Preparation method and application of difunctional cationic mesoporous silica nanomaterial loaded with abstinence sulfur

Abstract

The invention discloses a preparation method of a difunctional cationic mesoporous silica nano material loaded with drunkenness-quitting sulfur. The preparation method comprises the following steps: synthesizing SeSe-MSN; synthesis of the SeSe-MSNs modified by epoxy is carried out on the SeSe-MSNs; the preparation method comprises the following steps: synthesizing SeSe-MSN-Netil; and synthesizing the DSF (at) SeSeMSN-Netin. In the preparation method of the difunctional cationic mesoporous silica nano-material loaded with the drunkenness-quitting sulfur, different types of polycationic compounds are grafted on the surface of the mesoporous silica nano-material through an epoxy-opening reaction, and cations on the surface of the mesoporous silica material can be efficiently combined and remove free nucleic acid; activation of epidermis cells is inhibited, inflammatory response in psoriasis is reduced, and an efficient and safe anti-inflammatory effect is finally obtained; compared with a traditional psoriasis treatment medicine, the compound is used for anti-inflammatory treatment and is more efficient, safer and controllable, and a new thought and a new material are provided for efficient intervention of clinical psoriasis.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine carrier materials technology, and in particular to a method for preparing and applying a bifunctional cationic mesoporous silica nanomaterial loaded with disulfiram. Background Technology

[0002] Psoriasis is a prevalent chronic inflammatory disease characterized by well-defined, erythematous plaques with white or silvery scales that can affect any part of the skin. In addition to skin involvement, some patients may also suffer from psoriatic arthritis, accompanied by joint pain and deformities. Approximately 125 million people worldwide suffer from psoriasis. Because the pathogenesis of this disease is not fully understood, there is currently a lack of targeted treatments, and clinical treatment primarily focuses on symptom control. This leads to the disease being chronic and prone to relapses, causing significant physical and psychological suffering for patients. Therefore, further elucidation of the pathogenesis of psoriasis and the development of new therapeutic drugs are urgently needed. However, current technologies lack highly effective, safe, and controllable anti-inflammatory treatment routes and new materials.

[0003] Current research reports that psoriasis patients exhibit significant NET (neutrophil extracellular traps) deposition in their skin lesions, and local epidermal cells can undergo pyroptosis, releasing large amounts of inflammatory factors, creating a positive feedback loop that exacerbates psoriatic inflammation. Therefore, clearing NETs and inhibiting epidermal cell pyroptosis to reduce the inflammatory response have become effective strategies for treating psoriasis. Disulfiram is currently an effective inhibitor of pyroptosis. Therefore, this invention aims to propose a method for preparing and applying a disulfiram-loaded bifunctional cationic mesoporous silica nanomaterial that simultaneously clears NETs and inhibits pyroptosis, in order to address the aforementioned problems. Summary of the Invention

[0004] This invention addresses the problem that the pathogenesis of psoriasis is unclear and there are no highly effective, safe and controllable therapeutic drugs. It proposes a method for preparing and applying a bifunctional cationic mesoporous silica nanomaterial loaded with disulfiram.

[0005] The preparation method of the disulfide-loaded bifunctional cationic mesoporous silica nanomaterial of the present invention includes the following steps:

[0006] Synthesis of S1 and SeSe-MSN:

[0007] Hexadecyltrimethyl-p-toluenesulfonium (CTAT) and triethanolamine were dissolved in deionized water and stirred at 80°C for 2 hours. Then, a solution containing bis[3-(triethoxysilyl)propyl]selenide (BTESePD) and tetraethyl orthosilicate was added dropwise to the above reaction solution, and the mixture was stirred at 80°C for 4 hours. After centrifugation, the product was obtained, and the reaction mixture was repeatedly washed with ethanol. Finally, the mixture was refluxed in an ethanol solution containing ammonium nitrate for 12 hours to remove CTAT. After centrifugation and repeated washing with alternating ethanol and water, SeSe-MSN was collected.

