Silk fibroin microneedle transdermal patch for psoriasis and preparation method thereof

Abstract

The application discloses a silk fibroin microneedle transdermal patch suitable for psoriasis, which comprises a substrate layer and a microneedle layer, the microneedle layer comprises a base and a needle body extending outward from the base, and the substrate layer and the base are mutually attached; the substrate layer contains methotrexate and silk fibroin, and the mass ratio of the methotrexate to the silk fibroin in the substrate layer is 1-4:10; the microneedle layer contains methotrexate sodium salt and silk fibroin, and the mass ratio of the methotrexate sodium salt to the silk fibroin in the microneedle layer is 0.2-2:10. The silk fibroin microneedle patch suitable for psoriasis prepared by the application can effectively load drugs, improve the percutaneous penetration of methotrexate, quickly reach the drug effective concentration in the initial release stage of the microneedle, and ensure the continuous and stable release of methotrexate in the subsequent stage, thereby maintaining a high drug concentration in the body for a long time. Meanwhile, the microneedle transdermal patch avoids the liver first-pass effect and improves the bioavailability of methotrexate.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of transdermal drug delivery and microneedle, and particularly relates to a silk fibroin microneedle transdermal patch for psoriasis and a preparation method of the silk fibroin microneedle transdermal patch. BACKGROUND

[0002] Psoriasis, also widely known as "psoriasis", is a common polygenic hereditary skin disease, which is often manifested as localized or widely distributed scaly erythema or plaques. The disease is recurrent, and seriously affects the life and psychological condition of patients. According to research statistics, patients with a disease course of more than ten years account for as high as 65%, and an average of 8 relapses, and patients with more than 10 relapses account for nearly 60%.

[0003] Methotrexate, as an immunosuppressant and antimetabolite, has been widely used in the clinical treatment of psoriasis. However, the traditional methotrexate injection and oral drug delivery has low drug utilization rate, and the side effects caused by it are great, which limits its clinical application. Therefore, transdermal delivery of methotrexate is a good way of drug delivery.

[0004] The stratum corneum is the most important barrier of the human body, and it is very difficult for drugs to completely penetrate the skin surface, which leads to the fact that the treatment of psoriasis by transdermal drug delivery is always ineffective. When the microneedle is used for local drug delivery on the skin, it can effectively pierce the stratum corneum and does not touch the nerve, which not only avoids the adverse reactions caused by the drug through the gastrointestinal tract, but also does not produce pain, increases the bioavailability of the drug and improves the patient's compliance.

[0005] Silk fibroin, as a natural protein, has good mechanical properties and plasticity, good compatibility with human tissues, biodegradability, non-toxicity and easy processing. Silk fibroin microneedle is a potential transdermal immunization method. Compared with other microneedle production materials, silk fibroin has the ability to maintain the stability of certain biological macromolecules at a higher temperature.

[0006] In the prior art, a microneedle patch for treating psoriasis is disclosed in Chinese Patent No. 201910231045.2. The patch can effectively penetrate the stratum corneum and deliver the drug into the skin. However, since the psoriasis treatment drug is rapidly metabolized in the skin, it cannot meet the long-term treatment needs of patients with moderate to severe psoriasis.

[0007] For example, Chinese Patent No. 202110871605.8 discloses a silk fibroin microneedle patch for treating insomnia. The patch can effectively load melatonin and deliver it into the body to achieve a certain blood drug level. However, since the molecular weight of melatonin is 232, the addition of small molecule additives to change the swelling degree of the microneedle can only prolong the drug release to 6-8h, and cannot meet the long-term treatment needs of psoriasis.

[0008] The disclosure of the above Background Art is only used to assist in understanding the inventive concept and technical solutions of the present application, and does not necessarily belong to the prior art of the present patent application. In the absence of explicit evidence that the above background art has been disclosed before the filing date of the present patent application, the above background art should not be used to evaluate the novelty and inventiveness of the present application. SUMMARY

[0009] Therefore, in order to overcome the defects of the prior art, the purpose of the present application is to provide a silk fibroin microneedle transdermal patch for psoriasis and a preparation method thereof, which solves the technical problems that methotrexate is not easy to release due to its poor solubility in the material, and the drug is released too fast when it exists in the form of sodium salt. And it can maintain a high concentration of drug in the body for a long time.

[0010] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0011] A silk fibroin microneedle transdermal patch for psoriasis, comprising a base layer and a microneedle layer, the microneedle layer comprises a base and a needle body extending outward from the base, and the base layer and the base are mutually attached; the base layer contains methotrexate and silk fibroin, and the mass ratio of methotrexate to silk fibroin in the base layer is 1-4:10; the microneedle layer contains methotrexate sodium salt and silk fibroin, and the mass ratio of methotrexate sodium salt to silk fibroin in the microneedle layer is 0.2-2:10.

[0012] According to some preferred embodiments of the present application, the raw materials of the base layer include methotrexate, dimethylformamide and silk fibroin, and the mass ratio of dimethylformamide to silk fibroin in the base layer is 1-10:10.

[0013] Preferably, the base layer is a methotrexate-loaded layer, which is prepared from methotrexate, dimethylformamide and silk fibroin, and the microneedle layer is a methotrexate sodium salt-loaded layer, which is prepared from methotrexate sodium salt and silk fibroin.

[0014] According to some preferred embodiments of the present application, the silk fibroin in the microneedle layer is in a Silk I crystal structure; and / or, the silk fibroin in the base layer is in a Silk I crystal structure.

