Diaphragm type silk fibroin microneedle patch and preparation method thereof

Abstract

The diaphragm type silk fibroin microneedle patch sequentially comprises a medicine storage layer, a diaphragm layer and a blank microneedle layer, the blank microneedle layer is composed of silk fibroin and comprises a base body and a needle body extending outwards from the base body, and the side, away from the needle body, of the diaphragm layer is covered with the medicine storage layer; the medicine storage layer comprises silk fibroin and tranexamic acid, and the diaphragm layer is located between the medicine storage layer and the blank microneedle layer. According to the tranexamic acid-loaded diaphragm type silk fibroin microneedle patch containing the medicine storage layer, the release duration (14 h in vitro and 24 h in vivo) can be increased, a large tranexamic acid concentration difference can be formed between the microneedle and the skin, stable pushing force is provided for transdermal release of tranexamic acid, the stable medicine release rate is achieved after a certain time, and the transdermal effect of the tranexamic acid is improved. Therefore, the purpose of long-time stable administration is achieved.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutics, in particular to the field of transdermal drug delivery and silk fibroin microneedle technology, and specifically relates to a diaphragm-type silk fibroin microneedle patch containing a diaphragm layer and a preparation method of the diaphragm-type silk fibroin microneedle patch. Background Art

[0002] The incidence of melasma is higher in women with darker skin, reaching 9-50%. Melasma generally appears as brown to gray plaques, and its persistent and recurrent attacks can cause inflammatory stimulation and excessive pigmentation, further leading to an increase in the number of plaques and a deepening of color, which has a negative impact on the patient's appearance and quality of life. Clinically, melasma is caused by increased tyrosinase activity and a large amount of endothelin-1 (EF-1) expression, which increases the dendritic structure of melanocytes and promotes the transfer and deposition of new melanin.

[0003] A large number of studies have demonstrated the safety and effectiveness of tranexamic acid as a drug for the treatment of melasma. Tranexamic acid is mainly delivered by oral administration, injection, and transdermal administration. The more common transdermal preparations for melasma on the market are in the form of emulsions and creams, which cannot bypass the skin barrier and mainly rely on the diffusion behavior of the drug itself and the penetration ability of the penetration enhancer. Considering that tranexamic acid has the characteristics of high water solubility and small molecular weight, it is difficult to effectively break through the skin barrier. In order to solve this problem, microneedle transdermal drug delivery technology may be a better choice.

[0004] As an effective transdermal delivery carrier, microneedles have multiple physical parameters that can be adjusted to meet the long-term delivery requirements of tranexamic acid. Solid microneedles and dissolving microneedles are the two most widely used types of microneedles. Konisky et al. [Journal of Cosmetic Dermatology, 2023] used solid microneedles to deliver tranexamic acid, and the drug penetration time was only 30 minutes. Machekposhti et al. [Journal of Controlled Release, 2017] and Xing et al. [International Journal of Pharmaceutics, 2021] developed soluble microneedles, which were used in vitro and in rats, respectively, with a release amount of about 80%. However, the release duration and peak blood drug concentration are still not enough to achieve a better therapeutic effect. After the soluble microneedles penetrate the skin, there is still a certain risk of sensitization.

[0005] In the prior art, the Chinese invention patent with application number CN202211106981.9 discloses a method for preparing hyaluronic acid microneedles for treating chloasma. The penetration depth, penetration rate and biosafety of the microneedles were evaluated by loading rhodamine. However, the in vitro release of tranexamic acid was not evaluated. At the same time, sodium hyaluronate microneedles are soluble and do not have the characteristics of sustainable drug delivery compared to other insoluble microneedles.

[0006] Another example is the Chinese invention patent with application number CN202210357550.3, which discloses a method for preparing a sustained-release microneedle for removing chloasma. The Schiff base-protected tranexamic acid is released by ultraviolet irradiation. However, the microneedle has not been subjected to a transdermal experiment, so the actual transdermal sustained-release effect cannot be evaluated.

[0007] Another example is a Chinese patent with application number CN202311430684.4, which discloses a method for preparing a skeleton-type microneedle for removing chloasma. The microneedle contains only silk fibroin and tranexamic acid, wherein the silk fibroin has a Silk I crystal structure and belongs to an insoluble microneedle. The release of tranexamic acid is regulated by the network density of silk fibroin and the binding force between silk fibroin, which can achieve a certain sustained release, but the effect still needs to be improved.

[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 an improved tranexamic acid-loaded diaphragm silk fibroin microneedle patch having a diaphragm layer, which can achieve long-term slow release of tranexamic acid.

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

[0011] A diaphragm-type silk fibroin microneedle patch comprises a drug storage layer, a diaphragm layer and a blank microneedle layer in sequence, wherein the blank microneedle layer is composed of silk fibroin, comprising a matrix and a needle body extending outward from the matrix, and the drug storage layer covers a side of the diaphragm layer away from the needle body; the drug storage layer comprises silk fibroin and tranexamic acid, and the diaphragm layer is located between the drug storage layer and the blank microneedle layer.

[0012] According to some preferred implementation aspects of the present invention, the material of the diaphragm layer is cellulose acetate. Cellulose acetate as a diaphragm layer can hinder the diffusion process of water-soluble molecules and reduce the peak value of the release rate.

[0013] According to some preferred embodiments of the present invention, the thickness of the diaphragm layer is 10 μm-200 μm, and the thickness of the drug storage layer is 100 μm-2 mm.

[0014] According to some preferred implementation aspects of the present invention, the crystal structure of the silk fibroin in the drug storage layer is a Silk I crystal structure; and the crystal structure of the silk fibroin in the blank microneedle layer is a Silk I crystal structure.

[0015] According to some preferred embodiments of the present invention, the drug loading of tranexamic acid in the drug storage layer is 0.1-5 mg / cm 2 , that is, per unit area (1cm 2 ) The content of tranexamic acid in the drug storage layer is 0.1-5 mg, and can reach a maximum of 5 mg / cm2 of the drug storage layer.

[0016] According to some preferred embodiments of the present invention, the needle bodies of the blank microneedle layer are solid cones, the length of the needle bodies is 200-900 μm, the bottom diameter is 100-500 μm, and the spacing between the needle tips of adjacent needle bodies is 0.2-2 mm.

[0017] The present invention also provides a method for preparing the diaphragm silk fibroin microneedle patch as described above, comprising the following steps:

[0018] respectively preparing a silk fibroin solution, a membrane layer solution and a drug storage layer solution;

[0019] pouring the silk fibroin solution into the microneedle mold, degassing, and placing the degassing mold system under constant temperature and humidity conditions to dry, thereby obtaining the blank microneedle layer;

[0020] Casting the membrane layer solution on the substrate of the blank microneedle layer and drying it to obtain blank microneedles with the membrane layer;

[0021] pouring the drug storage layer solution onto the diaphragm layer and drying under constant temperature and humidity conditions to obtain a drug-loaded silk fibroin microneedle patch;

[0022] The drug-loaded silk fibroin microneedle patch is placed in an environment with a temperature of 20-50° C. and a relative humidity of 85-99% for 10-40 hours, taken out and dried to obtain the diaphragm-type silk fibroin microneedle patch.

[0023] According to some preferred embodiments of the present invention, the diaphragm layer solution is prepared according to the following method: cellulose acetate is dissolved in a polar volatile organic solvent to obtain a diaphragm layer solution with a concentration of 10-100 mg / mL.

[0024] According to some preferred embodiments of the present invention, the polar volatile organic solvent is acetone and / or chloroform.

