Soluble microcrystal with firming and anti-wrinkle effects as well as preparation method and application of soluble microcrystal

Abstract

The invention discloses a soluble microcrystal with tightening and anti-wrinkle effects as well as a preparation method and application thereof, and belongs to the technical field of beauty and skin care. The soluble microcrystal comprises the following components: collagen, hyaluronic acid, modified hyaluronic acid, melittin, nicotinamide, linoleic acid, phospholipid, cholesterol and the like. Hyaluronic acid is modified through EDTA and polyethylene glycol diamine, and modified hyaluronic acid is obtained. The modified hyaluronic acid is good in biocompatibility, has a multi-branch structure and can form a three-dimensional cross-linked network structure among molecules or with collagen, the mechanical strength of the microcrystal needle body is greatly improved, the microcrystal is prepared through the two steps of conventional drying and freeze-drying, and the rapid water solubility and drug release property of the microcrystal are guaranteed. Liposome formed by linoleic acid, phospholipid and cholesterol contains moisture and effective components, and transdermal absorption of the effective components can be greatly promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of beauty and skin care, and in particular to soluble microcrystals with firming and anti-wrinkle effects, and a preparation method and application thereof. Background Art

[0002] As we age and are exposed to increased UV rays, the amount of hyaluronic acid (HA) in our skin decreases, leading to a range of issues such as aging, decreased elasticity, and wrinkles. To combat these skin issues, HA is often added to cosmetics to improve skin quality through its unique moisturizing and repairing properties.

[0003] Hyaluronic acid, a substance naturally present in the skin, possesses a powerful water-absorbing capacity, capable of absorbing and retaining hundreds of times its own weight in water. When added to the skin in cosmetics, hyaluronic acid is absorbed through the skin, increasing its hydration. This moisturizing effect not only keeps the skin's surface moisturized but also penetrates deeper into the skin, providing ample moisture and nutrients to skin cells, thereby nourishing the skin and improving its texture.

[0004] Hyaluronic acid of varying molecular weights differs in its transdermal absorption capacity. Low-molecular-weight hyaluronic acid (typically less than 300kDa) has excellent transdermal absorption, as its smaller molecular structure allows it to more easily penetrate the skin's stratum corneum and sebum membrane, reaching the epidermis and even the dermis. Soluble patches with an array of microcrystalline protrusions (i.e., microneedle bodies) made with sodium hyaluronate or hyaluronic acid as the primary excipient and loaded with active ingredients are called soluble microcrystalline patches, which promote scalp absorption.

[0005] Human skin is divided into the epidermis and dermis. The epidermis includes the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale. The stratum corneum, the outermost layer of the epidermis, serves as the primary barrier to transdermal transport of most substances. Microneedles facilitate penetration of the stratum corneum and promote transdermal absorption.

[0006] Prior art CN118846363A discloses a microneedle for transdermal delivery of macromolecular drugs. The microneedle tip layer is prepared by repeatedly injection-molding and drying a 40 mg / mL pure protein solution. A 15% hyaluronic acid backing layer is then injected to increase the drug loading capacity of the protein transdermal microcrystals and avoid the safety risks associated with the use of high-molecular-weight polymers. Repeated injection-molding and drying indicate that the microneedles are susceptible to shrinkage and deformation during drying. Furthermore, even fully dried products still suffer from the technical issue of strong moisture absorption from the skin, which contradicts the hydration objective of cosmetic skincare.

[0007] It can be seen that the existing technology still has some unresolved technical problems, which are summarized as follows: 1. The backing of the soluble microneedle patch is made of water-soluble material. In order to ensure the strength of the microcrystals, the existing microcrystal (or microneedle) preparation technology adopts measures to fully dry the microcrystal patch. However, the backing has low water content after drying, and its adhesion to the skin is poor. It is not easy for human skin to absorb the active ingredients in the backing material; 2. In order to ensure the absorbability of the microcrystal needle tip, the needle tip needs to have a certain water content, but it will lead to low strength of the needle body. In order to ensure the strength of the needle tip and maintain the shape of the needle body, insoluble or high molecular excipients are added during the production process, resulting in poor absorption performance of the soluble material. 3. When microcrystals are stored at room temperature, it is easy for the needle tip to become passivated due to changes in temperature. For example, high temperatures in summer can easily cause the microcrystals to soften, which is not convenient for storage at room temperature. Summary of the Invention

[0008] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a soluble microcrystal with firming and anti-wrinkle effects, and a preparation method and application thereof. The soluble microcrystals have the advantages of high microcrystal hardness, not easy to soften at room temperature, low irritation, and easy solubility, and have the technical effects of moisturizing and whitening, and can be used to prepare cosmetics, skin care products, and medical beauty products.

