Hyaluronic acid nanospheres, method for preparing same and use thereof

Abstract

This application belongs to the field of biomedical technology, specifically relating to hyaluronic acid nanospheres, their preparation methods, and applications. This application provides a method for preparing hyaluronic acid nanospheres, which involves the interaction of nucleic acids with hyaluronic acid salts in a specific ratio. The preparation process is optimized, resulting in hyaluronic acid nanospheres with high hyaluronic acid content, strong skin permeability, and good stability, meeting the application requirements for transdermal drug delivery and skincare products. Furthermore, this application uses a non-covalent bonding method to prepare the nanospheres, simplifying the preparation process, effectively improving the gelling properties of hyaluronic acid salts, reducing stickiness at high concentrations, and broadening the application scenarios of hyaluronic acid in skincare products.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to hyaluronic acid nanospheres, their preparation methods, and applications. Background Technology

[0002] Hyaluronic acid is widely used in cosmetics and skincare, primarily in moisturizing products. Sodium hyaluronate is a high-molecular-weight polymer with a wide molecular weight range (from several thousand to several million Daltons). However, while a larger molecular weight of sodium hyaluronate results in better film-forming and moisturizing properties, it cannot effectively penetrate the stratum corneum of the skin, remaining only on the skin surface and unable to reach the stratum corneum or even the basal layer to exert its skincare effects. Currently, in the field of medical aesthetics, high-molecular-weight sodium hyaluronate is typically injected subcutaneously or into the dermis to improve facial wrinkles (such as nasolabial folds and tear troughs).

[0003] Small-molecule hyaluronic acid, marketed for its ability to "penetrate," typically undergoes complex manufacturing processes, requiring enzymatic hydrolysis and separation. During secondary processing, sodium hyaluronate itself acts as an excellent "culture medium" for microorganisms, making it highly susceptible to bacterial growth and spoilage. Therefore, extremely high standards are placed on the production environment and product preservation systems, significantly increasing raw material costs. Furthermore, while the processed small-molecule hyaluronic acid partially improves hyaluronic acid's permeability, its reduced molecular weight significantly diminishes its moisturizing and skin-smoothing effects in cosmetics, resulting in a significantly reduced user experience.

[0004] On the other hand, sodium hyaluronate has extremely strong water absorption properties, and even at very low concentrations (such as 0.1%), it can form a highly viscous gel. This can cause raw materials or skincare products containing sodium hyaluronate to easily pill, feel heavy, and sticky on the skin, severely affecting the user experience. Simply pursuing high concentration while neglecting the skin feel will make the product difficult to be accepted by the market.

[0005] Therefore, the amount of sodium hyaluronate added to skin care products is often very low, which limits its efficacy. Summary of the Invention

[0006] Based on this, one embodiment of this application provides hyaluronic acid nanospheres, their preparation method, and their applications.

[0007] This application provides a method for preparing hyaluronic acid nanospheres, comprising:

[0008] The nucleic acid and solvent are first mixed and heated to prepare a mixture;

[0009] Hyaluronic acid is mixed with the mixture for a second time, the pH is adjusted to 5-7, homogenized, and then cooled to 20℃-40℃ to prepare phase A;

[0010] Lysine, arginine, and histidine were dissolved in water, and the pH was adjusted to 5-7 to prepare phase B; and,

[0011] Phase A and Phase B are mixed for the third time, homogenized, and then an additive is added. The mixture is heated to 75℃-90℃ and stirred. Then the temperature is lowered to 20℃-40℃ to prepare the hyaluronic acid nanospheres.

[0012] The hyaluronic acid nanospheres comprise the following components by mass percentage: 0.01%-4% nucleic acid, 0.01%-2% hyaluronic acid salt, 0.01%-1.2% arginine, 0.01%-1.2% lysine, 0.01%-1.0% histidine, 0.1%-5% of the auxiliary agent, and 85.6%-99.85% water.

[0013] In some embodiments, the nucleic acid includes one or both of DNA and DNA derivatives.

[0014] In some embodiments, the DNA derivative includes one or more of sodium DNA, calcium DNA, and magnesium DNA.

[0015] In some embodiments, the hyaluronic acid salt comprises sodium hyaluronate; the molecular weight of the sodium hyaluronate ranges from 800 Da to 2,000,000 Da.

[0016] In some embodiments, the mass ratio of the nucleic acid to the total amino acids is 1:(0.5-2.0).

[0017] In some embodiments, the mass ratio of arginine to lysine is 1:(0.8-30).

[0018] In some of these embodiments, the pH-adjusting agent includes citric acid.

[0019] In some embodiments, the additives include one or both of thickeners and preservatives.

[0020] In some embodiments, the thickener includes one or two of hydroxyethyl cellulose, Carrageenan extract, xanthan gum, Sclerotium sclerotium gum, and ammonium acryloyldimethyl taurate and VP copolymer.

