Bird's nest peptide microneedle patch capable of resisting skin photoaging and preparation method and application of bird's nest peptide microneedle patch

Abstract

The invention provides an anti-skin photoaging cubilose peptide microneedle patch and a preparation method and application thereof, the anti-skin photoaging cubilose peptide microneedle patch comprises a base and a plurality of microneedles, and the plurality of microneedles are distributed on the base; the microneedle comprises a functional component and a supporting component; the functional component comprises cubilose peptide, and the supporting component comprises polyvinylpyrrolidone. The cubilose peptide is combined with a microneedle patch technology, so that the delivery efficiency and bioavailability of the active cubilose peptide are improved, and the application potential of the cubilose peptide in the aspect of anti-skin photoaging repair is enhanced. Moreover, the cubilose peptide microneedle patch disclosed by the invention has good stability, biocompatibility and targeting property, and a new solution is provided for resisting skin photoaging.

Description

Technical Field

[0001] The present application relates to the biological field. Specifically, the present application relates to a bird's nest peptide microneedle patch for resisting skin photoaging, and its preparation method and application. Background Art

[0002] In today's society, with the improvement of people's living standards and the pursuit of health and beauty, the research and development of anti-aging products and technologies has attracted widespread attention. Photoaging is one of the main factors contributing to skin aging. It is caused by factors such as long-term exposure to ultraviolet radiation, environmental pollution, and lifestyle. Photoaging can lead to cosmetic problems such as wrinkles, sagging, and hyperpigmentation. Therefore, the development of effective anti-photoaging products has significant market prospects.

[0003] Small molecule active peptides play a key role in anti-aging cosmetics and pharmaceuticals due to their unique bioactivity and excellent skin permeability. These peptides, typically composed of a few amino acid residues, possess characteristics such as low molecular weight, simple structure, ease of absorption, and high bioavailability. They can penetrate the skin barrier and act directly on the epidermis and dermis, exerting their biological effects, such as antioxidant, anti-inflammatory, and promotion of collagen synthesis, thereby combating photoaging.

[0004] However, despite the theoretical potential of small-molecule active peptides, several challenges remain in their practical application. First, the stability and activity of small-molecule peptides are easily affected by external environmental factors during storage and use. Second, how to effectively deliver small-molecule peptides deep into the skin to maximize their effectiveness at the target location is another issue that needs to be addressed. Furthermore, the structure and physicochemical properties of small-molecule peptides are closely related to their ability to penetrate the skin barrier.

[0005] To overcome these challenges, researchers have been searching for more effective carrier systems and preparation methods to achieve stable delivery and efficient utilization of small molecule active peptides. As a novel transdermal drug delivery system, microneedle patches can penetrate the stratum corneum through their tiny needles and deliver drugs directly to the epidermis and dermis, thereby improving drug bioavailability and efficacy. However, existing microneedle patch technology still needs to be improved in terms of drug loading, delivery efficiency, and biocompatibility. Summary of the Invention

[0006] This application aims to at least partially address the technical problems existing in the prior art. To this end, this application proposes a bird's nest peptide microneedle patch for combating skin photoaging, as well as its preparation method and application. By combining bird's nest peptide with microneedle patch technology, this application not only improves the delivery efficiency and bioavailability of active bird's nest peptides, but also enhances their potential for application in combating skin photoaging and repair. Furthermore, the bird's nest peptide microneedle patch of this application has good stability, biocompatibility, and targeting, providing a new solution for combating skin photoaging.

[0007] In one aspect, the present application provides a bird's nest peptide microneedle patch for anti-skin photoaging. According to an embodiment of the present application, the bird's nest peptide microneedle patch for anti-skin photoaging comprises: a base and a plurality of microneedles, the plurality of microneedles being distributed on the base; the microneedles comprising a functional component and a supporting component; the functional component comprising a bird's nest peptide, and the supporting component comprising polyvinyl pyrrolidone.

[0008] This application combines bird's nest peptide with microneedle patch technology to improve the delivery efficiency and bioavailability of active bird's nest peptide, thereby improving its anti-skin photoaging and repair effects. Polyvinylpyrrolidone (PVP) is a high molecular polymer with a chain vinyl structure. It has good amphiphilicity, complexation and biocompatibility and does not cause any irritation to the skin, mucous membranes, etc. Polyvinylpyrrolidone can form a complex with bird's nest peptide through chemical bond interaction to achieve the purpose of delivery. In addition, using polyvinylpyrrolidone (PVP) as a microneedle supporting component, the microneedle needle rate is high, the color is transparent, and the needle body shape is basically intact. The deformation degree is low during the drying process, the needle body has no obvious cavitation, the needle body is strong, and it is not easy to break. In addition, the dissolution characteristics of the microneedle are strong, the swelling performance is stable, the efficiency of transdermal delivery of peptides is high, and the rate is stable, thereby achieving the rapid release of bird's nest peptide in the interstitial fluid of the skin tissue, improving the delivery efficiency of bird's nest peptide, and ensuring the stability and effectiveness of bird's nest peptide.

[0009] According to the embodiments of the present application, the anti-skin photoaging bird's nest peptide microneedle patch may also have the following additional technical features:

[0010] According to an embodiment of the present application, the method for preparing the bird's nest peptide includes: subjecting the bird's nest to an enzymatic hydrolysis treatment, wherein the enzyme used in the enzymatic hydrolysis treatment includes at least one of alkaline protease, neutral protease and flavor protease.

[0011] According to an embodiment of the present application, the enzymes used in the enzymatic hydrolysis treatment include: a combination of alkaline protease and neutral protease, or a combination of alkaline protease and flavor protease.

[0012] According to an embodiment of the present application, in the combination of alkaline protease and neutral protease, the mass ratio of alkaline protease to neutral protease is 1:(0.1-5).

[0013] According to an embodiment of the present application, in the combination of alkaline protease and flavor protease, the mass ratio of alkaline protease to flavor protease is 1:(0.1-5).

[0014] According to an embodiment of the present application, the temperature of the enzymatic hydrolysis treatment is 45 to 65° C., and the time is 2 to 8 hours.

[0015] According to an embodiment of the present application, the preparation method of the bird's nest peptide includes: (a) crushing and sieving the bird's nest, soaking the obtained crushed material with water to obtain a bird's nest liquid; (b) discarding the supernatant of the bird's nest liquid, adding water, and sequentially performing microwave heating treatment and ultrasonic treatment to obtain a bird's nest protein aqueous solution; (c) performing the enzymatic hydrolysis treatment on the bird's nest protein aqueous solution to obtain the bird's nest peptide.

[0016] According to an embodiment of the present application, the protein content of the bird's nest protein aqueous solution is 55-65%; the amount of enzyme used in the enzymatic hydrolysis treatment is 2000-15000 U / g bird's nest protein aqueous solution.

[0017] According to an embodiment of the present application, the particle size of the pulverized material is 50-100 mesh.

[0018] According to an embodiment of the present application, the temperature of the microwave heating treatment is 100-200° C., the time is 5-20 minutes, and the power is 300-500W.