[0025] According to some preferred embodiments of the present invention, the drug reservoir solution is prepared according to the following method: a tranexamic acid solution and a silk fibroin solution are evenly mixed to form a drug reservoir solution; the concentration of silk fibroin in the drug reservoir solution is 20-50 mg / mL, and the concentration of tranexamic acid is 10-30 mg / mL.

[0026] According to some preferred embodiments of the present invention, the tranexamic acid solution is prepared according to the following method: weigh tranexamic acid powder, add deionized water, and obtain a tranexamic acid solution after the tranexamic acid is completely dissolved, wherein the concentration of tranexamic acid is 20-100 mg / mL, and the pH of the solution is adjusted to 6.5-7.5 using 0.1-1.0 mol / L sodium hydroxide.

[0027] According to some preferred implementation aspects of the present invention, the steps further include: taking out the dried diaphragm silk fibroin microneedles, cutting them, attaching a backing layer, and packaging them to obtain a tranexamic acid-loaded diaphragm silk fibroin microneedle patch product containing a diaphragm layer.

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

[0029] S1. Preparation of tranexamic acid solution

[0030] Weigh tranexamic acid powder, add deionized water to make the concentration of tranexamic acid in the solution 20-100 mg / mL, and use 0.1-1.0 mol / L sodium hydroxide to adjust the pH of the solution to 6.5-7.5.

[0031] S2. Preparation of diaphragm layer solution

[0032] Cellulose acetate is dissolved in a polar volatile organic solvent to obtain a diaphragm layer solution with a concentration of 10-100 mg / mL.

[0033] S3. Preparation of drug reservoir solution

[0034] The tranexamic acid solution is added to the silk fibroin solution, and the mixture is evenly mixed to form a drug reservoir solution. The concentration of the silk fibroin in the drug reservoir solution is 20-50 mg / mL, and the concentration of the tranexamic acid is 10-30 mg / mL.

[0035] S4. Preparation of blank microneedle layer

[0036] The silk fibroin solution is poured into the microneedle mold, and the bubbles in the solution and the microneedle template are removed by vacuuming to allow the solution to fully enter the needle cavity. The degassed mold system is placed under constant temperature and humidity conditions and dried for 4-24 hours in air circulation to obtain the blank microneedle layer.

[0037] S5. Preparation of diaphragm layer

[0038] The membrane layer solution prepared in S1 was poured onto the substrate of the blank microneedle layer, and dried for 1-10 min at room temperature under air circulation to obtain blank microneedles with a membrane layer.

[0039] S6. Preparation of drug reservoir

[0040] The drug storage layer solution prepared by S2 is poured onto the diaphragm layer, and dried for 2-20 hours in air circulation under constant temperature and humidity conditions to obtain a drug-loaded silk fibroin microneedle patch.

[0041] S7, post-processing of microneedle patches

[0042] The drug-loaded silk fibroin microneedle patch obtained in S5 was placed in a low-temperature and high-humidity environment for 10-40 hours, taken out and dried to obtain a diaphragm silk fibroin microneedle patch.

[0043] S8, backing layer

[0044] The diaphragm-type silk fibroin microneedle patch is affixed with a backing layer and packaged to obtain the silk fibroin tranexamic acid microneedle patch product containing a diaphragm layer.

[0045] According to some preferred embodiments of the present invention, the concentration of the silk fibroin solution in steps S3 and S4 is 30-80 mg / mL.

[0046] According to some preferred implementation aspects of the present invention, the polar volatile organic solvent in step S2 is acetone or chloroform.

[0047] According to some preferred implementation aspects of the present invention, the constant temperature and humidity conditions in steps S4 and S6 are: temperature of 20-40° C. and relative humidity of 55-75%.

[0048] According to some preferred implementation aspects of the present invention, the low temperature and high humidity environment in step S7 is: a temperature of 20-50° C. and a relative humidity of 85-99%.

[0049] The principle of the present invention is as follows: Silk fibroin, as a natural, partially crystallizable polymer, has good mechanical properties and biocompatibility. The treatment with low-temperature water vapor allows the silk fibroin to form a crystalline structure of Silk I, thereby achieving the insolubilization of the tranexamic acid-loaded silk fibroin microneedles. The insoluble microneedle drug release containing a diaphragm layer can be divided into two steps: the first step is that the blank microneedle body is used as a moisture conduction medium. After the microneedle pierces the skin, water molecules in the skin tissue fluid enter the microneedle and enter the drug storage layer along the pores of the microneedle, so that the tranexamic acid in the drug storage layer is dissolved, and then slowly released to the blank microneedle body through the diaphragm layer; the second step is that the tranexamic acid entering the blank microneedle is combined with the silk fibroin, and then released into the skin through the blank microneedle. First, the tranexamic acid in the drug storage layer combines with the silk protein of the Silk I crystal structure to delay the release; secondly, because the diaphragm layer can play a certain role in hindering the water-soluble small molecule drug tranexamic acid, the release rate of tranexamic acid is slowed down; thirdly, when tranexamic acid passes through the diaphragm layer to reach the blank microneedle, tranexamic acid combines with the hydrophilic silk protein of the Silk I crystal structure again, which delays the release of tranexamic acid again. The combined effect of the barrier effect of the diaphragm layer and the hydrophilic properties of silk protein can achieve a stable and long-term drug release.

[0050] The drug reservoir in the present invention is located above the diaphragm layer and the blank microneedle, and utilizes the barrier effect of the diaphragm layer and the hydrophilicity of the silk fibroin with the Silk I crystal structure to slow down the release rate of tranexamic acid, thereby releasing slowly, constantly and long-lastingly. The tranexamic acid-loaded silk fibroin microneedle of the present invention can delay the peak time of the drug release rate by 2 hours, and the overall release time can reach more than 14 hours.

[0051] Due to the adoption of the above technical scheme, the present invention has the following advantages compared with the prior art: the diaphragm-type silk fibroin microneedle patch of the present invention is treated with low-temperature water vapor so that the blank microneedles and the silk fibroin in the drug storage layer have a Silk I crystal structure, thereby achieving the insolubilization of the microneedles. The insoluble microneedles greatly reduce the sensitization after piercing the skin, provide a longer microneedle pinhole maintenance time, and ensure the cumulative release rate of the microneedle drug. The addition of the diaphragm layer slows down the release rate of the drug, thereby delaying the peak release rate of the drug. The drug can achieve a longer release time and maintain a sufficiently high blood drug concentration, which has a more positive significance for the treatment of chloasma. The microneedles are easy to prepare, and there is no residue when they are pulled out after use, and they have good biological safety.

[0052] The tranexamic acid-loaded diaphragm silk fibroin microneedle patch containing a drug storage layer of the present invention can achieve an increase in release time (14 hours in vitro, 24 hours in vivo), and can form a large tranexamic acid concentration difference between the microneedles and the skin, providing a stable driving force for the transdermal release of tranexamic acid, 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. 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.

[0054] Figure 1 This is a schematic diagram of the structure of a diaphragm-type silk fibroin microneedle patch in a preferred embodiment of the present invention;

[0055] Figure 2 is the XRD curve diagram of silk fibroin in Example 7 of the present invention;

[0056] Figure 3 A cumulative drug release rate curve of tranexamic acid-loaded silk fibroin microneedles provided in Examples 3-9 of the present invention and Comparative Examples 1-2;

[0057] Figure 4 The drug release rate curve of the tranexamic acid-loaded silk fibroin microneedles provided in Examples 3-9 and Comparative Examples 1-2 of the present invention;

[0058] Figure 5 This is a comparison chart of the in vivo drug release rate curve of the diaphragm silk fibroin microneedle patch provided in Example 7 of the present invention and the injection group. DETAILED DESCRIPTION

[0059] 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.