[0021] In some embodiments, the preservative includes one or two of hexanediol, pentanediol, phenoxyethanol, and ethylhexylglycerin.

[0022] In some embodiments, the thickener is present in the hyaluronic acid nanospheres at a concentration of 0.1 wt% to 0.3 wt%.

[0023] In some embodiments, the preservative content in the hyaluronic acid nanospheres is 1wt%-5wt%.

[0024] In some embodiments, the solvent includes water.

[0025] In some embodiments, the temperature of the first mixing is 60°C-80°C.

[0026] In some embodiments, the temperature of the second mixing is 50°C-70°C.

[0027] In some embodiments, the temperature of the third mixture is 20°C-40°C.

[0028] In some embodiments, the third mixing includes adding phase B to phase A and stirring at 2000 rpm to 10000 rpm for 5 min to 30 min.

[0029] In some embodiments, the homogenization process includes high-pressure homogenization or high-pressure microfluidic processing.

[0030] In some embodiments, the cooling rate after homogenization of the second mixture is ≤1℃ / 5min.

[0031] In some embodiments, the stirring time is 30 min to 120 min.

[0032] In some embodiments, the conditions for the high-pressure homogenization process include: homogenization temperature of 40°C-60°C; homogenization pressure of 100 bar-2000 bar; and homogenization cycles of 2-20.

[0033] The conditions for the high-pressure microjets treatment include: pressure of 1400Psi-15000Psi, and treatment times of 1-5 times.

[0034] In some embodiments, the hyaluronic acid nanospheres comprise, by weight percentage, the following components: 0.01%-4% nucleic acid, 0.01%-2% sodium hyaluronate, 0.01%-1.2% arginine, 0.01%-1.2% lysine, 0.01%-1.0% histidine, 0.1%-0.5% thickener, 0.1%-0.3% preservative, and water.

[0035] Another aspect of this application provides a method for preparing hyaluronic acid nanospheres, resulting in hyaluronic acid nanospheres.

[0036] This application also provides the use of the aforementioned hyaluronic acid nanospheres in the preparation of transdermal drug delivery products.

[0037] In some embodiments, the product includes cosmetics.

[0038] In some embodiments, the cosmetic includes one or more of the following: facial mask liquid, skin care lotion, serum, spray, and lotion.

[0039] This application also provides a cosmetic product whose raw materials include hyaluronic acid nanospheres prepared by the aforementioned preparation method.

[0040] This application provides a method for preparing hyaluronic acid nanospheres. The method involves the interaction of nucleic acids with hyaluronic acid salts in a specific ratio to prepare hyaluronic acid nanospheres. The preparation process is optimized, resulting in hyaluronic acid nanospheres with high hyaluronic acid content, strong skin permeability, and good stability, meeting the application requirements for transdermal drug delivery and skincare products. Furthermore, this application uses a non-covalent bonding method to prepare the nanospheres, simplifying the preparation process, effectively improving the gelling properties of hyaluronic acid salts, reducing stickiness at high concentrations, and broadening the application scenarios of hyaluronic acid in skincare products. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 Comparative images of Example 1 and Comparative Example 7;

[0043] Figure 2 SEM images of Example 1 and Comparative Example 7 after freeze-drying;

[0044] Figure 3 Low-pressure TEM electron microscope images of Example 1 and Comparative Example 3;

[0045] Figure 4 Reconstructed Raman pseudocolor images of skin Raman penetration detection in Example 1 and Comparative Example 7;

[0046] Figure 5 The total integral intensity graphs of sodium hyaluronate content for Example 1 and Comparative Example 7 are shown.

[0047] Figure 6 Cell migration graphs for Example 1 and Comparative Example 7;

[0048] Figure 7 This diagram illustrates the molecular interaction between nucleic acids and sodium hyaluronate. Detailed Implementation

[0049] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0050] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0051] the term

[0052] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0053] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0054] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0055] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0056] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0057] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0058] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0059] In this application, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions containing the listed features.

[0060] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0061] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0062] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0063] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0064] In this application, the terms "phase A" and "phase B" refer to the mixed state of different components, and "A," "B," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0065] The first aspect of this application provides a method for preparing hyaluronic acid nanospheres, comprising the following steps:

[0066] The nucleic acid and solvent are first mixed and heated to prepare a mixture.

[0067] Hyaluronic acid is mixed with the mixture for a second time, the pH is adjusted to 5-7, homogenized, and then cooled to 20℃-40℃ to prepare phase A.

[0068] Lysine, arginine, and histidine were dissolved in water, and the pH was adjusted to 5-7 to prepare phase B.

[0069] Phase A and Phase B are mixed for the third time, homogenized, and then an additive is added. The mixture is heated to 75℃-90℃ and stirred. Then the temperature is lowered to 20℃-40℃ to prepare the hyaluronic acid nanospheres.