[0019] According to an embodiment of the present application, the temperature of the ultrasonic treatment is 30-40° C., the time is 20-40 min, and the frequency is 40-50 Hz.

[0020] According to the embodiments of the present application, the enzyme dosage for the enzymatic hydrolysis treatment is 2000 to 15000 U / g of bird's nest protein aqueous solution.

[0021] According to an embodiment of the present application, the bird's nest peptide is provided in the form of a peptide mixture, in which peptides with a molecular weight of less than 3000Da account for more than 70%, and peptides with a molecular weight of less than 1000Da account for 65% to 70%; at least some of the peptides in the peptide mixture have hydrophobic amino acids at the C-terminus and / or N-terminus.

[0022] According to an embodiment of the present application, the preparation method of the bird's nest peptide further includes: drying the bird's nest peptide to obtain a bird's nest peptide powder; the particle size of the bird's nest peptide powder is 65 to 75 nm, and the PDI value is less than 0.5.

[0023] According to an embodiment of the present application, the functional component accounts for 1% to 5% of the mass of the microneedle.

[0024] According to an embodiment of the present application, the length of the microneedle is 400 to 800 μm.

[0025] According to an embodiment of the present application, the microneedle includes a top and a bottom, and the bottom is connected to the base; the distance between the tops of two adjacent microneedles is 250 to 650 μm; and / or the diameter of the bottom is 200 to 600 μm.

[0026] According to an embodiment of the present application, the microneedle is pyramidal, conical or quasi-conical in shape.

[0027] According to an embodiment of the present application, the surface of the microneedle is covered with a pH-responsive sealing layer.

[0028] According to an embodiment of the present application, the pH-responsive sealing layer includes at least one of methacrylic acid copolymer type C, polyvinyl acid resin II, and polyvinyl acid resin III.

[0029] According to an embodiment of the present application, the base has the same composition as that of the microneedle.

[0030] According to an embodiment of the present application, the base and the microneedle are integrally formed.

[0031] In another aspect of the present application, a method for preparing the aforementioned bird's nest peptide microneedle patch for anti-skin photoaging is proposed. According to an embodiment of the present application, the method comprises: providing a microneedle slurry containing the functional component and the supporting component and a microneedle template; pouring the microneedle slurry onto the mold cavity of the microneedle template so that the microneedle slurry completely covers the mold cavity; performing a vacuum treatment and a drying treatment, and demolding to obtain the bird's nest peptide microneedle patch.

[0032] According to an embodiment of the present application, the method includes: 1) dissolving the bird's nest peptide powder in water to obtain a bird's nest peptide aqueous solution; 2) stirring the bird's nest peptide aqueous solution and polyvinyl pyrrolidone until the polyvinyl pyrrolidone is completely dissolved, and then performing equilibrium dissolution to obtain a dissolving solution, wherein the temperature of the equilibrium dissolution is 35-40°C, the time is 20-40 minutes, and the concentration of polyvinyl pyrrolidone in the dissolving solution is 30% by mass to 60% by mass; 3) pouring the dissolving solution on the mold cavity of the microneedle template so that the microneedle liquid completely covers the mold cavity, vacuum treating for 10-30 minutes, removing excess liquid and bubbles with bubbles on the surface, re-adding the dissolving solution and vacuum treating for 1-5 times; 4) drying the assembled microneedle template at 37-50°C for 2-6 hours, then demolding and drying to obtain the bird's nest peptide microneedle patch.

[0033] In another aspect of the present application, the present application proposes the use of the aforementioned anti-skin photoaging bird's nest peptide microneedle patch in anti-skin photoaging.

[0034] The bird's nest peptide microneedle patch of the present application has a simple preparation process, stable chemical properties, can effectively penetrate the stratum corneum of the skin, and dissolve in the interstitial fluid of the epidermis and the upper dermis, releasing the bird's nest peptide to the epidermis and dermis, thereby combating skin photoaging damage, playing a repair and anti-aging role, and providing a new solution for anti-skin photoaging repair. Importantly, the microneedle substrate (PVP-K30, with high viscosity) selected in this application is a supporting material for a specific bird's nest peptide (segment), and the bird's nest peptide prepared by a specific method is characterized by a high small molecule peptide content (<1000Da accounts for 65-70%), uniform particle size, and a high distribution of hydrophobic amino acids such as Leu, Val and Phe at the C-terminus and N-terminus. Therefore, the present application utilizes PVP molecules to combine with the hydrophobic amino acids at the C-terminus or N-terminus of the prepared bird's nest peptide segment to form an amphiphilic copolymer, thereby achieving effective delivery of the bird's nest peptide. The bird's nest peptide microneedle patch can be used in the fields of medicine and beauty. It is prepared by biosoluble microneedles of small molecule substances that are treated by percutaneous or transdermal intervention, thereby broadening its application range and providing the possibility of personalized treatment and precision medicine.

[0035] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0037] Figure 1 This is a flow chart for the preparation of the bird's nest peptide microneedle patch with anti-skin photoaging and repair activity in this application.

[0038] Figure 2 Soluble microneedles prepared from polyvinyl pyrrolidone (PVP, top) and hyaluronic acid (HA, bottom) in Example 1 and Comparative Example 1.

[0039] Figure 3 This is a bar graph showing the results of the anti-skin cell photoaging repair activity of bird's nest peptides hydrolyzed by single enzyme and combined enzyme in Example 2 and Example 3.

[0040] Figure 4 This is a graph showing the cytotoxicity and anti-skin cell photoaging repair activity results of the bird's nest peptide prepared by alkaline-neutral protease hydrolysis in Example 4.

[0041] Figure 5The polyvinyl pyrrolidone-bird's nest peptide (PVP-bird's nest peptide) soluble microneedle (top) and hyaluronic acid-bird's nest peptide (HA-bird's nest peptide) soluble microneedle (bottom) in Example 5 and Comparative Example 2.

[0042] Figure 6 The morphology and dissolution and swelling characteristics of the HA-bird's nest peptide microneedles in Comparative Example 2 and the PVP-bird's nest peptide microneedles in Example 5.

[0043] Figure 7 This is a bar graph showing the peptide concentration in the receptor pool (A) and the diffusion efficiency of the PVP-bird's nest peptide microneedles (B) at different time points in Example 6.

[0044] Figure 8 This is a bar graph of the diffusion rate per unit area (A) and the cumulative amount of permeated peptide per unit area (B) of the PVP-bird's nest peptide microneedles in Example 6. DETAILED DESCRIPTION

[0045] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

[0046] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of this application, unless otherwise specified, "plurality" means two or more.

[0047] The endpoints of the ranges and any values ​​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 endpoints of each range, the endpoints of each range and 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 to be specifically disclosed herein.

[0048] In this document, the terms "include" or "comprising" are open expressions, that is, including the contents specified in this application, but not excluding other contents.

[0049] The present application proposes a bird's nest peptide microneedle patch for resisting skin photoaging, a preparation method thereof, and an application thereof, which will be described in detail below.

