A diaphragm type silk fibroin microneedle patch and a preparation method thereof

Abstract

This invention discloses a diaphragm-type silk fibroin microneedle patch, comprising a drug storage layer, a diaphragm layer, and a blank microneedle layer. The blank microneedle layer is composed of silk fibroin and includes a matrix and needles extending outward from the matrix. The drug storage layer covers the side of the diaphragm layer away from the needles. 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. This invention's tranexamic acid-loaded diaphragm-type silk fibroin microneedle patch with a drug storage layer can increase the release time (14 hours in vitro, 24 hours in vivo) and create a large tranexamic acid concentration gradient between the microneedles and the skin, providing a stable driving force for the transdermal release of tranexamic acid, achieving a stable drug release rate after a certain period, thereby achieving long-term stable drug delivery.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical science, particularly the field of transdermal drug delivery and silk fibroin microneedle technology. Specifically, it relates to a tranexamic acid-loaded diaphragm-type silk fibroin microneedle patch with a diaphragm layer and a method for preparing the diaphragm-type silk fibroin microneedle patch. Background Technology

[0002] Melasma has a higher incidence rate among women with darker skin tones, reaching 9-50%. Melasma typically manifests as brown to gray patches. Its persistent and recurring flare-ups cause inflammation and hyperpigmentation, further increasing the number and deepening the color of the patches, negatively impacting the patient's appearance and quality of life. Clinically, melasma is caused by increased tyrosinase activity and excessive expression of endothelin-1 (EF-1), leading to an increase in the dendritic structure of melanocytes and promoting the transfer and deposition of new melanin.

[0003] Numerous studies have demonstrated the safety and efficacy of tranexamic acid as a treatment for melasma. Tranexamic acid is primarily delivered orally, by injection, and transdermally. Common transdermal formulations for melasma are in the form of emulsions and creams, which cannot bypass the skin barrier and rely mainly on the drug's diffusion behavior and the penetration-enhancing abilities of penetration enhancers. Considering tranexamic acid's high water solubility and small molecular weight, making it difficult to effectively penetrate the skin barrier, microneedle transdermal delivery technology may be a better option to address this issue.

[0004] Microneedles, as an effective transdermal delivery carrier, offer several adjustable physical parameters to meet the long-term delivery requirements of tranexamic acid. Solid microneedles and soluble microneedles are the two most widely used types. Konisky et al. [Journal of Cosmetic Dermatology, 2023] used solid microneedles to deliver tranexamic acid, achieving a penetration time of 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 vivo in rats, respectively, with a release rate of approximately 80%. However, the release duration and peak plasma concentration are still insufficient to achieve satisfactory therapeutic effects. Furthermore, soluble microneedles pose a certain risk of sensitization after skin insertion.

[0005] In the prior art, Chinese invention patent application number CN202211106981.9 discloses a method for preparing hyaluronic acid microneedles for treating melasma. The skin penetration depth, penetration rate, and biocompatibility of the microneedles were evaluated by loading rhodamine. However, the in vitro release of tranexamic acid was not evaluated, and since sodium hyaluronate microneedles are soluble, they do not possess the characteristic of sustainable drug delivery compared to other insoluble microneedles.

[0006] For example, Chinese invention patent application number CN202210357550.3 discloses a method for preparing sustained-release microneedles for treating melasma. Tranexamic acid protected by a Schiff base is released through ultraviolet irradiation. However, the microneedles did not undergo transdermal testing, so the actual transdermal sustained-release effect cannot be evaluated.

[0007] For example, Chinese patent application number CN202311430684.4 discloses a method for preparing a skeletal microneedle for treating melasma. The microneedles contain only silk fibroin and tranexamic acid, with the silk fibroin having a Silk I crystalline structure and belonging to the insoluble microneedle type. The release of tranexamic acid is jointly regulated by the network density of silk fibroin and the binding force between the two, which can achieve a certain degree of sustained release, but the effect still needs to be improved.

[0008] The above background information is provided only to assist in understanding the inventive concept and technical solution of this invention. It does not necessarily belong to the prior art of this patent application. In the absence of clear evidence that the above information was disclosed before the filing date of this patent application, the above background information should not be used to evaluate the novelty and inventiveness of this application. Summary of the Invention

[0009] In view of this, in order to overcome the shortcomings of the prior art, the purpose of this invention is to provide an improved tranexamic acid-loaded membrane-type silk fibroin microneedle patch, which has a membrane layer and can achieve long-term slow release of tranexamic acid.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] A diaphragm-type silk fibroin microneedle patch comprises, in sequence, a drug storage layer, a diaphragm layer, and a blank microneedle layer. The blank microneedle layer is composed of silk fibroin and includes a matrix and needles extending outward from the matrix. The drug storage layer covers the side of the diaphragm layer away from the needles. 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.

[0012] According to some preferred embodiments of the present invention, the membrane layer is made of cellulose acetate. As a membrane layer, cellulose acetate can hinder the diffusion process of water-soluble molecules, thereby reducing the peak release rate.

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

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

[0015] According to some preferred embodiments of the present invention, the tranexamic acid loading in the drug reservoir is 0.1-5 mg / cm³. 2 That is, unit area (1cm²) 2 The content of tranexamic acid in the drug storage layer is 0.1-5 mg, with a maximum of 5 mg / cm² in the drug storage layer.