[0070] The hyaluronic acid nanospheres comprise the following components by mass percentage: 0.01%-4% nucleic acid, 0.01%-2% hyaluronic acid salt, 0.01%-1.2% arginine, 0.01%-1.2% lysine, 0.01%-1.0% histidine, 0.1%-5% of the auxiliary agent, and 85.6%-99.85% water.

[0071] Understandably, the above-mentioned mass percentages are calculated in this application without the amount of pH adjuster, i.e., the amount of raw materials other than pH adjuster is taken as 100%.

[0072] In a specific example, the pH values ​​of phase A and phase B are 4, 4.5, 5, 5.5, 6, and any value in between.

[0073] In one specific example, the amount of nucleic acid used is 0.01-4%, including but not limited to 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, or 4%, and any value in between. Understandably, this application directly utilizes the interaction between the amino acid composition and the negatively charged nucleic acid, and achieves co-compression of hyaluronic acid during the compression of the nucleic acid spatial structure.

[0074] In one specific example, the nucleic acid includes DNA and DNA derivatives.

[0075] In one specific example, the DNA derivative includes one or more of sodium DNA, calcium DNA, and magnesium DNA, such as sodium DNA. The nucleic acid contains 80% or more DNA, and the number of base pairs is 30-5000 bp, for example, 30 bp, 100 bp, 500 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp, etc. DNA and its derivatives interact well with hyaluronic acid, effectively forming nanosphere structures and improving the permeability of hyaluronic acid.

[0076] In one specific example, the additives include at least one of thickeners and preservatives; there is no particular limitation on the specific types of thickeners and preservatives.

[0077] In one specific example, the hyaluronic acid salt is selected from sodium hyaluronate, which is the sodium salt form of hyaluronic acid. Sodium hyaluronate has good biocompatibility and moisturizing properties and is a commonly used ingredient in cosmetics and pharmaceuticals. The molecular weight of the sodium hyaluronate ranges from 800 Da to 2,000,000 Da.

[0078] In one specific example, the mass ratio of the nucleic acid to the total amino acids is 1:(0.5-2.0). For example, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or 1:2.0, and any value in between.

[0079] In a specific example, the mass ratio of arginine to lysine is 1:(0.8-30). For example, 1:0.8, 1:0.9, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, or 1:30, and any value in between.

[0080] In one specific example, the thickener includes at least one of hydroxyethyl cellulose, Chondrus crispus (Carrageenan) Extract, xanthan gum, sclerotium gum, and AVC; used in amounts of 0.1-0.3 wt%, such as 0.1 wt%, 0.11 wt%, 0.15 wt%, 0.2 wt%, or 0.3 wt%, and any intermediate values. These thickeners and preservatives exhibit good safety and stability, making them suitable for use in the cosmetic and pharmaceutical industries.

[0081] In one specific example, the preservative includes at least one of hexanediol, pentanediol, and ethylhexylglycerin; and is used in an amount of 1 wt% to 5 wt%, such as 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, or 5 wt%, and any value in between.

[0082] In one specific example, the pH-adjusting reagent includes citric acid.

[0083] In one specific example, the additives include one or both of thickeners and preservatives.

[0084] In one specific example, the temperature of the first mixture is 60°C-80°C, such as 60°C, 65°C, 70°C, 75°C, 80°C, and any value in between.

[0085] In one specific example, the homogenization process and the homogenization process each independently include high-pressure homogenization or high-pressure microfluidic processing.

[0086] In a specific example, the conditions for the high-pressure homogenization process include: homogenization temperature of 40℃-60℃, such as 40℃, 45℃, 50℃, 55℃, 60℃, etc.; homogenization pressure of 100 bar-2000 bar, such as 100 bar, 500 bar, 1000 bar, 1500 bar, 2000 bar, etc.; and homogenization cycles of 2-20 times, such as 2 times, 5 times, 10 times, 15 times, 20 times, etc.

[0087] In a specific example, the conditions for the high-pressure microjets treatment include: pressure of 1400 Psi - 15000 Psi, such as 1400 Psi, 2500 Psi, 5000 Psi, 7500 Psi, 10000 Psi, 12500 Psi, 15000 Psi, etc., and the number of treatments is 1-5 times, such as 1 time, 2 times, 3 times, 4 times, 5 times, etc.

[0088] In one specific example, the temperature of the second mixture is 50°C-70°C, such as 50°C, 55°C, 60°C, 65°C, 70°C, and any value in between.

[0089] In one specific example, the temperature of the third mixture is 20°C-40°C, such as 20°C, 25°C, 30°C, 35°C, 40°C, and any value in between.

[0090] In a specific example, the conditions for the high-pressure homogenization process include: homogenization temperature of 40℃-60℃; homogenization pressure of 100 bar-2000 bar; and homogenization cycles of 2-20. For example, the homogenization temperature can be 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, or 60℃, or any value in between.