[0050] Bird's nest peptide microneedle patch for anti-skin photoaging

[0051] In one aspect, the present application provides a bird's nest peptide microneedle patch for anti-skin photoaging. According to an embodiment of the present application, the bird's nest peptide microneedle patch for anti-skin photoaging comprises: a base and a plurality of microneedles, the plurality of microneedles being distributed on the base; the microneedles comprising a functional component and a supporting component; the functional component comprising a bird's nest peptide, and the supporting component comprising polyvinyl pyrrolidone.

[0052] This application combines bird's nest peptide with microneedle patch technology to improve the delivery efficiency and bioavailability of active bird's nest peptide, thereby improving its anti-skin photoaging and repair effects. Polyvinylpyrrolidone (PVP) is a high molecular polymer with a chain vinyl structure. It has good amphiphilicity, complexation and biocompatibility and does not cause any irritation to the skin, mucous membranes, etc. Polyvinylpyrrolidone can form a complex with bird's nest peptide through chemical bond interaction to achieve the purpose of delivery. In addition, using polyvinylpyrrolidone (PVP) as a microneedle supporting component, the microneedle needle rate is high, the color is transparent, and the needle body shape is basically intact. The deformation degree is low during the drying process, the needle body has no obvious cavitation, the needle body is strong, and it is not easy to break. In addition, the dissolution characteristics of the microneedle are strong, the swelling performance is stable, the efficiency of transdermal delivery of peptides is high, and the rate is stable, thereby achieving the rapid release of bird's nest peptide in the interstitial fluid of the skin tissue, improving the delivery efficiency of bird's nest peptide, and ensuring the stability and effectiveness of bird's nest peptide.

[0053] According to an embodiment of the present application, the method for preparing a bird's nest peptide comprises: subjecting the bird's nest to an enzymatic hydrolysis treatment, wherein the enzyme employed in the enzymatic hydrolysis treatment comprises at least one of alkaline protease, neutral protease, and flavor protease. The bird's nest peptide obtained by using these three proteases individually or in combination can enhance the viability and proliferation of HaCaT cells damaged by ultraviolet radiation, thereby effectively improving the repair activity of photoaging skin. Of these, the combination of alkaline protease and neutral protease, and the combination of alkaline protease and flavor protease, are particularly effective in repairing photoaging skin. In some embodiments, in the combination of alkaline protease and neutral protease, the mass ratio of alkaline protease to neutral protease is 1:(0.1-5), for example, 1:0.1, 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5; in other embodiments, in the combination of alkaline protease and flavor protease, the mass ratio of alkaline protease to flavor protease is 1:(0.1-5), for example, 1:0.1, 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5.

[0054] According to an embodiment of the present application, the temperature of the enzymatic hydrolysis treatment is 45-65°C, for example, 45°C, 50°C, 55°C, 60°C, 65°C, and the time is 2-8 hours, for example, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours. Thus, the bird's nest peptide obtained under the above enzymatic hydrolysis conditions has a better effect on repairing skin photoaging.

[0055] According to an embodiment of the present application, the preparation method of the bird's nest peptide comprises: (a) crushing and sieving the bird's nest, soaking the obtained crushed material with water to obtain a bird's nest liquid; (b) discarding the supernatant of the bird's nest liquid, adding water, and sequentially performing microwave heating treatment and ultrasonic treatment to obtain a bird's nest protein aqueous solution; (c) subjecting the bird's nest protein aqueous solution to the enzymatic hydrolysis treatment to obtain the bird's nest peptide. Treatments in different physical fields will affect the yield of bird's nest protein hydrolysis, product activity, etc. Microwave energy causes molecular motion through ion conduction and dipole rotation, causing the temperature and pressure of the reaction system to increase and the protein structure to change; ultrasonic waves can generate mechanical vibrations and induce conformational changes in proteins. Therefore, the combined pretreatment of microwave heating and ultrasonic waves can effectively improve the extraction efficiency of bird's nest peptides.

[0056] According to an embodiment of the present application, the protein content of the bird's nest protein aqueous solution is 55-65%, for example, 55%, 56%, 58%, 60%, 62%, 64%, and 65%; the amount of enzyme used in the enzymatic hydrolysis treatment is 2000-15000 U / g bird's nest protein aqueous solution, for example, 2000, 3000, 5000, 8000, 10000, 12000, and 15000 U / g bird's nest protein aqueous solution. In this way, the enzyme can fully act on the bird's nest protein, and the enzymatic hydrolysis can produce bird's nest peptides with high anti-skin photoaging activity.

[0057] According to an embodiment of the present application, the particle size of the crushed material is 50-100 mesh, for example, 50 mesh, 60 mesh, 70 mesh, 80 mesh, 90 mesh, and 100 mesh. This is conducive to the enzyme fully acting on the bird's nest peptide, so that it is enzymatically hydrolyzed to obtain a bird's nest peptide with high anti-skin photoaging activity.

[0058] According to an embodiment of the present application, the temperature of the microwave heating treatment is 100-200°C, for example, 100°C, 120°C, 140°C, 150°C, 160°C, 180°C, and 200°C; the time is 5-20 minutes, for example, 5 minutes, 6 minutes, 8 minutes, 10 minutes, 12 minutes, 15 minutes, 18 minutes, and 20 minutes; the power is 300-500W, for example, 300W, 350W, 400W, 450W, and 500W. Thus, the microwave heating pretreatment improves the solubility of the bird's nest protein in the enzymatic hydrolysis system by efficiently depolymerizing the colloidal network structure of the bird's nest protein.

[0059] According to an embodiment of the present application, the temperature of the ultrasonic treatment is 30-40°C, for example, 30°C, 32°C, 34°C, 35°C, 36°C, 38°C, and 40°C; the time is 20-40 minutes, for example, 20 minutes, 25 minutes, 30 minutes, 35 minutes, and 40 minutes; and the frequency is 40-50 Hz, for example, 40 Hz, 42 Hz, 44 Hz, 45 Hz, 46 Hz, 48 Hz, and 50 Hz. Thus, the ultrasonic pretreatment utilizes the transient cavitation shear effect to expose key enzyme cleavage sites in the hydrophobic core of the bird's nest protein, thereby improving the efficiency of enzymatic hydrolysis of the bird's nest peptide.

[0060] According to the embodiments of the present application, the bird's nest peptide is provided in the form of a peptide mixture, wherein peptides with a molecular weight of less than 3000Da account for more than 70%, and peptides with a molecular weight of less than 1000Da account for 65% to 70%. As a result, the bird's nest peptide obtained by the above method has a high proportion of small molecular weight peptides, which is more conducive to the body's absorption and utilization, and better exerts its efficacy.

[0061] According to an embodiment of the present application, at least some of the peptides in the peptide mixture have hydrophobic amino acids at their C-terminus and / or N-terminus. As a result, the PVP molecules can combine with the hydrophobic amino acids at the C-terminus and / or N-terminus of the bird's nest peptide to form an amphiphilic copolymer, thereby achieving effective delivery of the bird's nest peptide. Specifically, the C-terminus and / or N-terminus of the peptide are rich in hydrophobic amino acids such as Leu, Val, and Phe.