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

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

[0018] Prepare silk fibroin solution, diaphragm layer solution and drug storage layer solution separately;

[0019] The silk fibroin solution was poured into the microneedle mold, degassed, and the degassed mold system was dried under constant temperature and humidity conditions to obtain the blank microneedle layer.

[0020] The membrane layer solution was poured onto the substrate of the blank microneedle layer and dried to obtain blank microneedles with a membrane layer.

[0021] The drug storage layer solution was poured onto the diaphragm layer and dried under constant temperature and humidity conditions to obtain a drug-loaded silk fibroin microneedle patch.

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

[0023] According to some preferred embodiments of the present invention, the membrane layer solution is prepared by dissolving cellulose acetate in a polar volatile organic solvent to obtain a membrane layer solution with a concentration of 10-100 mg / mL.

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

[0025] According to some preferred embodiments of the present invention, the drug storage layer solution is prepared by the following method: tranexamic acid solution and silk fibroin solution are mixed evenly to form a drug storage layer solution; the concentration of silk fibroin in the drug storage layer 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 by the following method: tranexamic acid powder is weighed, deionized water is added, and the tranexamic acid is completely dissolved to obtain a tranexamic acid solution, 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 embodiments of the present invention, the steps further include: removing the dried diaphragm-type silk fibroin microneedles, cutting them, attaching a backing layer, and packaging them to obtain a tranexamic acid-loaded diaphragm-type silk fibroin microneedle patch product containing a diaphragm layer.

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

[0029] Preparation of S1, tranexamic acid solution

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

[0031] S2, Preparation of the diaphragm layer solution

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

[0033] S3. Preparation of the drug reservoir solution

[0034] Tranexamic acid solution is added to silk fibroin solution and mixed thoroughly 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.

[0035] S4. Preparation of blank microneedle layer

[0036] The silk fibroin solution was poured into a microneedle mold, and a vacuum was applied to remove air bubbles from the solution and the microneedle template, allowing the solution to fully enter the needle cavity. The degassed mold system was then placed under constant temperature and humidity conditions and dried in a well-ventilated area for 4-24 hours to obtain the blank microneedle layer.

[0037] S5. Preparation of the diaphragm layer

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

[0039] S6. Preparation of the drug storage layer

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

[0041] S7, Microneedle Patch Post-Processing

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

[0043] S8, backing layer

[0044] A backing layer is attached to the diaphragm-type silk fibroin microneedle patch, and then packaged to obtain the silk fibroin tranexamic acid microneedle patch product containing the 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 embodiments of the present invention, the polar volatile organic solvent in step S2 is acetone or chloroform.

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

[0048] According to some preferred embodiments 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 this invention is as follows: Silk fibroin, as a natural, partially crystallizable polymer, possesses excellent mechanical properties and biocompatibility. Low-temperature steam treatment causes silk fibroin to form a crystalline structure of Silk I, achieving the insolubility of tranexamic acid-loaded silk fibroin microneedles. Drug release from the insoluble microneedles containing a diaphragm layer can be divided into two steps: First, the blank microneedle body acts as a water conduction medium. After the microneedle is inserted into the skin, water molecules from the skin tissue fluid enter the microneedle and flow along the microneedle's channels into the drug reservoir, dissolving the tranexamic acid in the reservoir layer. This tranexamic acid is then slowly released through the diaphragm layer into the blank microneedle body. Second, the tranexamic acid entering the blank microneedle binds to the silk fibroin and is subsequently released into the skin via the blank microneedle. First, tranexamic acid in the drug reservoir binds to silk fibroin with a Silk I crystalline structure, delaying its release. Second, the membrane layer acts as a barrier to the release of the water-soluble small-molecule drug tranexamic acid, slowing its release rate. Third, when tranexamic acid passes through the membrane layer to the blank microneedle, it binds again to the hydrophilic silk fibroin with a Silk I crystalline structure, further delaying its release. The combined effect of the membrane layer's barrier function and the hydrophilic properties of silk fibroin results in a stable and prolonged drug release.

[0050] In this invention, the drug reservoir is located above the diaphragm layer and the blank microneedles. Utilizing the barrier effect of the diaphragm layer and the hydrophilicity of silk fibroin with a Silk I crystal structure, the release rate of tranexamic acid is slowed, resulting in a slow, constant, and long-lasting release. The tranexamic acid-loaded silk fibroin microneedles of this invention can delay the peak drug release time by 2 hours, and the overall release duration can reach over 14 hours.

[0051] Due to the adoption of the above technical solutions, this invention has the following advantages compared with the prior art: The diaphragm-type silk fibroin microneedle patch of this invention, through low-temperature water vapor treatment, enables the silk fibroin in the blank microneedles and drug storage layer to have a Silk I crystalline structure, achieving microneedle insolubility. Insoluble microneedles significantly reduce skin sensitization after insertion, provide a longer microneedle puncture maintenance time, and ensure the cumulative drug release rate. The addition of the diaphragm layer slows down the drug release rate, thereby delaying the peak drug release rate, allowing the drug to achieve a longer release duration and maintain a sufficiently high blood drug concentration, which is more significant for the treatment of melasma. The microneedles are easy to prepare, leave no residue after use, and have good biocompatibility.

