A carrier, composition, use and cosmetic based on an association network

Abstract

The present application belongs to the field of new daily chemical materials and discloses a carrier based on an association network, wherein the carrier is a nano-scale or micron-scale particle in water; the carrier is a network carrier composed of arginine components and hyaluronic acid components associated by hydrogen bonds and / or ionic bonds; the carrier is based on the electrostatic binding force and hydrogen bonding force of hyaluronic acid and its derivatives, arginine and its polyhydroxy derivatives, to achieve effective binding for one or more small molecule polar active substances, and can promote the effective size reduction of hyaluronic acid and its salt derivatives in the carrier matrix material, thereby improving its own effective skin permeability; at the same time, further improving the physicochemical stability, transdermal transmissibility, sustained release and skin care effects of other active ingredients loaded by the carrier. At the same time, the present invention also provides a composition and the use and cosmetics of the carrier and the composition.

Description

Technical Field

[0001] The present invention relates to the field of new daily chemical materials, and in particular to a carrier, composition, use and cosmetics based on an association network. Background Art

[0002] Hyaluronic acid and its derivatives are widely recognized multifunctional skin care actives, but due to the limitation of their molecular weight, hyaluronic acid and its salt derivatives with a molecular weight above 10kDa are difficult to achieve active transdermal absorption.

[0003] Essendoubi et al. (Skin Research & Technology, 2016) used Raman spectroscopy to study the transdermal absorption of hyaluronic acid. They applied hyaluronic acid of varying molecular weights to the skin surface and used Raman spectroscopy to measure skin permeability. They found that high-molecular-weight hyaluronic acid (over 1,000,000) primarily penetrated the stratum corneum, while medium-molecular-weight hyaluronic acid (100,000-300,000) primarily penetrated the superficial epidermis. Low-molecular-weight hyaluronic acid (20,000-50,000) penetrated slightly deeper, reaching the deeper layers of the epidermis and the upper dermis.

[0004] But generally speaking, it is necessary to improve the transdermal absorption effect of hyaluronic acid and its derivatives, especially those with medium and high molecular weight. Common technical methods include direct chemical modification, including esterification modification, grafting modification, hydrophobic modification, etc. In addition, the carboxyl, amino and reducing ends of hyaluronic acid are amidated and esterified and then coupled with anti-tumor drugs to form drug conjugates, which can prolong the retention time of the prodrug in the body and enhance the water solubility and tumor targeting of the drug. For example, the acetylation modification of hyaluronic acid improves the transdermal efficiency of the skin and the biological activity of active substances by improving the fat solubility of hyaluronic acid, but this method requires complex chemical reactions to achieve, and is still limited by the molecular weight of hyaluronic acid and its derivatives themselves.

[0005] In addition, hyaluronic acid and its derivatives are also being used as carrier matrix materials to achieve the encapsulation and targeted delivery of active ingredients to the skin. Hyaluronic acid and its derivative nanogels generally refer to hydrogel particles composed of chemically or physically cross-linked polymer networks. Due to their high loading capacity and stability, they can be used as a novel drug carrier. Common methods are chemical or physical cross-linking. For example, chemical cross-linking of hyaluronic acid involves intermolecular cross-linking reactions between hyaluronic acid and cross-linking agents with relevant functional groups, or intramolecular cross-linking reactions using cross-linking agents as catalysts, resulting in molecular network structures with varying degrees of cross-linking. This enhances the viscoelasticity of the hyaluronic acid molecules, relatively reduces their water solubility, and improves their mechanical strength, thereby achieving a certain degree of encapsulation of active ingredients. Common chemical cross-linking methods include hydrazide cross-linking, disulfide cross-linking, polyethylene glycol cross-linking, aldehyde cross-linking, and carbodiimide cross-linking. However, the preparation of carriers of hyaluronic acid and its derivatives through this covalent chemical modification method requires the use of chemical crosslinkers and chemical reactions. This method is inflexible and carries the risk of toxicity from residual crosslinkers or incomplete reaction of crosslinking groups. Some studies have shown that as the molecular weight of hyaluronic acid increases, the longer the molecular chain, the more difficult it is to form effective crosslinks, the higher the probability of exposing unreacted crosslinking functional groups, and the higher the risk of toxicity and instability. On the other hand, effective physical crosslinking also has certain application limitations because it requires weaker physical interactions such as suitable non-covalent bonds.

[0006] As mentioned above, the above-mentioned HA modification or carrier technology involves complex chemical reactions, carries toxicity risks, and is still limited by the molecular weight of hyaluronic acid and its derivatives themselves.

[0007] Numerous technologies exist for encapsulating active ingredients, the most commonly used being liposomes, microcapsules, and nano-microgels. Due to their molecular polarity, water-soluble active ingredients require carrier delivery systems for efficient transdermal absorption. However, effective liposome encapsulation of water-soluble active ingredients, especially small molecules, is difficult to achieve using traditional processes such as high-pressure averaging and high-pressure microfluidization.

[0008] In addition, some small molecule active substances with metal ion complexes or positive charges, such as blue copper peptides and arginine / lysine peptides, cannot achieve good encapsulation efficiency and compatibility stability through commonly used encapsulation carriers such as liposomes. Among them, blue copper peptide, English name GHK-Cu, is a small molecule polypeptide composed of a tripeptide composed of glycyl, histidyl and lysine, complexed with copper (Cu) ions. GHK-Cu can promote collagen production, improve blood vessel growth, promote epithelial cell growth and differentiation, and promote hair follicle proliferation in skin repair. In addition, GHK-Cu is also an excellent antioxidant. Studies have shown that GHK-Cu is more effective than recognized antioxidants, retinoic acid and vitamin C in promoting collagen synthesis. The skin repair and antioxidant effects of GHK-Cu require three basic conditions: the first is that the coordination state of copper ions and tripeptides must be maintained. When the copper ions and tripeptides lose their complexation, GHK-Cu will lose its biological activity; the second is that GHK-Cu needs to effectively penetrate the epidermis and enter the dermis to exert its skin repair function; and the third is that GHK-Cu needs to be slowly released into the skin to ensure a gradual repair effect over a period of time. To meet these three basic application conditions for GHK-Cu, the technical means currently adopted by researchers is to encapsulate GHK-Cu with highly biocompatible nanocarriers, reducing the particle size of GHK-Cu so that it can penetrate the epidermis while being protected by the nanocarrier. Depending on the carrier used, it can be divided into types such as nanoparticle carriers and ionic liquid carriers. These technical means all have two very obvious disadvantages: (1) The carrier has weak protection for the stability of GHK-Cu. The negative charge carried by the main lecithin material of some carriers such as liposomes even destroys the complex stability between copper ions and tripeptides. (2) The prepared carrier has low encapsulation efficiency and different particle sizes, which affects the stable addition of GHK-Cu in cosmetics and the skin penetration efficiency; (3) The prepared carrier lacks the slow release effect of GHK-Cu, that is, it does not meet the third condition for GHK-Cu to exert its efficacy. CN111840125A discloses a nano-encapsulated skin repair agent containing blue copper peptide and a preparation method thereof, which comprises the following raw materials in mass percentage: oil phase: 9-90%, aqueous phase: 10-91%; the oil phase comprises the following raw materials in mass percentage: propylene glycol: 5-30%, lecithin: 1-10%, inulin lauryl carbamate: 0.5-10%, acetyl dipeptide-1 cetyl ester: 1-10%, bisabolol: 1-10%, copaiba resin: 1-20%; the aqueous phase comprises the following raw materials in mass percentage: blue copper peptide: 1-10%, decapeptide-4: 1-10%, and the balance is water.It utilizes a nanocarrier system (NDS) to encapsulate water-insoluble acetyl dipeptide-1 cetyl ester, bisabolol, and copaiba resin within a 10-100nm particle size carrier of lecithin and inulin lauryl carbamate. This carrier also incorporates a hydrophilic continuous phase as an external layer, allowing the active ingredients to penetrate the skin more easily and reach the target area. This solution utilizes a technology similar to microencapsulation to achieve stable delivery of the active ingredients.