[0008] S2. Synthesis of epoxy-modified SeSe-MSNs:

[0009] SeSe-MSNS was dispersed in toluene using ultrasound and refluxed at 80°C for 4 h. The temperature was then lowered to room temperature, and 3-glycidyl etheroxypropyltrimethoxysilane (epoxysilane) was added. The mixture was stirred at room temperature for 3 h, and then refluxed at 80°C for 24 h to obtain epoxy-modified SeSe-MSN. The mixture was then centrifuged and washed once with alternating ethanol and water to purify the epoxy-modified SeSe-MSN.

[0010] Synthesis of S3, SeSe-MSN-Netil:

[0011] Epoxy-modified SeSe-MSN was added to a netilmicin aqueous solution containing triethylamine and reacted at room temperature for 24 h. The mixture was then centrifuged, washed, and dried for later use.

[0012] Synthesis of S4, DSF@SeSeMSN-Netil:

[0013] Replace the dispersion medium of SeSeMSN-Netil in water with DMSO and sonicate thoroughly to ensure uniform dispersion. Weigh the amide DSF in a round-bottom flask, add dimethyl sulfoxide (DMSO), stir to dissolve, add SeSeMSN-Netil to the DMSO solution of DSF, add DMSO to bring the volume to a final volume, stir at room temperature for 12 hours, centrifuge and discard the supernatant, and repeatedly wash the reaction mixture with H2O to remove unloaded DSF and DMSO. Finally, add H2O and sonicate to resuspend the mixture to obtain DSF@SeSeMSN-Netil. Take a small amount, dry and weigh it for quantification, and store at 4°C in the dark.

[0014] In a preferred embodiment of the present invention, the synthesis of SeSe-MSN in S1 is as follows: 2.0 g of hexadecyltrimethyl-p-toluenesulfonium (CTAT) and 0.8 g of triethanolamine are dissolved in 100 mL of deionized water and stirred at 80 °C for 2 hours; then, a solution containing 1.0 g of bis[3-(triethoxysilyl)propyl]selenide (BTESePD) and 4.0 g of tetraethyl orthosilicate is added dropwise to the above reaction solution, stirred at 80 °C for 4 hours, centrifuged to obtain the product, and the reaction mixture is repeatedly washed with ethanol; finally, the mixture is refluxed in an ethanol solution containing 1% w / v ammonium nitrate for 12 hours to remove CTAT; after centrifugation, the mixture is washed repeatedly with alternating ethanol and water, and SeSe-MSN is collected.

[0015] Further, the synthesis of epoxy-modified SeSe-MSNs in S2: 1.0 g SeSe-MSNs were dispersed in 250 mL toluene by sonication and refluxed at 80 °C for 4 h. The temperature was then lowered to room temperature, and 1 mL of 3-glycidyl etheroxypropyltrimethoxysilane (epoxysilane) was added. The mixture was stirred at room temperature for 3 h, and then refluxed at 80 °C for 24 h to obtain epoxy-modified SeSe-MSNs. The mixture was centrifuged and washed once with alternating ethanol and water to purify the epoxy-modified SeSe-MSNs.

[0016] Further, the synthesis of SeSe-MSN-Netil in S3: 250 mg of epoxy-modified SeSe-MSN was added to 250 mL of netilmicin aqueous solution containing 1 mg / mL triethylamine. The pH of the reaction solution was 10, and the reaction was carried out at room temperature for 24 h. The mixture was then centrifuged, washed, and dried for later use.

[0017] Further, the synthesis of DSF@SeSeMSN-Netil in S4: 300 mg of SeSeMSN-Netil in water was replaced with DMSO as the dispersion medium and thoroughly ultrasonicated to ensure uniform dispersion; 300 mg of amide DSF was weighed into a 150 mL round-bottom flask, and 30 mL of dimethyl sulfoxide (DMSO) was added and stirred to dissolve. SeSeMSN-Netil was added to the DMSO solution of DSF, and then DMSO was added to bring the volume to 75 mL. The mixture was stirred at room temperature for 12 h, centrifuged and the supernatant was discarded. The reaction mixture was repeatedly washed with H2O to remove unloaded DSF and DMSO; finally, 10 mL of H2O was added and the mixture was ultrasonically resuspended to obtain DSF@SeSeMSN-Netil. A small amount was dried, weighed, and quantified, and stored at 4 °C in the dark.

