DNA-sodium nano-liposome as well as preparation method and application thereof

Abstract

The invention relates to a DNA-sodium nano-liposome as well as a preparation method and application thereof. The DNA-sodium nano-liposome has a W / O / W multiple emulsion structure and is mainly prepared from the following components in percentage by mass: (1) an inner water phase: 0.05-0.5% of DNA-sodium and 5-15% of water; (2) an oil phase: 3%-7% of phospholipid and 0.05%-0.2% of a first emulsifier; (3) an external water phase: 0.5%-1.8% of a second emulsifier, 5%-20% of glycerol and 55.5%-86.4% of water; the first emulsifier comprises cholesterol; and the second emulsifier comprises beheneth-25. The DNA-sodium nano-liposome has good solubility in water and good stability, has good anti-oxidation, anti-aging and anti-inflammatory effects, shows excellent stability in severe environments such as normal temperature, refrigeration, freezing, heating, freezing and thawing, illumination and light shielding, has a good slow release effect, and is suitable for the field of cosmetics.

Description

Technical Field

[0001] The present invention belongs to the technical field of liposomes, and particularly relates to a DNA-sodium nano-liposome, a preparation method thereof, and an application thereof. Background Art

[0002] DNA-sodium is a derivative of deoxyribonucleic acid, with a molecular weight between 50 - 1500 KDa and a chain length between 50 - 2000 bases. DNA-sodium binds to adenosine A 2a receptors, induces the synthesis of vascular endothelial growth factor, and promotes wound healing; it exerts an anti-inflammatory effect by downregulating inflammatory cytokines and inhibiting apoptosis. Moreover, DNA-sodium can be cleaved into nucleotides by enzymes in cells, stimulate the synthesis of nucleic acid DNA through the salvage pathway, reactivate the proliferation of normal cells, and enable rapid tissue recovery. In addition, DNA-sodium can also promote tissue repair, antioxidation, and anti-aging effects. Coupled with excellent safety and biocompatibility, it has become an ideal active ingredient for developing new skin care products.

[0003] Although DNA-sodium has many effects such as anti-inflammatory, antioxidative, anti-aging, and promoting wound healing, its application in skin care products faces some problems, specifically as follows: (1) In the physiological environment, it is generally used by injection or application. Because the half-life of DNA-sodium is only 3 hours, it is easily oxidized and decomposed when exposed to the environment, resulting in reduced activity and efficacy. (2) As a macromolecular nucleic acid derivative, DNA-sodium generally has a molecular weight between 50 - 1500 KDa and has poor permeability in the skin, which limits its effective penetration and accumulation in deep skin tissues. Therefore, the application of DNA-sodium in skin care products is restricted to a certain extent, hindering its application in skin repair and anti-aging. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to provide a DNA-sodium nano-liposome with good solubility, good transdermal permeability, good sustained-release effect, and good stability, which can be applied to cosmetics.

[0005] The technical solution is as follows:

[0006] A DNA-sodium nano-liposome having a W / O / W multiple emulsion structure is mainly made of the following components by mass percentage:

[0007] (1) Inner aqueous phase:

[0008] DNA-sodium 0.05% - 0.5%,

[0009] Water 5% - 15%;

[0010] (2) Oil phase:

[0011] Phospholipids: 3% - 7%

[0012] First emulsifier: 0.05% - 0.2%

[0013] (3) Outer aqueous phase:

[0014] Second emulsifier: 0.5% - 1.8%

[0015] Glycerol: 5% - 20%

[0016] Water: 55.5% - 86.4%

[0017] The first emulsifier includes cholesterol;

[0018] The second emulsifier includes behenyl alcohol polyether - 25.

[0019] In one embodiment, the phospholipids are selected from one or a combination of two of soy lecithin S45 and soy lecithin S75.

[0020] In one embodiment, the oil phase further includes an antioxidant.

[0021] In one embodiment, the antioxidant is pentaerythrityl tetra(bis - tert - butylhydroxyhydrocinnamate).

[0022] In one embodiment, the outer aqueous phase further includes a first preservative.

[0023] In one embodiment, the first preservative is pentylene glycol.

[0024] In one embodiment, the DNA - sodium nanoliposome further includes a second preservative.

[0025] In one embodiment, the second preservative is selected from one or a mixture of two of p - hydroxyacetophenone and pentylene glycol.

[0026] In one embodiment, the DNA - sodium nanoliposome is mainly made of the following components by mass percentage:

[0027] (1) Inner aqueous phase:

[0028] DNA - sodium: 0.05% - 0.5%

[0029] Water: 5% - 15%

[0030] (2) Oil phase:

[0031] Phospholipids: 3% - 7%

[0032] First emulsifier: 0.05% - 0.2%

[0033] Antioxidant: 0.05% - 0.1%;

[0034] (3) External aqueous phase:

[0035]

[0036] (4) Second preservative: 0.05% - 1.5%.

[0037] In one embodiment, the particle size of the DNA-sodium nanoliposomes is 90 nm - 150 nm, the PDI is 0.01 - 0.3, and the encapsulation efficiency is ≥60%.

[0038] The present invention also provides a preparation method of the DNA-sodium nanoliposomes as described above, comprising the following steps:

[0039] Mix the phospholipid and the first emulsifier in an alcohol solvent to prepare an oil-phase mixture;

[0040] Mix the DNA-sodium with water to prepare a first aqueous-phase mixture;

[0041] Mix the second emulsifier and glycerol in water to prepare a second aqueous-phase mixture;

[0042] Mix the oil-phase mixture with the first aqueous-phase mixture to prepare a primary emulsion, and the primary emulsion has a W / O structure;

[0043] Mix the primary emulsion with the second aqueous-phase mixture to prepare a first multiple emulsion, and the first multiple emulsion has a W / O / W structure;

[0044] Remove the alcohol solvent from the first multiple emulsion, and add water to make up the mass of the alcohol solvent to prepare a second multiple emulsion, and the second multiple emulsion has a W / O / W structure.

[0045] In one embodiment, the process parameters for preparing the first aqueous-phase mixture include: the pH of the system is 5.0 - 6.0, and the temperature is 65°C - 75°C.

[0046] In one embodiment, the process parameters for preparing the primary emulsion include: the emulsification speed is 5000 rpm - 9000 rpm, and the time is 5 min - 20 min.

[0047] In one embodiment, the process parameters for preparing the first multiple emulsion include: the emulsification speed is 3000 rpm - 5000 rpm, and the time is 5 min - 20 min.

[0048] In one embodiment, the alcohol solvent is ethanol.

[0049] In one embodiment, the process parameters for preparing the second multiple emulsion include: vacuum concentration, with a temperature of 40°C to 60°C and a time of 10 min to 30 min.

[0050] The present invention also provides the application of the DNA-sodium nano-liposome as described above. The technical solution is as follows:

[0051] A cosmetic, characterized in that it comprises the DNA-sodium nano-liposome as described above.

[0052] In one embodiment, the cosmetic is selected from at least one of a facial mask, eye cream, facial cream, primer, essence, lotion, and skin softener.

