Silk fibroin microneedle transdermal patch suitable for psoriasis and preparation method of silk fibroin microneedle transdermal patch

Abstract

The invention discloses a silk fibroin microneedle transdermal patch suitable for psoriasis, the silk fibroin microneedle transdermal patch comprises a matrix layer and a microneedle layer, the microneedle layer comprises a base part and a needle body extending outwards from the base part, and the matrix layer and the base part are attached to each other; the matrix layer contains methotrexate and silk fibroin, and the mass ratio of methotrexate to silk fibroin in the matrix 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 applicable to psoriasis prepared by the invention can be used for effectively loading a drug and improving the percutaneous penetration of methotrexate, the drug onset concentration can be quickly reached at the initial stage of microneedle release, the methotrexate can be continuously and stably released subsequently, and the drug can be kept at relatively high concentration in vivo for a long time. Meanwhile, the microneedle transdermal patch avoids the first-pass effect of the liver and improves the bioavailability of methotrexate.

Description

Technical Field

[0001] The invention belongs to the field of transdermal drug delivery and microneedle technology, and particularly relates to a silk fibroin microneedle transdermal patch suitable for psoriasis and a preparation method of the silk fibroin microneedle transdermal patch. Background Art

[0002] Psoriasis, also widely known as "psoriasis", is a common multi-gene inherited skin disease, often manifested as localized or widespread scaly erythema or plaques. The disease recurs repeatedly, seriously affecting the patient's life and psychological condition. According to research statistics, the proportion of patients with a course of more than ten years is as high as 65%, with an average of 8 relapses, and the proportion of patients with more than 10 relapses is close to 60%.

[0003] As an immunosuppressant and antimetabolite, methotrexate has been widely used in the clinical treatment of psoriasis. However, the traditional injection and oral administration of methotrexate have low drug utilization rate and great side effects, which limit its clinical application. Therefore, transdermal delivery of methotrexate is a good way to administer the drug.

[0004] The stratum corneum is the most important barrier of the human body. It is difficult for drugs to penetrate completely when applied directly to the skin surface, which leads to poor results in transdermal drug administration for psoriasis. When microneedles are administered topically to the skin, they can effectively penetrate the stratum corneum of the skin without touching the nerves. This not only avoids adverse reactions caused by drugs through the gastrointestinal tract, but also does not cause pain, increases the bioavailability of drugs, and improves patient compliance.

[0005] As a natural protein, silk fibroin has good mechanical properties and plasticity, good compatibility with human tissues, is biodegradable, non-toxic, and easy to process and prepare. Silk fibroin microneedles are a potential means of transdermal immunization. Compared with other microneedle production materials, silk fibroin has the ability to maintain the stability of certain biomacromolecules at higher temperatures.

[0006] In the prior art, for example, Chinese patent application number 201910231045.2 discloses a microneedle patch for the treatment of psoriasis, which can effectively penetrate the stratum corneum of the skin and deliver drugs into the skin. However, since psoriasis treatment drugs are rapidly metabolized in the skin, they cannot meet the long-term treatment needs of moderate to severe patients.

[0007] Another example is a Chinese patent with application number 202110871605.8, which discloses a silk protein microneedle patch for the treatment of insomnia. The patch can effectively load melatonin and deliver it to the body to reach a certain blood drug level. However, since the molecular weight of melatonin is 232, changing the swelling degree of the microneedle by small molecule additives can only extend the drug release to 6-8 hours, which cannot meet the long-term treatment needs of psoriasis.

[0008] The disclosure of the above background technology content is only used to assist in understanding the inventive concept and technical solution of the present invention. It does not necessarily belong to the prior art of this patent application. In the absence of clear evidence that the above content has been disclosed before the filing date of this patent application, the above background technology should not be used to evaluate the novelty and creativity of the present application. Summary of the invention

[0009] In view of this, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a silk fibroin microneedle transdermal patch suitable for psoriasis and a preparation method thereof, which solves the technical problem that methotrexate is difficult to release due to poor solubility in the material and the drug is released too quickly when it exists in the form of sodium salt. And it can maintain a high concentration of the drug in the body for a long time.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] A silk fibroin microneedle transdermal patch suitable for psoriasis comprises a base layer and a microneedle layer, wherein the microneedle layer comprises a base and a needle body extending outward from the base, and the base layer and the base are bonded to each other; the base layer contains methotrexate and silk fibroin, and the mass ratio of methotrexate to the 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 the silk fibroin in the microneedle layer is 0.2-2:10.