[0009] As mentioned above, the aforementioned coated carriers suffer from issues such as poor encapsulation, poor stability, poor sustained-release properties, and poor particle size uniformity, which in turn affect the efficacy of the blue copper peptide. These issues are not limited to GHK-Cu peptides; similar problems also exist with other ionic peptides or water-soluble active ingredients.

[0010] The technical problems solved in this case are:

[0011] How to promote the effective skin permeability of hyaluronic acid and its salt derivatives, especially for hyaluronic acid and its salt derivatives with large and medium molecular weight that are difficult to penetrate the skin;

[0012] How to simultaneously achieve effective and efficient encapsulation of skin care active ingredients, especially small molecule amino acids and their derivatives, small molecule peptides, etc., while the carrier can further improve the physicochemical stability, transdermal properties, sustained release and skin care effects of the active ingredients. Summary of the Invention

[0013] The purpose of the present invention is to provide a carrier based on an association network. The carrier is based on the electrostatic binding force and hydrogen bonding force of hyaluronic acid and its derivatives, arginine and its polyhydroxy derivatives, to achieve effective binding of one or more small molecule polar active substances, and can promote the effective size reduction of hyaluronic acid and its salt derivatives in the carrier matrix material, thereby improving its own effective skin permeability; at the same time, it further improves the physicochemical stability, transdermal permeability, sustained release and skin care effect of other active ingredients loaded by the carrier.

[0014] At the same time, the present invention also provides a composition, and uses and cosmetics of the carrier and the composition.

[0015] Unless otherwise specified in the present invention, nM represents nanomole / L, μM represents micromole / L, mM represents millimole / L, and M represents mole / L. In the present invention, unless otherwise specified, the reaction temperature is room temperature.

[0016] To achieve the above objectives, this application discloses:

[0017] A carrier based on an association network, wherein the carrier is a nano-scale or micron-scale particle in water; the carrier is a network carrier composed of arginine components and hyaluronic acid components associated through hydrogen bonds and / or ionic bonds;

[0018] The arginine component is arginine and / or a polyhydroxy derivative of arginine;

[0019] The hyaluronic acid component is hyaluronic acid and / or a salt derivative of hyaluronic acid.

[0020] The COO- of the hyaluronic acid component and at least one NH 2+ Association to form COO--NH 2+ and / or the hyaluronic acid component associates with at least one hydroxyl group of the arginine component to form an OH-H hydrogen bond; and / or the hyaluronic acid component associates with at least one N in the arginine component to form an OH-N hydrogen bond.

[0021] In the present invention, an associative network carrier is formed based on arginine and its polyhydroxyarginine derivatives and the hyaluronic acid and its salt derivatives. There are electrostatic interactions and hydrogen bond interactions between these two substances. Using a complex non-covalent bond-dominated network action system, i.e., these two weak interactions, this method successfully retains small molecule active substances such as ionic peptides and polar small molecules within the network of nanocarriers, effectively protecting the ionicity and physicochemical stability of the active substances, while increasing the transdermal performance of the carrier matrix material hyaluronic acid and its salt derivatives and the loaded active substances. Compared with existing technologies, this method has better active substance packaging and stability, and this stability advantage has been verified by physical and chemical experiments. Compared with existing technologies, this method has better transdermal efficiency, and the advantages of this transdermal performance have been verified by in vitro and human transdermal experiments. When the small molecule active substances affected by weak interactions enter the skin layer, for example, from the stratum corneum to the epidermis to the dermis, due to changes in the ionic strength and pH in the cell microenvironment, the active substances slowly release the carrier network over a period of time, achieving the gradual release of the active substances in the skin layer. Compared with existing technologies, this method has good slow release ability, and this slow release ability has been verified through in vitro sustained release experiments.

[0022] In the above-mentioned carrier based on the association network, the molar ratio of the arginine unit in the arginine component to the hyaluronic acid unit in the hyaluronic acid component is 0.05-2:0.2-2;

[0023] Preferably, the molar ratio of the arginine unit in the arginine component to the hyaluronic acid unit in the hyaluronic acid component is 0.36-3:1.

[0024] In some embodiments of the present invention, the molar ratio is: 0.05:2, 0.1:2, 0.2:2, 0.3:2, 0.4:2, 0.5:2, 0.6:2, 0.8:2, 1.0:2, 1.2:2, 1.5:2, 1.7:2, 1.9:2, 2:2, 2:1.8, 2:1.6, 2:1.4, 2:1.2, 2:1.0, 2:0.8, 2:0.6, 2:0.5, 2:0.4, 2:0.3 or 2:0.2;

[0025] In the above-mentioned carrier based on the association network, the salt derivative of hyaluronic acid is one or more of hydrolyzed hyaluronic acid, sodium hyaluronate, zinc hyaluronate, acetylated hyaluronic acid, hydroxypropyltrimethylammonium chloride hyaluronic acid, etc.;

[0026] The molecular weight of the hyaluronic acid component is 2 to 600 kDa.

[0027] In some embodiments of the present invention, the molecular weight of the hyaluronic acid component can be selected from: 2kDa, 5kDa, 10kDa, 20kDa, 30kDa, 40kDa, 60kDa, 80kDa, 100kDa, 120kDa, 150kDa, 200kDa, 230kDa, 250kDa, 280kDa, 300kDa, 300kDa, 330kDa, 350kDa, 380kDa, 400kDa, 430kDa, 450kDa, 480kDa, 500kDa, 600kDa, etc.;

[0028] In the above-mentioned carrier based on the association network, the structure of the polyhydroxy derivative of arginine is shown in Formula I:

[0029]

[0030] In formula I, X is a hydrogen atom or -CH2CH(OH)CH2(OH); Y is a hydrogen atom, an alkali metal, ammonium, an organic ammonium or -CH2CH(OH)CH2(OH).

[0031] Preferably, the carrier is a nano-scale or micron-scale particle with a diameter of 10 to 1000 nm in water, preferably a nano-scale particle with a diameter of 100 to 1000 nm.

[0032] At the same time, the present invention also discloses a composition comprising any of the carriers described above and an active ingredient that can be used in cosmetics and is loaded in the carrier; the active ingredient has the characteristics of an amino acid monomer, an amino acid derivative, or a polyamino acid (polypeptide). Specifically, the acidic group (carboxylic acid) or basic group (amino group) in the amino acid can form hydrogen bonds or ionic bonds (such as) with the functional groups (carboxyl groups, amino groups) of hyaluronic acid or its salt derivatives, and the functional groups (hydroxyl groups, amino groups) of arginine and its polyhydroxy derivatives in the carrier network. Figure 1 shown).

[0033] - In the above composition, the active ingredient is one or more of an amino acid, an amino acid derivative, or a polypeptide; the molecular weight of the active ingredient does not exceed 1500 Da, preferably does not exceed 1000 Da, and preferably does not exceed 500 Da.

[0034] In the above composition, the molar ratio of the arginine unit in the arginine component, the hyaluronic acid unit in the hyaluronic acid component, and the active ingredient is 0.05-2:0.2-2:1.

[0035] In the above composition, the amino acids are one or more combinations of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, histidine, etc.;

[0036] The amino acid derivative is one or more combinations of ergothioneine, nicotinamide, tetrahydropyrimidine carboxylic acid, acetylhydroxyproline, and N-acetylneuraminic acid;

[0037] The polypeptide is one or more combinations of arginine / lysine polypeptide, blue copper peptide, glutathione, nonapeptide-1, carnosine, acetyl tetrapeptide-2, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, palmitoyl tripeptide-5, snake venom peptide, etc.