[0053] The present invention has at least the following beneficial effects:

[0054] The DNA-sodium nano-liposome provided by the present invention has a W / O / W multiple emulsion structure. Its inner aqueous phase comprises DNA-sodium and water in specific mass percentages, the oil phase comprises phospholipids and a first emulsifier in specific mass percentages, the outer aqueous phase comprises a second emulsifier, glycerol, and water in specific mass percentages, and the first emulsifier comprises cholesterol, and the second emulsifier comprises behenyl polyether-25.

[0055] With the specific W / O / W multiple emulsion structure of the present invention, while ensuring good solubility of DNA-sodium in water, the stability of the DNA-sodium nano-liposome is significantly improved. Moreover, with the combined action of the second emulsifier and other raw materials in the present invention, the encapsulation rate of DNA-sodium can be significantly increased, and the particle size and PDI of the DNA-sodium nano-liposome can be significantly reduced, which can prolong the release time of DNA-sodium to achieve the purpose of slow release. In addition, the DNA-sodium nano-liposome provided by the present invention contains no harmful substances and is gentle to the human body.

[0056] It has been confirmed that the DNA-sodium nano-liposome provided by the present invention has good solubility in water, is gentle to the human body, has good skin physiological effects of antioxidant, anti-aging, and anti-inflammatory, and exhibits excellent stability under 7 different harsh environments of normal temperature, refrigeration, freezing, heating, freeze-thaw, light, and dark, slowly releases DNA-sodium, and improves its dose-effect relationship and bioavailability. Due to the above-mentioned many advantages of the DNA-sodium nano-liposome, it can be well used in the field of cosmetics.

[0057] In addition, the preparation method of the DNA-sodium nano-liposome described in the present invention is simple, has high repeatability, short preparation time, no harmful substance residues, high encapsulation rate, and high mechanization degree in the whole process, making the product quality and process have good reproducibility and stability and being easy for industrial production. Description of the Drawings

[0058] Figure 1TEM results of DNA-sodium nanoliposomes prepared for Experimental Group No. 3 of Example 5 Figure 1 In (a), the magnification is 15,000x and the scale bar is 200 nm; in (b), the magnification is 50,000x and the scale bar is 100 nm.

[0059] Figure 2 Trend graph of the in vitro slow release amount of DNA-sodium nanoliposomes prepared for Experimental Group No. 3 of Example 5

[0060] Figure 3 Trend graph of the particle size and PDI changes of DNA-sodium nanoliposomes prepared for Experimental Group No. 1 of Example 4 under 7 different environments

[0061] Figure 4 Trend graph of the particle size and PDI changes of DNA-sodium nanoliposomes prepared for Experimental Group No. 1 of Example 5 under 7 different environments

[0062] Figure 5 Trend graph of the particle size and PDI changes of DNA-sodium nanoliposomes prepared for Experimental Group No. 2 of Example 5 under 7 different environments

[0063] Figure 6 Trend graph of the particle size and PDI changes of DNA-sodium nanoliposomes prepared for Experimental Group No. 3 of Example 5 under 7 different environments Detailed implementation manners

[0064] The present invention will be further described in detail below in conjunction with specific embodiments. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0066] The terms "preferably", "more preferably", "more preferably", "even more preferably", etc. in the present invention refer to embodiments of the present invention that can provide certain beneficial effects in certain cases. However, in the same or other cases, other embodiments may also be preferred. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not available, nor is it intended to exclude other embodiments from the scope of the present invention. That is, in the present invention, "preferably", "more preferably", "more preferably", "even more preferably", etc. are only used to describe embodiments or examples with better effects, but do not constitute a limitation on the protection scope of the present invention.

[0067] In the present invention, "further", "even further", "especially", etc. are used for descriptive purposes and indicate differences in content, but should not be construed as a limitation on the protection scope of the present invention.

[0068] In the present invention, "above" and "below" both include the number itself. For example, "below 1" means ≥1.

[0069] In the present invention, the meaning of "at least one" is more than one, such as one, two or more. The meaning of "multiple" or "several" is at least two, such as two, three, etc. The meaning of "multiple layers" is at least two layers, such as two layers, three layers, etc., unless otherwise specifically defined. In the description of the present invention, the meaning of "several" is at least one, such as one, two, etc., unless otherwise specifically defined.

[0070] When a numerical range is disclosed in the present invention, the above range is considered continuous and includes the minimum and maximum values of the range, as well as every value between such minimum and maximum values. Further, when the range refers to an integer, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0071] If there is no special instruction, all steps of the present invention can be carried out in sequence or randomly. For example, the method includes steps (a) and (b), which means that the method can include steps (a) and (b) carried out in sequence, or can also include steps (b) and (a) carried out in sequence. For example, when it is mentioned that the method may further include step (c), it means that step (c) can be added to the method in any order. For example, the method can include steps (a), (b) and (c) in sequence, or can also include steps (a), (c) and (b), or can also include steps (c), (a) and (b), etc.

[0072] In the present invention, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one such feature.

[0073] In the present invention, "comprising", "including", "containing", "having" or other variants are intended to cover non-closed inclusion, and no distinction is made among these terms. The term "including" means that other steps and components can be added without affecting the final result. The compositions and methods / processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional components, ingredients, steps, or limitations described herein.

[0074] In the present invention, no distinction is made among the terms "efficacy", "performance", "effect", and "function".

[0075] The weights of the relevant components mentioned in the specification of the embodiments of the present invention not only can refer to the specific contents of each component, but also can represent the weight ratio relationship among the components. Therefore, as long as the contents of the relevant components in the specification of the embodiments of the present invention are enlarged or reduced in proportion, they are within the scope disclosed in the specification of the embodiments of the present invention. Specifically, the weights described in the specification of the embodiments of the present invention can be mass units well-known in the chemical industry such as μg, mg, g, kg, etc.

[0076] In the present invention, for temperature parameters, unless otherwise specifically defined, both constant temperature treatment and treatment within a certain temperature range are allowed. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. The room temperature referred to in the present invention means 0 - 40 °C, preferably 10 °C - 35 °C, and more preferably 20 °C - 30 °C.

[0077] Unless otherwise mentioned, terms in the singular form may include the plural form and should not be construed as having a quantity of one.

[0078] In addition, the drawings of the present invention are not drawn to a scale of 1:1, and the relative sizes of the components are only drawn by way of example in the drawings for the convenience of understanding the present invention, but are not necessarily drawn to the actual scale. The scale in the drawings does not constitute a limitation to the present invention.

[0079] DNA-sodium is a derivative of deoxyribonucleic acid, with a molecular weight between 50 - 1500 KDa and a chain length between 50 - 2000 bases. It is gentle to the human body and has good skin physiological effects of antioxidant, anti-aging, and anti-inflammatory. However, DNA-sodium has a relatively high molecular weight and poor solubility in water, which not only makes it difficult to be absorbed by the skin but also is the main reason for poor membrane permeability and poor bioavailability.