[0091] For example, the homogeneous pressure can be 100 bar, 200 bar, 300 bar, 400 bar, 500 bar, 600 bar, 700 bar, 800 bar, 900 bar, 1000 bar, 1100 bar, 1200 bar, 1300 bar, 1400 bar, 1500 bar, 1600 bar, 1700 bar, 1800 bar, 1900 bar, or 2000 bar, or any value in between.

[0092] In a specific example, the conditions for the high-pressure microjets treatment include: a pressure of 1400 Psi - 15000 Psi, and 1 to 5 treatment cycles. For example, the pressure can be 1400 Psi, 2400 Psi, 3400 Psi, 4400 Psi, 5400 Psi, 6400 Psi, 7400 Psi, 8400 Psi, 9400 Psi, 10400 Psi, 11400 Psi, 12400 Psi, 13400 Psi, 14400 Psi, or 15000 Psi, or any value in between.

[0093] In one specific example, the hyaluronic acid nanospheres comprise the following components by mass percentage: 0.01%-4% nucleic acid, 0.01%-2% sodium hyaluronate, 0.01%-1.2% arginine, 0.01%-1.2% lysine, 0.01%-1.0% histidine, 0.1%-0.5% thickener, 0.1%-0.3% preservative, and water.

[0094] This application also provides hyaluronic acid nanospheres prepared by the above-mentioned method. The hyaluronic acid nanospheres provided by this application exhibit good stability and uniform nanoscale particle size, resulting in better permeability, reduced stickiness at high concentrations, and the ability to promote cell migration, allowing them to penetrate deep into the skin to exert skin repair effects.

[0095] This application also provides the use of the aforementioned hyaluronic acid nanospheres in the preparation of transdermal drug delivery products.

[0096] In one specific example, the product includes cosmetics.

[0097] In one specific example, the cosmetic product includes at least one of facial mask liquid, skin care lotion, serum, spray and lotion.

[0098] This application also provides a cosmetic product whose raw material includes hyaluronic acid nanospheres prepared by the above-described preparation method. The hyaluronic acid nanospheres prepared using the method of this application, when used as a cosmetic raw material, can effectively improve the gelling properties of sodium hyaluronate and reduce the stickiness at high concentrations.

[0099] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0100] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. For temperature and time parameters, acceptable deviations due to instrument testing accuracy or operational precision are permissible.

[0101] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0102] Unless otherwise specified, the raw materials used in the following experiments can be purchased from the market.

[0103] The exemplary descriptions of the raw materials used in the embodiments and comparative examples are as follows:

[0104] Sodium hyaluronate: Sodium hyaluronate (average molecular weight 1,000,000-1,500,000 Da), purchased from Bloomage Biotechnology; (batch number: 20-C051504).

[0105] Sodium hyaluronate: Sodium hyaluronate (average molecular weight 1000 Da), purchased from Giant Microorganisms; (batch number: D2025072401).

[0106] Nucleic acid: Sodium DNA, DNA content ≥80%, purchased from Beijing Weiye Innovation Technology Co., Ltd. (Batch No.: 20250311).

[0107] Lysine, purity ≥95%, purchased from Zhangjiagang Huachang Pharmaceutical Co., Ltd. (batch number: SC52-20250501).

[0108] Arginine, purity ≥95%, purchased from Zhangjiagang Huachang Pharmaceutical Co., Ltd. (batch number: SC14-20250401).

[0109] Histidine, purity ≥95%, purchased from Zhangjiagang Huachang Pharmaceutical Co., Ltd. (batch number: SC38-20250601).

[0110] Thickener: Carrageenan extract, purchased from Spico Inc., USA (batch number: SK30056964).

[0111] Preservative: Hexanediol, commercially available; (batch number: RP-0194472000-2702).

[0112] Other raw materials are commercially available.

[0113] Example 1

[0114] Preparation of hyaluronic acid nanospheres:

[0115] Based on a total of 100wt% of raw materials excluding pH adjuster, the formula is as follows: 1.0wt% of nucleic acid (sodium DNA), 0.5wt% of sodium hyaluronate (molecular weight 150w Da), 0.6wt% of lysine, 0.5wt% of arginine, 0.4wt% of histidine, 0.3wt% of thickener (Chlorella vulgaris extract), 2wt% of preservative (hexanediol), and the balance being water.

[0116] The preparation process is as follows:

[0117] Nucleic acid was dissolved in 90 wt% water (final product weight), heated to 50°C, and after complete dissolution without any aggregated particles, sodium hyaluronate was added and stirred (stirred at 6000 rpm for 30 min). After complete dissolution without any aggregated particles, the pH was adjusted to 5.0 with anhydrous citric acid solution, and the mixture was homogenized under high pressure at 500 bar for 5 cycles. The mixture was then cooled to 40°C at a cooling rate of 1°C / 5 min to obtain phase A.

[0118] Lysine, arginine, and histidine were dissolved in the remaining water (50°C) in a ratio of 6:5:4. After the solution was completely dissolved, the pH was adjusted to 5.0 with anhydrous citric acid solution to obtain phase B.