[0018] In a preferred embodiment of the present invention, the DSF@SeSeMSN-Neti prepared by the above method can be applied to the drug treatment of psoriasis.

[0019] Implementing this invention has the following beneficial effects:

[0020] The cationic mesoporous silica nanomaterial loaded with disulfiram in this invention can utilize the disulfiram loaded with disulfiram to target psoriatic lesions and exert its therapeutic effect, inhibiting epidermal cell pyroptosis. The mesoporous silica is grafted with polycations that have a strong binding capacity to nucleic acids, which can effectively bind to NETs and achieve anti-inflammatory effects by clearing NETs. It plays an anti-inflammatory role from multiple aspects, and the disulfiram loading dose is much lower than the dose previously reported to be effective in treating psoriasis, ultimately achieving a highly efficient and safe therapeutic effect.

[0021] In the preparation method of cationic mesoporous silica nanomaterials loaded with disulfide in this invention, different kinds of polycationic compounds are grafted onto the surface of the mesoporous silica nanomaterials through an epoxy-opening reaction. The cations on the surface of the mesoporous silica material can efficiently bind to and remove free nucleic acids, inhibit the activation of epidermal cells, reduce the inflammatory response in psoriasis, and ultimately obtain a highly efficient and safe anti-inflammatory effect.

[0022] This invention provides a cationic mesoporous silica nanomaterial loaded with disulfide, which is more efficient, safe and controllable for anti-inflammatory treatment compared with traditional psoriasis drugs, providing new ideas and new materials for efficient clinical intervention of psoriasis. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Transmission electron microscopy (TEM) image of the cationic mesoporous silica nanomaterial (DSF@SeSeMSN-N) prepared in Example 1, which has dual functions of NET scavenging and loading disulfiram.

[0025] Figure 2 The image shows the scanning electron microscope (SEM) results of the cationic mesoporous silica nanomaterial (DSF@SeSeMSN-N) prepared in Example 1, which has dual functions of NET removal and loading disulfiram.

[0026] Figure 3 A graph showing the results of DSF@SeSeMSN-N clearing NETs;

[0027] Figure 4 Figure showing the inhibitory effect of DSF@SeSeMSN-N on epidermal cell pyroptosis;

[0028] Figure 5Gross image of the skin of a mouse model of psoriasis treated with DSF@SeSeMSN-N;

[0029] Figure 6 PASI score of DSF@SeSeMSN-N treated psoriasis model mice;

[0030] Figure 7 HE staining results of skin tissue sections from mice with psoriasis model treated with DSF@SeSeMSN-N. Detailed Implementation

[0031] 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.

[0032] The preparation method of this bifunctional cationic mesoporous silica nanomaterial loaded with disulfide for alcohol addiction includes the following steps:

[0033] Synthesis of S1 and SeSe-MSN: Cetyltrimethyl-p-toluenesulfonium (CTAT) and triethanolamine were dissolved in deionized water and stirred at 80 °C for 2 hours. Subsequently, a solution containing bis[3-(triethoxysilyl)propyl]selenide (BTESePD) and tetraethyl orthosilicate was added dropwise to the above reaction solution, and the mixture was stirred at 80 °C for 4 hours. After centrifugation, the product was obtained, and the reaction mixture was repeatedly washed with ethanol. Finally, the mixture was refluxed in an ethanol solution containing ammonium nitrate for 12 hours to remove CTAT. After centrifugation, the mixture was washed repeatedly with alternating ethanol and water, and SeSe-MSN was collected.

[0034] S2. Synthesis of epoxy-modified SeSe-MSNs: SeSe-MSNs were dispersed in toluene by ultrasound and refluxed at 80°C for 4 h. The temperature was then lowered to room temperature, and 3-glycidyl etheroxypropyltrimethoxysilane (epoxysilane) was added. The mixture was stirred at room temperature for 3 h, and then refluxed at 80°C for 24 h to obtain epoxy-modified SeSe-MSNs. The mixture was centrifuged and washed once with ethanol and water alternately to purify the epoxy-modified SeSe-MSNs.