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

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

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

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

[0016] The present invention also provides a method for preparing the silk fibroin microneedle transdermal patch as described above, comprising the following steps:

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

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

[0019] Casting the second mixed solution into a microneedle mold, vacuum degassing, and placing the degassed mold system under constant temperature and humidity conditions to dry to form a microneedle layer;

[0020] Casting the first mixed solution on the microneedle layer, placing the mold system under constant temperature and humidity conditions to dry to form a matrix layer, and obtaining a silk fibroin microneedle transdermal patch after demoulding;

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

[0022] According to some preferred embodiments of the present invention, the methotrexate solution is prepared by the following method: adding methotrexate into a dimethylformamide aqueous solution and dissolving it to obtain a methotrexate solution.

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

[0024] According to some preferred embodiments of the present invention, the methotrexate sodium salt aqueous solution is prepared by the following method: adding methotrexate sodium salt into deionized water and dissolving it to obtain the methotrexate sodium salt aqueous solution.

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

[0026] According to some preferred embodiments of the present invention, the methotrexate sodium salt aqueous solution and / or the methotrexate solution is stored at 2-8° C. and protected from light.

[0027] According to some preferred implementation aspects of the present invention, the drying under constant temperature and humidity conditions is drying for 4-12 hours in a constant temperature and humidity room (20-40° C., 55-75%) with air circulation.

[0028] In some embodiments of the present invention, the method for preparing an anti-psoriatic silk fibroin microneedle transdermal patch uses silk as a raw material, and obtains a silk fibroin aqueous solution by degumming, dissolving, and dialysis, comprising the following steps:

[0029] (1) Preparation of methotrexate solution

[0030] Add methotrexate to the dimethylformamide aqueous solution, stir thoroughly, and dissolve evenly to obtain a methotrexate solution having a concentration of 30-80 mg / mL.

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

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

[0033] Add methotrexate sodium salt into deionized water, stir thoroughly, and dissolve evenly to obtain a methotrexate sodium salt aqueous solution having a concentration of 10-50 mg / mL.

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

[0035] The solution obtained in step (1) is added to a 30-80 mg / mL silk fibroin aqueous solution, and mixed evenly to obtain a first mixed solution, wherein 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] The solution obtained in step (2) is added to a 30-60 mg / mL silk fibroin aqueous solution, and mixed evenly to obtain a second mixed solution, wherein the concentration of methotrexate sodium salt in the mixed solution is 1-10 mg / mL.

[0038] (5) Preparation of microneedle patches

[0039] Casting the second mixed solution into the microneedle mold, vacuum degassing, placing the degassed mold system in a constant temperature and humidity room, and drying it for 4-12 hours under air circulation to form a microneedle tip part (microneedle layer) after drying;

[0040] Then cast the first mixed solution on it, place the mold system in a constant temperature and humidity room, and dry it for 3-10 hours under air circulation to form the microneedle matrix part (matrix layer), and after demoulding, the silk fibroin microneedle transdermal patch is obtained. The conditions of the constant temperature and humidity room are: temperature: 20-40°C, relative humidity: 55-75%.

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

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

[0043] Preferably, the silk fibroin aqueous solution is obtained from Bombyx mori silk by degumming, dissolving, and dialysis purification. 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 needle 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 stably release methotrexate for 20-50 hours, and the release rate of methotrexate per unit time is 20 μg / cm 2 ·h or more.