[0038] At the same time, the present invention also discloses a method for preparing any of the above-described compositions, wherein a multi-microchannel high-speed mixer is used to mix a solution containing arginine components, a solution containing hyaluronic acid components, and a solution containing active ingredients after passing through multiple microchannels of the multi-microchannel high-speed mixer.

[0039] In the above preparation method, the concentration of the solution containing the hyaluronic acid component is 1 to 30 mg / mL;

[0040] The concentration of the solution containing arginine components is 0.01 to 50 mg / mL;

[0041] The concentration of the solution containing the active ingredient is 1 to 100 mg / mL.

[0042] In the above preparation method, the mixing time of the solution containing arginine components, the solution containing hyaluronic acid components, and the solution containing active ingredients is less than 0.1 second; and the Reynolds number Re during mixing is greater than 4000.

[0043] At the same time, the present invention also discloses the use of any of the above carriers to load active ingredients that can be used in cosmetics; the active ingredients have amino groups.

[0044] And, use of any of the above compositions in preparing cosmetics.

[0045] Finally, the present invention also discloses a cosmetic comprising any one of the above compositions.

[0046] Preferably, in cosmetics, the amount of the composition is 0.0001 wt% to 100 wt%;

[0047] The cosmetics described in the present invention are products such as essences, gels, emulsions, creams, etc.

[0048] This application has at least the following beneficial effects:

[0049] In the present invention, an associative network carrier is formed based on arginine and its polyhydroxyarginine derivatives and the hyaluronic acid and its salt derivatives. There are electrostatic interactions and hydrogen bond interactions between these two substances. Using a complex non-covalent bond-dominated network action system, i.e., these two weak interactions, this method successfully retains small molecule active substances such as ionic peptides and polar small molecules within the network of nanocarriers, effectively protecting the ionicity and physicochemical stability of the active substances, while increasing the transdermal performance of the carrier matrix material hyaluronic acid and its salt derivatives and the loaded active substances. Compared with existing technologies, this method has better active substance packaging and stability, and this stability advantage has been verified by physical and chemical experiments. Compared with existing technologies, this method has better transdermal efficiency, and the advantages of this transdermal performance have been verified by in vitro and human transdermal experiments. When the small molecule active substances affected by weak interactions enter the skin layer, for example, from the stratum corneum to the epidermis to the dermis, due to changes in the ionic strength and pH in the cell microenvironment, the active substances slowly release the carrier network over a period of time, achieving the gradual release of the active substances in the skin layer. Compared with existing technologies, this method has good slow release ability, and this slow release ability has been verified through in vitro sustained release experiments.

[0050] The present invention is based on the electrostatic binding force and hydrogen bonding force of hyaluronic acid and its derivatives, arginine and its polyhydroxy derivatives to achieve effective binding of one or more small molecule polar active substances, and can promote the effective size reduction of hyaluronic acid and its salt derivatives in the carrier matrix material, thereby improving its own effective skin permeability; at the same time, it further improves the physicochemical stability, transdermal permeability, sustained release and skin care effect of other active ingredients loaded on the carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a schematic diagram of the carrier loading ergothioneine and blue copper peptide;

[0052] Figure 2 is the electron microscope image of sample 1;

[0053] Figure 3 is the electron microscope image of sample 9;

[0054] Figure 4 The figure is a test result diagram of the encapsulation stability of the active ingredient of the samples of the present invention and the comparative samples;

[0055] Figure 5 This is a comparison chart of the cumulative permeation amount of active substances per unit area in the samples of the present invention and the comparative samples;

[0056] Figure 6 The content distribution image of sample 17 penetrating into different depths of the skin over time;

[0057] Figure 7 is the cumulative release rate of the active ingredient of Examples and Comparative Examples;

[0058] Figure 8 This is a comparison chart of cell migration rates after cell scratch repair in the example and the comparative example.

[0059] Specific embodiment

[0060] The present invention will be described clearly and completely below in conjunction with the examples of the present invention. In the description of the present invention, it should be noted that, where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0061] Part I. Examples and Comparative Examples

[0062] Example 1

[0063] Preparation of blank carriers of hyaluronic acid-arginine polyhydroxy derivatives association network by multi-microchannel high-speed mixing

[0064] Step 1: Prepare 10 mg / mL of 300 kDa hyaluronic acid solution. Add water slowly during the dissolution process while stirring gradually to promote hydration and allow complete swelling and dispersion.

[0065] Step 2: Prepare a dihydroxypropyl arginine solution with a concentration of 21.18 mg / mL.

[0066] Step 3: Inject the prepared hyaluronic acid and dihydroxypropyl arginine solutions into two opposing pumps of the mixer, respectively, at a flow rate of 40 mL / min, to ensure a molar ratio of repeating units of dihydroxypropyl arginine to hyaluronic acid of 3. Simultaneously inject deionized water into two other opposing pumps at a flow rate of 9 mL / min to obtain Sample 1.

[0067] Note: The supplier of the mixer is: Microfluidics Technology Co., Ltd.; the model is: MF-X1 multi-microchannel vortex mixer, and this device is used in subsequent examples.

[0068] Example 2

[0069] The same as Example 1, except that the molar ratio of the repeating units of dihydroxypropyl arginine and hyaluronic acid was adjusted to 0.2, 0.8, and 1.5;

[0070] During this process, the hyaluronic acid concentration was maintained at 10 mg / mL, and the concentration of dihydroxypropylarginine was changed to 1.41 mg / mL, 5.65 mg / mL, and 10.59 mg / mL.

[0071] The molar ratio of the repeating units of dihydroxypropylarginine to hyaluronic acid was 0.2, to obtain sample 2;

[0072] The molar ratio of the repeating units of dihydroxypropylarginine to hyaluronic acid was 0.8, to obtain sample 3;

[0073] The molar ratio of the repeating units of dihydroxypropylarginine to hyaluronic acid was 1.5, and sample 4 was obtained.

[0074] Example 3

[0075] The process is substantially the same as Example 1, except that the molecular weight of hyaluronic acid is adjusted to 5 kDa, 25 kDa, and 600 kDa.

[0076] The molecular weight of hyaluronic acid was adjusted to 5 kDa to obtain sample 5.

[0077] The molecular weight of hyaluronic acid was adjusted to 25 kDa to obtain sample 6.

[0078] The molecular weight of hyaluronic acid was adjusted to 600 kDa to obtain sample 7.

[0079] Example 4

[0080] The method is substantially the same as Example 1, except that hyaluronic acid is replaced by sodium hyaluronic acid; dihydroxypropyl arginine is replaced by arginine; and the molar ratio of the repeating units of arginine and sodium hyaluronate is 3, thereby obtaining Sample 8.

[0081] Example 5

[0082] The method is similar to Example 1, except that the active substance, blue copper peptide, is encapsulated in the blank carrier.

[0083] The method for preparing the blue copper peptide-hyaluronic acid-arginine polyhydroxy derivative association network carrier by multi-microchannel high-speed mixing method is as follows:

[0084] Step 1: Prepare 10 mg / mL of 300 kDa hyaluronic acid solution. Add water slowly during the dissolution process and stir gradually to promote dissolution.

[0085] Step 2: Prepare a dihydroxypropyl arginine solution with a concentration of 21.18 mg / mL, ensuring that the molar ratio of repeating units of dihydroxypropyl arginine to hyaluronic acid is 3.

[0086] Step 3: Prepare a 13.6 wt% blue copper peptide solution, and perform ultrasonic treatment for 60 min during the preparation process to ensure that the blue copper peptide is completely dissolved.

[0087] Step 4: Inject the prepared hyaluronic acid and dihydroxypropyl arginine solutions into two opposing pumps of the mixer, respectively, maintaining a consistent flow rate of 40 mL / min. Simultaneously inject the prepared blue copper peptide solution into two other opposing pumps at a flow rate of 9 mL / min, yielding Sample 9. The final concentration of the solution was 2.5 wt% blue copper peptide, 8.64 mg / mL dihydroxypropyl arginine, and 4.08 mg / mL hyaluronic acid.

