Selenium-containing soluble composite microneedle as well as preparation method and application thereof

Abstract

The invention discloses a selenium-containing soluble composite microneedle as well as a preparation method and application thereof, and belongs to the technical field of biological materials and biological medicines, a microneedle forming matrix is selenized hyaluronic acid and medicine-loaded tetrahedral framework nucleic acid, and a substrate part is polyvinyl alcohol; the preparation method comprises the following steps: step 1, preparation of selenized hyaluronic acid; step 2, preparation of tetrahedral framework nucleic acid and drug loading; and step 3, preparation of the selenium-containing and drug-loaded microneedle patch. The selenized hyaluronic acid is synthesized through a specific chemical method, the selenized hyaluronic acid is used as a substrate to load the medicine-carrying tetrahedral framework nucleic acid, and the advantages of high permeability, good biocompatibility, editability and the like of the tetrahedral framework nucleic acid are utilized, so that the sustained and controlled release capacity of the microneedle is improved, efficient transdermal delivery of the medicine is realized, and the application prospect is broad. And pain and discomfort possibly caused by a traditional administration mode are reduced. The invention provides a novel and effective drug delivery system in the field of psoriasis treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials and biomedicine, and specifically relates to a selenium-containing soluble composite microneedle for treating psoriasis, and a preparation method and application thereof. Background Art

[0002] Psoriasis is a chronic inflammatory skin disease, and its treatment has made significant progress in recent years. Current treatments mainly include topical therapy, phototherapy, systemic immunomodulators and biologics. These methods aim to relieve symptoms and improve the quality of life of patients. However, there are still some challenges, such as adverse reactions, treatment resistance, high costs, and differences in individual responses. In recent years, with the in-depth study of the pathogenesis of psoriasis, the development of new drugs has made rapid progress. Traditional treatments include drug therapy, phototherapy and biologics, which can relieve symptoms to a certain extent, but have problems such as limited efficacy, large side effects and high recurrence rate.

[0003] Microneedle therapy provides a new treatment idea. It uses tiny needles to create tiny channels on the skin surface to promote drug penetration and skin repair. As an innovative physical therapy method, microneedle therapy has shown unique advantages and application prospects in the treatment of psoriasis. Patent CN202110836016.6 uses pH-responsive polymer micelles coated with human keratinocyte membranes to construct an epidermal active-targeted drug delivery system, and uses microneedle technology to assist in the transdermal delivery of the anti-inflammatory drug shikonin, thereby prolonging the duration of action. Patent CN202010809588.0 discloses a soluble microneedle preparation that effectively realizes the programmed release of psoriasis treatment drugs, leaving the sustained-release particles in the skin to maintain a long-term and effective drug treatment effect. However, in the currently disclosed patents, microneedles are only used as drug delivery systems, and the microneedles themselves lack functionalization.

[0004] Patients with psoriasis have high levels of oxidative stress. Oxidative stress refers to the excessive production of free radicals in the body or decreased antioxidant capacity, which leads to damage to cells and tissues. The level of free radicals in the skin and blood of patients with psoriasis is elevated, and these free radicals can damage skin cells and aggravate inflammatory responses. As an essential trace element, selenium plays an important role in the treatment of psoriasis, and its principles mainly involve three aspects: anti-oxidation, immunomodulation, and cell protection. Selenium can enhance the function of the body's endogenous antioxidant system, effectively remove free radicals and peroxides by increasing the activity of antioxidant enzymes such as glutathione peroxidase, and reduce the damage of oxidative stress to the skin. The immunomodulatory effect of selenium is reflected in its ability to balance the imbalance of Th1 / Th2 cell subsets, inhibit the release of inflammatory factors such as IFN-γ caused by excessive activation of Th1 cells, and promote the production of anti-inflammatory cytokines such as IL-4, thereby alleviating skin inflammation in patients with psoriasis. In addition, selenium can directly act on skin cells, protect cell membranes from oxidative damage, maintain the integrity of cell structure and function, help inhibit abnormal proliferation and differentiation of epidermal cells, and improve the skin lesions of psoriasis patients. So far, no selenium-containing microneedles have been used to treat psoriasis. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a selenium-containing soluble composite microneedle and a preparation method thereof and its application in the treatment of psoriasis. The selenium-containing soluble composite microneedle provided by the present invention can exert antioxidant effects, effectively remove reactive oxygen, regulate the immune microenvironment, reduce inflammation, and control the chronic inflammatory state of psoriasis. At the same time, it can carry drugs and nanoformulations, and has a good therapeutic effect on psoriasis. At the same time, in order to improve the sustained and controlled release effect of the drug, the present invention also provides a tetrahedral framework nucleic acid (tFNA) as a drug delivery carrier to achieve the purpose of sustained and controlled release of the drug and realize efficient transdermal delivery of the drug.