[0009] The present invention is achieved through the following technical solution: providing a soluble microcrystal, characterized in that it includes the following components, calculated by weight: 20-30 parts of collagen, 10-20 parts of hyaluronic acid, 20-30 parts of modified hyaluronic acid, 3-5 parts of bee venom peptide, 1-3 parts of whitening component, 5-10 parts of linoleic acid, 10-20 parts of phospholipids, and 3-5 parts of cholesterol.

[0010] The modified hyaluronic acid has the following structure:

[0011] The ratio of m:n is 1:(1-10), wherein m and n in the structural formula only represent the number of grafted carboxyl groups of hyaluronic acid, and are not used as a limitation on the position of the grafted carboxyl groups in hyaluronic acid, and do not indicate that the modified hyaluronic acid is a block compound, wherein z is an integer of 1-100.

[0012] The modified hyaluronic acid preparation method is as follows:

[0013] S1: EDTA-modified polyethylene glycol diamine (NH2-PEG9-NH2): EDTA (10 mmol) was dissolved in 1 L of pH 5.5 phosphate buffer, EDC (10 mmol) and NHS (11 mmol) were added, and stirred at room temperature for 2 hours to form an EDTA-NHS ester solution.

[0014] S2: Dissolve 10 mmol of polyethylene glycol diamine (NH2-PEG9-NH2) in a phosphate buffer solution at pH 7.4, add EDTA-NHS ester solution dropwise under stirring, and continue the reaction for 24 hours to obtain an EDTA-PEG-NH2 solution.

[0015] S3: Dissolve hyaluronic acid (HA) (50 mmol carboxyl groups) in 3 L of phosphate buffer (pH 6.0), add EDC (10 mmol) and NHS (10 mmol), and stir at room temperature for 1 hour to form an HA-NHS ester solution. Add the EDTA-PEG-NH2 solution to the HA-NHS ester solution and stir for 24 hours. Transfer the reaction solution to a dialysis bag (MWCO 3 kDa) and dialyze it against deionized water for purification; after dialysis, dry and pulverize to obtain modified hyaluronic acid powder.

[0016] The molar ratio of EDTA, EDC and NHS is 1:(0.9-1.1):(0.9-1.2), preferably 1:1:1.1.

[0017] The molar ratio of the polyethylene glycol diamine to EDTA is 1:0.9-1.1, preferably 1:1.

[0018] The molar ratio of hyaluronic acid to polyethylene glycol diamine calculated based on the carboxyl content is (1-10):1.

[0019] The phospholipid is selected from at least one of sodium dioleoylphosphatidylserine (DOPS), egg yolk lecithin, soybean lecithin, dioleoyl L-α-phosphatidylethanolamine (DOPE), 1,2-dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC).

[0020] The whitening component is selected from at least one of niacinamide, vitamin C, ursolic acid, azelaic acid, and glutathione.

[0021] The present invention also provides a method for preparing soluble microcrystals: a mixed aqueous solution is prepared by adding a prescribed amount of collagen, hyaluronic acid, modified hyaluronic acid, and melittin to sterile deionized water. Phospholipids and cholesterol are dissolved in ethanol under low-temperature water bath conditions. Linoleic acid and a whitening ingredient are added to the phospholipid ethanol solution and rapidly stirred. An equal volume of sterile deionized water is added and stirred at high speed to form a suspension. An equal volume of sterile deionized water is added to the suspension and high-pressure homogenization is performed to prepare liposomes containing the whitening ingredient. The liposomes are added to the prepared mixed aqueous solution and mixed uniformly to obtain a microcrystal preparation solution. The microcrystal preparation solution is injected into a microcrystal mold under vacuum conditions in an amount that completely covers the needle tip area of the mold. The solution is allowed to stand at room temperature until it dries into an elastic gel-like state and is freeze-dried for 0.5-2 hours to obtain microcrystals. The microcrystals prepared by the above method include a basal layer and a needle body, the needle body having a length of 50-600 μm, preferably 100-500 μm, or 200-500 μm.

