Supported hyaluronic acid particle, cosmetic, preparation method and application

Abstract

The invention discloses a supported hyaluronic acid particle, a cosmetic, a preparation method and an application. The preparation method comprises the following steps: S1, carrying out ultrasonic treatment on a hyaluronic acid aqueous solution and a cationic polymer solution, and drying to obtain powder; and S2, in the presence of a buffer solution and a cross-linking agent, carrying out a cross-linking reaction on the powder and the effective components, and carrying out ball milling to prepare the supported hyaluronic acid particles. According to the method, the surface of the hyaluronic acid is coated with the cationic polymer, the active ingredients are loaded, the loaded hyaluronic acid particles are prepared through ball milling and are in a corner angle shape, the skin permeability can be remarkably improved, the stability of the loaded active ingredients is improved, and meanwhile the dual effects of moisturizing and skin barrier strengthening of the hyaluronic acid are achieved. According to the cosmetic containing the supported hyaluronic acid particles, the active ingredients can stably exist under various conditions, and the activity can maintain the stability for a long time.

Description

Technical Field

[0001] The present invention relates to loaded hyaluronic acid microparticles, cosmetics, and preparation methods and applications. Background Art

[0002] Hyaluronic acid (HA) is a natural polysaccharide (glycosaminoglycan) found in various human tissues, particularly in the skin, connective tissue, and vitreous humor. It plays a vital role as a lubricant and shock absorber. Due to its excellent water absorption and moisturizing properties, HA is crucial for maintaining skin hydration and elasticity. Therefore, it is widely used in cosmetics and medicine for wrinkle improvement, moisturizing, arthritis treatment, and eye dryness relief.

[0003] Hyaluronic acid is a linear biomacromolecule composed of N-acetyl-D-glucosamine and D-glucuronic acid. It has excellent biocompatibility, biodegradability, and adhesion properties. It can promote the adhesion and proliferation of mammalian cells and has anti-inflammatory effects. Unlike other glycosaminoglycans, hyaluronic acid is stable under various physiological conditions because it does not undergo sulfate.

[0004] The skin surface usually carries a negative charge, and the hyaluronic acid molecular structure contains carboxyl groups. When dissolved in water, it is partially ionized to form negatively charged carboxylate ions, causing the hyaluronic acid molecule to carry a negative charge as a whole, making it difficult to adsorb on the skin surface.

[0005] The skin is composed of the epidermis, dermis, and subcutaneous fat, performing functions such as protection, barrier function, temperature regulation, excretion, and respiration. The epidermis is the outermost layer, composed of keratinocytes and melanocytes. The dermis, which accounts for 95% of the skin, is responsible for moisturizing and protecting the skin. It contains proteins such as collagen and elastin, which are distributed in a reticular structure to maintain skin elasticity (wrinkles). It also contains blood vessels, nerves, mast cells involved in allergic reactions, and natural moisturizing factors such as sodium-polyacrylamide (Na-PCA) and hyaluronic acid.

[0006] Typically, cosmetics are only applied to the outer surface of the epidermis, making it difficult for active ingredients to be delivered to the inner skin to take effect. As consumers' expectations for cosmetic effects increase, researchers are committed to developing technologies that can deliver active ingredients to the epidermis, dermis, and even deeper layers. Among them, technologies using liposomes are relatively typical. However, compared with other drug delivery methods such as oral or injection, liposomes loaded with active ingredients in cosmetics have difficulty penetrating the skin's stratum corneum and delivering them to skin cells. Therefore, the development of carriers with excellent skin permeability and particle stability is currently an urgent need. Summary of the Invention

[0007] The technical problem actually solved by the present invention is to overcome the defects of the prior art cosmetics such as poor skin permeability, difficulty in delivering active ingredients to the skin to exert their effects, or poor stability of carrier particles. The present invention provides a loaded hyaluronic acid microparticle, cosmetics, preparation method, and application. The loaded hyaluronic acid microparticles of the present invention not only have high skin permeability but can also stably load active ingredients. Cosmetic formulations containing these microparticles can maintain the stability of the active ingredients over a long period of time.

[0008] The present invention solves the above technical problems through the following technical solutions.

[0009] The present invention provides a method for preparing loaded hyaluronic acid microparticles, which comprises the following steps:

[0010] S1. Ultrasonic treatment of hyaluronic acid aqueous solution and cationic polymer solution, drying to obtain powder;

[0011] S2 In the presence of a buffer solution and a cross-linking agent, the powder and the active ingredient are cross-linked and then ball-milled to obtain loaded hyaluronic acid microparticles.