[0006] The invention discloses a selenium-containing soluble composite microneedle, wherein the microneedle forming matrix is ​​selenized hyaluronic acid, loaded with drug-carrying tetrahedral framework nucleic acid, and the base part is polyvinyl alcohol with good biocompatibility.

[0007] The present invention also provides a method for preparing the above-mentioned selenium-containing soluble composite microneedle, comprising the following steps: Step 1, preparation of selenized hyaluronic acid: Add 1-[3-dimethylaminopropyl]-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the hyaluronic acid solution and stir for 0.5h to 24h for activation. Continue to add small molecule compounds containing selenium to the solution and react in the dark under inert gas protection for 8h to 48h. After the reaction is completed, dialyze 2 to 3 times with distilled water using a dialysis bag (Mw 3000), and obtain selenized hyaluronic acid by freeze-drying at -60℃ for 40h to 48h.

[0008] Step 2: Preparation of tetrahedral framework nucleic acid and drug loading: (1) Sequence design: Design four single-stranded DNAs, each containing three complementary regions and one non-complementary region. The length of the complementary region is 20-30 bases to ensure stable double-strand formation, and the length of the non-complementary region is 5-10 bases to form the vertices of the tetrahedron; (2) Annealing assembly: Dissolve the single-stranded DNA in a buffer containing 10 mM Tris-HCl and 50 mM MgCl2, then maintain the mixture at 80°C to 105°C for 5 min to 15 min, slowly cool to 2°C to 8°C and maintain for 10 min to 60 min to allow the DNA to self-assemble into a tetrahedral framework structure. The product is stored at 4°C. (3) Drug loading: Slowly add the DMSO solution of the hydrophobic drug to the prepared tetrahedral framework nucleic acid solution, stir and mix for 1 h to 24 h in the dark, and centrifuge through an ultrafiltration tube (10 kDa to 50 kDa) to remove the unloaded drug to obtain the drug-loaded tetrahedral framework nucleic acid.

[0009] Step 3: Preparation of selenium-containing and drug-loaded microneedle patch: (1) In a clean bench, weigh the selenized hyaluronic acid obtained in step 1 into a sterile container, mix it with the drug-loaded tetrahedral framework nucleic acid obtained in step 2, add ultrapure water and ultrasonically dissolve it to prepare a solution as the needle layer gel solution; (2) Weighing a polyvinyl alcohol (PVA) solution dissolved in pure water as a backing layer gel solution; (3) placing both the needle layer gel solution and the backing layer gel solution in a centrifuge for degassing; (4) Add the degassed needle layer gel solution into the microneedle holes of the microneedle mold using a syringe, place it in a vacuum drying oven to evacuate, centrifuge the mold, and scrape off excess colloid on the surface; (5) Add the needle layer gel solution again and repeat steps (4) to (5) until each needle hole is filled, and then place it in a vacuum drying oven or a constant temperature and humidity chamber for drying; (6) Add the backing layer gel solution into the groove of the microneedle mold as the backing layer of the microneedle, then place it at 37°C to dry, and demold it to obtain the microneedle patch.

[0010] The above-mentioned method for preparing a selenium-containing soluble composite microneedle, wherein: Furthermore, in step 1, the solvent of the hyaluronic acid solution is selected from water, DMSO, DMF or a mixture thereof in any proportion.

