A nanoenzyme complex and its application

Abstract

The present invention provides a nanozyme complex and its application. The nanozyme complex comprises a Mn3O4 nanozyme and a small interfering RNA (siRNA) loaded onto the Mn3O4 nanozyme; the siRNA interferes with Irf5 gene expression. The present invention combines the artificial nanozyme Mn3O4 with the siRNA to effectively promote the repair of spinal cord injury. Given that many physiological and disease states are accompanied by high levels of reactive oxygen species and inflammation, the multifunctional integrated nanozyme strategy developed in this invention may have broad applications in a range of disease models, including those of the nervous system, cardiovascular and immune systems, skeletal muscle and metabolic control, and aging.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a nanoenzyme complex and applications thereof. Background Art

[0002] Vascular rupture after spinal cord injury leads to insufficient oxygen supply, which further produces reactive oxygen species, which is one of the main inhibitory factors of the spinal cord injury microenvironment. Due to the high content of polyunsaturated fatty acids and relatively low antioxidant capacity, spinal cord neurons are particularly susceptible to oxidative and electrophilic stress. At the same time, reactive oxygen species can cause severe diffusion damage by mediating lipid peroxidation, protein nitration and nitric oxide consumption. At present, antioxidant therapeutic drugs used for spinal cord injury repair mainly include free radical scavengers (such as glutathione GSH, vitamin E, vitamin C, superoxide dismutase SOD, melatonin and edaravone), lipid peroxidation inhibitors (such as methylprednisolone and tilirapa), and natural plant extracts (such as curcumin, resveratrol and baicalin).

[0003] Neuroinflammation is an important factor that inhibits neuroregeneration at the injury site after spinal cord injury. Spinal cord injury triggers a strong immune response, which is partly characterized by the coordinated infiltration of peripheral leukocytes (especially M1 macrophages) and the synthesis of cytokines and chemokines. Continued inflammatory cell infiltration leads to further cell death and the formation of cystic microcavities. Macrophages, as the main effector cells, are the main target cells for regulating neuroinflammation after spinal cord injury. Several studies have shown that the transition from an inflammatory phenotype (M1) to a repair phenotype (M2) is crucial for the resolution of inflammation. Groundbreaking research on macrophages has identified several interferon regulatory factors (IRFs), which act as central switches to activate a family of factors that activate pro-inflammatory (M1) or anti-inflammatory (M2) genes, thereby promoting polarization.

[0004] However, current antioxidant drugs used for spinal cord injury repair have a short half-life and are rapidly cleared from the body, failing to exert a stable and long-lasting antioxidant effect, resulting in unsatisfactory therapeutic results. Furthermore, due to the dynamic and complex nature of the neuroinhibitory microenvironment, the therapeutic effects of single antioxidant and anti-inflammatory agents are limited. Therefore, providing a stable drug with enhanced substrate affinity for potently scavenging the high levels of reactive oxygen species following spinal cord injury while also achieving anti-inflammatory effects is of great value. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a nanozyme complex and its application. The present invention constructs a multifunctional nanozyme combined with SiRNA (small interfering RNA) technology, which exhibits important synergistic anti-inflammatory and antioxidant effects in the pathological neural environment of spinal cord injury in vitro and in vivo. The nanozyme shows synergistic ability in catalyzing the conversion of high levels of ROS into O2 without causing biological toxicity. In addition, the continuous release of oxygen stimulates the formation of endothelial blood vessels and angiogenesis. High levels of ROS and inflammation, as two major inhibitory factors, have steadily broadened the understanding of the autoimmune regulatory system and the damaged microenvironment in many injury scenarios. Given that many physiology and diseases are accompanied by ROS and inflammation, the multifunctional integrated nanozyme strategy developed by the present invention may have a wide range of applications in a series of disease models, including the nervous system, cardiovascular and immune systems, skeletal muscle and metabolic control, and aging.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a nanozyme complex, characterized in that the nanozyme complex comprises a Mn3O4 nanozyme and a small interfering RNA loaded on the Mn3O4 nanozyme;

[0008] The small interfering RNA is a small interfering RNA that interferes with the expression of the Irf5 gene.

[0009] In the present invention, the nanozyme complex includes a Mn3O4 nanozyme and a small interfering RNA loaded on the Mn3O4 nanozyme; the present invention designs an artificial nanozyme Mn3O4 through price engineering, and the nanozyme can effectively simulate natural superoxidase, while also simulating catalase and glutathione peroxidase, and the above three enzymes constitute a cascade reaction; some sequences are also designed by small interfering RNA technology for specifically silencing the expression of IRF-5; the artificial nanozyme Mn3O4 is combined with small interfering RNA to obtain a nanozyme complex that can be used to reshape the microenvironment of spinal cord injury; the nanozyme prepared by the present invention has good stability and higher affinity for the substrate, and is used for the potent removal of high levels of reactive oxygen species after spinal cord injury, thereby accelerating nerve regeneration. In the present invention, the combination of artificial nanozyme Mn3O4 and small interfering RNA can effectively reshape the microenvironment of spinal cord injury, and a suitable microenvironment will promote the regeneration of nerves and blood vessels and promote the repair of spinal cord injury.

[0010] In the present invention, the catalytic effect of the Mn3O4 nanozyme simulating the natural superoxide dismutase is achieved by the metal ion Mn 3+ (oxidation state) and Mn 2+ The Mn3O4 prepared by the present invention can effectively simulate natural superoxidase.

[0011] Preferably, the Mn3O4 nanozyme presents a tetrahedral hausmannite crystal form, and the diameter of the Mn3O4 nanozyme is 130-170 nm, for example, 130 nm, 150 nm or 170 nm.

[0012] Preferably, the Mn3O4 nanozyme presents a nanoflower-like structure.

[0013] The prepared Mn3O4 nanoparticles were characterized by transmission electron microscopy and scanning electron microscopy, revealing a uniform particle size of approximately 150 nm with no apparent agglomeration. Furthermore, the prepared nanozymes exhibited excellent enzymatic reaction kinetics, with higher substrate affinity and maximum reaction rate than natural antioxidant enzymes.

[0014] Preferably, the loading ratio of the small interfering RNA on the Mn3O4 nanozyme in the nanozyme complex is (4.5-5.5):2000, for example, it can be 4.5:2000, 5:2000 or 5.5:2000, etc.

[0015] Preferably, the sense strand of the small interfering RNA comprises the nucleotide sequence shown in SEQ ID No: 5, and the antisense strand of the small interfering RNA comprises the nucleotide sequence shown in SEQ ID No: 6, wherein two TT bases are added to the sense strand and the antisense strand as bases of the protruding tail.

[0016] The present invention designs a series of small interfering RNAs and screens out the small interfering RNA sequences with the highest silencing efficiency. The transition from inflammatory type (M1) to repair type (M2) is a crucial step in resolving inflammation. Breakthrough research on macrophages has identified interferon regulatory factors (IRFs) as the activation center, switching subsets of M1 or M2 genes to promote polarization. Therefore, by specifically reducing the expression of IRF-5, the polarization state of macrophages can be effectively controlled, causing macrophages with an early pro-inflammatory phenotype to reverse more towards the M2 repair phenotype, thereby controlling the inflammatory microenvironment after spinal cord injury.

[0017] The small interfering RNA designed in the present invention includes SiRNA1, SiRNA2 or SiRNA3; the sense chain of SiRNA1 is SEQ ID No: 1, and the antisense chain is SEQ ID No: 2; the sense chain of SiRNA2 is SEQ ID No: 3, and the antisense chain is SEQ ID No: 4; the sense chain of SiRNA3 is SEQ ID No: 5, and the antisense chain is SEQ ID No: 6.

[0018] The small interfering RNA used in the present invention can effectively silence the expression of IRF-5 in vitro, wherein the silencing efficiency of SiRNA1, SiRNA2 or SiRNA3 is -47.57%, 28.94% and 76.86% respectively, among which SiRNA3 has the strongest silencing efficiency and can effectively reverse the phenotypic conversion of macrophages. The reversal efficiency of CD206 was determined by flow cytometry. - CD86 + The nanozyme complex can effectively reduce oxidative stress and inflammation after spinal cord injury and is a potential target for spinal cord injury repair.

[0019] Preferably, the surface of the nanozyme complex further contains a coating material.

[0020] Preferably, the coating material comprises any one of biological membranes, monoclonal antibodies or macromolecules with biological activity, or a combination of at least two of them.

[0021] Preferably, the biological membrane comprises a cell membrane.

[0022] Preferably, the bioactive macromolecule comprises hyaluronic acid.