[0035] Synthesis of S3 and SeSe-MSN-Netil: Epoxy-modified SeSe-MSN was added to a netilmicin aqueous solution containing triethylamine, reacted at room temperature for 24 h, centrifuged, washed, and dried for later use;

[0036] Synthesis of S4 and DSF@SeSeMSN-Netil: SeSeMSN-Netil in water was replaced with DMSO as the dispersion medium and thoroughly ultrasonicated to ensure uniform dispersion. Amid DSF was weighed from a round-bottom flask, and dimethyl sulfoxide (DMSO) was added and stirred to dissolve. SeSeMSN-Netil was added to the DMSO solution of DSF, and then DMSO was added to bring the volume to a final depth. The mixture was stirred at room temperature for 12 hours, centrifuged, and the supernatant was discarded. The reaction mixture was repeatedly washed with H2O to remove unloaded DSF and DMSO. Finally, H2O was added and the mixture was ultrasonically resuspended to obtain DSF@SeSeMSN-Netil. A small amount was dried, weighed, and quantified. The mixture was stored at 4°C in the dark.

[0037] The DSF@SeSeMSN-Neti prepared by the above method can be used for the drug treatment of psoriasis.

[0038] Example 1

[0039] A method for preparing a cationic mesoporous silica nanomaterial loaded with dual functions of alcohol quercetin includes the following steps:

[0040] (1) Synthesis of SeSe-MSN: 2.0 g of hexadecyltrimethyl-p-toluenesulfonium (CTAT) and 0.8 g of triethanolamine were dissolved in 100 mL of deionized water and stirred at 80 °C for 2 h. Subsequently, a solution containing 1.0 g of bis[3-(triethoxysilyl)propyl]selenide (BTESePD) and 4.0 g of tetraethyl orthosilicate was added dropwise to the above reaction solution, and the mixture was stirred at 80 °C for 4 h. After centrifugation, the product was obtained, and the reaction mixture was repeatedly washed with ethanol. Finally, the mixture was refluxed in an ethanol solution containing 1% (wv) ammonium nitrate for 12 h to remove CTAT. Finally, the mixture was centrifuged, washed alternately with ethanol and water, and SeSeMSN was collected.

[0041] (2) Synthesis of epoxy-modified SeSe-MSNs: 1.0 g of SeSe-MSNs was dispersed in 250 mL of toluene using ultrasound and refluxed at 80 °C for 4 h. Then, the mixture was cooled to room temperature, and 1 mL of 3-glycidyl etheroxypropyltrimethoxysilane (epoxysilane) was added. The mixture was stirred at room temperature for 3 h, and then refluxed at 80 °C for 24 h to obtain epoxy-modified SeSe-MSNs. The mixture was purified by centrifugation and washing once with alternating ethanol and water.

[0042] (3) Synthesis of SeSe-MSN-Netil: 250 mg of epoxy-modified SeSe-MSN was added to 250 mg netilmicin aqueous solution (1 mg / m) containing triethylamine (reaction solution pH-10), and reacted at room temperature for 24 h. The mixture was then centrifuged, washed, and dried for later use.

[0043] (4) Synthesis of DSF@SeSeMSN-Netil: Take 300 mg of SeSeMSN-Netil in water and replace the dispersion medium with DMSO. Sonicate thoroughly to disperse evenly. Take 300 mg of DSF in a 150 mL round-bottom flask, add about 30 mL of DMSO, stir to dissolve, add SeSeMSN-Netil to the DMSO solution of DSF, add DMSO to make up to 75 mL, stir at room temperature for 12 h, centrifuge and discard the supernatant, and wash the reaction mixture repeatedly with H2O to remove unloaded DSF and DMSO. Finally, add 10 mL of H2O and sonicate to resuspend. Take a small amount, dry and weigh for quantification, and store at 4 °C in the dark.