[0052] The tranexamic acid-loaded diaphragm-type silk fibroin microneedle patch of the present invention, containing a drug storage layer, can increase the release time (14h in vitro, 24h in vivo) and form a large tranexamic acid concentration difference between the microneedle and the skin, providing a stable driving force for the transdermal release of tranexamic acid, so that a stable drug release rate is reached after a certain period of time, thereby achieving the purpose of long-term stable drug administration. Attached Figure Description

[0053] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

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

[0056] Figure 3 The cumulative drug release rate curves of the tranexamic acid-loaded silk fibroin microneedles provided in Examples 3-9 and Comparative Examples 1-2 of this invention are shown.

[0057] Figure 4 The drug release rate curves of the tranexamic acid-loaded silk fibroin microneedles provided in Examples 3-9 and Comparative Examples 1-2 of this invention are shown.

[0058] Figure 5 The in vivo drug release rate curve of the diaphragm-type silk fibroin microneedle patch provided in Example 7 of the present invention is compared with that of the injection group. Detailed Implementation

[0059] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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 in this embodiment includes the following steps:

[0061] Preparation of S1, tranexamic acid solution

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

[0063] S2, Preparation of the diaphragm layer solution

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

[0065] The polar volatile organic solvents are acetone or chloroform.

[0066] S3. Preparation of the drug reservoir solution

[0067] Tranexamic acid solution is added to silk fibroin solution and mixed thoroughly 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. The concentration of silk fibroin solution is 30-80 mg / mL.

[0068] S4. Preparation of blank microneedle layer

[0069] The silk fibroin solution was poured into a microneedle mold, and air bubbles were removed from both the solution and the microneedle template by vacuuming, ensuring the solution fully entered the needle cavity. The degassed mold system was then dried under constant temperature and humidity conditions with air circulation for 4-24 hours to obtain a blank microneedle layer. The concentration of the silk fibroin solution was 30-80 mg / mL. The constant temperature and humidity conditions were: temperature 20-40℃ and relative humidity 55-75%.

[0070] S5. Preparation of the diaphragm layer

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

[0072] S6. Preparation of the drug storage layer

[0073] The drug-loaded silk fibroin microneedle patch was prepared by pouring the drug-loaded solution prepared by S2 onto the diaphragm layer and drying it under constant temperature and humidity conditions with air circulation for 2-20 hours to obtain the patch. The constant temperature and humidity conditions were: temperature 20-40℃ and relative humidity 55-75%.

[0074] S7, Microneedle Patch Post-Processing

[0075] The drug-loaded silk fibroin microneedle patches obtained from S5 were placed in a low-temperature, high-humidity environment for 10-40 hours, then removed and dried to obtain septum-type silk fibroin microneedle patches. The low-temperature, high-humidity environment was defined as a temperature of 20-50℃ and a relative humidity of 85-99%, i.e., low-temperature steam treatment.

[0076] S8, backing layer

[0077] The dried diaphragm-type silk fibroin microneedles are removed, cut, backed, and packaged to obtain a tranexamic acid-loaded diaphragm-type silk fibroin microneedle patch product containing a diaphragm layer.

[0078] The silk fibroin solution mentioned above was obtained from silkworm silk through degumming, lithium bromide dissolution, and dialysis purification, with a concentration of 30-80 mg / mL.

[0079] The diaphragm-type silk fibroin microneedle patch prepared by the above steps includes, in sequence, a drug storage layer, a diaphragm layer, and a blank microneedle layer. The blank microneedle layer is composed of silk fibroin and includes a matrix and needles extending outward from the matrix. The drug storage layer covers the side of the diaphragm layer away from the needles. The drug storage layer includes silk fibroin and tranexamic acid. The diaphragm layer is located between the drug storage layer and the blank microneedle layer.

[0080] The membrane layer is made of cellulose acetate. The thickness of the membrane layer is 10μm-200μm, and the thickness of the drug storage layer is 100μm-2mm. Cellulose acetate has a certain degree of hydrophobicity, which, as a membrane layer, can reduce the diffusion rate of water-soluble molecules, thereby reducing the peak release rate.

[0081] The silk fibroin in the drug storage layer has a Silk I crystalline structure; the silk fibroin in the blank microneedle layer has a Silk I crystalline structure.

[0082] The tranexamic acid loading in the drug reservoir is 0.1-5 mg / cm³. 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 / cm2 of drug storage layer.

[0083] The needles in the blank microneedle layer are solid cones with a length of 200-900μm, a bottom diameter of 100-500μm, and a distance of 0.2-2mm between the tips of adjacent needles.

[0084] The aforementioned diaphragm-type silk fibroin microneedle patch consists of a drug reservoir layer, a diaphragm layer, and blank microneedles. It employs a low-temperature steam treatment process to form silk fibroin-loaded tranexamic acid microneedles with a Silk I crystal structure. Targeting the high water solubility and low molecular weight of tranexamic acid, a hydrophobic diaphragm layer is designed to slow its release. The diaphragm layer directly inhibits the release rate of tranexamic acid, resulting in tranexamic acid microneedles with a longer sustained-release time, improved bioavailability, and increased efficacy.

