Recombinant collagen microcrystal patch with anti-fine wrinkles effect and preparation method thereof

Abstract

The application discloses recombinant collagen microcrystal patches with anti-wrinkle effect and a preparation method thereof. The recombinant collagen microcrystal patch comprises a substrate and a plurality of needle bodies arranged on the substrate. Each needle body comprises a first needle body and a second needle body. The second needle body is connected with the first needle body and covers the outer layer of the first needle body. The first needle body is integrally formed with the substrate. The volume ratio of the first needle body to the second needle body is less than or equal to 0.8. The first needle body and the substrate are non-biodegradable. The second needle body is biodegradable and comprises active ingredients. The active ingredients comprise recombinant collagen. The microcrystal patch provided by the application can simultaneously meet the requirements of high mechanical performance, high drug loading and long service life.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and more specifically, to a recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects and its preparation method. Background Technology

[0002] With social development and improved living standards, people's pursuit of healthy and youthful skin is increasing. Skin aging, especially the formation of facial wrinkles, is a core concern in this field. Skin aging is mainly caused by endogenous aging and exogenous aging (such as ultraviolet radiation). Its fundamental mechanism includes the loss and degradation of extracellular matrix such as collagen and elastin in the dermis, leading to the collapse of the skin's supporting structure and moisture loss, thus forming signs of aging such as wrinkles and sagging skin.

[0003] Collagen, as the main structural protein of the dermis, accounts for about 70-80% of its dry weight and is key to maintaining skin plumpness, firmness and elasticity. Therefore, supplementing collagen has always been the core direction of anti-aging and wrinkle reduction strategies. At present, the mainstream methods of supplementing collagen on the market include: (1) Topical cosmetics: such as serums and creams containing collagen. However, due to the huge molecular weight of collagen, it is difficult to penetrate the stratum corneum barrier of the skin epidermis. Its bioavailability is extremely low, and it can only play a surface moisturizing role and cannot effectively reach the dermis to exert biological functions; (2) Oral collagen peptides: after being broken down into small molecule peptides and amino acids through the digestive tract, they are absorbed. Its mechanism of action is indirect, the efficiency varies from person to person, and it requires long-term persistence and is slow to take effect; (3) Medical injection fillers: collagen or other fillers are directly introduced into the dermis through injection, and the effect is immediate. However, this method is an invasive operation and has risks such as pain, allergies, infection, high cost and short-lasting effect (usually only lasting for a few months), which limits its popular application. To overcome the limitations of the aforementioned technologies, transdermal drug delivery technology, especially microneedle technology, has received widespread attention in recent years. Microneedle arrays can briefly and minimally invasively open channels in the stratum corneum of the skin, forming micron-sized pores, thereby efficiently delivering macromolecular active ingredients to the target skin layer, combining the convenience and safety of topical preparations with the high efficiency of injection.

[0004] Recombinant collagen is prepared using genetic engineering methods and has the advantages of high safety, controllable quality, and strong functionality. Combining recombinant collagen with microcrystalline transdermal technology can theoretically create a new type of highly efficient, safe, and convenient anti-aging and wrinkle-reducing product. However, achieving this combination faces a series of technical challenges, including: (1) how to ensure that the bioactivity of recombinant collagen is not destroyed by high temperature, organic solvents, or shear force during the preparation process of microcrystalline patches (such as mixing, drying, and curing); (2) how to effectively load sufficient recombinant collagen into microcrystals and ensure that it can continuously and completely dissolve and release after being inserted into the skin; (3) microcrystals need to have sufficient mechanical strength to pierce the stratum corneum, but not too hard to avoid pain or breakage; (4) in order to achieve synergistic effects, it is often necessary to compound other functional ingredients (such as hyaluronic acid, peptides, growth factors, etc.), and how to design a stable and compatible matrix formulation system is the key.

[0005] Therefore, this invention is proposed. Summary of the Invention

[0006] This invention provides a recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects and its preparation method. The microcrystalline patch has suitable mechanical strength and can exert the effect of transdermal anti-aging and wrinkle-reducing active substances for a long time, thereby maximizing the bioavailability of the active substances.

[0007] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects, the recombinant collagen microcrystalline patch comprising: a substrate and a plurality of needles disposed on the substrate; each needle comprising a first needle and a second needle, the second needle being connected to the first needle and covering the outer layer of the first needle, the first needle being integrally formed with the substrate; the volume ratio of the first needle to the second needle being less than or equal to 0.8; the first needle and the substrate being non-biodegradable; the second needle being biodegradable and comprising an active ingredient, the active ingredient comprising recombinant collagen.

[0008] Recombinant collagen is a popular ingredient in anti-aging and wrinkle-reducing cosmetics, but its large molecular weight makes it difficult for the skin to absorb transdermally. Theoretically, microneedling could create a new, highly effective, safe, and convenient anti-aging and wrinkle-reducing product. However, microneedle patches for facial skincare suffer from poor transdermal delivery, poor delivery persistence, and low bioavailability. On one hand, for safety reasons, the height and strength of the microneedles cannot be too high, making it difficult to open skin channels and resulting in poor delivery. On the other hand, to increase the effective ingredient loading, current microneedle patches typically use biodegradable materials to prepare the needles. Once the needles degrade, it becomes difficult to maintain the transdermal delivery effect, leading to poor delivery persistence and low bioavailability. Furthermore, in the field of anti-aging and wrinkle reduction, delivering large molecular weight active ingredients like collagen requires a sustained and long-term process to achieve results. Therefore, developing a microneedle system capable of sustained delivery is beneficial for improving anti-aging and wrinkle-reducing effects.

[0009] This invention provides a recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects. It has a dual-layer structure comprising a non-biodegradable first needle and a biodegradable second needle. When applied to the skin, the first needle is gradually exposed as the second needle degrades, acting as a second needle tip to continuously stimulate the skin, maintain open stratum corneum channels, continuously deliver active ingredients, improve the utilization rate of active ingredients, and extend the lifespan of the microcrystalline patch. In the microcrystalline patch provided by this invention, the second needle is connected to and covers the first needle, and the volume ratio of the first needle to the second needle is less than or equal to 0.8. Therefore, the second needle can provide sufficient space to load active substances, increasing the loading capacity of active ingredients. The two-layer structure of the needles works together to continuously deliver active substances, achieving the anti-aging and wrinkle-reducing effect.

[0010] It should be noted that biodegradability means that after the needle is applied to the skin, it can dissolve and dissolve into a transdermal absorbable substance over time, and the needle structure eventually collapses or even disappears; conversely, non-biodegradability means that after the needle is applied to the skin, its structure does not collapse over time.

[0011] In some embodiments, the recombinant collagen microcrystal patch contains 5% to 15% recombinant collagen.

[0012] In some embodiments, the loading of recombinant collagen in the recombinant collagen microcrystal patch is 0.02~0.1 mg / needle.

[0013] The microcrystalline patch provided by this invention can simultaneously achieve high mechanical properties, high drug loading capacity, and long service life, thereby achieving a better anti-aging and wrinkle-reducing effect.

[0014] In some embodiments, the first needle body is a cone or cone-like body with an average height of 200-450 μm and a bottom diameter of 80-160 μm.