[0044] Performance testing:

[0045] 1. In Example 1, the cationic mesoporous silica nanomaterial DSF@SeSeMSN-N, which was prepared to remove NETs and loaded with disulfide, was subjected to transmission electron microscopy (TEM) and scanning electron microscopy (SEM) tests. The TEM and SEM results are shown in the figures below. Figure 1 , Figure 2 As shown.

[0046] from Figure 1 , Figure 2 It can be seen that DSF@SeSeMSN-N has a uniform spherical morphology with a uniform particle size of about 50-60 nm, and the pores are highly ordered and have a uniform pore size.

[0047] 2. NETs combined with experiments:

[0048] The DSF@SeSeMSN-N prepared in Example 1 was mixed with 1640 complete culture medium to obtain a solution of 48 ug / mL.

[0049] NETs induce:

[0050] (1) 15 ml of peripheral blood was drawn from healthy volunteers and placed in three 5 mL EDTA vacuum collection containers.

[0051] (2) Use a dropper to add 15 mL of neutrophil separation solution into three 15 mL centrifuge tubes in several portions (take out the neutrophil separation solution in advance and equilibrate to room temperature).

[0052] (3) Tilt the centrifuge tube and slowly add the blood sample along the tube wall so that the blood is above the surface of the separation liquid; be careful to add it slowly to keep the interface between the two clear; the separation liquid and the blood should be of equal volume.

[0053] (4) Centrifugation: Carefully and steadily transfer the centrifuge tube containing the blood sample and separation solution to the centrifuge, centrifuge at 500-550g at room temperature for 35 minutes; (if the separation is not complete after 35 minutes, the speed can be adjusted to 600g and the time can be extended to 60 minutes).

[0054] (5) After centrifugation, remove the centrifuge tube gently and do not shake it. At this time, two layers of white floating cell bands can be seen in the centrifuge tube: the upper layer is mononuclear cells (PBMCs); the lower layer is neutrophils (PMNs).

[0055] (6) Gently insert the pipette into the lower PMN band and aspirate it into a new 15mL centrifuge tube, and label it; be careful to aspirate as little separation liquid as possible; replace the pipette tip with a new one to aspirate the upper PBMC band, place it into another new 15mL centrifuge tube, and label it.

[0056] (7) Add PBS solution to 10 mL in each of the above centrifuge tubes, mix thoroughly, and centrifuge again at 400 g for 10 minutes at room temperature.

[0057] (8) After centrifugation, discard the supernatant and add red blood cell lysis buffer. The volume of lysis buffer is determined according to the situation, usually 2-5 mL. Mix thoroughly and react at 4°C in the dark for 10 minutes. After the reaction is complete, add PBS solution to 10 mL, mix thoroughly and centrifuge at 1000 rpm for 5 minutes at room temperature.

[0058] (9) After centrifugation, discard the supernatant, add 10 mL of PBS solution to resuspend, and centrifuge again at 100 g for 10 minutes to remove platelets.

[0059] (10) After centrifugation, discard the supernatant and add 2 mL of 1640 whole culture to fully resuspend the cells.

[0060] (11) Prepare a blood cell counting chamber. Take 10 μL of cell suspension and add it to the counting chamber. Be careful to remove air bubbles and prevent leakage. Otherwise, prepare a new PMN suspension and count the cells.

[0061] (12) Calculation: Total volume (μL) = Cell suspension volume (μL) + 24ug / mLDSF@SeSeMSN-N volume (μL) + Supplemented 1640 total culture volume (μL).

[0062] (13) The collected neutrophils were resuspended in 1640 complete medium, with 1×10^5 neutrophils per well, and 100 μL of the system was added to a 96-well cell plate.

[0063] (14) Grouping: blank control group, PMA treatment group, PMA+DSF@SeSeMSN-N solution treatment group; each group has 3 replicates.

[0064] (15) PMA induction: 25 nM tetradecanoylphorbol acetate (phorbol ester, PMA) was added to the corresponding well for induction. The induced neutrophils were cultured in a cell culture incubator at 37°C with 5% CO2 concentration for 4 h.