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

[0086] See Figure 1 As shown, the diaphragm-type silk fibroin microneedle patch of this embodiment includes three parts from top to bottom: a drug storage layer 1, a diaphragm layer 2, and blank microneedles 3. The blank microneedles 3 are composed solely of silk fibroin. The diaphragm 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] Blank microneedles comprise a matrix and needles extending outward from the matrix. The silk fibroin has a Silk I crystalline structure. The needles are solid cones with a length of 200-900 μm, a base diameter of 100-500 μm, and a spacing of 0.2-2 mm between the needles.

[0088] In the drug storage layer, tranexamic acid is uniformly mixed with silk fibroin solution in aqueous solution and dried to form a film. The loading of tranexamic acid is 0.1-5 mg / cm³. 2 .

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

[0090] In some embodiments, the cellulose acetate of the diaphragm layer 2 may be one of cellulose monoacetate, cellulose diacetate, and cellulose triacetate.

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

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

[0093] Weigh 3g NaHCO3 and 1g Na2CO3 and dissolve them in 4000mL of deionized water. Heat to boiling, add 80g of silkworm silk, and keep it simmering for 30 minutes. Remove and rinse thoroughly in deionized water.

[0094] After repeating the above steps three times, place the product in a 60℃ oven to dry and obtain degummed silk fibroin fibers.

[0095] Prepare a 9.3M LiBr aqueous solution. Take 100mL of the LiBr aqueous solution and heat it to 65℃ in a water bath. Add 15g of degummed silk fibroin fiber and stir to dissolve. Continue heating and stirring for 40min. After cooling, transfer the solution into a dialysis bag and dialyze it in deionized water for 72h, changing the deionized water every 2h.

[0096] After dialysis, the solution is filtered to obtain a silk fibroin solution, which is then stored 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 includes the following steps:

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

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

[0101] Preparation of cellulose diacetate solution: Weigh a certain amount of cellulose diacetate powder and dissolve it in acetone solution to make the concentration of cellulose diacetate in the solution 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 applied three times to remove air bubbles from the solution and the microneedle template, ensuring that the solution fully entered the needle cavity. The degassed mold system was then dried in a constant temperature and humidity chamber (25℃, 60%) with good air circulation for 10 hours to obtain blank microneedles with a length of 600 μm, a bottom diameter of 250 μm, and a spacing of 0.56 mm between the tips of adjacent needles.

[0106] 4) Preparation of the membrane layer

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

[0108] 5) Preparation of drug storage layer

[0109] Take the silk fibroin tranexamic acid solution prepared in step (2) and slowly pour it onto the surface of the diaphragm layer prepared in step (4). Place it in a constant temperature and humidity room (25℃, 60%) with air circulation and dry for 12 hours. Demold to obtain silk fibroin microneedles loaded with tranexamic acid containing the diaphragm layer.

[0110] 6) Low-temperature steam treatment

[0111] Place 0.05% of the volume of deionized water at the bottom of the vacuum drying oven. Lay 50g of pure cotton nonwoven fabric flat on the bottom of the petri dish, and then cover it with a 45g layer of pure cotton nonwoven fabric.

[0112] Place the silk fibroin tranexamic acid microneedles obtained in step (5) into the chamber. Close the chamber door, evacuate to a vacuum degree of -0.09 MPa, maintain a temperature of 37°C and a humidity of 85%, process for 14 hours, then remove and dry to obtain insoluble silk fibroin tranexamic acid microneedles.

[0113] 7) Take out the dried silk fibroin tranexamic acid microneedles, cut them, attach a backing layer, and package them 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 includes the following steps:

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

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

[0118] Preparation of cellulose diacetate solution: Weigh a certain amount of cellulose diacetate powder and dissolve it in acetone solution to make the concentration of cellulose diacetate in the solution 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 applied three times to remove air bubbles from the solution and the microneedle template, ensuring that the solution fully entered the needle cavity. The degassed mold system was then dried in a constant temperature and humidity chamber (27℃, 55%) with good air circulation for 6 hours to obtain blank microneedles with a length of 400 μm, a bottom diameter of 200 μm, and a spacing of 0.87 mm between the tips of adjacent needles.

[0123] 4) Preparation of the membrane layer

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

[0125] 5) Preparation of drug storage layer

[0126] Take the silk fibroin tranexamic acid solution prepared in step (2) and slowly pour it onto the surface of the diaphragm layer prepared in step (4). Place it in a constant temperature and humidity room (27℃, 55%) with air circulation and dry for 14 hours. Demold to obtain silk fibroin microneedles loaded with tranexamic acid containing the diaphragm layer.

[0127] 6) Low-temperature steam treatment

[0128] Place 0.07% of the volume of deionized water at the bottom of the vacuum drying oven. Spread 60g of pure cotton nonwoven fabric on the bottom of the petri dish, and cover it with a 50g layer of pure cotton nonwoven fabric. Place the silk fibroin tranexamic acid microneedles obtained in step (5) inside. Close the oven door, evacuate to a vacuum degree of -0.08Mpa, maintain a temperature of 40℃ and a humidity of 89%, and process for 20h. After processing, remove the microneedles and dry them to obtain insoluble silk fibroin tranexamic acid microneedles.