[0015] In some embodiments, the needle body is a cone or cone-like shape with an average height of 400-800 μm and a bottom diameter of 120-300 μm.

[0016] In some embodiments, the area of ​​the largest cross-section of the first needle body is smaller than the area of ​​the largest cross-section of the needle body.

[0017] In some embodiments, the distance between the tip of the first needle body and the tip of the second needle body is less than 200 μm.

[0018] In some embodiments, the second needle body further includes a template material, the concentration of which gradually decreases from the needle tip to the substrate. The gradual decrease in template material concentration from the needle tip to the substrate in the second needle body provided by this invention helps to balance the mechanical properties and drug loading capacity of the microneedle, thereby achieving continuous delivery of active substances. Specifically, in the initial stage of use, the needle tip of the second needle body pierces the skin as a switch to open the channel, thus requiring a needle tip with high mechanical properties, resulting in a relatively high template material content at the needle tip. Over time, the needle tip of the second needle body degrades and loses its stimulating effect on the skin, replaced by the first needle body. Therefore, the upper part of the second needle body does not need excessive strength, reducing the template material content and thus increasing the loading capacity of the active ingredient.

[0019] In some embodiments, the microcrystalline recombinant collagen microcrystal patch further includes a backing, which is adhered to the substrate and disposed on a surface away from the needle.

[0020] In some embodiments, the template material is selected from at least one of polyvinylpyrrolidone, hyaluronic acid or its salts, chitosan and hydroxypropyl methylcellulose.

[0021] In some embodiments, the active ingredient further includes at least one of carnosine, asiaticoside, hydroxyasiaticoside, Centella Asiatica extract, arginine / lysine polypeptide, acetyl hexapeptide-8, hyaluronic acid or a salt thereof, hydroxypropyl tetrahydropyranotriol, tetrahydromethylpyrimidine carboxylic acid, and ascorbic acid.

[0022] Carnosine protects proteins such as collagen and elastin from glycation, thus delaying skin aging and brightening the complexion. It also neutralizes free radicals, providing dual anti-aging protection. Asiaticoside and asiaticoside are the core active monomers of Centella Asiatica, significantly promoting wound healing, stimulating collagen synthesis, and inhibiting inflammatory responses, making them ideal for repairing damaged skin barriers, soothing sensitivity, and improving redness and acne scars. Centella Asiatica extract contains a complex of multiple ingredients, including asiaticoside and asiaticoside, comprehensively enhancing the skin's self-repair capabilities and overall health. Arginine / lysine peptides act as signal peptides, penetrating the epidermis to send signals to skin cells to accelerate collagen and elastin production, thereby reducing wrinkles, firming the skin, and achieving anti-aging effects. Acetyl hexapeptide-8 is a neurotransmitter inhibitory peptide that can locally block the transmission of muscle contraction signals, thereby reducing the formation of expression lines (such as crow's feet, forehead wrinkles, and frown lines) and making the skin surface smoother. Hyaluronic acid or its salts are natural moisturizing factors in the skin. They achieve deep hydration, fill fine lines, and make the skin plump and moisturized by forming a moisturizing film on the skin surface and absorbing moisture from the air. Hydroxypropyl tetrahydropyranotriol (Pro-Xylane) can stimulate the synthesis of various substances (such as collagen IV and VII) at the junction of the dermis and epidermis (DEJ), thereby improving skin firmness and elasticity, improving sagging, and making the skin plumper. Tetrahydromethylpyrimidine carboxylic acid (Ectoin) can form a "protective hydration shell" around cells, protecting cells from environmental stressors such as ultraviolet rays, pollution, and stress, and has extremely strong repairing, stabilizing, and anti-inflammatory capabilities. Ascorbic acid can neutralize free radicals and prevent photoaging of the skin; it can inhibit the production of melanin and reduce existing melanin, thereby brightening the skin tone and fading dark spots; it can also help synthesize collagen and play an anti-aging role.

[0023] In some embodiments, the backing is selected from at least one of polyethylene, polypropylene, nonwoven fabric, polyurethane, and hydrogel.

[0024] In some embodiments, the first needle body and the matrix comprise a crosslinker of an acrylamide functional substance and a photoinitiator; the acrylamide functional substance is selected from at least one of methacrylic acid, methacrylate, acrylic acid, acrylate, polyethylene glycol methacrylate, and methacrylamide gelatin.

[0025] In some embodiments, the first needle body and the matrix further include fiber fillers, which include at least one of polylactic acid fiber, cellulose fiber and silk fibroin fiber; the addition of fiber fillers is beneficial to improving the mechanical properties of the first needle body and the rheological properties (viscoelasticity) of the first needle body solution used to prepare the first needle body, thereby facilitating the preparation of the first needle body by the extraction method.

[0026] In some embodiments, the compression modulus of the first needle body is 250~400MPa.

[0027] In some embodiments, the first needle body and the matrix comprise, by weight percentage, the following components: 0-14% fiber filler, 85-99% crosslinked acryloyl functional material, and 0.5-2% photoinitiator.

[0028] In some embodiments, the second needle body comprises, by weight percentage, the following components: 80%–85% sodium hyaluronate, 14%–18% recombinant collagen, 0–0.6% carnosine, 0–0.1% asiaticoside, 0–0.1% asiaticoside, 0–0.1% centella asiatica extract, 0–0.05% arginine / lysine polypeptide, 0–0.05% acetyl hexapeptide-8, 0–1% hydroxypropyl tetrahydropyranotriol, 0–1% tetrahydromethylpyrimidine carboxylic acid, and 0–0.1% ascorbic acid.

[0029] Secondly, the present invention provides a method for preparing the recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects according to any of the above embodiments, comprising the following steps: (1) Obtaining a mold: The first mold is used to form the base and the first needle body; the second mold is used to form the second needle body; the first mold is a copper plate with a plurality of protrusions arranged in a matrix on one side, each of the protrusions corresponding to each of the first needle bodies; the second mold is a silicone template with a plurality of grooves arranged in a matrix on one side, each of the grooves corresponding to each of the second needle bodies; (2) Preparation of needle body solutions: The first needle body solution is used to prepare the matrix and the first needle body; the second needle body solution and the third needle body solution are used to prepare the second needle body; the first needle body solution is prepared as follows: the acrylamide functional substance, photoinitiator and fiber filler are dissolved in the first solvent, dispersed evenly and defoamed; the second needle body solution is prepared as follows: the active ingredient of the second needle body and the first template material are dissolved in ultrapure water in sequence, stirred to dissolve and defoamed; the third needle body solution is prepared as follows: the active ingredient of the second needle body and the second template material are dissolved in ultrapure water in sequence, stirred to dissolve and defoamed. (3) Molding the substrate and the first needle body: The first mold is brought close to the first needle body solution so that the protrusion is immersed in the first needle body solution. Then the first mold is lifted in the vertical direction so that the protrusion leaves the first needle body solution. The mold is left to stand for UV curing to obtain the unmolded substrate and the first needle body. (4) Preparation of recombinant collagen microcrystal patch: Add the second needle body solution to the second mold and perform the first drying; then add the third needle body solution, and bring the undemolded substrate and the first needle body close to the second mold so that the first needle body is inserted into the corresponding groove of the second mold and perform the second drying; remove the first mold and the second mold in sequence to obtain the recombinant collagen microcrystal patch.