[0119] Phase B was added to Phase A, and the high-pressure homogenization process was performed at a pressure of 500 bar twice. Thickener and preservative were added, and the mixture was heated to 80°C and stirred for 60 min. The mixture was then cooled to 35°C to obtain a hyaluronic acid nanosphere composition.

[0120] After being placed at 25℃ for 7 days, the product becomes transparent and clear with good fluidity. Figure 1 As shown in 'a'.

[0121] Example 2

[0122] Replace the sodium hyaluronate with sodium hyaluronate with a molecular weight of 1000 Da, and the rest is the same as in Example 1.

[0123] Example 3

[0124] The amount of nucleic acid (sodium DNA) was adjusted to 2 wt%, and the rest remained the same as in Example 1. The specific formulation is as follows:

[0125] Nucleic acid (sodium DNA) 2.0wt%, sodium hyaluronate (molecular weight 150w Da) 0.5wt%, lysine 0.6wt%, arginine 0.5wt%, histidine 0.4wt%, thickener (Chlorella vulgaris extract) 0.3wt%, preservative (hexanediol) 2wt%, and water balance.

[0126] Example 4

[0127] Based on Example 1, after adding the sodium hyaluronate to the nucleic acid solution, the high-pressure homogenization process was performed at a pressure of 1000 bar for one cycle, with the rest being the same as in Example 1.

[0128] Example 5

[0129] Based on Example 1, phase B was added to phase A, and the high-pressure homogenization treatment was performed at a pressure of 1000 bar for one cycle, with the rest being the same as in Example 1.

[0130] Example 6

[0131] Based on Example 1, the pH was adjusted to 6.0 with anhydrous citric acid solution when preparing phase A and phase B, and the rest was the same as in Example 1.

[0132] Example 7

[0133] Based on Example 1, the pH was adjusted to 7.0 with anhydrous citric acid solution when preparing phase A and phase B, and the rest was the same as in Example 1.

[0134] Example 8

[0135] The arginine content remains unchanged, but the arginine:lysine ratio is adjusted to 10:1.

[0136] The formula proportions are as follows: 1.0wt% nucleic acid (sodium DNA), 0.5wt% sodium hyaluronate (molecular weight 150w Da), 0.05wt% lysine, 0.5wt% arginine, 0.4wt% histidine, 0.3wt% thickener (Chlorella vulgaris extract), 2wt% preservative (hexanediol), and the balance being water.

[0137] Example 9

[0138] When preparing phase A, the cooling rate after high-pressure homogenization was 1℃ / 1min to 40℃, except that the rest was the same as in Example 1.

[0139] Comparative Example 1

[0140] The formula is as follows: 10wt% nucleic acid (sodium DNA), 0.5wt% sodium hyaluronate (molecular weight 150w Da), 0.6wt% lysine, 0.5wt% arginine, 0.4wt% histidine, 0.3wt% thickener (Chlorella vulgaris extract), 2wt% preservative (hexanediol), and the balance being water;

[0141] The preparation process is the same as in Example 1.

[0142] Comparative Example 2

[0143] The formula is as follows: 1 wt% of nucleic acid (sodium DNA), 0.5 wt% of sodium hyaluronate (molecular weight 150w Da), 0.001 wt% of lysine, 0.001 wt% of arginine, 0.001 wt% of histidine, 0.3 wt% of thickener (Chlorella vulgaris extract), 2 wt% of preservative (hexanediol), and the balance of water;

[0144] The preparation process is the same as in Example 1.

[0145] Comparative Example 3

[0146] Except for phases A and B, which do not undergo pH adjustment, the rest are the same as in Example 1.

[0147] Comparative Example 4

[0148] Except for the absence of homogenization treatment during the preparation of phase A, the rest is the same as in Example 1.

[0149] Comparative Example 5

[0150] Except that no homogenization process is performed after phase B is added to phase A, the rest is the same as in Example 1.

[0151] Comparative Example 6

[0152] When preparing phase A, except for homogenization and cooling to 50°C, the rest is the same as in Example 1.

[0153] Comparative Example 7

[0154] Add 1.0 wt% of nucleic acid (sodium DNA), 0.5 wt% of sodium hyaluronate (molecular weight 150 w Da), 0.6 wt% of lysine, 0.5 wt% of arginine, 0.4 wt% of histidine, 0.3 wt% of thickener (Chlorella vulgaris extract), and 2 wt% of preservative (hexanediol) to water. Dissolve and stir at 80°C (6000 rpm) for 100 min, then cool to 35°C to obtain the prepared sample. After standing for 1 h, a gel forms, as shown... Figure 1 As shown in b in the figure.

[0155] Test Example 1

[0156] The particle size, PDI, and Zeta potential of the nucleic acid microspheres in the examples and comparative examples were determined using a ZETASIZER PRO particle size analyzer. Particle size and PDI were tested according to the methods specified in GB / T 19077-2016 "Laser Diffraction Method for Particle Size Distribution". Zeta potential was measured according to GB / Z 42353-2023 "Guideline for Zeta Potential Determination". The results are shown in Table 1.

