments, UK). Disperse ZnS@BSA in ultrapure water, physiological saline, and Dulbecco's modified Eagle medium to judge the effect of the solution on the particle size of ZnS@BSA. The results are as Figures 2 to 9 shown.

[0077] From Figure 2 A in Figure 3 and 3 COO) 2 it can be seen that the particle size of ZnS@BSA is about 100 nm, showing a uniform spherical structure and excellent crystallinity, which is confirmed by transmission electron microscopy (TEM). X-ray diffraction (XRD) analysis reveals the crystal structure of ZnS@BSA and confirms the main diffraction peaks of ZnS. The results show that by the self-assembly method, co-loading Zn(CH Figure 1 ) and NaHS with BSA protein can form ZnS@BSA nanomaterials ( 3 COO) 2 . That is, Zn(CH 2+ is combined with BSA through hydrophilic and hydrophobic interactions to form a BSA solution chelating Zn 2 . Subsequently, NaHS is added to the solution as a typical H Figure 2As can be seen from B in [reference], when the cross-linking reaction is carried out without using a cross-linking agent, the obtained product is non-uniform and spherical nanoparticles cannot be formed. Therefore, the formation of ZnS@BSA requires the presence of a cross-linking agent.

[0078] As can be seen from Figure 4 that ZnS@BSA exhibits excellent dispersibility and stability in ultrapure water, physiological saline, and Dulbecco's modified Eagle medium solution, and the particle size distribution is uniform.

[0079] Figure 5 Compared with Figure 6 The UV-vis-NIR spectra of [reference] show that the methylene blue (MB) probe used for detecting hydrogen sulfide exhibits H 2 2S gas generation ability in a concentration-dependent and time-dependent manner. H 2 2S, as an endogenous gas signaling molecule, is involved in key physiological processes and disease states, especially in the development of central nervous system diseases. This indicates that the H 2 2S-mediated signaling pathway has the potential to treat SCI. The UV-vis-NIR spectra reveal that the peak of ZnS appears at a wavelength of 300 nm. According to the absorbance of ZnS at different molar concentrations, a formula for calculating the molar concentration of ZnS corresponding to different absorbances was determined ( Figure 7 ). The zeta potential indicates ( Figure 9 ) that ZnS@BSA carries partial charges in physiological saline and DMEM solution. Although there is no statistically significant difference, the cell membrane shows the characteristics of positive charge inside and negative charge outside, which indicates that the charge characteristics of ZnS@BSA may enhance its adsorption to the cell membrane, thereby promoting membrane coating. ZnS@BSA targeting the SCI site requires a stable particle size in the inflammatory microenvironment. Figure 8 As shown in [figure], by evaluating the diameter change of ZnS@BSA in solutions with different pH values, its stability under pathological conditions was confirmed. It was observed that ZnS@BSA remained stable in solutions with different pH values, and the particle size decreased slightly in the acidic environment, which may be due to the degradation of the material caused by the release of hydrogen sulfide.

[0080] 2.2 Hydrogen sulfide (H 2 2S) release of ZnS@BSA

[0081] Qualitative analysis of H 2 2S generation was carried out using the MB probe. First, 200 μL of FeCl 2 (1 mg / mL) and 200 μL of H 2 2O 2(10 mM) was added to 10 mL of deionized water, and then 50 μL of MB (0.5 mM) was added to the solution. The mixture was used to detect the H2S release of ZnS@BSA under different concentrations and different treatment times. The change in the absorbance of MB at 664 nm indicated the H2S release of ZnS@BSA. 2 In addition, the WSP-1 probe (50 μM, 1 mL) was used to quantitatively analyze the H2S release in cells, and the fluorescence intensity was measured using a microplate reader (Ex = 465 nm, Em = 515 nm). 2 In addition, to evaluate the ability of ZnS@BSA to release H2S, lead acetate test paper was used. A piece of lead acetate ((CH3COO)2Pb) test paper was placed above a small petri dish containing ZnS@BSA, and the reaction was carried out at 37 °C for 3 h. The color change of the test paper was observed, and the results are shown in 2 3 2 Figure 10