[0030] In some embodiments, each groove includes two through cavities, wherein the upper cavity is an inverted trapezoidal cylinder and the lower cavity is a cone; preferably, the total depth of each groove is 400-800 μm, the diameter of the maximum cross-section of the lower cavity is 120-300 μm, and the height of the lower cavity is 90% of the total depth of the groove. The upper cavities facilitate the drainage of excess third-needle solution from the grooves or the replenishment of third-needle solution during the drying process to fill the grooves; each upper cavity is interconnected and communicates with the outside.

[0031] In some embodiments, each of the protrusions is a cylinder or a cone; preferably, each of the protrusions has a height of 60-80 μm and a bottom diameter of 10 μm.

[0032] In some embodiments, the chemical compositions of the first template material and the second template material are the same or different, and the mass concentration of the first template material in the second needle solution is greater than the mass concentration of the second template material in the third needle solution; preferably, the mass concentration of the first template material in the second needle solution is 20% to 30%, and the mass concentration of the second template material in the third needle solution is 6% to 9%.

[0033] In some embodiments, the viscosity of the first needle solution at 25°C is 20~50 Pa·s.

[0034] In some embodiments, the volume ratio of the second needle solution to the third needle solution is 1:2 to 15.

[0035] In some embodiments, the first drying refers to drying under static conditions at 20~35°C and negative pressure for 3~5 hours.

[0036] In some embodiments, the second drying refers to drying at 20~35°C under negative pressure for 12~24 hours.

[0037] In some embodiments, the UV curing includes irradiating with UV light for 0.25 to 1 hour, followed by drying at 20 to 35°C and 40% to 60% humidity for 1 to 2 days.

[0038] In some embodiments, the step of removing the first mold and the second mold in sequence specifically includes: removing the first mold to expose the back of the substrate, then attaching a backing to the back, and finally removing the second mold.

[0039] The present invention has the following beneficial effects: (1) This invention provides a recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects. The recombinant collagen loading of the microcrystalline patch is as high as 0.0987 mg / needle. Furthermore, it has a double-layer structure. During use, the insoluble first needle body is gradually exposed as the second needle body dissolves, acting as a second needle head to continuously stimulate the skin, maintain the open stratum corneum channels, continuously deliver active ingredients, improve the utilization rate of active ingredients, and extend the service life of the microcrystalline patch. The microcrystalline patch provided by this invention can simultaneously achieve high mechanical properties, high drug loading, and long service life.

[0040] (2) The recombinant collagen microcrystal patch provided by the present invention contains a variety of active ingredients, which can synergistically enhance the anti-aging and wrinkle-reducing effects of recombinant collagen through skin absorption.

[0041] (3) The present invention also provides a method for preparing recombinant collagen microcrystal patches. The preparation method uses an extraction method combined with ultraviolet curing to prepare a first needle with high mechanical strength and small volume, thereby providing a large space for the loading of recombinant collagen while taking into account mechanical strength and service life. In addition, the second needle is prepared by using the principle of concentration difference and solid-liquid diffusion to prepare a second needle with a template material concentration that gradually decreases from the needle tip to the matrix, thereby ensuring that the needle tip of the second needle has greater mechanical strength, while the needle can load more active ingredients. Attached Figure Description

[0042] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0043] Figure 1 This is a schematic diagram of the structure of the microcrystalline patch in Example 1; Figure 2 The images shown are actual pictures of the microcrystalline patch from Example 1, where A is a microscope image of the first needle body; and B is an actual picture of the microcrystalline patch. Figure 3 These are schematic diagrams of the first mold and the second mold in Example 1, where A is a schematic diagram of the first mold and B is a schematic diagram of the second mold. Figure 4The images shown are actual product images of Comparative Example 2 and Comparative Example 3, where A is an actual product image of Comparative Example 2 and B is an actual product image of Comparative Example 3. Figure 5 This is a graph showing the change in the release of recombinant collagen delivered transdermally over time in Test Example 4. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0045] In the description of the embodiments of this application, the technical terms "first" and "second" are used only to distinguish different objects and should not be construed as indicating or implying relative importance or implicitly indicating the number, specific order, or primary and secondary relationship of the indicated technical features.

[0046] The term "embodiment" in this document means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment that is mutually exclusive with other embodiments.

[0047] In the embodiments of this application, the term "or / and" is only a description of the relationship between related objects, indicating that there can be three kinds of relationships. For example, A or / and B can represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

[0048] Additionally, the character " / " in this article generally indicates that the objects before and after it are in an "or" relationship.

[0049] In the embodiments of this application, "multiple" means two or more (including two), similarly, "multiple groups" means two or more (including two groups), and "multiple layers" means two or more (including two layers), unless otherwise explicitly specified and limited.

[0050] In the embodiments of this application, "at least one" means one or more.

[0051] In the embodiments of this application, the technical terms "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," and "horizontal," etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the embodiments of this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation or be constructed in a specific orientation, etc., and should not be construed as a limitation on the embodiments of this application. Those skilled in the art can understand the specific meaning of the above terms in the embodiments of this application according to the specific circumstances.

[0052] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0053] Example 1 This embodiment provides a recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects, the structural diagram of which is attached. Figure 1 As shown. From Figure 1 As can be seen, the microcrystalline substrate includes a backing layer, a substrate connected to the backing layer, and multiple cone-shaped needles disposed on the other side of the substrate; each needle includes a first needle body and a second needle body, the second needle body being connected to and covering the first needle body, and the first needle body being integrally formed with the substrate; the volume of the first needle body is smaller than the volume of the second needle body. Optical fiber mirror images of the substrate and the first needle body are attached. Figure 2 As shown in Figure A, the average height of the first needle body is 422 μm, and the average bottom diameter is 95 μm; a physical image of the needle body is attached. Figure 2 As shown in Figure B, the total height of the needle body is 600 μm, and the diameter of the bottom of the cone is 200 μm; the distance between the needle tip of the first needle body and the needle tip of the second needle body is 178 μm.

[0054] The backing layer is a polypropylene film (0.5 mm thick).

[0055] The matrix and the first needle body, by mass percentage, comprise the following components: 8.451% polylactic acid ultrashort fibers (3 mm in length, purchased from Yisheng New Materials Co., Ltd.); 90.141% crosslinked acrylamide functionalized material; and 1.408% photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP). The crosslinked acrylamide functionalized material is obtained by crosslinking methacrylic acid, hydroxyethyl methacrylate, N,N-methylenebisacrylamide, and polyethylene glycol dimethacrylate.

[0056] The second injection, by weight percentage, comprises the following components: 82.832% sodium hyaluronate, 16.928% recombinant type III human collagen (CS90, Jiangsu Hengrui Biomedical Technology Co., Ltd.), 0.06% asiaticoside, 0.06% hydroxyasiaticoside, 0.06% Centella Asiatica extract, and 0.06% ascorbic acid.