[0065] (16) Prepare SytoxGreen working solution: Add 2 μL of SytoxGreen working solution to every 100 μL of system, calculate the total volume, and dilute with PBS solution at a ratio of 1:20.

[0066] (17) After 4 hours, remove the 96-well plate and add 2 μL of SytoxGreen working solution.

[0067] (18) Vibrating plate: 300 rpm, 5 minutes.

[0068] (19) Preheat the microplate reader with excitation light at 480nm and emission light at 520nm for 3 minutes.

[0069] (20) Detect fluorescence intensity.

[0070] Please see the results. Figure 3 , Figure 3 A graph showing the NET binding rates of DSF@SeSeMSN-N; from Figure 3 It can be seen that DSF@SeSeMSN-N has good NET binding force.

[0071] 3. Verification that DSF@SeSeMSN-N inhibits pyroptosis:

[0072] The specific operating steps of the experiment are as follows:

[0073] (1) After digesting HaCaT cells in good condition with trypsin, centrifuge them, resuspend them in MEM-a complete medium, count them, and seed them into 12-well plates at 5×105 / 200ul / well; divide them into 3 groups: control group, LPS+ATP group, LPS+ATP+nanomaterial DSF@SeSeMSN-N group, with 2 replicates in each group.

[0074] (2) Place in a 37℃, 5% CO2 incubator and incubate for 8 hours. After the cells are completely attached, discard the supernatant. Add 1ug / ml of LPS to the LPS+ATP group and the LPS+ATP+nanomaterial DSF@SeSeMSN-N group. Add 12ug / ml of nanomaterial DSF@SeSeMSN-N to the LPS+ATP+nanomaterial DSF@SeSeMSN-N group. Mix well and place in an incubator to continue incubation for 6 hours.

[0075] (3) After 6 hours, the LPS+ATP group and the LPS+ATP+nanomaterial DSF@SeSeMSN-N group were given 5 mM ATP and stimulated for another 0.5-2 hours.

[0076] (4) After incubation, place the cells under an Olympus inverted fluorescence microscope to take pictures and record the cell morphology as soon as possible within 1 hour.

[0077] The results are as follows Figure 4 As shown, Figure 4 Electron micrograph of DSF@SeSeMSN-N inhibiting pyroptosis. From Figure 4 It can be seen that LPS+ATP can induce pyroptosis, while DSF@SeSeMSN-N can significantly inhibit LPS+ATP-induced pyroptosis.

[0078] The therapeutic effects of the DSF@SeSeMSN-N prepared above on a mouse model of psoriasis:

[0079] (I) Establishment of a mouse model of psoriasis and application of drugs:

[0080] (1) Fifteen 8-week-old female Balb / c mice (20g±2g) were purchased from Guangdong Zhiyuan Biomedical Technology Co., Ltd. and divided into three groups of five mice each (n=5): control group, imiquimod group, and imiquimod+DSF@SeSeMSN-N group.

[0081] (2) Mice were anesthetized by intraperitoneal injection of chloral hydrate. The hair on the back of the mice was shaved off with a razor. Fine hairs were removed with depilatory cream (Veet), exposing a skin area of ​​about 2cm x 3cm.

[0082] (3) After shaving the mice, the model was established starting from the second day. The control group applied 62.5 mg of petroleum jelly ointment to the hair removal area every day, the imiquimod group applied 62.5 mg of imiquimod ointment to the hair removal area every day, and the imiquimod + DSF@SeSeMSN-N group applied 62.5 mg of imiquimod ointment every day. After the ointment was absorbed, 100 μL of 180 μg / ml DSF@SeSeMSN-N solution was injected into the tail vein. The degree of inflammation of the mice's skin lesions was observed and photographed every day for 7 consecutive days.

[0083] (II) Collection of psoriatic skin lesion samples from mice

[0084] (1) Mice were general anesthetized with isoflurane;

[0085] (2) After the mice are completely anesthetized, use sterile scissors and tweezers to cut along the edge of the lesion. When cutting the lesion, cut perpendicular to the skin surface and along the skin lines to obtain a sample of uniform thickness.