[0062] According to an embodiment of the present application, the preparation method of the bird's nest peptide further includes: drying the bird's nest peptide to obtain a bird's nest peptide powder; the particle size of the bird's nest peptide powder is 65 to 75 nm, for example, 65 nm, 68 nm, 70 nm, 72 nm, 74 nm, 75 nm, and the PDI value is less than 0.5, indicating that the prepared bird's nest peptide powder system has high uniformity, which is conducive to its in vitro transdermal absorption rate or the composite efficiency with the microneedle delivery system.

[0063] According to an embodiment of the present application, the functional ingredient accounts for 1% to 5% of the mass of the microneedles, for example, 1%, 2%, 3%, 4%, or 5%. Thus, the loading amount of the functional ingredient in the microneedles is appropriate, which helps to better exert the anti-photoaging skin repair effect.

[0064] According to an embodiment of the present application, the length of the microneedle is 400 to 800 μm, for example, 400 μm, 500 μm, 600 μm, 700 μm, or 800 μm. This helps the bird's nest peptide to better penetrate into the bottom of the dermis and improve the delivery efficiency of the bird's nest peptide.

[0065] According to an embodiment of the present application, the microneedle includes a top and a bottom, the bottom being connected to the base; the distance between the tops of two adjacent microneedles is 250 to 650 μm, for example, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm; and / or the diameter of the bottom is 200 to 600 μm, for example, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm. This facilitates better penetration of the bird's nest peptide into the bottom of the dermis and improves the delivery efficiency of the bird's nest peptide.

[0066] According to an embodiment of the present application, the microneedle is pyramidal, conical or quasi-conical in shape.

[0067] According to an embodiment of the present application, the surface of the microneedle is covered with a pH-responsive seal. The pH-responsive seal can dissolve or degrade under a specific pH environment (such as the weakly acidic environment of the skin surface, pH 5.5-7.0), thereby triggering the release of the bird's nest peptide from the microneedle. Thus, it can be ensured that the bird's nest peptide is released only after reaching the epidermis and dermis of the skin, avoiding premature release in other non-target areas, thereby increasing the local concentration and bioavailability of the drug, enhancing its anti-skin photoaging repair activity, while reducing the potential side effects of the drug in other parts, and achieving more accurate drug delivery and therapeutic effects.

[0068] According to an embodiment of the present application, the pH-responsive sealing layer comprises at least one of methacrylic acid copolymer type C, polyvinyl acid resin II, and polyvinyl acid resin III, thereby facilitating the precise release of bird's nest peptides.

[0069] According to an embodiment of the present application, the base has the same composition as that of the microneedle.

[0070] According to an embodiment of the present application, the base and the microneedle are integrally formed.

[0071] Method for preparing bird's nest peptide microneedle patch for resisting skin photoaging

[0072] In another aspect of the present application, the present application proposes a method for preparing the aforementioned anti-skin photoaging bird's nest peptide microneedle patch. According to the embodiments of the present application, see Figure 1 The method includes: providing a microneedle liquid containing the functional components and the supporting components and a microneedle template; pouring the microneedle liquid onto the mold cavity of the microneedle template so that the microneedle liquid completely covers the mold cavity, then performing vacuum treatment and drying treatment, and demolding to obtain the bird's nest peptide microneedle patch.

[0073] According to an embodiment of the present application, the method includes:

[0074] 1) dissolving the bird's nest peptide powder in water to obtain a bird's nest peptide aqueous solution.

[0075] 2) Stirring the bird's nest peptide aqueous solution and polyvinyl pyrrolidone until the polyvinyl pyrrolidone is completely dissolved, and then performing equilibrium dissolution to obtain a solution.

[0076] In some embodiments, the temperature of the balanced dissolution is 35-40°C and the time is 20-40 minutes. This ensures that the polyvinyl pyrrolidone is evenly distributed in the peptide aqueous solution, avoiding the phenomenon of uneven concentration. Through balanced dissolution, the physical and chemical properties of the solution can be stabilized, which is beneficial to the subsequent microneedle production process. Bubbles may be generated during the stirring process, and balanced dissolution can help these bubbles escape, avoiding the formation of voids in the microneedle structure.

[0077] In some embodiments, the concentration of polyvinylpyrrolidone in the dissolving solution is 30% to 60% by mass, for example, 30%, 42%, 45%, 48%, 50%, 52%, 55%, 58%, or 60% by mass. Thus, the strong water solubility and high viscosity of PVP are fully utilized, effectively increasing the viscosity of the microneedle preparation, improving the texture and plasticity of the bird's nest peptide, and making the bird's nest peptide microneedles more stable and easy to use.

[0078] 3) pouring the dissolving solution onto the mold cavity of the microneedle template so that the microneedle material liquid completely covers the mold cavity, vacuum treating for 10 to 30 minutes, removing excess liquid and bubbles with bubbles on the surface, and re-adding the dissolving solution 1 to 5 times.

[0079] 4) Drying the assembled microneedle template at 37-50° C. for 2-6 hours, then demolding and drying to obtain the bird's nest peptide microneedle patch.

[0080] Thus, the bird's nest peptide microneedle patch prepared using the method of the present application not only improves the delivery efficiency and bioavailability of active bird's nest peptides, but also enhances their application potential in anti-photoaging and skin repair. Furthermore, the bird's nest peptide microneedle patch of the present application has good stability, biocompatibility, and targeting. Furthermore, the method is simple to operate, low-cost, and suitable for large-scale production.

[0081] It should be noted that the characteristics and advantages described above for the bird's nest peptide microneedle patch for anti-skin photoaging are also applicable to this preparation method and will not be repeated here.

[0082] application

[0083] In another aspect of the present application, the present application proposes the use of the aforementioned bird's nest peptide microneedle patch for anti-skin photoaging in anti-skin photoaging. By combining bird's nest peptide with microneedle patch technology, the present application not only improves the delivery efficiency and bioavailability of active bird's nest peptide, but also enhances its application potential in anti-skin photoaging and repair. In addition, the bird's nest peptide microneedle patch of the present application has good stability, biocompatibility and targeting, providing a new solution for anti-skin photoaging.

[0084] It should be noted that the characteristics and advantages described above for the bird's nest peptide microneedle patch for anti-skin photoaging are also applicable to this application and will not be repeated here.

[0085] Below in conjunction with embodiment, the scheme of the application will be explained. Those skilled in the art will appreciate that the following examples are merely for illustration of the application and should not be considered as limiting the scope of the application. Where specific techniques or conditions are not indicated in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents used or instruments not indicated by the manufacturer are conventional products that can be obtained commercially.