[0088] Example 6

[0089] The process was substantially the same as Example 5, except that the molar ratio of the repeating units of dihydroxypropyl arginine to hyaluronic acid was adjusted to 0.2, 0.8, and 1.5. During this process, the hyaluronic acid concentration was maintained at 10 mg / mL, and the concentration of dihydroxypropyl arginine was changed to 1.41 mg / mL, 5.65 mg / mL, and 10.59 mg / mL.

[0090] The molar ratio of the repeating units of dihydroxypropylarginine to hyaluronic acid was 0.2, to obtain sample 10;

[0091] The molar ratio of the repeating units of dihydroxypropylarginine to hyaluronic acid was 0.8, to obtain sample 11;

[0092] The molar ratio of the repeating units of dihydroxypropylarginine to hyaluronic acid was 1.5, and sample 12 was obtained.

[0093] Example 7

[0094] Same as Example 5, except that the molecular weight of hyaluronic acid was adjusted to 5 kDa, 25 kDa, and 600 kDa.

[0095] The molecular weight of hyaluronic acid was adjusted to 5 kDa to obtain sample 13.

[0096] The molecular weight of hyaluronic acid was adjusted to 25 kDa to obtain sample 14.

[0097] The molecular weight of hyaluronic acid was adjusted to 600 kDa to obtain sample 15.

[0098] Example 8

[0099] The method is substantially the same as Example 5, except that the molar ratio of dihydroxypropyl arginine / hyaluronic acid / blue copper peptide is adjusted to 5:1:1 to obtain Sample 16.

[0100] Example 9

[0101] The method is substantially the same as Example 5, except that the ratio of dihydroxypropyl arginine / hyaluronic acid / blue copper peptide is adjusted to 0.05:0.2:1 to obtain Sample 17.

[0102] Example 10

[0103] The method is substantially the same as Example 5, except that ergothioneine of the same mass is used instead of blue copper peptide; dihydroxypropyl arginine is replaced by arginine, and the molar ratio of the repeating units of arginine and sodium hyaluronate is 3, thereby obtaining Sample 18.

[0104] The schematic diagram of the mechanism of action of Examples 5 to 9 is as follows Figure 1 .

[0105] The mechanism of action of Example 10 is equivalent Figure 1 .

[0106] Example 11

[0107] The method is substantially the same as Example 5, except that the same mass of nonapeptide-1 is used instead of blue copper peptide to obtain sample 19.

[0108] Example 12

[0109] The present invention is substantially the same as Example 10, except that ergothionein is adjusted to other active ingredients, niacinamide 0.050%, thiamine hydrochloride 0.050%, inositol 0.050%, pyridoxine 0.050%, chlorhexidine digluconate 0.025%, N-hydroxysuccinimide, glutamyl 0.0041%, leucine 0.0035%, phenylalanine 0.0029%, lysine hydrochloride 0.0028%, proline 0.0024%, aspartic acid 0.0024%, valine 0.0022%, glycine 0.0021%, isoleucine 0.0021%, threonine 0.0017%, histidine 0.0015%, serine 0.00 palmitoyl tripeptide-1 0.0013%, potassium sorbate

[0110] 0.0013%, 5,7-dihydroxyflavone 0.0013%, alanine 0.0012%, methionine 0.0012%, biotin 0.0008%, tryptophan 0.0007%, palmitoyl tetrapeptide-7 0.0006%, tyrosine 0.0006%, folic acid 0.0005%, vitamin B12 0.0005%, tocopheryl acetate 0.0005%, bioflavonoids 0.0005%, ascorbic acid 0.0005%, menadione

[0111] 0.0005%, riboflavin 0.0005%, glutathione 0.0005%, cysteine ​​0.0002%, taurine 0.0001%, carnitine 0.00005%, and a sample of 20 was obtained.

[0112] Comparative Example 1

[0113] Preparation of comparative sample 1

[0114] Step 1: Prepare 10 mg / mL of 300 kDa hyaluronic acid solution. Add water slowly during the dissolution process and stir gradually to promote dissolution.

[0115] Step 2: Inject the prepared hyaluronic acid solution into one of the pumps of the mixer at a flow rate of 40 mL / min. Simultaneously inject deionized water into the other three pumps at a flow rate of 9 mL / min to obtain comparative sample 1.

[0116] Preparation of comparative samples 2 to 4

[0117] The preparation method is basically the same as that of comparative sample 1, except that the molecular weight of hyaluronic acid is adjusted to 5 kDa, 25 kDa, and 600 kDa, respectively. The samples are named comparative samples 2 to 4.

[0118] Comparative Example 2

[0119] A blue copper peptide solution with a concentration of 2.5 wt % was prepared, and ultrasonic treatment was performed for 60 min during the preparation process to ensure that the blue copper peptide was completely dissolved, thereby obtaining comparative sample 5.

[0120] Comparative Example 3

[0121] According to the ratio of hyaluronic acid, dihydroxypropyl arginine, blue copper peptide and water in the final finished solution described in Example 5, they were mixed in a laboratory glass reactor with a stirring speed of 1500 r / min and a stirring time of 30 min to obtain comparative sample 6.

[0122] Comparative Example 4

[0123] Blue copper peptide common lipid carrier: weigh 6.0 parts by weight of lecithin, 0.4 parts by weight of cholesterol, and 1.0 parts by weight of Tween 80, add 30 ml of a mixed organic solvent of methanol and dichloromethane (the volume ratio of methanol to dichloromethane is 5:2) until completely dissolved.

[0124] A mixture of lecithin, cholesterol, Tween 80, and an organic solvent was placed in a flask of a rotary evaporator, and the organic solvent was removed by rotary evaporation under reduced pressure (pressure: gradient reduction starting from normal pressure to finally reach 50 mbar; temperature: 65° C.) to form a dry film.

[0125] 30 parts by weight of a polyol and an aqueous solution containing 2.5 parts by weight of a blue copper peptide were weighed and added to a rotary evaporation flask, and hydration was performed until a thin film was eluted to obtain a liposome suspension.

[0126] The liposome suspension was homogenized using a high-pressure homogenizer or a high-pressure microfluidizer at a pressure of 800 bar or higher to obtain ordinary liposomes containing 2.5% blue copper peptide (total amount of 100 parts by weight), which is comparative sample 7.

[0127] Comparative Example 5

[0128] A lysine-substituted blue copper peptide-hyaluronic acid-lysine complex was prepared by a multi-microchannel high-speed mixing method: first, a 10 mg / mL hyaluronic acid (HA) solution with a molecular weight of 300 kDa was prepared. Note that water was added slowly during the dissolution process, and stirring was performed gradually to promote dissolution. Then a lysine solution (LYS) was prepared to ensure that the molar ratio of the repeating units of LYS and HA was 3. Finally, a blue copper peptide solution with a concentration of 13.6 wt% was prepared. Ultrasonic treatment was used for 60 minutes during the preparation process to ensure that all the blue copper peptides were dissolved. The prepared HA and LYS solutions were injected into the two opposite pumps of the mixer respectively, and the flow rate of the two pumps was kept consistent at 40 mL / min. The prepared blue copper peptide solution was injected into the other two opposite pumps at the same time, and the flow rate of the pump was 9 mL / min. The blue copper peptide concentration in the final sample was guaranteed to be 2.5 wt%, and comparative sample 8 was obtained.