[0080] In addition, DNA-sodium causes problems such as irreversible oxidation and degradation in aqueous solution with the increase of time and temperature, which limits its application to a certain extent. Therefore, improving the stability of DNA-sodium, slowing down the release rate of DNA-sodium and prolonging the release time are the prerequisites for its wide application.

[0081] To solve the above problems, the present invention provides a DNA-sodium nano-liposome with good solubility, good transdermal permeability, good stability, high encapsulation efficiency and slow release effect, which can be applied to cosmetics.

[0082] The technical solution is as follows:

[0083] A DNA-sodium nano-liposome with a W / O / W multiple emulsion structure is mainly composed of the following components by mass percentage:

[0084] (1) Inner aqueous phase:

[0085] DNA-sodium 0.05% - 0.5%,

[0086] Water 5% - 15%;

[0087] (2) Oil phase:

[0088] Phospholipid 3% - 7%,

[0089] The first emulsifier 0.05% - 0.2%;

[0090] (3) Outer aqueous phase:

[0091] The second emulsifier 0.5% - 1.8%,

[0092] Glycerol 5% - 20%,

[0093] Water 55.5% - 86.4%;

[0094] The first emulsifier includes cholesterol;

[0095] The second emulsifier includes behenyl alcohol polyether-25.

[0096] Through a specific W / O / W multiple emulsion structure, while ensuring good solubility of DNA-sodium in water, the stability of DNA-sodium nano-liposome is significantly improved. Moreover, under the combined action of the second emulsifier and other raw materials in the present invention, the encapsulation efficiency of DNA-sodium can be significantly improved, and the particle size and PDI of DNA-sodium nano-liposome can be significantly reduced, and the release time of DNA-sodium can be prolonged to achieve the purpose of slow release. In addition, the DNA-sodium nano-liposome provided by the present invention contains no harmful substances and is gentle to the human body.

[0097] It has been verified that the DNA-sodium nano-liposomes provided by the present invention have good solubility in water (good solubility in both cold and hot water), are gentle to the human body, have good antioxidant, anti-aging, and anti-inflammatory skin physiological effects, and exhibit excellent stability under 7 different harsh environments including normal temperature, refrigeration, freezing, heating, freeze-thawing, light exposure, and light avoidance, slowly release DNA-sodium, and improve its dose-effect relationship and bioavailability. Given the above-mentioned many advantages of the DNA-sodium nano-liposomes, they can be well used in the cosmetic field.

[0098] (1) In the present invention, the DNA-sodium nano-liposomes have a W / O / W multiple emulsion structure, wherein the inner aqueous phase comprises components in the following mass percentages: 0.05% to 0.5% of DNA-sodium and 5% to 15% of water. Setting DNA-sodium in the inner aqueous phase is beneficial to stably preserving DNA-sodium in the liposome vesicles and avoiding direct leakage due to vesicle rupture.

[0099] It can be understood that, calculated by mass percentage of the DNA-sodium nano-liposomes, the inner aqueous phase of the DNA-sodium nano-liposomes contains 0.05% to 0.5% of DNA-sodium, including but not limited to 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%. Preferably, calculated by mass percentage, the inner aqueous phase of the DNA-sodium nano-liposomes contains 0.3% of DNA-sodium.

[0100] It can be understood that, calculated by mass percentage of the DNA-sodium nano-liposomes, the inner aqueous phase of the DNA-sodium nano-liposomes contains 5% to 15% of water, including but not limited to 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. Preferably, calculated by mass percentage of the DNA-sodium nano-liposomes, the inner aqueous phase of the DNA-sodium nano-liposomes contains 15% of water.

[0101] (2) In the present invention, the DNA-sodium nano-liposomes have a W / O / W multiple emulsion structure, wherein the oil phase comprises components in the following mass percentages: 3% to 7% of phospholipids and 0.05% to 0.2% of the first emulsifier. Setting phospholipids and the first emulsifier in the oil phase completely dissolves the liposome preparation membrane material in advance so as to form a closed vesicle structure after encountering water to encapsulate DNA-sodium.

[0102] Understandably, based on the mass percentage of the DNA-sodium nanoliposomes, the oil phase of the DNA-sodium nanoliposomes contains 3% to 7% of phospholipids, including but not limited to 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5% or 7%. Preferably, based on the mass percentage, the oil phase of the DNA-sodium nanoliposomes contains 3% to 10% of phospholipids. Further preferably, based on the mass percentage of the DNA-sodium nanoliposomes, the oil phase of the DNA-sodium nanoliposomes contains 6% of phospholipids.

[0103] In one embodiment, the phospholipid is selected from one or a combination of two of soy lecithin S45 and soy lecithin S75. Preferably, the phospholipid is soy lecithin S75. The prepared DNA-sodium nanoliposomes are a translucent light yellow solution, and the PDI is significantly smaller than that of liposomes prepared with other phospholipids, and the particle size (90 nm to 130 nm) and PDI (0.01 to 0.3) are significantly more stable than those of liposomes prepared with other phospholipids. Further, when using other types of lecithin, the encapsulation efficiency of the DNA-sodium nanoliposomes is 60% to 70%, while the encapsulation efficiency of the DNA-sodium nanoliposomes using soy lecithin S75 as the phospholipid is above 72%, and in some embodiments, it is above 80%.

[0104] Understandably, based on the mass percentage of the DNA-sodium nanoliposomes, the oil phase of the DNA-sodium nanoliposomes contains 0.05% to 0.2% of the first emulsifier, including but not limited to 0.05%, 0.075%, 0.1%, 0.125%, 0.15%, 0.175% or 0.2%.

[0105] In the present invention, the first emulsifier includes cholesterol. In one embodiment, the first emulsifier is cholesterol.

[0106] In one embodiment, the oil phase of the DNA-sodium nanoliposomes further contains an antioxidant. Adding the antioxidant further enhances the antioxidant property and stability of the DNA-sodium nanoliposomes and improves the stability problem of DNA-sodium.

[0107] In one embodiment, based on the mass percentage of the DNA-sodium nanoliposomes, the oil phase of the DNA-sodium nanoliposomes contains 0.05% to 0.1% of the antioxidant, including but not limited to 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095% or 0.1%.

[0108] In one embodiment, the antioxidant is pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamate).

[0109] (3) In the present invention, the DNA-sodium nanoliposome has a W / O / W multiple emulsion structure, wherein the outer aqueous phase comprises components in the following mass percentages: 0.5% to 1.8% of a second emulsifier, 5% to 20% of glycerol, and 55.5% to 86.4% of water. The second emulsifier includes behenyl polyether-25 (BA-25). Setting the second emulsifier and glycerol in the outer aqueous phase can enable the second emulsifier to adhere well to the vesicle surface, enhance the interaction force between particles in the solution, and enhance the stability of the solution. At the same time, glycerol can improve the freeze-thaw resistance property, enabling the vesicles and the solution to be placed in an environment of -15°C, which can play a protective role for the vesicles and the solution. The present invention selects behenyl polyether-25 as the emulsifier, which exhibits excellent stability under 7 different harsh environments including normal temperature, refrigeration, freezing, heating, freeze-thaw, light, and dark.

