Supramolecular nano-enzyme antagonist with multi-effect synergistic effect as well as preparation method and application of supramolecular nano-enzyme antagonist

Abstract

The invention discloses a supramolecular nano enzyme antagonist with a multi-effect synergistic effect as well as a preparation method and application thereof, and belongs to the technical field of biological nano engineering materials. The preparation method comprises the following steps: firstly driving the formation of a nano assembly through Mannich reaction, in the process, an amino group on an aminoglycoside compound only forms a benzoxazine structure with an A ring of EGCG, while a polyphenol part with metal ion chelating ability on the EGCG still exists, and then introducing selenium ions into a reaction system, so that the selenium-rich nano assembly is obtained. According to the preparation method, selenium ions are added into the nano-assembly and are strongly coordinated with residual polyphenol groups in the nano-assembly, the selenium ions are adsorbed to the surface of the nano-assembly, finally, the selenium ions are reduced into elemental selenium in situ under the reduction of ascorbic acid, and meanwhile, nano-selenium particles are formed in situ due to a nano-confinement effect; therefore, the supramolecular nano-enzyme antagonist with efficient cfDNA capture and ROS removal capabilities at the same time is constructed.

Description

Technical Field

[0001] This invention belongs to the field of bio-nanotechnology, and particularly relates to a supramolecular nanoenzyme antagonist with multiple synergistic effects, its preparation method, and its application. Background Technology

[0002] Psoriasis is a common inflammatory skin disease characterized by thickened epidermis, erythematous plaques, and silvery-white, hardened scales covering the skin. It affects approximately 2-3% of the global population. Currently, clinical treatment for psoriasis primarily relies on medication to control the inflammatory response, including corticosteroids, vitamin D derivatives, targeted biologics, folic acid antagonists, and calcineurin inhibitors. However, long-term use of these medications may lead to immunosuppression, increasing the risk of infection and skin cancer. Therefore, there is an urgent need to develop new drug alternative therapies to effectively alleviate the inflammatory response of psoriasis and reduce its side effects.

[0003] In recent years, researchers have developed a series of cationic nanomaterials to regulate inflammatory responses by clearing nucleic acids released from dead cells. Therefore, designing cationic nanomaterials to capture cell-free DNA (cfDNA) to block inflammatory signaling pathways has become a potential treatment strategy for psoriasis. For example, the team led by Chen Yongming (Z. Liu, Y. Wang, Y. Zhang, et al. Biguanide chitosan microneedles with cell-free DNA scavenging ability for psoriasis therapy [J]. Bioactive Materials: 2024, 33: 497-505.) designed and synthesized a series of poly(2-(dimethylamino)methacrylate) (PDMA)-grafted hairy silica particles (cSPs) as cfDNA scavengers, which can be used to clear cfDNA in the dermis and have shown good therapeutic effects on psoriasis. On the other hand, reactive oxygen species (ROS), such as superoxide anions (•O2), are also a potential target for cfDNA scavenging. -Inflammatory substances such as hydroxyl radicals (ROS) and hydrogen peroxide (H2O2) can activate the mTOR-NF-κB pathway in keratinocytes and stimulate the release of inflammatory factors, thereby initiating and maintaining the progression of psoriasis. In psoriasis patients, an imbalance in the redox balance leads to excessive accumulation of reactive oxygen species (ROS), which in turn increases oxidative damage to keratinocytes. Dead keratinocytes release more cfDNA, creating a vicious cycle of inflammation. Therefore, clearing excessive ROS accumulation at inflammatory sites and reducing oxidative stress levels in skin tissue is considered a potential strategy for treating psoriasis. For example, the team of Zhu Jintao / Liu Yijing at Huazhong University of Science and Technology (D. Bi, F. Qu, W. Xiao, et al. Reactive oxygen species-responsive gel-based microneedle patches for prolonged and intelligent psoriasis management [J]. ACS Nano: 2023, 17 (5):4346-4357) developed a gel microneedle patch that can simultaneously deliver the anti-proliferative drug methotrexate (MTX) and the ROS scavenging drug epigallocatechin gallate (EGCG) for the combined treatment of psoriasis. The release of MTX can inhibit the proliferation of keratinocytes, while the continuous release of EGCG can scavenge intracellular ROS and inhibit the activation of NF-κB, thereby achieving an anti-inflammatory effect. The human body has a natural antioxidant system, mainly composed of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). These enzymes scavenge intracellular ROS through catalytic reactions to maintain redox homeostasis. However, studies have found that these antioxidant enzymes are underexpressed in the skin tissue of psoriasis patients, leading to the inability to promptly clear accumulated ROS in the skin and exacerbating the condition. Therefore, utilizing nanomaterials with antioxidant enzyme-like activity to remove excess ROS in psoriatic skin and restore its redox homeostasis is a highly promising treatment option.