[0023] In the present invention, the surface of the nanozyme complex has rich amino functional groups and positive charges, which are beneficial for further molecular modification, such as coating with other homologous cell membranes to achieve drug delivery to different organs; or modifying monoclonal antibodies with targeted effects for precise drug delivery; in addition to the effective coating with cell membranes, the surface of the nanozyme complex can also be modified and wrapped with a wide range of bioactive macromolecules, such as hyaluronic acid coating.

[0024] In the present invention, the surface of the nanozyme complex is coated with a cell membrane, and the effective coating of the neutrophil-like membrane is used to achieve targeted delivery of small interfering RNA to macrophages, specifically reversing the inflammatory phenotype of macrophages, thereby reducing inflammation after spinal cord injury.

[0025] In a second aspect, the present invention provides a method for preparing the nanozyme complex described in the first aspect, the preparation method comprising the following steps:

[0026] (a) The precursor MnO2 is prepared by chemical coprecipitation and calcined at high temperature to obtain mixed-valence Mn3O4 nanozymes;

[0027] (b) Using the Mn3O4 nanozyme as a carrier, Mn3O4 and polyethyleneimine are cross-linked by an electrostatic conjugation method, and grafted polyethyleneimine is modified on the carrier as an intermediate medium layer. Small interfering RNA is loaded by utilizing the coordination effect of the amino group of polyethyleneimine on the Mn3O4 nanozyme carrier.

[0028] Preferably, in step (b), after loading the small interfering RNA, the step further includes coating the nanozyme loaded with the small interfering RNA with a coating material.

[0029] Preferably, the step of coating with cell membrane includes: subjecting the nanozyme loaded with small interfering RNA and repeatedly frozen and thawed cell fragments to repeated extrusion by a liposome extruder to achieve effective coating of the cell membrane.

[0030] Preferably, in step (a), the Mn3O4 nanozyme is prepared by a method comprising the following steps: stirring and mixing KMnO4 with water, then adding oleic acid and stirring and mixing to obtain a black precipitate and washing it, drying the washed black precipitate, and calcining it to obtain the Mn3O4 nanozyme.

[0031] Preferably, the mass volume ratio of the KMnO4 mixed with water is 1:(450-550), for example, it can be 1:450, 1:480, 1:500, 1:520 or 1:550.

[0032] Preferably, the KMnO4 and water are stirred and mixed for 25-35 min, for example, 25 min, 28 min, 30 min, 32 min or 35 min, and the rotation speed is 180-220 rpm, for example, 180 rpm, 200 rpm or 220 rpm.

[0033] Preferably, the volume ratio of oleic acid to water is 10:(450-550), for example, it can be 10:450, 10:480, 10:500, 10:520 or 10:550.

[0034] Preferably, the time for adding oleic acid and stirring and mixing is 12-24 hours, for example, 12 hours, 16 hours, 20 hours or 24 hours, etc., and the rotation speed is 180-220 rpm, for example, 180 rpm, 200 rpm or 220 rpm, etc.

[0035] Preferably, the cleaning is performed alternately with water and ethanol, and the number of cleaning times is 6-8 times, for example, 6, 7 or 8 times.

[0036] Preferably, the drying is carried out by vacuum drying, the vacuum drying temperature is 30-45°C, for example, it can be 30°C, 35°C, 40°C or 45°C, etc., the vacuum drying time is 12-18h, for example, it can be 12h, 14h, 16h or 18h, etc., and the vacuum degree of the vacuum drying is -0.085 to -0.095, for example, it can be -0.085 or -0.095, etc.

[0037] Preferably, the calcination temperature is 180-200°C, for example, 180°C, 185°C, 190°C, 195°C or 200°C, etc., the calcination time is 5-6h, for example, 5h, 5.5h or 6h, etc., and the calcination atmosphere is air.

[0038] Preferably, in step (b), the Mn3O4 and polyethyleneimine are cross-linked using a method comprising the following steps: mixing Mn3O4 with PBS buffer, ultrasonically crushing, adding polyethyleneimine and incubating to obtain Mn3O4 modified with polyethyleneimine.

[0039] Preferably, the mass volume ratio of Mn3O4 to PBS buffer is 1 mg:(0.8-1.2 mL), for example, it can be 1 mg:0.8 mL, 1 mg:1 mL or 1 mg:1.2 mL.

[0040] Preferably, the mass ratio of Mn3O4 to polyethyleneimine is 1:(2-5), for example, it can be 1:2, 1:3, 1:4 or 1:5.

[0041] Preferably, the incubation temperature is 35-38°C, for example, it can be 35°C, 36°C, 37°C or 38°C, etc., the incubation time is 10-14h, for example, it can be 10h, 12h or 14h, etc., and the incubation speed is 200-240rpm, for example, it can be 200rpm, 220rpm or 240rpm, etc.

[0042] As a preferred technical solution of the present invention, the preparation method comprises the following steps:

[0043] (a) The precursor MnO2 was prepared by chemical coprecipitation method, and the precursor MnO2 was calcined at high temperature to obtain mixed-valence Mn3O4 nanozyme.

[0044] KMnO4 and water are stirred and mixed at 180-220 rpm for 25-35 minutes, and the mass volume ratio of KMnO4 to water is 1:(450-550); oleic acid is then added and stirred at 180-220 rpm for 12-24 hours, and the volume ratio of oleic acid to water is 10:(450-550) to obtain a black precipitate; the black precipitate is alternately washed with water and ethanol for 6-8 times, and the washed black precipitate is vacuum dried for 12-18 hours, the vacuum drying temperature is 30-45°C, and the vacuum degree is -0.085 to -0.095; calcined at 180-200°C for 5-6 hours, and the calcination atmosphere is air to obtain the Mn3O4 nanozyme.

[0045] (b) Using the Mn3O4 nanozyme as a carrier, Mn3O4 and polyethyleneimine are cross-linked by an electrostatic conjugation method, and the grafted polyethyleneimine is modified on the carrier as an intermediate medium layer. The small interfering RNA is loaded by utilizing the coordination effect of the amino group of polyethyleneimine on the Mn3O4 nanozyme carrier; the cell membrane is coated to obtain the nanozyme complex.

[0046] Mn3O4 is mixed with PBS buffer at a mass-to-volume ratio of 1 mg:(0.8-1.2 mL) and ultrasonically disrupted until the nanoparticles are completely dispersed in PBS; polyethyleneimine is added and incubated at 35-38°C and 200-240 rpm for 10-14 hours, and the mass ratio of the Mn3O4 to polyethyleneimine is 1:(2-5) to obtain Mn3O4 modified with polyethyleneimine; small interfering RNA is loaded on the polyethyleneimine-modified Mn3O4, and the loading ratio of the small interfering RNA on the Mn3O4 nanozyme is (4.5-5.5):2000; the above-mentioned nanozyme loaded with small interfering RNA and repeatedly frozen and thawed cell fragments are repeatedly extruded in a liposome extruder to achieve effective coating of the cell membrane.

[0047] In a third aspect, the present invention provides the use of the nanozyme complex described in the first aspect in the preparation of a drug for reshaping the spinal cord injury microenvironment.

[0048] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the nanozyme complex described in the first aspect.

[0049] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0050] The numerical range described in the present invention includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] (1) The synthesized manganese tetraoxide (Mn3O4) has a high specific surface area as a nanoflower-like structure, which helps to provide more surface active sites and siRNA attachment sites;

[0053] (2) Valence-engineered nanozymes can effectively mimic the metal ion catalytic active sites of natural antioxidant enzymes and simulate the catalytic process of natural enzymes;

[0054] (3) Artificially designed and screened SiRNA effectively regulates the phenotype of macrophages and controls inflammation, thereby playing a role in spinal cord injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 yes IRF-5 Characterization results of SiRNA / M@pMn nanozyme complex;

[0056] Figure 2 These are the X-ray photoelectron spectroscopy and X-ray diffraction characterization results of Mn3O4;

[0057] Figure 3 It is the characterization result of the multiple enzymatic cascade reactions of nanozymes;

[0058] Figure 4 yes IRF-5 Cell biological functional experimental results of SiRNA / M@pMn on macrophages (targeting ability);

[0059] Figure 5 yes IRF-5 Cell biological function experimental results of SiRNA / M@pMn on macrophages (anti-inflammatory and antioxidant abilities);

[0060] Figure 6 This is the preparation and characterization result of nanoparticle composite hydrogel;

[0061] Figure 7 This is the result of the in vitro biocompatibility test of the hydrogel;

[0062] Figure 8 It is the test results of treatment regimen and short-term lesion site in animal models;

[0063] Figure 9 It is the result of testing the anti-inflammatory effect of short-term treatment regimen in animal models;

[0064] Figure 10 It is the motor recovery function evaluation and staining results of long-term treatment in animal models;

[0065] Figure 11 is qPCR analysis of neuronal and vascular marker expression. DETAILED DESCRIPTION

[0066] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0067] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0068] Example 1

[0069] This example provides small interfering RNAs (siRNAs) that interfere with Irf5 gene expression. These small interfering RNAs include siRNA1 (corresponding to the gene Irf5-Rat-289), siRNA2 (corresponding to the gene Irf5-Rat-482), and siRNA3 (corresponding to the gene Irf5-Rat-818). The coding sequence of Irf5 (Gene ID: 296953) was obtained from the National Center for Biotechnology Information (NCBI). Antisense sequences were created by screening potential silencing sites. The sense and antisense strands of the small interfering RNAs are shown in Table 1. Two TT bases were added to the sense and antisense strands as overhanging tails. The nucleotide sequences of the aforementioned RNAs are shown in Table 1.