[0011] Furthermore, in step 1, the selenium-containing small molecule compound is selected from selenocystamine, Se-(methyl)selenocysteine, selenomethionine, selenocysteine ​​and their corresponding hydrochlorides. The molar ratio of hyaluronic acid to the selenium-containing small molecule compound is (0.1-5): 1. Hyaluronic acid is selected from one of the molecular weights of 3kDa-10kDa, 10kDa-100kDa, 200kDa-400kDa, and 1100kDa-1300kDa.

[0012] Furthermore, in step 2 (1), the four complementary single-stranded DNA sequences (5′→3′) are as shown in SEQ ID NOs. 1 to 4.

[0013] Furthermore, in step 2 (2), the molar ratio of the four single-stranded DNAs is 1:1:1:1. The pH value of the buffer is 7.5-8.5, and the optimal value is 8.0. The mixture is heated at a temperature of 80°C to 105°C, and the optimal value is 95°C. The heating time is 5min to 15min, and the optimal value is 10min. The mixture is slowly cooled to 2°C to 8°C, and the optimal value is 4°C. The cooling rate is 1°C / min to 3°C / min, and the holding time is 10min to 60min, and the optimal value is 30min.

[0014] Furthermore, in step 2 (3), the hydrophobic drug mainly refers to a drug containing an aromatic ring that can treat psoriasis, including but not limited to methotrexate, tofacitinib, apremilast, tacrolimus, tazarotene, rapamycin, and shikonin.

[0015] Furthermore, in step 2 (3), the molar ratio of the drug to the tetrahedral framework nucleic acid is (100-1000):1. The light-proof stirring temperature is 4°C-25°C, preferably 4°C, and the stirring time is 1h-24h, preferably 6h. The molecular weight cutoff of the ultrafiltration tube is 10kDa-50kDa, and the appropriate size is selected according to the molecular weight of the drug.

[0016] Furthermore, in step 3 (1), the concentration of the selenized hyaluronic acid is 5% to 50% (w / v). The ratio of the drug in the drug-loaded tetrahedral framework nucleic acid to the selenized hyaluronic acid is (1 to 10):100.

[0017] Furthermore, in step 3 (2), the concentration of polyvinyl alcohol is 5% to 30% (w / v).

[0018] Furthermore, in step 3 (3), the rotation speed of the centrifugal degassing is 2000 rpm to 5000 rpm, preferably 3000 rpm, and the degassing time is 2 min to 3 min.

[0019] Furthermore, in step 3 (4), the vacuum pressure is 0.09 MPa to 0.1 MPa, the standing time is 2 min to 3 min, the centrifugal adjustment speed is 2000 rpm to 5000 pm, the optimal is 4000 rpm, and the centrifugal time is 2 min to 3 min.

[0020] Furthermore, in step 3 (5), the drying time is 6 hours to 24 hours.

[0021] Furthermore, in step 3 (6), the drying can be carried out in a vacuum drying oven or in a constant temperature and humidity oven, and the drying time is 24 hours to 72 hours.

[0022] The invention discloses an application of a selenium-containing soluble composite microneedle in preparing a therapeutic patch for psoriasis.

[0023] The selenium-containing soluble composite microneedle of the present invention can also be used to prepare drugs for treating other skin diseases caused by oxidative stress, such as ulcers and infected wound healing drugs.

[0024] The key points of the present invention are: The present invention synthesizes selenized hyaluronic acid by a specific chemical method, and prepares microneedles with selenized hyaluronic acid as the substrate. Compared with traditional microneedle materials, the microneedles prepared by the present invention not only have the function of drug delivery, but also have significant antioxidant capacity, can effectively remove reactive oxygen and improve the immune environment. At the same time, by utilizing the advantages of high permeability, good biocompatibility, editability, high stability and easy preparation of tetrahedral framework nucleic acids, the sustained-controlled release capacity of microneedles is improved, efficient transdermal delivery of drugs is achieved, and the pain and discomfort that may be caused by traditional drug administration methods are reduced.