[0129] 7) Take out the dried silk fibroin tranexamic acid microneedles, cut them, attach a backing layer, and package them 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 includes the following steps:

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

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

[0134] Preparation of cellulose diacetate solution: Weigh a certain amount of cellulose diacetate powder and dissolve it in acetone solution to make the concentration of cellulose diacetate in the solution 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 applied three times to remove air bubbles from the solution and the microneedle template, ensuring that the solution fully entered the needle cavity. The degassed mold system was then dried in a constant temperature and humidity chamber (38℃, 58%) with good air circulation for 8 hours to obtain blank microneedles with a length of 500 μm, a bottom diameter of 300 μm, and a 1.7 mm spacing between the tips of adjacent needles.

[0139] 4) Preparation of the membrane layer

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

[0141] 5) Preparation of drug storage layer

[0142] Take the silk fibroin tranexamic acid solution prepared in step (2) and slowly pour it onto the surface of the diaphragm layer prepared in step (4). Place it in a constant temperature and humidity room (38℃, 58%) with air circulation and dry for 18 hours. Demold to obtain silk fibroin microneedles loaded with tranexamic acid containing the diaphragm layer.

[0143] 6) Low-temperature steam treatment

[0144] Place 0.08% of the volume of deionized water at the bottom of the vacuum drying oven. Spread 80g of pure cotton nonwoven fabric on the bottom of the petri dish, and cover it with a 60g layer of pure cotton nonwoven fabric. Place the silk fibroin tranexamic acid microneedles obtained in step (5) inside. Close the oven door, evacuate to a vacuum degree of -0.06Mpa, maintain a temperature of 42℃ and a humidity of 92%, and treat for 15h. After treatment, remove the microneedles and dry them to obtain insoluble silk fibroin tranexamic acid microneedles.

[0145] 7) Take out the dried silk fibroin tranexamic acid microneedles, cut them, attach a backing layer, and package them to obtain a silk fibroin tranexamic acid microneedle patch containing a diaphragm layer.

[0146] Preparation method of Example 6

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

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

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

[0150] Preparation of cellulose triacetate solution: Weigh a certain amount of cellulose triacetate powder and dissolve it in chloroform solution to make the concentration of cellulose triacetate in the solution 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 applied three times to remove air bubbles from the solution and the microneedle template, ensuring that the solution fully entered the needle cavity. The degassed mold system was then dried in a constant temperature and humidity chamber (29℃, 68%) with air circulation for 10 hours to obtain blank microneedles with a length of 700 μm, a bottom diameter of 350 μm, and a 0.50 mm interval between the tips of adjacent needles.

[0155] 4) Preparation of the membrane layer

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

[0157] 5) Preparation of drug storage layer

[0158] Take the silk fibroin tranexamic acid solution prepared in step (2) and slowly pour it onto the surface of the diaphragm layer prepared in step (4). Place it in a constant temperature and humidity room (29℃, 68%) with air circulation and dry for 17 hours. Demold to obtain silk fibroin microneedles loaded with tranexamic acid containing the diaphragm layer.

[0159] 6) Low-temperature steam treatment

[0160] Place 0.05% of the volume of deionized water at the bottom of the vacuum drying oven. Spread 80g of pure cotton nonwoven fabric on the bottom of the petri dish, and cover it with a 60g layer of pure cotton nonwoven fabric. Place the silk fibroin tranexamic acid microneedles obtained in step (5) inside. Close the oven door, evacuate to a vacuum degree of -0.06Mpa, maintain a temperature of 41℃ and a humidity of 87%, and treat for 18h. After treatment, remove the microneedles and dry them to obtain insoluble silk fibroin tranexamic acid microneedles.

[0161] 7) Take out the dried silk fibroin tranexamic acid microneedles, cut them, attach a backing layer, and package them 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 includes the following steps:

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

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

[0166] Preparation of cellulose triacetate solution: Weigh a certain amount of cellulose triacetate powder and dissolve it in chloroform solution to make the concentration of cellulose triacetate in the solution 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. Vacuum was applied three times to remove air bubbles from the solution and the microneedle template, ensuring the solution fully entered the needle cavity. The degassed mold system was then dried in a constant temperature and humidity chamber (35℃, 52%) with good air circulation for 7 hours to obtain blank microneedles with a length of 800 μm, a bottom diameter of 440 μm, and a 0.95 mm spacing between the tips of adjacent needles.

[0171] 4) Preparation of the membrane layer

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

[0173] 5) Preparation of drug storage layer

[0174] Take the silk fibroin tranexamic acid solution prepared in step (2) and slowly pour it onto the surface of the diaphragm layer prepared in step (4). Place it in a constant temperature and humidity room (35℃, 52%) with air circulation and dry for 18 hours. Demold to obtain silk fibroin microneedles loaded with tranexamic acid containing the diaphragm layer.

[0175] 6) Low-temperature steam treatment

[0176] Place 0.06% of the volume of deionized water at the bottom of the vacuum drying oven. Spread 50g of pure cotton nonwoven fabric on the bottom of the petri dish, and cover it with a 45g layer of pure cotton nonwoven fabric. Place the silk fibroin tranexamic acid microneedles obtained in step (5) inside. Close the oven door, evacuate to a vacuum degree of -0.10Mpa, maintain a temperature of 37℃ and a humidity of 85%, and treat for 19h. After treatment, remove the microneedles and dry them to obtain insoluble silk fibroin tranexamic acid microneedles.