[0015] According to some preferred embodiments of the present application, the length of the needle body is 300-900 μm, the diameter of the bottom is 100-500 μm, and the distance between the tips of adjacent needle bodies is 0.2-2 mm. The skin of psoriasis is thicker, and longer microneedles are needed.

[0016] The present application also provides a preparation method of the silk fibroin microneedle transdermal patch as described above, comprising the following steps:

[0017] The methotrexate solution is mixed with the silk fibroin aqueous solution to obtain a first mixed solution, and the concentration of methotrexate in the first mixed solution is 5-20 mg / mL.

[0018] The methotrexate sodium salt aqueous solution is mixed with the silk fibroin aqueous solution to obtain a second mixed solution, and the concentration of methotrexate sodium salt in the second mixed solution is 1-10 mg / mL.

[0019] The second mixed solution is cast into a microneedle mold, vacuum degassing is performed, and the degassed mold system is dried under constant temperature and humidity conditions to form a microneedle layer.

[0020] The first mixed solution is cast on the microneedle layer, and the mold system is dried under constant temperature and humidity conditions to form a substrate layer, and the silk fibroin microneedle transdermal patch is obtained after demolding.

[0021] The silk fibroin microneedle transdermal patch is treated in an environment with a temperature of 20-50℃ and a relative humidity of 80-99% for 20-50h, and is dried after being taken out, to obtain a silk fibroin microneedle transdermal patch suitable for psoriasis.

[0022] According to some preferred embodiments of the present application, the methotrexate solution is prepared by adding methotrexate to an aqueous dimethylformamide solution to dissolve and obtain the methotrexate solution.

[0023] According to some preferred embodiments of the present application, when preparing the methotrexate solution, the concentration of the aqueous dimethylformamide solution used is 100-600 mg / mL.

[0024] According to some preferred embodiments of the present application, the methotrexate sodium salt aqueous solution is prepared by adding methotrexate sodium salt to deionized water to dissolve and obtain the methotrexate sodium salt aqueous solution.

[0025] According to some preferred embodiments of the present application, the concentration of the methotrexate solution is 30-80 mg / mL, and the concentration of the methotrexate sodium salt aqueous solution is 10-50 mg / mL.

[0026] According to some preferred embodiments of the present application, the methotrexate sodium salt aqueous solution and / or the methotrexate solution are stored in the dark at 2-8℃.

[0027] According to some preferred embodiments of the present application, the drying under constant temperature and humidity conditions is drying under the condition of being placed in a constant temperature and humidity chamber (20-40℃, 55-75%) with air circulation for 4-12h.

[0028] In some embodiments of the present invention, the preparation method of the anti-psoriasis silk fibroin microneedle transdermal patch, using silkworm silk as raw material, obtains an aqueous solution of silk fibroin through degumming, dissolution, and dialysis, including the following steps:

[0029] (1) Preparation of methotrexate solution

[0030] Methotrexate was added to an aqueous solution of dimethylformamide and stirred thoroughly until dissolved to obtain a methotrexate solution. The concentration of the methotrexate solution was 30-80 mg / mL.

[0031] The concentration of dimethylformamide in the aqueous solution used was 100-600 mg / mL.

[0032] (2) Preparation of methotrexate sodium salt aqueous solution

[0033] Add methotrexate sodium salt to deionized water, stir thoroughly until dissolved, to obtain a methotrexate sodium salt aqueous solution. The concentration of the methotrexate sodium salt aqueous solution is 10-50 mg / mL.

[0034] (3) Preparation of the first mixed solution

[0035] Add the solution obtained in step (1) to a silk fibroin aqueous solution with a concentration of 30-80 mg / mL, mix well to obtain the first mixed solution, so that the concentration of methotrexate in the mixed solution is 5-20 mg / mL, and the mass ratio of dimethylformamide to silk fibroin is 1-10:10.

[0036] (4) Preparation of the second mixed solution

[0037] Add the solution obtained in step (2) to a silk fibroin aqueous solution with a concentration of 30-60 mg / mL, mix well to obtain a second mixed solution, so that the concentration of methotrexate sodium salt in the mixed solution is 1-10 mg / mL.

[0038] (5) Preparation of microneedle patches

[0039] The second mixed solution is poured into a microneedle mold, degassed under vacuum, and the degassed mold system is placed in a constant temperature and humidity room and dried under air circulation for 4-12 hours. After drying, the microneedle tip part (microneedle layer) is formed.

[0040] The first mixed solution is then poured onto the mold, which is then placed in a temperature- and humidity-controlled chamber with good air circulation to dry for 3-10 hours, forming the microneedle matrix (matrix layer). After demolding, the silk fibroin microneedle transdermal patch is obtained. The conditions of the temperature- and humidity-controlled chamber are: temperature: 20-40℃, relative humidity: 55-75%.

[0041] (6) Post-processing of microneedle patch

[0042] The prepared silk fibroin microneedle transdermal patch is placed in a low-temperature and high-humidity environment for 20-50 h, and then taken out and dried to obtain a silk fibroin microneedle transdermal patch suitable for psoriasis. The low-temperature and high-humidity environment is: the temperature is 20-50℃, and the relative humidity is 80-99%.

[0043] Preferably, the silk fibroin aqueous solution is obtained by degumming, dissolving, and dialysis purification of domestic silkworm silk. The concentration of the silk fibroin aqueous solution is 30-80 mg / mL.

[0044] The concave hole of the microneedle mold is conical, with a length of 300-900 μm, a bottom diameter of 100-500 μm, and a spacing between the tips of adjacent needle bodies of 0.2-2 mm. The microneedle mold is made of silicone rubber.