[0025] Beneficial effects of the present invention: The present invention improves the drug loading capacity and delivery efficiency by combining selenized hyaluronic acid and tetrahedral framework nucleic acid, so that the drug can reach the target site more effectively. The antioxidant and immunomodulatory effects of selenized hyaluronic acid help alleviate the inflammatory response of psoriasis and improve the therapeutic effect. Soluble microneedle technology reduces the pain and discomfort that may be caused by traditional drug administration methods and improves patient compliance. The painless transdermal drug delivery system improves the patient's comfort and makes the treatment process more humane. The advantages of tetrahedral framework nucleic acid such as high permeability, good biocompatibility, editability, high stability and easy preparation improve the drug loading capacity and delivery efficiency.

[0026] The technical solution of the present invention combines a variety of advanced technologies and provides a new and effective drug delivery system in the field of psoriasis treatment, which is expected to improve existing treatment methods, enhance treatment effects, reduce side effects, and improve the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the Fourier transform infrared spectra of hyaluronic acid and selenized hyaluronic acid in the embodiments of the present invention; Figure 2 The whole scanning electron microscope image (a) and the local magnified image (b) of the microneedle in the embodiment of the present invention; Figure 3 It is a microneedle element mapping image in an embodiment of the present invention; Figure 4 The mechanical properties and in vitro transdermal release effects of the microneedle patch in the embodiment of the present invention; wherein (a) is the mechanical properties of the selenium-containing composite microneedles and the hyaluronic acid matrix microneedles, and (b) is the in vitro transdermal release of shikonin from the microneedle patch; Figure 5 The uptake effect of shikonin by cells in the embodiment of the present invention (red: shikonin, blue: cell nucleus, green: cell membrane); Figure 6 The following are the effects of SeHA on scavenging reactive oxygen species in RAW 264.7 cells and HacaT cells under oxidative stress in the embodiments of the present invention; wherein (a) is the effect of SeHA on scavenging reactive oxygen species in RAW 264.7 cells under oxidative stress, and (b) is the effect of SeHA on scavenging reactive oxygen species in HacaT cells under oxidative stress; Figure 7 The results of the experiment of SeHA scavenging reactive oxygen species in the examples of the present invention; wherein (a) is the protective effect of SeHA on RAW 264.7 cells under oxidative stress in the MTT experiment, and (b) is the protective effect of SeHA on HacaT cells under oxidative stress in the MTT experiment; Figure 8 The results of quantitatively detecting the levels of IL-6 and TNF-α in the RAW cell model of oxidative stress induced by H2O2 in the embodiment of the present invention; wherein (a) is the result of detecting the levels of IL-6, and (b) is the result of detecting the levels of TNF-α; Fig. 9 The figures show the treatment effects of different treatment groups on imiquimod-induced psoriasis mice in the examples of the present invention; (a) shows the skin surface of different treatment groups after treatment, and (b) shows the PASI score of psoriasis skin. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0029] Unless otherwise specified, the instruments, reagents and materials used in the present invention are commercially available.

[0030] Example 1 The preparation of selenized hyaluronic acid comprises: (1) Selenocysteine-modified hyaluronic acid: 100 mg of hyaluronic acid (10-100 kDa) was dissolved in 50 mL of DMSO, and 251 mg of 1-[3-dimethylaminopropyl]-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 151 mg of N-hydroxysuccinimide (NHS) were added in a molar ratio of 1:1, and stirred at room temperature for 6 hours. Slowly add selenocystamine dihydrochloride to the above solution, and react in the dark under inert gas protection for 48 hours. After the reaction is completed, dialyze three times with distilled water using a dialysis bag (Mw 3000), and selenocystamine-modified hyaluronic acid is obtained by freeze-drying at -60°C for 48 hours. Results are shown in Figure 1 Fourier transform infrared spectroscopy, infrared spectrum shows C-Se (771cm -1 ) and Se-Se(615cm -1 ) peak confirmed the presence of diselenide, confirming that selenocysteine ​​was successfully modified on HA.