[0110] Understandably, calculated by mass percentage of the DNA-sodium nanoliposome, the outer aqueous phase of the DNA-sodium nanoliposome contains 0.5% to 1.8% of the second emulsifier, including but not limited to 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, or 1.8%.

[0111] Understandably, calculated by mass percentage of the DNA-sodium nanoliposome, the outer aqueous phase of the DNA-sodium nanoliposome contains 5% to 20% of glycerol, including but not limited to 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. Preferably, calculated by mass percentage of the DNA-sodium nanoliposome, the outer aqueous phase of the DNA-sodium nanoliposome contains 6% to 12% of glycerol.

[0112] Understandably, calculated by mass percentage of the DNA-sodium nanoliposome, the outer aqueous phase of the DNA-sodium nanoliposome contains 55.5% to 86.4% of water, including but not limited to 55.5%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85.8%, or 86.4%. Preferably, calculated by mass percentage of the DNA-sodium nanoliposome, the outer aqueous phase of the DNA-sodium nanoliposome contains 55.5% to 85.8% of water.

[0113] In one embodiment, the outer aqueous phase of the DNA-sodium nanoliposome further contains a first preservative. Further, the mass content of the first preservative in the DNA-sodium nanoliposome is 0.5% to 1.5%. Further, the first preservative in the outer aqueous phase is pentylene glycol.

[0114] In one embodiment, based on the mass percentage of the DNA-sodium nanoliposome, the outer aqueous phase of the DNA-sodium nanoliposome contains 0.5% to 1.5% of pentylene glycol, including but not limited to 0.5%, 0.7%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4% or 1.5%. Preferably, based on the mass percentage of the DNA-sodium nanoliposome, the outer aqueous phase of the DNA-sodium nanoliposome contains 1% of pentylene glycol.

[0115] In one embodiment, the DNA-sodium nanoliposome further includes a second preservative.

[0116] Further, based on the mass percentage of the DNA-sodium nanoliposome, the DNA-sodium nanoliposome contains 0.05% to 1.05% of the second preservative, including but not limited to 0.05%, 0.06%, 0.07%, 0.075%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or 1.05%.

[0117] In one embodiment, the second preservative is selected from one or a mixture of two of hydroxyacetophenone and pentylene glycol. Further, the second preservative is a mixture of hydroxyacetophenone and pentylene glycol.

[0118] In one embodiment, based on the mass percentage of the DNA-sodium nanoliposome, the DNA-sodium nanoliposome contains 0.01% to 0.1% of hydroxyacetophenone, including but not limited to 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.075%, 0.08%, 0.09% or 0.1%. Preferably, based on the mass percentage, the DNA-sodium nanoliposome contains 0.05% of hydroxyacetophenone.

[0119] In one embodiment, based on the mass percentage of the DNA-sodium nanoliposomes, the second preservative in the DNA-sodium nanoliposomes contains 0.01% to 1% of pentylene glycol, including but not limited to 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.075%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%. Preferably, based on the mass percentage, the second preservative in the DNA-sodium nanoliposomes contains 1% of pentylene glycol.

[0120] In one embodiment, the DNA-sodium nanoliposomes comprise the following components in mass percentages:

[0121] (1) Inner aqueous phase:

[0122] DNA-sodium 0.05% to 0.5%,

[0123] Water 5% to 15%;

[0124] (2) Oil phase:

[0125] Phospholipid 3% to 7%,

[0126] First emulsifier 0.05% to 0.2%,

[0127] Antioxidant 0.05% to 0.1%;

[0128] (3) Outer aqueous phase:

[0129]

[0130] (4) Second preservative 0.05% to 1.5%;

[0131] The first emulsifier includes cholesterol;

[0132] The second emulsifier includes behenyl alcohol polyether-25.

[0133] In one embodiment, the DNA-sodium nanoliposomes comprise the following components in mass percentages: (1) Inner aqueous phase:

[0134] DNA-sodium 0.05%,

[0135] Water 5% to 15%;

[0136] (2) Oil phase:

[0137] Phospholipid 3% to 7%,

[0138] First emulsifier 0.05% to 0.2%,

[0139] Antioxidant: 0.05% - 0.1%;

[0140] (3) Outer aqueous phase:

[0141]

[0142] (4) Second preservative: 0.05% - 1.5%;

[0143] The first emulsifier is cholesterol;

[0144] The second emulsifier includes behenyl alcohol polyether - 25.

[0145] In one embodiment, the DNA - sodium nanoliposome comprises components in the following mass percentages: (1) Inner aqueous phase:

[0146] DNA - sodium 0.1%,

[0147] Water 5% - 15%;

[0148] (2) Oil phase:

[0149] Phospholipid 3% - 7%,

[0150] First emulsifier 0.05% - 0.2%,

[0151] Antioxidant 0.05% - 0.1%;

[0152] (3) Outer aqueous phase:

[0153]

[0154] (4) Second preservative 0.05% - 1.5%;

[0155] The first emulsifier is cholesterol;

[0156] The second emulsifier includes behenyl alcohol polyether - 25.

[0157] In one embodiment, the DNA - sodium nanoliposome comprises components in the following mass percentages: (1) Inner aqueous phase:

[0158] DNA - sodium 0.2%,

[0159] Water 5% - 15%;

[0160] (2) Oil phase:

[0161] Phospholipid 3% - 7%,

[0162] First emulsifier 0.05% - 0.2%,

[0163] Antioxidant: 0.05% - 0.1%;

[0164] (3) Outer aqueous phase:

[0165]

[0166] (4) Second preservative: 0.05% - 1.5%;

[0167] The first emulsifier is cholesterol;

[0168] The second emulsifier includes behenyl alcohol polyether - 25.

[0169] In one embodiment, the DNA - sodium nanoliposome comprises the following components by mass percentage: (1) Inner aqueous phase:

[0170] DNA - sodium: 0.3%,

[0171] Water: 5% - 15%;

[0172] (2) Oil phase:

[0173] Phospholipid: 3% - 7%,

[0174] First emulsifier: 0.05% - 0.2%,

[0175] Antioxidant: 0.05% - 0.1%;

[0176] (3) Outer aqueous phase:

[0177]

[0178] (4) Second preservative: 0.05% - 1.5%;

[0179] The first emulsifier is cholesterol;

[0180] The second emulsifier includes behenyl alcohol polyether - 25.

[0181] In one embodiment, the particle size of the DNA - sodium nanoliposome is 90nm - 150nm, the PDI is 0.01 - 0.3, and the encapsulation efficiency is ≥70%. Further, the particle size of the DNA - sodium nanoliposome is 90nm - 130nm, the PDI is 0.01 - 0.3, and the encapsulation efficiency is ≥80%.

[0182] In one embodiment, the particle size of the DNA - sodium nanoliposome is 90nm - 135nm, the PDI is 0.01 - 0.3, and the encapsulation efficiency is ≥70%. Further, the particle size of the DNA - sodium nanoliposome is 90nm - 130nm, the PDI is 0.01 - 0.3, and the encapsulation efficiency is ≥80%.