[0045] The microneedle patch prepared by the above preparation method can continuously and stably release methotrexate for 20-50 h, and the release rate of methotrexate per unit time is more than 20 μg / cm 2 ·h.

[0046] Invention principle: Methotrexate for treating psoriasis is difficult to mix uniformly with a silk fibroin solution because it is poorly soluble in water, while the organic solvent dimethylformamide is a good solvent for methotrexate. However, the addition of an organic solvent can cause a decrease in biocompatibility and may cause cytotoxicity. Therefore, dimethylformamide is transferred to the base layer of the microneedle, which does not contact the skin tissue, preventing possible decreases in biocompatibility. At the same time, dimethylformamide can induce silk fibroin to form a crystalline structure, constructing a silk fibroin base layer that is insoluble in water, thereby reducing the transdermal release rate of methotrexate.

[0047] After methotrexate reacts with sodium hydroxide to form a sodium salt, the interaction with water molecules is improved, and it can be dissolved in water and blended with a silk fibroin solution to make a microneedle. However, the hydrophilic sodium salt will have a burst release phenomenon in the early stage of release, and the formed microneedle tips will dissolve, thereby causing the microneedle to fail to achieve long-term stable drug delivery. Therefore, low-temperature water vapor treatment is used to induce silk fibroin to form a silk I crystalline structure, thereby reducing the dissolution and loss rate of the microneedle tips, forming a certain swelling pore size, and thereby controlling the release rate of methotrexate sodium salt.

[0048] In this way, by loading methotrexate sodium salt in the microneedle layer and methotrexate in the base layer, the methotrexate sodium salt in the microneedle layer can be relatively rapidly released, and the effective concentration can be rapidly achieved through the skin. The methotrexate in the base layer is slowly released. Ultimately, the methotrexate can reach a relatively high effective concentration at the psoriasis site for a short time, and can maintain a sustained and stable release, so that the concentration at the psoriasis site can be maintained at a relatively high effective treatment concentration, thereby achieving the purpose of improving the curative effect. By adjusting the different proportions of drugs in the microneedle layer and the base layer and by physical modification (silk I crystal structure is formed by low-temperature water vapor treatment), the drug release rate can be improved or regulated, the bioavailability of methotrexate can be further improved, the drug efficacy can be enhanced, and thus the amount of added drug can be reduced.

[0049] Compared with the prior art, the technical scheme of the present application has the following advantages: the silk fibroin microneedle patch prepared by the present application can effectively load drugs, improve the transdermal penetration of methotrexate, rapidly reach the effective concentration of the drug in the initial release stage of the microneedle, and ensure the sustained and stable release of methotrexate, thereby maintaining a relatively high concentration of the drug in the body for a long time. At the same time, the microneedle transdermal patch avoids the first-pass effect of the liver and improves the bioavailability of methotrexate. BRIEF DESCRIPTION OF DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the following embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0051] Figure 1 The cross-sectional structure of the silk fibroin microneedle transdermal patch prepared in Example 3-6 of the present application is shown in the figure.

[0052] Figure 2 The microscope photo of the silk fibroin microneedle transdermal patch prepared in Example 3 of the present application is shown in the figure.

[0053] Figure 3 The X-ray diffraction (XRD) curves of the needle body of the silk fibroin microneedle transdermal patch prepared in Example 3 of the present application before and after low-temperature water vapor treatment are shown in the figure.

[0054] Figure 4 The XRD curves of the upper base layer of the silk fibroin microneedle transdermal patch prepared in Examples 3-6 and Comparative Example 2 of the present application are shown in the figure.

[0055] Figure 5 The in-vitro dissolution diagram of dimethylformamide of the silk fibroin microneedle transdermal patch prepared in Examples 3-6 and Comparative Example 2 of the present application is shown in the figure.

[0056] Figure 6 The in-vitro cumulative transdermal drug release rate curve of the silk fibroin microneedle transdermal patch prepared in the present application examples 3-6 and comparative examples 1-2.

[0057] Figure 7 The in-vitro transdermal drug release rate curve of the silk fibroin microneedle transdermal patch prepared in the present application examples 3-6 and comparative examples 1-2. DETAILED DESCRIPTION

[0058] In order to make the personnel in the technical field better understand the technical solutions of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by the person of ordinary skill in the art without creative labor should belong to the protection scope of the present application.

[0059] The present application designs a double-layer microneedle patch, which loads two different solubility substances, i.e. drugs and sodium salt of drugs, in the upper and lower layers to achieve the effects of early rapid release and later maintenance of drug concentration. After dissolving methotrexate in dimethylformamide, the first mixed solution is obtained by adding the silk fibroin aqueous solution. The second mixed solution is obtained by dissolving methotrexate sodium salt in water and adding the silk fibroin aqueous solution. The second mixed solution is cast into a microneedle mold, and after defoaming and drying, the microneedle tip part is formed. Then, the first mixed solution is cast on the top, and the base part of the microneedle bottom is dried to form the base part. The two parts are combined to form the silk fibroin microneedle transdermal patch suitable for psoriasis. The silk fibroin microneedle transdermal patch prepared by the present application has good mechanical properties, can quickly reach the effective concentration of drugs in the early release stage, and can maintain the sustained and stable release of methotrexate in the later stage.