[0060] like Figure 1-5 As shown, the preparation method of the diaphragm type silk fibroin microneedle patch of this embodiment comprises the following steps:

[0061] S1. Preparation of tranexamic acid solution

[0062] Weigh tranexamic acid powder, add deionized water to make the concentration of tranexamic acid in the solution 20-100 mg / mL, and use 0.1-1.0 mol / L sodium hydroxide to adjust the pH of the solution to 6.5-7.5.

[0063] S2. Preparation of diaphragm layer solution

[0064] Cellulose acetate is dissolved in a polar volatile organic solvent to obtain a diaphragm layer solution with a concentration of 10-100 mg / mL.

[0065] The polar volatile organic solvent is acetone or chloroform.

[0066] S3. Preparation of drug reservoir solution

[0067] Add the tranexamic acid solution to the silk fibroin solution, mix well, and form a drug reservoir solution. The concentration of the silk fibroin in the drug reservoir solution is 20-50 mg / mL, and the concentration of the tranexamic acid is 10-30 mg / mL. The concentration of the silk fibroin solution is 30-80 mg / mL.

[0068] S4. Preparation of blank microneedle layer

[0069] Pour the silk fibroin solution into the microneedle mold, and remove the bubbles in the solution and the microneedle template by vacuuming, so that the solution can fully enter the needle body cavity. Place the degassed mold system under constant temperature and humidity conditions, and dry it for 4-24 hours in air circulation to obtain a blank microneedle layer. The concentration of the silk fibroin solution is 30-80 mg / mL. The constant temperature and humidity conditions are: temperature 20-40°C, relative humidity 55-75%.

[0070] S5. Preparation of diaphragm layer

[0071] The membrane layer solution prepared in S1 was poured onto the substrate of the blank microneedle layer, and dried for 1-10 min at room temperature under air circulation to obtain blank microneedles with a membrane layer.

[0072] S6. Preparation of drug reservoir

[0073] The drug storage layer solution prepared in S2 is poured onto the diaphragm layer, and dried for 2-20 hours in air circulation under constant temperature and humidity conditions to obtain a drug-loaded silk fibroin microneedle patch. The constant temperature and humidity conditions are: temperature of 20-40°C and relative humidity of 55-75%.

[0074] S7, post-processing of microneedle patches

[0075] The drug-loaded silk fibroin microneedle patch obtained in S5 is placed in a low-temperature and high-humidity environment for 10-40 hours, taken out and dried, and a diaphragm-type silk fibroin microneedle patch is obtained. The low-temperature and high-humidity environment is: a temperature of 20-50° C. and a relative humidity of 85-99%, that is, low-temperature water vapor treatment.

[0076] S8, backing layer

[0077] The dried diaphragm silk protein microneedle is taken out, cut, attached with a backing layer, and packaged to obtain a diaphragm silk protein microneedle patch product containing a diaphragm layer.

[0078] The silk fibroin solution is obtained from Bombyx mori silk by degumming, dissolving with lithium bromide, and dialysis purification, with a concentration of 30-80 mg / mL.

[0079] The diaphragm silk fibroin microneedle patch prepared by the above steps comprises a drug storage layer, a diaphragm layer and a blank microneedle layer in sequence, wherein the blank microneedle layer is composed of silk fibroin, including a matrix and a needle body extending outward from the matrix, and the drug storage layer covers the side of the diaphragm layer away from the needle body; the drug storage layer comprises silk fibroin and tranexamic acid, and the diaphragm layer is located between the drug storage layer and the blank microneedle layer.

[0080] The material of the diaphragm layer is cellulose acetate. The thickness of the diaphragm layer is 10μm-200μm, and the thickness of the drug storage layer is 100μm-2mm. Cellulose acetate has a certain hydrophobicity. As a diaphragm layer, it can reduce the diffusion rate of water-soluble molecules and reduce the peak release rate.

[0081] The structure of the silk fibroin in the drug storage layer is the Silk I crystal structure; the structure of the silk fibroin in the blank microneedle layer is the Silk I crystal structure.

[0082] The drug loading of tranexamic acid in the drug reservoir is 0.1-5 mg / cm 2 , that is, the content of tranexamic acid in the drug storage layer per unit area (1cm2) is 0.1-5mg, and the maximum can reach 5mg / square centimeter of drug storage layer.

[0083] The needle body of the blank microneedle layer is a solid cone, the length of the needle body is 200-900 μm, the bottom diameter is 100-500 μm, and the spacing between the needle tips of adjacent needle bodies is 0.2-2 mm.

[0084] The above-mentioned diaphragm-type silk fibroin microneedle patch is composed of a drug storage layer, a diaphragm layer and a blank microneedle body. The silk fibroin-loaded tranexamic acid microneedle with a Silk I crystal structure is formed by low-temperature steam treatment. The diaphragm layer with hydrophobic properties is designed to slow down the release of tranexamic acid due to the strong water solubility and low molecular weight of tranexamic acid. The diaphragm layer directly hinders the release rate of tranexamic acid, thereby obtaining a tranexamic acid microneedle with a longer sustained release time, improving bioavailability and increasing drug efficacy.

[0085] Example 1: Tranexamic acid-loaded silk fibroin microneedle structure with a diaphragm layer

[0086] See also Figure 1 As shown, the membrane-type silk fibroin microneedle patch containing a membrane layer of this embodiment includes three parts from top to bottom, namely, a drug storage layer 1, a membrane layer 2 and a blank microneedle 3. Among them, the blank microneedle 3 is composed only of silk fibroin. The membrane layer 2 is composed of cellulose acetate. The drug storage layer 1 is composed of silk fibroin and the drug tranexamic acid. Preferably, a backing layer as described above is also provided on the drug storage layer 1.

[0087] The blank microneedle comprises a matrix and a needle body extending outward from the matrix, and the crystal structure of the silk fibroin is the crystal structure of Silk I. The needle body is a solid cone, the needle body length is 200-900 μm, the bottom diameter is 100-500 μm, and the interval between the needle bodies is 0.2-2 mm.

[0088] In the drug storage layer, tranexamic acid is mixed evenly with the silk fibroin solution in the form of an aqueous solution and dried to form a film. The loading amount of tranexamic acid is 0.1-5 mg / cm 2 .

[0089] See also Figure 2 As shown, the X-ray diffraction results of the microneedles of this embodiment (prepared by the preparation method in Example 7) show that the silk fibroin has characteristic peaks at 12.2° and 19.7°, and it can be seen that the crystal structure of the silk fibroin is the Silk I crystal structure.

[0090] In some embodiments, the cellulose acetate of the separator layer 2 may be one of monoacetyl cellulose, diacetyl cellulose, and triacetyl cellulose.

[0091] Example 2 Preparation of microneedle raw material-silk fibroin solution

[0092] The preparation method of the silk fibroin solution in this embodiment comprises the following steps:

[0093] Weigh 3g NaHCO3 and 1g Na2CO3 and dissolve them in 4000mL deionized water. Heat to boiling, add 80g mulberry silk, keep it boiling for 30min, take it out and wash it in deionized water.

[0094] The above steps were repeated three times and then placed in a 60° C. oven to dry to obtain degummed silk fibroin fibers.

[0095] Prepare 9.3M LiBr aqueous solution, take 100mL of LiBr aqueous solution and heat it to 65℃ in a water bath, add 15g of degummed silk fiber and stir to dissolve, continue heating and stirring for 40min. After cooling, put the solution into a dialysis bag and place it in deionized water for 72h, and replace the deionized water every 2h.