[0177] 7) Take out the dried silk fibroin tranexamic acid microneedles, cut them, attach a backing layer, and package them 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 includes the following steps:

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

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

[0182] Preparation of cellulose triacetate solution: Weigh a certain amount of cellulose triacetate powder and dissolve it in chloroform solution to make the concentration of cellulose triacetate in the solution 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 applied three times to remove air bubbles from the solution and the microneedle template, ensuring that the solution fully entered the needle cavity. The degassed mold system was then dried in a constant temperature and humidity chamber (23℃, 47%) with good air circulation for 9 hours, resulting in blank microneedles with a length of 550 μm, a bottom diameter of 400 μm, and a 1.1 mm interval between the tips of adjacent needles.

[0187] 4) Preparation of the membrane layer

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

[0189] 5) Preparation of drug storage layer

[0190] Take the silk fibroin tranexamic acid solution prepared in step (2) and slowly pour it onto the surface of the diaphragm layer prepared in step (4). Place it in a constant temperature and humidity room (23℃, 47%) with air circulation and dry for 14 hours. Demold to obtain silk fibroin microneedles loaded with tranexamic acid containing the diaphragm layer.

[0191] 6) Low-temperature steam treatment

[0192] Place 0.1% of the volume of deionized water at the bottom of the vacuum drying oven. Spread 56g of pure cotton nonwoven fabric on the bottom of the petri dish, and cover it with a 38g layer of pure cotton nonwoven fabric. Place the silk fibroin tranexamic acid microneedles obtained in step (5) inside. Close the oven door, evacuate to a vacuum degree of -0.09Mpa, maintain a temperature of 38℃ and a humidity of 90%, and process for 20h. After processing, remove the microneedles and dry them to obtain insoluble silk fibroin tranexamic acid microneedles.

[0193] 7) Take out the dried silk fibroin tranexamic acid microneedles, cut them, attach a backing layer, and package them 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 includes the following steps:

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

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

[0198] Preparation of cellulose monoacetate solution: Weigh a certain amount of cellulose monoacetate powder and dissolve it in acetone solution to make the concentration of cellulose monoacetate in the solution 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 applied three times to remove air bubbles from the solution and the microneedle template, ensuring that the solution fully entered the needle cavity. The degassed mold system was then dried in a constant temperature and humidity chamber (39℃, 50%) with good air circulation for 5 hours to obtain blank microneedles with a length of 700 μm, a bottom diameter of 400 μm, and a spacing of 0.75 mm between the tips of adjacent needles.

[0203] 4) Preparation of the membrane layer

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

[0205] 5) Preparation of drug storage layer

[0206] Take the silk fibroin tranexamic acid solution prepared in step (2) and slowly pour it onto the surface of the diaphragm layer prepared in step (4). Place it in a constant temperature and humidity room (39℃, 50%) with air circulation and dry for 10 hours. Demold to obtain silk fibroin microneedles loaded with tranexamic acid containing the diaphragm layer.

[0207] 6) Low-temperature steam treatment

[0208] Place 0.03% of the volume of deionized water at the bottom of the vacuum drying oven. Spread 62g of pure cotton nonwoven fabric on the bottom of the petri dish, and cover it with a 30g layer of pure cotton nonwoven fabric. Place the silk fibroin tranexamic acid microneedles obtained in step (5) inside. Close the oven door, evacuate to a vacuum degree of -0.08Mpa, maintain a temperature of 36℃ and a humidity of 91%, and process for 24h. After processing, remove the microneedles and dry them to obtain insoluble silk fibroin tranexamic acid microneedles.

[0209] 7) Take out the dried silk fibroin tranexamic acid microneedles, cut them, attach a backing layer, and package them 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 its preparation method includes the following steps:

[0212] 1) Preparation of tranexamic acid solution

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

[0214] 2) Prepare a tranexamic acid-silk fibroin 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 vacuumed three times to remove air bubbles from 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℃, 60%) with air circulation and dried for 16 h to obtain silk fibroin tranexamic acid microneedles without a diaphragm layer. The needle length was 500 μm, the bottom diameter was 300 μm, and the needle tip of adjacent needles was spaced 0.5 mm apart.

[0218] 4) Low-temperature steam treatment

[0219] Place 0.05% of the volume of deionized water at the bottom of the vacuum drying oven. Spread 53g of pure cotton nonwoven fabric on the bottom of the petri dish, and cover it with a 40g layer of pure cotton nonwoven fabric. Place the skeleton-type microneedles obtained in step (3) inside. Close the oven door, evacuate to a vacuum degree of -0.09Mpa, maintain a temperature of 37℃ and a humidity of 90%, and process for 24h. After processing, remove the microneedles and dry them to obtain insoluble silk fibroin microneedles.

[0220] 5) Remove the dried skeleton-shaped microneedles, cut them, attach a backing layer, and package them to obtain a silk fibroin tranexamic acid microneedle patch without a diaphragm layer.