[0129] Comparative Example 6

[0130] A multi-microchannel high-speed mixing method was used to prepare a blue copper peptide-hyaluronic acid-arginine polyhydroxy derivative complex with a molecular weight higher than 600kDa: first, a 10mg / mL hyaluronic acid solution with a molecular weight of 1200kDa was prepared. Note that water was added slowly during the dissolution process, and stirring was gradually performed to promote dissolution. Then, a dihydroxypropyl arginine solution with a concentration of 21.18mg / mL was prepared to ensure that the molar ratio of repeating units of dihydroxypropyl arginine to hyaluronic acid was 3. Finally, a blue copper peptide solution with a concentration of 13.6wt% was prepared. Ultrasonic treatment was used for 60min during the preparation process to ensure that the blue copper peptide was completely dissolved. The prepared hyaluronic acid and dihydroxypropyl arginine solutions were respectively injected into the two opposite pumps of the mixer, and the flow rate of the two pumps was kept consistent at 40mL / min. The prepared blue copper peptide solution was injected into the other two opposite pumps at the same time, and the flow rate of the pump was 9mL / min to obtain comparative sample 9.

[0131] Comparative Example 7

[0132] A multi-microchannel high-speed mixing method was used to prepare a blue copper peptide-arginine polyhydroxy derivative complex that does not contain hyaluronic acid: a dihydroxypropyl arginine solution with a concentration of 21.18 mg / mL was prepared; a blue copper peptide solution with a concentration of 13.6 wt% was prepared, and ultrasonic treatment was performed for 60 minutes during the preparation process to ensure that the blue copper peptide was completely dissolved. The prepared water and dihydroxypropyl arginine solution were injected into two opposite pumps of the mixer respectively, and the flow rate of the two pumps was kept consistent at 40 mL / min. The prepared blue copper peptide solution was injected into two other opposite pumps at the same time, and the pump flow rate was 9 mL / min to obtain comparative sample 10.

[0133] Comparative Example 8

[0134] A multi-microchannel high-speed mixing method was used to prepare a blue copper peptide-hyaluronic acid complex that does not contain arginine polyhydroxy derivatives: first, a 10 mg / mL hyaluronic acid solution with a molecular weight of 300 kDa was prepared. Note that water was added slowly during the dissolution process, and stirring was performed gradually to promote dissolution. A blue copper peptide solution with a concentration of 13.6 wt% was prepared, and ultrasonic treatment was performed for 60 minutes during the preparation process to ensure that all the blue copper peptides were dissolved. The prepared hyaluronic acid and water were injected into the two opposite pumps of the mixer respectively, and the flow rate of the two pumps was kept consistent at 40 mL / min. The prepared blue copper peptide solution was injected into the other two opposite pumps at the same time, and the flow rate of the pump was 9 mL / min to obtain comparison sample 11.

[0135] Comparative Example 9

[0136] The active ingredient solution is the same as that of Example 12, niacinamide 0.050%, thiamine hydrochloride 0.050%, inositol 0.050%, pyridoxine 0.050%, chlorhexidine digluconate 0.025%, N-hydroxysuccinimide, glutamyl 0.0041%, leucine 0.0035%, phenylalanine 0.0029%, lysine hydrochloride 0.0028%, proline 0.0024%, aspartic acid 0.0024%, valine 0.0022%, glycine 0.0021%, isoleucine 0.0021%, threonine 0.0017%, histidine 0.0015%, and serine 0.000 palmitoyl tripeptide-1 0.0013%, potassium sorbate 0.0013%, 5,7-dihydroxyflavone 0.0013%, alanine 0.0012%, methionine 0.0012%, biotin 0.0008%, tryptophan 0.0007%, palmitoyl tetrapeptide-7 0.0006%, tyrosine 0.0006%, folic acid 0.0005%, vitamin B12 0.0005%, tocopheryl acetate 0.0005%, bioflavonoids 0.0005%, ascorbic acid 0.0005%, menadione 0.0005%, riboflavin 0.0005%, glutathione 0.0005%, cysteine ​​0.0002%, taurine 0.0001%, carnitine 0.00005%; the above active ingredients were mixed to obtain sample 12.

[0137] Part II Performance Testing

[0138] 1. Particle size and potential experiments

[0139] Dynamic light scattering (DLS), a cutting-edge analytical method, enables high-precision measurement of particle size and surface potential. A Malvern particle size analyzer was used to characterize the particle size and surface potential of the Examples and Comparative Examples. The particle size and surface potential data for the Examples are shown in Table 1, while those for the Comparative Examples are shown in Table 2.

[0140] Table 1 Particle size and potential data of the examples

[0141]

[0142]

[0143] Table 2 Particle size and potential data of comparative examples

[0144]

[0145]

[0146] 2. Hyaluronic acid-arginine association network empty vector:

[0147] 2.1 Effective reduction in the molecular size of hyaluronic acid components:

[0148] Comparative Samples 1-4 in Table 2 show that hyaluronic acid components, in solution, have very large molecular sizes and highly negatively charged surfaces. As the molecular weight of hyaluronic acid increases (5-600 kDa), the particle size also increases significantly (1000-10000 nm) and the surface negative charge also increases (-50--80 mV), consistent with the conclusion that medium- and high-molecular-weight hyaluronic acids have difficulty penetrating the skin.

[0149] From the changes in particle size and potential of Sample 1 and Sample 5 to Sample 7 in Table 1, it can be seen that the associated network carrier matrix material of the present invention, the hyaluronic acid component and the arginine component can effectively form a nanoscale complex (200 to 400 nm) with a significantly reduced molecular size through a multi-microchannel high-speed mixing process, and the surface potential of the complex (-20 to -40 mV) is significantly lower than that of the hyaluronic acid solution of equal molecular weight of comparative samples 1 to 4. That is, the combined matrix material of the present invention can achieve the effective reduction of the molecular size of hyaluronic acid and its salt derivatives to the nanoscale, providing the possibility of increasing its own transdermal efficiency.

[0150] 2.2 Active ingredient - hyaluronic acid component - arginine component association network carrier:

[0151] As shown in Table 1, Examples 5 to 12, and the particle size and potential changes of samples 9 to 17, the associated network carrier matrix material of the present invention, the hyaluronic acid component and the arginine component are effectively mixed with skin care actives through a multi-microchannel high-speed mixing process, including small molecule peptides such as blue copper peptides, nonapeptide-1, water-soluble ergothioneine, and multiple small molecule amino acids. Active substances form a nanoscale complex (200 to 400 nm) with a significantly reduced molecular size, and the surface potential of the complex (-20 to -40 mV) is significantly lower than that of the hyaluronic acid solution of the same molecular weight of the control sample, that is, the combined matrix material of the present invention can achieve the effective reduction of the molecular size of hyaluronic acid and its salt derivatives to the nanoscale, thereby providing a possibility for improving the protection stability and transdermal efficiency of the active substance.

[0152] 3. Microstructure electron microscope image

[0153] Transmission electron microscopy (TEM) is a high-resolution characterization technique specifically used to observe the microstructure of nano- and micron-sized materials.

[0154] Figure 2 This is an electron micrograph of the carrier of sample 1. As shown in the figure, the matrix material hyaluronic acid component and arginine component of the present invention can effectively form a nanometer-scale carrier morphology through a multi-microchannel high-speed mixing process, which is consistent with the particle size analyzer results.

[0155] Figure 3 This is an electron micrograph of the copper peptide-hyaluronic acid-arginine network carrier in Sample 9. As shown, the hyaluronic acid and arginine components of the present invention, through a multi-microchannel high-speed mixing process, effectively encapsulate the copper peptide active ingredient, forming a nanometer-scale carrier morphology, consistent with the particle size analyzer results.

[0156] 4. Stability and active ingredient encapsulation and protection experiments

[0157] 4.1 Appearance stability

[0158] The samples of the embodiment and the comparative example were placed under light, 4°C, 25°C, and 48°C for one month, and then the appearance was compared by macroscopic observation to determine whether there was any delamination, precipitation, etc. The results are recorded in Tables 1 and 2.

[0159] As shown in Table 1, after the samples of the examples were subjected to a 4-week stability challenge under different storage conditions, their appearance did not show any unstable phenomena such as precipitation and stratification, and their appearance color did not change significantly.