[0096] After dialysis, the solution is filtered to obtain a silk fibroin solution, which is then placed in a refrigerator for later use.

[0097] Example 3 Preparation method

[0098] The preparation method of the silk fibroin tranexamic acid microneedle patch containing a diaphragm layer in this embodiment comprises the following steps:

[0099] 1) Prepare tranexamic acid solution and cellulose diacetate solution separately

[0100] Preparation of tranexamic acid solution: Take tranexamic acid powder in a centrifuge tube, add deionized water to make the tranexamic acid concentration in the solution 30 mg / mL, and use 0.7 mol / L sodium hydroxide to adjust the pH of the solution to 7.4.

[0101] Preparation of cellulose diacetate solution: Weigh a certain amount of cellulose diacetate powder and dissolve it in acetone solution so that the concentration of cellulose diacetate in the solution is 20 mg / mL.

[0102] 2) Preparation of drug reservoir solution

[0103] The tranexamic acid solution prepared above was slowly added to the silk fibroin solution prepared in Example 2 while stirring, so that the concentration of silk fibroin was 25 mg / mL and the concentration of tranexamic acid was 15 mg / mL.

[0104] 3) Preparation of blank microneedle layer

[0105] 1 mL of silk fibroin solution was poured into a polydimethylsiloxane microneedle mold, and vacuum was drawn three times to remove bubbles in the solution and the microneedle template, so that the solution could fully enter the needle cavity. The degassed mold system was placed in a constant temperature and humidity room (25°C, 60%) with air circulation and dried for 10 hours to obtain a blank microneedle with a needle body length of 600 μm, a bottom diameter of 250 μm, and a spacing of 0.56 mm between the needle tips of adjacent needle bodies.

[0106] 4) Preparation of diaphragm layer

[0107] Take 0.5 mL of the cellulose diacetate solution prepared in step (1) and evenly pour it on the surface of the blank microneedle substrate obtained by drying in step (3), and dry it again at room temperature for 4 minutes to obtain a blank microneedle containing a diaphragm layer.

[0108] 5) Preparation of drug reservoir layer

[0109] The silk fibroin tranexamic acid solution prepared in step (2) is slowly poured onto the surface of the diaphragm layer prepared in step (4), and placed in a constant temperature and humidity room (25°C, 60%) with air circulation to dry for 12 hours, and demolded to obtain tranexamic acid-loaded silk fibroin microneedles containing a diaphragm layer.

[0110] 6) Low temperature steam treatment

[0111] Deionized water accounting for 0.05% of the volume of the vacuum drying oven is placed at the bottom of the vacuum drying oven. 50g of pure cotton non-woven fabric is spread flat on the bottom of the culture dish, and covered with a layer of 45g of pure cotton non-woven fabric.

[0112] The silk fibroin tranexamic acid microneedles obtained in step (5) were placed therein, the chamber door was closed, and the vacuum was evacuated to a vacuum degree of -0.09 MPa, the temperature was maintained at 37°C, and the humidity was maintained at 85%, and the chamber was taken out after 14 hours of treatment, and the insoluble silk fibroin tranexamic acid microneedles were obtained after drying.

[0113] 7) The dried silk fibroin tranexamic acid microneedles are taken out, cut, attached with a backing layer, and packaged to obtain a silk fibroin tranexamic acid microneedle patch containing a diaphragm layer.

[0114] Example 4 Preparation method

[0115] The preparation method of the silk fibroin tranexamic acid microneedle patch containing a diaphragm layer in this embodiment comprises the following steps:

[0116] 1) Prepare tranexamic acid solution and cellulose diacetate solution separately

[0117] Preparation of tranexamic acid solution: Take tranexamic acid powder in a centrifuge tube, add deionized water to make the tranexamic acid concentration in the solution 40 mg / mL, and use 0.5 mol / L sodium hydroxide to adjust the pH of the solution to 7.0.

[0118] Preparation of cellulose diacetate solution: Weigh a certain amount of cellulose diacetate powder and dissolve it in acetone solution so that the concentration of cellulose diacetate in the solution is 22 mg / mL.

[0119] 2) Preparation of drug reservoir solution

[0120] The tranexamic acid solution prepared above was slowly added to the silk fibroin solution prepared in Example 2 while stirring, so that the concentration of silk fibroin was 26 mg / mL and the concentration of tranexamic acid was 20 mg / mL.

[0121] 3) Preparation of blank microneedle layer

[0122] 1 mL of silk fibroin solution was poured into a polydimethylsiloxane microneedle mold, and vacuum was drawn 3 times to remove bubbles in the solution and the microneedle template, so that the solution could fully enter the needle cavity. The degassed mold system was placed in a constant temperature and humidity room (27°C, 55%) with air circulation and dried for 6 hours to obtain a blank microneedle with a needle body length of 400 μm, a bottom diameter of 200 μm, and a spacing of 0.87 mm between the needle tips of adjacent needle bodies.

[0123] 4) Preparation of diaphragm layer

[0124] Take 0.5 mL of the cellulose diacetate solution prepared in step (1) and evenly pour it on the surface of the blank microneedle substrate obtained by drying in step (3), and dry it again at room temperature for 5 minutes to obtain a blank microneedle containing a diaphragm layer.

[0125] 5) Preparation of drug reservoir layer

[0126] The silk fibroin tranexamic acid solution prepared in step (2) is slowly poured onto the surface of the diaphragm layer prepared in step (4), and placed in a constant temperature and humidity room (27°C, 55%) with air circulation to dry for 14 hours, and demolded to obtain tranexamic acid-loaded silk fibroin microneedles containing a diaphragm layer.

[0127] 6) Low temperature steam treatment

[0128] Deionized water accounting for 0.07% of the volume of the drying vacuum drying oven is placed at the bottom of the drying vacuum drying oven. A 60g cotton non-woven fabric is spread flat on the bottom of the culture dish, and a layer of 50g cotton non-woven fabric is covered thereon, and the silk fibroin tranexamic acid microneedle obtained in step (5) is placed therein. The door of the oven is closed, and the vacuum is evacuated to a vacuum degree of -0.08Mpa, the temperature is maintained at 40°C, and the humidity is maintained at 89%. After 20h of treatment, the microneedle is taken out and dried to obtain insoluble silk fibroin tranexamic acid microneedle.

[0129] 7) The dried silk fibroin tranexamic acid microneedles are taken out, cut, attached with a backing layer, and packaged to obtain a silk fibroin tranexamic acid microneedle patch containing a diaphragm layer.

[0130] Example 5 Preparation method

[0131] The preparation method of the silk fibroin tranexamic acid microneedle patch containing a diaphragm layer in this embodiment comprises the following steps:

[0132] 1) Prepare tranexamic acid solution and cellulose diacetate solution separately

[0133] Preparation of tranexamic acid solution: Take tranexamic acid powder in a centrifuge tube, add deionized water to make the concentration of tranexamic acid in the solution 45 mg / mL, and use 0.2 mol / L sodium hydroxide to adjust the pH of the solution to 6.9.

[0134] Preparation of cellulose diacetate solution: Weigh a certain amount of cellulose diacetate powder and dissolve it in acetone solution so that the concentration of cellulose diacetate in the solution is 24 mg / mL.

[0135] 2) Preparation of drug reservoir solution

[0136] The tranexamic acid solution prepared above was slowly added to the silk fibroin solution prepared in Example 2 while stirring, so that the concentration of silk fibroin was 28 mg / mL and the concentration of tranexamic acid was 25 mg / mL.