[0057] This embodiment provides a method for preparing the above-mentioned recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects, including the following steps: (1) Obtaining the mold: The first mold is used to form the base and the first needle; the second mold is used to form the second needle; the first mold is a copper plate, one side of which has protrusions arranged in a matrix, each protrusion corresponding to each of the first needles; the second mold is a silicone mold, one side of which has grooves arranged in a matrix, each groove corresponding to each of the second needles, each groove including two through layers, wherein the upper cavity is an inverted trapezoidal prism and the lower cavity is a cone; the partial structure of the copper plate and the silicone mold is as follows Figure 3 (A represents the first mold, and B represents part of the second mold) As shown, the protrusions on the copper plate are cylindrical, with a height of 80μm and a diameter of 10μm; the grooves in the silicone mold have a total height of 600μm, and the bottom diameter of the lower cone is 200μm. The height of the cone is 90% of the total height of the groove. Figure 3 For clarity, only 4×2 groove structures are shown in section B. In reality, the number of grooves is the same as the number of cylindrical protrusions in the first mold and their positions correspond.

[0058] (2) Preparation of needle body solutions: The first needle body solution is used to prepare the matrix and the first needle body; the second needle body solution and the third needle body solution are used to prepare the second needle body; the first needle body solution is prepared as follows: the acrylamide functional substance, photoinitiator and fiber filler are dissolved in the first solvent, dispersed evenly and defoamed; the second needle body solution is prepared as follows: the active ingredient of the second needle body and the first template material are dissolved in ultrapure water in sequence, stirred to dissolve and defoamed; the third needle body solution is prepared as follows: the active ingredient of the second needle body and the second template material are dissolved in ultrapure water in sequence, stirred to dissolve and defoamed. The first needle solution, by mass percentage, comprises the following components: 6% polylactic acid cellulose; 10% methacrylic acid; 15% hydroxyethyl methacrylate; 4% N,N-methylenebisacrylamide; 15% polyethylene glycol dimethacrylate (average Mn = 750 g / mol, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.); 20% polyethylene glycol dimethacrylate (average Mn = 10000 g / mol, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.); 1% initiator LAP; and the balance being water. The viscosity of the first needle solution at 25°C is 38.7 Pa·s.

[0059] The second injection solution, by mass percentage, comprises the following components: 26% sodium hyaluronate (average Mn = 200,000 Da), 1.58% sodium hyaluronate (average Mn = 8 kDa), 1.292% recombinant type III human collagen, 0.01% asiaticoside, 0.01% hydroxyasiaticoside, 0.01% Centella Asiatica extract, 0.01% ascorbic acid, and the balance being water. The first template materials used in the second injection solution are sodium hyaluronate (average Mn = 200,000 Da) and sodium hyaluronate (average Mn = 8 kDa), with a combined mass concentration of 27.58% in the second injection solution.

[0060] The third injection solution, by mass percentage, comprises the following components: 8.545% sodium hyaluronate (average Mn = 80,000 Da), 3.415% recombinant type III human collagen, 0.01% asiaticoside, 0.01% asiaticoside II, 0.01% Centella Asiatica extract, 0.01% ascorbic acid, with the balance being water. The second template material used in the third injection solution is sodium hyaluronate (average Mn = 8 kDa), with a total mass concentration of 8.545% in the solution.

[0061] (3) Molding the substrate and the first needle body: The fixture with the first mold is brought close to the solution of the first needle body so that the protrusion is immersed in the solution of the first needle body. Then the fixture is lifted so that the first mold leaves the solution of the first needle body with liquid. The substrate and the first needle body are left to stand for UV curing for 20 minutes, and then left to stand (35℃, 55% humidity) for 2 days to dry, so as to obtain the unmolded substrate and the first needle body.

[0062] (4) Preparation of recombinant collagen microcrystal patch: Add the second needle body solution to the second mold, and use scraping and vacuuming to make the second needle body solution enter the groove for the first drying (drying at 35℃ and -0.06MPa for 4h); add the third needle body solution in the same way, let stand for 20min, and then bring the undemolded substrate and the first needle body close to the second mold so that the first needle body is inserted into the corresponding groove of the second mold for the second drying (drying at 35℃ and -0.06MPa for 24h); remove the first mold to expose the back side of the substrate; attach a backing layer coated with pressure-sensitive adhesive to the back side of the substrate, remove the second mold, and the recombinant collagen microcrystal patch is obtained; The volume ratio of the amount of the third needle solution added to the amount of the second needle solvent added is 2.6:1.

[0063] To demonstrate that the template material in the second needle prepared by the above process gradually decreases in size from the needle tip to the needle base, a small amount of carotene (200 mg / L) was added to the solution of the second needle. The final microcrystalline substrate image is attached. Figure 2 B. It can be seen that the pigment gradually decreases from the tip to the bottom of the needle, indicating that the needle tip component in the second needle body after the first drying will diffuse into the third needle body solution in a small amount during the subsequent preparation process, thus causing the template material to gradually decrease from the tip to the bottom of the needle.

[0064] Example 2

[0065] This embodiment provides a recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects. The difference from Embodiment 1 is that the template material concentration in the second needle body is the same, that is, the second needle body includes the following components by mass percentage: sodium hyaluronate 82.832%, recombinant type III human collagen (CS90, Jiangsu Hengrui Biomedical Technology Co., Ltd.) 16.928%, asiaticoside 0.06%, hydroxyasiaticoside 0.06%, Centella Asiatica extract 0.06%, and ascorbic acid 0.06%.

[0066] The preparation method of the above-mentioned recombinant collagen microcrystalline patch differs from that in Example 1 in that: the second needle solution in step (2) comprises the following components by mass percentage: sodium hyaluronate (average Mn = 200,000 Da) 7.22%, sodium hyaluronate (average Mn = 8 kDa) 0.44%, sodium hyaluronate (average Mn = 80,000 Da) 6.17%, recombinant type III human collagen 2.83%, asiaticoside 0.01%, hydroxyasiaticoside 0.01%, Centella Asiatica extract 0.01%, ascorbic acid 0.01%, and the remainder is water. The template materials used in the second needle solution are sodium hyaluronate (average Mn = 200,000 Da), sodium hyaluronate (average Mn = 8 kDa), and sodium hyaluronate (average Mn = 80,000 Da), with a total mass concentration of 13.83% in the second needle solution.

[0067] Step (4) Add the second needle solution to the second mold. The second needle solution is made to enter the groove by scraping and vacuuming. After standing for 20 minutes, bring the undemolded substrate and the first needle close to the second mold so that the first needle is inserted into the corresponding groove of the second mold and perform (drying at 35℃ and -0.06MPa for 16 hours); remove the first mold to expose the back of the substrate; attach a backing layer coated with pressure-sensitive adhesive to the back of the substrate, remove the second mold, and you will get the recombinant collagen microcrystal patch.

[0068] Example 3 This embodiment provides a recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects. The difference from Embodiment 1 is that the second needle body, by weight percentage, comprises the following components: sodium hyaluronate 82.898%, recombinant type III human collagen (CS90, Jiangsu Hengrui Biomedical Technology Co., Ltd.) 14.64%, carnosine 0.6%, asiaticoside 0.06%, hydroxyasiaticoside 0.06%, Centella Asiatica extract 0.04%, arginine / lysine polypeptide (purchased from Langbowan Biomedical Co., Ltd.) 0.01%, acetyl hexapeptide-8 (purchased from Huatai Biotechnology Co., Ltd.) 0.01%, hydroxypropyl tetrahydropyranotriol 0.82%, tetrahydromethylpyrimidine carboxylic acid 0.82%, and ascorbic acid 0.06%.