[0031] (2) Se-(methyl)selenocysteine-modified hyaluronic acid: 50 mg of hyaluronic acid (3-10 kDa) was dissolved in 50 mL of DMSO / water (1:1, v / v), and 126 mg of 1-[3-dimethylaminopropyl]-3-ethylcarbodiimide (EDC·HCl) and 76 mg of N-hydroxysuccinimide (NHS) were added at a molar ratio of 1:1, and stirred at room temperature for 8 h. Se-(methyl)selenocysteine ​​hydrochloride was slowly added to the above solution, and the reaction was carried out in the dark under inert gas protection for 48 h. After the reaction was completed, the hyaluronic acid modified with Se-(methyl)selenocysteine ​​was obtained by lyophilization at -60 °C for 48 h.

[0032] Example 2 The preparation of tetrahedral framework nucleic acid comprises the following steps: (1) Sequence design: Four single-stranded DNAs are designed, each of which contains three complementary regions and one non-complementary region. The length of the complementary region is 20-30 bases to ensure stable double-strand formation, and the length of the non-complementary region is 5-10 bases to form the vertices of the tetrahedron. The four complementary single-stranded DNA sequences (5′→3′) designed by the present invention are shown in SEQ ID NOs. 1 to 4.

[0033] (2) Annealing assembly: Four single-stranded DNAs (final concentration of 10,000 nM) were dissolved in a buffer containing 10 mM Tris-HCl and 50 mM MgCl2. The molar ratio of the four single-stranded DNAs was 1:1:1:1, and the pH value of the buffer was 8.0. The mixture was then maintained at 95°C for 10 min, and then slowly cooled to 4°C for 30 min at a cooling rate of 2°C / min to allow the DNA to self-assemble into a tetrahedral framework structure. The resulting tetrahedral framework nucleic acid product was stored at 4°C for further use.

[0034] Example 3 Tetrahedral framework nucleic acid drug loading: 50 mL of shikonin DMSO solution (500 mM) was slowly added dropwise to the tetrahedral framework nucleic acid solution prepared in Example 2, and the mixture was stirred and mixed at 4° C. in the dark for 6 h. The unloaded drug was removed by centrifugation through an ultrafiltration tube (30 kDa) to obtain a drug-loaded tetrahedral framework nucleic acid.

[0035] Example 4 Preparation of tetrahedral framework nucleic acid-loaded selenium composite microneedles, the specific steps are as follows: (1) In a clean bench, weigh 0.8 g of selenocystamine-modified hyaluronic acid prepared in Example 1, add it to 1 mL of the drug-loaded tetrahedral framework nucleic acid solution prepared in Example 3 in three batches, and dissolve it by ultrasonication to obtain a needle-layer gel solution.

[0036] (2) Weigh polyvinyl alcohol (PVA) and dissolve it in pure water to prepare a 20% polyvinyl alcohol solution as the backing layer gel solution.

[0037] (3) Centrifuge the needle layer gel solution and the backing layer gel solution at 3000 rpm for 3 min to remove bubbles.

[0038] (4) Add the degassed needle layer gel solution into the microneedle holes of the microneedle mold using a syringe, place it in a vacuum drying oven and evacuate the air (0.09 MPa to 0.1 MPa) for 2 min, place the mold in a centrifuge (4000 rpm, 3 min), and scrape off excess colloid on the surface.

[0039] (5) Add the needle layer gel solution again and repeat steps (4)-(5) 1-2 times until each needle hole is filled, and then place it in a vacuum drying oven at 37°C for static drying for 24 hours.

[0040] (6) Add the backing layer gel solution into the groove of the microneedle mold as the backing layer of the microneedle, and then place it in a static drying state at 37°C for 72 hours. Demolding the microneedle patch loaded with tetrahedral framework nucleic acid and containing selenium is obtained.

[0041] Characterization and performance: The microneedle patch prepared in this example has a size of 14.5×14.5 mm and is composed of a 15×15 array of regular quadrangular pyramids. The bottom side of each needle is 360 μm long and 800 μm high, and the needle tip spacing is 720 μm.