[0070] Table 1

[0071]

[0072] SiRNA1: GGGCUAAAGAGACAGGGAATT;UUCCCUGUCUCUUUAGCCCTT.

[0073] SiRNA2: GCCCACAGAUGAUUACGUUTT; AACGUAAUCAUCUGUGGGCTT.

[0074] SiRNA3: GCUGCCUUUGACUGACCUATT;UAGGUCAGUCAAAGGCAGCTT.

[0075] The sequences with the highest potential for silencing efficiency were screened from the above sequences for cellular silencing efficiency testing. The screening steps are as follows:

[0076] Preparation of transfection complex and transfection: Before use, place the commercial cationic transfection agent GP-transfect-Mate at room temperature and gently shake before use. Add 100 μL of serum-free culture medium to a 1.5 mL sterile centrifuge tube, add 2 μL of GP-transfect-Mate, mix gently with a pipette, and incubate at room temperature for 5 minutes. Simultaneously, add 50 μL of serum-free culture medium to another sterile centrifuge tube, add 10 pmol of siRNA, mix gently with a pipette, and incubate at room temperature for 5 minutes. Combine the mixtures in the two centrifuge tubes, mix gently with a pipette, and incubate at room temperature for 15-20 minutes. Add the mixture to 0.5 mL of prewarmed fresh culture medium, add to adherent rat primary macrophages, and gently shake the plate. 24 hours after transfection, perform quantitative PCR to screen for the small interfering RNA sequences with the highest silencing efficiency. The results of the screening are shown in Table 2.

[0077] Table 2

[0078] Small interfering RNA Target protein IRF-5 expression level (%) Silencing efficiency (%) Nonsense sequence (siRNA-NC) 108.8±27.15％ - SiRNA1 147.5±24.21％ -47.57±24.21％ SiRNA2 71.05±28.96％ 28.94±28.96％ SiRNA3 23.14±23.40％ 76.86±23.40％

[0079] The silencing efficiencies of siRNA1, siRNA2, and siRNA3 were -47.57%, 28.94%, and 76.86%, respectively. By specifically reducing IRF-5 expression, the small interfering RNA effectively controlled the polarization state of macrophages, causing the early pro-inflammatory phenotype of macrophages to reverse toward the M2 repair phenotype, thereby controlling the inflammatory microenvironment after spinal cord injury.

[0080] Example 2

[0081] This embodiment provides a nanozyme complex, comprising a Mn3O4 nanozyme and a small interfering RNA loaded on the Mn3O4 nanozyme, wherein the small interfering RNA is the small interfering RNA combination that interferes with Irf5 gene expression as described in Example 1. The preparation method of the nanozyme complex comprises the following steps:

[0082] (a) The precursor MnO2 was prepared by chemical coprecipitation method, and the precursor MnO2 was calcined at high temperature to obtain mixed-valence Mn3O4 nanozyme.

[0083] KMnO4 and ultrapure water were stirred and mixed at 220 rpm for 30 minutes, with the mass volume ratio of KMnO4 to ultrapure water being 1:500; oleic acid was added dropwise to the stirred mixture, with the volume ratio of oleic acid to ultrapure water being 10:500, and stirred and mixed in air at 220 rpm for 18 hours to obtain a black precipitate.

[0084] The black precipitate was washed alternately with ultrapure water and ethanol 7 times, and the black precipitate was collected by centrifugation at 12000 rpm for 20 minutes; the obtained black precipitate was vacuum dried for 12 hours, the drying temperature was 45°C, and the vacuum degree was -0.085; and the Mn3O4 nanozyme was obtained by calcining at 190°C for 5.5 hours in an air atmosphere.

[0085] (b) Using the Mn3O4 nanozyme as a carrier, Mn3O4 and polyethyleneimine are cross-linked by an electrostatic conjugation method, and the grafted polyethyleneimine is modified on the carrier as an intermediate medium layer. The small interfering RNA is loaded by utilizing the coordination effect of the amino group of polyethyleneimine on the Mn3O4 nanozyme carrier; the cell membrane is coated to obtain the nanozyme complex.

[0086] The specific steps for loading small interfering RNA are as follows:

[0087] Mn3O4 was mixed with PBS buffer at a mass-to-volume ratio of 1 mg:1 mL and ultrasonically crushed. The reaction was carried out under the conditions of a Φ6 operating rod, 20% power, and ultrasonication for 20 minutes (working 4 seconds, stopping 4 seconds) in an ultrasonic crusher until the nanoparticles were completely dispersed in PBS; polyethyleneimine was added and incubated at 37°C and 220 rpm for 12 hours. The mass ratio of the Mn3O4 to polyethyleneimine was 1:3 to obtain Mn3O4 modified with polyethyleneimine; small interfering RNA was loaded on the Mn3O4 modified with polyethyleneimine, and the loading ratio of the small interfering RNA on the Mn3O4 nanozyme was 5.32:2000.

[0088] The specific steps for coating the cell membrane are as follows:

[0089] Cell membrane fragment extraction: Neutrophil-like cell membranes were obtained using freeze-thaw cycles and physical disruption. The collected cells were frozen and thawed three times in liquid nitrogen. The resulting cells were then placed in an ultrasonic bath for 5 minutes, centrifuged (700 g, 10 minutes, 4°C) to obtain the supernatant, and then centrifuged (14,000 g, 30 minutes) to obtain membrane fragments. Protein content was then quantified using conventional BCA assay. Finally, the membrane fragments were lyophilized and stored at -80°C for future use.

[0090] Cell membranes and nanoparticles were added to the solution at a 1:1 mass ratio and mixed under an ice-cold ultrasonic bath for 30 minutes. The nanoparticles were then physically extruded 21 times sequentially through porous polycarbonate membranes (1 μm and 400 nm) using a mini-extruder (Avanti, USA). Finally, the resulting nanoparticles were washed three times with PBS by centrifugation and quantified using BCA protein.

[0091] Example 3

[0092] This embodiment provides a nanozyme complex, comprising a Mn3O4 nanozyme and a small interfering RNA loaded on the Mn3O4 nanozyme, wherein the small interfering RNA is the small interfering RNA combination that interferes with Irf5 gene expression as described in Example 1. The preparation method of the nanozyme complex comprises the following steps:

[0093] (a) The precursor MnO2 was prepared by chemical coprecipitation method, and the precursor MnO2 was calcined at high temperature to obtain mixed-valence Mn3O4 nanozyme.

[0094] KMnO4 and ultrapure water were stirred and mixed at 180 rpm for 35 minutes, with the mass volume ratio of KMnO4 to ultrapure water being 1:450; oleic acid was added dropwise to the stirred mixture, with the volume ratio of oleic acid to ultrapure water being 10:450, and stirred and mixed in air at 180 rpm for 24 hours to obtain a black precipitate.

[0095] The black precipitate was washed alternately with ultrapure water and ethanol for 6 times, and the black precipitate was collected by centrifugation at 12000 rpm for 20 minutes; the obtained black precipitate was vacuum dried for 12 hours, the drying temperature was 40°C, and the vacuum degree was -0.095; and the Mn3O4 nanozyme was obtained by calcining at 180°C for 6 hours in an air atmosphere.

[0096] (b) Using the Mn3O4 nanozyme as a carrier, Mn3O4 and polyethyleneimine are cross-linked by an electrostatic conjugation method, and the grafted polyethyleneimine is modified on the carrier as an intermediate medium layer. The small interfering RNA is loaded by utilizing the coordination effect of the amino group of polyethyleneimine on the Mn3O4 nanozyme carrier; the cell membrane is coated to obtain the nanozyme complex.