[0046] Principle of the invention: Methotrexate used to treat psoriasis is poorly soluble in water and difficult to mix evenly with silk protein solution, while the organic solvent dimethylformamide is a good solvent for methotrexate. However, the addition of organic solvents will cause a decrease in biocompatibility and may cause cytotoxicity. Therefore, dimethylformamide is transferred to the basal layer of the microneedle without contacting the skin tissue to prevent the possible decrease in biocompatibility. At the same time, dimethylformamide can induce silk protein to form a crystalline structure, construct a water-insoluble silk protein basal layer, and reduce the transdermal release rate of methotrexate.

[0047] After methotrexate reacts with sodium hydroxide to form sodium salt, the interaction with water molecules is enhanced, and it can be dissolved in water, and can be blended with silk protein solution to form microneedles. However, hydrophilic sodium salts will have a burst release phenomenon in the early stage of release, and the formed microneedle tip will dissolve, resulting in the inability of the microneedle to achieve long-term stable drug delivery. Therefore, low-temperature water vapor treatment is used to induce silk protein to form a crystalline structure of silk I, so as to reduce the dissolution loss rate of the microneedle tip and form a certain swelling pore size, thereby controlling the release rate of methotrexate sodium salt.

[0048] In this way, by carrying methotrexate sodium salt in the microneedle layer and carrying methotrexate in the matrix layer, the methotrexate sodium salt in the microneedle layer can be released relatively quickly, and the effective concentration can be reached through the skin quickly. The methotrexate in the matrix layer is slowly released. Finally, the methotrexate is made to reach a higher effective concentration in the psoriasis part in a short time, and the sustained and stable release can be maintained, so that the concentration of the psoriasis part is maintained at a higher effective treatment concentration all the time, and the purpose of improving the curative effect is achieved. By adjusting the different ratios of the drugs in the microneedle layer and the matrix layer and modifying by physical methods (using low-temperature water vapor to process the crystalline structure of silk I), the drug release rate can be improved or regulated, the bioavailability of methotrexate is further improved, and the drug effect is enhanced, thereby reducing the amount of drug added.

[0049] The technical solution of the present invention has the following advantages over the prior art: the silk fibroin microneedle patch suitable for psoriasis prepared by the present invention can effectively load drugs and improve the transdermal penetration of methotrexate. The microneedle release can quickly reach the drug effective concentration in the early stage, and can subsequently ensure the continuous and stable release of methotrexate, and maintain a 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 THE DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0051] Figure 1 Schematic diagram of the cross-sectional structure of the silk fibroin microneedle transdermal patch prepared in Examples 3-6 of the present invention.

[0052] Figure 2 This is a microscope photograph of the silk fibroin microneedle transdermal patch prepared in Example 3 of the present invention.

[0053] Figure 3 This is the X-ray diffraction (XRD) curve of the needle body of the silk fibroin microneedle transdermal patch prepared in Example 3 of the present invention before and after low-temperature water vapor treatment.

[0054] Figure 4 This is the XRD curve of the upper substrate layer of the silk fibroin microneedle transdermal patch prepared in Examples 3-6 and Comparative Example 2 of the present invention.

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

[0056] Figure 6 It is a curve diagram of the in vitro transdermal cumulative drug release rate of the silk fibroin microneedle transdermal patch prepared in Examples 3-6 of the present invention and Comparative Examples 1-2.

[0057] Figure 7 It is a curve chart of the in vitro transdermal drug release rate of the silk fibroin microneedle transdermal patch prepared in Examples 3-6 of the present invention and Comparative Examples 1-2. DETAILED DESCRIPTION

[0058] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0059] The present invention designs a double-layer microneedle patch, and two substances with different solubility, a drug and a sodium salt of the drug, are loaded in the upper and lower layers respectively, so as to achieve the effect of early rapid release and maintenance of drug concentration in the later stage. After dissolving methotrexate by dimethylformamide, it is added to a silk fibroin aqueous solution to obtain a first mixed solution; the sodium salt of methotrexate is dissolved in water and added to the silk fibroin aqueous solution to obtain a second mixed solution. The second mixed solution is cast into a microneedle mold, and after degassing and drying, a microneedle needle tip part is formed, and then the first mixed solution is cast on it, and dried to form a matrix part at the bottom of the microneedle. The two parts are compositely constituted to be suitable for a psoriasis silk fibroin microneedle transdermal patch. The silk fibroin microneedle transdermal patch prepared by the present invention has good mechanical properties, can quickly reach the drug effective concentration in the early stage of release, and can subsequently maintain a sustained and stable release of methotrexate.