[0022] The present invention also provides a soluble microcrystal, characterized in that it comprises the following components, calculated by weight: 20-30 parts of collagen, 10-20 parts of hyaluronic acid, 20-30 parts of modified hyaluronic acid, 5-10 parts of linoleic acid, 10-20 parts of phospholipids, and 3-5 parts of cholesterol; 0.01-20 parts of the following functional components can be optionally added, and the functional components are selected from at least one of melittin DNA sodium, acetyl hexapeptide-8, arginine / lysine polypeptide, milk extract, grape fruit cell extract, and exosomes.

[0023] Beneficial effects

[0024] The present invention provides a soluble microcrystal and a preparation method thereof. The modified hyaluronic acid is uniquely used as a microcrystal matrix. The modified hyaluronic acid is prepared by grafting ethylenediaminetetraacetic acid (EDTA) onto hyaluronic acid through polyethylene glycol diamine. Both polyethylene glycol diamine and EDTA are highly hydrophilic biocompatible components. The modified hyaluronic acid is rich in N and O side chains, which can promote the formation of intermolecular forces such as hydrogen bonds between polymers, thereby forming a three-dimensional network structure and significantly improving the mechanical strength and elastic modulus of the material as well as its water retention and water absorption properties. The modified hyaluronic acid can also form effective intermolecular forces with collagen and melittin to form a three-dimensional network structure, thereby improving water retention and the strength of the microcrystal, reducing the use of microcrystal matrix materials, and greatly increasing drug loading.

[0025] The introduction of EDTA gives HA a strong metal ion chelating ability, which can remove Fe in the skin cell microenvironment. 2+ 、Cu 2+ Metal ions such as copper ions can inhibit the generation of free radicals and oxidation reactions. For example, copper ions in competitive chelated tyrosinase can effectively inhibit the activity of tyrosinase, reduce the production of melanin, and have a whitening effect.

[0026] The present invention provides a secondary drying method. First, after the microcrystalline component solution is molded, it is dried at room temperature to a gel-like state. This facilitates the uniform solidification of the microcrystalline matrix, forming a three-dimensional network structure and improving mechanical strength. Second, the secondary drying method uses freeze drying. The freezing process helps maintain the shape of the microcrystalline matrix and prevents shrinkage and deformation due to water loss during the drying process. At the same time, the vacuum pumping time during the freeze drying process is controlled to ensure that the microcrystalline retains some water while maintaining sufficient hardness. After freeze drying, the microcrystals have a relative mesoporous structure, which is more conducive to water absorption and dissolution, promoting the release of active ingredients.

[0027] The microcrystals described in the present invention are not completely dry; water is still contained within the liposomes and within the microcrystal matrix. Linoleic acid, a fluid liquid at room temperature, penetrates between the phospholipid bilayers and is highly hydrophobic. This helps increase the density of the liposome membrane, improves encapsulation capacity, reduces water loss during the freeze-drying process, and reduces the microcrystals' absorption of skin interstitial fluid, thereby improving applicability. The biocompatibility of the liposomes facilitates the entry of the active ingredients contained within cells, promoting transdermal absorption.

[0028] The microcrystals of the present invention have the technical effects of high microcrystal hardness, not easy to soften at room temperature, low irritation, easy solubility, moisturizing and whitening. The microcrystals can be used to prepare microcrystal needles and microcrystal backings at one time, and are easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Chemical structure of modified hyaluronic acid.

[0030] Figure 2 Infrared spectrum of modified hyaluronic acid (amide bond peak position 1655cm -1 ).

[0031] Figure 3 Electron microscope images of microcrystals of Example 4 and Comparative Example 5. DETAILED DESCRIPTION

[0032] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. The described embodiments are only some preferred embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] In the examples, the experimental methods used are conventional or common methods in the art unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0034] The raw materials used in the embodiments and comparative examples are now described as follows:

[0035] Collagen: purchased from Chenguang Biotechnology;

[0036] Hyaluronic acid: Hunan Bloomage Biotechnology (300kDa);

[0037] Polyethylene glycol diamine (NH2-PEG4-NH2, NH2-PEG9-NH2, NH2-PEG 19 -NH2): Shanghai Yanfen Biochemical Technology Co., Ltd.;

[0038] Melittin: purchased from North China Pharmaceutical Co., Ltd. (for injection);

[0039] EDTA (diethylaminetetraacetic acid): Xi'an Tianyi Biological;

[0040] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was purchased from Jier Biochemical (Shanghai) Co., Ltd.