[0097] The specific steps for loading small interfering RNA are as follows:

[0098] Mn3O4 was mixed with PBS buffer at a mass-to-volume ratio of 1 mg:0.8 mL and ultrasonically crushed. The reaction was carried out under the conditions of a Φ6 operating rod, 20% power, and ultrasonication for 20 minutes (working 4 seconds, stopping 4 seconds) in an ultrasonic crusher until the nanoparticles were completely dispersed in PBS; polyethyleneimine was added and incubated at 35°C and 240 rpm for 14 hours. The mass ratio of the Mn3O4 to polyethyleneimine was 1:2 to obtain Mn3O4 modified with polyethyleneimine; small interfering RNA was loaded on the Mn3O4 modified with polyethyleneimine, and the loading ratio of the small interfering RNA on the Mn3O4 nanozyme was 5:2000.

[0099] The specific steps of coating the cell membrane are shown in Example 2.

[0100] Example 4

[0101] This embodiment provides a nanozyme complex, comprising a Mn3O4 nanozyme and a small interfering RNA loaded on the Mn3O4 nanozyme, wherein the small interfering RNA is the small interfering RNA combination that interferes with Irf5 gene expression as described in Example 1. The preparation method of the nanozyme complex comprises the following steps:

[0102] (a) The precursor MnO2 was prepared by chemical coprecipitation method, and the precursor MnO2 was calcined at high temperature to obtain mixed-valence Mn3O4 nanozyme.

[0103] KMnO4 and ultrapure water were stirred and mixed at 200 rpm for 25 minutes, with the mass volume ratio of KMnO4 to ultrapure water being 1:550; oleic acid was added dropwise to the stirred mixture, with the volume ratio of oleic acid to ultrapure water being 10:550, and stirred and mixed in air at 200 rpm for 20 hours to obtain a black precipitate.

[0104] The black precipitate was washed alternately with ultrapure water and ethanol 8 times, and the black precipitate was collected by centrifugation at 12000 rpm for 20 minutes; the obtained black precipitate was vacuum dried for 18 hours, the drying temperature was 30°C, and the vacuum degree was -0.085; and the Mn3O4 nanozyme was obtained by calcining at 200°C for 5 hours in an air atmosphere.

[0105] (b) Using the Mn3O4 nanozyme as a carrier, Mn3O4 and polyethyleneimine are cross-linked by an electrostatic conjugation method, and the grafted polyethyleneimine is modified on the carrier as an intermediate medium layer. The small interfering RNA is loaded by utilizing the coordination effect of the amino group of polyethyleneimine on the Mn3O4 nanozyme carrier; the cell membrane is coated to obtain the nanozyme complex.

[0106] The specific steps for loading small interfering RNA are as follows:

[0107] Mn3O4 was mixed with PBS buffer at a mass-to-volume ratio of 1 mg:1 mL and ultrasonically crushed until the nanoparticles were completely dispersed in PBS; polyethyleneimine was added and incubated at 38°C and 200 rpm for 10 hours, with the mass ratio of Mn3O4 to polyethyleneimine being 1:5 to obtain polyethyleneimine-modified Mn3O4; small interfering RNA was loaded on the polyethyleneimine-modified Mn3O4, and the loading ratio of the small interfering RNA on the Mn3O4 nanozyme was 5.5:2000.

[0108] The specific steps of coating the cell membrane are shown in Example 2.

[0109] Example 5

[0110] This embodiment provides a nanozyme complex. The only difference between this embodiment and Example 2 is that in the preparation method of the complex, in step (a), the calcination temperature is 150° C., and the remaining steps refer to Example 2.

[0111] Example 6

[0112] This embodiment provides a nanozyme complex. The only difference between this embodiment and Example 2 is that in the preparation method of the complex, in step (a), the calcination temperature is 250° C., and the remaining steps refer to Example 2.

[0113] The enzymatic activity of the Mn3O4 nanozymes in Examples 2-6 was detected, and the results showed that the enzymatic activity of the Mn3O4 nanozymes in Examples 5-6 was low. In the present invention, MnO2 is first synthesized by a hydrothermal method, and the valence of Mn is controlled by controlling the calcination temperature and atmosphere. Since the active center site of the natural antioxidant enzyme superoxide dismutase changes between the oxidized state and the reduced state between divalent and trivalent manganese, the present invention simulates the catalytic process of the natural enzyme by controlling the valence of Mn. When the calcination temperature is lower than 180°C, the lower temperature will lead to the formation of a less crystalline structure (layered), and the weaker crystallization may affect the sustained and stable catalytic performance of manganese tetraoxide as a nanozyme. At the same time, when the calcination temperature is too high, for example, when it exceeds 250°C, the layered self-assembled flower-like structure of manganese tetraoxide will be destroyed, and the loss of the layered structure will cause the specific surface area of ​​manganese tetraoxide to decrease, and the active sites of the catalytic reaction that can be provided and the ability to load siRNA will be reduced.

[0114] Example 7

[0115] In this example, the nanozyme complex prepared in Example 2 was characterized, wherein the samples characterized included: pMn: PEI-modified Mn3O4 nanozyme; M@pMn: PEI-modified Mn3O4 nanozyme coated with cell membrane; IRF-5 SiRNA / pMn: small interfering RNA loaded on pMn; IRF-5 SiRNA / M@pMn, in IRF-5 SiRNA / pMn is coated on the cell membrane.

[0116] The properties of the prepared nanoparticle composites were determined using different methods, including:

[0117] (1) The morphology of the prepared Mn3O4 and M@pMn was characterized using scanning electron microscopy (SEM, HITACHI, Japan).

[0118] (2) Unbound siRNA was quantitatively analyzed by agarose gel electrophoresis, and the related imaging was stained with Goldview (Yeasen, China).

[0119] (3) Fourier transform infrared spectroscopy (FTIR, Thermo Scientific Nicolet iS5, USA) was used to record the FTIR spectra of Mn3O4, pMn, and M@pMn nanoparticles.

[0120] (4) Quantitative proteomic analysis of membrane proteins was performed using TMT (tandem mass tag) technology. All raw data were analyzed in the database homo_sapiens_uniprot_2022_1_27.fasta.fasta (203,746 sequences) was searched using the library search software Proteome Discoverer 2.4.

[0121] (5) Western blot was used to further analyze the membrane protein content.

[0122] (6) Dynamic laser scattering (DLS) Malvern Zetasizer Nano Series (Malvern, USA) was used to determine Mn3O4, pMn, IRF-5 SiRNA / pMn, M@pMn and IRF-5 Zeta potential and hydrated diameter of SiRNA / M@pMn.

[0123] The characterization results of IRF-5SiRNA / M@pMn are shown in Figure 2. Figure 1 As shown, Figure 1 In the figure, AB are SEM images of Mn3O4. C is the result of agarose gel electrophoresis on the pMn / SiRNA nanoparticles in the presence of different volumes of SiRNA (0 μL, 4 μL, 8 μL, 12 μL, 16 μL, 20 μL of 20 μM SiRNA) and 2 μL of 1 mg / mL pMn. D is IRF-5 TEM images of siRNA / M@pMn. E: FTIR spectra of Mn3O4, pMn, and M@pMn nanoparticles. F: Heat map of membrane protein expression in HL-60 and neutrophil-like cells. G: Western blot analysis of CXCR1 / 2 protein levels in M@pMn. H: Diameters of different nanoparticles. I: Zeta potential of nanoparticles synthesized in each step (n = 3).

[0124] Hereinafter referred to as “ Figure 1 A" means Figure 1 A in " Figure 1 B" means Figure 1 The following other figures are described similarly.

[0125] Figure 1 A and Figure 1 B shows a clear nanoflower-like structure. From the inserted image in A, it can be seen that the average diameter is about 150 nm. The flower-like structure with high specific surface area enhances the diffusion of guest molecules, exposes more active sites for catalytic reactions, and provides more surface binding sites for siRNA loading.

[0126] Agarose gel electrophoresis results showed that the highest binding amount of nanoparticles and siRNA was 20 μL (1 mg / mL) and 2 μL (20 μM), respectively ( Figure 1 C). Transmission electron microscope images show that, compared with Mn3O4( Figure 1 A) Compared to M@pMn, the surface roughness and topography increased, but the size did not significantly expand, indicating effective cell membrane encapsulation ( Figure 1 D).

[0127] Fourier transform infrared spectroscopy (FTIR) analysis results ( Figure 1 E) shows that the three samples have -1 The Mn-O stretching vibration peaks were detected at 1536cm -1 and 861cm -1 The stretching at 1157 cm corresponds to the deformation vibration peak and the rocking vibration peak of the primary amine NH of PEI. -1 The peak at 1236 cm in M@pMn is the CN stretching vibration of PEI, indicating that PEI is successfully modified. -1 and 1543cm -1 The peak at was identified as the amide II band generated by the CN stretching or bending vibration of CHN, indicating effective cell membrane coverage. Overall, the loading of siRNA and cell membrane coating in the integrated nanozyme were successful through direct and indirect observations.