[0004] Currently, while both cfDNA capture strategies based on cationic nanomaterials and ROS scavenging strategies based on antioxidant enzyme-like nanomaterials have shown some efficacy in alleviating psoriasis symptoms, the complex inflammatory microenvironment of psoriasis means that nanomaterials with single mechanisms of action still face challenges in terms of efficacy limitations. Therefore, constructing hybrid nanomaterials with both highly efficient cfDNA capture and ROS scavenging capabilities has become a pressing technical problem to be solved in this field. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a supramolecular nanozyme antagonist with multiple synergistic effects, its preparation method, and its applications. The supramolecular nanozyme antagonist prepared by this invention possesses both highly efficient cfDNA capture and ROS scavenging capabilities. Through synergistic action of multiple mechanisms, it blocks the vicious cycle of psoriatic skin inflammation, thereby achieving synergistic relief and treatment of the symptoms.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing a supramolecular nanoenzyme antagonist with multiple synergistic effects, comprising the following steps: (1) Mix the aqueous solution of epigallocatechin gallate and the aqueous solution of formaldehyde and react them. Then add the aqueous solution of aminoglycoside compound to carry out the Mannich reaction to obtain the Mannich nano-assembly. (2) The Mannich nanoassembly is dispersed in water, sodium selenite aqueous solution is added, and after stirring evenly, ascorbic acid solution is added to carry out a reduction reaction to obtain the supramolecular nanoenzyme antagonist.

[0007] Further, in step (1), the concentration of the epigallocatechin gallate aqueous solution is 5~20 mmol / L; the concentration of the formaldehyde aqueous solution is 12.3 mmol / L; and the volume ratio of the epigallocatechin gallate aqueous solution to the formaldehyde aqueous solution is 200:(1~20).

[0008] Furthermore, in step (1), the reaction time is 5 to 30 minutes.

[0009] Further, in step (1), the aminoglycoside compound in the aqueous solution of the aminoglycoside compound is selected from at least one of hygromycin, netilmicin, kanamycin, tobramycin and neomycin; the concentration of the aqueous solution of the aminoglycoside compound is 5~20 mmol / L; and the volume ratio of the aqueous solution of the aminoglycoside compound to the aqueous solution of epigallocatechin gallate is 1:1.

[0010] Furthermore, in step (1), the dropping rate is 5~10s / drop; the time for the Mannich reaction after the dropping is completed is 2~12h.

[0011] Further, in step (2), the concentration of the sodium selenite aqueous solution is 10~100 mmol / L; the volume ratio of the sodium selenite aqueous solution to the Mannich nanoassembly is 1:5.

[0012] Further, in step (2), the concentration of the ascorbic acid solution is 40 mmol / L; the volume ratio of the ascorbic acid solution to the Mannich nanoassembly is 2:5.

[0013] Furthermore, in step (2), the reduction reaction takes 12 to 24 hours.

[0014] This invention provides a supramolecular nanoenzyme antagonist with multiple synergistic effects, which is prepared according to the preparation method described in the above technical solution.

[0015] This invention also provides the application of a supramolecular nanoenzyme antagonist with synergistic effects as described above in the preparation of drugs to alleviate psoriasis.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: This invention first drives the formation of nanoassemblies through the Mannich reaction. During this process, the amino group on the aminoglycoside compound forms a benzoxazine structure only with the A ring of EGCG, while the polyphenolic part of EGCG with metal ion chelating ability remains. Then, selenium ions are introduced into the reaction system to strongly coordinate with the residual polyphenolic groups in the nanoassemblies. The selenium ions are adsorbed onto the surface of the nanoassemblies. Finally, under the reduction of ascorbic acid, the selenium ions are reduced to elemental selenium in situ. At the same time, due to the nano-constraint effect, nano-selenium particles are formed in situ, thereby constructing a supramolecular nanozyme antagonist with both highly efficient cfDNA capture and ROS scavenging capabilities.

[0017] This invention employs a synergistic strategy of Mannich reaction-driven assembly and confined in-situ reaction to prepare a multi-functional supramolecular nanoenzyme antagonist via a rapid one-pot method using aminoglycoside compounds, epigallocatechin gallate (EGCG), and sodium selenite. The supramolecular nanoenzyme antagonist prepared in this invention can effectively capture cfDNA through multiple non-covalent interactions, compete for TLR receptors, block the activation of inflammatory pathways, and rapidly scavenge ROS through its highly efficient antioxidant enzyme activity, thereby restoring skin redox homeostasis, alleviating the inflammatory microenvironment, and promoting skin tissue repair. Furthermore, the reduction in reactive oxygen species levels in skin tissue can further inhibit excessive proliferation of keratinocytes and alleviate psoriasis symptoms.