[0086] Example 1

[0087] Taking the microneedle patch as an example, a polydimethylsiloxane (PDMS) microneedle template is used. The mold is a PDMS mold with a diameter of 2 cm. The needle body is conical in shape, the needle body length is 400 μm, the diameter of the needle body bottom is 500 μm, and the distance between the needle bodies (measured at the needle tip) is 450 μm. When preparing the microneedles, the prepared 30% polyvinyl pyrrolidone (PVP) microneedle substrate solution is dripped on the mold surface, and then placed in a vacuum drying oven for 5 minutes. After taking it out, the excess liquid with bubbles on the surface is poured off and some stubborn bubbles are removed with a scraper. Then, new substrate solution is dripped again. This is repeated three times. After the fourth substrate solution is added, the mold assembled with the substrate solution is placed in an oven at 45°C and dried for 4 hours until the microneedles are completely dry. The demoulded microneedle patch is stored in a desiccator.

[0088] Comparative Example 1

[0089] Taking the microneedle patch as an example, a polydimethylsiloxane (PDMS) microneedle template is used. The mold is a PDMS mold with a diameter of 2 cm. The needle body is conical in shape, the needle body length is 400 μm, the bottom diameter of the needle body is 500 μm, and the distance between the needle bodies is 450 μm. When preparing the microneedles, the prepared 10% hyaluronic acid (HA) microneedle substrate solution is dripped onto the mold surface, and then placed in a vacuum drying oven to evacuate for 5 minutes. After taking it out, the excess liquid with bubbles on the surface is poured off and some stubborn bubbles are removed with a scraper. Then, new substrate solution is dripped again. This is repeated three times. After the fourth addition of substrate solution, the mold assembled with the substrate solution is placed in an oven at 45°C and dried for 4 hours until the microneedles are completely dry. The demoulded microneedle patch is stored in a desiccator.

[0090] Figure 2 The soluble microneedles are prepared from polyvinyl pyrrolidone (PVP, top) and hyaluronic acid (HA, bottom) in Example 1 and Comparative Example 1. The 10% HA solution has a low viscosity and strong fluidity, and produces fewer stubborn bubbles during vacuum assembly. The HA microneedles after drying have a high forming rate, transparent color, and the basic shape of the needle body is intact. The lining layer is relatively thin and easy to adhere to the skin. However, due to the low viscosity and low dry matter content, the HA microneedles are more deformable during drying, and the volume shrinks sharply during drying, resulting in cavitations in the needle body and uneven distribution of bird's nest peptides. The 30% PVP solution has a higher viscosity and relatively low fluidity, so there is more foam on the surface during vacuum assembly. The PVP microneedles after drying have a high needle forming rate, transparent color, and the needle body shape is basically intact. The degree of deformation is low during the drying process, there are no obvious cavitations in the needle body, the needle body is strong, and it is not easy to break.

[0091] Example 2

[0092] Taking Indonesian edible bird's nest as an example, the raw bird's nest is crushed, passed through a 60-mesh sieve, and soaked overnight in distilled water at a material-to-liquid ratio of 1:100 g / mL to fully swell. The supernatant is then discarded, and distilled water is added at a material-to-liquid ratio of 1:50 g / mL. After microwave heating (400W, 120°C, 10 minutes) and ultrasonic treatment (45Hz, 37°C, 30 minutes), a bird's nest protein aqueous solution with a protein content of 60% is obtained.

[0093] For single-enzyme hydrolysis, using alkaline protease as an example: Weigh a certain amount of alkaline protease, dissolve it in deionized water, and preheat it at 37°C for 10 minutes to activate the enzyme. Then, add the activated alkaline protease solution to the bird's nest sample preheated to 55°C, so that the final enzyme dosage is 10,000U / g protein. After shaking evenly, place the entire system in a 55°C water bath shaker for 4 hours. After the enzymatic hydrolysis is complete, remove the bird's nest enzymatic hydrolysis sample bottle and heat it at 100°C for 10 minutes to inactivate the enzyme. After cooling to room temperature, centrifuge it at 8,000g for 15 minutes. The supernatant is the single-enzyme enzymatic hydrolysis bird's nest peptide.

[0094] After revival, human immortalized HaCaT cells were cultured in a complete medium consisting of 89% Dulbecco's Modified Eagle Medium, 10% fetal bovine serum, and 1% penicillin-streptomycin. The culture environment was maintained at 37°C and 5% CO2. Subsequent experiments were performed after the cells reached the logarithmic phase. HaCaT cells grown to the logarithmic phase were trypsinized, centrifuged, resuspended, and plated at 3×10 4 The cells were seeded at a density of 100 cells / well in a 96-well plate. After 12 hours of cell attachment, the original culture medium was discarded, and after washing with PBS twice, it was replaced with serum-free DMEM complete culture medium (99% DMEM basal culture medium + 1% double antibody) for 24 hours of cell serum starvation. Then, the culture medium was replaced with PBS solution and UVB irradiation damage was performed. Tin foil was used to cover the blank group that did not require modeling damage. The UVB lamp was 30 cm away from the cell plate, the irradiation time was 1.5 h, and the irradiation intensity was 102 μW / cm 2 , the total irradiation dose is 0.55J / cm 2 .

[0095] After UV irradiation modeling, the original culture medium was discarded, and the model group and the blank group were added with 37°C preheated DMEM complete culture medium. Bird's nest peptide intervention groups (1000 μg / mL) with different single enzyme hydrolysis (neutral protease, trypsin, flavor protease, alkaline protease and papain) and positive controls (glutathione 500 μg / mL and sialic acid 500 μg / mL) were added to the UV-damaged cell wells for intervention and cultured for 24 hours (n=5). The supernatant was discarded, and the absorbance of each well was measured by MTT assay to calculate the cell survival rate.

[0096] Example 3

[0097] Multi-enzyme complex hydrolysis, taking alkaline protease and neutral protease as examples: alkaline protease and neutral protease were weighed in different centrifuge tubes respectively, added with deionized water to dissolve and placed at 37°C for activation for 10 minutes, and then the alkaline protease solution and neutral protease solution were added to the bird's nest sample preheated to 55°C, so that the total enzyme amount was 10,000U / g protein, and the enzyme activity contribution mass ratio was alkaline protease: neutral protease 1:1. After enzymatic hydrolysis at 55°C for 4 hours, the sample was taken out and heated at 100°C for 10 minutes to inactivate the enzyme. After cooling to room temperature, it was centrifuged at 8,000g for 15 minutes, and the supernatant was the complex enzymatic hydrolyzed bird's nest peptide.

[0098] HaCaT cells grown to the logarithmic phase were trypsinized, centrifuged and resuspended at 3×10 4The cells were seeded at a density of 100 cells / well in a 96-well plate. After 12 hours of cell attachment, the original culture medium was discarded, and after washing with PBS twice, it was replaced with serum-free DMEM complete culture medium (99% DMEM basal culture medium + 1% double antibody) for 24 hours of cell serum starvation. Then, the culture medium was replaced with PBS solution and UVB irradiation damage was performed. Tin foil was used to cover the blank group that did not require modeling damage. The UVB lamp was 30 cm away from the cell plate, the irradiation time was 1.5 h, and the irradiation intensity was 102 μW / cm 2 , the total irradiation dose is 0.55J / cm 2 .