[0157] Table 1. Summary of the performance of nucleic acid microspheres in the examples and comparative examples.

[0158]

[0159] As shown in Table 1, Examples 1-8 prepared hyaluronic acid nanospheres with small particle size, uniform particle size distribution, and high stability. In contrast, the nanospheres prepared in other comparative examples outside the defined range had significantly larger particle sizes and poorer uniformity. The particle sizes of Examples 1-8 were between 231-459 nm, PDI between 0.19-0.41, and Zeta potentials between -28 and -35 mV, indicating the formation of a uniform and stable nanosphere structure. In contrast, the particle sizes of Comparative Examples 1-7 were between 823-1320 nm, PDI between 0.70 and 1, and Zeta potentials between -0.3 and -8.2 mV, indicating a failure to form a satisfactory nanosphere structure.

[0160] Furthermore, in Example 9, when preparing phase A, the cooling rate after high-pressure homogenization was 1℃ / 1min to the target temperature. Although it could also prepare nanospheres, the rapid cooling process was not conducive to the formation of supramolecular polymers between nucleic acids and hyaluronic acid, resulting in imperfect polymer interactions and a loose structure. The final nanospheres obtained would have a particle size much larger than 389nm in Example 1, and the product might become cloudy, precipitate, or separate during storage, with a low absolute value of the Zeta potential.

[0161] In Comparative Example 1, an excessively high nucleic acid content (10%) disrupts the balance of charge and spatial structure, leading to system overload and preventing the formation of stable nanospheres, making aggregation or gelation highly likely. In Comparative Example 2, the total amount of cationic amino acids is too low to neutralize and bridge the negatively charged HA and DNA, resulting in incomplete complexation and system instability. If the pH is not adjusted (Comparative Example 3), it will alter the charge state of each component, potentially weakening electrostatic interactions and causing microspheres to fail to form or exhibit poor stability. High-pressure homogenization (Comparative Examples 4 and 5): This step is crucial for obtaining uniformly sized and stable nanospheres. Phase A homogenization ensures thorough mixing of the HA and DNA solutions and pre-shrinks aggregates. For example, Comparative Example 4, lacking the homogenization step, cannot achieve thorough mixing and pre-shrinking of aggregates. Homogenization after mixing is essential to ensure that phases A and B are thoroughly and uniformly mixed to form homogeneous nanospheres. For example, in Comparative Example 5, the absence of this step prevents the formation of uniform nanospheres. It is evident that omitting any step leads to coarse particles or uneven distribution. Furthermore, in Comparative Example 6, the cooling temperature after homogenization is too high. At higher temperatures (e.g., above 50°C), the polymer chains (sodium hyaluronate and DNA) exhibit excessive mobility, becoming overly extended and active. This hinders their ability to form stable and compact pre-assembled structures through intermolecular forces (such as hydrogen bonds and electrostatic interactions).

[0162] Test Example 2: SEM Electron Microscopy Observation

[0163] The samples prepared in Example 1 and Comparative Example 7 were freeze-dried and then subjected to SEM detection.

[0164] Test samples were sputter-coated with gold for approximately 120 seconds and then tested using a field emission scanning electron microscope (Thermo Fisher Quattro S). The accelerating voltage was 10 kV, the magnification range was 3-30 kV, and the test mode was secondary electron mode. The results are as follows: Figure 2 As shown in the SEM images, the spherical compressed microsphere structure can be observed in Example 1, while in Comparative Example 7, the microspheres are not compressed and exhibit a network structure.

[0165] Test Example 3: TEM Electron Microscopy Observation of the Morphology of Hyaluronic Acid Nanospheres

[0166] The hyaluronic acid nanospheres prepared in Example 1 and Comparative Example 3 were subjected to TEM detection.

[0167] Weigh a sample and drop it onto a copper mesh. After a few seconds, gently remove the sample with tweezers. Use filter paper to absorb excess liquid along one side. After it dries slightly, place the copper mesh on a drop of 2% phosphotungstic acid staining solution for 60 seconds. Remove it with tweezers and again absorb excess liquid along one side with filter paper. Place the mesh face up on filter paper to dry. Observe and photograph using a transmission electron microscope. Results are as follows: Figure 3 In Example 1, a clearly identifiable microsphere structure is present, while in Comparative Example 7, no obvious spherical structure is observed; instead, a loose network structure is formed.

[0168] SEM and TEM observations showed that spherical compressed microspheres were observed in Example 1, while Comparative Example 7 was not compressed and exhibited a network structure. Clearly identifiable microsphere structures were present in Example 1, while no obvious spherical structure was observed in Comparative Example 3; instead, a loose network structure was observed. This indicates that the preparation method of this application can successfully prepare nanosphere structures.