[0018] The supramolecular nanozyme antagonist prepared in this invention has good biocompatibility, antioxidant enzyme-like activity, and cfDNA capture performance. At the lesion site of psoriasis, the supramolecular nanozyme antagonist effectively captures cfDNA through multiple non-covalent interactions, blocks its binding to TLR receptors, thereby inhibiting the activation of related pathways. It can also further inhibit the excessive proliferation of keratinocytes by scavenging reactive oxygen species, thus alleviating psoriasis symptoms. This provides a promising nano-alternative therapy for alleviating psoriasis-related symptoms. Attached Figure Description

[0019] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The preparation process, 1H NMR spectrum, and mass spectrometry of the model reaction product EmAM-ol are shown, where a represents the preparation process, b represents the 1H NMR spectrum, and c represents the mass spectrometry. Figure 2 Transmission electron microscopy (TEM) images and EDS elemental mapping images of the supramolecular nanozyme antagonists prepared in Examples 1-5; Figure 3 The antioxidant capacity of EmNeo-Se prepared in Example 5 is given by: a) scavenging ability of ABTS free radicals, b) scavenging ability of DPPH free radicals, and c) scavenging ability of •O2. - The ability, where d represents the ability to scavenge •OH; Figure 4 This demonstrates the ability of EmNeo-Se prepared in Example 5 to capture and adsorb nucleic acid molecules. Figure 5 The evaluation results of EmNeo-Se regulating the intracellular oxidative stress process of keratinocytes prepared in Example 5 are shown in Figure a, where a represents the fluorescence effect and b represents the fluorescence intensity. Figure 6 The results of evaluating the regulation of intracellular oxidative stress and abnormal proliferation behavior of keratinocytes by EmNeo-Se prepared in Example 5 under an in vitro psoriasis simulation environment are shown in the figures. a is a DCFH fluorescence flow cytometry, b is the quantitative statistics of the flow cytometry results, c is the HaCat cell proliferation results detected by the EdU method, and d is the HaCat cell proliferation results detected by the CCK-8 method. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] This invention provides a method for preparing a supramolecular nanozyme antagonist with multiple synergistic effects, comprising the following steps: (1) An aqueous solution of epigallocatechin gallate (EGCG) and an aqueous solution of formaldehyde were mixed and reacted, and then an aqueous solution of aminoglycoside compound was added dropwise to carry out the Mannich reaction to obtain Mannich nanoassemblies. (2) The Mannich nanoassembly is dispersed in water, sodium selenite aqueous solution is added, and after stirring evenly, ascorbic acid solution is added to carry out a reduction reaction to obtain the supramolecular nanoenzyme antagonist.

[0023] In a preferred embodiment, in step (1), the concentration of the epigallocatechin gallate aqueous solution is 5-20 mmol / L, more preferably 10-20 mmol / L; the concentration of the formaldehyde aqueous solution is 12.3 mmol / L; and the volume ratio of the epigallocatechin gallate aqueous solution to the formaldehyde aqueous solution is 200:(1-20), more preferably 200:2. In this invention, epigallocatechin gallate provides antioxidant activity and DNA binding capacity, while formaldehyde is used to connect EGCG with aminoglycoside compounds to construct amphiphilic molecules and promote self-assembly.

[0024] In a preferred embodiment, in step (1), the reaction time is 5 to 30 minutes, the reaction temperature is room temperature, and the reaction is carried out under stirring at a speed of 500 rpm.

[0025] In a preferred embodiment, in step (1), the aminoglycoside compound in the aqueous solution is selected from at least one of hygromycin (Hyg), netilmicin (Net), kanamycin (Kan), tobramycin (Tob), and neomycin (Neo), more preferably neomycin (Neo); the concentration of the aqueous solution of the aminoglycoside compound is 5-20 mmol / L, more preferably 10-20 mmol / L; the volume ratio of the aqueous solution of the aminoglycoside compound to the aqueous solution of epigallocatechin gallate is 1:1. The aminoglycoside compound in this invention is used to introduce a positive charge, enhancing the electrostatic adsorption with negatively charged cfDNA.

[0026] In a preferred embodiment, in step (1), the dropping rate is 5-10 s / drop, more preferably 5 s / drop; the time for the Mannich reaction after the dropping is completed is 2-12 h, more preferably 7 h; the temperature of the Mannich reaction is room temperature. During the Mannich reaction, formaldehyde reacts with aminoglycoside compounds to generate imine ions, which then react with the A ring of EGCG to form a benzoxazine structure, generating a Mannich base. The Mannich base molecules self-assemble into nanoparticles through non-covalent interactions such as π-π stacking, hydrogen bonding, and hydrophobic interactions.

[0027] In a preferred embodiment, step (1) further includes centrifugation, washing and drying after the Mannich reaction is completed.

[0028] In a preferred embodiment, in step (2), the concentration of the sodium selenite aqueous solution is 10-100 mmol / L, more preferably 10 mmol / L; the volume ratio of the sodium selenite aqueous solution to the Mannich nanoassemblies is 1:5. This invention introduces selenium ions into the reaction system, where they strongly coordinate with the residual polyphenol groups in the nanoassemblies. The selenium ions are adsorbed onto the surface of the nanoassemblies, and finally, under the reduction of ascorbic acid, the selenium ions are reduced in situ to elemental selenium. Simultaneously, due to the nano-constraint effect, nano-selenium particles are formed in situ, thereby constructing a supramolecular nanozyme antagonist that simultaneously possesses highly efficient cfDNA capture and ROS scavenging capabilities.

[0029] In a preferred embodiment, in step (2), the concentration of the ascorbic acid solution is 40 mmol / L; the volume ratio of the ascorbic acid solution to the Mannich nanoassembly is 2:5.