[0041] N-hydroxysuccinimide (NHS): purchased from Hubei Xinghengkang Chemical Technology Co., Ltd.;

[0042] Phospholipids (HSPE): purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.;

[0043] Linoleic acid: purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.

[0044] Cholesterol: purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.

[0045] Unless otherwise specified, the components and raw materials used in the examples and comparative examples of the present invention are all commercially available raw materials, and the components and raw materials used in each parallel experiment are all of the same kind.

[0046] The preparation method of modified hyaluronic acid is as follows:

[0047] S1: EDTA-modified polyethylene glycol diamine (NH2-PEG9-NH2): EDTA (10 mmol) was dissolved in 1 L of pH 5.5 phosphate buffer, EDC (10 mmol) and NHS (11 mmol) were added, and stirred at room temperature for 2 hours to form an EDTA-NHS ester solution.

[0048] S2: Dissolve 10 mmol of polyethylene glycol diamine (NH2-PEG9-NH2) in a phosphate buffer solution at pH 7.4, add EDTA-NHS ester solution dropwise under stirring, and continue the reaction for 24 hours to obtain an EDTA-PEG-NH2 solution.

[0049] S3: Dissolve HA (50 mmol carboxyl groups) in 3 L of phosphate buffer (pH 6.0), add EDC (10 mmol) and NHS (10 mmol), and stir at room temperature for 1 hour to form a HA-NHS ester solution. Add the EDTA-PEG-NH2 solution to the HA-NHS ester solution and stir for 24 hours. Transfer the reaction solution to a dialysis bag (MWCO 3 kDa) and dialyze against deionized water for purification; after dialysis, dry and pulverize to obtain modified hyaluronic acid powder for later use.

[0050] Example 1

[0051] Preparation method of soluble microcrystals:

[0052] Prescription: 20 parts of collagen, 10 parts of hyaluronic acid, 20 parts of modified hyaluronic acid, 3 parts of bee venom peptide, 1 part of niacinamide, 5 parts of linoleic acid, 10 parts of phospholipids, and 3 parts of cholesterol.

[0053] Step 1: Add 20 equivalents of collagen, 10 equivalents of hyaluronic acid, 20 equivalents of modified hyaluronic acid, and 3 equivalents of melittin to sterile deionized water to prepare a mixed aqueous solution (the mass concentration of modified hyaluronic acid is 1%).

[0054] Step 2: Under low temperature 50°C water bath heating conditions, 10 equivalents of phospholipids (HSPE) and 3 equivalents of cholesterol were dissolved in ethanol (the amount used was such that the phospholipid concentration was 1 g / ml), 5 equivalents of linoleic acid and 1 equivalent of nicotinamide were added to the phospholipid ethanol solution, and the mixture was rapidly stirred. An equal volume of sterile deionized water was added, and the mixture was stirred with a high-speed stirring wall breaker to form a suspension. An equal volume of sterile deionized water was added to the above suspension, and the mixture was homogenized with a high-pressure homogenizer (model: ATS AH-D150). The homogenization cycle was repeated 3 times to prepare liposomes containing the active ingredients linoleic acid and nicotinamide.

[0055] Step 3: Add the liposomes from step 2 to the mixed aqueous solution prepared in step 1 and mix well to obtain a microcrystal preparation solution. Under vacuum conditions, inject the microcrystal preparation solution into the microneedle mold, injecting enough to completely cover the needle tip area of the mold. Let it stand at room temperature for 2.5 hours until it dries into an elastic gel. Freeze-dry, slowly cool to -20°C, and vacuum dry for 0.5 hours to obtain microcrystals.

[0056] Examples 2-4

[0057] The preparation methods of Examples 2-4 are the same as those of Example 1, with the only difference being that the amounts of the components are adjusted.

[0058] Comparative Examples 1-5

[0059] Comparative Examples 1 and 2 are prepared in the same manner as Example 1 and Example 4, respectively, except that the modified hyaluronic acid is replaced with an equal amount of hyaluronic acid.