[0060] Specifically, the preparation method of the silk fibroin microneedle transdermal patch for psoriasis in this embodiment uses silk as a raw material, obtains a silk fibroin aqueous solution by degumming, dissolving, and dialysis, and comprises the following steps:

[0061] (1) Preparation of methotrexate solution

[0062] Add methotrexate to the dimethylformamide aqueous solution, stir thoroughly, and dissolve evenly to obtain a methotrexate solution having a concentration of 30-80 mg / mL.

[0063] The concentration of dimethylformamide in the dimethylformamide aqueous solution used is 100-600 mg / mL. Directly adding pure dimethylformamide to silk fibroin will cause the silk fibroin to denature, so it is first dissolved in water to form an aqueous solution, then methotrexate is dissolved, and then added to the silk fibroin.

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

[0065] Add methotrexate sodium salt into deionized water, stir thoroughly, and dissolve evenly to obtain a methotrexate sodium salt aqueous solution having a concentration of 10-50 mg / mL.

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

[0067] The solution obtained in step (1) is added to a 30-80 mg / mL silk fibroin aqueous solution, and mixed evenly to obtain a first mixed solution, wherein 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 30-60 mg / mL silk fibroin aqueous solution, and mixed evenly to obtain a second mixed solution, wherein the concentration of methotrexate sodium salt in the mixed solution is 1-10 mg / mL.

[0070] (5) Preparation of microneedle patches

[0071] Casting the second mixed solution into the microneedle mold, vacuum degassing, placing the degassed mold system in a constant temperature and humidity room, and drying it for 4-12 hours under air circulation to form a microneedle tip part (microneedle layer) after drying;

[0072] Then cast the first mixed solution on it, place the mold system in a constant temperature and humidity room, and dry it for 3-10 hours under air circulation to form the microneedle matrix part (matrix layer), and after demoulding, the silk fibroin microneedle transdermal patch is obtained. The conditions of the constant temperature and humidity room are: temperature: 20-40°C, relative humidity: 55-75%.

[0073] (6) Post-processing of microneedle patches

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

[0075] The silk fibroin aqueous solution is obtained by degumming, dissolving and dialysis purification of silkworm silk. The concentration of the silk fibroin aqueous solution is 30-80 mg / mL. 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 needle tips of adjacent needle bodies of 0.2-2 mm. The microneedle mold is made of silicone rubber.

[0076] The silk fibroin microneedle patch suitable for psoriasis prepared by the above preparation method can form a large methotrexate concentration difference between the microneedles and the skin, providing a stable driving force for the transdermal release of methotrexate, so that a stable drug release rate is achieved after a certain period of time, thereby achieving the purpose of long-term stable drug administration; and the methotrexate can be released stably and continuously for 20-50 hours.

[0077] Example 1 Structure of silk fibroin microneedles with methotrexate and methotrexate sodium salt

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

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

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

[0081] Example 2 Preparation of Silk Fibroin Solution

[0082] Weigh 3g NaHCO3 and 1g Na2CO3 and dissolve them in 4000mL deionized water, heat to boiling, put in 80g silk, keep boiling at 98℃ for 30min, take out and wash with deionized water.

[0083] The above steps were repeated three times and then placed in a 60° C. oven to dry to obtain silk fibroin fibers.

[0084] Prepare 9.3 mol / L LiBr solution, take 100 mL of LiBr solution and heat it to 65°C in a water bath, add 15 g of degummed silk fibroin several times and stir to dissolve, then continue heating and stirring for 40 min.

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

[0086] Example 3

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

[0088] (1) Preparation of methotrexate solution

[0089] Weigh methotrexate powder into a centrifuge tube, add 0.8 mL of dimethylformamide aqueous solution, and shake to mix until the concentration of methotrexate in the solution is 50 mg / mL. The concentration of the dimethylformamide aqueous solution used is 600 mg / ml. The methotrexate solution is stored at 4°C in the dark.