[0183] In one embodiment, the DNA-sodium nanoliposomes have a particle size of 90 nm to 125 nm, a PDI of 0.01 to 0.3, and an encapsulation efficiency of ≥70%. Further, the DNA-sodium nanoliposomes have a particle size of 90 nm to 125 nm, a PDI of 0.01 to 0.3, and an encapsulation efficiency of ≥80%.

[0184] The present invention provides a method for preparing DNA-sodium nanoliposomes, which has high repeatability, short preparation time, no residue of harmful substances, and high encapsulation efficiency. By encapsulating DNA-sodium with this method, the release rate of DNA-sodium can be prolonged, its solubility, encapsulation efficiency, and stability under 7 different harsh environments of normal temperature, refrigeration, freezing, heating, freeze-thaw, light, and dark can be improved.

[0185] The technical solution is as follows:

[0186] A method for preparing DNA-sodium nanoliposomes as described above, comprising the following steps:

[0187] Mix the phospholipid and the first emulsifier in an alcohol solvent to prepare an oil-phase mixture;

[0188] Mix the DNA-sodium with water to prepare a first aqueous-phase mixture;

[0189] Mix the second emulsifier and glycerol in water to prepare a second aqueous-phase mixture;

[0190] Mix the oil-phase mixture and the first aqueous-phase mixture to prepare a primary emulsion, and the primary emulsion has a W / O structure;

[0191] Mix the primary emulsion and the second aqueous-phase mixture to prepare a first multiple emulsion, and the first multiple emulsion has a W / O / W structure;

[0192] Remove the alcohol solvent in the first multiple emulsion, and add water to make up the mass of the alcohol solvent to prepare a second multiple emulsion, and the second multiple emulsion has a W / O / W structure.

[0193] In one embodiment, the alcohol solvent is ethanol. The present invention uses the multiple emulsion method to prepare DNA-sodium nanoliposomes. The preparation method is simple, rapid, and the process is simple, which is suitable for large-scale industrial production.

[0194] Further, the mass percentage of ethanol in the DNA-sodium nanoliposomes is 20% to 30%, including but not limited to 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%. Preferably, the mass percentage of ethanol in the DNA-sodium nanoliposomes is 24%.

[0195] In one embodiment, the process parameters for preparing the first aqueous phase mixture include: the pH of the system is 5.0 - 6.0, and the temperature is 65°C - 75°C.

[0196] In one embodiment, the DNA-sodium is mixed with water to prepare the first aqueous phase mixture; the steps include:

[0197] Using a 0.1 mol / L citric acid solution in water to adjust the pH to 5.0 - 6.0, heating to 65°C - 75°C and dissolving the DNA-sodium for 10 min to obtain the first aqueous phase mixture.

[0198] In one embodiment, the process parameters for preparing the primary emulsion include: the emulsification rotation speed is 5000 rpm - 9000 rpm, and the time is 5 min - 20 min. It can be understood that in the step of preparing the primary emulsion, the emulsification rotation speed includes but is not limited to 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm or 9000 rpm; the time includes but is not limited to 5 min, 6 min, 8 min, 10 min, 12 min or 15 min. Preferably, in the step of preparing the primary emulsion, the emulsification rotation speed is 7000 rpm and the emulsification time is 10 min.

[0199] In one embodiment, mixing the oil phase mixture with the first aqueous phase mixture to prepare a primary emulsion with a W / O structure includes the following steps:

[0200] Adding the first aqueous phase mixture dropwise to the oil phase mixture and emulsifying for 5 min - 20 min at a rotation speed of 5000 rpm - 9000 rpm to form a W / O system and obtain the primary emulsion.

[0201] In one embodiment, the process parameters for preparing the first multiple emulsion include: the emulsification rotation speed is 3000 rpm - 5000 rpm, and the time is 5 min - 20 min. It can be understood that in the step of preparing the first multiple emulsion, the emulsification rotation speed includes but is not limited to 3000 rpm, 4000 rpm or 5000 rpm; the time includes but is not limited to 5 min, 10 min, 15 min or 20 min. Preferably, in the step of preparing the first multiple emulsion, the emulsification rotation speed is 5000 rpm and the emulsification time is 10 min.

[0202] In one embodiment, mixing the primary emulsion with the second aqueous phase mixture to prepare a first multiple emulsion with a W / O / W structure includes the following steps:

[0203] The colostrum is dropped into the second aqueous phase mixture and emulsified for 5 min to 20 min under the condition that the rotation speed is 3000 rpm to 5000 rpm to form a W / O / W system, and the first multiple emulsion is obtained.

[0204] In one of the embodiments, the process parameters for preparing the second multiple emulsion include: vacuum concentration, the temperature is 40°C to 60°C, and the time is 15 min to 30 min. It can be understood that in the step of preparing the second multiple emulsion, the temperature includes but is not limited to 40°C, 45°C, 50°C, 55°C or 60°C; the time includes but is not limited to 15 min, 16 min, 18 min, 20 min, 22 min, 25 min or 30 min.

[0205] In one of the embodiments, removing the alcohol solvent in the first multiple emulsion and adding water to make up the mass of the alcohol solvent, and preparing the second multiple emulsion with a W / O / W structure includes the following steps:

[0206] The first multiple emulsion is rotary evaporated at 40°C to 60°C for 15 min to 30 min by a rotary evaporator to remove absolute ethanol, and pure water is used to make up the weight difference before and after rotary evaporation.

[0207] It can be understood that for the DNA-sodium nanoliposomes in which the outer aqueous phase contains the first preservative (such as pentylene glycol), in the step of preparing the second aqueous phase mixture, it also includes the step of adding the first preservative and mixing it with the emulsifier, glycerol and water.

[0208] It can be understood that for the DNA-sodium nanoliposomes that contain (1) inner aqueous phase, (2) oil phase, (3) outer aqueous phase, and also contain (4) second preservative, after the step of preparing the second multiple emulsion, it also includes the step of mixing the second multiple emulsion and the second preservative.

[0209] The present invention also provides the application of the DNA-sodium nanoliposomes as described above. The technical solution is as follows:

[0210] A cosmetic, characterized in that it includes the DNA-sodium nanoliposomes as described above.

[0211] In one of the embodiments, the cosmetic is selected from at least one of facial masks, eye creams, facial creams, primer creams, essences, lotions and skin softeners.

[0212] The following is the specific embodiment part.

[0213] In the following embodiments, the antioxidant is pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamate).

[0214] Unless otherwise specified, all raw materials are commercially available products.

[0215] (1) The room temperature in the following examples is 20°C to 30°C, and the sum of the raw material dosages in the examples is 100%.