[0060] Specifically, the preparation method of the silk fibroin microneedle transdermal patch suitable for psoriasis in the present embodiment uses domestic silkworm silk as raw material, and obtains silk fibroin aqueous solution through degumming, dissolution and dialysis, including the following steps:

[0061] (1) Preparation of methotrexate solution

[0062] Methotrexate is added to dimethylformamide aqueous solution, stirred thoroughly, and dissolved uniformly to obtain methotrexate solution. The concentration of the methotrexate solution is 30-80 mg / mL.

[0063] The concentration of dimethylformamide in the used dimethylformamide aqueous solution is 100-600 mg / mL. Pure dimethylformamide is directly added to silk fibroin, which can cause denaturation of silk fibroin. Therefore, dimethylformamide is first dissolved in water to form an aqueous solution, then methotrexate is dissolved, and then the silk fibroin is added.

[0064] (2) Preparation of methotrexate sodium salt aqueous solution

[0065] The methotrexate sodium salt is added to deionized water, stirred thoroughly, and dissolved uniformly to obtain a methotrexate sodium salt aqueous solution. The concentration of the methotrexate sodium salt aqueous solution is 10-50 mg / mL.

[0066] (3) Preparation of the first mixed solution

[0067] The solution obtained in step (1) is added to a silk fibroin aqueous solution with a concentration of 30-80 mg / mL, mixed uniformly, to obtain a first mixed solution, so that the concentration of methotrexate in the mixed solution is 5-20 mg / mL, and the mass ratio of dimethylformamide to silk fibroin is 1-10:10.

[0068] (4) Preparation of the second mixed solution

[0069] The solution obtained in step (2) is added to a silk fibroin aqueous solution with a concentration of 30-60 mg / mL, mixed uniformly, to obtain a second mixed solution, so that the concentration of methotrexate sodium salt in the mixed solution is 1-10 mg / mL.

[0070] (5) Preparation of the microneedle patch

[0071] The second mixed solution is cast into a microneedle mold, vacuum degassed, and the degassed mold system is placed in a constant temperature and humidity chamber, dried for 4-12 h in an air flow, and after drying, a microneedle tip part (microneedle layer) is formed;

[0072] The first mixed solution is then cast on top, the mold system is placed in a constant temperature and humidity chamber, dried for 3-10 h in an air flow, and a microneedle base part (base layer) is formed, and after demolding, a silk fibroin microneedle transdermal patch is obtained. The conditions of the constant temperature and humidity chamber are: temperature: 20-40℃, relative humidity: 55-75%.

[0073] (6) Post-processing of the microneedle patch

[0074] The above prepared silk fibroin microneedle transdermal patch is placed in a low temperature and high humidity environment for 20-50 h, taken out and dried, and a silk fibroin microneedle transdermal patch suitable for psoriasis is obtained. The low temperature and high humidity environment is: temperature: 20-50℃, relative humidity: 80-99%.

[0075] The silk fibroin aqueous solution is obtained by degumming, dissolving, and dialysis purification of domestic silkworm silk. The concentration of the silk fibroin aqueous solution is 30-80 mg / mL. The concave holes of the microneedle mold are conical, with a length of 300-900 μm, a bottom diameter of 100-500 μm, and a spacing between the tips of adjacent needle bodies of 0.2-2 mm. The microneedle mold is made of silicone rubber.

[0076] The fibroin microneedle patch prepared by the above preparation method can form a larger methotrexate concentration difference between the microneedle and the skin, provide a stable driving force for the transdermal release of methotrexate, so that a stable drug release rate is reached after a certain period of time, thereby achieving the purpose of long-term stable drug delivery; the methotrexate can be continuously and stably released for 20-50 h.

[0077] Structure of the fibroin microneedle of Example 1 with methotrexate and methotrexate sodium salt

[0078] As shown in Figure 1 , the fibroin microneedle patch of the present embodiment comprises a base layer and a microneedle layer, the microneedle layer comprises a base and a needle body extending downward from the base, the upper base layer is a methotrexate-loaded layer, which is composed of methotrexate, dimethylformamide and fibroin; wherein the mass ratio of methotrexate to fibroin in the layer is 1-4:10, and the mass ratio of dimethylformamide to fibroin in the layer is 1-10:10. The lower microneedle layer is a methotrexate sodium salt-loaded layer, which is composed of fibroin and methotrexate sodium salt, wherein the mass ratio of methotrexate sodium salt to fibroin in the layer is 0.2-2:10.

[0079] As shown in Figure 2 , the needle body of the microneedle layer is a solid cone, the length of the microneedle is 300-900 μm, the diameter of the bottom is 100-500 μm, and the microneedles are spaced apart by 0.5-2 mm.

[0080] In the methotrexate sodium salt-loaded layer, the content of methotrexate sodium salt is 100-400 μg, which is 2-20% of the mass of fibroin in the layer. The content of methotrexate in the methotrexate-loaded layer is 600-900 μg, which is 10-40% of the mass of fibroin in the layer.

[0081] Preparation of fibroin solution

[0082] Weigh 3g of NaHCO3 and 1g of Na2CO3 and dissolve them in 4000mL of deionized water. Heat to boiling and put in 80g of silk. Keep the temperature at 98℃ and boil for 30min. Then take out and wash in deionized water.

[0083] Repeat the above steps three times and then dry in a 60℃ oven to obtain fibroin fibers.

[0084] Prepare a 9.3mol / L LiBr solution. Take 100mL of LiBr solution and heat it in a water bath to 65℃. Put in 15g of degummed fibroin in several portions and stir to dissolve. Continue heating and stirring for 40min.