[0086] (3) Collect a 0.5cm*1cm skin sample from the center of the lesion and fix it in a 15ml centrifuge tube containing paraformaldehyde;

[0087] (4) Collect the remaining skin lesions and place them in cryovials, then keep them in liquid nitrogen.

[0088] (III) Histopathological HE staining:

[0089] (1) After the tissue is dehydrated and embedded, paraffin blocks are prepared. The paraffin blocks are cut into 4-micrometer-thick slices and spread on a warm water bath. Then, they are transferred to glass slides and fixed by baking.

[0090] (2) Dewax the slices in xylene (twice, 10 minutes each time), then rehydrate them gradually with a gradient of ethanol (100%, 95%, 90%, 80%, 70%) for 5 minutes each time, and finally rinse them in distilled water.

[0091] (3) Immerse the slices in hematoxylin staining solution for 5 minutes, then rinse with tap water until the rinsing solution becomes clear.

[0092] (4) Differentiate the sections in the differentiation solution for 5 seconds, then rinse with tap water. Place the sections in the blueing solution for 3 minutes, then rinse with tap water.

[0093] (5) Immerse the slices in eosin staining solution for 1 minute, then rinse with tap water.

[0094] (6) The slices were dehydrated sequentially by a gradient of ethanol (70%, 80%, 90%, 95%, 100%) for 5 minutes each time.

[0095] (7) Clear the dehydrated sections in xylene (twice, 5 minutes each time). Finally, add mounting medium to the sections and cover with a coverslip. Observe and photograph under an electron microscope.

[0096] Figure 5 Gross image of the skin of mice with a psoriasis model treated with DSF@SeSeMSN-N. Mice in the imiquimod ointment group showed significantly thickened skin with more scales, while mice in the DSF@SeSeMSN-N treatment group showed reduced skin lesions. Figure 6 Line graph showing PASI scores in mice with a psoriasis model treated with DSF@SeSeMSN-N. Higher PASI scores indicate successful establishment of the psoriasis model. Mice in the tail vein injection group of DSF@SeSeMSN-N showed significantly reduced severity of dorsal skin lesions, less scaling, and lower PASI scores. Figure 7 Image showing HE staining results of skin tissue sections from a mouse psoriasis model after DSF@SeSeMSN-N treatment. In conclusion, DSF@SeSeMSN-N treatment significantly improves the condition of mouse psoriasis models.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a bifunctional cationic mesoporous silica nanomaterial loaded with disulfiram, characterized in that, Includes the following steps: Synthesis of S1 and SeSe-MSN: Hexadecyltrimethyl-p-toluenesulfonium (CTAT) and triethanolamine were dissolved in deionized water and stirred at 80°C for 2 hours. Then, a solution containing bis[3-(triethoxysilyl)propyl]selenide (BTESePD) and tetraethyl orthosilicate was added dropwise to the above reaction solution, and the mixture was stirred at 80°C for 4 hours. After centrifugation, the product was obtained, and the reaction mixture was repeatedly washed with ethanol. Finally, the mixture was refluxed in an ethanol solution containing ammonium nitrate for 12 hours to remove CTAT. After centrifugation and repeated washing with alternating ethanol and water, SeSe-MSN was collected. S2. Synthesis of epoxy-modified SeSe-MSNs: SeSe-MSNS was dispersed in toluene using ultrasound and refluxed at 80°C for 4 h. The temperature was then lowered to room temperature, and 3-glycidyl etheroxypropyltrimethoxysilane (epoxysilane) was added. The mixture was stirred at room temperature for 3 h, and then refluxed at 80°C for 24 h to obtain epoxy-modified SeSe-MSN. The mixture was then centrifuged and washed once with alternating ethanol and water to purify the epoxy-modified SeSe-MSN. Synthesis of S3, SeSe-MSN-Netil: Epoxy-modified SeSe-MSN was added to a netilmicin aqueous solution containing triethylamine and reacted at room temperature for 24 h. The mixture was then centrifuged, washed, and dried for later use. Synthesis of S4, DSF@SeSeMSN-Netil: Replace the dispersion medium of SeSeMSN-Netil in water with DMSO, and sonicate thoroughly to ensure uniform dispersion. Weigh the amide DSF in a round-bottom flask, add dimethyl sulfoxide (DMSO), and stir to dissolve. Add SeSeMSN-Netil to the DMSO solution of DSF, and then add DMSO to bring the volume to a final volume. Stir at room temperature for 12 hours, centrifuge and discard the supernatant. Wash the reaction mixture repeatedly with H2O to remove unloaded DSF and DMSO. Finally, add H2O and sonicate to resuspend the mixture to obtain DSF@SeSeMSN-Netil. Take a small amount, dry it, weigh it, and quantify it. Store it at 4°C in the dark.