[0160] The results show that the empty carrier or the carrier encapsulated with active ingredients of the present invention has excellent stability.

[0161] As shown in Table 2, after four weeks of stability testing under various storage conditions, many of the comparative samples, particularly those containing active ingredients such as blue copper peptides, exhibited instability, including precipitation and stratification, and their color also changed significantly. These comparative examples demonstrate the stability advantages of the carriers described herein.

[0162] 4.2 Particle size and potential stability

[0163] Some samples prepared in the examples and comparative examples were placed under light, 4°C, 25°C, and 48°C for one month, and then their particle size and zeta potential were measured using a Malvern particle size analyzer. The particle size and potential stability results are shown in Table 3.

[0164] Table 3 Particle size and potential stability test results of samples of Examples and Comparative Examples after storage

[0165]

[0166] As shown in Table 3, the particle size and zeta potential of Samples 1 and 9 did not change much after being challenged with different storage conditions, indicating that the microstructure of the empty carrier or the carrier encapsulated with the active ingredient described in the present invention is excellent. However, most of the comparative examples have already become unstable at the macro level, and therefore the microscopic particle size and zeta potential do not need to be further examined.

[0167] 4.3 Advantages of active substance encapsulation efficiency and protection stability:

[0168] The encapsulation efficiency test method of the active substance is as follows: the encapsulation efficiency (EE) of the carrier active substance is tested by ultrafiltration centrifugation.

[0169] 5 mL of the prepared example or comparative example sample was added to an ultrafiltration centrifuge tube (molecular weight cutoff MW 10KD), centrifuged at 8000 rpm for 5 min, and the filtrate was collected. The content of the active ingredient, such as a small molecule peptide, was determined by HPLC external standard method, and the encapsulation efficiency was calculated according to the following formula:

[0170]

[0171] Where Wt is the total input, Wf is the free drug content, and Wl is the membrane material (carrier) content.

[0172] The active substance encapsulation efficiency of some examples and comparative examples is shown in Table 4.

[0173] As shown in Table 4, the encapsulation efficiency of Sample 9 is significantly better than that of the free small molecule peptide shown in Comparative Sample 5, the complex prepared by the conventional mixing method shown in Comparative Sample 6, and the small molecule peptide liposome shown in Comparative Sample 7. The embodiment significantly improves the encapsulation efficiency of small molecule peptides, especially blue copper peptides, and provides a basis for achieving better protection of drug activity.

[0174] For the samples encapsulating active ingredients, some samples prepared in the examples and comparative examples were placed under light, 4°C, 25°C, and 48°C for 1 month, and then the changes in their active ingredient content before and after were further detected by high performance liquid HPLC. The active ingredient content results are recorded in Tables 4 and Figure 4 shown.

[0175] Table 4 Active ingredient content changes

[0176]

[0177] As shown in Table 4 and Figure 4 As shown, the content of small molecule peptides in Sample 9 of Example 5, Sample 5 of Comparative Example 2, Sample 6 of Comparative Example 3, and Sample 7 of Comparative Example 4 changes after being challenged with different storage conditions. Sample 9 of Example 5 is significantly better than the free small molecule peptide of Sample 5 of Comparative Example 2, the complex of the same components of the Example prepared by the conventional mixing method of Sample 6 of Comparative Example 3, and the small molecule peptide liposome of Sample 7 of Comparative Example 4. The Examples significantly improve the thermal stability and photostability of the small molecule peptide solution, achieving better protection of drug activity.

[0178] 5. Skin permeability and sustained release test

[0179] 5.1 In vitro skin permeability

[0180] use The membrane was used to simulate skin in a vertical Franz diffusion cell for transdermal testing. First, an intact, undamaged synthetic membrane was fixed between the receiving cell and the supply cell (with the shiny side facing up). 8 mL of a buffer solution was injected into the receiving cell, and 37°C constant-temperature circulating water was introduced, stirring at 300 rpm at 37°C. Then, 1.5 mL of the liposome sample was injected into the diffusion cell to begin the transdermal test. At 1, 3, 7, 11, and 24 hours, 1 mL of the receiving solution was aspirated and supplemented with 1 mL of isothermal blank receiving solution. The water-soluble drug content in the receiving solution was determined using high-performance liquid chromatography. The cumulative drug permeation at each time point was calculated and plotted against time to understand the drug's in vitro transdermal permeation effect.

[0181] The free small molecule peptide of Example 5 Sample 9, Comparative Example 2 Comparative Example 5, the complex of the same components as those of Example prepared by the ordinary mixing method of Comparative Example 3 Comparative Example 6, the small molecule peptide liposome of Comparative Example 4 Comparative Example 7, the small molecule peptide-dihydroxypropyl arginine complex containing high molecular weight hyaluronic acid of Comparative Example 6 Comparative Example 9, the small molecule peptide-dihydroxypropyl arginine complex not containing hyaluronic acid of Comparative Example 7 Comparative Example 10, and the small molecule peptide-hyaluronic acid complex not containing arginine polyhydroxy derivatives of Comparative Example 8 Comparative Example 11 were subjected to transdermal tests according to the above method. The specific results are shown in Tables 5 and 6. Figure 5 shown.

[0182] The formula for calculating the cumulative drug permeation is:

[0183] Where Qn is the cumulative drug permeation, Cn is the drug concentration measured at the nth time, Ci is the drug concentration measured at the ith point, V0 is the volume of the diffusion cell (i.e., the amount of release medium added), and Vi is the amount of each sample taken. The cumulative permeation per unit area, Q, = Qn / S, where S is the area of ​​the diffusion cell.

[0184] Table 5 Drug cumulative permeation test results

[0185]

[0186]

[0187] As can be seen from Table 5, the cumulative permeation of Example 5 Sample 9 and Comparative Example 2 Comparative Sample 5, Comparative Example 3 Comparative Sample 6, Comparative Example 4 Comparative Sample 7, Comparative Example 7 Comparative Sample 10, and Comparative Example 8 Comparative Sample 11 over 24 hours is 0.03693 mg / cm 2 、0.1572mg / cm 2 (free small molecule peptide), 0.3368 mg / cm2 (complex with the same components as in Example prepared by conventional mixing method), 0.2413 mg / cm 2 (small molecule peptide liposomes), 0.11230 mg / cm 2 (Small molecule peptide-dihydroxypropyl arginine complex containing high hyaluronic acid), 1653mg / cm 2 (small molecule peptide-dihydroxypropyl arginine complex without hyaluronic acid), and 0.1765mg / cm 2 (Small molecule peptide-hyaluronic acid complex without dihydroxypropyl arginine).

[0188] The cumulative skin permeation of the small molecule peptide active ingredient in Example 5 described in this project was significantly higher than that of the control group. This demonstrates that the carrier technology provided by this invention enables high concentrations of skin care active ingredients to penetrate the stratum corneum and deliver the drug to the skin care area, thereby improving skin care efficacy.

[0189] 5.2 Human skin permeability

[0190] Two subjects used the test sample Example 12 Sample 17 and Comparative Example 9 Comparative Sample 12, and adopted the human (in vivo) Raman non-invasive optical (LabRAM Odyssey high-speed and high-resolution micro-confocal Raman spectrometer HORIBA, temperature: 22°C ± 2°C; humidity: 50% RH ± 10% RH) testing method to study the penetration effect of the test sample in human skin 0.5h, 1h, 2h, and 4h after using the sample.

[0191] A 3cm x 3cm area was selected for testing on the front of the forearm. After the subject arrived, they cleaned the skin on the inside of their arm with water and sat quietly in a constant temperature and humidity chamber for 30 minutes. After 30 minutes, the subjects were given the sample according to the test instructions. Testing was performed on the skin in the test area 0.5, 1, 2, and 4 hours after application, and the relative permeability of the test sample was calculated.