[0082] As Figure 10 known, 20 μM of ZnS@BSA was dispersed in PBS with different pH values to simulate the inflammatory microenvironment. The depth of the solution color represented the generated H2S concentration. The darker the color, the higher the concentration. At the same time, ZnS@BSA gradually degraded in the PBS solution at pH 5.5, and the color of the lead acetate test paper significantly deepened, indicating that the acidic microenvironment promoted the H2S release. ZnS@BSA exhibited a pH-responsive ability and released H2S under the action of an acidic microenvironment. 2 2 2

[0083] 2.3 Quantitative analysis of ·OH elimination

[0084] Excessive ROS are considered to be the main cause of the formation of an inhibitory microenvironment after SCI. ROS can trigger oxidative stress, cytotoxic nerve activation, and lead to subsequent severe inflammatory reactions. H2S with the ability to eliminate excessive ROS can effectively regulate the harmful microenvironment. The anti-inflammatory properties of ZnS@BSA were comprehensively studied. The antioxidant properties and scavenging efficiency of ZnS@BSA were evaluated using 2,2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). 2

[0085] The decomposition of ·OH by ZnS@BSA under the catalysis of H2O2 was detected using the TMB colorimetric method. First, 4 μL of TMB (80 mM) was added to 1 mL containing 100 μM H2O2 2 2 2 2 ​​​​​​​​​​​In the ZnS@BSA solution. Monitor the absorbance change at 654 nm to evaluate the elimination of ·OH. In addition, use the ABTS colorimetric method to measure the change of ABTS + The change. Evaluate the antioxidant capacity of ZnS@BSA by observing the absorbance change at 414 nm of a UV-vis spectrophotometer, and the results are as Figures 11 to 13 shown.

[0086] It can be seen from Figure 11 that as the concentration of ZnS@BSA increases, the color of the ABTS + solution gradually fades and finally becomes clear, and the ABTS radical scavenging efficiency increases, indicating that ZnS@BSA has excellent ROS scavenging ability. It can be seen from Figure 12 that as the treatment time extends, ZnS@BSA at a concentration of 0.5 μM also effectively scavenges ROS. The scavenging effect of ZnS@BSA on hydroxyl radicals (·OH) was quantified using the ROS probe 3,3',5,5'-tetramethylbenzidine (TMB). The characteristic absorption peak of TMB gradually decreases, usually around 652 nm, indicating that ZnS@BSA releases H 2 S to scavenge ·OH.

[0087] Natural products have always been the richest source for drug development and are applied in the fields of infection, cardiovascular diseases, inflammation, etc. However, the limited water solubility and bioavailability of natural products often limit their wider application. To verify the ROS scavenging ability of ZnS@BSA, existing ROS scavengers (hesperetin, curcumin, vitamin C, and quercetin) were used as controls for comparison, and the results are as Figure 13 shown.

[0088] It can be seen from Figure 13 that compared with natural ROS scavengers such as hesperetin, curcumin, vitamin C, and quercetin, at equimolar concentrations, ZnS@BSA exhibits significantly stronger ROS scavenging ability, exceeding that of hesperetin and curcumin.

[0089] 2.4 Regulation of the inflammatory response of microglia by ZnS@BSA

[0090] Filter the ZnS@BSA nanomaterial through a 0.2 μm filter to remove bacteria, and then co-culture it with microglia. BV2 microglia (2.0×10 4Cells / well) were purchased from Wuhan Punosai Biotechnology Co., Ltd., seeded in 24-well plates with DMEM medium containing 10% FBS, and treated with LPS (100 ng / mL) at specific times. After stimulation, the samples were stained with CD86 (Abcam, ab239075, USA), CD206 (Abcam, ab125028, USA), and DAPI (Invitrogen, 62247, USA). The phenotypic polarization of microglia was observed using a confocal laser scanning microscope (CLSM, LSM 800 with Airyscan, ZEISS, Germany).