[0099] After UV irradiation modeling, the original culture medium was discarded, and the model group and the blank group were added with 37°C preheated DMEM complete culture medium. Different composite enzymatic hydrolysis (alkaline-neutral protease, neutral-flavor protease, alkaline-flavor protease) bird's nest peptide intervention groups (10, 100, 1000 μg / mL) and positive controls (glutathione 10, 100, 1000 μg / mL and sialic acid 10, 100, 1000 μg / mL) were added to the UV-damaged cell wells for intervention and cultured for 24 hours (n=5). The supernatant was discarded, and the absorbance of each well was measured by MTT assay to calculate the cell survival rate.

[0100] Figure 3 The following is a bar graph showing the results of the anti-skin cell photoaging repair activity of bird's nest peptides hydrolyzed by single enzymes and combined enzymes in Examples 2 and 3. In Example 2, after UVB damage modeling, cell viability was significantly reduced (p < 0.0001), indicating that the UVB damage model was successfully constructed. Compared with the model group, bird's nest peptides prepared by single enzyme hydrolysis of alkaline protease, flavor protease and neutral protease were able to significantly promote the improvement of HaCaT cell viability after intervention (p < 0.05), indicating that they have repair activity on UVB-damaged HaCaT cells. In Example 3, the HaCaT cell repair activity of the enzymatic hydrolysis products of different complex enzymes (alkaline-neutral protease, neutral-flavor protease, alkaline-flavor protease) was evaluated. The results showed that all three bird's nest peptides had a certain degree of repair activity, which could improve the viability of HaCaT cells damaged by UVB. The cell proliferation activity had a certain dose effect. As the concentration increased from 10 μg / mL to 1000 μg / mL, the cell viability gradually increased. 1000 μg / mL alkaline-neutral protease enzymatic peptide, 100 and 1000 μg / mL alkaline-flavor protease enzymatic peptides all significantly promoted the improvement of cell viability, indicating that bird's nest peptides have potential cell repair activity.

[0101] Example 4

[0102] Taking Indonesian edible bird's nest as an example, the raw bird's nest is crushed, passed through a 60-mesh sieve, and soaked overnight in distilled water at a material-to-liquid ratio of 1:100 g / mL to allow it to fully swell. The supernatant is then discarded, and distilled water is added at a material-to-liquid ratio of 1:50 g / mL. After microwave heating (400W, 120°C, 10 minutes) and ultrasonic treatment (45Hz, 37°C, 30 minutes), a bird's nest protein aqueous solution is obtained. Bird's nest peptides are then prepared using an alkaline-neutral protease complex enzymatic hydrolysis temperature of 55°C, a hydrolysis time of 4 hours, and an enzyme dosage of 10,000 U / g protein.

[0103] HaCaT cells grown to the logarithmic phase were trypsinized, centrifuged and resuspended at 5×10 3 HaCaT cells were seeded at a density of 100 cells / well in 96-well cell culture plates. After overnight attachment, they were treated with bird's nest peptides at varying concentrations (0.5, 5, 50, 100, 200, 400, 500, 2000, and 5000 μg / mL). The positive control group (glutathione and sialic acid) received 500 μg / mL. After 24 hours of treatment, absorbance at 570 nm was measured using the MTT assay, and cell viability was calculated.

[0104] After UV irradiation modeling, the original culture medium was discarded, and the model group and the blank group were added with 37°C preheated DMEM complete culture medium. Different concentrations of bird's nest peptide intervention groups (10, 100, and 500 μg / mL) and positive controls (glutathione 500 μg / mL and sialic acid 500 μg / mL) were added to the UV-damaged cell wells for intervention and cultured for 24 hours (n=5). The supernatant was discarded, and the absorbance of each well was measured by MTT assay to calculate the cell survival rate.

[0105] Figure 4 The following figure shows the results of the cytotoxicity and anti-photoaging repair activity of the bird's nest peptide prepared by alkaline-neutral protease hydrolysis in Example 4. The degree of hydrolysis of the bird's nest peptide prepared by alkaline-neutral protease hydrolysis was 15-18%, and its peptide composition showed that peptides with a molecular weight of less than 3000 Da accounted for more than 70%, and peptides with a molecular weight of less than 1000 Da accounted for 65% to 70%. Excessive UVB damage to the skin can lead to skin aging, tissue redness and swelling, and collagen loss. Therefore, repairing UVB damage is of great significance to skin homeostasis and health. The experimental results showed that alkaline-neutral protease hydrolyzed bird's nest peptide had no inhibitory effect on HaCaT cell activity at a concentration of 0-500 μg / mL and had a certain activity in promoting cell viability and cell proliferation. After the cells were damaged by UVB, the bird's nest peptide at a concentration of 500 μg / mL was able to significantly promote cell activity (p < 0.0001), indicating that it can promote the repair of UVB damage in skin epidermal cells.

[0106] Example 5

[0107] Taking polyvinyl pyrrolidone (PVP) as an example, the PVP-bird's nest peptide microneedle patch is prepared by casting and drying the PDMS microneedle mold. The microneedle design height is 400μm, the microneedle base diameter is 350μm, the microneedle bottom spacing is 300μm, and the needle density is 1cm 2 The microneedle array contains a 20×20 microneedle array. The specific preparation method is as follows: Bird's nest peptide prepared by enzymatic hydrolysis with alkaline-neutral protease (1:1) according to the method of Example 3 is dissolved in deionized water to a final peptide solution concentration of 10 mg / mL. Then, 3 g of PVP powder is mixed with 10 mL of the peptide solution to a final PVP concentration of 30%. The solution is stirred continuously with a magnetic stirrer to ensure complete dissolution of the PVP in the water. After the PVP is completely dissolved, the solution is equilibrated at 37°C for 30 minutes to obtain the final needle solution. During preparation, the prepared microneedle solution is dripped onto the mold surface and placed in a vacuum drying oven for 5 minutes. After removal, excess liquid with bubbles on the surface is decanted and stubborn bubbles are removed with a spatula. Fresh substrate solution is then added dropwise. This process is repeated three times. After the fourth addition of substrate solution, the mold containing the substrate solution is placed in an oven at 45°C for 4 hours to dry the microneedles completely. The demolded microneedles are stored in a desiccator.

[0108] Agarose powder was prepared into a 3% agarose solution by mass and poured into a clean, sterile Petri dish to form a 1 cm thick agarose gel layer, which served as an artificial skin simulation to evaluate the dissolution and swelling properties of the microneedles. A PVP-bird's nest peptide microneedle patch was inserted into the simulated skin and pressed for 0.5-1.0 minutes. The peptide microneedle patch was then removed every 30 seconds. The morphological changes of the microneedles were observed under a stereomicroscope and photographed. ImageJ software was used to measure the average width of the microneedles during the statistical process and calculate the changes in the lateral swelling rate of the peptide microneedles.