[0216] (2) The method for testing the encapsulation efficiency of DNA-sodium nanoliposomes is as follows:

[0217] Accurately weigh 0.0005 g of DNA-sodium standard product, dissolve it in a 10 mL volumetric flask with pure water, and prepare a reference substance with a concentration of 0.05 g / mL. Respectively take 0, 200, 400, 500, 600, 800, 1000 μL of the reference substance solution, make up to 1 mL with pure water and shake well to prepare a series of solutions with concentrations of 0, 0.01, 0.02, 0.025, 0.03, 0.04, 0.05 μg / mL. Use an ultraviolet spectrophotometer to measure the ratio relationship between the absorbance value of DNA-sodium and the DNA-sodium concentration at 290 nm, and draw a standard curve y = 19.43351x - 0.01743, R 2 = 0.99807.

[0218] The ultrafiltration centrifugation method is used to measure the encapsulation efficiency of DNA-sodium nanoliposomes. First, take 400 μL of DNA-sodium nanoliposomes and place them in an ultrafiltration centrifugal tube with a molecular cut-off of 3000. After ultrafiltration centrifugation at 10000 rpm for 30 min, take out 100 μL of the filtrate from the outer tube and dilute it to 1 mL. Use an ultraviolet spectrophotometer to measure the absorbance value as the content of free DNA-sodium. Take another 100 μL of DNA-sodium nanoliposomes, add methanol to demulsify and then dilute it to 1 mL. After centrifugation at 10000 rpm for 30 min, let it stand for a while and take the supernatant to pass through a 0.22 μm organic membrane. Measure the absorbance value as the total DNA-sodium content. Calculate the total DNA-sodium content C1 and the free DNA-sodium content C0 respectively according to the linear regression equation of the standard curve. Finally, calculate the encapsulation efficiency according to the calculation formula: Encapsulation efficiency = [(C1 - C0) / C1] × 100%.

[0219] (3) The method for testing the particle size of DNA-sodium nanoliposomes is as follows:

[0220] Respectively take 15 μL of the DNA-sodium nanoliposomes prepared in the examples, add pure water to 3000 μL and mix evenly, and then measure the particle size of the sample with a ZSU 3200 nanoparticle size analyzer.

[0221] Example 1

[0222] Prepare samples according to the ratios in Table 1, and investigate the types and contents of the oil-phase emulsifiers (the first emulsifiers) suitable for the present invention, specifically as follows:

[0223] Dissolve lecithin, oil-phase emulsifier, and antioxidant in absolute ethanol to obtain the oil phase. Adjust the pH of the water in the inner aqueous phase to 5.0 - 6.0 with 0.1 mol / L citric acid solution, stir and dissolve sodium DNA for 10 min to obtain the inner aqueous phase. Then, adjust the pH of the water in the outer aqueous phase to 5.0 - 6.0 with 0.1 mol / L citric acid solution, and heat to 50 °C to dissolve glycerol, emulsifier (1%), and pentanediol to obtain the outer aqueous phase. Add the inner aqueous phase to the oil phase and emulsify at 7000 rpm for 10 min to form the primary emulsion, then add the primary emulsion to the outer aqueous phase and emulsify at 5000 rpm for 10 min to form the multiple emulsion. Rotate evaporate the multiple emulsion at 40 °C - 60 °C for 15 min - 30 min using a rotary evaporator to remove absolute ethanol. After compensating for the weight difference before and after rotary evaporation with pure water, add a preservative and mix well to obtain each sample. Observe the initial appearance of the sample and the appearance after standing at room temperature for 15 d. The results are shown in Table 1.

[0224] Table 1

[0225]

[0226]

[0227] As can be seen from Table 1, compared with sodium cholate and polyoxyethylene (21) stearyl ether, cholesterol is more suitable as the oil-phase emulsifier of the present invention. In addition, the dosage of cholesterol needs to be controlled at 0.05% - 0.2%. If it is lower than 0.05% or higher than 0.2%, the stability of the prepared sodium DNA nanoliposomes is not ideal, and the appearance will change significantly after standing at room temperature for 15 d.

[0228] Example 2

[0229] Prepare samples according to the ratios in Table 2 to investigate the types of aqueous-phase emulsifiers (secondary emulsifiers) suitable for the present invention, as follows:

[0230] Dissolve lecithin, cholesterol, and antioxidant in absolute ethanol to obtain the oil phase. Adjust the pH of the water in the inner aqueous phase to 5.0 - 6.0 with 0.1 mol / L citric acid solution, stir and dissolve sodium DNA for 10 min to obtain the inner aqueous phase. Then, adjust the pH of the water in the outer aqueous phase to 5.0 - 6.0 with 0.1 mol / L citric acid solution, and heat to 50 °C to dissolve glycerol, secondary emulsifier, and pentanediol to obtain the outer aqueous phase. Add the inner aqueous phase to the oil phase and emulsify at 7000 rpm for 10 min to form the primary emulsion, then add the primary emulsion to the outer aqueous phase and emulsify at 5000 rpm for 10 min to form the multiple emulsion. Rotate evaporate the multiple emulsion at 40 °C - 60 °C for 15 min - 30 min using a rotary evaporator to remove absolute ethanol. After compensating for the weight difference before and after rotary evaporation with pure water, add a preservative and mix well to obtain each sample. Observe the initial appearance of the sample and the appearance after standing at room temperature for 15 d. The results are shown in Table 2.

[0231] Table 2

[0232]

[0233]

[0234] As can be seen from Table 2, compared with GLUCATE SS, GTCC, cetearyl glucoside, poloxamer 407, and monoglyceride, BA-25 is more suitable as the external aqueous phase emulsifier of the present invention. Moreover, the compounding of BA-25 with the above types of emulsifiers will all result in the failure of formula preparation or poor short-term stability, making it impossible to conduct further stability investigations and subsequent application research.

[0235] Example 3

[0236] Samples were prepared according to the ratios in Table 3 to investigate the dosage of the aqueous phase emulsifier (the second emulsifier) suitable for the present invention, as follows:

[0237] Dissolve lecithin, cholesterol, and antioxidant in absolute ethanol to obtain the oil phase. Adjust the pH of the water in the inner aqueous phase to 5.0 - 6.0 with 0.1 mol / L citric acid solution, stir and dissolve DNA-sodium for 10 min to obtain the inner aqueous phase. Then adjust the pH of the water in the outer aqueous phase to 5.0 - 6.0 with 0.1 mol / L citric acid solution, heat to 50 °C to dissolve glycerol, the second emulsifier, and pentylene glycol to obtain the outer aqueous phase. Add the inner aqueous phase to the oil phase and emulsify at 7000 rpm for 10 min to form the primary emulsion, then add the primary emulsion to the outer aqueous phase and emulsify at 5000 rpm for 10 min to form the multiple emulsion. Rotate and evaporate the multiple emulsion on a rotary evaporator at 40 °C - 60 °C for 15 min - 30 min to remove absolute ethanol. After compensating for the weight difference before and after rotary evaporation with pure water, add preservatives and mix well to obtain each sample. Observe the initial appearance of the samples and the appearance after standing at room temperature for 15 d. The results are shown in Table 3.