[0030] In a preferred embodiment, in step (2), the reduction reaction takes 12 to 24 hours, more preferably 16 hours; the reduction reaction takes place at room temperature.

[0031] In a preferred embodiment, step (2) further includes centrifugation, washing and drying after the reduction reaction is completed.

[0032] This invention provides a supramolecular nanoenzyme antagonist with multiple synergistic effects, which is prepared according to the preparation method described in the above technical solution.

[0033] This invention also provides the application of a supramolecular nanoenzyme antagonist with synergistic effects as described above in the preparation of drugs to alleviate psoriasis.

[0034] In this embodiment of the invention, room temperature refers to "25±2℃".

[0035] Unless otherwise specified, all raw materials used in the embodiments of this invention were purchased through commercial channels.

[0036] Example 1 A method for preparing a supramolecular nanoenzyme antagonist with multiple synergistic effects (EmHyg-Se), the specific steps of which are as follows: (1) Add 20 μL of formaldehyde aqueous solution with a concentration of 12.3 mmol / L to 2 mL of EGCG aqueous solution (concentration of 20 mmol / L), and stir at 500 rpm for 30 min at room temperature; then add 2 mL of Hyg aqueous solution (concentration of 20 mmol / L) dropwise at a rate of 5 s / drop, and continue stirring at room temperature for 7 h after the addition is completed; then collect the white solid precipitate by centrifugation, and wash and dry the white solid precipitate to obtain the Mannich nanoassemblies EmHyg; (2) The Mannig nanoassembly EmHyg obtained in step (1) was redispersed in 5 mL of water, and 1 mL of sodium selenite aqueous solution (concentration of 10 mmol / L, volume ratio of sodium selenite aqueous solution to Mannig nanoassembly of 1:5) was added dropwise. After stirring and mixing thoroughly, 2 mL of ascorbic acid aqueous solution (concentration of 40 mmol / L, volume ratio of ascorbic acid solution to Mannig nanoassembly of 2:5) was added. The reaction was carried out at room temperature for 16 h. The reaction solution gradually turned red. After the reaction was completed, the red solid precipitate was collected by centrifugation. The red solid precipitate was washed and dried to obtain the supramolecular nanoenzyme antagonist EmHyg-Se with multiple synergistic effects.

[0037] Example 2 A method for preparing a multi-effect synergistic supramolecular nanozyme antagonist (EmNet-Se), the specific steps of which are as follows: (1) Add 20 μL of formaldehyde aqueous solution with a concentration of 12.3 mmol / L to 2 mL of EGCG aqueous solution (concentration of 20 mmol / L), and stir at 500 rpm for 30 min at room temperature; then, add 2 mL of Net aqueous solution (concentration of 20 mmol / L) dropwise at a rate of 5 s / drop, and continue stirring at room temperature for 7 h after the addition is completed; then, collect the white solid precipitate by centrifugation, and wash and dry the white solid precipitate to obtain the Mannich nanoassembly EmNet; (2) The Mannich nanoassembly EmNet obtained in step (1) was redispersed in 5 mL of water, and 1 mL of sodium selenite aqueous solution (concentration of 10 mmol / L, volume ratio of sodium selenite aqueous solution to Mannich nanoassembly of 1:5) was added dropwise. After stirring and mixing thoroughly, 2 mL of ascorbic acid aqueous solution (concentration of 40 mmol / L, volume ratio of ascorbic acid solution to Mannich nanoassembly of 2:5) was added. The reaction was carried out at room temperature for 16 h. The reaction solution gradually turned red. After the reaction was completed, the red solid precipitate was collected by centrifugation. The red solid precipitate was washed and dried to obtain EmNet-Se, a supramolecular nanoenzyme antagonist with multiple synergistic effects.

[0038] Example 3 A method for preparing a multi-effect synergistic supramolecular nanoenzyme antagonist (EmKan-Se), the specific steps of which are as follows: (1) Add 20 μL of formaldehyde aqueous solution with a concentration of 12.3 mmol / L to 2 mL of EGCG aqueous solution (concentration of 20 mmol / L), and stir at 500 rpm for 30 min at room temperature; then add 2 mL of Kan aqueous solution (concentration of 20 mmol / L) dropwise at a rate of 5 s / drop, and continue stirring at room temperature for 7 h after the addition is completed; then collect the white solid precipitate by centrifugation, and wash and dry the white solid precipitate to obtain the Mannich nanoassembly EmKan; (2) The Mannich nanoassembly EmKan obtained in step (1) was redispersed in 5 mL of water, and 1 mL of sodium selenite aqueous solution (concentration of 10 mmol / L, volume ratio of sodium selenite aqueous solution to Mannich nanoassembly of 1:5) was added dropwise. After stirring and mixing thoroughly, 2 mL of ascorbic acid aqueous solution (concentration of 40 mmol / L, volume ratio of ascorbic acid solution to Mannich nanoassembly of 2:5) was added. The reaction was carried out at room temperature for 16 h. The reaction solution gradually turned red. After the reaction was completed, the red solid precipitate was collected by centrifugation. The red solid precipitate was washed and dried to obtain the supramolecular nanoenzyme antagonist EmKan-Se with multiple synergistic effects.