[0169] Test Example 4: Confocal Raman Skin Permeability Analysis

[0170] The permeability of hyaluronic acid in human skin was studied using confocal Raman spectroscopy in Examples 1 and 7 of this application. Raman signals from the test areas were acquired using a confocal Raman spectrometer before sample use and at 2h, 4h, 6h, 8h, and 24h after sample use. The Raman spectra of the skin before and after sample use were analyzed, and the relative transdermal absorption of hyaluronic acid in the sample was semi-quantitatively determined using a mathematical model.

[0171] Witec data analysis software was used to obtain relatively clear spectra through methods such as spectral range selection, cosmic ray removal, polynomial fitting to remove baseline, and Savitzky-Golay spectrum smoothing. The Raman spectral dataset was analyzed using univariate analysis to extract the compositional information of the experimental samples. Witec software performed univariate imaging, i.e., Raman spectroscopy imaging. A pseudo-color image was reconstructed using the combined intensity of characteristic peaks of a substance in the sample's scanned spectrum. The intensity of the characteristic peaks is related to the brightness of the Raman image color. Brightness is directly related to the Raman signal intensity. Higher signal intensity corresponds to brighter colors; lower signal intensity corresponds to darker colors. Red typically indicates higher signal intensity, and blue typically indicates lower signal intensity.

[0172] Transdermal tests were conducted on Examples 1 and 7 of this application. The test results can be found in [reference needed]. Figure 4 , Figure 5 .

[0173] from Figure 4The reconstructed Raman pseudocolor images after a large-scale scan show that the penetration of sodium hyaluronate in Example 1 is far superior to that in Comparative Example 7. From... Figure 5 The total integral intensity of sodium hyaluronate shows that the hyaluronic acid permeation in Example 1 is significantly higher than that in Comparative Example 7. These experimental results are consistent with the permeation process of hyaluronic acid. Clearly, the hyaluronic acid nanospheres of this application have the effect of improving the transdermal permeation of hyaluronic acid, thus improving the bioavailability of transdermal drug delivery products, promoting the absorption and utilization efficiency of pharmaceutical products, and enhancing the skincare effects of cosmetic products.

[0174] Test Example 5: Cell Migration Test

[0175] (1) Inoculation: First, draw two vertical lines on the back of the 6-well plate with a pen and a ruler, then inoculate the cells into the 6-well plate and incubate overnight at 37°C in a 5% CO2 incubator.

[0176] (2) Solution preparation: Prepare the test substance and positive control according to Table 2.

[0177] (3) Scratching: After the cells in the 6-well plate have grown for 18-24 hours, use a 1m pipette tip to make two scratches in each well, perpendicular to the horizontal line on the back, with the tip aligned with a ruler. Discard the supernatant, wash three times with PBS to remove the scratched cells.

[0178] (4) Drug administration and photography: The prepared samples (Example 1, Comparative Example 7) were added to the corresponding positions of the 6-well plate for drug administration, and photographed under a 5× microscope at 0h (immediately). The culture plate was placed at 37℃ and 5% CO2 and cultured for 18-24h.

[0179] (5) Photographing and image analysis: After 24 hours of cell culture, take a picture again under a 5× microscope. The position of the picture should be the same as that at 0 hours. Then perform migration area analysis.

[0180] (6) Cell migration rate calculation: The cell migration rate is calculated according to the cell migration rate formula, which is as follows:

[0181]

[0182] Where A0 refers to the blank area when the photo was taken at 0h, A 24 This refers to the blank area of ​​a photograph taken within 24 hours.

[0183] (7) Data processing: All data obtained in the experiment were processed and plotted using Excel software. Statistical analysis was performed using SPSS 17.0. One-way ANOVA was used for comparisons between groups. A p-value < 0.05 was considered statistically significant.

[0184] Test results are as follows Figure 6It can be seen that the test sample of Example 1 has a stronger effect on cell migration than that of Comparative Example 7, indicating that the sodium hyaluronate microspheres with the special structure have a significant effect on promoting cell migration.

[0185] Test Example 6: Verification Test of the Interaction Force between Sodium Hyaluronate and Nucleic Acids

[0186] By querying the DNA sequence information of the DNA-derived material, the characteristic repetitive sequences were used to generate the base sequence of PDRN (sodium DNA) from the web.x3dna.org website. A PDRN model was constructed, and a PDB format file was downloaded. A PDB format file was also created by querying the structural units of sodium hyaluronate. AutoDockTools-1.5.6 software was opened, and preprocessing was performed on both DNA and sodium hyaluronate. The DNA was treated as the acceptor, undergoing dehydration, hydrogenation, and charge addition, and set as the acceptor, saved as a PDBQT file. Sodium hyaluronate was set as the ligand and also saved as a PDBQT file. The preprocessing was then completed. Clicking "Docking" and then "Output" was performed, and 10 docking attempts were executed. The program was run, and the docking diagram and docking force results were awaited.