[0221] Comparative Example 2

[0222] This comparative example is a silk fibroin tranexamic acid microneedle containing only blank microneedles and a drug storage layer, without a diaphragm layer. The preparation method includes the following steps:

[0223] 1) Preparation of tranexamic acid solution

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

[0225] 2) Prepare a tranexamic acid-silk fibroin 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 applied three times to remove air bubbles from the solution and the microneedle template, ensuring that the solution fully entered the needle cavity. The degassed mold system was then dried in a constant temperature and humidity chamber (30℃, 65%) with good air circulation for 7 hours to obtain blank microneedles with a length of 500 μm, a bottom diameter of 300 μm, and a 0.5 mm spacing between the tips of adjacent needles.

[0229] 4) Preparation of the drug storage layer

[0230] Take the silk fibroin tranexamic acid solution prepared in step (2) and slowly pour it onto the surface of the blank microneedles prepared in step (3). Place them in a constant temperature and humidity room (30℃, 65%) with air circulation and dry for 13 hours. Demold to obtain the separated silk fibroin microneedles loaded with tranexamic acid.

[0231] 5) Low-temperature steam treatment

[0232] Place 0.05% of the volume of deionized water at the bottom of the vacuum drying oven. Spread 48g of pure cotton nonwoven fabric on the bottom of the petri dish, and cover it with another 42g of pure cotton nonwoven fabric. Place the skeleton-type microneedles obtained in step (3) inside. Close the oven door, evacuate to a vacuum degree of -0.09Mpa, maintain a temperature of 37℃ and a humidity of 90%, and process for 24h. After processing, remove the microneedles and dry them to obtain insoluble silk fibroin microneedles.

[0233] 5) Remove the dried microneedles, cut them, attach a backing layer, and package them to obtain a separate silk fibroin tranexamic acid microneedle patch without a diaphragm layer.

[0234] Tests and Results Discussion

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

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

[0237] Frozen bullfrog skin was thawed in physiological saline, and then the abdominal portion was cut into small pieces. A Franz diffusion cell was used as the in vitro drug receiving cell, with deionized water as the simulated body fluid; the receiving cell volume was 6 mL. Microneedles as used in Examples 3-9 and Comparative Examples 1-2 were inserted into the bullfrog skin. The bullfrog skin was then smoothly placed over the circular receiving chamber of the Franz diffusion cell, ensuring the dermis of the bullfrog skin was in contact with the solution in the receiving cell. The donor and receiver chambers were secured with stainless steel clamps.

[0238] (2) Acquisition of the test solution

[0239] In the drug release device pool of the above steps, 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 solutions: 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 sample introduction.

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

[0242] (5) Detection of the sustained-release effect of microneedles by high performance liquid chromatography

[0243] The sample solutions obtained in step (2) are injected sequentially into the liquid chromatograph, and the chromatograms are recorded. The corresponding concentrations are calculated based on the standard curve of tranexamic acid concentration versus peak area obtained in step (4). A drug release rate curve is plotted, as shown below. Figure 3 and 4 As shown.

[0244] II. Pharmacokinetic analysis of the diaphragm-type silk fibroin microneedles prepared in Example 7 in guinea pigs with brown spots:

[0245] (1) Application of microneedle patches

[0246] Shave the back of the guinea pig model with brown spots using a razor to remove the fine downy hairs, and rinse off any remaining depilatory cream with running water. Cut a medical dressing to a size of 10cm × 3cm, place the microneedle patch in the center of the dressing, and then place the microneedle patch on the guinea pig's back, pressing the patch with a force of 3N. Ensure the microneedles are firmly inserted into the skin.

[0247] (2) Pharmacokinetic detection

[0248] The microneedle group and the injection group received the same single-dose administration. In the microneedle group, tail vein blood was collected at 0, 1, 2, 4, 6, 8, 10, and 12 hours after microneedle patch application (N=3). In the injection group, tail vein blood was collected at 0, 0.17, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 hours after injection (N=3). Each blood sample was approximately 10 μL. Chromatographic grade methanol was added at a ratio of blood sample to methanol of 1:5 (v:v), vortexed for 1 min to mix thoroughly, and the supernatant was collected by centrifugation for liquid chromatography analysis.

[0249] The cumulative release rate curves of the microneedle patches prepared in Examples 3-9 and Comparative Examples 1-2 are shown below. Figure 3As shown. Compared with Comparative Examples 1-2, the cumulative drug release rate of the microneedles prepared in Example 9 was lower, but the drug release time of Example 9 was significantly increased. The release effect of the microneedles prepared in Examples 3-4 and 6-8 was better than that of Example 9. The microneedles prepared in Examples 3-5 were loaded with cellulose diacetate membranes, and the cumulative release rate decreased with the increase of microneedle spacing. When the microneedle spacing is too large, the number of microneedles per unit area is small, which is not conducive to the transport of water molecules and tranexamic acid. As in Example 5, the cellulose diacetate membrane has a strong ability to permeate water molecules, and the change in microneedle spacing becomes the biggest influencing factor on the release of tranexamic acid. The microneedles prepared in Examples 6-8 were loaded with cellulose triacetate membranes, and the cumulative release rate was all above 80%, which was higher than that of Comparative Example 1. The change in microneedle spacing did not have a significant impact on the microneedles prepared in Examples 6-8, and the microneedles had 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. Comparative Example 1 showed a peak release rate at 0.5 h, and the release rate at around 4 h was lower than that of the microneedles prepared in the examples, indicating that the drug release rate decreased significantly with time. Comparative Example 2, based on Comparative Example 1, controlled the microneedle tip to not be loaded with drug, successfully delaying the peak release rate to 1 h, and the peak release rate at around 4 h was still lower than that of the microneedles prepared in the examples. In the examples, by adding a cellulose acetate membrane layer to the back of the silk fibroin microneedles, the microneedles had a certain slow release effect, and the drug release time was extended to 10 h. Examples 3-5, loaded with cellulose diacetate, compared with Comparative Example 1, maintained a certain release rate value and extended the peak release rate to 2 h (203 μg / h·cm). 2 The membrane layer is made of cellulose triacetate. Compared with Examples 3-5, Examples 6-8 also ensured a certain release rate and an extended peak release rate (2h, 279μg / h·cm). 2 As the distance between the needle tips in the microneedles increases, the microneedles can maintain a stable and relatively close release rate curve, with a release duration of up to 14 hours.