[0039] Example 4 A method for preparing a supramolecular nanozyme antagonist (EmTob-Se) with multiple synergistic effects, the specific steps of which are as follows: (1) Add 20 μL of formaldehyde aqueous solution with a concentration of 12.3 mmol / L to 2 mL of EGCG aqueous solution (concentration of 20 mmol / L), and stir at 500 rpm for 30 min at room temperature; then, add 2 mL of Tob aqueous solution (concentration of 20 mmol / L) dropwise at a rate of 5 s / drop, and continue stirring at room temperature for 7 h after the addition is completed; then, collect the white solid precipitate by centrifugation, and wash and dry the white solid precipitate to obtain Mannig nano-assemblies EmTob; (2) The Mannich nanoassembly EmTob obtained in step (1) was redispersed in 5 mL of water, and 1 mL of sodium selenite aqueous solution (concentration of 10 mmol / L, volume ratio of sodium selenite aqueous solution to Mannich nanoassembly of 1:5) was added dropwise. After stirring and mixing thoroughly, 2 mL of ascorbic acid aqueous solution (concentration of 40 mmol / L, volume ratio of ascorbic acid solution to Mannich nanoassembly of 2:5) was added. The reaction was carried out at room temperature for 16 h. The reaction solution gradually turned red. After the reaction was completed, the red solid precipitate was collected by centrifugation. The red solid precipitate was washed and dried to obtain the supramolecular nanoenzyme antagonist EmTob-Se with multiple synergistic effects.

[0040] Example 5 A method for preparing a multi-effect synergistic supramolecular nanozyme antagonist (EmNeo-Se), the specific steps of which are as follows: (1) Add 20 μL of formaldehyde aqueous solution with a concentration of 12.3 mmol / L to 2 mL of EGCG aqueous solution (concentration of 20 mmol / L), and stir at 500 rpm for 30 min at room temperature; then add 2 mL of Neo aqueous solution (concentration of 20 mmol / L) dropwise at a rate of 5 s / drop, and continue stirring at room temperature for 7 h after the addition is completed; then collect the white solid precipitate by centrifugation, and wash and dry the white solid precipitate to obtain the Mannich nanoassembly EmNeo; (2) The Mannich nanoassemblies EmNeo obtained in step (1) were redispersed in 5 mL of water, and 1 mL of sodium selenite aqueous solution (concentration of 10 mmol / L, volume ratio of sodium selenite aqueous solution to Mannich nanoassemblies of 1:5) was added dropwise. After stirring and mixing thoroughly, 2 mL of ascorbic acid aqueous solution (concentration of 40 mmol / L, volume ratio of ascorbic acid solution to Mannich nanoassemblies of 2:5) was added. The reaction was carried out at room temperature for 16 h. The reaction solution gradually turned red. After the reaction was completed, the red solid precipitate was collected by centrifugation. The red solid precipitate was washed and dried to obtain the supramolecular nanoenzyme antagonist EmNeo-Se with multiple synergistic effects.

[0041] To elucidate the mechanism of the Mannich reaction-driven assembly of EGCG with aminoglycosides, aminocyclohexanol (4-Am-ol), with a structure similar to the aminocyclic alcohol ring of aminoglycosides, was selected as a model molecule. The preparation process of Example 1 was repeated to obtain the model reaction product EmAM-ol, and its 1H NMR and mass spectrometry were analyzed. The results are shown in [Figure 1]. Figure 1 .

[0042] Figure 1 The preparation process, 1H NMR spectrum, and mass spectrometry of the model reaction product EmAM-ol are shown below. In the figure, a represents the preparation process, b represents the 1H NMR spectrum, and c represents the mass spectrometry. The 1H NMR spectrum of EmAM-ol shows characteristic peaks for OCH2N and PhCH2N, while the H1 and H2 signals of EGCG disappear, suggesting that the A ring of EGCG forms a benzoxazine structure through Mannich condensation with the amino group. Furthermore, mass spectrometry analysis further confirms the molecular ion peak of the product ([EmAM-ol + H)). + , m / z 597.18). Figure 1 The results showed that EGCG and aminoglycosides achieved covalent coupling through the Mannich reaction, which changed their hydrophilicity and hydrophobicity, driving their supramolecular self-assembly to form nanoparticles.

[0043] 1 mg of the supramolecular nanoenzyme antagonist prepared in Examples 1-5 was added to 10 mL of deionized water and ultrasonically dispersed to obtain a supramolecular nanoenzyme antagonist dispersion system. This system was then dropped onto a copper grid. After drying, the microstructure and elemental distribution of the supramolecular nanoenzyme antagonist were observed using transmission electron microscopy. The results... Figure 2 .

[0044] Figure 2 Transmission electron microscopy (TEM) images and EDS elemental mapping diagrams of the supramolecular nanozyme antagonists prepared in Examples 1-5. Figure 2 It can be seen that the supramolecular nanoenzyme antagonists prepared in Examples 1-5 are spherical in shape with different sizes. Further elemental distribution scanning revealed that the nano-selenium is uniformly distributed on the outer surface of the spheres, thus confirming that the synergistic strategy of Mannig-driven assembly and confined in-situ reaction can effectively prepare nanostructures similar to "raspberry".