[0137] 3) Preparation of blank microneedle layer

[0138] 1 mL of silk fibroin solution was poured into a polydimethylsiloxane microneedle mold, and vacuum was drawn 3 times to remove bubbles in the solution and the microneedle template, so that the solution could fully enter the needle cavity. The degassed mold system was placed in a constant temperature and humidity room (38°C, 58%) with air circulation and dried for 8 hours to obtain a blank microneedle with a needle body length of 500 μm, a bottom diameter of 300 μm, and a spacing of 1.7 mm between the needle tips of adjacent needle bodies.

[0139] 4) Preparation of diaphragm layer

[0140] Take 0.5 mL of the cellulose diacetate solution prepared in step (1) and evenly pour it on the surface of the blank microneedle substrate obtained by drying in step (3), and dry it again at room temperature for 2 minutes to obtain a blank microneedle containing a diaphragm layer.

[0141] 5) Preparation of drug reservoir layer

[0142] The silk fibroin tranexamic acid solution prepared in step (2) is slowly poured onto the surface of the diaphragm layer prepared in step (4), and placed in a constant temperature and humidity room (38°C, 58%) with air circulation to dry for 18 hours, and demolded to obtain tranexamic acid-loaded silk fibroin microneedles containing a diaphragm layer.

[0143] 6) Low temperature steam treatment

[0144] Deionized water accounting for 0.08% of the volume of the drying vacuum drying oven is placed at the bottom of the drying vacuum drying oven. 80g of pure cotton non-woven fabric is spread flat on the bottom of the culture dish, and a layer of 60g of pure cotton non-woven fabric is covered thereon, and the silk fibroin tranexamic acid microneedle obtained in step (5) is placed therein. The door of the oven is closed, and the vacuum is evacuated to a vacuum degree of -0.06Mpa, the temperature is maintained at 42°C, and the humidity is maintained at 92%. After 15h of treatment, the microneedle is taken out and dried to obtain insoluble silk fibroin tranexamic acid microneedle.

[0145] 7) The dried silk fibroin tranexamic acid microneedles are taken out, cut, attached with a backing layer, and packaged to obtain a silk fibroin tranexamic acid microneedle patch containing a diaphragm layer.

[0146] Example 6 Preparation method

[0147] The preparation method of the silk fibroin tranexamic acid microneedle patch containing a diaphragm layer in this embodiment comprises the following steps:

[0148] 1) Prepare tranexamic acid solution and cellulose triacetate solution separately

[0149] Preparation of tranexamic acid solution: Take tranexamic acid powder in a centrifuge tube, add deionized water to make the concentration of tranexamic acid in the solution 50 mg / mL, and use 0.4 mol / L sodium hydroxide to adjust the pH of the solution to 6.8.

[0150] Preparation of cellulose triacetate solution: Weigh a certain amount of cellulose triacetate powder and dissolve it in chloroform solution so that the concentration of cellulose triacetate in the solution is 21 mg / mL.

[0151] 2) Preparation of drug reservoir solution

[0152] The tranexamic acid solution prepared above was slowly added to the silk fibroin solution prepared in Example 2 while stirring, so that the concentration of silk fibroin was 30 mg / mL and the concentration of tranexamic acid was 22 mg / mL.

[0153] 3) Preparation of blank microneedle layer

[0154] 1 mL of silk fibroin solution was poured into a polydimethylsiloxane microneedle mold, and vacuum was drawn three times to remove bubbles in the solution and the microneedle template, so that the solution could fully enter the needle cavity. The degassed mold system was placed in a constant temperature and humidity room (29°C, 68%) with air circulation and dried for 10 hours to obtain a blank microneedle with a needle body length of 700 μm, a bottom diameter of 350 μm, and a spacing of 0.50 mm between the needle tips of adjacent needle bodies.

[0155] 4) Preparation of diaphragm layer

[0156] Take 0.5 mL of the cellulose diacetate solution prepared in step (1) and evenly pour it on the surface of the blank microneedle substrate obtained by drying in step (3), and dry it again at room temperature for 3 minutes to obtain a blank microneedle containing a diaphragm layer.

[0157] 5) Preparation of drug reservoir layer

[0158] The silk fibroin tranexamic acid solution prepared in step (2) was slowly poured onto the surface of the diaphragm layer prepared in step (4), and dried for 17 hours in a constant temperature and humidity room (29°C, 68%) with air circulation, and demolded to obtain tranexamic acid-loaded silk fibroin microneedles containing a diaphragm layer.

[0159] 6) Low temperature steam treatment

[0160] Deionized water accounting for 0.05% of the volume of the drying vacuum drying oven is placed at the bottom of the drying vacuum drying oven. 80g of pure cotton non-woven fabric is spread flat on the bottom of the culture dish, and a layer of 60g of pure cotton non-woven fabric is covered thereon, and the silk fibroin tranexamic acid microneedle obtained in step (5) is placed therein. The door of the oven is closed, and the vacuum is evacuated to a vacuum degree of -0.06Mpa, the temperature is maintained at 41°C, and the humidity is 87%. After processing for 18 hours, it is taken out and dried to obtain insoluble silk fibroin tranexamic acid microneedle.

[0161] 7) The dried silk fibroin tranexamic acid microneedles are taken out, cut, attached with a backing layer, and packaged to obtain a silk fibroin tranexamic acid microneedle patch containing a diaphragm layer.

[0162] Example 7 Preparation method

[0163] The preparation method of the silk fibroin tranexamic acid microneedle patch containing a diaphragm layer in this embodiment comprises the following steps:

[0164] 1) Prepare tranexamic acid solution and cellulose triacetate solution separately

[0165] Preparation of tranexamic acid solution: Take tranexamic acid powder in a centrifuge tube, add deionized water to make the tranexamic acid concentration in the solution 55 mg / mL, and use 0.6 mol / L sodium hydroxide to adjust the pH of the solution to 7.2.

[0166] Preparation of cellulose triacetate solution: Weigh a certain amount of cellulose triacetate powder and dissolve it in chloroform solution so that the concentration of cellulose triacetate in the solution is 23 mg / mL.

[0167] 2) Preparation of drug reservoir solution

[0168] The tranexamic acid solution prepared above was slowly added to the silk fibroin solution prepared in Example 2 while stirring, so that the concentration of silk fibroin was 32 mg / mL and the concentration of tranexamic acid was 18 mg / mL.

[0169] 3) Preparation of blank microneedle layer

[0170] 1 mL of silk fibroin solution was poured into a polydimethylsiloxane microneedle mold, and vacuum was drawn 3 times to remove bubbles in the solution and the microneedle template, so that the solution could fully enter the needle cavity. The degassed mold system was placed in a constant temperature and humidity room (35°C, 52%) with air circulation and dried for 7 hours to obtain a blank microneedle with a needle body length of 800 μm, a bottom diameter of 440 μm, and a spacing of 0.95 mm between the needle tips of adjacent needle bodies.

[0171] 4) Preparation of diaphragm layer

[0172] Take 0.5 mL of the cellulose diacetate solution prepared in step (1) and evenly pour it on the surface of the blank microneedle substrate obtained by drying in step (3), and dry it again at room temperature for 6 minutes to obtain a blank microneedle containing a diaphragm layer.

[0173] 5) Preparation of drug reservoir layer

[0174] The silk fibroin tranexamic acid solution prepared in step (2) was slowly poured onto the surface of the diaphragm layer prepared in step (4), and placed in a constant temperature and humidity room (35°C, 52%) with air circulation to dry for 18 hours, and demolded to obtain tranexamic acid-loaded silk fibroin microneedles containing a diaphragm layer.