[0060] Comparative Example 3 was prepared in the same manner as Example 2, except that linoleic acid was reduced in the components.

[0061] Comparative Example 4 was prepared in the same manner as Example 3, except that melittin was reduced as a component.

[0062] Comparative Example 5 has the same components as Example 4, except that: after the microcrystal preparation liquid is injected into the microneedle mold, it is allowed to stand at room temperature for 2.5 hours, dried into an elastic gel, and then dried at room temperature for another 4 hours.

[0063] Table 1 Components and mass parts of Examples 1 to 4 and Comparative Examples 1 to 5

[0064]

[0065]

[0066] Performance test verification of Examples 1-4 and Comparative Examples 1-5

[0067] (1) Skin irritation test:

[0068] Skin irritation tests were conducted on the whitening skin care products of the Examples and Comparative Examples. The test subjects were nude BALB / c-nu mice. Ten mice (five male and five female mice) were randomly assigned to each sample. Sample microcrystals (0.5 cm × 0.5 cm) were applied to the backs of the mice (0.5 cm × 0.5 cm) for 0.5 h, once in the morning and once in the evening (9:00 and 18:00). The mice's behavior and skin condition were observed daily for four consecutive weeks. Scoring was performed according to the following table:

[0069] Mouse skin condition Rating / points Normal skin 5 No irritation, normal skin color 4 Mild irritation (redness, etc.) 3 Rash and edema, 2 Ulcers, blisters, loss of mobility, death 0

[0070] Table 2 Results of skin irritation test after 4 weeks

[0071]

[0072] After 4 weeks of testing, the above Table 2 shows that the microcrystals prepared in Examples 1-4 and Comparative Examples 1-6 have good biosafety and do not cause irritation to the skin of mice.

[0073] Safety evaluation of human trial test: The human skin trial test is used for testing. The specific test methods refer to the "Technical Specifications for Safety of Cosmetics" (2015 edition), see the table below for details.

[0074] Grading Skin reactions 0 No response 1 Faint erythema 2 Erythema, infiltration, and papules 3 Erythema, infiltration, edema, papules, blisters 4 Erythema, edema, bullae

[0075] A total of 180 subjects, aged 18-56 years (mean age 32 years), were randomly divided into 9 groups. Sample 1 of Examples 1-4 and Comparative Examples 1-5 was applied to the outer side of the forearm for 0.5 hours once daily for 4 weeks. The test results (see Table 3) show that all samples of Examples 1-4 and Comparative Examples 1-5 showed a negative reaction (grade 0), indicating no erythema reaction on the skin.

[0076] Table 3 Test results

[0077] Project Number Level 0 Level 1 Level 2 Level 3 Level 4 Example 1 20 0 0 0 0 Example 2 20 0 0 0 0 Example 3 20 0 0 0 0 Example 4 20 0 0 0 0 Comparative Example 1 20 0 0 0 0 Comparative Example 2 20 0 0 0 0 Comparative Example 3 20 0 0 0 0 Comparative Example 4 20 0 0 0 0 Comparative Example 5 20 0 0 0 0

[0078] (2) Determination of water content of microcrystals

[0079] The microcrystals described in Examples 1-4 and Comparative Examples 1-5 were fully dried in a drying oven at 50°C for 48 hours, and the weight before drying W1 and the weight after drying W2 were recorded. The water content of the microcrystals was: (W1-W2) / W1; the experimental results are shown in Table 4.

[0080] Table 4 Microcrystalline water content (%)

[0081]

[0082] Examples 1-4 use liposomes to encapsulate an effective combination of water and water, which greatly improves the water content of the microcrystals. The water content is positively correlated with the usage ratio of the liposome components. Compared with traditional microcrystals (microneedles), there is no need to use a completely dry method to ensure the strength of the needle body, which can effectively reduce the absorption of moisture in the skin cells by the microcrystals during use; Compared with Example 2, the water content of Comparative Example 3 is reduced, which is related to the lack of the component linoleic acid, which is related to the fact that linoleic acid can improve the encapsulation performance of the liposome bilayer.