[0251] The diaphragm-type silk fibroin microneedle patch of this invention is a transdermal drug delivery technology that avoids the first-pass effect in the liver and gastrointestinal inactivation associated with oral administration, thus improving bioavailability. It can replace injection to achieve higher bioavailability, reduce patient discomfort, and improve patient compliance. Figure 5 As shown, the diaphragm-type microneedle patch prepared in Example 7 was applied to mice, and a single microneedle patch could maintain a tranexamic acid blood concentration for 24 hours. Compared to injection, the bioavailability of the microneedle patch prepared in Example 7 reached 264% relative to the injection group.

[0252] The silk fibroin tranexamic acid microneedle patch of the present invention comprises, from top to bottom, three parts: a drug reservoir layer, a diaphragm layer, and blank microneedles. The blank microneedles are composed solely of silk fibroin; the drug reservoir layer consists of silk fibroin and tranexamic acid, wherein the tranexamic acid loading is 0.1-5 mg / cm³. 2 The diaphragm layer is composed of any one of cellulose acetate, cellulose diacetate, or cellulose triacetate. The corresponding preparation method includes solution preparation, mold casting, vacuum degassing, casting of the diaphragm layer and drug storage layer, and drying and molding. The tranexamic acid diaphragm-type silk fibroin microneedle patch prepared by this invention can prolong the release time of the microneedle patch, ensure the release amount reaches the therapeutic level, and solve the burst release phenomenon of tranexamic acid. The microneedles are easy to prepare and use. The blank microneedles, diaphragm layer, and drug storage layer form a whole, eliminating the need for replacement or external drug storage devices, reducing unnecessary trouble.

[0253] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

[0254] The endpoints and any values ​​of the ranges disclosed herein 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 the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

Claims

1. A diaphragm-type silk fibroin microneedle patch, characterized in that, The device comprises, in sequence, a drug storage layer, a diaphragm layer, and a blank microneedle layer. The blank microneedle layer is composed of silk fibroin and includes a matrix and needles extending outward from the matrix. The drug storage layer covers the side of the diaphragm layer away from the needles. The drug storage layer includes silk fibroin and tranexamic acid. The diaphragm layer is located between the drug storage layer and the blank microneedle layer. The diaphragm layer is made of cellulose acetate.

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

3. The diaphragm-type silk fibroin microneedle patch according to claim 1, characterized in that, The crystalline structure of the silk fibroin in the drug storage layer is the SilkI crystalline structure; and / or, the crystalline structure of the silk fibroin in the blank microneedle layer is the SilkI crystalline structure.

4. The diaphragm-type silk fibroin microneedle patch according to claim 1, characterized in that, The tranexamic acid loading in the drug reservoir is 0.1-5 mg / cm³. 2 .

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

6. A method for preparing a diaphragm-type silk fibroin microneedle patch as described in any one of claims 1-5, characterized in that, Includes the following steps: Prepare silk fibroin solution, diaphragm layer solution and drug storage layer solution separately; The silk fibroin solution was poured into the microneedle mold, degassed, and the degassed mold system was dried under constant temperature and humidity conditions to obtain the blank microneedle layer. The membrane layer solution was poured onto the substrate of the blank microneedle layer and dried to obtain blank microneedles with a membrane layer. The drug storage layer solution was poured onto the diaphragm layer and dried under constant temperature and humidity conditions to obtain a drug-loaded silk fibroin microneedle patch. The drug-loaded silk fibroin microneedle patch was placed in an environment with a temperature of 20-50℃ and a relative humidity of 85-99% for 10-40 hours, then removed and dried to obtain the diaphragm-type silk fibroin microneedle patch.

7. The preparation method according to claim 6, characterized in that, The membrane layer solution is prepared by dissolving cellulose acetate in a polar volatile organic solvent to obtain a membrane layer solution with a concentration of 10-100 mg / mL.

8. The preparation method according to claim 6, characterized in that, The drug storage layer solution is prepared by mixing tranexamic acid solution and silk fibroin solution evenly to form a drug storage layer solution; the concentration of silk fibroin in the drug storage layer solution is 20-50 mg / mL, and the concentration of tranexamic acid is 10-30 mg / mL.

9. The preparation method according to claim 8, characterized in that, The tranexamic acid solution is prepared as follows: tranexamic acid powder is weighed, deionized water is added, and the tranexamic acid is completely dissolved to obtain a tranexamic acid solution, 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.

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