[0012] In S1, the hyaluronic acid aqueous solution can be prepared by conventional methods in the art, for example, by dissolving hyaluronic acid in purified water. The concentration of the hyaluronic acid aqueous solution can be 1%.

[0013] In S1, the molecular weight of the hyaluronic acid in the hyaluronic acid aqueous solution is preferably 50,000-2,000,000 Da, more preferably 500,000-1,500,000 Da, such as 1,300,000 Da.

[0014] In a preferred embodiment, hyaluronic acid with a molecular weight of 1,300,000 Da is dissolved in water at a concentration of 1% to form a hyaluronic acid aqueous solution.

[0015] In S1, the cationic polymer solution can be prepared by conventional methods in the art, such as dissolving the cationic polymer in a solvent.

[0016] In S1, in the cationic polymer solution, the cationic polymer is preferably selected from one or more of collagen, chitosan, polyethyleneimine, polylysine, polydiallyldimethylammonium chloride, polyallylamine, polyornithine, polyvinylamine hydrochloride, poly(2-dimethylaminoethyl methacrylate) and polyamidoamine.

[0017] In a preferred embodiment, the cationic polymer may be chitosan.

[0018] In a preferred embodiment, chitosan is dissolved in 1% acetic acid solution to form a chitosan solution.

[0019] In S1, during the ultrasonic treatment, the cationic polymer can be adsorbed on the surface of the hyaluronic acid.

[0020] In S1, the frequency of the ultrasonic treatment is preferably 20-30 kHz.

[0021] In S1, the ultrasonic treatment time is preferably 5-30 min, for example 20 min.

[0022] In S2, it is preferred that the powder is first dissolved in the buffer solution, then the active ingredient is added for dispersion, and finally a cross-linking agent is added for cross-linking reaction.

[0023] In S2, the pH value of the buffer solution is preferably 6-9.

[0024] In S2, the buffer solution can be conventional in the art, for example, can be selected from phosphate buffered saline (PBS) or MES buffer.

[0025] In S2, the cross-linking agent can be conventional in the art, and preferably can be selected from one or more of 1,4-butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), butanediol diglycidyl ether (BCDI), diepoxyoctane (DEO), hexamethylenediamine (HMDA), polyethylene glycol diglycidyl ether (PEG), ethylene glycol diglycidyl ether (EGDE), hexamethylene diisocyanate (HDI), propylene glycol diglycidyl ether (PGDE), triphenylphosphine (TPP), glyoxal and carbodiimide, such as 1,4-butanediol diglycidyl ether (BDDE).

[0026] In S2, the temperature of the cross-linking reaction may be 40-60°C, for example 50°C.

[0027] In S2, the cross-linking reaction time may be 0.5-3 h, for example 2 h.

[0028] In a preferred embodiment, 1,4-butanediol diglycidyl ether (BDDE) is used as a crosslinking agent and reacted at 37°C for 2 hours. After the reaction is completed, the residual BDDE and reaction by-products are removed by adjusting the pH to neutral (7).

[0029] In S2, the ball milling operation and conditions can be conventional in the art, and a ball mill is generally used.

[0030] In S2, the ball milling speed is preferably 100-250 rpm, for example 200 rpm.

[0031] In S2, the active ingredients generally refer to ingredients in the product that have specific biological activities and can produce physiological effects on the skin (such as moisturizing, whitening, anti-aging, etc.), including but not limited to asiaticoside, madecassoside, asiatic acid, madecassoside, bacchusol, salicylic acid (BHA), adenosine, elastin, collagen, peptides, vitamins and their derivatives, ceramide, glycolic acid (AHA), niacinamide, caffeine, allantoin, panthenol, coenzyme Q10, glutathione , arbutin, astaxanthin, ethyl ascorbate, retinyl palmitate, squalene, vegetable oils (such as coconut oil and argan oil), pigments, resveratrol, polyphenols, polyglutamic acid, ferulic acid, hydroquinone, β-glucan, ascorbyl palmitate, phytosterols, pullulan, epicatechin gallate (EGCG), mannose, trehalose, α-bisphenol, ascorbyl glucoside, proline, oleic acid, quercetin, exosomes, PDRN and growth factors, etc. One or more.