[0175] 6) Low temperature steam treatment

[0176] Deionized water accounting for 0.06% of the volume of the drying vacuum drying oven is placed at the bottom of the drying vacuum drying oven. A 50g cotton non-woven fabric is spread flat on the bottom of the culture dish, and a layer of 45g cotton non-woven fabric is covered thereon, and the silk fibroin tranexamic acid microneedle obtained in step (5) is placed therein. The door of the oven is closed, and the vacuum is evacuated to a vacuum degree of -0.10Mpa, maintained at a temperature of 37°C and a humidity of 85%, and taken out after processing for 19h, and insoluble silk fibroin tranexamic acid microneedle is obtained after drying.

[0177] 7) The dried silk fibroin tranexamic acid microneedles are taken out, cut, attached with a backing layer, and packaged to obtain a silk fibroin tranexamic acid microneedle patch containing a diaphragm layer.

[0178] Example 8 Preparation method

[0179] The preparation method of the silk fibroin tranexamic acid microneedle patch containing a diaphragm layer in this embodiment comprises the following steps:

[0180] 1) Prepare tranexamic acid solution and cellulose triacetate solution separately

[0181] Preparation of tranexamic acid solution: Take tranexamic acid powder in a centrifuge tube, add deionized water to make the concentration of tranexamic acid in the solution 25 mg / mL, and use 0.9 mol / L sodium hydroxide to adjust the pH of the solution to 7.3.

[0182] Preparation of cellulose triacetate solution: Weigh a certain amount of cellulose triacetate powder and dissolve it in chloroform solution so that the concentration of cellulose triacetate in the solution is 20 mg / mL.

[0183] 2) Preparation of drug reservoir solution

[0184] The tranexamic acid solution prepared above was slowly added to the silk fibroin solution prepared in Example 2 while stirring, so that the concentration of silk fibroin was 29 mg / mL and the concentration of tranexamic acid was 28 mg / mL.

[0185] 3) Preparation of blank microneedle layer

[0186] 1 mL of silk fibroin solution was poured into a polydimethylsiloxane microneedle mold, and vacuum was drawn 3 times to remove bubbles in the solution and the microneedle template, so that the solution could fully enter the needle cavity. The degassed mold system was placed in a constant temperature and humidity room (23°C, 47%) with air circulation and dried for 9 hours to obtain a blank microneedle with a length of 550 μm, a bottom diameter of 400 μm, and a spacing of 1.1 mm between the tips of adjacent needles.

[0187] 4) Preparation of diaphragm layer

[0188] Take 0.5 mL of the cellulose diacetate solution prepared in step (1) and evenly pour it on the surface of the blank microneedle substrate obtained by drying in step (3), and dry it again at room temperature for 5 minutes to obtain a blank microneedle containing a diaphragm layer.

[0189] 5) Preparation of drug reservoir layer

[0190] The silk fibroin tranexamic acid solution prepared in step (2) is slowly poured onto the surface of the diaphragm layer prepared in step (4), and placed in a constant temperature and humidity room (23°C, 47%) with air circulation for drying for 14 hours, and demolding is performed to obtain tranexamic acid-loaded silk fibroin microneedles containing a diaphragm layer.

[0191] 6) Low temperature steam treatment

[0192] Deionized water accounting for 0.1% of the volume of the drying vacuum drying oven is placed at the bottom of the drying vacuum drying oven. A 56g cotton non-woven fabric is spread flat on the bottom of the culture dish, and a layer of 38g cotton non-woven fabric is covered thereon, and the silk fibroin tranexamic acid microneedle obtained in step (5) is placed therein. The door of the oven is closed, and the vacuum is evacuated to a vacuum degree of -0.09Mpa, the temperature is maintained at 38°C, and the humidity is 90%. After processing for 20 hours, the microneedle is taken out and dried to obtain an insoluble silk fibroin tranexamic acid microneedle.

[0193] 7) The dried silk fibroin tranexamic acid microneedles are taken out, cut, attached with a backing layer, and packaged to obtain a silk fibroin tranexamic acid microneedle patch containing a diaphragm layer.

[0194] Example 9 Preparation method

[0195] The preparation method of the silk fibroin tranexamic acid microneedle patch containing a diaphragm layer in this embodiment comprises the following steps:

[0196] 1) Prepare tranexamic acid solution and cellulose acetate solution separately

[0197] Preparation of tranexamic acid solution: Take tranexamic acid powder in a centrifuge tube, add deionized water to make the tranexamic acid concentration in the solution 60 mg / mL, and use 1.0 mol / L sodium hydroxide to adjust the pH of the solution to 7.0.

[0198] Preparation of cellulose acetate solution: Weigh a certain amount of cellulose acetate powder and dissolve it in acetone solution so that the concentration of cellulose acetate in the solution is 20 mg / mL.

[0199] 2) Preparation of drug reservoir solution

[0200] The tranexamic acid solution prepared above was slowly added to the silk fibroin solution prepared in Example 2 while stirring, so that the concentration of silk fibroin was 27 mg / mL and the concentration of tranexamic acid was 26 mg / mL.

[0201] 3) Preparation of blank microneedle layer

[0202] 1 mL of silk fibroin solution was poured into a polydimethylsiloxane microneedle mold, and vacuum was drawn 3 times to remove bubbles in the solution and the microneedle template, so that the solution could fully enter the needle cavity. The degassed mold system was placed in a constant temperature and humidity room (39°C, 50%) with air circulation and dried for 5 hours to obtain a blank microneedle with a needle body length of 700 μm, a bottom diameter of 400 μm, and a spacing of 0.75 mm between the needle tips of adjacent needle bodies.

[0203] 4) Preparation of diaphragm layer

[0204] Take 0.5 mL of the cellulose diacetate solution prepared in step (1) and evenly pour it on the surface of the blank microneedle substrate obtained by drying in step (3), and dry it again at room temperature for 8 minutes to obtain a blank microneedle containing a diaphragm layer.

[0205] 5) Preparation of drug reservoir layer

[0206] The silk fibroin tranexamic acid solution prepared in step (2) is slowly poured onto the surface of the diaphragm layer prepared in step (4), and placed in a constant temperature and humidity room (39°C, 50%) with air circulation to dry for 10 hours, and demolded to obtain tranexamic acid-loaded silk fibroin microneedles containing a diaphragm layer.

[0207] 6) Low temperature steam treatment

[0208] Deionized water accounting for 0.03% of the volume of the drying vacuum drying oven is placed at the bottom of the drying vacuum drying oven. A 62g cotton non-woven fabric is spread flat on the bottom of the culture dish, and a layer of 30g cotton non-woven fabric is covered thereon, and the silk fibroin tranexamic acid microneedle obtained in step (5) is placed therein. The door of the oven is closed, and the vacuum is evacuated to a vacuum degree of -0.08Mpa, the temperature is maintained at 36°C, and the humidity is maintained at 91%. After 24h of treatment, the microneedle is taken out and dried to obtain insoluble silk fibroin tranexamic acid microneedle.

[0209] 7) The dried silk fibroin tranexamic acid microneedles are taken out, cut, attached with a backing layer, and packaged to obtain a silk fibroin tranexamic acid microneedle patch containing a diaphragm layer.

[0210] Comparative Example 1

[0211] This comparative example is a silk fibroin tranexamic acid microneedle without a diaphragm layer, and the preparation method comprises the following steps:

[0212] 1) Preparation of tranexamic acid solution

[0213] Preparation of tranexamic acid solution: Take tranexamic acid powder in a centrifuge tube, add deionized water to make the tranexamic acid concentration in the solution 25 mg / mL, and use 0.7 mol / L sodium hydroxide to adjust the pH of the solution to 7.4.