[0042] The microneedle patch prepared in this example was characterized by scanning electron microscopy. Figure 2 Scanning electron microscopy shows that the microneedles are complete in shape and are regular tetrahedral pyramids. Figure 3 The elemental mapping image showed the presence of C, N, O, and Se elements at the tip, indicating that selenohyaluronic acid had been successfully integrated into the microneedle.

[0043] The mechanical properties of the microneedle patch prepared in this example were tested by an electronic universal material testing machine. Figure 4 (a). The force exhibited by the microneedle patch at a displacement of 800 μm is greater than 0.3 N / needle, which has sufficient properties to penetrate the skin. Compared with the traditional hyaluronic acid matrix microneedles (blue HA), the selenium-containing composite microneedles (red tFNAs / shikonin-SeHA) prepared in the example have better mechanical strength.

[0044] The microneedle patch prepared in this example was subjected to an external transdermal release experiment to study its sustained-release effect. Figure 4 (b). As time goes by, the cumulative release percentage of shikonin gradually increases. In the first 12 hours, the release rate of shikonin is fast, and the cumulative release percentage increases rapidly to about 40%. Subsequently, the release rate gradually slows down, and the cumulative release percentage reaches about 80% at 72 hours. These results show that the prepared selenium-containing composite microneedle patch can effectively release drugs in vitro in a simulated in vivo environment.

[0045] In summary, the selenium-containing composite microneedle patch of the present invention has good mechanical properties and drug release characteristics, achieves the purpose of sustained and controlled release of drugs, and can realize efficient transdermal delivery of drugs.

[0046] Application Example 1 Testing the effect of tetrahedral framework nucleic acid (tFNA) on promoting cellular uptake of shikonin:

[0047] HacaT cells were treated with shikonin or the drug-loaded tetrahedral framework nucleic acid (tFNAs / shikonin) prepared in Example 3 for 8 h, and then confocal microscopy was used to examine how tFNA effectively helped shikonin penetrate HacaT cells. Figure 5 As shown, in the presence of tFNA, the fluorescence of tFNAs / shikonin is significantly stronger than that of shikonin (red), indicating that more shikonin enters the cells.

[0048] Application Example 2 Test the effect of selenized hyaluronic acid in clearing intracellular reactive oxygen species and reducing intracellular inflammatory factors:

[0049] As evaluated by reactive oxygen species probe and MTT assay, selenocystamine-modified hyaluronic acid (SeHA) prepared in Example 1 showed a protective effect on RAW264.7 cells and HacaT cells subjected to oxidative stress.

[0050] Under the stress condition induced by 100 μM H2O2, a model of high reactive oxygen levels in Hacat and RAW 264.7 cells was obtained. By staining with 2',7'-dichlorofluorescein (DCF), the changes in DCF fluorescence intensity in different treatment groups can be observed. The results showed that 400 μM SeHA was able to significantly reduce the increase in DCF fluorescence intensity, while the control group 400 μM HA (without Se-Se bond) did not. The results are shown in Figure 6 Further, the MTT experiment was used to verify the protective effect of SeHA on the cell viability of the two cell types. The cell viability of the SeHA-treated group was significantly higher than that of the HA-treated group, indicating that SeHA has a protective effect on cells, further confirming the effective elimination of reactive oxygen species. The results are shown in Figure 7 .

[0051] In addition, we also examined the antioxidant effect of SeHA in H2O2-induced RAW 264.7 cells by measuring the levels of interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) in the cell supernatant. Figure 8 , SeHA significantly reduced the production of IL-6 and TNF-α induced by H2O2, and the HA treatment group had no significant therapeutic effect, indicating that SeHA can not only scavenge reactive oxygen species, but also reduce the production of inflammatory factors induced by oxidative stress.

[0052] In summary, the selenocystamine-modified hyaluronic acid prepared in Example 1 can effectively remove ROS induced by H2O2 and significantly reduce the level of intracellular inflammatory factors, indicating the application potential of selenized hyaluronic acid as a potential antioxidant in cell protection.