[0069] The preparation method of the above-mentioned recombinant collagen microcrystalline patch differs from that in Example 1 in that: the second needle solution in step (2) comprises the following components by mass percentage: 26% sodium hyaluronate (average Mn = 200,000 Da), 1.58% sodium hyaluronate (average Mn = 8 kDa), 1% recombinant type III human collagen, 0.1% carnosine, 0.01% asiaticoside, 0.01% hydroxyasiaticoside, 0.001% arginine / lysine polypeptide, 0.001% acetyl hexapeptide-8, 0.1% hydroxypropyl tetrahydropyranotriol, 0.1% tetrahydromethylpyrimidine carboxylic acid, and 0.01% ascorbic acid, with the remainder being water. The first template material used in the second needle solution is sodium hyaluronate (average Mn = 200,000 Da) and sodium hyaluronate (average Mn = 8 kDa), with a total mass concentration of 27.58% in the second needle solution.

[0070] The third injection solution, by mass percentage, comprises the following components: sodium hyaluronate (average Mn = 80,000 Da) 8.558%, recombinant type III human collagen 3%, carnosine 0.1%, asiaticoside 0.01%, hydroxyasiaticoside 0.01%, Centella Asiatica extract 0.01%, arginine / lysine peptide 0.001%, acetyl hexapeptide-8 0.001%, hydroxypropyl tetrahydropyranotriol 0.15%, tetrahydromethylpyrimidine carboxylic acid 0.15%, and ascorbic acid 0.01%, with the balance being water. The second template material used in the third injection solution is sodium hyaluronate (average Mn = 8 kDa), with a total mass concentration of 8.558% in the solution.

[0071] Example 4 This embodiment provides a recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects. The difference from Embodiment 1 is that the distance between the tip of the first needle and the tip of the second needle is 213 μm, the average height of the first needle is 387 μm, and the average bottom diameter is 152 μm.

[0072] The matrix and the first needle body, by mass percentage, comprise the following components: silk fibroin fiber (Zhejiang Xingyue Biotechnology Co., Ltd.) 12.903%, cross-linked acrylamide functional material cross-linker 85.484%, and initiator LAP 1.613%; the cross-linked acrylamide functional material cross-linked from methacrylic acid, hydroxyethyl methacrylate, methacrylamide gelatin (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), and polyethylene glycol dimethacrylate (average Mn=10000g / mol, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0073] The preparation method of the above-mentioned recombinant collagen microcrystalline patch differs from that of Example 1 in that: the first needle solution in step (2) comprises the following components by mass percentage: 8% silk fibroin fiber, 10% methacrylic acid, 15% hydroxyethyl methacrylate, 8% methacrylamide gelatin, 20% polyethylene glycol dimethacrylate (average Mn = 10000 g / mol), and 1% initiator LAP, with the balance being water. The viscosity of the first needle solution (at 25°C) is 42.6 Pa·s.

[0074] Example 5 This embodiment provides a recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects. The difference from Embodiment 1 is that the total height of the needle body is 550 μm, and the diameter of the cone base is 150 μm. The distance between the tips of the first and second needle bodies is 128 μm.

[0075] The preparation method of the above-mentioned recombinant collagen microcrystal patch differs from that of Example 1 in that: in step (1), the groove of the second mold has a total height of 550 μm, the bottom diameter of the lower cone is 150 μm, and the height of the cone is 90% of the total height of the groove.

[0076] Example 6 This embodiment provides a recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects. The difference from Embodiment 1 is that the first needle does not contain fiber filler, the average height of the first needle is 327 μm, and the average bottom diameter is 98 μm; the total height of the microneedles in the microcrystalline patch is 500 μm, the bottom diameter of the cone is 120 μm, and the distance between the tips of the first needle and the second needle is 173 μm.

[0077] The matrix and the first needle body, by mass percentage, comprise the following components: 98.592% cross-linked acrylamide functional material; 1.408% photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP); the cross-linked acrylamide functional material is obtained by cross-linking methacrylic acid, hydroxyethyl methacrylate, N,N-methylenebisacrylamide, and polyethylene glycol dimethacrylate.

[0078] The preparation method of the above-mentioned recombinant collagen microcrystalline patch differs from that of Example 1 in that: the first needle solution in step (2) includes the following components by mass percentage: 10% methacrylic acid, 15% hydroxyethyl methacrylate, 4% N,N-methylenebisacrylamide, 15% polyethylene glycol dimethacrylate (average Mn=750g / mol, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), 26% polyethylene glycol dimethacrylate (average Mn=10000g / mol, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.); 1% initiator LAP, and the remainder is water; the viscosity of the first needle solution at 25°C is 27.4 Pa·s.

[0079] Example 7 This embodiment provides a recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects. The difference from Embodiment 1 is that the average height of the first needle is 487μm, the average bottom diameter is 103μm, and the distance between the tips of the first and second needles is 173μm.

[0080] The matrix and the first needle body, by mass percentage, comprise the following components: 5.634% polylactic acid ultrashort fibers (3 mm in length, purchased from Yisheng New Materials Co., Ltd.); 92.958% crosslinked acrylamide functionalized material; and 1.408% photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP). The crosslinked acrylamide functionalized material is obtained by crosslinking methacrylic acid, hydroxyethyl methacrylate, N,N-methylenebisacrylamide, and polyethylene glycol dimethacrylate.

[0081] The preparation method of the above-mentioned recombinant collagen microcrystalline patch differs from that of Example 1 in that: the first needle solution in step (2) includes the following components by mass percentage: polylactic acid fiber 4%; methacrylic acid 12%; hydroxyethyl methacrylate 15%; N,N-methylenebisacrylamide 5%; polyethylene glycol dimethacrylate (average Mn=750g / mol, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) 19%; polyethylene glycol dimethacrylate (average Mn=10000g / mol, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) 15%; initiator LAP 1%; and the remainder is water; the viscosity of the first needle solution at 25°C is 30.62 Pa·s.

[0082] Example 8 This embodiment provides a recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects, comprising a backing layer, a substrate connected to the backing layer, and a plurality of cone-shaped needles disposed on the other side of the substrate; each needle includes a first needle body and a second needle body, the second needle body being connected to and covering the first needle body, the first needle body being integrally formed with the substrate; the volume of the first needle body is smaller than the volume of the second needle body. The total height of the needles is 400 μm, and the diameter of the cone base is 100 μm. The distance between the tip of the first needle body and the tip of the second needle body is 192 μm.

[0083] The backing layer is a TPU polyurethane film (0.5mm thick).