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

[0091] Weigh methotrexate sodium salt powder into a centrifuge tube, add 2 mL of deionized water, and shake to mix until the concentration of methotrexate sodium salt in the solution is 10 mg / mL. The methotrexate sodium salt aqueous solution is stored at 4°C away from light.

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

[0093] The methotrexate solution obtained in step (1) was added to a 80 mg / mL silk fibroin aqueous solution. After thorough mixing, the silk fibroin concentration was 50 mg / mL and the methotrexate concentration was 20 mg / mL. A first mixed solution was obtained and stored at 4°C away from light.

[0094] (4) Preparation of the Second Mixed Solution

[0095] The methotrexate sodium salt aqueous solution obtained in step (2) was slowly added to the 60 mg / mL silk fibroin aqueous solution. After thorough mixing, the silk fibroin concentration was 50 mg / mL and the methotrexate sodium salt concentration was 2 mg / mL. A second mixed solution was obtained and stored at 4°C away from light.

[0096] (5) Preparation of microneedle patches

[0097] Take 1mL of the second mixed solution and cast it into the silicone rubber microneedle mold. Vacuum degassing is performed 3 times to remove bubbles in the solution and the microneedle template, so that the solution can fully enter the mold cavity. The degassed mold system is placed in a constant temperature and humidity room (20°C, 55%), and dried for 8 hours under air circulation to form the microneedle tip part (microneedle layer); continue to add 2mL of the first mixed solution, cast it on the needle tip, place the mold system in a constant temperature and humidity room, and dry it for 10 hours under air circulation to form the matrix part (matrix layer) at the bottom of the microneedle.

[0098] (6) Post-processing of microneedle patches

[0099] The silk fibroin microneedle transdermal patch was placed in a low temperature and high humidity environment (20° C., 90%) for 48 hours, 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 embodiment comprises the following steps:

[0102] (1) Preparation of methotrexate solution

[0103] Weigh methotrexate powder into a centrifuge tube, add 0.7 mL of dimethylformamide aqueous solution, and shake to mix until the concentration of methotrexate in the solution is 40 mg / mL. The concentration of the dimethylformamide aqueous solution used is 500 mg / ml. The methotrexate solution is stored at 4°C in the dark.

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

[0105] Weigh methotrexate sodium salt powder into a centrifuge tube, add 2 mL of deionized water, and shake to mix until the concentration of methotrexate sodium salt in the solution is 20 mg / mL. The methotrexate sodium salt aqueous solution is stored at 4°C away from light.

[0106] (3) Preparation of the first mixed solution

[0107] The methotrexate solution obtained in step (1) was added to a 70 mg / mL silk fibroin aqueous solution. 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 away from light.

[0108] (4) Preparation of the Second Mixed Solution

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

[0110] (5) Preparation of microneedle patches

[0111] Take 1mL of the second mixed solution and cast it into the silicone rubber microneedle mold. Vacuum degassing is performed 3 times to remove bubbles in the solution and the microneedle template, so that the solution can fully enter the needle body. The degassed mold system is placed in a constant temperature and humidity room (25°C, 60%), and dried for 5 hours under air circulation to form the microneedle tip part (microneedle layer); continue to add 2mL of the first mixed solution, cast it on the needle tip, place the mold system in a constant temperature and humidity room (25°C, 60%), and dry it for 9 hours under air circulation to form the matrix part (matrix layer) at the bottom of the microneedle.

[0112] (6) Post-processing of microneedle patches

[0113] The silk fibroin microneedle transdermal patch was placed in a low temperature and high humidity environment (22° C., 93%) for 40 hours, 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, and shake to mix until the concentration of methotrexate in the solution is 35 mg / mL. The concentration of the dimethylformamide aqueous solution used is 450 mg / ml. Store the methotrexate solution at 4°C 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, and shake to mix until the concentration of methotrexate sodium salt in the solution is 30 mg / mL. The methotrexate sodium salt aqueous solution is stored at 4°C in the dark.