[0083] (3) Test microcrystal hardness

[0084] The microcrystal strength was tested using a Shimadzu universal materials testing machine. The microcrystals were fixed to a horizontal test bench and a P / 6 flat-head stainless steel cylindrical probe was used to apply vertical pressure along the microneedle axis at a constant rate of 0.1 mm / s and an excitation force of 0.01 N. The entire mechanical response, from the moment the probe contacted the needle tip until it reached the predetermined end height (i.e., the full length of the 400 μm microcrystal), was recorded in detail to evaluate the mechanical properties, deformation characteristics, and product stability of the microcrystals. The microneedle strength of the test samples was measured at 30°C and 40°C on day 0, and after four months of sealed storage at 30°C.

[0085] Table 5 Strength of microcrystals at different temperatures (N)

[0086]

[0087]

[0088] Comparison of Comparative Examples 1 and 2 with Examples 1-4 shows that Examples 1-4 use modified hyaluronic acid, which can greatly improve the strength of the microneedles, and at 40 ° C, still have good strength, which shows that the microcrystals of the present invention still have extremely strong strength at high temperatures and can be effectively delivered transdermally; however, at 40 ° C, the strength of the microcrystals of Comparative Examples 1 and 2 is significantly reduced, and the transdermal performance of the skin is reduced. The human body temperature is close to 37 ° C. In summer, when the temperature is high or the human body contacts the microcrystals, it may seriously affect the use of hyaluronic acid as a matrix of microcrystals. The results of the microcrystal strength at 30 ° C after 4 months show that the microcrystal strength of Examples 1-4 is consistent with the strength at 0 days, showing that the microcrystals of the present invention have good placement stability, are easy to store at room temperature, and are easy to transport. The strength of the microcrystals of Comparative Example 3 is significantly reduced after 4 months, showing that linoleic acid can effectively stabilize the encapsulation performance of liposomes in liposomes, and can prevent water from leaking from the liposomes, resulting in a decrease in the strength of the microcrystal needle body. This may be due to the easy flow and strong hydrophobicity of linoleic acid, which enhances the encapsulation performance of the liposomes. Compared with Example 4, the strength of the microcrystals in Comparative Example 5 is enhanced, but the needle body of the microcrystals in Comparative Example 5 shrinks after drying, resulting in the needle body being short and fat, so the strength is slightly increased.

[0089] (4) Microcrystal solubility test

[0090] The microcrystal patch was immersed in a constant temperature 37℃ water bath to simulate the process of microcrystal dissolution in the human body, and the time (min) taken for the microcrystal needle part to be completely dissolved was recorded.

[0091] Table 6 Dissolution time of microcrystals

[0092]

[0093] As shown in Table 6, the dissolution time of the microcrystals is similar when comparing Comparative Example 1 with Example 1, and Comparative Example 2 with Example 4. This indicates that after conventional drying and freeze-drying, the modified hyaluronic acid and hyaluronic acid used as the microcrystal matrix maintain their original shape and molecular interpenetrating network structure, increasing the intermolecular gaps and improving the dissolution rate. However, the microcrystals in Comparative Example 5, which were dried using only conventional drying methods, shrank, resulting in tighter intermolecular bonding and less water penetration into the microcrystals, significantly extending the dissolution time.

[0094] (5) Human skin anti-wrinkle test

[0095] 90 volunteers aged 30-50 were selected as test subjects. Microcrystals according to Examples 1-4 and Comparative Examples 1-5 were applied to the left outer canthus of the test subjects' eyes. The microcrystals were applied for 10 minutes before removal. This was repeated twice daily, morning and evening, for four weeks. The wrinkle depth (D1) before and after application and the wrinkle depth (D2) after four weeks of application were recorded. The wrinkle improvement rate was then calculated using the formula: Improvement rate (%) = (D1-D2) / D1×100%.

[0096] Table 7 Anti-wrinkle improvement rate of microcrystals

[0097]

[0098]

[0099] As shown in Table 7, the wrinkle improvement rates of Comparative Examples 1 and 2 are similar to those of Examples 1 and 4, respectively. The skin improvement rate of Comparative Example 3 is slightly lower than that of Comparative Example 2. This may be due to the low water content in the microcrystals, which increases the absorption of moisture from the skin by the microcrystals and reduces the transdermal absorption of active ingredients such as melittin by cells. The wrinkle improvement rate of Comparative Example 4 is significantly lower than that of Example 3, indicating that the combination of melittin with collagen and hyaluronic acid can significantly improve the anti-wrinkle effect. The anti-wrinkle effect of Comparative Example 5 is lower than that of Example 4, which is related to the drying method of the microcrystals. The microcrystals in Comparative Example 5 shrink due to drying, and the length of the microcrystal needles is significantly reduced, resulting in a significant decrease in transdermal penetration depth and reduced transdermal delivery performance.