[0084] The matrix and the first needle body, by mass percentage, comprise the following components: silk fibroin fiber (Zhejiang Xingyue Biotechnology Co., Ltd.) 12.903%, cross-linked acrylamide functional material cross-linker 85.484%, and initiator LAP 1.613%; the cross-linked acrylamide functional material cross-linked from methacrylic acid, hydroxyethyl methacrylate, methacrylamide gelatin (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), and polyethylene glycol dimethacrylate (average Mn=10000g / mol, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0085] The second injection, by weight percentage, comprises the following components: sodium hyaluronate 82.898%, recombinant type III human collagen (CS90, Jiangsu Hengrui Biomedical Technology Co., Ltd.) 14.64%, carnosine 0.6%, asiaticoside 0.06%, hydroxyasiaticoside 0.06%, Centella Asiatica extract 0.04%, arginine / lysine polypeptide (purchased from Langbowan Biomedical Co., Ltd.) 0.01%, acetyl hexapeptide-8 (purchased from Huatai Biotechnology Co., Ltd.) 0.01%, hydroxypropyl tetrahydropyranotriol 0.82%, tetrahydromethylpyrimidine carboxylic acid 0.82%, and ascorbic acid 0.06%.

[0086] This embodiment provides a method for preparing the above-mentioned recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects, including the following steps: (1) Obtaining molds: The first mold is used to form the base and the first needle; the second mold is used to form the second needle; the first mold is a copper plate, one side of which is provided with protrusions arranged in a matrix, each of the protrusions corresponding to each of the first needles; the second mold is a silicone mold, one side of which is provided with grooves arranged in a matrix, each of the grooves corresponding to each of the second needles, each groove including two through layers, wherein the upper cavity is an inverted trapezoidal column and the lower cavity is a cone; the protrusions on the copper plate are cones with a height of 60μm and a bottom diameter of 10μm; the grooves of the silicone mold have a total height of 400μm, the bottom diameter of the lower cone is 150μm, and the height of the cone is 90% of the total height of the groove.

[0087] (2) Preparation of needle body solutions: The first needle body solution is used to prepare the matrix and the first needle body; the second needle body solution and the third needle body solution are used to prepare the second needle body; the first needle body solution is prepared as follows: the acrylamide functional substance, photoinitiator and fiber filler are dissolved in the first solvent, dispersed evenly and defoamed; the second needle body solution is prepared as follows: the active ingredient of the second needle body and the first template material are dissolved in ultrapure water in sequence, stirred to dissolve and defoamed; the third needle body solution is prepared as follows: the active ingredient of the second needle body and the second template material are dissolved in ultrapure water in sequence, stirred to dissolve and defoamed. The first injection solution, by mass percentage, comprises the following components: 8% silk fibroin fiber, 10% methacrylic acid, 15% hydroxyethyl methacrylate, 8% methacrylated gelatin, 20% polyethylene glycol dimethacrylate (average Mn = 10000 g / mol), and 1% initiator LAP, with the balance being water. The viscosity of the first injection solution (25°C) is 42.6 Pa·s.

[0088] The second injection solution, by mass percentage, comprises the following components: 26% sodium hyaluronate (average Mn = 200,000 Da), 1.58% sodium hyaluronate (average Mn = 8 kDa), 1% recombinant type III human collagen, 0.1% carnosine, 0.01% asiaticoside, 0.01% hydroxyasiaticoside, 0.001% arginine / lysine peptide, 0.001% acetyl hexapeptide-8, 0.1% hydroxypropyl tetrahydropyranotriol, 0.1% tetrahydromethylpyrimidine carboxylic acid, and 0.01% ascorbic acid, with the balance being water. The first template materials used in the second injection solution are sodium hyaluronate (average Mn = 200,000 Da) and sodium hyaluronate (average Mn = 8 kDa), with a combined mass concentration of 27.58% in the second injection solution.

[0089] The third injection solution, by mass percentage, comprises the following components: sodium hyaluronate (average Mn = 80,000 Da) 8.558%, recombinant type III human collagen 3%, carnosine 0.1%, asiaticoside 0.01%, hydroxyasiaticoside 0.01%, Centella Asiatica extract 0.01%, arginine / lysine peptide 0.001%, acetyl hexapeptide-8 0.001%, hydroxypropyl tetrahydropyranotriol 0.15%, tetrahydromethylpyrimidine carboxylic acid 0.15%, and ascorbic acid 0.01%, with the balance being water. The second template material used in the third injection solution is sodium hyaluronate (average Mn = 8 kDa), with a total mass concentration of 8.558% in the solution.

[0090] (3) Molding the substrate and the first needle body: The fixture with the first mold is brought close to the solution of the first needle body so that the protrusion is immersed in the solution of the first needle body. Then the fixture is lifted so that the first mold with liquid leaves the solution of the first needle body. The substrate and the first needle body are cured by UV light for 20 minutes and then dried for 2 days to obtain the undemolded substrate and the first needle body. The average height of the first needle body is 208 μm and the average bottom diameter is 84 μm, as observed and statistically obtained by microscope.

[0091] (4) Preparation of recombinant collagen microcrystal patch: Add the second needle body solution to the second mold, and use scraping and vacuuming to make the second needle body solution enter the groove for the first drying (drying at 35℃ and -0.06MPa for 4h); add the third needle body solution in the same way, let stand for 20min, and then bring the undemolded substrate and the first needle body close to the second mold so that the first needle body is inserted into the corresponding groove of the second mold for the second drying (drying at 35℃ and -0.06MPa for 24h); remove the first mold to expose the back side of the substrate; attach a backing layer coated with pressure-sensitive adhesive to the back side of the substrate, remove the second mold, and the recombinant collagen microcrystal patch is obtained; The volume ratio of the amount of the third needle solution added to the amount of the second needle solvent added is 10:1.

[0092] Comparative Example 1 This comparative example provides a microcrystalline patch that differs from Example 1 in that it does not contain a first needle.

[0093] The preparation method of the above-mentioned microcrystalline patch is as follows: A second needle-body solution is added to a second mold (silicone mold), and the solution is allowed to enter the groove by scraping and vacuuming. A first drying process is then performed (drying at 35℃ and -0.06MPa for 4 hours). A third needle-body solution is added in the same manner, and after standing for 20 minutes, a second drying process is performed (drying at 35℃ and -0.06MPa for 24 hours). A backing layer coated with pressure-sensitive adhesive is applied to the surface, and the second mold is removed to obtain the microcrystalline patch. The volume ratio of the third needle-body solution to the second needle-body solution is 2.6:1. The components of the second mold, the second needle-body solution, and the third needle-body solution are the same as in Example 1.

[0094] Comparative Example 2 This comparative example provides a microcrystalline patch, which differs from Example 1 in that the first needle body is prepared by casting, and its structure is shown in the attached figure. Figure 4 As shown in Figure A, the first needle body prepared by casting is relatively large, similar in volume to the second needle body. Therefore, the second needle body cannot cover the first needle body, which makes the connection between the first and second needle bodies prone to breakage.

[0095] The preparation method of the above-mentioned microcrystalline patch is as follows: A first needle-like solution was added to a first silicone mold (an array of conical grooves with a height of 400 μm and a bottom diameter of 200 μm). The solution was then applied by scraping and vacuuming to allow it to penetrate the grooves. UV curing was performed for 0.25 h, followed by drying at 35 °C and -0.06 MPa for 24 h. Demolding yielded an assembly of the matrix and the first needle. A second needle-like solution was added to a second silicone mold (an array of conical grooves with a height of 400 μm and a bottom diameter of 120 μm). The solution was then applied by scraping and vacuuming to allow the solution to penetrate the grooves. The second needle solution is introduced into the groove and subjected to the first drying (drying at 35°C and -0.06MPa for 4 hours); the third needle solution is added in the same manner, and after standing for 20 minutes, the substrate and the assembly of the first needle are inserted into the corresponding grooves of the second silicone mold and subjected to the second drying (drying at 35°C and -0.06MPa for 24 hours); a backing layer coated with pressure-sensitive adhesive is attached to the back of the substrate, and the second silicone mold is removed to obtain the microcrystalline patch; the first needle solution, the second needle solution, and the third needle solution are the same as in Example 1.