[0128] The present invention performs tandem mass tag (TMT) quantitative proteomics analysis on neutrophils to further study their membrane protein composition. Figure 1 As shown in Figure F, DMSO-treated HL-60 cells exhibit excellent neutrophil chemotaxis and interaction with the immune system. Proteins CD14, CD89, and CD48 are involved in neutrophil chemotaxis. TMT membrane analysis revealed the presence of chemokine receptors (CXCR2, CXCR4, and CCR1), a family of seven-transmembrane protein receptors linked to guanine nucleotide-binding proteins (G proteins). Their seven-transmembrane structure effectively prevents protein loss during membrane extraction.

[0129] The prepared nanoparticles were analyzed by dynamic light scattering (DLS). Figure 1 H, hydrated Mn3O4, pMn, IRF-5SiRNA / pMn, M@pMn and IRF-5 The average particle sizes of SiRNA / M@pMn were 140.6nm, 171.3nm, 138.5nm, 238.0nm and 256.3nm respectively. IRF-5 The particle size of SiRNA / pMn was slightly reduced, which may be due to the negative charge loading on the surface of the nanoparticles, resulting in a decrease in the strength of ionic interactions in the solution. IRF-5 SiRNA / pMn, M@pMn and IRF-5 The zeta potential of SiRNA / M@pMn was approximately -26.43±1.29mV, +27.40±0.44mV, +21.83±0.35mV, -23.00±1.42mV and -24.33±0.60mV( Figure 1 I).

[0130] Example 8

[0131] In this example, the synthesized Mn3O4 was characterized by X-ray photoelectron spectroscopy and X-ray diffraction.

[0132] The crystal structure of Mn3O4 was analyzed by X-ray diffraction (XRD, Rigaku SmartLab SE, Japan). The synthesized precursors MnO2 and Mn3O4 were characterized by X-ray photoelectron spectroscopy (XPS) using a Thermo Scientific K-Alpha spectrometer.

[0133] The energy spectrum characterization results of Mn3O4 are as follows Figure 2 As shown, Figure 2 In the figure, A is the XPS spectrum of MnO2. B is the high-resolution Mn 2p spectrum, Mn 4+ 2p 3 / 2 and Mn 4+ 2p 1 / 2 The peaks are centered at 642.4eV and 653.9eV, and Mn 4+ The atomic percentage is 100%. C is the high-resolution O1s spectrum, with the peaks of Mn-O-Mn and Mn-OH centered at 529.9eV and 531.62eV. D is the XPS survey spectrum of Mn3O4. E is the high-resolution Mn 2p spectrum, with Mn 2+ 2p 3 / 2 , Mn 2+ 2p 1 / 2 , Mn 3+ 2p 3 / 2 and Mn 3+ 2p 3 / 2 The peak values ​​are 641.5eV, 652.9eV, 642.6eV and 654.2eV.2+ and Mn 3+ The atomic percentages are 65.32% and 34.68%. F is a high-resolution O1s spectrum, with the peaks of Mn-O-Mn and Mn-OH centered at 529.6 eV and 531.2 eV.

[0134] The catalytic action of natural SOD is through alternating electron gain and metal ion Mn 3+ (oxidation state) and Mn 2+ Inspired by natural SOD, the valence of Mn was changed from +4 to +3 and +2 by valence engineering strategy. High-resolution X-ray photoelectron spectroscopy (XPS) showed that there were two main spin-orbit lines on the Mn 2p peak. 4+ 2p3 / 2 and Mn 4+ The binding energy peaks of 2p1 / 2 elements are 642.4eV and 653.9eV respectively. 4+ The atomic percentage of Mn is 100%. The same analysis was performed on calcined Mn3O4. The results were convolved into two pairs of double peaks by Gaussian curve fitting, and the Mn 2+ 2p3 / 2、Mn 2+ 2p1 / 2、Mn 3+ 2p3 / 2 and Mn 3+ The binding energy peaks of 2p3 / 2 are 641.5eV, 652.9eV, 642.6eV and 654.2eV respectively. The quantitative analysis results show that Mn 2+ and Mn 3+ The atomic percentages of Mn and MnO2 are 65.32% and 34.68% respectively. The valence of Mn changes from +4 to +2 and +3, which confirms that MnO2 is calcined to form Mn3O4 ( Figure 2 AF).

[0135] The present invention anticipates that biomimetic technology will mimic the catalytic ability of natural enzymes. X-ray diffraction (XRD, Figure 2 G) Six broad peaks were observed at 2θ angles of 18.1°, 29.4°, 32.4°, 36.2°, 60.2°, and 65.2°, corresponding to the (101), (112), (103), (211), (224), and (314) planes (Mn3O4, JCPDS 80-0382), respectively, confirming the XPS results.

[0136] Example 9

[0137] This example conducts theoretical research and characterization of multiple enzymatic cascade reactions of nanozymes using the following methods:

[0138] (1) The Fridovich method was used to determine the SOD activity of nanozymes and natural superoxide dismutase (SOD). The detection steps were as follows: 20 μL of xanthine (25 mM) and 20 μL of color developer NBT (15 mM) were added to PBS, 10 μL of nanozyme (1 mg / mL), and then a portion of PBS (0.1 M, pH 7.4) was added to obtain 900 μL of sample. The centrifuge tube was then rotated for 5 seconds. Finally, 100 μL of xanthine oxidase (1.5 mg / mL) was added to the sample before vortexing for 5 seconds. The absorbance change over time was recorded at a wavelength of 565 nm for 5 minutes. The inhibition curve was constructed by plotting the effect of the inhibitory effect of each sample on the free radical-NBT reaction on the final nanozyme concentration in the corresponding sample.

[0139] (2) Catalase activity assay: The CAT activity of the nanozyme and natural CAT was measured using a portable dissolved oxygen analyzer, Seven2Go Pro (Mettler, Toledo, China). The generated O2 was used to measure the CAT activity of the nanozyme and natural CAT. Kinetic assays of nanozyme (0-4 μg / mL) and CAT (positive control, 0-40 μg / mL) were performed in 1.0 mL of buffer (0.1 M PBS, pH 7.4) using H2O2 as the substrate. The generated O2 was measured after 30 s of reaction.

[0140] (3) Glutathione peroxidase activity assay: The classic glutathione reductase (GR) coupling method was used. In the kinetic mode, the GPx-like activity of nanozyme (10 μg / mL) and GPx (positive control, 10 μg / mL) was determined as the concentration of nicotinamide adenine dinucleotide phosphate (NADPH) decreased at 340 nm (the molar extinction coefficient of NADPH: ). In a typical experiment (total volume of 1 mL), nanozyme (10 μg / mL), reduced glutathione (5 mM), NADPH (0.2 mM), GR (3 units), and H2O2 (0-200 mM) were reacted in PBS in a 37°C water bath for 5 min.

[0141] The ability of nanozymes to scavenge ROS was further tested by electron paramagnetic resonance (EPR) experiments. The xanthine (X) / xanthine oxidase (XO) system was used to produce superoxide anions (O2 - ). O2 - The characteristic four-line peak (relative intensity of 1:1:1:1:1) gradually weakened, indicating that the nanozyme - The scavenging activity was concentration-dependent ( Figure 3 A). Then, the effect of nanozyme in scavenging hydroxyl radicals was calculated based on its spin spectrum, and the intensity of the spin spectrum was 1:2:2:1. As the Mn concentration increased, the signal intensity decreased ( Figure 3 B), with O2 -The results for the peaks were similar.

[0142] SOD converts superoxide into H2O2 and O2. When X reacts with XO, superoxide is formed, which reduces nitro blue tetrazolium (NBT) to purple crystals with a maximum absorbance of 565nm. Figure 3 As shown in Figure C (using natural SOD as a positive control), the absorbance decreased significantly with the increase of nanozyme concentration, indicating that under physiological conditions, the catalytic ability of SOD mimics is positively correlated with concentration. Figure 3 As shown in D and E, the decomposition of H2O2 catalyzed by the nanozyme follows Michaelis-Menten kinetics; the typical GR-coupling method is used to detect the GPx-mimicking activity of the nanozyme.

[0143] The catalytic function and performance of the nanozyme were studied using typical Michaelis-Menten steady-state kinetics. Nanozyme assays were performed using 0.16 and 0.20 M H2O2 as reactants. The Beer-Lambert law (Formula A) was used to convert the average initial rate of absorbance change into the initial rate (v0) of generating O2 or reducing NADPH, which was then plotted against the appropriate concentration and fitted with a Michaelis-Menten curve (Formula B). The Michaelis-Menten constant (K) was then calculated using a linear double reciprocal curve (Formula C). m ) and maximum speed (V max ).