[0091] It can be seen from Figure 14 that LPS-stimulated microglia highly expressed CD86, indicating M1 polarization, but ZnS@BSA inhibited M1-like polarization. Similarly, the fluorescence of CD86 in the NaHS group decreased, indicating that H 2 S could delay the polarization of M1 microglia.

[0092] Western blot analysis: BV2 microglia were cultured in 6-well plates and treated as described above. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Then the membranes were incubated with primary antibodies (IκBα, phosphorylated-IκBα, NF-κBp65, phosphorylated-NF-κB p65) and secondary antibody (goat anti-rabbit IgG H+L HRP). Protein bands were detected using enhanced chemiluminescence reagents (BiYuntian, Wuxi, China) and quantified using ImageJ software. The levels of TNF-α, IL-1β, and IL-4 secreted by microglia were quantified using an ELISA kit (mlbio, Shanghai, China), and the results are shown in Figure 15 .

[0093] It can be seen from Figure 15 that the CD86 level in the LPS treatment group was significantly higher than that in the control group, indicating an increase in CD86 expression in the inflammatory state induced by LPS. Treatment with ZnS@BSA significantly reduced the CD86 level. In addition, LPS significantly increased the expression of phosphorylated IκBα (p-IκBα) and p-p65, while ZnS@BSA partially alleviated this phenomenon. In summary, ZnS@BSA may inhibit M1 polarization and promote M2 microglia polarization through the NF-κB pathway.

[0094] 2.5 ROS Scavenging of ZnS@BSA in BV2 Cells

[0095] To verify the antioxidant performance of ZnS@BSA in microglia, the ROS probe DCFH-DA (D6883, Sigma) was used. The ROS level in BV2 cells was quantitatively analyzed based on the fluorescence intensity of DCFH-DA. BV2 microglia were cultured in 100 μM H 2 O 2 for 12 h. After stimulation, the cells were stained with 20 μM 2',7'-dichlorofluorescein diacetate (DCFH-DA) for 30 min to qualitatively analyze the ROS scavenging efficiency, and the results are shown in Figure 16 .

[0096] As Figure 16 can be seen, the H 2 O 2 group showed a significant DCFH-DA signal, indicating that inflammatory mediators induced ROS production in microglia and triggered a strong inflammatory response. In contrast, the ROS level decreased in the NaHS and ZnS@BSA groups under H 2 O 2 stimulation. This indicates that the H 2 S released by ZnS@BSA effectively inhibited the production of ROS in neuroimmune cells.

[0097] 2.6 ZnS@BSA nanoparticles promote the proliferation and axon growth of PC12 cells

[0098] The neurite outgrowth function was evaluated using PC12 cells. To further compare the differences in neurite outgrowth among different groups, low-differentiated PC12 cells were incubated with PBS, LPS, LPS+NaHS, LPS+Zn 2+ , LPS+ZnS@BSA groups, respectively. 2.0×10 4 PC12 cells / mL were seeded in 24-well plates with DMEM medium containing 10% FBS. Cells were treated with different concentrations of ZnS@BSA, and then stained with F-actin (FITC-phalloidin, Invitrogen, B1370, USA) and DAPI to select a suitable experimental concentration. PC12 cells were treated with ZnCl 2 , NaHS and ZnS@BSA, and then stained with FITC-phalloidin (Invitrogen, B1370, USA) and DAPI. PC12 cells were incubated with LPS (100 ng / mL) for 24 h, and the changes in neurite length under pathological conditions were detected by CLSM after staining, and the results are shown in Figure 17 .