[0085] The liquid was loaded into the dialysis bag and placed in deionized water for dialysis for 72 h, and the deionized water was replaced every 2 h. After dialysis, the solution was filtered to obtain the silk fibroin solution.

[0086] Example 3

[0087] The preparation method of the silk fibroin microneedle transdermal patch for psoriasis in this embodiment includes the following steps:

[0088] (1) Preparation of methotrexate solution

[0089] Methotrexate powder was weighed into a centrifuge tube, 0.8 mL of dimethylformamide aqueous solution was added, and the solution was shaken and mixed to make the concentration of methotrexate in the solution 50 mg / mL. The concentration of the dimethylformamide aqueous solution used was 600 mg / ml, and the methotrexate solution was stored at 4°C in the dark.

[0090] (2) Preparation of methotrexate sodium salt aqueous solution

[0091] Methotrexate sodium salt powder was weighed into a centrifuge tube, 2 mL of deionized water was added, and the solution was shaken and mixed to make the concentration of methotrexate sodium salt in the solution 10 mg / mL. The methotrexate sodium salt aqueous solution was stored at 4°C in the dark.

[0092] (3) Preparation of the first mixed solution

[0093] The methotrexate solution obtained in step (1) was added to the silk fibroin aqueous solution with a concentration of 80 mg / mL, and after thorough mixing, the silk fibroin concentration was 50 mg / mL and the methotrexate concentration was 20 mg / mL, obtaining the first mixed solution, which was stored at 4°C in the dark.

[0094] (4) Preparation of the second mixed solution

[0095] The methotrexate sodium salt aqueous solution obtained in step (2) was slowly added to the silk fibroin aqueous solution with a concentration of 60 mg / mL, and after thorough mixing, the silk fibroin concentration was 50 mg / mL and the methotrexate sodium salt concentration was 2 mg / mL, obtaining the second mixed solution, which was stored at 4°C in the dark.

[0096] (5) Preparation of microneedle patch

[0097] 1 mL of the second mixed solution was cast into the silicone rubber microneedle mold, vacuum degassing was performed 3 times to remove bubbles in the solution and the microneedle template, so that the solution fully entered the mold cavity. The degassed mold system was placed in a constant temperature and humidity chamber (20°C, 55%) with air circulation and dried for 8 h to form the microneedle tip part (microneedle layer); 2 mL of the first mixed solution was further added and cast on the tip, and the mold system was placed in a constant temperature and humidity chamber with air circulation and dried for 10 h to form the base part of the microneedle bottom (base layer).

[0098] (6) Post-treatment of microneedle patch

[0099] The silk fibroin microneedle transdermal patch was placed in a low-temperature and high-humidity environment (20°C, 90%) for 48 h, and then taken out and dried to obtain a silk fibroin microneedle transdermal patch suitable for psoriasis.

[0100] Example 4

[0101] The preparation method of the silk fibroin microneedle transdermal patch suitable for psoriasis in this example comprises the following steps:

[0102] (1) Preparation of methotrexate solution

[0103] Methotrexate powder was weighed into a centrifuge tube, 0.7 mL of dimethylformamide aqueous solution was added, and the solution was shaken and mixed to make the concentration of methotrexate in the solution 40 mg / mL. The concentration of the dimethylformamide aqueous solution used was 500 mg / mL, and the methotrexate solution was stored at 4°C in the dark.

[0104] (2) Preparation of methotrexate sodium salt aqueous solution

[0105] Methotrexate sodium salt powder was weighed into a centrifuge tube, 2 mL of deionized water was added, and the solution was shaken and mixed to make the concentration of methotrexate sodium salt in the solution 20 mg / mL. The methotrexate sodium salt aqueous solution was stored at 4°C in the dark.

[0106] (3) Preparation of first mixed solution

[0107] The methotrexate solution obtained in step (1) was added to the silk fibroin aqueous solution with a concentration of 70 mg / mL, and after thorough mixing, the silk fibroin concentration was 50 mg / mL and the methotrexate concentration was 15 mg / mL. The first mixed solution was stored at 4°C in the dark.

[0108] (4) Preparation of second mixed solution

[0109] The methotrexate sodium salt aqueous solution obtained in step (2) was slowly added to the silk fibroin aqueous solution with a concentration of 60 mg / mL, and after thorough mixing, the silk fibroin concentration was 50 mg / mL and the methotrexate sodium salt concentration was 4 mg / mL. The second mixed solution was stored at 4°C in the dark.

[0110] (5) Preparation of microneedle patch

[0111] Take 1 mL of the second mixed solution to cast into the silicone rubber microneedle mold, vacuum degassing 3 times, remove bubbles in the solution and microneedle template, so that the solution fully into the needle body, the degassed mold system is placed in a constant temperature and humidity chamber (25℃, 60%), air circulation under dry 5h to form the microneedle tip part (microneedle layer); continue to add 2 mL of the first mixed solution, cast on the tip, the mold system is placed in a constant temperature and humidity chamber (25℃, 60%), air circulation under dry 9h, forming the base of the microneedle base part (base layer).

[0112] (6) Microneedle patch post-processing

[0113] The silk fibroin microneedle transdermal patch is placed in a low temperature and high humidity environment (22℃, 93%) for 40h, and then taken out and dried to obtain a silk fibroin microneedle transdermal patch suitable for psoriasis.