[0214] 2) Preparation of tranexamic acid silk protein mixed solution

[0215] The tranexamic acid solution prepared above was slowly added to the silk fibroin solution prepared in Example 2 while stirring, so that the concentration of silk fibroin was 50 mg / mL and the concentration of tranexamic acid was 16 mg / mL.

[0216] 3) Preparation of silk fibroin tranexamic acid microneedles without a diaphragm layer

[0217] 3 mL of the mixed solution prepared in step (2) was poured into the microneedle mold, and vacuum was drawn 3 times to remove bubbles in the solution and the microneedle template, so that the solution fully entered the needle body cavity. The degassed mold system was placed in a constant temperature and humidity room (25° C., 60%) with air circulation and dried for 16 hours to obtain a silk fibroin tranexamic acid microneedle without a diaphragm layer, with a needle body length of 500 μm, a bottom diameter of 300 μm, and a spacing of 0.5 mm between the needle tips of adjacent needle bodies.

[0218] 4) Low temperature steam treatment

[0219] Deionized water accounting for 0.05% of the volume of the vacuum drying oven is placed at the bottom of the vacuum drying oven. 53g of pure cotton non-woven fabric is spread flat on the bottom of the culture dish, and a layer of 40g of pure cotton non-woven fabric is covered on it, and the skeleton-type microneedle obtained in step (3) is placed therein. Close the door of the oven, evacuate to a vacuum degree of -0.09Mpa, maintain a temperature of 37°C and a humidity of 90%, take out after processing for 24 hours, and obtain insoluble silk protein microneedles after drying.

[0220] 5) The dried skeleton-type microneedles are taken out, cut, attached with a backing layer, and packaged to obtain a silk fibroin tranexamic acid microneedle patch without a diaphragm layer.

[0221] Comparative Example 2

[0222] This comparative example does not contain a diaphragm layer, but only contains blank microneedles and a drug storage layer. The preparation method comprises the following steps:

[0223] 1) Preparation of tranexamic acid solution

[0224] Preparation of tranexamic acid solution: Take tranexamic acid powder in a centrifuge tube, add deionized water to make the concentration of tranexamic acid in the solution 35 mg / mL, and use 0.7 mol / L sodium hydroxide to adjust the pH of the solution to 7.1.

[0225] 2) Preparation of tranexamic acid silk protein mixed solution

[0226] The tranexamic acid solution prepared above was slowly added to the silk fibroin solution prepared in Example 2 while stirring, so that the concentration of silk fibroin was 38 mg / mL and the concentration of tranexamic acid was 16 mg / mL.

[0227] 3) Preparation of blank microneedle layer

[0228] 1 mL of silk fibroin solution was poured into a polydimethylsiloxane microneedle mold, and vacuum was drawn 3 times to remove bubbles in the solution and the microneedle template, so that the solution could fully enter the needle cavity. The degassed mold system was placed in a constant temperature and humidity room (30°C, 65%) with air circulation and dried for 7 hours to obtain a blank microneedle with a needle body length of 500 μm, a bottom diameter of 300 μm, and a spacing of 0.5 mm between the needle tips of adjacent needle bodies.

[0229] 4) Preparation of drug reservoir layer

[0230] The silk fibroin tranexamic acid solution prepared in step (2) was slowly poured onto the surface of the blank microneedles prepared in step (3), and placed in a constant temperature and humidity room (30°C, 65%) with air circulation for drying for 13 hours, and demolded to obtain separated tranexamic acid-loaded silk fibroin microneedles.

[0231] 5) Low temperature steam treatment

[0232] Deionized water accounting for 0.05% of the volume of the vacuum drying oven is placed at the bottom of the vacuum drying oven. 48g of pure cotton non-woven fabric is spread flat on the bottom of the culture dish, and a layer of 42g of pure cotton non-woven fabric is covered on it, and the skeleton-type microneedle obtained in step (3) is placed therein. Close the door of the oven, evacuate to a vacuum degree of -0.09Mpa, maintain a temperature of 37°C and a humidity of 90%, take out after processing for 24 hours, and obtain insoluble silk protein microneedles after drying.

[0233] 5) The dried microneedles are taken out, cut, attached with a backing layer, and packaged to obtain a separated silk fibroin tranexamic acid microneedle patch without a diaphragm layer.

[0234] Test and Results Discussion

[0235] 1. In vitro transdermal drug release test of the silk fibroin microneedles prepared in Examples 3-9 and Comparative Examples 1-2:

[0236] (1) Assembly of in vitro drug release device

[0237] Take frozen bullfrog skin and place it in physiological saline to thaw, take out and cut the abdominal bullfrog skin into small pieces. Use Franz diffusion cell as in vitro drug receiving cell, deionized water as simulated body fluid, and the receiving cell volume is 6mL. The microneedles of Examples 3-9 and Comparative Examples 1-2 are inserted into bullfrog skin. The bullfrog skin is flatly covered on the circular receiving chamber of Franz diffusion cell, so that the dermis of the bullfrog skin contacts the solution in the receiving cell, and the donor chamber and the receiving chamber are fixed with stainless steel clips.

[0238] (2) Obtaining the test solution

[0239] In the drug release device pool of the above step, 1 mL of solution was taken out at different times, and 1 mL of deionized water was added. The taken out solution was filtered through a 0.45 μm filter, and the tranexamic acid content was detected by high performance liquid chromatography.

[0240] (3) Preparation of standard solution: Dissolve tranexamic acid to prepare a 1 mg / mL tranexamic acid solution. Further dilute the 1 mg / mL tranexamic acid solution to the following concentrations: 100, 50, 20, 10, 5, and 3 μg / mL, and filter using a 0.45 μm filter before adding the sample.

[0241] (4) Chromatographic conditions: The chromatographic conditions of this experiment were phosphate buffer: methanol = 60:40 (v:v) for isocratic elution. The retention time was 23.3 min, the flow rate was 0.5 mL / min, the injection was 20 μL, the detection wavelength was 220 nm, the chromatographic column was a C18 reverse phase column, and the column temperature was set to 35 °C. The characteristic peak area of ​​tranexamic acid was calculated and linearly fitted with the tranexamic acid concentration to obtain the standard curve of tranexamic acid concentration-peak area.

[0242] (5) Detection of sustained-release effect of microneedles by HPLC

[0243] The sample solution obtained in step (2) is sequentially injected into a liquid chromatograph, and the chromatogram is recorded. The corresponding concentration is calculated according to the standard curve of tranexamic acid concentration-peak area obtained in step (4). A drug release rate curve is drawn, such as Figure 3 and 4 shown.

[0244] 2. Pharmacokinetic test of the diaphragm silk fibroin microneedles prepared in Example 7 in chloasma guinea pigs:

[0245] (1) Application of microneedle patch

[0246] The back of the chloasma model guinea pig was shaved and the small hairs were shaved with a razor, and the residual depilatory cream was cleaned with running water. The medical dressing was cut into 10cm×3cm specifications, and the microneedle patch was placed in the center of the medical dressing. Then the microneedle patch was placed on the back of the guinea pig and pressed with a force of 3N. Make sure that the microneedle penetrates the skin.

[0247] (2) Pharmacokinetic testing

[0248] The microneedle group and the injection group had the same single dose. The microneedle group was subjected to tail vein tail cutting and blood collection at 0, 1, 2, 4, 6, 8, 10, and 12 hours after the use of the microneedle patch (N=3), and the injection group was subjected to tail vein tail cutting and blood collection at 0, 0.17, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 hours after the injection (N=3). The blood volume was about 10 μL each time, and chromatographic grade methanol was added at a ratio of blood sample: methanol = 1:5 (v:v), vortexed for 1 min to mix evenly, and the supernatant was centrifuged for liquid chromatography detection.