[0144] A=kbc(A)

[0145]

[0146]

[0147] K of nanozymes CAT and GPx m (Substrate concentration is V max half of the K of natural CAT) were 0.04813M and 0.07136M, respectively, which are higher than the K of natural CAT. m (0.01158M) and GPx (0.02292M). Oxidative stress occurs at high H2O2 concentrations (10-1000μM), leading to inflammatory responses, growth arrest, and cell death in the central nervous system (CNS). The endogenous H2O2 concentration at the site of spinal cord injury increases sharply, suggesting that nanozymes may have sufficient therapeutic effects. In addition, once all active sites of the cascade nanozyme are occupied, the nanozyme can catalyze H2O2 at a maximum rate of 17.09M / s (CAT) and 4.701M / s (GPx), with a synergistic and stable therapeutic effect.

[0148] Based on the basic catalytic properties of nanozymes, this invention demonstrates an efficient cascade reaction without the need for additional enzymes. - SOD rapidly catalyzes the generation of H2O2 (step 1), which is then consumed through synergistic catalysis (CAT and GPx, step 2). The catalytic efficiency of the cascade reaction is significantly improved due to its high local reactant concentration, low intermediate breakdown rate, and high mass transfer efficiency. Therefore, the intelligent artificial "enzyme" Mn3O4 came into being.

[0149] Example 10

[0150] This embodiment is carried out IRF-5 Study on the biological functions of siRNA / M@pMn on BMDM (including targeting inflammatory macrophages, scavenging ROS, and alleviating inflammation). The samples used include: FAM-IRF-5 SiRNA / RBITC pMn、M@ FAM-IRF-5 SiRNA / RBITC pMn.

[0151] FAM-IRF-5 SiRNA / RBITC pMn refers to IRF-5 Based on SiRNA / pMn, nanoparticles are labeled with rhodamine (RBITC), making the nanoparticles emit red fluorescence, and FAM labels SiRNA, making it emit green fluorescence;

[0152] M@ FAM-IRF-5 SiRNA / RBITC pMn refers to IRF-5 Based on SiRNA / M@pMn, nanoparticles are labeled with rhodamine (RBITC), making the nanoparticles emit red fluorescence, and FAM labels SiRNA, making it emit green fluorescence.

[0153] The experimental steps are as follows:

[0154] (1) Determination of the targeting ability of nanoparticles: Chemotaxis experiments were performed using a transwell chamber with a polycarbonate membrane (Corning, USA) with a pore size of 8 μm. Human umbilical vein endothelial cells (HUVECs) were cultured at a rate of 1×10 5 The number of cells was implanted into the upper chamber, while the lower chamber was used for rat primary macrophages (BMDMs). IRF- 5 SiRNA / pMn, M@pMn, IRF-5SiRNA / M@pMn) was added to the upper chamber. After 24 hours, the cells remaining above the filter were removed with a cotton swab. BMDMs in the lower chamber were collected for flow cytometry (FACS) and confocal laser scanning microscopy (CLSM) analysis. At the same time, the obtained BMDMs were treated with 2.5% glutaraldehyde and then eluted with a graded series of alcohol gradients. The ethanol in the sample was replaced with anhydrous propylene oxide or acetone, and then the epoxy resin was infiltrated, and the catalyst was added to the epoxy resin at a ratio of 1.5-2% and mixed. Then it was polymerized at 35°C for 12 hours, 45°C for 12 hours, and 60°C for 24 hours. The slices were ultrathin and finally observed with a transmission electron microscope (HT7700, Tokyo, Japan).

[0155] (2) Intracellular oxidative stress experiment: BMDMs were placed at 1×10 5 Cells were seeded into 24-well plates at a density of 10 μM / mL. After 6 hours of nanoparticle treatment, the culture medium was replaced with 1 mL of DCFH-DA (10 μM / mL) and incubated at 37°C in the dark for 20 minutes. The cells were rinsed three times with PBS (pH 7.4) and then observed using CLSM.

[0156] After the same treatment, BMDMs were collected and labeled with 10 μg / mL rhodamine 123 at 37°C for 10 min, and then fluorescence intensity was measured using a FACS Calibur flow cytometer (Coulter Beckman, USA). Debris was filtered using SSC / FSC gating, and 30,000 cells were captured.

[0157] (3) Study of macrophage polarization phenotype: BMDMs were gently scraped on ice using a cell scraper, rinsed three times with pre-chilled DPBS, and blocked for 45 min in DPBS containing 1% FBS and FcRblock (Biolegend, USA). The cells were then stained for CD11b and CD86 for 30 min at 4°C. The cells were fixed and permeabilized using a BD fixation / permeabilization kit (BD Bioscience, USA), and then stained with CD206 for 30 min. The fluorescence intensity of the cells was then measured using a FACS Calibur flow cytometer (CoulterBeckman, USA). Debris was filtered using SSC / CD11b gating, and 30,000 cells were collected.

[0158] IRF-5 The biological function experimental results of SiRNA / M@pMn on BMDMs (macrophages) are as follows Figure 4 and Figure 5 shown. Figure 4 In Figure 2, A is the fluorescence of pMn and M@pMn in non-inflammatory and inflammatory BMDMs measured by FACS (n=5). B is the fluorescence of BMDM and FAM-IRF-5SiRNA / RBITC pMn and M@ FAM-IRF-5 SiRNA / RBITC Representative confocal microscopy images of pMn with or without LPS and IFN-γ stimulation (blue, nuclei; red, RBITC pMn; green, FAM siRNA). Figure 5 Figure 5 (E) shows fluorescence imaging of BMDMs stained with a DCFH-DA probe. Figure 6 (F) shows quantitative analysis of MMPs by flow cytometry (n = 3). Figure 7 (G) shows IRF-5 mRNA expression by qPCR (n = 5). Figure 8 (H) shows flow cytometry and quantification of BMDM polarization (n = 3). M1 and M2 subtypes were distinguished by the number of CD86 (primarily in Q3) and CD206 (primarily in Q1) cell populations.

[0159] Due to the interaction between CXCR1 / 2 (neutrophil receptor) and chemokines, membrane-coated NPs are easily driven to inflammatory M1 macrophages. The in vitro established Transwell model facilitates the study of this targeting ability, and HUVECs mimic cells that are not expected to be taken up during SCI.

[0160] As expected, in pro-inflammatory BMDMs, the mean fluorescence intensity (MFI) of RBITC (rhodamine B isothiocyanate) detected by flow cytometry in M@pMn-treated cells was 303% higher than that in pMn-treated cells ( Figure 4 A). Meanwhile, confocal laser scanning microscopy (CLSM) was used to further analyze the targeting efficiency and SiRNA delivery ability of M@pMn ( Figure 4 B). Green fluorescence ( FAM SiRNA) and red fluorescence ( RBITC The fluorescence of M@pMn was overlapped, indicating that M@pMn effectively delivered SiRNA drugs into cells.

[0161] Cellular esterases cleave the DCFH-DA probe (reactive oxygen fluorescence probe) to produce DCFH, which is then oxidized by ROS to produce highly fluorescent 2',7'-dichlorofluorescein (DCF). The fluorescence intensity gradually decreases ( Figure 5 E) shows that nanozymes exhibit good catalytic performance in cells. High levels of oxidative stress can also cause depolarization of mitochondrial membrane potential (MMP). Our results show that Mn3O4, IRF-5 SiRNA / pMn and IRF-5 SiRNA / M@pMn reverses the process of mitochondrial membrane potential depolarization by scavenging ROS ( Figure 5 F), which also prevents the imbalance of apoptosis and autophagy.

[0162] IRF-5 SiRNA / pMn group and IRF-5 The expression of IRF-5 mRNA in the SiRNA / M@pMn group decreased sharply, suggesting that SiRNA can form a silencing induction complex (RISC) in macrophages and reduce the expression of target genes ( Figure 5 G). Fluorescence activated cell sorting (FACS) results showed that the polarization ratio of macrophages in the treatment group changed ( Figure 5 H). In CD11b + In cell populations, IRF- 5 SiRNA / pMn group and IRF-5 CD206 in the SiRNA / M@pMn group + CD86 - The proportion of macrophages in the treated group was slightly higher than that in the untreated group (1.48%), which were 6.02% and 7.35% respectively. IRF-5 SiRNA / pMn-treated group and IRF-5 CD206 in the SiRNA / M@pMn group - CD86 + The proportion of macrophages (25.0%) was significantly lower than that in the untreated group (48.5%), indicating that the nanoparticles could successfully reprogram inflammatory macrophages to a repair phenotype.