[0114] Example 5

[0115] The preparation method of the silk fibroin microneedle transdermal patch suitable for psoriasis in this embodiment comprises the following steps:

[0116] (1) Preparation of methotrexate solution

[0117] Weigh methotrexate powder into a centrifuge tube, add 0.6 mL of dimethylformamide aqueous solution, shake and mix well to make the concentration of methotrexate in the solution 35 mg / mL, the concentration of dimethylformamide aqueous solution used is 450 mg / mL, and the methotrexate solution is stored at 4℃ in the dark.

[0118] (2) Preparation of methotrexate sodium salt aqueous solution

[0119] Weigh methotrexate sodium salt powder into a centrifuge tube, add 2.5 mL of deionized water, shake and mix well to make the concentration of methotrexate sodium salt in the solution 30 mg / mL, and the methotrexate sodium salt aqueous solution is stored at 4℃ in the dark.

[0120] (3) Preparation of the first mixed solution

[0121] Add the methotrexate solution obtained in step (1) to the silk fibroin aqueous solution with a concentration of 65 mg / mL, mix well to make the concentration of silk fibroin 50 mg / mL and the concentration of methotrexate 10 mg / mL, and obtain the first mixed solution which is stored at 4℃ in the dark.

[0122] (4) Preparation of the second mixed solution

[0123] Slowly add the methotrexate sodium salt aqueous solution obtained in step (2) to the silk fibroin aqueous solution with a concentration of 55 mg / mL, mix well to make the concentration of silk fibroin 45 mg / mL and the concentration of methotrexate sodium salt 6 mg / mL, and obtain the second mixed solution which is stored at 4℃ in the dark.

[0124] (5) Preparation of microneedle patch

[0125] Take 1 mL of the second mixed solution and cast it into the silicone rubber microneedle mold. Vacuum degassing for 3 times, remove the bubbles in the solution and microneedle template, so that the solution fully enters the needle body. Place the degassed mold system in a constant temperature and humidity chamber (35°C, 65%) with air circulation and dry for 6h to form the microneedle tip part (microneedle layer). Continue to add 2 mL of the first mixed solution and cast it on the tip. Place the mold system in a constant temperature and humidity chamber (35°C, 65%) with air circulation and dry for 8h to form the base part of the microneedle bottom (base layer).

[0126] (6) Post-processing of microneedle patch

[0127] Place the silk fibroin microneedle transdermal patch in a low-temperature and high-humidity environment (25°C, 95%) for 35h, and then take it out and dry it to obtain a silk fibroin microneedle transdermal patch suitable for psoriasis.

[0128] Example 6

[0129] The preparation method of the silk fibroin microneedle transdermal patch suitable for psoriasis in this example includes the following steps:

[0130] (1) Preparation of methotrexate solution

[0131] Weigh the methotrexate powder into a centrifuge tube, add 0.6 mL of dimethylformamide aqueous solution with a concentration of 400 mg / mL, shake and mix well to make the concentration of methotrexate in the solution 30 mg / mL. The methotrexate solution is stored at 4°C in the dark.

[0132] (2) Preparation of methotrexate sodium salt aqueous solution

[0133] Weigh the methotrexate sodium salt powder into a centrifuge tube, add 3 mL of deionized water, shake and mix well to make the concentration of methotrexate sodium salt in the solution 40 mg / mL. The methotrexate sodium salt aqueous solution is stored at 4°C in the dark.

[0134] (3) Preparation of the first mixed solution

[0135] Add the methotrexate solution obtained in step (1) to the silk fibroin aqueous solution with a concentration of 60 mg / mL. After mixing well, the concentration of silk fibroin is 50 mg / mL and the concentration of methotrexate is 5 mg / mL. The first mixed solution is stored at 4°C in the dark.

[0136] (4) Preparation of the second mixed solution

[0137] The methotrexate sodium salt aqueous solution obtained in step (2) was slowly added to the 60 mg / mL aqueous solution of silk fibroin with a concentration of 50 mg / mL, and the concentration of methotrexate sodium salt was 8 mg / mL after mixing. A second mixed solution was obtained and stored at 4°C in the dark.

[0138] (5) Preparation of microneedle patch

[0139] 1.5 mL of the second mixed solution was cast into the silicone rubber microneedle mold, vacuum degassing was performed 3 times, and bubbles in the solution and microneedle template were removed to allow the solution to fully enter the needle body. The degassed mold system was placed in a constant temperature and humidity chamber (40°C, 70%) with air circulation and dried for 7 h to form the microneedle tip part (microneedle layer). Then, 2 mL of the first mixed solution was added and cast on the tip, and the mold system was placed in a constant temperature and humidity chamber (40°C, 70%) with air circulation and dried for 7 h to form the base part of the microneedle bottom (base layer).

[0140] (6) Post-processing of microneedle patch

[0141] The silk fibroin microneedle transdermal patch was treated in a low-temperature and high-humidity environment (30°C, 99%) for 30 h, and then dried to obtain a silk fibroin microneedle transdermal patch suitable for psoriasis.

[0142] Comparative Example 1

[0143] In this comparative example, only methotrexate sodium salt was loaded into the silk fibroin microneedle, and the preparation method was as follows:

[0144] (1) Preparation of methotrexate sodium salt solution

[0145] Methotrexate sodium salt powder was weighed into a centrifuge tube, 2 mL of deionized water was added, and the mixture was shaken to obtain a solution with a concentration of 35 mg / mL of methotrexate sodium salt. The methotrexate sodium salt aqueous solution was stored at 4°C in the dark.

[0146] (2) Preparation of mixed solution

[0147] The above methotrexate sodium salt aqueous solution was slowly added to the 60 mg / mL aqueous solution of silk fibroin with a concentration of 50 mg / mL, and the concentration of methotrexate sodium salt was 10 mg / mL after mixing. The mixed solution was stored at 4°C in the dark.