[0045] The selenium nanoparticles attached to the outer surface of the supramolecular nanozyme antagonist possess strong antioxidant capabilities. To verify the antioxidant capacity of the supramolecular nanozyme antagonist, EmNeo-Se, prepared in Example 5 with the highest selenium nanoparticle loading, was selected for the experiment. The concentrations of EmNeo-Se were 4 μg / mL, 8 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 100 μg / mL. Free radical scavenging experiments were conducted using 1,1-diphenyl-2-picrylhydrazine (DPPH) and 2,2'-aminobis(3-ethylbenzothiazol-6-sulfonic acid) (ABTS). The specific experimental procedures are as follows: ABTS free radical scavenging experiment: 7 mM ABTS was mixed with 2.45 mM K2S2O8 and incubated in the dark for 16 h to obtain the ABTS radical reagent stock solution. The ABTS radical reagent stock solution was diluted with 0.01 M PBS at 734 nm to A = 0.7 ± 0.02 to obtain the ABTS radical reagent working solution. 750 μL of the ABTS radical reagent working solution was mixed with 250 μL of EmNeo-Se (final concentrations of EmNeo-Se were 4 μg / mL, 8 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 100 μg / mL; the control group received an equal volume of solvent). The mixture was reacted at 37 °C in the dark for 10 min, centrifuged at 12000 rpm for 10 min, and 200 μL of the supernatant was measured at 734 nm using a microplate reader (SpectraMax ID3, Molecular Devices, USA). The ABTS scavenging rate was calculated using the following formula: ABTS + ·scavenging ratio (%) = ((A) control -A sample ) / Acontrol ) 100%, of which A control A represents the absorbance of the control group. sample This indicates the absorbance of the experimental group.

[0046] DPPH radical scavenging experiment: DPPH was added to DMSO to obtain a DPPH radical stock solution (concentration of 0.25 mg / mL). The DPPH radical stock solution was diluted with deionized water at a volume ratio of 1:4 to obtain a working solution. 750 μL of the working solution was mixed with 250 μL of EmNeo-Se at different concentrations (final concentrations of EmNeo-Se were 4 μg / mL, 8 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 100 μg / mL; the control group was added with an equal volume of solvent) and incubated at 37℃ in the dark for 45 min. After the reaction was completed, 200 μL of the supernatant was collected by centrifugation at 12000 rpm for 10 min and the absorbance was measured at 517 nm using a microplate reader. The scavenging rate was calculated according to the following formula: DPPH·scavengingratio(%) = ((A control -A sample ) / A control ) 100%, of which A control A represents the absorbance of the control group. sample This indicates the absorbance of the experimental group.

[0047] Two representative biorelevant reactive oxygen species, O2, were selected. - The reactive oxygen species scavenging activity of EmNeo-Se was studied using •OH, and the experimental results are shown in [Figure number missing]. Figure 3 The specific experimental procedure is as follows: •OH removal experiment: 500 μg / mL EmNeo-Se was dispersed in deionized water, vortexed, and then sonicated for 1 min. This process was repeated three times to obtain the EmNeo-Se dispersion. Control group: 50 μL of 30% hydrogen peroxide was mixed with 150 μL of deionized water, followed by 100 μL of 100 mM DMPO solution. After irradiation with 365 nm UV light for 10 min, the sample was loaded and sealed using a capillary tube. Experimental group: 50 μL of 30% hydrogen peroxide was mixed with 150 μL of EmNeo-Se dispersion, followed by 100 μL of 100 mM DMPO solution. After irradiation with 365 nm UV light for 10 min, the sample was loaded and sealed using a capillary tube. The sealed tubes were then placed in sample tubes, and tests were performed and data collected using electron paramagnetic resonance (EPR) (A300-10 / 12, Bruker, Germany).

[0048] •O2- Elimination experiment: The final concentrations of EmNeo-Se dispersions were 4 μg / mL, 8 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 100 μg / mL. An equal volume of solvent was added to the control group. The reaction was performed according to the instructions of the kit for inhibiting and generating superoxide anion radicals. After the reaction was complete, the sample was centrifuged at 12000 rpm for 10 min, and 200 μL of the supernatant was measured at 550 nm using a microplate reader. The scavenging rate was calculated using the following formula: •O 2- scavenging ratio (%) = ((A) control -A sample ) / A control ) 100%, of which A control A represents the absorbance of the control group. sample This indicates the absorbance of the experimental group.

[0049] Figure 3 The antioxidant capacity of EmNeo-Se prepared in Example 5 is given by: a) scavenging ability of ABTS free radicals, b) scavenging ability of DPPH free radicals, and c) scavenging ability of •O2. - The ability, d, represents the ability to scavenge •OH. From Figure 3 It can be seen that after treatment with 100 μg / mL EmNeo-Se, approximately 90% of ABTS radicals and 80% of DPPH radicals were eliminated (see...). Figure 3 (Parts a-b in the original text). EmNeo-Se exhibits significant ROS scavenging activity, and this activity is concentration-dependent. 100 μg / mL of EmNeo-Se can scavenge approximately 40% of •O2. - (See Figure 3 (See part c in the text), indicating that EmNeo-Se possesses superoxide dismutase (SOD)-like activity. Furthermore, EmNeo-Se exhibits a significant scavenging effect on •OH (see [section c]). Figure 3 (part d in the original text). The above experimental results confirm that EmNeo-Se has strong antioxidant capabilities.