[0053] Application Example 3 Anti-psoriasis effect of tetrahedral framework nucleic acid-loaded selenium-containing composite microneedles prepared in Example 4:

[0054] Establishment of animal model: 1) Twenty-five C58BL / 6 mice aged 6-8 weeks were selected and all mice were randomly divided into 5 groups: control group, imiquimod treatment group (model group), shikonin cream group (SHI group), selenohyaluronic acid microneedle patch (SeHA group), and tetrahedral framework nucleic acid-loaded shikonin-selenium composite microneedle patch (tFNAs / shikonin-SeHA) group. There were 5 mice in each group. The backs of the mice were depilated; 2) The control group received no treatment; the mice in the imiquimod model group were applied with 62.5 mg imiquimod cream daily; the mice in the shikonin cream group were applied with 62.5 mg imiquimod cream daily, and then applied with 62.5 mg shikonin cream dissolved in vaseline daily; the mice in the SeHA group were applied with 62.5 mg imiquimod cream daily, and then given a microneedle patch; the mice in the tFNAs / shikonin-SeHA group were applied with 62.5 mg imiquimod cream daily, and then given a microneedle patch, for 7 consecutive days; 3) The mice were euthanized on the 8th day, and the back skin of the mice was collected for subsequent detection.

[0055] PASI scoring of mice: The back skin of mice was photographed daily to observe local erythema, scaling, and skin thickening. The modified psoriasis severity index score (PASI) was used to score the erythema, scaling, and thickening of the mouse skin lesions as follows: 0, no symptoms; 1, mild; 2, moderate; 3, severe; 4, extremely severe. The clinical photos of mice after treatment in different groups are shown in Fig. 9 (a), PASI scores of mice in different groups Fig. 9 (b) Erythema score, scaled score, skin thickness score, and PASI total score.

[0056] Experimental results: Fig. 9 It can be seen that imiquimod cream caused obvious psoriasis symptoms such as erythema, scaling and epidermal thickening in mice, and successfully induced a psoriasis mouse model. The normal group did not show erythema, scaling and thickening of the skin during the 1d-7d experimental period; while the other experimental groups all showed skin lesions of varying degrees. The model group began to show obvious erythema symptoms within 2d of modeling, accompanied by mild scaling and keratin thickening. Thereafter, the PASI score increased sharply and reached the maximum value on the 6th day. The topical shikonin, selenohyaluronic acid microneedle patch and tetrahedral framework nucleic acid-loaded shikonin-containing selenium composite microneedle patch given to mice with imiquimod-induced skin inflammation can reduce the manifestations of epidermal thickening, scaling and erythema in psoriasis model mice. Fig. 9It can be seen that the appearance and clinical scores of mice in the shikonin cream group were better than those in the SeHA group, which proved that shikonin had a certain therapeutic effect, but the treatment effect of mice in the tFNAs / shikonin-SeHA group was better.

Claims

1. A selenium-containing soluble composite microneedle, characterized in that: The microneedle forming matrix is ​​selenized hyaluronic acid, loaded with drug-carrying tetrahedral framework nucleic acid, and the base part is polyvinyl alcohol.