[0099] As Figure 17It can be seen that under pathological conditions, LPS inhibited the neurite outgrowth of PC12 cells (about 5 μm). However, in the LPS+ZnCl 2 (about 50 μm) and LPS+ZnS@BSA (about 115 μm) groups, the neurite length was still higher than that of other groups. Although H 2 S derived from NaHS could alleviate the inflammatory response, it could not promote the axonal growth of neurons. Overall, the results confirmed the role of ZnS@BSA in neurite growth. Therefore, ZnS@BSA could establish a suitable environment to stimulate the proliferation of nerve cells and the growth of neurites.

[0100] Example 2 Preparation and verification of ZnS@BSA nanoparticles coated with biomimetic microglial cell membranes

[0101] 1. Preparation of ZnS@BSA nanoparticles coated with biomimetic microglial cell membranes

[0102] ZnS@BSA@MM was synthesized by the extrusion method ( Figure 18 ). The BV2 microglial cell line was used as the source of cell membranes. The BV2 cells were collected and washed twice with physiological saline. Then the cells were resuspended in a hypotonic solution (a mixture of 0.25 M sucrose, 1 mM EDTA, 20 mM HEPES-NaOH, and serine protease inhibitors). The cells were sonicated in an ice bath using an ultrasonic processor (ultrasonic power 200 W, sonication for 3 s, interval 7 s, 25 times). The mixture was centrifuged at 4000 rpm for 10 min to remove larger cell debris. The supernatant was collected and centrifuged again at 14800 rpm for 20 min. The precipitate was collected, washed twice with physiological saline, and centrifuged again at 14800 rpm for 20 min. The cell membrane precipitate was resuspended. The cell membrane suspension was mixed with 400 mM ZnS@BSA and sonicated in an ice bath for 2 h. It was passed through a 200-nm polycarbonate porous membrane 15 times, and the passed product was collected, that is, ZnS@BSA nanoparticles coated with biomimetic microglial cell membranes (ZnS@BSA@MM) were obtained and stored in physiological saline containing protease inhibitors for later use.

[0103] 2. Verification of ZnS@BSA nanoparticles coated with biomimetic microglial cell membranes

[0104] 2.1 Characterization of ZnS@BSA nanoparticles coated with biomimetic microglial cell membranes

[0105] The effectiveness of the cell membrane-coated nanoparticles was evaluated by transmission electron microscopy ( Figure 19 ) and flow cytometry ( Figure 20)。The expression of CD9, CD63, IBA-1 and β-actin proteins in microglia, cell membranes and ZnS@BSA@MM was detected by Western blot (WB) analysis( Figure 21 )。

[0106] The morphology of microglial cell membranes and ZnS@BSA@MM was observed by transmission electron microscopy (TEM)( Figure 19 )。The microglial cell membrane showed a layered structure, while ZnS@BSA@MM presented an obvious core-shell structure, indicating that the ZnS@BSA@MM obtained in the present invention was a membrane-coated nanoparticle. To verify the successful encapsulation of the nanomaterial by the cell membrane, ZnS@BSA@MM was stained with zinc ion fluorescent probe (Zinquin) and hydrogen sulfide fluorescent probe (WSP-1) respectively( Figure 20 )。Flow cytometry analysis showed that the cell membrane effectively encapsulated ZnS@BSA, showing the ability to release hydrogen sulfide and zinc ions.

[0107] Western blot analysis showed( Figure 21 ) that the above groups all significantly expressed cell membrane protein (CD9 and CD63) markers. However, the activation marker IBA1 was not significantly expressed in each group, indicating that the extracted cell membrane was derived from resting microglia. However, the expression of the reference protein (β-actin) was low in the microglial cell membrane and ZnS@BSA@MM groups, which might be due to the fact that β-actin was mainly expressed in the cytoplasm and was almost absent in the cell membrane. It can be seen that the ZnS@BSA@MM constructed in the present invention was a cell membrane-coated zinc sulfide nanomaterial.