[0163] Example 11

[0164] This example investigates the characterization and biocompatibility analysis of gelatin hydrogels that align with the native healing process of the spinal cord. The experimental steps are as follows:

[0165] (1) Synthesis of nanoparticle-doped gelatin hydrogels. To prepare gelatin hydrogels, nanozymes (10 μL, 10 mg / mL) were mixed with a polymer solution of 10% w / v cross-linked gelatin and 0.15% w / v photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP). The hydrogels were then rapidly formed by irradiation with a UV curing lamp (Engineering For Life, China) for 40 s.

[0166] (2) The structure and morphology of the samples were characterized. The morphology and microstructure of the hydrogel were measured using a field emission scanning electron microscope (Hitachi Regulus 8100, Japan). The rheological properties of the prepared 80 mm parallel plate structure hydrogel were studied using a rheometer (Kinexus pro+Instrument). The compression properties of the hydrogel were measured using a universal material testing machine (Instron, 3400). The degradation D was calculated according to the formula r =W0-W t / W0, where Dr is the degradation ratio, and W t and W0 are the dry weights of the scaffolds at 37°C and after initial incubation, respectively. The entire process was carried out in constant temperature (37°C) PBS.

[0167] (3) Determination of the biocompatibility of the hydrogel: Primary neural stem cells were prepared into a cell suspension and co-cultured with gelatin hydrogel. Three days later, the morphology was observed by laser confocal microscopy after immunofluorescence staining.

[0168] The characterization and biocompatibility analysis results of gelatin hydrogels that match the native healing process of the spinal cord are shown in Figure 2. Figure 6 and Figure 7 shown. Figure 6 Figure 1: (A) Schematic diagram of the prepared nanozyme mixed with hydrogel. (B) SEM images of different hydrogels. (C) Rheological analysis of the cross-linking of the prepolymer solution. (D) Representative stress-strain curves of different hydrogels. The inset image shows the compression modulus (n = 3). (E) Degradation rate of the hydrogel after 1, 2, 3, and 4 weeks of incubation in saline (n = 3). (F) Sustained release behavior of the Mn3O4 hydrogel (n = 4). Figure 7 In (G), cells adhere to the gelatin hydrogel surface. In (H), NSCs exhibit high cell viability after 5 days of culture (green: live cells; red: dead cells). In (I), representative images and quantitative analysis of Tuj-1 (neurons) and GFAP (astrocytes) expression, showing the growth of neural stem cells on gelatin hydrogels after 7 days of culture.

[0169] GelMA-based hydrogels are suitable for spinal cord implantation applications. In the presence of a photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl) phosphonate, LAP), GelMA mixed with nanozymes undergoes photoinitiated free radical polymerization under UV irradiation to generate covalently cross-linked hydrogels for implantation into the SCI site, e.g. Figure 6 Figure A. The morphological characteristics of the hydrogels were observed using scanning electron microscopy. The results showed that the hydrogels contained an interconnected porous network with a diameter of approximately 150 nm, demonstrating their ability to serve as a living space for cells and support the movement of nutrients and waste. The addition of nanozymes to the gelatin-based hydrogels significantly reduced the pore size of the nanocomposite hydrogels, which may be due to the increased cross-linking density generated by the nanoparticles ( Figure 6 B), and subsequent rheological and compression modulus tests confirmed this conclusion. The gelatin-based hydrogel was enhanced by adding nanoparticles as cross-linking centers. Rheological tests showed that the hydrogel could form a covalently cross-linked network within 40 seconds, which can be used for further in vivo applications ( Figure 6 C). The compressive modulus showed similar mechanical properties to the natural spinal cord (<10 kPa) ( Figure 6 D).

[0170] Figure 6 E is the degradation rate of hydrogel after incubation in saline for 1, 2, 3 and 4 weeks (n=3). The results showed that the degradation rates of Gel group, G group and Gel-IRF-5 The SiRNA / M@pMn group degraded by more than 60% after 4 weeks, which is consistent with the spinal cord repair process in the SCI model, and the hydrogel material met the mechanical property requirements of the implant material.

[0171] Figure 6 The results of F showed that the drug release capacity of the hydrogel matched the degradation process of the gelatin hydrogel, and the supernatant contents released into the solution after 14 days were 38.54%, 68.21% and 60.38%, respectively.

[0172] The porous hydrogel prepared in this example simulates the extracellular matrix and has mechanical properties that match those of the native spinal cord and degradation capabilities suitable for spinal cord tissue regeneration. Therefore, the prepared gelatin-based hydrogel can be used as a candidate natural material for spinal cord injury repair. The ability of cells to attach, grow, and differentiate within functional hydrogels is crucial for neuronal regeneration in the spinal cord. In this example, cell viability and differentiation were determined by identifying cells adhered to the surface of different nanoparticle hydrogels. Figure 7 G, Gel, Gel-Mn3O4, Gel-IRF-5 SiRNA / pMn and Gel-IRF-5 SiRNA / M@pMn).

[0173] Figure 7 H and Figure 7 I shows the inherent biocompatibility of different hydrogels, and the biomaterials do not affect the differentiation direction of neurons and astrocytes. These results indicate that the prepared hydrogels mixed with different nanoparticles have good in vitro biocompatibility and can be used as implant materials in the rat spinal cord injury model.

[0174] Example 12

[0175] Evaluation of short-term treatment efficacy in animal models. The experimental steps are as follows:

[0176] Rats were anesthetized with 10% chloral hydrate (0.5 g / kg). In order to expose the T9-T10 vertebra, a vertical incision was made on the dorsal muscles on both sides of the vertebra. After the dorsal laminectomy, a T9 full lesion cavity with a length of 4±0.5 mm appeared. When the bleeding stopped, the hydrogel was injected into the spinal cavity and cured in situ with a UV-curing laser for 40 seconds. The dorsal muscles and skin were sutured with degradable sutures. Manual bladder massage was performed twice a day until the bladder resumed automatic urination. The experiment lasted for 7 days, and 8 animals were randomly selected from each group. The relevant frozen section steps were carried out according to immunofluorescence staining and immunohistochemistry.

[0177] Results of short-term treatment effect evaluation in animal models Figure 8 and Figure 9 shown. Figure 8 Figure 2 (A) shows a representative fluorescent staining image of DHE and immunohistochemical image of HIF-1α in injured spinal cord tissue. Figure 2 (B) shows a quantitative analysis of DHE levels. Figure 2 (C) shows a quantitative analysis of HIF-1α levels. Figure 9 A shows Western blot analysis (n = 3) and quantification, and B shows qPCR analysis of IRF-5 expression levels (n = 5). C shows qPCR analysis of CXCL1, CXCL2, CXCL3, CXCL4, CXCL6, and CXCL7 expression levels. D and E show representative immunohistochemical images of CD68 and F4 / 80-positive cells in the spinal cord injury site, respectively.

[0178] The accumulation of electrons during hypoxia leads to the accumulation of O2 in mitochondria. - Dihydroethidium (DHE) is a commonly used fluorescent probe for detecting superoxide anion levels in phagocyte respiratory burst. Figure 8 AC showed a decrease in the mean fluorescence intensity of DHE and the mean optical density (MOD) of HIF-1α, indicating that nanozymes can catalyze the conversion of toxic ROS into non-toxic O2, effectively reducing oxidative stress and alleviating local hypoxia.

[0179] Figure 9 The results of A and B showed that the expression of IRF-5 protein and mRNA decreased by 73.08% and 63.10%, respectively, which is consistent with the IRF-5 The increased expression of IRF-5 in the gelatin hydrogel group may be related to the acute inflammation after the implantation of gelatin hydrogel.

[0180] The attenuation of neutrophil infiltration was confirmed by the reduced expression of CXCL family proteins (CXCL1, CXCL2, CXCL3, CXCL4, CXCL6, and CXCL7). Figure 9 C), which to some extent explains the ability to regulate inflammation. In addition, the expression of inflammatory markers was reduced at the SCI site, including CD68 and F4 / 80, indicating that the activation, expansion and infiltration of inflammatory macrophages were gradually reduced ( Figure 9 D and E).

[0181] Example 13

[0182] The experimental steps for evaluating the long-term treatment efficacy of animal models are as follows:

[0183] The animal modeling process followed the steps described in Example 12. An open field (120 × 200 cm) was used to test and measure hindlimb motor function and perform the BBB motor function score. This was repeated weekly for 8 weeks after surgery. Two independent observers observed each rat for at least 5 minutes, and the observation group was blinded.

[0184] In long-term animal experiments, the functional hydrogel has a good recovery effect, and the test results are as follows Figure 10 and Figure 11 shown. Figure 10 Figure 2 (A) shows the BBB scores of rats from surgery to 8 weeks after injury (n = 8). Figure 2 (B) shows rat footprints (forepaw, blue ink; hindpaw, red ink). Figure 2 (C) shows representative spinal cord images after SCI repair. Figure 2 (D) shows representative immunofluorescence images of ChAT, calbindin, and Brn3a staining. Figure 2 (E) shows the quantification of ChAT staining. Figure 2 (F) shows the quantification of Calbindin staining. Figure 2 (G) shows the quantification of Brn3a staining. Figure 11 qPCR analysis of neuronal and vascular marker expression (n=5).