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

[0121] The methotrexate solution obtained in step (1) was added to a 65 mg / mL silk fibroin aqueous solution. After thorough mixing, the silk fibroin concentration was 50 mg / mL and the methotrexate concentration was 10 mg / mL. The first mixed solution was stored at 4°C away from light.

[0122] (4) Preparation of the Second Mixed Solution

[0123] The methotrexate sodium salt aqueous solution obtained in step (2) was slowly added to the 55 mg / mL silk fibroin aqueous solution. After thorough mixing, the silk fibroin concentration was 45 mg / mL and the methotrexate sodium salt concentration was 6 mg / mL. A second mixed solution was obtained and stored at 4°C away from light.

[0124] (5) Preparation of microneedle patches

[0125] Take 1mL of the second mixed solution and cast it into the silicone rubber microneedle mold. Vacuum degassing 3 times to remove the bubbles in the solution and the microneedle template, so that the solution can fully enter the needle body, and place the degassed mold system in a constant temperature and humidity room (35°C, 65%), and dry it for 6 hours under air circulation to form the microneedle tip part (microneedle layer); continue to add 2mL of the first mixed solution, cast it on the needle tip, place the mold system in a constant temperature and humidity room (35°C, 65%), and dry it for 8 hours under air circulation to form the matrix part (matrix layer) at the bottom of the microneedle.

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

[0127] The silk fibroin microneedle transdermal patch was placed in a low temperature and high humidity environment (25° C., 95%) for 35 hours, taken out and dried 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 embodiment comprises the following steps:

[0130] (1) Preparation of methotrexate solution

[0131] Weigh methotrexate powder into a centrifuge tube, add 0.6 mL of dimethylformamide aqueous solution, and shake to mix until the concentration of methotrexate in the solution is 30 mg / mL. The concentration of the dimethylformamide aqueous solution used is 400 mg / ml. Store the methotrexate solution at 4°C away from light.

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

[0133] Weigh methotrexate sodium salt powder into a centrifuge tube, add 3 mL of deionized water, and shake to mix until the concentration of methotrexate sodium salt in the solution is 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] The methotrexate solution obtained in step (1) was added to a 60 mg / mL silk fibroin aqueous solution. After thorough mixing, the silk fibroin concentration was 50 mg / mL and the methotrexate concentration was 5 mg / mL. The first mixed solution was stored at 4°C away from light.

[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 silk fibroin aqueous solution. After thorough mixing, the silk fibroin concentration was 50 mg / mL and the methotrexate sodium salt concentration was 8 mg / mL. A second mixed solution was obtained and stored at 4°C away from light.

[0138] (5) Preparation of microneedle patches

[0139] Take 1.5mL of the second mixed solution and cast it into the silicone rubber microneedle mold. Vacuum degassing 3 times to remove the bubbles in the solution and the microneedle template, so that the solution can fully enter the needle body, and place the degassed mold system in a constant temperature and humidity room (40°C, 70%), and dry it for 7 hours under air circulation to form the microneedle tip part (microneedle layer); continue to add 2mL of the first mixed solution, cast it on the needle tip, place the mold system in a constant temperature and humidity room (40°C, 70%), and dry it for 7 hours under air circulation to form the matrix part (matrix layer) at the bottom of the microneedle.

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

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

[0142] Comparative Example 1

[0143] The comparative example is a silk fibroin microneedle loaded with only methotrexate sodium salt, and the preparation method thereof is as follows:

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

[0145] Weigh methotrexate sodium salt powder into a centrifuge tube, add 2 mL of deionized water, and shake to mix until the concentration of methotrexate sodium salt in the solution is 35 mg / mL. The methotrexate sodium salt aqueous solution is stored at 4°C away from light.

[0146] (2) Preparation of mixed solution

[0147] The above-mentioned methotrexate sodium salt aqueous solution was slowly added into the 60 mg / mL silk fibroin aqueous solution. After mixing, the concentration of silk fibroin was 50 mg / mL, and the concentration of methotrexate sodium salt was 10 mg / mL. The mixed solution was stored at 4°C away from light.