[0108] 2.2 Fluorescence analysis of ZnS@BSA nanoparticles coated with biomimetic microglial cell membranes

[0109] Fluorescent ZnS@BSA@MM was prepared using Cy5.5-labeled ZnS@BSA and Dil-labeled microglial cell membranes and co-incubated with microglial cells to observe the cellular phagocytosis efficiency of ZnS@BSA@MM at different concentrations. Although Cy5.5- and Dil-labeled NPs (red and orange-red) could be detected in cells, the intracellular trafficking pathways and organelle distributions of the nanoparticles still needed to be evaluated. The antibodies used included anti-EEA1 (ab2900, Abcam, Cambridge, MA, USA) for early endosomes, anti-Rab7 (ab126712, Abcam) for late endosomes, and anti-GM130 (ab169276, Abcam) for the Golgi apparatus. Goat anti-rabbit IgG H&L (Alexa Fluor 488, green) secondary antibody (ab150077, Abcam) was used to label ZnS@BSA@MM in the organelles. All samples were detected using a confocal microscope, and the results are shown in Figure 22 .

[0110] As Figure 22 shown, after 6 h of cellular uptake, immunofluorescence staining showed co-localization of red fluorescence (ZnS@BS) and orange-red fluorescence (cell membrane) on the microglial cell membrane and in the cytoplasm. Notably, increasing the concentration of ZnS@BSA@MM did not cause organelle swelling, cell membrane rupture, or cytoplasmic and nuclear degradation. This indicates that ZnS@BSA@MM has good biocompatibility with microglial cells, highlighting its potential in biomedical applications.

[0111] During the endocytosis of ZnS@BSA@MM, the present invention explored the localization of H 2 S and Zn 2+ in organelles, using cell markers related to the endocytic pathway, including Rab5, Rab7, and GM130. Microglial cells were exposed to 10 μM ZnS@BSA@MM for 30 min and 6 h, and the results are shown in Figure 23 .

[0112] As Figure 23It can be seen that initially, ZnS@BSA@MM co-localized with Rab5 within 30 min, indicating that ZnS@BSA@MM entered early endosomes, and the endocytosis efficiency within 30 min exceeded 70%. The co-localization of Rab7 (a marker of late endosomes and autophagosomes) increased significantly after 6 h, indicating that ZnS@BSA@MM was transported to late endosomes after endosome maturation and then localized in larger organelles. In addition, the present invention found that ZnS@BSA@MM co-localized with GM130, indicating that ZnS@BSA@MM might leave the cell through the endoplasmic reticulum-Golgi pathway after entering late endosomes and might affect other neuronal cells. These findings suggest that the co-localization of ZnS@BSA@MM might be related to exocytosis.

[0113] 2.3 Animal experiments on spinal cord injury

[0114] A mouse spinal cord transection model was used to evaluate spinal cord tissue regeneration, and the animal experiment was approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University. To evaluate the therapeutic effect of ZnS@BSA@MM in vivo, it was injected into mice with spinal cord transection via the tail vein. Forty-five six-week-old female C57BL / 6J mice (20±1 g, provided by the Experimental Animal Center of Soochow University) were divided into five groups. Sham-operated mice served as the normal control group, and spinal cord injury mice receiving saline tail vein injection were used as the SCI group. Spinal cord injury mice receiving tail vein injection of ZnS@BSA, cell membrane (MM), and ZnS@BSA@MM were classified into the ZnS@BSA, MM, and ZnS@BSA@MM groups, respectively. The nanoparticle solution was filtered and sterilized using a 0.2 μm filter.

[0115] The mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (125 μL / 20 g). Microsurgery was performed on C57BL / 6J mice using a digital stereomicroscope. The skin was incised 1.5 cm at the T9-T10 level to expose the spinous process and lamina. A complete transection injury was created at the T9 spinal cord level using a scalpel to establish a spinal cord transection injury model. The muscle and skin were sutured layer by layer, and each mouse was intramuscularly injected with antibiotics within 3 days after the operation. After SCI, ZnS@BSA, MM, ZnS@BSA@MM nanoparticles (150 μL, 2 mg / mL) or normal saline (150 μL) were injected via the tail vein. Injection was performed three times a week until the 14th week, and the results are shown in Figure 24 .