2. A method for preparing the selenium-containing soluble composite microneedle according to claim 1, characterized in that: The following steps are involved: Step 1, preparation of selenized hyaluronic acid: Add 1-[3-dimethylaminopropyl]-3-ethylcarbodiimide and N-hydroxysuccinimide to the hyaluronic acid solution, stir for 0.5h to 24h for activation, continue to add selenium-containing small molecule compounds to the solution, react in the dark under inert gas protection conditions for 8h to 48h, dialyze 2 to 3 times after the reaction is completed, and obtain selenized hyaluronic acid by freeze-drying at -60°C; Step 2: Preparation of tetrahedral framework nucleic acid and drug loading: (1) Sequence design: Design four single-stranded DNAs, each containing three complementary regions and one non-complementary region. The length of the complementary region is 20-30 bases, and the length of the non-complementary region is 5-10 bases, forming the vertices of a tetrahedron; (2) Annealing assembly: Dissolve the single-stranded DNA in a buffer containing 10 mM Tris-HCl and 50 mM MgCl2, then maintain the mixture at 80°C to 105°C for 5 min to 15 min, slowly cool to 2°C to 8°C and maintain for 10 min to 60 min to allow the DNA to self-assemble into a tetrahedral framework structure. The product is stored at 4°C. (3) Drug loading: Slowly add the hydrophobic drug solution to the prepared tetrahedral framework nucleic acid solution, stir and mix for 1 h to 24 h in the dark, and remove the unloaded drug by ultrafiltration tube centrifugation to obtain the drug-loaded tetrahedral framework nucleic acid; Step 3: Preparation of selenium-containing and drug-loaded microneedle patch: (1) In a clean bench, weigh the selenized hyaluronic acid obtained in step 1 into a sterile container, mix it with the drug-loaded tetrahedral framework nucleic acid obtained in step 2, add ultrapure water and ultrasonically dissolve it to prepare a solution as the needle layer gel solution; (2) Weighing a polyvinyl alcohol solution prepared by dissolving polyvinyl alcohol in pure water as a backing layer gel solution; (3) placing both the needle layer gel solution and the backing layer gel solution in a centrifuge for degassing; (4) Add the degassed needle layer gel solution into the microneedle holes of the microneedle mold using a syringe, place it in a vacuum drying oven to evacuate, centrifuge the mold, and scrape off excess colloid on the surface; (5) re-add the needle layer gel solution and repeat steps (4)-(5) until each needle hole is filled and then dry; (6) Add the backing layer gel solution into the groove of the microneedle mold as the backing layer of the microneedle, then place it at 37°C to dry, and demold it to obtain the microneedle patch.

3. The method for preparing a selenium-containing soluble composite microneedle according to claim 2, characterized in that: In step 1, the selenium-containing small molecule compound is selected from selenocystamine, Se-(methyl)selenocysteine, selenomethionine, selenocysteine ​​and their corresponding hydrochlorides; the molar ratio of hyaluronic acid to the selenium-containing small molecule compound is (0.1-5):

1.

4. The method for preparing a selenium-containing soluble composite microneedle according to claim 2, characterized in that: In step 2 (1), the four complementary single-stranded DNA sequences are shown as SEQ ID NOs. 1 to 4.

5. The method for preparing a selenium-containing soluble composite microneedle according to claim 2, characterized in that: In step 2 (2), the molar ratio of the four single-stranded DNAs is 1:1:1:

1.

6. The method for preparing a selenium-containing soluble composite microneedle according to claim 2, characterized in that: In step 2 (3), the hydrophobic drug includes but is not limited to methotrexate, tofacitinib, apremilast, tacrolimus, tazarotene, rapamycin, and shikonin; the molar ratio of the drug to the tetrahedral framework nucleic acid is (100-1000):1; and the light-proof stirring temperature is 4°C-25°C.

7. The method for preparing a selenium-containing soluble composite microneedle according to claim 2, characterized in that: In step 3 (1), the concentration of the selenized hyaluronic acid is 5% to 50%; the ratio of the drug in the drug-loaded tetrahedral framework nucleic acid to the selenized hyaluronic acid is (1 to 10):100; in step 3 (2), the concentration of the polyvinyl alcohol is 5% to 30%.

8. The method for preparing a selenium-containing soluble composite microneedle according to claim 2, characterized in that: In step 3 (3), the speed of centrifugal degassing is 2000rpm~5000rpm, and the degassing time is 2min~3min; in step 3 (4), the vacuum pressure is 0.09MPa~0.1MPa, and the standing time is 2min~3min; the centrifugal adjustment speed is 2000rpm~5000pm, and the centrifugal time is 2min~3min.

9. Use of the selenium-containing soluble composite microneedle according to claim 1 in preparing a therapeutic patch for psoriasis.

10. Use of the selenium-containing soluble composite microneedle according to claim 1 in preparing a drug for treating skin diseases caused by oxidative stress.

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