[0238] Table 3

[0239]

[0240]

[0241] As can be seen from Table 3, compared with the DNA-sodium nanoliposomes containing 0.5% behenyl alcohol polyether-25, the DNA-sodium nanoliposomes containing 1% behenyl alcohol polyether-25 have more excellent room temperature stability, and smaller particle size and PDI. For the DNA-sodium nanoliposomes containing 2% and 4% behenyl alcohol polyether-25, although the initial particle size and PDI are smaller than those of the DNA-sodium nanoliposomes containing 1% behenyl alcohol polyether-25, their room temperature stability is inferior to that of the DNA-sodium nanoliposomes containing 1% behenyl alcohol polyether-25.

[0242] Example 4

[0243] Prepare samples according to the ratios in Table 4 as follows:

[0244] Dissolve lecithin, cholesterol, and antioxidant in absolute ethanol to obtain the oil phase. Adjust the pH of the water in the inner aqueous phase to 5.0 - 6.0 with 0.1 mol / L citric acid solution, stir and dissolve DNA-sodium in water for 10 min to obtain the inner aqueous phase. Then adjust the pH of the water in the outer aqueous phase to 5.0 - 6.0 with 0.1 mol / L citric acid solution, heat to 50 °C and dissolve glycerol, polyoxyethylene 25 behenyl ether, and pentylene glycol to obtain the outer aqueous phase. Add the inner aqueous phase to the oil phase and emulsify at 7000 rpm for 10 min to form the primary emulsion, then add the primary emulsion to the outer aqueous phase and emulsify at 5000 rpm for 10 min to form the double emulsion. Rotate and evaporate the double emulsion at 40 °C - 60 °C for 15 min - 30 min with a rotary evaporator to remove absolute ethanol. After making up the weight difference before and after rotary evaporation with pure water, add preservatives and mix well to obtain the samples. The appearance and particle size results are shown in Table 4.

[0245] Table 4

[0246]

[0247] Specification

[0248]

[0249] As can be seen from Table 4, the DNA-sodium nanoliposomes prepared from soy lecithin S45 and soy lecithin S75 are both light yellow, but the DNA-sodium nanoliposomes made from S45 soy lecithin have higher transparency. The initial particle sizes of the two DNA-sodium nanoliposomes are close, but the DNA-sodium nanoliposomes made from soy lecithin S75 have a smaller PDI, better system stability and homogeneity. And through 90-day stability test, it shows that compared with soy lecithin S75, the degree of increase in the particle size of the liposomes made from soy lecithin S45 as phospholipid is larger. Therefore, soy lecithin S75 is preferably used as the raw material of lecithin in the DNA-sodium nanoliposome system.

[0250] Example 5

[0251] Prepare samples according to the ratios in Table 5 as follows:

[0252] Dissolve soy lecithin S75, cholesterol, and antioxidant in absolute ethanol to obtain the oil phase. Adjust the pH of the water in the inner aqueous phase to 5.0 - 6.0 with 0.1 mol / L citric acid solution, stir and dissolve DNA-sodium for 10 min to obtain the inner aqueous phase. Then adjust the pH of the water in the outer aqueous phase to 5.0 - 6.0 with 0.1 mol / L citric acid solution, heat to 50 °C and dissolve glycerol, polyoxyethylene 25 behenate, and pentylene glycol to obtain the outer aqueous phase. Add the inner aqueous phase to the oil phase and emulsify at 7000 rpm for 10 min to form the primary emulsion, then add the primary emulsion to the outer aqueous phase and emulsify at 5000 rpm for 10 min to form the multiple emulsion. Rotate evaporate the multiple emulsion at 40 °C - 60 °C for 15 min - 30 min using a rotary evaporator to remove absolute ethanol, supplement the weight difference before and after rotary evaporation with pure water, add a preservative and mix well to obtain the sample.

[0253] Table 5

[0254]

[0255] As can be seen from Table 5, when the mass fraction of DNA-sodium in DNA-sodium nano-liposomes is 0.3%, the encapsulation efficiency of DNA-sodium nano-liposomes is the highest, the drug loading is the highest, ΔBS is the smallest, and the stability is the strongest. Therefore, it is preferred that the mass fraction of DNA-sodium in DNA-sodium nano-liposomes is 0.3%.

[0256] Example 6

[0257] Prepare samples according to the ratios in Table 6 to investigate the types and dosages of polyols suitable for the outer aqueous phase of the present invention, as follows:

[0258] Dissolve soy lecithin S75, cholesterol, and antioxidant in absolute ethanol to obtain the oil phase. Adjust the pH of the water in the inner aqueous phase to 5.0 - 6.0 with 0.1 mol / L citric acid solution, stir and dissolve DNA-sodium for 10 min to obtain the inner aqueous phase. Then adjust the pH of the water in the outer aqueous phase to 5.0 - 6.0 with 0.1 mol / L citric acid solution, heat to 50 °C and dissolve polyol, polyoxyethylene 25 behenate to obtain the outer aqueous phase. Add the inner aqueous phase to the oil phase and emulsify at 7000 rpm for 10 min to form the primary emulsion, then add the primary emulsion to the outer aqueous phase and emulsify at 5000 rpm for 10 min to form the multiple emulsion. Rotate evaporate the multiple emulsion at 40 °C - 60 °C for 15 min - 30 min using a rotary evaporator to remove absolute ethanol, supplement the weight difference before and after rotary evaporation with pure water, add a preservative and mix well to obtain the sample.

[0259] Table 6

[0260]

[0261]

[0262] As can be seen from Table 6, compared with pentanediol, glycerol is more suitable as the external aqueous phase emulsifier of the present invention.

[0263] Test

[0264] (1) The DNA-sodium nanoliposomes prepared in Experiment No. 3 of Example 5 were detected by TEM as follows:

[0265] Weigh the sample, drop it on the copper mesh. After a few seconds, gently pick up the copper mesh sample with forceps, and absorb the excess liquid along one side with filter paper. After it is slightly dried, place the copper mesh on a 2% phosphotungstic acid staining solution drop for floating staining for 60 s. After picking it up with forceps, also absorb the excess liquid along one side with filter paper, place it on the filter paper with the membrane side up to dry, and observe and take pictures with a transmission electron microscope. The results are as Figure 1 shown, the particles are spherical-like and the distribution is relatively uniform.

[0266] (2) The DNA-sodium nanoliposomes prepared in Experiment No. 3 of Example 5 were tested for in vitro slow release as follows:

[0267] For the in vitro release test of the prepared DNA-sodium nanoliposomes, the DNA-sodium in the release medium was measured by PBS dialysis. Put 5 mL of the sample into a dialysis bag (cut-off molecular weight: 6000 - 8000), and place the dialysis bag into a 250 mL beaker containing 200 mL of the release medium. The beaker was incubated in a 37 °C shaking water bath thermostat and shaken at 100 rpm by a magnetic stirrer. At predetermined time intervals (1, 2, 4, 6, 8, 12, 24, 48, 72, 96 hours after the start of the incubation), take out all the release medium (3 mL) and replace it with the same volume of fresh release medium (3 mL). The release amount of DNA-sodium was determined by HPLC and a slow release curve was plotted. The results are shown in Figure 2 .