[0096] Comparative Example 3 This comparative example provides a microcrystalline patch, which differs from Example 1 in that the first needle body is prepared by casting using a silicone mold (an array of conical grooves with a height of 400 μm and a bottom diameter of 100 μm). Its structure is shown in the attached figure. Figure 4As shown in Figure B, the microneedles have poor morphology, indicating that the first needle body with a slender and small size cannot be prepared by casting.

[0097] Test Example 1: Mechanical Performance Evaluation Mechanical performance evaluation includes the following aspects: A universal testing machine is used. The sample to be tested is placed on the stage with the needle tip facing upwards, and the sample is compressed at a speed of 0.5 mm / min. The load-displacement curve is recorded. The load of a single microneedle is calculated as load / number of microneedles. The stress of a single microneedle is calculated using the cross-sectional area at half the height of the microneedle (distance between the needle tip and the substrate) as the cross-sectional area, and the strain is calculated as displacement / microneedle height, thus obtaining the stress-strain curve of a single microneedle. The compressive modulus is calculated using the data range of 0~10% strain. The integral area of ​​the stress-strain curve is used as the energy absorption to evaluate the toughness of the sample. The compressive modulus and energy absorption of the first needle assembly (intermediate product, obtained after removing the first mold from the un-demolded substrate and the first needle) and the compressive modulus of the microcrystalline laminate without a backing layer are shown in Table 1.

[0098] Table 1

[0099] As shown in Table 1, the first needle body provided in this embodiment of the invention has a large compressive modulus and energy absorption. Therefore, when the second needle body melts, it can act as a substitute to continuously stimulate the skin and promote the absorption of active substances. Simultaneously, its large energy absorption indicates that the first needle body possesses a certain degree of toughness, preventing breakage and residue within the skin. The first needle body in Comparative Example 2 was prepared by casting, resulting in a larger volume that cannot meet the structural requirements of the second needle body covering the first needle body. This leads to the connection point between the first and second needle bodies becoming a stress concentration point (see...). Figure 4 This reduces the compressive modulus of the microcrystalline film without a backing layer.

[0100] Test Example 2: Security Test The microcrystalline patch was immersed in 1×PBS solution and allowed to stand for 48 hours to dissolve. After removal, it was observed under a microscope and the breakage rate of the first needle was counted. The breakage rate was recorded as: breakage rate = number of broken needles ÷ total number of needles × 100%. The microneedle array was 10 × 10, and the total number of needles was 100. Five groups of equilibrium tests were conducted for each sample and the average was calculated.

[0101] Fresh pigskin with intact stratum corneum after hair removal was fixed on the base of the texture analyzer. The microcrystalline patch was fixed to the probe using double-sided tape. The probe was set to descend at a speed of 0.5 mm / s, and the microcrystalline patch was applied to the pigskin until the force sensor displayed a load of 30 N. The position was held for 2 minutes, then the load was slowly released to 0 and held still for 24 hours. The probe was then raised at a speed of 0.5 mm / s, the microcrystalline patch was removed, and the breakage rate of the first needle was observed and calculated using a microscope. The breakage rate was calculated as follows: (Broken needles ÷ Total number of needles) × 100% - Preparation breakage rate. The microneedle array consisted of 10 × 10 needles, with a total of 100 needles. Five sets of equilibrium tests were conducted for each sample, and the average was calculated. The results are shown in Table 2.

[0102] Table 2

[0103] As can be seen from the data in Table 2, the microcrystalline patch provided in the embodiments of the present invention has a low needle breakage rate, especially with a low needle breakage rate, and is highly safe to use.

[0104] Test Example 3: Recombinant Collagen Loading Test Microcrystalline patches without backing were prepared using recombinant collagen containing fluorescent labeling as raw material according to the specific implementation method. The microcrystalline patches were immersed in 1×PBS solution and allowed to stand for 48 hours to dissolve. The content of recombinant collagen was quantitatively analyzed according to the fluorescence intensity. Each group of experiments was performed in parallel three times. The recombinant collagen loading per unit mass of microcrystalline patch (loading rate, %) and the loading of a single microneedle (loading, mg / needle) were calculated. The results are shown in Table 3.

[0105] The specific procedures for fluorescently labeled recombinant collagen are as follows: Recombinant collagen was dissolved in ultrapure water to prepare a solution of 0.01 g / mL, and fluorescein isothiocyanate (FITC) was dissolved in carbonate buffer at pH 9 to prepare a solution of 0.5 mg / mL. The two solutions were mixed at a volume ratio of 1:1 and reacted at 4°C in the dark for 24 h. Then, the solution was poured into a dialysis bag with a molecular weight cutoff of 8000 Da and dialyzed with PBS buffer for 3 days, with the PBS buffer being changed every 12 h. The dialyzed protein was freeze-dried to obtain fluorescently labeled recombinant collagen and stored in the dark.

[0106] Table 3

[0107] Compared to Example 4, Example 4 had a larger first needle body volume due to the higher viscosity of the first needle body solution, thus reducing the loading of recombinant collagen. Example 2 and Example 1 had similar loading amounts, but based on the data in Table 1, Example 1 had a higher compressive modulus, indicating that the gradual distribution of the template material in the second needle body is beneficial for balancing loading amount and improving the mechanical properties of the microcrystalline patch.

[0108] Test Example 4: In vitro transdermal experiment Microcrystalline patches were prepared using recombinant collagen containing fluorescent labeling as raw material according to the method of the specific implementation method (the method is the same as in test example 3), and then in vitro transdermal experiments were conducted using a Franz diffusion cell.

[0109] Eight mL of PBS solution (pH 7.4) was added to the receiving chamber of the Franz diffusion cell as the receiving solution. The same operator pressed the microcrystal patches obtained in each specific implementation method onto mouse skin (abdominal skin of C57 mice, after hair removal and removal of subcutaneous fat and blood vessels) with similar force. The skin was then fixed between the supply and receiving chambers, with the skin layer facing the supply chamber. A stainless steel cylinder was placed on top of the microcrystal patch to ensure full adhesion between the microneedles and the skin. Air bubbles were removed, ensuring complete contact between the receiving solution surface and the mouse skin. The receiving chamber was maintained at 37°C and magnetically stirred at 750 r / min. Experimental groups were set up for 1, 2, 4, 6, 12, and 24 h. At the corresponding time points, the microcrystal patches were removed, the mouse skin surface was washed with water, and a skin homogenate was prepared. Both the skin homogenate and the removed receiving solution were centrifuged at 3000 r / min for 10 min. The recombinant collagen content was quantitatively analyzed based on fluorescence intensity. Each group of experiments was conducted in parallel three times. A release curve was plotted with the release rate (release amount ÷ load amount × 100%) on the ordinate and the release time on the x-axis. Figure 5 As shown.