2. The method for preparing the disulfiram-loaded bifunctional cationic mesoporous silica nanomaterial according to claim 1, characterized in that, Synthesis of SeSe-MSN in S1: 2.0 g of hexadecyltrimethyl-p-toluenesulfonium (CTAT) and 0.8 g of triethanolamine were dissolved in 100 mL of deionized water and stirred at 80 °C for 2 hours. Subsequently, a solution containing 1.0 g of bis[3-(triethoxysilyl)propyl]selenide (BTESePD) and 4.0 g of tetraethyl orthosilicate was added dropwise to the above reaction solution, stirred at 80 °C for 4 hours, centrifuged to obtain the product, and the reaction mixture was repeatedly washed with ethanol. Finally, the mixture was refluxed in an ethanol solution containing 1% w / v ammonium nitrate for 12 hours to remove CTAT. After centrifugation, the mixture was washed repeatedly with alternating ethanol and water, and SeSe-MSN was collected.

3. The method for preparing the disulfiram-loaded bifunctional cationic mesoporous silica nanomaterial according to claim 2, characterized in that, Synthesis of epoxy-modified SeSe-MSNs in S2: 1.0 g SeSe-MSNs were dispersed in 250 mL toluene by ultrasonication and refluxed at 80 °C for 4 h. The temperature was then lowered to room temperature, and 1 mL of 3-glycidyl etheroxypropyltrimethoxysilane (epoxysilane) was added. The mixture was stirred at room temperature for 3 h, and then refluxed at 80 °C for 24 h to obtain epoxy-modified SeSe-MSNs. The mixture was centrifuged and washed once with alternating ethanol and water to purify the epoxy-modified SeSe-MSNs.

4. The method for preparing the disulfiram-loaded bifunctional cationic mesoporous silica nanomaterial according to claim 3, characterized in that, Synthesis of SeSe-MSN-Netil in S3: 250 mg of epoxy-modified SeSe-MSN was added to 250 mL of netilmicin aqueous solution containing 1 mg / mL triethylamine, and the reaction was carried out at room temperature for 24 h. The mixture was then centrifuged, washed, and dried for later use.

5. The method for preparing the disulfiram-loaded bifunctional cationic mesoporous silica nanomaterial according to claim 4, characterized in that, Synthesis of DSF@SeSeMSN-Netil in S4: Take 300 mg of SeSeMSN-Netil in water and replace the dispersion medium with DMSO. Sonicate thoroughly to ensure uniform dispersion. Weigh 300 mg of amide DSF in a 150 mL round-bottom flask, add 30 mL of dimethyl sulfoxide (DMSO), stir to dissolve, add SeSeMSN-Netil to the DSF DMSO solution, and then add DMSO to bring the volume to 75 mL. Stir at room temperature for 12 h, centrifuge and discard the supernatant, and repeatedly wash the reaction mixture with H2O to remove unloaded DSF and DMSO. Finally, add 10 mL of H2O and sonicate to resuspend, obtaining DSF@SeSeMSN-Netil. Take a small amount, dry and weigh for quantification, and store at 4 °C protected from light.

6. The method for preparing the disulfiram-loaded bifunctional cationic mesoporous silica nanomaterial according to any one of claims 1-5, characterized in that, The prepared DSF@SeSeMSN-Neti was used in the drug treatment of psoriasis.

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