[0116] The Basso Mouse Scale (BMS) was used to evaluate the motor behavior of the mice. By Figure 24It can be seen that on the 0th day, the BMS scores of all SCI mice decreased to 0, confirming the successful establishment of the SCI model. Within 14 weeks, the mice in the SCI, ZnS@BSA, and MM groups remained hindlimb paralyzed, while the ZnS@BSA@MM group showed signs of motor function recovery. The BMS score of the ZnS@BSA@MM group was significantly higher than those of the PBS (0.75), ZnS@BSA (1.75), and MM (2.20) groups. In addition, compared with other groups, the body weight of the mice in the ZnS@BSA@MM group increased, indicating an improvement in malnutrition caused by SCI-induced hindlimb paralysis.

[0117] 2.4 Functional recovery and footprint analysis of SCI mice

[0118] The hindlimb recovery was evaluated using the Basso Mouse Scale (BMS) score, which ranges from 0 to 9, to measure the changes in hindlimb movement. The BMS scores of the mice were recorded weekly within 14 weeks. In addition, the movement of the treatment groups was videotaped to capture their hindlimb movement and weight-bearing ability when walking on a horizontal plane. The evaluation was conducted by observers using the double-blind method and recorded five times. All observers were trained before scoring consistency. Before footprint assessment, the mice were allowed to move freely in an open field for 5 min. For footprint analysis, the forelimbs were dipped into blue ink and the hindlimbs were dipped into red ink. Then the mice were guided to walk in a straight line on a white paper, and the results are shown in Figure 25 .

[0119] It can be seen from Figure 25 that the footprint analysis at 14 weeks post-operation revealed obvious differences among the groups. The hindlimb footprints of the control group always showed stable landing and the longest forefoot stride. In contrast, the hindfootprints of the PBS, ZnS@BSA, and MM groups showed a reduced footprint area and a significant lack of stride amplitude, indicating a significant decline in motor function. However, ZnS@BSA@MM treatment significantly improved the area of the hindfootprint and extended the forefoot stride, indicating a significant recovery of motor function.

[0120] 2.5 Louisville Swim Score (LSS) of SCI mice

[0121] At 14 weeks after SCI, the swimming performance was evaluated using LSS. The mice were placed in a transparent rectangular pool (50×30×10 cm) with a water temperature of 30 °C and a water depth sufficient to prevent the mice from touching the bottom of the pool. Each animal received a 4-min swimming training once in each evaluation. Two unaware observers scored the performance. In addition, a third party was responsible for animal handling and recorded a 1-min segment of each training using a digital video camera.

[0122] Their movements were recorded by cameras mounted on rails along the length of the pool. The joint angle dispersion and the motor function of the ankle and knee joints of ZnS@BSA@MM mice were significantly improved, especially significantly at 14 weeks after surgery.

[0123] 2.6 Motor electrophysiology

[0124] At 14 weeks after SCI, nerve function was evaluated by electrophysiological tests. Mice were deeply anesthetized with pentobarbital, and motor evoked potentials (MEPs) were recorded using an electrophysiological device. The motor cortex was activated by applying a single stimulus of 10 mA, and the results are shown in Figure 26 .

[0125] As can be seen from Figure 26 , at 14 weeks after surgery, ZnS@BSA@MM significantly shortened the latency and significantly increased the amplitude of motor evoked potentials (MEPs).

[0126] 2.7 Imaging of ZnS@BSA@MM targeting spinal cord injury

[0127] Fluorescence imaging: ZnS@BSA@MM-Cy5.5 NSs (150 μL, 2 mg / mL) or Cy5.5 solution was injected intravenously. After 48 h, the mice were sacrificed, and the spinal cord, heart, liver, spleen, lung, and kidney were collected for imaging, and the results are shown in Figure 27 .