[0050] To verify the ability of EmNeo-Se prepared in Example 5 to scavenge nucleic acid molecules in solution, calf thymus DNA (ssDNA) and CpG1826 (dsDNA) were selected as model DNAs, and a systematic evaluation of the scavenging effect was conducted. The experimental results are shown in [Figure 5]. Figure 4 The specific experimental steps are as follows: DNA clearance capacity assessment experiment: For dsDNA, calf thymus DNA (ctDNA, final concentration 5 μg / mL) and different concentrations of EmNeo-Se (final concentrations of 0 μg / mL, 4 μg / mL, 8 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 100 μg / mL, with an equal volume of solvent added to the control group) were incubated in Tris-HCl (10 mM, pH 7.4) buffer at 37°C for 2 h in the dark. After centrifugation (12000 rpm, 10 min), 199 μL of the supernatant was added to 1 μL of ethidium bromide (EtBr, final concentration 0.5 μg / mL), and the mixture was shaken (60 rpm, 15 min) in the dark and allowed to stand for 2 min. After the readings stabilized, the fluorescence intensity was measured at Ex / Em = 520 / 620 nm using a microplate reader. The fluorescence intensity was then substituted into the standard curve to obtain the dsDNA concentration. For ssDNA, FITC-CpG was used. 1826 (final concentration 5 μg / mL) was co-incubated with different concentrations of EmNeo-Se (final concentrations of 0 μg / mL, 4 μg / mL, 8 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 100 μg / mL, respectively; the control group was incubated with an equal volume of solvent). After centrifugation, 200 μL of the supernatant was taken, and the fluorescence intensity was measured at Ex / Em=488 / 525 nm using a microplate reader. The concentration of ssDNA was obtained by substituting the values ​​into the standard curve. The DNA binding efficiency of EmNeo-Se is calculated using the following formula: DNA binding efficiency (%) = ((C control -C sample ) / C control ) 100%, of which C control The fluorescence intensity of the control group is represented by C. sample This indicates the fluorescence intensity of the experimental group.

[0051] Figure 4 This demonstrates the ability of EmNeo-Se prepared in Example 5 to capture and adsorb nucleic acid molecules. Figure 4 The results confirmed that the EmNeo-Se prepared in Example 5 could effectively remove DNA in a concentration-dependent manner.

[0052] To investigate the antioxidant activity of EmNeo-Se prepared in Example 5 on keratinocytes under oxidative stress, co-culture of tert-butyl hydrogen peroxide (tBHP) and keratinocytes (HaCaT) was used to simulate the physiological environment of oxidative stress in psoriasis. Simultaneously, intracellular oxidative stress status was assessed using DCFH-DA (a probe that emits green fluorescence in the presence of reactive oxygen species). The tests were conducted in four groups: a normal group (Control), a model group (tBHP), an EmNeo group, and an EmNeo-Se group. The experimental results are shown below. Figure 5 The specific experimental steps are as follows: HaCat intracellular ROS clearance assay: HaCat cells were seeded at 200,000 cells / well in 12-well plates for 24 hours. The normal group only had its culture medium changed. The model group, EmNeo group, and EmNeo-Se group had their original culture medium replaced with medium containing 100 μM tBHP. The EmNeo group was given 25 μg / mL EmNeo, and the EmNeo-Se group was given 25 μg / mL EmNeo-Se. After incubation for 4 hours, the cells were stained with Hoechst 33342 for 15 minutes, loaded with the DCFH-DA probe for 20 minutes, and then photographed using an FV3000 multispectral rapid laser confocal microscope.

[0053] Figure 5 The results of evaluating the regulation of intracellular oxidative stress in keratinocytes by EmNeo-Se prepared in Example 5 are shown, where a represents fluorescence effect and b represents fluorescence intensity. Figure 5 As can be seen, HaCaT cells treated with tBHP in the model group exhibited strong intracellular green fluorescence, indicating an increased level of intracellular oxidative stress. Subsequent treatment with EmNeo showed a lesser reduction in intracellular green fluorescence, presumably due to the lack of antioxidant enzyme activity inherent in nano-selenium materials, thus failing to effectively alleviate the intracellular oxidative stress environment. Cells treated with EmNeo-Se showed almost no intracellular green fluorescence. These experimental results demonstrate that EmNeo-Se can effectively alleviate the intracellular oxidative stress environment of keratinocytes.