[0110] from Figure 5 It can be seen that, compared with Comparative Example 1, Example 1 was able to continuously deliver recombinant collagen percutaneously due to the presence of the first needle, while the percutaneous delivery rate of Comparative Example 1 slowed down after 6 hours because the tip of the second needle melted and could not continuously stimulate the skin. In addition, compared with Example 4, Example 4 had a poorer ability to continuously deliver recombinant collagen percutaneously because the distance between the tips of the first and second needles was too large.

[0111] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing recombinant collagen microcrystalline patches with anti-aging and wrinkle-reducing effects, characterized in that, The recombinant collagen microcrystal patch includes: a substrate and a plurality of needles disposed on the substrate; each needle includes a first needle and a second needle, the second needle being connected to the first needle and covering the outer layer of the first needle, the first needle being integrally formed with the substrate; the volume ratio of the first needle to the second needle is less than or equal to 0.8; the first needle and the substrate are insoluble, the second needle is soluble and includes an active ingredient, the active ingredient including recombinant collagen; The preparation method includes the following steps: (1) Obtaining a mold: The first mold is used to form the base and the first needle body; the second mold is used to form the second needle body; the first mold is a copper plate with a plurality of protrusions arranged in a matrix on one side, each of the protrusions corresponding to each of the first needle bodies; the second mold is a silicone template with a plurality of grooves arranged in a matrix on one side, each of the grooves corresponding to each of the second needle bodies; (2) Preparation of needle body solutions: The first needle body solution is used to prepare the matrix and the first needle body; the second needle body solution and the third needle body solution are used to prepare the second needle body; the first needle body solution is prepared as follows: the acrylamide functional substance, photoinitiator and fiber filler are dissolved in the first solvent, dispersed evenly and defoamed; the second needle body solution is prepared as follows: the active ingredient of the second needle body and the first template material are dissolved in ultrapure water in sequence, stirred to dissolve and defoamed; the third needle body solution is prepared as follows: the active ingredient of the second needle body and the second template material are dissolved in ultrapure water in sequence, stirred to dissolve and defoamed. (3) Molding the substrate and the first needle body: The first mold is brought close to the first needle body solution so that the protrusion is immersed in the first needle body solution. Then the first mold is lifted in the vertical direction so that the protrusion leaves the first needle body solution. The mold is left to stand for UV curing to obtain the unmolded substrate and the first needle body. (4) Preparation of recombinant collagen microcrystal patch: Add the second needle body solution to the second mold and perform the first drying; then add the third needle body solution, and bring the undemolded substrate and the first needle body close to the second mold so that the first needle body is inserted into the corresponding groove of the second mold and perform the second drying; remove the first mold and the second mold in sequence to obtain the recombinant collagen microcrystal patch; The first template material and the second template are independently selected from at least one of polyvinylpyrrolidone, hyaluronic acid or its salts, chitosan and hydroxypropyl methylcellulose; The acrylamide functional substance is selected from at least one of methacrylic acid, methacrylate, acrylic acid, acrylate, polyethylene glycol methacrylate and methacrylamide gelatin; The fiber filler includes at least one of polylactic acid fiber, cellulose fiber, and silk fibroin fiber.

2. The preparation method of the recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects according to claim 1, characterized in that, The recombinant collagen microcrystalline patch contains 5% to 15% recombinant collagen. And / or, the loading of recombinant collagen in the recombinant collagen microcrystal patch is 0.02~0.1 mg / needle; And / or, the first needle body is a cone or cone-like shape with an average height of 200~450μm and a bottom diameter of 80~160μm; And / or, the needle body is a cone or cone-like shape with an average height of 400~800μm and a bottom diameter of 120~300μm; And / or, the area of ​​the largest cross-section of the first needle body is smaller than the area of ​​the largest cross-section of the needle body; And / or, the distance between the tip of the first needle body and the tip of the second needle body is less than 200 μm.

3. The method for preparing the recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects according to claim 1 or 2, characterized in that, The microcrystalline recombinant collagen microcrystal patch also includes a backing, which is adhered to the substrate and disposed on the surface away from the needle.

4. The preparation method of the recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects according to claim 3, characterized in that, The active ingredients also include at least one of carnosine, asiaticoside, hydroxyasiaticoside, centella asiatica extract, arginine / lysine polypeptide, acetyl hexapeptide-8, hyaluronic acid or its salt, hydroxypropyl tetrahydropyranotriol, tetrahydromethylpyrimidine carboxylic acid and ascorbic acid. And / or, the backing is selected from at least one of polyethylene, polypropylene, nonwoven fabric, polyurethane and hydrogel.

5. The preparation method of the recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects according to claim 4, characterized in that, The compression modulus of the first needle body is 250~400MPa; And / or, the first needle body and the matrix, by weight percentage, comprise the following components: 5.634%~14% fiber filler, 85%~99% crosslinked acryloyl functional material, and 0.5%~2% photoinitiator; And / or, the second needle body comprises, by weight percentage, the following components: 80%–85% sodium hyaluronate, 14%–18% recombinant collagen, 0–0.6% carnosine, 0–0.1% asiaticoside, 0–0.1% hydroxyasiaticoside, 0–0.1% centella asiatica extract, 0–0.05% arginine / lysine polypeptide, 0–0.05% acetyl hexapeptide-8, 0–1% hydroxypropyl tetrahydropyranotriol, 0–1% tetrahydromethylpyrimidine carboxylic acid, and 0–0.1% ascorbic acid.

6. The preparation method of the recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects according to claim 1, characterized in that, Each of the grooves includes two through cavities, wherein the upper cavity is an inverted trapezoidal cylinder and the lower cavity is a cone; And / or, each of the protrusions is a cylinder or a cone.

7. The method for preparing the recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects according to claim 6, characterized in that, Each of the protrusions has a height of 60~80μm and a bottom diameter of 10μm; And / or, the total depth of each groove is 400~800μm, the diameter of the maximum cross-section of the lower cavity is 120~300μm, and the height of the lower cavity is 90% of the total depth of the groove.

8. The preparation method of the recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects according to claim 1, characterized in that, The first template material and the second template material may have the same or different chemical compositions, and the mass concentration of the first template material in the second needle solution is greater than the mass concentration of the second template material in the third needle solution; And / or, the volume ratio of the second needle solution to the third needle solution is 1:2~15; And / or, the viscosity of the first needle solution at 25°C is 20~50 Pa·s; And / or, the mass concentration of the first template material in the second needle solution is 20% to 30%; the mass concentration of the second template material in the third needle solution is less than or equal to 10%.

9. The method for preparing the recombinant collagen microcrystalline patch with anti-aging and wrinkle-reducing effects according to claim 1, characterized in that, The first drying refers to drying under static conditions at 20~35℃ and negative pressure for 3~5 hours; And / or, the second drying refers to drying at 20~35℃ under negative pressure for 12~24h; And / or, the UV curing includes irradiating with UV light for 0.25 to 1 hour, and then drying by standing at 20 to 35°C and 40% to 60% humidity for 1 to 2 days; And / or, the step of sequentially removing the first mold and the second mold specifically includes: removing the first mold to expose the back of the substrate, then attaching a backing to the back, and finally removing the second mold.

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