[0032] In a preferred embodiment, the active ingredient may be asiaticoside.

[0033] The present invention also provides loaded hyaluronic acid microparticles prepared by the preparation method of the loaded hyaluronic acid microparticles.

[0034] In the present invention, the particle size of the loaded hyaluronic acid microparticles may be 0.5-15.0 μm, for example, 1 μm.

[0035] In the present invention, the morphology of the loaded hyaluronic acid microparticles can be angular.

[0036] The present invention also provides an application of the loaded hyaluronic acid microparticles in the field of cosmetics.

[0037] In the present invention, the amount of the loaded hyaluronic acid microparticles is preferably 0.1-30.0%, for example 10%, where the above percentage is the weight percentage of the loaded hyaluronic acid microparticles in the cosmetic.

[0038] The present invention also provides a cosmetic containing the loaded hyaluronic acid microparticles, wherein the amount of the loaded hyaluronic acid microparticles is 0.1-30.0%, for example 10%, of the total weight of the cosmetic.

[0039] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0040] The reagents and raw materials used in the present invention are commercially available.

[0041] The positive progress effect of the present invention is:

[0042] The present invention coats a cationic polymer on the surface of hyaluronic acid, loads an active ingredient, and ball-mills to prepare loaded hyaluronic acid microparticles in an angular shape. This method can significantly enhance skin permeability and improve the stability of the loaded active ingredient, while simultaneously exerting the dual effects of hyaluronic acid in moisturizing and strengthening the skin barrier.

[0043] Cosmetics containing loaded hyaluronic acid particles can maintain the stability of active ingredients under various conditions and their activity can be maintained for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a microscope image of the loaded hyaluronic acid microparticles prepared in Example 1 of the present invention.

[0045] Figure 2 This is a diagram showing the cytotoxicity results of the products of Example 1 and Comparative Examples 1-2 evaluated by MTT experiments.

[0046] Figure 3 This is a graph showing the results of evaluating the anti-wrinkle effects of the products of Example 1 and Comparative Examples 1-2 through COL1A1 expression.

[0047] Figure 4 This is a graph showing the results of evaluating the anti-wrinkle effects of the products of Example 1 and Comparative Examples 1-2 by inhibiting MMP-1 expression. DETAILED DESCRIPTION

[0048] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0049] Example 1: Preparation of asiatically loaded hyaluronic acid microparticles

[0050] Dissolving hyaluronic acid and cationic polymer: dissolving hyaluronic acid with a molecular weight of 1,300,000 Da in purified water at a concentration of 1% to form a hyaluronic acid aqueous solution; dissolving chitosan in a 1% acetic acid solution to form a chitosan solution;

[0051] Adsorption of cationic polymers: Hyaluronic acid aqueous solution and chitosan solution were mixed and kept in a uniformly dispersed state, and then treated with an ultrasonic frequency of 30 kHz for 20 minutes to allow chitosan to be adsorbed on the surface of hyaluronic acid.

[0052] Drying: The above solution is dried to prepare chitosan-coated hyaluronic acid powder.

[0053] Loading active ingredients: Dissolve the dried chitosan-coated hyaluronic acid powder in a pH 6.0 buffer solution, dissolve asiatically in 20% ethanol, and then slowly add it to the hyaluronic acid solution and mix at 50°C for 1 hour to evenly disperse the asiatically indole.

[0054] Cross-linking reaction: 1,4-Butanediol diglycidyl ether (BDDE) was used as a cross-linking agent and reacted at 37°C for 2 hours. After the reaction was completed, the residual BDDE and reaction by-products were removed by adjusting the pH to neutral (7).

[0055] Preparation of microparticles by ball mill: The above mixture was ground into a particle size of about 1 μm using a ball mill at a grinding speed of 200 rpm.

[0056] Comparative Example 1: Preparation of liposomes loaded with asiaticoside

[0057] For comparison with Example 1, asiaticoside-loaded liposomes were prepared using a conventional method, which specifically included the following steps:

[0058] 70% glycerol and 15% medium-chain triglycerides were mixed and heated to 70-75°C. Asiaticoside was added to completely dissolve the mixture. 3% lecithin was added for emulsification, and finally 10% purified water was added. A high-pressure homogenizer was used to homogenize the mixture three times at 1000 bar to prepare asiaticoside-loaded liposomes.