[0192] Raman spectroscopy imaging data processing includes spectral preprocessing and data analysis: Spectral preprocessing includes cosmic ray removal, spectral smoothing, background noise removal, baseline calibration, and spectral normalization. Univariate data analysis primarily analyzes the Raman spectral data for the biochemical substance corresponding to a specific peak position, revealing the distribution of that substance within human skin. Data analysis uses Labspec5 software to perform baseline calibration and confirm the position of characteristic peaks on the Raman spectra. The obtained Raman spectra are then calculated, including peak intensity, peak shift, peak area, and half-width. Labspec6 software is also used to numerically analyze the peak intensities at different depths and plot their spatial distribution. The permeability behavior of the sample is determined by using the sample's characteristic Raman signal, which is distinct from the skin's intrinsic signal, to confirm its distribution at different skin depths.

[0193] Relative permeability (%) = standard deviation (test value before test sample use - test value after test sample use) × 100% According to the data processing results, the relative permeability of the test samples in the skin at different time points is shown in Table 6:

[0194] Table 6 Relative permeability results of samples in skin

[0195]

[0196] As can be seen from Table 6, after using sample 17 of test example 12 on human skin, the relative permeabilities of the test sample were 1.57%, 3.15%, 5.01% and 5.28% at 0.5h, 1h, 2h and 4h, respectively. In contrast, sample 12 of comparative example 9 was a free active ingredient combination without the matrix carrier of the present invention, and the relative permeabilities were 0.19%, 1.41%, 2.03% and 3.08%, respectively. This result indicates that the carrier of the embodiment of the present invention can effectively improve the human permeability of various skin care active ingredients.

[0197] Through Figure 6 Depth analysis of the Raman image, using test sample Example 12, shows the distribution of the sample at different depths in the human skin after penetration: within 0.5 hours, the sample penetrated the stratum corneum to a depth of 20 μm; within 1 hour, the sample continued to penetrate the stratum corneum and penetrated the stratum corneum into the viable epidermis to a depth of 45 μm; within 2 hours, the sample continued to penetrate the stratum corneum and viable epidermis, and the content entering the stratum corneum and viable epidermis increased, reaching a depth of 65 μm; within 4 hours, the sample continued to penetrate the stratum corneum and viable epidermis, and a small amount broke through the viable epidermis to enter the dermis, reaching a depth of 95 μm. This result shows that the carrier of the embodiment of the present invention can efficiently deliver sodium hyaluronate and various skin care actives to the human dermis.

[0198] 5.4 In vitro skin sustained release

[0199] The slow-release performance of the active ingredient was tested as follows: 5 mL of an example or comparative example sample was aspirated and added to a dialysis bag with a cutoff of 100 kDa; the dialysis bag was immersed in 300 mL of deionized water for sustained release of the drug; 1 mL of the sustained-release liquid was aspirated from the receiving cell at 0, 1, 3, 6, 9, 12, and 24 hours, and 1 mL of deionized water was added to the receiving cell simultaneously; the active ingredient content in the sustained-release liquid in the receiving cell was determined using HPLC; the cumulative release rate was calculated as follows: Cumulative release rate = (active ingredient mass concentration measured at time t * receiving liquid volume + sum of active ingredient mass concentration measured from time (t-1) onwards * receiving liquid volume) / total mass of active ingredient in the sample;

[0200] The test results are shown in Table 7 and Figure 7 shown.

[0201] Table 7 In vitro skin sustained release test results

[0202]

[0203] From Table 7 and Figure 7 It can be seen that the sustained-release performance of Example 5 Sample 9 is significantly better than that of Comparative Example 2 Comparative Sample 5 (free small molecule peptide), Comparative Example 3 Comparative Sample 6 (complex of the same embodiment components prepared by ordinary mixing method), Comparative Example 4 Comparative Sample 7 (small molecule peptide-dihydroxypropyl arginine complex containing high hyaluronic acid), Comparative Example 7 Comparative Sample 10 (small molecule peptide-dihydroxypropyl arginine complex without hyaluronic acid), Comparative Example 8 Comparative Sample 11 (small molecule peptide-hyaluronic acid complex without dihydroxypropyl arginine). This shows that the carrier technology provided by the present invention can improve the sustained-release performance of the active ingredient, thereby achieving a more lasting skin care effect.

[0204] 5.5 Advantages of Cell Scratch Skin Anti-aging Repair

[0205] The efficacy of skin repair and anti-aging treatments was evaluated using a mesenchymal stem cell migration test. Human umbilical cord mesenchymal stem cells (HUCMSCs) are unique precursor cells found in the Wharton's jelly and perivascular tissues of the umbilical cord, possessing self-renewal and multidirectional differentiation potential. Numerous scientific studies have shown that HUCMSCs can achieve anti-aging and repair effects by regenerating and repairing aging cells, tissues, and organs.

[0206] After the HUCMSCs cells were digested with trypsin solution, culture medium was added to terminate the digestion. After centrifugation, the supernatant was discarded and the cells were resuspended in complete culture medium. The HUCMSCs cell solution with a concentration of 5000,000 cells / cell was seeded into a 12-well plate at a volume of 40,000 / cm2 and cultured for 1 day. After the cells covered the bottom of the plate, a 20 μL pipette tip was used to make cell scratches perpendicular to the well plate, trying to ensure that the width of each scratch was consistent. The cell culture medium was aspirated and the well plate was rinsed three times with PBS to wash away the cell debris generated by the scratches. The Example and Comparative Example were prepared into a drug culture medium at a concentration of 1 wt%, and the blank control group was added to the well plate and photographed. The culture plate was placed in an incubator for culture and taken out every 2-4 hours for photography. The experimental results were analyzed based on the collected image data. The scratch area of ​​each photographing time period was calculated using Image J, and the cell migration rate was calculated using the following formula:

[0207]

[0208] The specific results are shown in Table 8 and Figure 8 .

[0209] Table 8 Cell scratch skin anti-aging repair results

[0210]

[0211] From Table 8 and Figure 8 It can be seen that the cell scratch performance of Example 5 Sample 9 is significantly better than that of Comparative Example 2 Comparative Sample 5 (free small molecule peptide), Comparative Example 3 Comparative Sample 6 (complex of the same embodiment components prepared by ordinary mixing method), Comparative Example 4 Comparative Sample 7 (small molecule peptide-dihydroxypropyl arginine complex containing high hyaluronic acid), Comparative Example 7 Comparative Sample 10 (small molecule peptide-dihydroxypropyl arginine complex without hyaluronic acid), Comparative Example 8 Comparative Sample 11 (small molecule peptide-hyaluronic acid complex without dihydroxypropyl arginine). This shows that the carrier technology provided by the present invention can improve the skin care, repair and anti-aging effects of active substances.

[0212] Part III Results and Discussion

[0213] Comparative Example 1 Comparative Samples 1 to 4 are solutions of sodium hyaluronate with different molecular weights. The hyaluronic acid molecules are in an irregular polymer state, and the hydrated particle size is much larger than that of the carriers of the same molecular weight in Sample 1 of Example 1 and Samples 5 to 7 of Example 3. The surface negative charge is high, and there is a certain repulsion with the negatively charged skin, and therefore, it does not have the potential for transdermal performance.

[0214] Comparative Example 3, Comparative Sample 6, is a composite prepared using a conventional mixing method. Compared to Example 5, Sample 9 exhibits multiple particle size peaks and a broadened distribution due to the lack of extensive microchannel high-speed mixing. Furthermore, the effective average particle size and surface potential could not be measured. This demonstrates the necessity of the multi-microchannel high-speed mixing method described herein for preparing the carrier and its superior properties. Furthermore, due to the lower encapsulation efficiency of the conventional method compared to Example 5, the in vitro skin permeability, sustained-release performance, and skin repair efficacy were all reduced.