[0054] To investigate the effects of EmNeo-Se prepared in Example 5 on abnormal oxidative stress and proliferation of keratinocytes in the abnormal microenvironment of psoriasis, HaCaT cells were treated with M5 (a mixture of five pro-inflammatory factors, including IL-22, IL-17A, IL-1α, TNF-α, and tumor suppressor protein M) and then further systematically evaluated. The test was conducted in three groups: a normal control group, an M5 group, and an M5+EmNeo-Se group. The experimental results are shown in [Figure 1]. Figure 6 The specific experimental steps are as follows: M5-induced intracellular ROS clearance assay in HaCat cells: HaCat cells were seeded at 100,000 cells / well in 12-well plates for 24 hours. The normal group only had its culture medium changed. The M5 group and the M5+EmNeo-Se group had their original culture medium replaced with a medium containing 10 ng / mL of M5 factor. The M5+EmNeo-Se group had 25 μg / mL of EmNeo-Se added. After incubation for 48 hours, the cells were collected, loaded with the DCFH-DA probe for 20 minutes, and then analyzed by flow cytometry.

[0055] M5-induced HaCat cell proliferation inhibition assay (EdU method): HaCat cells were seeded at 10,000 per well in 96-well plates and incubated overnight until they returned to normal. The normal group only had its culture medium changed. The M5 group and the M5+EmNeo-Se group had their original culture medium replaced with a medium containing 10 ng / mL of M5 factor. The M5+EmNeo-Se group had 25 μg / mL of EmNeo-Se added. When the confluence of cells reached 70% in the control group, the absorbance was measured at 370 nm using an EdU microplate reader.

[0056] M5-induced inhibition of HaCat cell proliferation assay (CCK-8 assay): HaCat cells were seeded at 10,000 per well in 96-well plates. After 24 hours, the cells were allowed to recover to normal. The normal group only had its culture medium changed. The M5 group and the M5+EmNeo-Se group had their original culture medium replaced with a medium containing 10 ng / mL of M5 factor. The M5+EmNeo-Se group had 25 μg / mL of EmNeo-Se added. The absorbance was measured at 450 nm using a CCK-8 assay kit at 0, 24, 48, and 72 hours.

[0057] Figure 6 The results of evaluating the regulation of intracellular oxidative stress and abnormal proliferation behavior of keratinocytes by EmNeo-Se prepared in Example 5 under an in vitro psoriasis simulation environment are shown. In the figures, a is a DCFH fluorescence flow cytometry plot, b is the quantitative statistical analysis of the flow cytometry results, c is the HaCat cell proliferation results detected by the EdU method, and d is the HaCat cell proliferation results detected by the CCK-8 assay. Figure 6 It can be seen that M5-induced HaCaT cells exhibit strong intracellular oxidative stress and abnormal proliferation of keratinocytes. However, after treatment with EmNeo-Se, the intracellular oxidative stress process and the abnormal proliferation of keratinocytes can be effectively alleviated, indicating that EmNeo-Se has the potential to alleviate psoriasis-related symptoms.

[0058] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a supramolecular nanoenzyme antagonist with multiple synergistic effects, characterized in that, Includes the following steps: (1) Mix the aqueous solution of epigallocatechin gallate and the aqueous solution of formaldehyde and react them. Then add the aqueous solution of aminoglycoside compound to carry out the Mannich reaction to obtain the Mannich nano-assembly. (2) The Mannich nanoassembly is dispersed in water, sodium selenite aqueous solution is added, and after stirring evenly, ascorbic acid solution is added to carry out a reduction reaction to obtain the supramolecular nanoenzyme antagonist.

2. The preparation method according to claim 1, characterized in that, In step (1), the concentration of the epigallocatechin gallate aqueous solution is 5~20 mmol / L; the concentration of the formaldehyde aqueous solution is 12.3 mmol / L; and the volume ratio of the epigallocatechin gallate aqueous solution to the formaldehyde aqueous solution is 200:(1~20).

3. The preparation method according to claim 1, characterized in that, In step (1), the reaction time is 5 to 30 minutes.

4. The preparation method according to claim 1, characterized in that, In step (1), the aminoglycoside compound in the aqueous solution of the aminoglycoside compound is selected from at least one of hygromycin, netilmicin, kanamycin, tobramycin and neomycin; the concentration of the aqueous solution of the aminoglycoside compound is 5~20 mmol / L; and the volume ratio of the aqueous solution of the aminoglycoside compound to the aqueous solution of epigallocatechin gallate is 1:

1.

5. The preparation method according to claim 1, characterized in that, In step (1), the dropping rate is 5~10s / drop; the time for the Mannich reaction after the dropping is completed is 2~12h.

6. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the sodium selenite aqueous solution is 10~100 mmol / L; the volume ratio of the sodium selenite aqueous solution to the Mannich nanoassembly is 1:

5.

7. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the ascorbic acid solution is 40 mmol / L; the volume ratio of the ascorbic acid solution to the Mannich nanoassembly is 2:

5.

8. The preparation method according to claim 1, characterized in that, In step (2), the reduction reaction takes 12 to 24 hours.

9. A supramolecular nanoenzyme antagonist with multiple synergistic effects, characterized in that, It is prepared according to any one of claims 1 to 8.

10. The use of a supramolecular nanozyme antagonist with synergistic effects as described in claim 9 in the preparation of a drug to alleviate psoriasis.

Images