[0128] As can be seen from Figure 27 , there were fluorescent signals in the SCI regions of both the ZnS@BSA@MM and MM groups, indicating that the membrane-coated or encapsulated nanoparticles could be delivered to the injury site. In contrast, ZnS@BSA mainly accumulated in the liver region and was partially recognized by the spleen tissue and enriched in the spleen region. Therefore, ZnS@BSA failed to play a role in the SCI region to promote nerve function recovery, which was consistent with the BMS score results. Coating with microglial cell membranes reduced the uptake of ZnS@BSA@MM by innate immune phagocytes in the liver and spleen regions, enabling it to pass through the blood circulation and play a role in the SCI region.

[0129] Fluorescence imaging of the spinal cord was observed in the control group, ZnS@BSA group, MM group, and ZnS@BSA@MM group. The results showed that the fluorescent nanoparticles were significantly enriched in the cell membrane and membrane-coated ZnS@BSA@MM groups in the SCI region. This indicates that the targeting recognition ability of ZnS@BSA@MM was enhanced due to the presence of the cell membrane, thus amplifying the therapeutic effect of ZnS at the SCI site.

[0130] Histological analysis: At 14 weeks after SCI, the mice were deeply anesthetized and normal saline was injected intracardially, followed by injection of 4% paraformaldehyde. A 1.5-cm-long spinal cord tissue ([ Figure 29 ) surrounding the injury site was removed, longitudinally sectioned to 10 μm thickness with a cryostat, and prepared for various experimental analyses. Hematoxylin-eosin (HE) staining was used to evaluate the morphology and size of the injury cavity ([ Figure 30 ), and HE staining was used to evaluate the recovery of neurogenic bladder in bladder tissue. The results are shown in Figure 31 .

[0131] As can be seen from Figures 29 to 30 , transverse wounds were still visible at the injury sites in the SCI and ZnS@BSA groups, while obvious cavities and scar tissues were observed in the MM group. In contrast, in the ZnS@BSA@MM group, these structural defects were significantly reduced. In HE-stained sections, it was also confirmed that ZnS@BSA@MM promoted SCI repair, and no significant defects or scar areas were found compared with other groups.

[0132] As can be seen from Figure 31 , the thickness of the detrusor muscle and the changes in muscle fibers were used as evaluation parameters for the recovery of neurological function after SCI. HE staining showed that in the control group, the muscle fibers of the detrusor muscle showed disorder and atrophy after surgery, and the detrusor muscle morphology recovered better in the ZnS@BSA@MM group than in the control group.

[0133] Immunofluorescence analysis: At 14 weeks after surgery, the mice were deeply anesthetized and 50 mL of normal saline was injected intracardially, followed by injection of 50 mL of 4% paraformaldehyde. Then the spinal cord containing the lesion area was dissected and dehydrated in 30% sucrose solution. The spinal cords of each group were photographed with a digital camera and then embedded in OCT freezing medium. The samples were sectioned along the sagittal axis to 8 μm thickness. Double staining with β3-tubulin and GFAP was used to evaluate the distribution of neurons and astrocytes at the lesion site. Immunofluorescence staining was used to identify β3-tubulin (Abcam, ab78078, USA), GFAP (Abcam, ab7260, USA), NeuN (Abcam, ab177487, USA), ChAT (Affinity, DF6964, USA), Nestin (Affinity, DF7754, USA), and synaptophysin (Abcam, ab32127, USA) at the lesion site. The results are shown in Figure 32 .