[0148] (3) Preparation of microneedle patches

[0149] Pour 3 mL of the mixed solution into the silicone rubber microneedle mold and vacuumize for 3 times to remove bubbles in the solution and the microneedle template, so that the solution can fully enter the needle cavity. The degassed mold system is placed in a constant temperature and humidity room (25°C, 65%) with air circulation for 8 hours.

[0150] (4) Post-processing of microneedle patches

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

[0152] Comparative Example 2

[0153] The comparative example is a silk fibroin microneedle loaded with only methotrexate, and the preparation method thereof 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, and shake to mix until the concentration of methotrexate in the solution is 35 mg / mL. The concentration of the dimethylformamide aqueous solution used is 500 mg / ml. The methotrexate solution is stored at 4°C away from light.

[0156] (2) Preparation of mixed solution

[0157] The above methotrexate solution was slowly added to the 70 mg / mL silk fibroin aqueous solution. After mixing, the silk fibroin concentration was 50 mg / mL and the methotrexate concentration was 15 mg / mL. The mixed solution was stored at 4°C away from light.

[0158] (3) Preparation of microneedle patches

[0159] Pour 3 mL of the mixed solution into the silicone rubber microneedle mold and vacuumize for 3 times to remove bubbles in the solution and the microneedle template, so that the solution can fully enter the needle cavity. The degassed mold system is placed in a constant temperature and humidity room (25°C, 65%) with air circulation for 8 hours.

[0160] (4) Post-processing of microneedle patches

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

[0162] Tests and Results

[0163] (1) Figure 2 As shown, it is a microscope photo of the silk fibroin microneedle transdermal patch suitable for psoriasis prepared in Example 3, which shows that the microneedles prepared by the example method can be well filled in the silicone rubber microneedle mold and keep the morphology intact.

[0164] (2) Figure 3As shown, XRD detection was performed on the microneedles before and after the low temperature and high humidity environment treatment in step (6) of Example 3. From top to bottom, the XRD curves of the treated and untreated ones are shown. It can be seen that the untreated XRD curve has basically no characteristic peaks, only one large steamed bun peak, indicating that the silk fibroin is mainly a random curled non-crystalline structure. For the treated microneedles, the XRD curve has a new diffraction peak near 12.2° and a stronger new diffraction peak near 19.7°, indicating that the silk fibroin has formed a Silk I type crystalline structure. After treatment, the aggregated structure of the silk fibroin has changed, forming a silk I crystalline structure, and an insoluble silk fibroin microneedle is obtained.

[0165] (3) Figure 4 As shown, from top to bottom are the XRD curves of the upper substrate layer of the microneedle transdermal patch prepared in Examples 3, 4, 5, 6 and Comparative Example 2.

[0166] It can be seen that the microneedle transdermal patches prepared in Examples 3-6 and Comparative Example 2 have a weak diffraction peak near 12.2° and a strong diffraction peak near 19.7°, indicating that Silk I type crystal structure appears in the silk fibroin structure. 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, if Figure 5 As shown, the amount of dimethylformamide dissolved in Comparative Example 2 is significantly higher than that in Examples 3-6. Considering the poor biocompatibility of dimethylformamide, the dissolution of dimethylformamide in the needle tip will cause certain damage to the skin, so it is not a good choice. Therefore, the present invention places the matrix layer containing dimethylformamide above the microneedle layer, so that it does not directly contact the skin, so as to reduce possible damage to the skin.

[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 as follows: Figure 6 As shown, it can be seen from Examples 3-6 that the silk fibroin microneedles composed of two parts, methotrexate loaded on the matrix and methotrexate sodium salt loaded on the needle body, can obviously maintain the stability of the methotrexate drug release rate within a certain period of time. With the different ratios of the needle body and the matrix drug in the microneedle, the initial rate of drug release can be adjusted, and the subsequent release can still be stable, reaching a release time of 48 hours. However, Comparative Example 1 is released quickly in the early stage, and then rapidly decreases, making it difficult to achieve a sustained release effect. The cumulative drug release rate of Comparative Example 2 is less than 50%, and the effective utilization of the drug is low.