[0109] Comparative Example 2

[0110] Taking hyaluronic acid (HA) as an example, the HA-bird's nest peptide microneedle patch is prepared by casting and drying a PDMS microneedle mold. The microneedle design height is 400μm, the microneedle base diameter is 350μm, the microneedle bottom spacing is 300μm, and the needle density is 1cm 2The microneedle array contains a 20×20 microneedle array. The specific preparation method is as follows: Bird's nest peptide prepared by enzymatic hydrolysis with alkaline-neutral protease (1:1) according to the method of Example 3 is dissolved in deionized water to a final peptide solution concentration of 10 mg / mL. Then, 1 g of HA powder is mixed with 10 mL of the peptide solution to a final HA concentration of 10%. The solution is stirred continuously with a magnetic stirrer to ensure complete dissolution of the HA in the water. After the HA is completely dissolved, the solution is equilibrated at 37°C for 30 minutes to obtain the final needle solution. During preparation, the prepared microneedle solution is dripped onto the mold surface and placed in a vacuum drying oven for 5 minutes. After removal, excess liquid with bubbles on the surface is decanted and stubborn bubbles are removed with a spatula. Fresh substrate solution is then added dropwise. This process is repeated three times. After the fourth addition of substrate solution, the mold containing the substrate solution is placed in an oven at 45°C for 4 hours to dry the microneedles completely. The demolded microneedles are stored in a desiccator.

[0111] Agarose powder was prepared into a 3% agarose solution by mass and poured into a clean, sterile Petri dish to form a 1cm thick agarose gel layer, which served as an artificial skin simulation to evaluate the dissolution and swelling properties of the microneedles. An HA-bird's nest peptide microneedle patch was inserted into the simulated skin and pressed for 0.5-1.0 minutes. The peptide microneedle patch was then removed every 30 seconds. The morphological changes of the microneedles were observed under a stereomicroscope and photographed. ImageJ software was used to measure the average width of the microneedles during the statistical process and calculate the changes in the lateral swelling rate of the peptide microneedles.

[0112] Figure 5 Figure 5 shows the soluble microneedles of polyvinyl pyrrolidone-bird's nest peptide (PVP-bird's nest peptide) and hyaluronic acid-bird's nest peptide (HA-bird's nest peptide) in Example 5 and Comparative Example 2. During the preparation of the microneedle substrate solution, a white precipitate is produced after HA and bird's nest peptide are miscible. Preliminary analysis shows that the precipitate is the dissolved bird's nest peptide. This may be because the hyaluronic acid aqueous solution is negatively charged, and the osmotic pressure of the solution is high when the concentration is high, causing the bird's nest peptide to precipitate and dissolve. PVP and bird's nest peptide are miscible and have good stability, and the system is clear and bright. This experiment found that the HA-bird's nest peptide microneedle has a high needle formation rate and a complete needle body morphology. The addition of bird's nest peptide increases the dry matter content in the needle body solution, reduces the deformability of the needle during the drying process, and the obtained needle body has no obvious cavitation and is transparent in color. The PVP-bird's nest peptide microneedle obtained after drying has a high needle formation rate, a complete needle body structure morphology, a high degree of molding, and a sharp needle tip. After PVP and bird's nest peptide are mixed, the solution system is slightly yellowish, and the needle body formed after drying has a yellow luster. This may be because the high viscosity and strong reflectivity of the PVP solution enhance the light yellow color of the bird's nest peptide itself.

[0113] Example 6

[0114] The PVP-bird's nest peptide microneedles (400μm and 800μm) prepared in Example 5 were applied to mouse skin to evaluate their skin penetration ability. Mouse skin was taken from C57BL / 6J mice. After the mice were killed by dislocating their necks, the hair on the back was removed using pet electric clippers, and the fine hair on the epidermis was removed with a depilatory cream. The entire skin layer was then removed, and the fat layer at the bottom of the skin was carefully removed to avoid damage to the skin during the process. The removed skin tissue was trimmed into a 2.2cm×2.2cm square, wrapped individually with tin foil, and stored in a -80℃ refrigerator for use within 7 days.

[0115] Before use, the frozen skin tissue was placed in 37°C PBS for moist equilibrium for 1 hour, and then the bird's nest peptide microneedle patch was attached and pressed on the skin tissue for 30 seconds. The skin tissue with 400μm and 800μm bird's nest peptide microneedle patches was loaded into the skin membrane layer of the transdermal diffusion pool. The blank group used the same dose of bird's nest peptide solution loaded into the donor chamber.

[0116] Figure 6 The morphology and dissolution and swelling characteristics of the HA-bird's nest peptide microneedle in Comparative Example 2 and the PVP-bird's nest peptide microneedle in Example 5 are shown. The dissolution and swelling characteristics of soluble microneedles are important indicators reflecting the characteristics of microneedles, and can characterize the peptide release efficiency of the microneedle to a certain extent. In this experiment, 3% agarose was used to prepare artificial skin to simulate the water content and elasticity of skin tissue. The peptide microneedle samples were inserted into the simulated skin for different lengths of time to observe the degree of dissolution and swelling of the microneedle. The experimental results showed that after the HA-bird's nest peptide microneedle was inserted into the artificial skin, the needle body rapidly absorbed water and expanded. After 30 seconds, it had swelled to form an ellipsoidal morphology. After 1 minute of insertion into the body, the needle body had been completely absorbed by the artificial skin, and there was no obvious needle body on the surface of the microneedle, indicating that the HA-bird's nest peptide microneedle has strong dissolution and swelling properties and a fast transdermal absorption rate. After piercing the artificial skin, the PVP-bird's nest peptide microneedles rapidly dissolved and swelled. Within 30 seconds of dissolution, the needles had significantly reduced in volume, while maintaining their conical shape. After 1 minute, the needles essentially disappeared, leaving even less residue than the HA-bird's nest peptide microneedles, indicating complete absorption by the artificial skin. The experimental results demonstrate that the PVP-bird's nest peptide microneedles exhibit strong solubility characteristics, stable swelling properties, and high efficiency and stable rate of transdermal peptide delivery.

[0117] Figure 7The following is a bar graph showing the peptide concentration in the receiving pool (A) and the diffusion efficiency of the PVP-bird's nest peptide microneedle (B) at different time points in Example 6. The skin transmembrane permeation efficiency of the bird's nest peptide microneedle patch was studied using fresh mouse full-thickness skin in combination with a Franz vertical diffusion cell. Figure A shows the changes in peptide concentration in the receiving pool at different time points. It can be seen from the change curve that as the diffusion time increases, the peptide concentration gradually increases, indicating that the amount of bird's nest peptide penetrating the skin gradually increases. In addition, from the comparison between the groups, the penetration of the bird's nest peptide microneedle group (400μm and 800μm) is higher than that of the epidermal application group. The diffusion efficiency in Figure B reflects the efficiency of bird's nest peptide delivery by different administration methods. By comparing the efficiency of transdermal delivery of bird's nest peptide by microneedle and epidermal application, it was found that the delivery efficiency of the microneedle group was significantly higher than that of the epidermal application group, and the diffusion efficiency was increased by 7.94 times (400μm group: epidermal application group). Among the microneedle groups, the diffusion efficiency of the 400μm group was significantly higher than that of the 800μm group. This may be because the mass of 800μm itself is larger than that of 400μm. When the area of ​​mouse skin used in the experiment is fixed, the amount of peptide that can be penetrated and utilized by the skin is limited. Therefore, the excess bird's nest peptide in the microneedle of the 800μm group cannot be penetrated and utilized, resulting in a decrease in the overall diffusion efficiency.