[0268]

[0269] Qi: Cumulative release amount at the i-th sampling time point, %;

[0270] V: Volume of the release medium, mL;

[0271] ρ n : Concentration of DNA-sodium in the release medium released during the n-th sampling, μg / mL;

[0272] ρ i : Concentration of DNA-sodium in the release medium released during the i-th sampling, μg / mL;

[0273] Vi: Volume of the i-th sampling, mL;

[0274] m: mass of DNA-sodium in the nano-liposome sample, μg;

[0275] Figure 2 The in vitro permeation results of 0.3% DNA-sodium wrapped in a 14KD dialysis bag in different formulations in PBS solution are shown. Less than 1% of the 0.3% DNA-sodium aqueous solution was released in PBS, and almost none permeated through the dialysis bag due to its high molecular weight. The DNA-sodium nano-liposomes were well-released in PBS, not only improving the permeability of DNA-sodium, but also the release amount slowly increasing from 9.1% to 19.6% within 24 hours, showing a good sustained-release effect. Due to the influence of experimental conditions and the stability of PBS solution on DNA-sodium, DNA-sodium was oxidized and decomposed after being released from the liposome, so the DNA-sodium content showed a downward trend between 24 and 72 hours. The cumulative release amount of the DNA-sodium nano-liposome group decreased by 24% of the highest value, while the DNA-sodium aqueous solution group decreased by 42.7% of the highest value. Due to the protection and sustained-release ability of the DNA-sodium nano-liposome, DNA-sodium was still continuously released after 24 hours, reducing the decrease in the cumulative release amount of DNA-sodium in the PBS solution.

[0276] (3) The stability of the DNA-sodium nano-liposomes prepared in Experiment No. 1 of Example 4 and Experiments No. 1, 2, and 3 of Example 5 was investigated as follows:

[0277] Four kinds of samples were respectively filled into 7 vials and sealed, and stored in normal temperature, refrigeration (4°C), freezing (-15°C), heating (45°C), freeze-thaw (-15°C and 45°C alternating), dark, and light (28°C) environments. The particle size and PDI were tested at 7, 14, 30, 60, and 90 days respectively. The results are as Figures 3 - 6 shown successively, where Figure 3 is the test result of Experiment No. 1 of Example 4, Figure 4 is the test result of Experiment No. 1 of Example 5, Figure 5 is the test result of Experiment No. 2 of Example 5, Figure 6 is the test result of Experiment No. 3 of Example 5. It can be seen that the particle size and PDI changes of each sample are small. As the content of the encapsulated DNA-sodium increases, the particle size of the DNA-sodium nano-liposome increases from 105.2nm to 124.9nm, and the PDI increases from 0.06 to 0.12. Affected by the particle size, the transparency of the light yellow liposome solution gradually decreases. The PDI of all groups is less than 0.2, indicating that the distribution uniformity of each system is good. As the storage time becomes longer, the overall light yellow of the solution gradually fades and turns milky white. The particle size change value under each condition is small, and the PDI is less than 0.2, and the system distribution uniformity is good.

[0278] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as within the scope described in this specification.

[0279] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A DNA-sodium nanoliposome, characterized in that, It has a W / O / W multiple emulsion structure and is mainly made of components with the following mass percentages: (1) Inner aqueous phase: Sodium DNA 0.05% - 0.5%, Water 5% - 15%; (2) Oil phase: Phospholipid 3% - 7%, First emulsifier 0.05% - 0.2%; (3) Outer aqueous phase: Second emulsifier 0.5% - 1.8%, Glycerol 5% - 20%, Water 55.5% - 86.4%; The first emulsifier includes cholesterol; The second emulsifier includes behenyl alcohol polyether - 25.

2. The DNA-sodium nanoliposome according to claim 1, characterized in that, The phospholipid is selected from one or a combination of two of soy lecithin S45 and soy lecithin S75.

3. The DNA-sodium nanoliposome according to claim 1 or 2, characterized in that, Meet at least one of the following (1) - (3): (1) The oil phase further includes an antioxidant; (2) The outer aqueous phase further includes a first preservative; (3) The sodium DNA nanoliposome further includes a second preservative.

4. The DNA-sodium nanoliposome according to claim 3, wherein It is mainly made of components with the following mass percentages: (1) Inner aqueous phase: Sodium DNA 0.05% - 0.5%, Water 5% - 15%; (2) Oil phase: Phospholipid 3% - 7%, First emulsifier 0.05% - 0.2%, Antioxidant 0.05% - 0.1%; (3) Outer aqueous phase: and (4) Second preservative 0.05% - 1.5%.

5. The DNA-sodium nanoliposome according to claim 4, wherein Meet at least one of the following (1) - (3): (1) The antioxidant is pentaerythritol tetra(bis - tert - butylhydroxyhydrocinnamate); (2) The first preservative is pentylene glycol; (3) The second preservative is selected from one or a mixture of two of p - hydroxyacetophenone and pentylene glycol.

6. The DNA-sodium nanoliposome according to claim 1 or 2, characterized in that, The particle size of the sodium DNA nanoliposome is 90nm - 150nm, the PDI is 0.01 - 0.3, and the encapsulation efficiency is ≥60%.

7. A method for preparing the DNA-sodium nanoliposome according to any one of claims 1 to 6, characterized in that, It includes the following steps: Mix the phospholipid and the first emulsifier in an alcohol solvent to prepare an oil - phase mixture; Mix the sodium DNA and water to prepare a first aqueous - phase mixture; Mix the second emulsifier and glycerol in water to prepare a second aqueous - phase mixture; Mix the oil - phase mixture and the first aqueous - phase mixture to prepare a primary emulsion, and the primary emulsion has a W / O structure; Mix the primary emulsion and the second aqueous - phase mixture to prepare a first multiple emulsion, and the first multiple emulsion has a W / O / W structure; Remove the alcohol solvent in the first multiple emulsion and add water to make up for the mass of the alcohol solvent to prepare a second multiple emulsion, and the second multiple emulsion has a W / O / W structure.

8. The preparation method of the DNA-sodium nanoliposome according to claim 7, wherein Meet at least one of the following (1) - (5): (1) The process parameters for preparing the first aqueous - phase mixture include: the pH of the system is 5.0 - 6.0, and the temperature is 65°C - 75°C; (2) The process parameters for preparing the primary emulsion include: the emulsification speed is 5000rpm - 9000rpm, and the time is 5min - 20min; (3) The process parameters for preparing the first multiple emulsion include: the emulsification speed is 3000rpm - 5000rpm, and the time is 5min - 20min; (4) The alcohol solvent is ethanol; (5) The process parameters for preparing the second multiple emulsion include: vacuum concentration, with the temperature being 40°C to 60°C and the time being 10 min to 30 min.

9. A cosmetic, characterized in that, It includes the DNA-sodium nanoliposomes according to any one of claims 1 to 6.

10. The cosmetic according to claim 9, characterized in that, The cosmetic product is selected from at least one of a facial mask, eye cream, face cream, primer, essence, lotion, and skin softener.

Images