[0148] (3) Preparation of microneedle patch

[0149] 3 mL of the mixed solution was poured into the silicone rubber microneedle mold, vacuum degassing was performed 3 times, and bubbles in the solution and microneedle template were removed to allow the solution to fully enter the needle body cavity. The degassed mold system was placed in a constant temperature and humidity chamber (25°C, 65%) with air circulation and dried for 8 h.

[0150] (4) Post-treatment of microneedle patch

[0151] The silk fibroin microneedle transdermal patch was placed in a low-temperature, high-humidity environment (20℃, 99%) for 48 hours, then removed and dried to obtain a silk fibroin microneedle transdermal patch suitable for psoriasis.

[0152] Comparative Example 2

[0153] This comparative example uses silk fibroin microneedles loaded only with methotrexate, and its preparation method is as follows:

[0154] (1) Preparation of methotrexate solution

[0155] Weigh methotrexate powder into a centrifuge tube, add 0.6 mL of dimethylformamide aqueous solution, shake to mix well, so that the concentration of methotrexate in the solution is 35 mg / mL, and the concentration of the dimethylformamide aqueous solution used is 500 mg / mL. Store the methotrexate solution at 4°C protected from light.

[0156] (2) Preparation of mixed solutions

[0157] The above-mentioned methotrexate solution was slowly added to a 70 mg / mL silk fibroin aqueous solution. After mixing, the concentration of silk fibroin was 50 mg / mL and the concentration of methotrexate was 15 mg / mL. The mixed solution was stored at 4°C in the dark.

[0158] (3) Preparation of microneedle patches

[0159] Pour 3 mL of the mixed solution into a silicone rubber microneedle mold, and vacuum it three times to remove air bubbles from the solution and the microneedle template, ensuring the solution fully enters the needle cavity. Place the degassed mold system in a constant temperature and humidity chamber (25℃, 65%) with good air circulation to dry for 8 hours.

[0160] (4) Post-treatment of microneedle patch

[0161] The silk fibroin microneedle transdermal patch was placed in a low-temperature, high-humidity environment (20℃, 99%) for 48 hours, then removed and dried to obtain a silk fibroin microneedle transdermal patch suitable for psoriasis.

[0162] Detection and Results

[0163] (1) As Figure 2 As shown, it is a microscopic photograph of the transdermal patch of silk fibroin microneedles for psoriasis prepared in Example 3, which illustrates that the microneedles prepared by the method of the example can be well filled in the silicone rubber microneedle mold and maintain the integrity of the morphology.

[0164] (2) Figure 3The XRD curves of the microneedles prepared in Example 3, 4, 5, 6 and Comparative Example 2 from top to bottom are shown in Figure 6.

[0165] (3) As shown in Figure 5, the XRD curves of the microneedles prepared in Example 3, 4, 5, 6 and Comparative Example 2 from top to bottom are shown. Figure 4

[0166] As can be seen, the microneedle transdermal patches prepared in Examples 3-6 and Comparative Example 2 have weak diffraction peaks near 12.2° and strong diffraction peaks near 19.7°, indicating that Silk I type crystalline structure appears in the structure of silk fibroin. No other diffraction peaks appear in the XRD curve, indicating that methotrexate is well combined with silk fibroin and no phase separation structure is formed.

[0167] In addition, as shown in Figure 7, the dissolution amount of dimethylformamide in Comparative Example 2 is significantly higher than that in Examples 3-6. Considering that dimethylformamide has poor biocompatibility, the dissolution of dimethylformamide in the needle tip can cause certain damage to the skin, so it is not a good choice. Therefore, the matrix layer containing dimethylformamide is placed above the microneedle layer in the present application, so as not to directly contact the skin, so as to reduce the possible damage to the skin. Figure 5

[0168] (4) The in vitro cumulative release rate curves of the microneedle transdermal patches prepared in Examples 3-6 and Comparative Examples 1-2 are shown in Figure 8. Figure 6 As can be seen from Examples 3-6, the silk fibroin microneedles composed of two parts of matrix loaded with methotrexate and needle body loaded with methotrexate sodium salt can obviously maintain the stability of the methotrexate drug release rate within a certain time. With different drug ratios of the needle body and the matrix in the microneedle, the initial release rate of the drug can be adjusted, and the subsequent release can still be stable, reaching a release time of 48 hours. Comparative Example 1 has a rapid release in the early stage, and then rapidly decreases, making it difficult to achieve sustained release. The cumulative drug release rate of Comparative Example 2 is less than 50%, and the effective utilization of the drug is low.

[0169] (5) As shown in Figure 9, the in vitro release rate curves of the microneedle transdermal patches prepared in Examples 3-6 and Comparative Examples 1-2 are shown. Figure 7 ​​As shown, the comparative example 1 is a silk fibroin microneedle loaded with methotrexate sodium salt only, which has a faster release rate in the first 5 hours, but the high release rate is maintained for a relatively short time, and the drug release curve stability is relatively poor than the examples 3-6, which can only maintain the drug release for 10 hours. The drug release rate per unit time is reduced to 20 μg / cm 2 ·h. The release curve of the comparative example 2 is better than that of the comparative example 1, and the drug release time is extended to more than 24 hours, but due to the possible toxicity of dimethylformamide, the present application further studies the double-layer microneedle to achieve a longer stable drug release time and less damage to the skin. From Figure 7 It can be seen that the prepared double-layer microneedle examples 3-6 of the present application have a relatively stable drug release rate, and the high-speed release of 100 μg / cm 2 ·h in the early stage of 1-3 hours provides a therapeutic concentration, and the later sustained release has a drug release rate per unit time of 20 μg / cm 2 ·h at 48 hours, achieving the sustained release effect of long-term stable drug delivery.