[0059] Comparative Example 2: Preparation of Hyaluronic Acid Microparticles Not Loaded with Centella asiatica

[0060] Except for not loading asiaticoside, the remaining steps were the same as those in Example 1 to prepare hyaluronic acid microparticles.

[0061] Effect Example 1: Microscopic Observation of Hyaluronic Acid Microparticles

[0062] The morphology of the prepared loaded hyaluronic acid microparticles was observed using an optical microscope.

[0063] Figure 1 This is a microscope image of the loaded hyaluronic acid microparticles prepared in Example 1. Figure 1 The microparticles were angular and about 1 μm in size.

[0064] Effect Example 2: Stability Analysis of Hyaluronic Acid Microparticles

[0065] The analysis results after 12 weeks showed that compared with liposomes, the loaded hyaluronic acid microparticles were able to more stably maintain the content and raw material state of Centella asiatica.

[0066] Effect Example 3: Cytotoxicity Assessment

[0067] The cytotoxicity of Example 1 (asiaticoside-loaded hyaluronic acid microparticles), Comparative Example 1 (asiaticoside-loaded liposomes), and Comparative Example 2 (hyaluronic acid microparticles not loaded with asiaticoside) was evaluated using the MTT assay.

[0068] Test method: Human keratinocytes (HaCaT) were cultured at a rate of 1×10 5 Cells were seeded at a concentration of 10 cells / mL in a 96-well plate and incubated at 37°C, 5% CO2 for 18 hours. After incubation, the medium was removed, the cells were washed with PBS, and then the samples at different concentrations were added and incubated for another 24 hours. MTT solution (5 mg / mL) was added, and the formazan formed was dissolved with dimethyl sulfoxide (DMSO) after 4 hours. The absorbance was measured at 570 nm using a microplate reader.

[0069] Figure 2 This is a diagram showing the cytotoxicity evaluation results of the products of Example 1 and Comparative Examples 1-2 by MTT experiment. Figure 2 All samples showed no cytotoxicity at all concentrations.

[0070] Effect Example 4: Evaluation of Anti-Wrinkle Effect (COL1A1 Expression)

[0071] The anti-wrinkle effect of asiaticoside-loaded hyaluronic acid microparticles was evaluated by assessing COL1A1 expression. Human dermal fibroblasts (HDFa) were cultured, treated with various concentrations, and then irradiated with UVB (20 mJ / cm²). COL1A1 mRNA expression was analyzed by RT-qPCR.

[0072] Figure 3 This is a graph showing the results of evaluating the anti-wrinkle effects of the products of Example 1 and Comparative Examples 1-2 through COL1A1 expression. Figure 3 The results showed that compared with liposomes and hyaluronic acid microparticles not loaded with asiaticoside, the loaded hyaluronic acid microparticles of Example 1 could significantly increase the expression of COL1A1 mRNA.

[0073] Effect Example 5: Evaluation of Anti-Wrinkle Effect (MMP-1 Expression Inhibition)

[0074] The anti-wrinkle effect of the loaded hyaluronic acid microparticles was evaluated by evaluating the inhibition of MMP-1 expression. The experimental method was the same as that of Example 4.

[0075] Figure 4 This is a graph showing the results of evaluating the anti-wrinkle effects of the products of Example 1 and Comparative Examples 1-2 by inhibiting MMP-1 expression. Figure 4 It was shown that loaded hyaluronic acid microparticles could significantly inhibit the expression of MMP-1 mRNA.

[0076] Formulation Example: Preparation of Cream

[0077] According to the formulation in Table 1, a cream was prepared using the asiaticoside-loaded hyaluronic acid microparticles prepared in Example 1, and compared with the cream prepared using the liposomes of Comparative Example 1.

[0078] Table 1 Formula ingredients and content

[0079]

Claims

1. A method for preparing loaded hyaluronic acid microparticles, characterized in that: It includes the following steps: S1. Ultrasonic treatment of hyaluronic acid aqueous solution and cationic polymer solution, drying to obtain powder; S2 In the presence of a buffer solution and a cross-linking agent, the powder and the active ingredient are cross-linked and then ball-milled to obtain loaded hyaluronic acid microparticles.