[0118] Figure 8 The diffusion rate per unit area of ​​the PVP-bird's nest peptide microneedle (A) and the cumulative amount of peptide permeated per unit area (B) are bar graphs in Example 6. The diffusion rate per unit area reflects the amount of per cm per unit time. 2 The amount of bird's nest peptide that has penetrated the skin, from an overall perspective (Figure A), shows a fluctuating trend in the penetration process. The diffusion rates of the 400μm and 800μm microneedle groups in the early diffusion period of 0-3h are both higher than those of the epidermal application group, indicating that the mechanical penetration properties of the microneedles can quickly deliver the peptide to the dermis of the skin, greatly reducing the time it takes for the bird's nest peptide to penetrate through the epidermis into the dermis. Figure B reflects the cumulative amount of bird's nest peptide that has penetrated per unit area. The results show that the permeation per unit area of ​​the microneedle group is significantly higher than that of the epidermal application group. The cumulative permeation per unit area increased by 1.36 times (800μm group) and 1.27 times (400μm group), respectively. The permeation of the 800μm group is significantly higher than that of the 400μm group. This result may be attributed to the characteristics of the selected skin membrane itself. Compared with the 400μm group, the 800μm group can deliver more bird's nest peptides to the bottom of the dermis, therefore, it has a higher diffusion permeation per unit time.

[0119] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A bird's nest peptide microneedle patch for anti-skin photoaging, characterized in that: include: A base and a plurality of microneedles, wherein the plurality of microneedles are distributed on the base; The microneedle comprises a functional component and a supporting component; the functional component comprises bird's nest peptide, and the supporting component comprises polyvinyl pyrrolidone.

2. The bird's nest peptide microneedle patch according to claim 1, characterized in that The preparation method of the bird's nest peptide comprises: performing enzymatic hydrolysis on the bird's nest, wherein the enzyme used in the enzymatic hydrolysis comprises at least one of alkaline protease, neutral protease and flavor protease; Optionally, the enzymes used in the enzymatic treatment include: a combination of alkaline protease and neutral protease, or a combination of alkaline protease and flavor protease; Optionally, in the combination of alkaline protease and neutral protease, the mass ratio of alkaline protease to neutral protease is 1:(0.1-5); Optionally, in the combination of alkaline protease and flavor protease, the mass ratio of alkaline protease to flavor protease is 1:(0.1-5); Optionally, the temperature of the enzymatic hydrolysis treatment is 45 to 65° C., and the time is 2 to 8 hours.

3. The bird's nest peptide microneedle patch according to claim 2, characterized in that The preparation method of the bird's nest peptide comprises: (a) crushing and sieving the bird's nest, and soaking the crushed material in water to obtain a bird's nest liquid; (b) discarding the supernatant of the bird's nest liquid, adding water, and sequentially performing microwave heating and ultrasonic treatment to obtain a bird's nest protein aqueous solution; (c) subjecting the bird's nest protein aqueous solution to the enzymatic hydrolysis treatment to obtain the bird's nest peptide.

4. The bird's nest peptide microneedle patch according to claim 3, characterized in that The bird's nest protein aqueous solution has a protein content of 55-65%; the enzyme dosage for the enzymatic hydrolysis treatment is 2000-15000 U / g bird's nest protein aqueous solution; Optionally, the particle size of the pulverized material is 50 to 100 mesh; Optionally, the microwave heating treatment is performed at a temperature of 100 to 200° C., a time of 5 to 20 minutes, and a power of 300 to 500 W; Optionally, the temperature of the ultrasonic treatment is 30-40° C., the time is 20-40 min, and the frequency is 40-50 Hz.

5. The bird's nest peptide microneedle patch according to any one of claims 1 to 4, characterized in that: The bird's nest peptide is provided in the form of a peptide mixture, wherein peptides with a molecular weight of less than 3000 Da account for more than 70%, and peptides with a molecular weight of less than 1000 Da account for 65% to 70%; At least some of the peptides in the peptide mixture have hydrophobic amino acids at their C-termini and / or N-termini; Optionally, the preparation method of the bird's nest peptide further comprises: Drying the bird's nest peptide to obtain bird's nest peptide powder; The particle size of the bird's nest peptide powder is 65-75 nm, and the PDI value is less than 0.

5.

6. The bird's nest peptide microneedle patch according to claim 1, characterized in that The functional component accounts for 1% to 5% of the mass of the microneedle.

7. The bird's nest peptide microneedle patch according to claim 1, characterized in that The length of the microneedle is 400 to 800 μm; Optionally, the microneedle comprises a top end and a bottom end, and the bottom end is connected to the base; The distance between the top ends of two adjacent microneedles is 250 to 650 μm; and / or the diameter of the bottom ends is 200 to 600 μm; Optionally, the microneedle is pyramidal, conical or quasi-conical; Optionally, the base has the same composition as the microneedles; Optionally, the base and the microneedle are integrally formed.

8. A method for preparing the anti-skin photoaging bird's nest peptide microneedle patch according to any one of claims 1 to 7, characterized in that: include: Providing a microneedle material solution and a microneedle template containing the functional component and the supporting component; The microneedle material liquid is poured onto the mold cavity of the microneedle template so that the microneedle material liquid completely covers the mold cavity, and then vacuum treatment and drying treatment are performed, and demolding is performed to obtain the bird's nest peptide microneedle patch.

9. The method according to claim 8, characterized in that include: 1) dissolving the bird's nest peptide powder in water to obtain a bird's nest peptide aqueous solution; 2) stirring the bird's nest peptide aqueous solution and polyvinyl pyrrolidone until the polyvinyl pyrrolidone is completely dissolved, and then performing equilibrium dissolution to obtain a dissolving solution, wherein the equilibrium dissolution temperature is 35-40° C., the time is 20-40 min, and the concentration of polyvinyl pyrrolidone in the dissolving solution is 30% to 60% by mass; 3) pouring the dissolving solution onto the mold cavity of the microneedle template so that the microneedle material solution completely covers the mold cavity, vacuum treating for 10 to 30 minutes, removing excess liquid and bubbles on the surface, and re-adding the dissolving solution and vacuum treating for 1 to 5 times; 4) Drying the assembled microneedle template at 37-50° C. for 2-6 hours, then demolding and drying to obtain the bird's nest peptide microneedle patch.

10. Use of the bird's nest peptide microneedle patch for resisting skin photoaging according to any one of claims 1 to 7 in resisting skin photoaging.

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