[0169] (5) Figure 7As shown, comparative example 1 is a silk fibroin microneedle loaded with only methotrexate sodium salt, which can have a faster release rate in the first 5 hours, but the high release rate is maintained for a relatively short time, and the stability of the drug release curve is poorer than that of Examples 3-6, and can only maintain drug release for 10 hours. At 12 hours, the drug release rate per unit time is reduced to 20μg / cm 2 The release curve of comparative example 2 is better than that of comparative example 1, and the drug release time is extended to more than 24 hours. However, due to the possible toxicity of dimethylformamide, the present invention further studies the double-layer microneedles to achieve a longer stable drug release time and less damage to the skin. Figure 7 It can be seen that the release rate of the double-layer microneedle embodiments 3-6 prepared by the present invention is relatively stable, providing 100 μg / cm in the first 1-3 hours. 2 ·h high-speed drug release to obtain therapeutic concentration, and continued release in the later period, with the drug release rate per unit time still reaching 20μg / cm at 48 hours. 2 ·h, achieving a sustained-release effect for long-term stable drug delivery.

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

[0171] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

[0172] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

Claims

1. A silk fibroin microneedle transdermal patch suitable for psoriasis, characterized in that: It includes a substrate layer and a microneedle layer, wherein the microneedle layer includes a base and a needle body extending outward from the base, and the substrate layer and the base are bonded to each other; the substrate layer contains methotrexate and silk fibroin, and the mass ratio of 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 methotrexate sodium salt to the silk fibroin in the microneedle layer is 0.2-2:

10.

2. The silk fibroin microneedle transdermal patch according to claim 1, characterized in that: The raw materials of the matrix layer include methotrexate, dimethylformamide and silk fibroin, and the mass ratio of the dimethylformamide to the silk fibroin in the matrix layer is 1-10:

10.

3. The silk fibroin microneedle transdermal patch according to claim 1, characterized in that: The silk fibroin in the microneedle layer is a Silk I crystalline structure; and / or, the silk fibroin in the matrix layer is a Silk I crystalline structure.

4. 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 spacing between the needle tips of adjacent needle bodies is 0.2-2 mm.

5. A method for preparing the silk fibroin microneedle transdermal patch according to any one of claims 1 to 4, characterized in that: The steps include: The methotrexate solution and the silk fibroin aqueous solution are mixed evenly 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 and the silk fibroin aqueous solution are uniformly mixed to obtain a second mixed solution, wherein the concentration of the methotrexate sodium salt in the second mixed solution is 1-10 mg / mL; Casting the second mixed solution into a microneedle mold, vacuum degassing, and placing the degassed mold system under constant temperature and humidity conditions to dry to form a microneedle layer; Casting the first mixed solution on the microneedle layer, placing the mold system under constant temperature and humidity conditions to dry to form a matrix layer, and obtaining a silk fibroin microneedle transdermal patch after demoulding; The silk fibroin microneedle transdermal patch is placed in an environment with a temperature of 20-50° C. and a relative humidity of 80-99% for 20-50 hours, taken out and dried to obtain a silk fibroin microneedle transdermal patch suitable for psoriasis.

6. The preparation method according to claim 5, characterized in that: The methotrexate solution is prepared by the following method: adding methotrexate into a dimethylformamide aqueous solution and dissolving the methotrexate to obtain the methotrexate solution.

7. The preparation method according to claim 6, characterized in that: When preparing the methotrexate solution, the concentration of the dimethylformamide aqueous solution used is 100-600 mg / mL.

8. The preparation method according to claim 5, characterized in that: The methotrexate sodium salt aqueous solution is prepared by the following method: adding methotrexate sodium salt into deionized water and dissolving it to obtain the methotrexate sodium salt aqueous solution.

9. The preparation method according to claim 5, characterized in that: The concentration of the methotrexate solution is 30-80 mg / mL; the concentration of the methotrexate sodium salt aqueous solution is 10-50 mg / mL.

10. The preparation method according to claim 5, characterized in that: The methotrexate sodium salt aqueous solution and / or the methotrexate solution is stored at 2-8° C. in the dark.

Images