[0249] The cumulative release rate curves of the microneedle patches prepared in Examples 3-9 and Comparative Examples 1-2 are as follows: Figure 3As shown. Compared with Comparative Examples 1-2, the cumulative drug release rate of the microneedles prepared in Example 9 is reduced, but the drug release time of Example 9 is significantly increased, and the release effect of the microneedles prepared in Examples 3-4 and 6-8 is better than that of Example 9. The microneedles prepared in Example 3-5 are loaded with cellulose diacetate diaphragms, and the cumulative release rate decreases with the increase of the microneedle spacing. When the microneedle spacing is too large, the number of microneedles per unit area is small, which is not conducive to the transmission of water molecules and tranexamic acid. As in Example 5, the ability of cellulose diacetate diaphragms to penetrate water molecules is strong, and the change in the microneedle spacing becomes the biggest influencing factor for the release of tranexamic acid. The microneedles prepared in Examples 6-8 are loaded with cellulose triacetate diaphragms, and the cumulative release rate is above 80%, which is higher than Comparative Example 1. The change in the microneedle spacing does not have a significant effect on the microneedles prepared in Examples 6-8, and the microneedles have high stability.

[0250] The in vitro release rate curves of the microneedle patches prepared in Examples 3-9 and Comparative Examples 1-2 are as follows: Figure 4 As shown. In Comparative Example 1, the peak release rate appeared at 0.5h, and the release rate at about 4h was lower than that of the microneedles prepared in the example, indicating that the drug release rate decreased significantly with time. Comparative Example 2, based on Comparative Example 1, controlled the microneedle tip not to load drugs, and successfully delayed the peak release rate to 1h. Subsequently, the peak release rate at about 4h was still lower than that of the microneedles prepared in the example. In the example, a cellulose acetate diaphragm layer was added to the back of the silk microneedle, so that the microneedle had a certain slow release effect, and the drug release time was extended to 10h. Compared with Comparative Example 1, Examples 3-5 loaded with cellulose diacetate ensured a certain release rate value and extended the release rate peak to 2h (203μg / h·cm 2 The diaphragm layer is made of cellulose triacetate. Compared with Examples 3-5, Examples 6-8 also ensure a certain release rate value and a prolonged release rate peak value (2h, 279 μg / h·cm 2 ). As the distance between the microneedles increases, the microneedles can maintain a stable and relatively close release rate curve, and the release time reaches 14h.

[0251] The diaphragm type silk protein microneedle patch of the present invention is a transdermal drug delivery technology, which can avoid the first-pass effect of the liver and the gastrointestinal inactivation caused by oral administration, and improve the bioavailability. It can replace injection to achieve higher bioavailability, relieve the pain of patients, and improve patient compliance. Figure 5 As shown, the diaphragm microneedle patch prepared in Example 7 was applied to mice, and a single microneedle patch could maintain the blood concentration of tranexamic acid for 24 hours. Compared with injection, the bioavailability of the microneedle patch prepared in Example 7 relative to the injection group reached 264%.

[0252] The diaphragm-layered silk fibroin tranexamic acid microneedle patch of the present invention is composed of three parts from top to bottom, namely, a drug storage layer, a diaphragm layer and a blank microneedle. The blank microneedle is composed only of silk fibroin; the drug storage layer is composed of silk fibroin and tranexamic acid, and the loading amount of tranexamic acid is 0.1-5 mg / cm 2 ; The diaphragm layer is composed of any one of mono-, di-, and tri-acetyl cellulose. The corresponding preparation method includes solution configuration, mold casting, vacuum degassing, diaphragm layer and drug storage layer casting, and drying and molding. The tranexamic acid diaphragm silk protein microneedle patch prepared by the present invention can prolong the release time of the microneedle patch, ensure the release amount reaching the therapeutic level, and solve the sudden release phenomenon of tranexamic acid. The microneedles are easy to prepare and easy to use. The blank microneedles, the diaphragm layer, and the drug storage layer form a whole, and there is no need to replace or add an external drug storage device, which reduces unnecessary trouble.

[0253] 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.

[0254] 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 diaphragm type silk fibroin microneedle patch, characterized in that: The invention comprises a drug storage layer, a diaphragm layer and a blank microneedle layer in sequence. The blank microneedle layer is composed of silk fibroin, and comprises a matrix and a needle body extending outward from the matrix. The drug storage layer covers a side of the diaphragm layer away from the needle body. The drug storage layer comprises silk fibroin and tranexamic acid. The diaphragm layer is located between the drug storage layer and the blank microneedle layer.

2. The diaphragm silk fibroin microneedle patch according to claim 1, characterized in that: The material of the diaphragm layer is cellulose acetate.

3. The diaphragm silk fibroin microneedle patch according to claim 1, characterized in that: The thickness of the diaphragm layer is 10 μm-200 μm; the thickness of the drug storage layer is 100 μm-2 mm.

4. The diaphragm silk fibroin microneedle patch according to claim 1, characterized in that: The crystal structure of the silk fibroin in the drug storage layer is a Silk I crystal structure; and / or, the crystal structure of the silk fibroin in the blank microneedle layer is a Silk I crystal structure.

5. The diaphragm silk fibroin microneedle patch according to claim 1, characterized in that: The drug loading of tranexamic acid in the drug storage layer is 0.1-5 mg / cm 2 .

6. The diaphragm silk fibroin microneedle patch according to claim 1, characterized in that: The needle body of the blank microneedle layer is a solid cone, the length of the needle body is 200-900 μm, the bottom diameter is 100-500 μm, and the spacing between the needle tips of adjacent needle bodies is 0.2-2 mm.

7. A method for preparing a diaphragm silk fibroin microneedle patch according to any one of claims 1 to 6, characterized in that: The steps include: respectively preparing a silk fibroin solution, a membrane layer solution and a drug storage layer solution; pouring the silk fibroin solution into the microneedle mold, degassing, and placing the degassing mold system under constant temperature and humidity conditions to dry, thereby obtaining the blank microneedle layer; Casting the membrane layer solution on the substrate of the blank microneedle layer and drying it to obtain blank microneedles with the membrane layer; pouring the drug storage layer solution onto the diaphragm layer and drying under constant temperature and humidity conditions to obtain a drug-loaded silk fibroin microneedle patch; The drug-loaded silk fibroin microneedle patch is placed in an environment with a temperature of 20-50° C. and a relative humidity of 85-99% for 10-40 hours, taken out and dried to obtain the diaphragm-type silk fibroin microneedle patch.

8. The preparation method according to claim 7, characterized in that: The diaphragm layer solution is prepared according to the following method: cellulose acetate is dissolved in a polar volatile organic solvent to obtain a diaphragm layer solution with a concentration of 10-100 mg / mL.

9. The preparation method according to claim 7, characterized in that: The drug reservoir solution is prepared according to the following method: a tranexamic acid solution and a silk fibroin solution are evenly mixed to form a drug reservoir solution; the concentration of the silk fibroin in the drug reservoir solution is 20-50 mg / mL, and the concentration of tranexamic acid is 10-30 mg / mL.

10. The preparation method according to claim 9, characterized in that: The tranexamic acid solution is prepared according to the following method: weighing tranexamic acid powder, adding deionized water, and completely dissolving the tranexamic acid to obtain the tranexamic acid solution, wherein the concentration of the tranexamic acid is 20-100 mg / mL, and using 0.1-1.0 mol / L of sodium hydroxide to adjust the pH of the solution to 6.5-7.5.

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