2. The preparation method according to claim 1, wherein The preparation method of the loaded hyaluronic acid microparticles meets one or more of the following conditions: (1) In S1, the concentration of the hyaluronic acid aqueous solution is 0.5-1.5%; (2) In S1, the hyaluronic acid aqueous solution has a molecular weight of hyaluronic acid of 50,000-2,000,000 Da; (3) In S1, the cationic polymer in the cationic polymer solution is selected from one or more of collagen, chitosan, polyethyleneimine, polylysine, polydiallyldimethylammonium chloride, polyallylamine, polyornithine, polyvinylamine hydrochloride, poly(2-dimethylaminoethyl methacrylate) and polyamidoamine; (4) In S1, the frequency of the ultrasonic treatment is 20-30 kHz; and (5) In S1, the ultrasonic treatment time is 5-30 min.

3. The preparation method according to claim 1, wherein The preparation method of the loaded hyaluronic acid microparticles meets one or more of the following conditions: (1) In S1, the hyaluronic acid aqueous solution has a molecular weight of hyaluronic acid of 500,000-1,500,000 Da, for example, 1,300,000 Da; (2) In S1, the cationic polymer solution comprises chitosan; and (3) In S1, the ultrasonic treatment time is 20 min.

4. The preparation method according to claim 1, wherein The preparation method of the loaded hyaluronic acid microparticles meets one or more of the following conditions: (1) In S2, the powder is dissolved in the buffer solution, the active ingredient is added for dispersion, and a cross-linking agent is added for cross-linking reaction; (2) In S2, the pH value of the buffer solution is 6-9; (3) In S2, the buffer solution is selected from phosphate buffer or MES buffer; (4) In S2, the crosslinking agent is selected from one or more of 1,4-butanediol diglycidyl ether, divinyl sulfone, butanediol diglycidyl ether, diepoxyoctane, hexamethylenediamine, polyethylene glycol diglycidyl ether, ethylene glycol diglycidyl ether, hexamethylene diisocyanate, propylene glycol diglycidyl ether, triphenylphosphine, glyoxal and carbodiimide; (5) In S2, the temperature of the cross-linking reaction is 40-60°C; (6) In S2, the cross-linking reaction time is 0.5-3 hours; and, (7) In S2, the ball milling speed is 100-250 rpm.

5. The preparation method according to claim 1, wherein The preparation method of the loaded hyaluronic acid microparticles meets one or more of the following conditions: (1) In S2, the cross-linking agent is 1,4-butanediol diglycidyl ether; (2) In S2, the temperature of the cross-linking reaction is 50°C; (3) In S2, the cross-linking reaction time is 2 hours; and, (4) In S2, the ball milling speed is 200 rpm.

6. The preparation method according to claim 1, wherein In S2, the active ingredients include but are not limited to one or more of asiaticoside, madecassoside, asiatic acid, madecassoside, bacchusol, salicylic acid, adenosine, elastin, collagen, peptides, vitamins and their derivatives, ceramide, fruit acid, niacinamide, caffeine, allantoin, panthenol, coenzyme Q10, glutathione, arbutin, astaxanthin, ethyl ascorbate, retinyl palmitate, squalene, vegetable oil, pigment, resveratrol, polyphenols, polyglutamic acid, ferulic acid, hydroquinone, β-glucan, ascorbyl palmitate, phytosterols, pullulan, epicatechin gallate (EGCG), mannose, trehalose, α-bisphenol, ascorbyl glucoside, proline, oleic acid, quercetin, exosomes, PDRN and growth factors, such as asiaticoside.

7. Loaded hyaluronic acid microparticles prepared by the method for preparing loaded hyaluronic acid microparticles according to any one of claims 1 to 6.

8. The loaded hyaluronic acid microparticles according to claim 7, wherein The particle size of the loaded hyaluronic acid microparticles is 0.5-15.0 μm, for example, 1 μm; And / or, the loaded hyaluronic acid microparticles have an angular morphology.

9. Use of the loaded hyaluronic acid microparticles according to any one of claims 1 to 6 in the field of cosmetics; The amount of the loaded hyaluronic acid microparticles is preferably 0.1-30.0%, for example 10%, where the above percentage is the weight percentage of the loaded hyaluronic acid microparticles in the cosmetic.

10. A cosmetic containing the loaded hyaluronic acid microparticles according to any one of claims 1 to 6, characterized in that: The amount of the loaded hyaluronic acid microparticles used is 0.1-30.0% of the total weight of the cosmetic, for example 10%, where the above percentage is the weight percentage of the loaded hyaluronic acid microparticles in the cosmetic.

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