[0187] The simulated intermolecular interaction force is -14.6 kcal / mol. Generally, intermolecular interactions with forces below -3 kcal / mol indicate good interaction. Figure 7 It is known that there is a strong interaction between DNA and sodium hyaluronate molecules. After being homogenized under high pressure, they easily form a partially chimeric complex. It can be deduced that the formation of this structure is conducive to the formation of spherical sodium hyaluronate complexes, reducing the proportion of network structure and improving the spatial structure.

[0188] In summary, the hyaluronic acid nanosphere composition of this application, through the interaction of nucleic acids and amino acids in a specific ratio with sodium hyaluronate and optimized preparation process, successfully prepared nanospheres with good stability and uniform nanoscale particle size, exhibiting better permeability and meeting the application requirements in transdermal drug delivery and skin care products. Furthermore, this application employs a non-covalent bonding method to prepare the nanospheres, simplifying the preparation process and effectively improving the gelling properties of sodium hyaluronate, thus reducing the stickiness at high concentrations. In addition, the hyaluronic acid nanosphere composition of this application promotes cell migration, allowing it to penetrate deep into the skin to exert skin repair effects.

[0189] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing hyaluronic acid nanospheres, characterized in that, include: The DNA derivative is first mixed with a solvent and then heated to prepare a mixture; The hyaluronic acid salt is mixed with the mixture for a second time, the pH is adjusted to 5-7, homogenized, and then cooled to 20℃-40℃ to prepare phase A; Lysine, arginine, and histidine were dissolved in water, and the pH was adjusted to 5-7 to prepare phase B. as well as, Phase A and Phase B are mixed for the third time, homogenized, and then an additive is added. The mixture is heated to 75℃-90℃ and stirred. Then the temperature is lowered to 20℃-40℃ to prepare the hyaluronic acid nanospheres. The hyaluronic acid nanospheres comprise the following components by mass percentage: 1%-2% DNA derivative, 0.5%-2% hyaluronic acid salt, 0.5%-1.2% arginine, 0.05%-0.6% lysine, 0.4%-1.0% histidine, 0.1%-5% of the auxiliary agent, and the balance being water. The cooling rate after homogenization of the second mixture is ≤1℃ / 5min; The DNA derivative includes sodium DNA; the mass ratio of the DNA derivative to the total amount of arginine, lysine, and histidine is 1:(0.5-2.0). The hyaluronic acid salt includes sodium hyaluronate; The additives include one or both of thickeners and preservatives; The solvent includes water.

2. The method for preparing hyaluronic acid nanospheres according to claim 1, characterized in that, It meets one or more of the following conditions: (1) The molecular weight range of the sodium hyaluronate is 800 Da - 2,000,000 Da; (2) The mass ratio of arginine to lysine is 1:(0.8-30); (3) The pH-adjusting reagents include citric acid.

3. The method for preparing hyaluronic acid nanospheres according to claim 2, characterized in that, It meets one or more of the following conditions: (1) The thickener comprises one or two of hydroxyethyl cellulose, chondrus crispus extract, xanthan gum, sclerotium gum, and ammonium acryloyl dimethyl taurate and VP copolymer; (2) The preservatives include one or two of hexanediol, pentanediol, phenoxyethanol and ethylhexylglycerin; (3) The content of the thickener in the hyaluronic acid nanospheres is 0.1wt%-0.3wt%; (4) The content of the preservative in the hyaluronic acid nanospheres is 1wt%-5wt%.

4. The method for preparing hyaluronic acid nanospheres according to any one of claims 1 to 3, characterized in that, It meets one or more of the following conditions: (1) The temperature of the first mixture is 60℃-80℃; (2) The temperature of the second mixture is 50℃-70℃; (3) The temperature of the third mixture is 20℃-40℃; (4) The third mixing includes: adding phase B to phase A and stirring at 2000rpm-10000rpm for 5min-30min; (5) The homogenization process includes high-pressure homogenization or high-pressure microjet processing; (6) The stirring time is 30 min-120 min.

5. The method for preparing hyaluronic acid nanospheres according to claim 4, characterized in that, The conditions for the high-pressure homogenization process include: homogenization temperature 40℃-60℃; homogenization pressure 100 bar-2000 bar; homogenization cycles of 2-20; and / or The conditions for the high-pressure microjets treatment include: pressure of 1400Psi-15000Psi, and treatment times of 1-5 times.

6. Hyaluronic acid nanospheres prepared by the method according to any one of claims 1 to 5.

7. The use of the hyaluronic acid nanospheres according to claim 6 in the preparation of transdermal drug delivery products.

8. The application according to claim 7, characterized in that, The products include cosmetics.

9. The application according to claim 8, characterized in that, The cosmetics include one or more of the following: facial mask liquid, skin care water, serum, spray and lotion.

10. A cosmetic product, characterized in that, The raw materials include hyaluronic acid nanospheres prepared by the preparation method described in any one of claims 1 to 5.

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