[0100] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A soluble microcrystal, characterized in that The composition comprises the following components by weight: 20-30 parts of collagen, 10-20 parts of hyaluronic acid, 20-30 parts of modified hyaluronic acid, 3-5 parts of melittin, 1-3 parts of whitening component, 5-10 parts of linoleic acid, 10-20 parts of phospholipids, and 3-5 parts of cholesterol; The modified hyaluronic acid has the following structure: The ratio of m:n is 1:(1-10), wherein m and n in the structural formula only represent the number of grafted carboxyl groups of hyaluronic acid, and are not used as a limitation on the position of the grafted carboxyl groups in hyaluronic acid, and do not indicate that the modified hyaluronic acid is a block compound, wherein z is an integer of 1-100.

2. The soluble microcrystals according to claim 1, wherein The preparation method of the modified hyaluronic acid comprises the following steps: S1: EDTA-modified polyethylene glycol diamine: Dissolve EDTA in phosphate buffer, add EDC and NHS, and stir at room temperature to fully activate the carboxyl groups to form an EDTA-NHS ester solution; S2: dissolving polyethylene glycol diamine in phosphate buffer, adding EDTA-NHS ester solution dropwise under stirring, and continuing the reaction to obtain EDTA-PEG-NH2 solution; S3: Dissolve hyaluronic acid (HA) in phosphate buffer, add EDC and NHS, and stir at room temperature to form an HA-NHS ester solution; add EDTA-PEG-NH2 solution to the HA-NHS ester solution and stir to react; after the reaction is completed, transfer the reaction solution to a dialysis bag and dialyze with deionized water for purification; after dialysis, dry and pulverize to obtain modified hyaluronic acid powder.

3. The soluble microcrystals according to claim 2, wherein: The molar ratio of EDTA, EDC and NHS is 1:(0.9-1.1):(0.9-1.2).

4. The soluble microcrystals according to claim 2, wherein: The molar ratio of the polyethylene glycol diamine to EDTA is 1:(0.9-1.1).

5. The soluble microcrystals according to claim 2, wherein: The molar ratio of hyaluronic acid to polyethylene glycol diamine calculated based on the carboxyl content is (1-10):

1.

6. The soluble microcrystals according to claim 2, wherein: The phospholipid is selected from at least one of sodium dioleoylphosphatidylserine (DOPS), egg yolk lecithin, soybean lecithin, dioleoyl L-α-phosphatidylethanolamine (DOPE), 1,2-dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC).

7. The soluble microcrystals according to claim 2, wherein: The whitening component is selected from at least one of niacinamide, vitamin C, ursolic acid, azelaic acid, and glutathione.

8. The method for preparing soluble microcrystals according to any one of claims 1 to 7, wherein: S1: Collagen, hyaluronic acid, modified hyaluronic acid, and melittin are added with sterile deionized water to prepare a mixed aqueous solution; S2: Dissolve phospholipids and cholesterol in ethanol under water bath heating conditions, add linoleic acid and whitening ingredients to the phospholipid ethanol solution, stir rapidly, add an equal volume of sterile deionized water, stir at high speed to form a suspension, add an equal volume of sterile deionized water to the suspension, and homogenize under high pressure to prepare liposomes containing the whitening ingredients; S3: Add liposomes to the mixed aqueous solution prepared in S1 and mix well to obtain a microcrystal preparation solution; under vacuum conditions, inject the microcrystal preparation solution into a microcrystal mold, let it stand at room temperature, dry into an elastic gel, and freeze-dry to obtain soluble microcrystals.

9. The method for preparing soluble microcrystals according to claim 8, wherein: The microcrystal preparation liquid is injected into the microcrystal mold with an injection amount that completely covers the needle tip area of the mold. It is allowed to stand at room temperature for about 4-6 hours, and dried into an elastic gel. It is freeze-dried, slowly cooled to -20°C, and vacuum-dried for 0.5-2 hours to obtain microcrystals.

10. Use of the soluble microcrystals according to any one of claims 1 to 7 in the preparation of skin care products or medical beauty products.

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