[0134] As can be seen from Figure 32 , in the SCI and ZnS@BSA groups, GFAP +Cells were concentrated around the lesion, with fewer neurons. The MM group showed appropriate lesion healing but retained a significant cavity area. In contrast, SCI treated with ZnS@BSA@MM showed a significant increase in the number of neurons at the lesion site, with neurons evenly distributed in the spinal cord and a moderate presence of astrocytes. These findings suggest that ZnS@BSA@MM has the potential to optimize the neuron-astrocyte balance and promote spinal cord regeneration. The local accumulation of ZnS@BSA@MM nanoparticles promoted the neural differentiation of endogenous NSCs, resulting in a significantly increased number of NeuN + cell numbers compared to other experimental groups. In addition, synapses play a key role in nerve regeneration and axonal extension. Zn 2+ as a key synaptic signaling molecule affects the regulation of protein synthesis in neuronal dendrites. After treatment with ZnS@BSA@MM nanoparticles, the synaptic distribution at the lesion site increased significantly. ChAT + cells, as motor neurons in spinal cord tissue, were confirmed by immunofluorescence in the ZnS@BSA@MM and MM groups. This indicates that the recovery of mouse motor function is closely related to the number of motor neurons, which is consistent with the BMS score trend. Nestin identified in the SCI region + cells are neural stem cells with the potential to differentiate into neurons. The number of Nestin + cells increased significantly in the ZnS@BSA@MM group. In addition, the lesion area at the injury center decreased compared to other groups.

[0135] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A bionic microglial membrane-coated ZnS@BSA nanoparticle, characterized in that: The bionic microglial cell membrane-coated ZnS@BSA nanoparticles include microglial cell membranes and zinc sulfide nanoparticles of bovine serum albumin with a final concentration of 300 to 500 mM.

2. The bionic microglial membrane-coated ZnS@BSA nanoparticles according to claim 1, characterized in that: The preparation method of the bovine serum albumin zinc sulfide nanoparticles is as follows: (2.1) Dissolve bovine serum albumin in water, and add Zn(CH3COO)2 solution, NaHS solution and NaOH solution in sequence to obtain a mixture; (2.2) A cross-linking agent is added to the mixture, and after the cross-linking reaction, dialyzation is performed to obtain zinc sulfide nanoparticles of bovine serum albumin.

3. The biomimetic microglial membrane-coated ZnS@BSA nanoparticles according to claim 2, characterized in that: The cross-linking agent is a glutaraldehyde solution, and the volume ratio of the cross-linking agent to the mixture is 1:400-600.

4. The bionic microglial membrane-coated ZnS@BSA nanoparticles according to claim 3, characterized in that: The temperature of the cross-linking reaction is 20-30° C., and the time of the cross-linking reaction is 3-5 hours.

5. The method for preparing the bionic microglial membrane-coated ZnS@BSA nanoparticles according to claim 1, characterized in that: The following steps are involved: (5.1) resuspending microglia in a hypotonic solution and sonicating to obtain a mixture; (5.2) Centrifuge the mixture, collect the supernatant, and centrifuge again to obtain a precipitate; (5.3) washing the precipitate, centrifuging, and resuspending the precipitate to obtain a cell membrane suspension; (5.4) The cell membrane suspension is mixed with zinc sulfide nanoparticles of bovine serum albumin, sonicated, filtered, and the filtered components are collected to obtain biomimetic microglial cell membrane-coated ZnS@BSA nanoparticles.

6. The preparation method according to claim 5, characterized in that: The microglial cells are BV2 microglial cell line.

7. The preparation method according to claim 5, characterized in that: The hypotonic solution uses water as a solvent and contains sucrose with a final concentration of 0.2-0.3 M, EDTA of 0.5-2 mM, HEPES-NaOH of 15-30 mM, and protease inhibitor of 0.1-1.0 mmol / L.

8. The preparation method according to claim 7, characterized in that: The protease inhibitor is a serine protease inhibitor.

9. The preparation method according to claim 5, characterized in that: The power of the ultrasound in step (5.1) is 100 to 300 W, the time of ultrasound is 1 to 5 s, the number of ultrasounds is 20 to 30 times, and the time interval between two adjacent ultrasounds is 5 to 10 s.

10. Use of the bionic microglial membrane-coated ZnS@BSA nanoparticles according to any one of claims 1 to 4 or the bionic microglial membrane-coated ZnS@BSA nanoparticles obtained by the preparation method according to any one of claims 5 to 9 in the preparation of a preparation for treating spinal cord injury.

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