[0215] Comparative Example 4, Comparative Sample 7 is an ordinary blue copper peptide liposome. Compared with Sample 9 in Example 5, the chelating property of copper ions is incompatible with the strong anionic property of the liposome phospholipid material, resulting in obvious defects in carrier stability, and even precipitation in the comparative example. At the same time, the strong ionic effect causes the encapsulated blue copper peptide to have poor transdermal performance and sustained release performance. Furthermore, due to the inactivation of copper ions, the in vitro skin scratch repair and anti-aging efficacy is reduced.

[0216] Comparative Example 5 Comparative Sample 8 is a lysine-composite hyaluronic acid. Compared to Example 5 Sample 9, lysine contrasts arginine and its polyhydroxy derivatives lack hydrogen bonding sites and the side chain carbon chain grows. The sample presents multiple particle size peaks, the distribution becomes wider, the particle size is significantly larger, and the surface has a high negative charge, and after 1 month of stability storage, the sample becomes unstable. It can be seen that the reduction of hydrogen bonding sites weakens the ability of lysine to bind hyaluronic acid, the carrier particle size becomes larger, and the uneven distribution leads to reduced carrier stability; the lysine side chain carbon chain grows, the overall hydrophobic segment increases, and the carrier stability is reduced.

[0217] Comparative Example 6 Comparative Sample 9 is a blue copper peptide-high molecular weight hyaluronic acid-arginine polyhydroxy derivative complex. Compared with Sample 9 in Example 5, the high molecular weight hyaluronic acid has an increased molecular weight, which leads to an increase in the intermolecular friction of hyaluronic acid before high-speed mixing in multiple microchannels, and the overall solution viscosity is significantly improved. The mixing efficiency is greatly reduced, and uniform mixing cannot be achieved within the effective mixing time, thereby preparing an extremely heterogeneous sample (multiple peaks, wide particle size distribution, and extremely large average particle size), which has a great negative effect on the stability and skin permeability of the sample.

[0218] Comparative Example 7 Comparative Sample 10 is a blue copper peptide-arginine polyhydroxy derivative complex that does not contain hyaluronic acid prepared by a multi-microchannel high-speed mixing method. Compared with Sample 9 in Example 5, the product characteristics of the lack of hyaluronic acid are similar to those of a simple mixture of blue copper peptide and arginine polyhydroxy derivative. Arginine polyhydroxy derivatives and blue copper peptides are both small amounts of positively charged molecules under neutral conditions. The same charge properties lead to enhanced electrostatic repulsion, making it difficult for these two components to form a carrier. Arginine polyhydroxy derivatives and blue copper peptides cannot directly form a complex with measurable particle size and potential, resulting in the blue copper peptide not having the function of nanocarrier protection, thereby further losing the sustained-release effect of the comparative sample; the product's transdermal effect deteriorates; and the in vitro skin scratch repair and anti-aging efficacy is reduced. The skin permeability and sustained-release properties of this example will be lost.

[0219] Comparative Example 8, Sample 11, is a copper peptide-hyaluronic acid complex without an arginine polyhydroxy derivative. Compared to Sample 9 in Example 5, the properties of this comparative example, lacking the arginine polyhydroxy derivative, are similar to those of a simple mixture of copper peptide and hyaluronic acid. Direct charge neutralization between hyaluronic acid and copper peptide can lead to the risk of copper ion inactivation, further compromising the sustained-release effect of this comparative example; impaired transdermal penetration; and reduced in vitro scratch repair and anti-aging efficacy.

[0220] In summary, the carrier of the present invention is based on the non-covalent bond forces of hyaluronic acid and its salt derivatives, arginine and its polyhydroxy derivatives, including electrostatic binding forces and / or hydrogen bonding forces. Through a multi-microchannel high-speed mixing process, a non-covalent bond association network at the nanometer scale is realized, thereby achieving 1) effective reduction of the molecular size of hyaluronic acid and its salt derivatives, which can effectively enhance their skin permeability, especially for hyaluronic acid and its salt derivatives with large and medium molecular weight that are difficult to penetrate the skin; 2) effective combination of skin care active ingredients, especially small molecule amino acids and their derivatives, small molecule polypeptides, etc., which can ensure the biological activity and efficient association and encapsulation of the active substances. At the same time, the carrier can further improve the physicochemical stability, transdermal permeability, sustained release and repair of the active ingredients, and other excellent skin care effects.

[0221] The applicant declares that while the above-described embodiments illustrate the process of the present invention, the present invention is not limited to the above-described process steps, nor does it imply that the present invention must rely on the above-described process steps for implementation. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for the raw materials used, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims

1. A composition for preparing cosmetics, characterized in that, The invention comprises a carrier and an active ingredient loaded in the carrier and used in cosmetics; the active ingredient has an amino group; The carrier is a nanometer-scale or micrometer-scale particle in water; the carrier is a network carrier composed of arginine components and hyaluronic acid components linked by hydrogen bonds and / or ionic bonds; The arginine component is arginine and / or a polyhydroxy derivative of arginine; The hyaluronic acid component is hyaluronic acid and / or a salt derivative of hyaluronic acid; The molecular weight of the hyaluronic acid component is 2 to 600 kDa; The preparation method of the composition comprises: using a multi-microchannel high-speed mixer, mixing a solution containing an arginine component, a solution containing a hyaluronic acid component, and a solution containing an active ingredient after passing through multiple microchannels of the multi-microchannel high-speed mixer; The structure of the polyhydroxy derivative of arginine is shown in Formula I: Formula I In formula I, X is a hydrogen atom or -CH2CH(OH)CH2(OH); Y is a hydrogen atom, an alkali metal, ammonium, an organic ammonium or -CH2CH(OH)CH2(OH); The salt derivative of hyaluronic acid is sodium hyaluronate; The active ingredient is one or more of amino acids, amino acid derivatives, and polypeptides; the molecular weight of the active ingredient does not exceed 1500 Da; the amino acid derivative is one or more combinations of ergothioneine, nicotinamide, ectoine, acetylhydroxyproline, and N-acetylneuraminic acid.

2. The composition according to claim 1, characterized in that The molar ratio of the arginine unit in the arginine component to the hyaluronic acid unit in the hyaluronic acid component is 0.05-2:0.2-2.

3. The composition according to claim 2, characterized in that The molar ratio of the arginine unit in the arginine component to the hyaluronic acid unit in the hyaluronic acid component is 0.36-3:

1.

4. The composition according to claim 1, characterized in that The carrier is nanometer-scale or micrometer-scale particles with a diameter of 10 to 1000 nm in water.

5. The composition according to claim 1, characterized in that The molar ratio of the arginine unit in the arginine component, the hyaluronic acid unit in the hyaluronic acid component, and the active ingredient is 0.05-2:0.2-2:

1.

6. The composition according to claim 1, characterized in that The amino acids are one or more combinations of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, and histidine; The polypeptide is one or more combinations of arginine / lysine polypeptide, blue copper peptide, glutathione, nonapeptide-1, carnosine, acetyl tetrapeptide-2, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, palmitoyl tripeptide-5, and snake venom peptide.

7. A method for preparing the composition according to any one of claims 1 to 6, characterized in that: A multi-microchannel high-speed mixer is used to mix a solution containing arginine components, a solution containing hyaluronic acid components, and a solution containing active ingredients after passing through multiple microchannels of the multi-microchannel high-speed mixer.

8. The preparation method according to claim 7, characterized in that The concentration of the solution containing hyaluronic acid components is 1 to 30 mg / mL; The concentration of the solution containing arginine components is 0.01 to 50 mg / mL; The concentration of the solution containing the active ingredient is 1 to 100 mg / mL.

9. The preparation method according to claim 7, characterized in that The mixing time of the solution containing arginine components, the solution containing hyaluronic acid components, and the solution containing active ingredients is less than 0.1 second; and the Reynolds number Re during mixing is greater than 4000.

10. Use of the composition according to any one of claims 1 to 6 in the preparation of cosmetics.

11. A cosmetic, characterized in that: Contains the composition according to any one of claims 1 to 6.

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