[0170] The silk fibroin microneedle patch is a new transdermal drug delivery technology, which can replace injection to reduce the pain of patients and improve patient compliance. The microneedle transdermal drug delivery replaces oral drug delivery, avoids the possible liver first-pass effect and gastrointestinal inactivation, and improves the bioavailability. The silk fibroin microneedle patch of the present application is composed of a methotrexate sodium salt microneedle layer and a methotrexate base layer from bottom to top. In the methotrexate sodium salt microneedle layer, methotrexate sodium salt is uniformly dispersed in silk fibroin. The content of methotrexate sodium salt is 2-20% of the mass of silk fibroin. In the methotrexate base layer, the mass ratio of dimethylformamide to silk fibroin is 1-10:10, and the content of methotrexate is 10-40% of the mass of silk fibroin in the base layer. The preparation method includes solution preparation, modification treatment of silk fibroin, mold pouring, vacuum degassing, solution pouring, and drying and molding. The prepared methotrexate microneedle patch of the present application can maintain the stability of the methotrexate release rate for a long time, maintain the methotrexate concentration in the body for a long time (more than 48 hours), and the microneedle is convenient to prepare and use.

[0171] The above examples are only for illustrating the technical concept and characteristics of the present application, the purpose is to enable the person skilled in the art to understand the content of the present application and to implement it, and cannot limit the protection scope of the present application, any equivalent changes or modifications made according to the spirit and essence of the present application should be covered within the protection scope of the present application.

[0172] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as exactly that endpoint point. The endpoints of the ranges and any values are understood to be approximate values. For ranges having an upper and lower limit, the range can be understood to include each integer within the defined range. The upper and lower limits of the range can independently be included in the range, or independently excluded from the range. The range can also be understood to include single values within the range, which can be the upper or lower limit of the range. For ranges having an upper and lower limit, the range can be understood to include each integer within the defined range. The upper and lower limits of the range can independently be included in the range, or independently excluded from the range. The range can also be understood to include single values within the range, which can be the upper or lower limit of the range.

Claims

1. A silk fibroin microneedle transdermal patch for psoriasis, characterized by, The silk fibroin microneedle transdermal patch comprises a base layer and a microneedle layer, the microneedle layer comprises a base and a needle body extending outward from the base, and the base layer is in close contact with the base; the base layer contains methotrexate and silk fibroin, and the mass ratio of methotrexate to silk fibroin in the base layer is 1-4:10; the microneedle layer contains methotrexate sodium salt and silk fibroin, and the mass ratio of methotrexate sodium salt to silk fibroin in the microneedle layer is 0.2-2:10; the raw material of the base layer comprises methotrexate, dimethylformamide and silk fibroin, and the mass ratio of dimethylformamide to silk fibroin in the base layer is 1-10:10; the silk fibroin in the microneedle layer is in a Silk I crystal structure; and the silk fibroin in the base layer is in a Silk I crystal structure.

2. The silk fibroin microneedle transdermal patch according to claim 1, characterized in that, The length of the needle body is 300-900 μm, the bottom diameter is 100-500 μm, and the distance between the tips of adjacent needle bodies is 0.2-2 mm.

3. A method for preparing the silk fibroin microneedle transdermal patch according to any one of claims 1-2, characterized in that, The method comprises the following steps: The methotrexate solution is uniformly mixed with the silk fibroin aqueous solution to obtain a first mixed solution; the concentration of methotrexate in the first mixed solution is 5-20 mg / mL; The methotrexate sodium salt aqueous solution is uniformly mixed with the silk fibroin aqueous solution to obtain a second mixed solution, and the concentration of methotrexate sodium salt in the second mixed solution is 1-10 mg / mL; The second mixed solution is cast into a microneedle mold, vacuum degassing is performed, and the degassed mold system is dried under constant temperature and humidity conditions to form a microneedle layer; The first mixed solution is cast on the microneedle layer, and the mold system is dried under constant temperature and humidity conditions to form a base layer, and the silk fibroin microneedle transdermal patch is obtained after demolding; The silk fibroin microneedle transdermal patch is treated in an environment with a temperature of 20-50℃ and a relative humidity of 80-99% for 20-50 h, and then dried after being taken out, to obtain a silk fibroin microneedle transdermal patch suitable for psoriasis.

4. The production method according to claim 3, characterized by, The methotrexate solution is prepared by adding methotrexate into an aqueous dimethylformamide solution and dissolving to obtain the methotrexate solution.

5. The production method according to claim 4, characterized by, When preparing the methotrexate solution, the concentration of the aqueous dimethylformamide solution used is 100-600 mg / mL.

6. The preparation method according to claim 3, characterized in that, The methotrexate sodium salt aqueous solution is prepared by adding methotrexate sodium salt into deionized water and dissolving to obtain the methotrexate sodium salt aqueous solution.

7. The production method according to claim 3, characterized by, The concentration of the methotrexate solution is 30-80 mg / mL; and the concentration of the methotrexate sodium salt aqueous solution is 10-50 mg / mL.

8. The preparation method according to claim 3, characterized in that, The methotrexate sodium salt aqueous solution and / or the methotrexate solution are stored at 2-8℃ in the dark.

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