[0185] Short-term in vivo animal experiments confirmed that the load IRF-5 SiRNA-derived Mn3O4 nanozymes can effectively reduce oxidative damage, alleviate hypoxia, promote the expression of M2 macrophage phenotype, reduce the infiltration of neutrophils and macrophages, and attenuate the immune storm during spinal cord injury. We expect that the exogenous neural environment at the SCI site will be affected by Gel- IRF-5 SiRNA / M@pMn remodeling promoted neuronal regeneration by allowing endogenous neurons to survive, migrate, differentiate, and improve neovascularization, until the motor function of SCI model rats was restored.

[0186] Gel- IRF-5 The BBB score of rats in the siRNA / M@pMn group was 9.57±1.13, which was significantly improved compared with the BBB scores of rats in the other groups (the BBB scores of the control group were 5.14±1.46, 5.43±2.07 for Gel, 6.00±1.15 for Gel-Mn3O4, and 7.00±1.17 for Gel-Mn3O4). IRF- 5 SiRNA / pMn is 7.57±0.98, as Figure 10 A). Treatment group rats (Gel- IRF-5 SiRNA / M@pMn) footprints are clearer ( Figure 10 B), the treatment group was able to carry out weight-bearing movements on the palm surface of the paw even without coordinated movements of the front and back limbs. The above results confirmed the recovery of motor function in rats. Figure 10 This is also confirmed by the restoration of connections in the anatomical diagram of the injured spinal cord shown in C.

[0187] Diversification of neuronal subtypes is essential for the formation of functional neural circuits. Neurons in the spinal cord are functionally classified as sensory neurons, motor neurons, and interneurons. However, the molecular events underlying neuronal diversity after spinal cord injury remain largely unexplored. Several neuronal subtypes were analyzed as markers for post-treatment evaluation, depending on the time course of spinal cord injury.

[0188] The gene expression at the mRNA level in the damaged area was detected by qPCR ( Figure 11 The results showed that the expression of markers for immature neurons (Tuj1, Dcx, and Stmn1), mature neurons (Map2), sensory neurons (Sox10), motor neurons (Chat, Mnx1, and Isl2), and interneurons (Htr3a, Calb1, and Gad2) was significantly increased, and the formation of blood vessels (CD31 and Vcam-1) was also observed.

[0189] In summary, the present invention constructs a multifunctional nanozyme combined with SiRNA technology, which exhibits important synergistic anti-inflammatory and antioxidant effects in the pathological neural environment of spinal cord injury in vitro and in vivo. The appropriate exogenous neural environment induces the differentiation of various neuronal subtypes, thereby reducing inflammation, which also reduces scar formation. Nanozymes show synergistic ability in catalyzing the conversion of high levels of ROS into O2 without causing biological toxicity. In addition, the continuous release of oxygen stimulates the formation of endothelial tubes and angiogenesis. High levels of ROS and inflammation, as two major inhibitory factors, have steadily broadened the understanding of the autoimmune regulatory system and the damaged microenvironment in many injury scenarios. Given that many physiology and diseases are accompanied by ROS and inflammation, the multifunctional integrated nanozyme strategy developed by the present invention may have a wide range of applications in a range of disease models, including the nervous system, cardiovascular and immune systems, skeletal muscle and metabolic control, and aging.

[0190] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A nanozyme complex, characterized in that The nanozyme complex includes a Mn3O4 nanozyme and a small interfering RNA loaded on the Mn3O4 nanozyme; The small interfering RNA is a small interfering RNA that interferes with the expression of the Irf5 gene; The Mn3O4 nanozyme exhibits a tetrahedral hausmannite crystal form, and the diameter of the Mn3O4 nanozyme is 130-170 nm; The Mn3O4 nanozyme exhibits a nanoflower-like structure; The Mn3O4 nanozyme is a mixed-valence Mn3O4 nanozyme; The loading ratio of small interfering RNA on Mn3O4 nanozyme in the nanozyme complex is (4.5-5.5):2000; The surface of the nanozyme complex also contains a coating material; The coating material includes any one or a combination of at least two of a biofilm, a monoclonal antibody or a macromolecule with biological activity; The sense strand of the small interfering RNA includes the nucleotide sequence shown in SEQ ID No: 5, and the antisense strand of the small interfering RNA includes the nucleotide sequence shown in SEQ ID No: 6, wherein two TT bases are added to the sense strand and the antisense strand as bases of the protruding tail.

2. The nanozyme complex according to claim 1, characterized in that The biofilm includes a cell membrane; The bioactive macromolecules include hyaluronic acid.

3. The method for preparing the nanozyme complex according to any one of claims 1 to 2, characterized in that: The preparation method comprises the following steps: (a) The precursor MnO2 is prepared by chemical coprecipitation and calcined at high temperature to obtain mixed-valence Mn3O4 nanozymes; (b) Using the Mn3O4 nanozyme as a carrier, Mn3O4 and polyethyleneimine are cross-linked by an electrostatic conjugation method, and grafted polyethyleneimine is modified on the carrier as an intermediate medium layer. Small interfering RNA is loaded by utilizing the coordination effect of the amino group of polyethyleneimine on the Mn3O4 nanozyme carrier.

4. The method for preparing the nanozyme complex according to claim 3, characterized in that: In step (b), after loading the small interfering RNA, the method further includes coating the nanozyme loaded with the small interfering RNA with a coating material.

5. The method for preparing the nanozyme complex according to claim 4, characterized in that: The steps of cell membrane coating include: repeatedly squeezing the nanozyme loaded with small interfering RNA and repeatedly frozen and thawed cell fragments in a liposome squeezer to achieve effective cell membrane coating.

6. The method for preparing the nanozyme complex according to claim 3, characterized in that: In step (a), the Mn3O4 nanozyme is prepared by a method comprising the following steps: stirring and mixing KMnO4 with water, then adding oleic acid and stirring and mixing to obtain a black precipitate and washing it, drying the washed black precipitate, and calcining it to obtain the Mn3O4 nanozyme.

7. The method for preparing the nanozyme complex according to claim 6, characterized in that: The mass volume ratio of the KMnO4 mixed with water is 1:(450-550).

8. The method for preparing the nanozyme complex according to claim 7, characterized in that: The KMnO4 and water are stirred and mixed for 25-35 minutes at a rotation speed of 180-220 rpm.

9. The method for preparing the nanozyme complex according to claim 8, characterized in that: The volume ratio of oleic acid to water is 10:(450-550).

10. The method for preparing the nanozyme complex according to claim 9, characterized in that: The time for adding oleic acid and stirring and mixing is 12-24 hours, and the rotation speed is 180-220 rpm.

11. The method for preparing the nanozyme complex according to claim 10, characterized in that: The cleaning is performed alternately with water and ethanol, and the number of times of the cleaning is 6-8 times.

12. The method for preparing the nanozyme complex according to claim 11, characterized in that: The drying is performed by vacuum drying, the vacuum drying temperature is 30-45° C., and the vacuum drying time is 12-18 hours.

13. The method for preparing the nanozyme complex according to claim 12, characterized in that: The calcination temperature is 180-200° C., the calcination time is 5-6 hours, and the calcination atmosphere is air.

14. The method for preparing the nanozyme complex according to any one of claims 3 to 13, characterized in that: In step (b), the Mn3O4 and polyethyleneimine are cross-linked using a method comprising the following steps: mixing Mn3O4 with PBS buffer, ultrasonically crushing, adding polyethyleneimine and incubating to obtain Mn3O4 modified with polyethyleneimine.

15. The method for preparing the nanozyme complex according to claim 14, characterized in that: The mass volume ratio of the Mn3O4 to PBS buffer solution is 1 mg:(0.8-1.2 mL).

16. The method for preparing the nanozyme complex according to claim 15, characterized in that: The mass ratio of Mn3O4 to polyethyleneimine is 1:(2-5).

17. The method for preparing the nanozyme complex according to claim 16, characterized in that: The incubation temperature is 35-38° C., the incubation time is 10-14 h, and the incubation speed is 200-240 rpm.

18. Use of the nanozyme complex according to any one of claims 1-2 in the preparation of a drug for reshaping the microenvironment of spinal cord injury.

19. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the nanozyme complex according to any one of claims 1-2.

20. The pharmaceutical composition according to claim 19, characterized in that The pharmaceutical composition further includes pharmaceutically acceptable excipients.

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