Nucleic acid nano-microsphere as well as preparation method and application thereof

Abstract

The invention discloses nucleic acid nano microspheres as well as a preparation method and application thereof, and belongs to the technical field of nano delivery. The preparation method comprises the following steps: carrying out first mixing on nucleic acid and part of water, and carrying out pretreatment and curing to form nucleic acid semi-solid gel; melting the nucleic acid semi-solid gel at 45-80 DEG C, and adjusting the pH value to 4-6 to form a phase A; carrying out second mixing on the spermidine-containing plant extract and the remaining water, and adjusting the pH value to 4-6 to form a phase B; and carrying out third mixing on the phase A, the phase B and an auxiliary agent to form the nucleic acid nano-microsphere, the nucleic acid nano-microsphere is prepared from the following components in percentage by mass: 0.5 to 8 percent of nucleic acid, 0.1 to 7.5 percent of a plant extract containing spermidine, 0 to 6 percent of an auxiliary agent and 78.5 to 99.4 percent of water, the mass ratio of the nucleic acid to the plant extract containing spermidine is (1-10): 1. The nucleic acid nano-microsphere has improved transdermal effect and stability, and can be used for transdermal drug delivery.

Description

Technical Field

[0001] The present application relates to the technical field of nano-delivery technology, and particularly to a nucleic acid nano-microsphere, a preparation method thereof, and an application thereof. Background Art

[0002] Nucleic acids are a class of important biomolecules in organisms, and they play a key role in storing and transmitting genetic information in cells. Currently, commonly used nucleic acid raw materials include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and their salts. Among them, sodium DNA, as a bioactive molecule similar to a growth factor, is one of the raw materials for generating DNA in cells and has natural biocompatibility. It has been widely used in pharmaceutical fields such as small nucleic acid drugs, mRNA vaccines, and gene therapy, and plays a powerful anti-inflammatory and repair effect in aspects such as wound healing, scar repair, and joint injury, and has a positive promoting effect on angiogenesis, collagen synthesis, and the cell activities of fibroblasts and osteoblasts.

[0003] However, most nucleic acids have a short half-life and a short shelf life. For example, the half-life of sodium DNA is only about 3 hours, with poor stability and low bioavailability; nucleic acids carry negative charges on their surfaces, and have high molecular weight and high hydrophilicity, making it difficult to penetrate cell membranes to reach intracellular sites to play their roles; nucleic acids have weak targeting in the body and are prone to off-target effects. The above characteristics greatly limit the application of nucleic acids in products such as transdermal drug delivery.

[0004] Microspheres are spherical particles with very small diameters, made of natural or synthetic polymer materials, and can encapsulate various substances such as drugs, bioactive substances, dyes, and catalysts inside. Microspheres have a highly uniform spherical structure, which makes them have good stability and repeatability in physical and chemical properties. Their surfaces can be modified to change their hydrophilicity, hydrophobicity, charge properties, etc., so as to achieve specific functions. Preparing nucleic acids into nucleic acid microspheres can improve the stability, bioavailability, and targeting of nucleic acids.

[0005] However, the current nucleic acid microspheres have limited stability and insufficient drug loading capacity. Summary of the Invention

[0006] Based on this, the main purpose of the present application is to provide a preparation method of a nucleic acid nano-microsphere, so as to prepare a nucleic acid nano-microsphere with improved transdermal effect and stability, which can effectively load nucleic acids and meet the application requirements in products such as transdermal drug delivery.

[0007] In the first aspect of the present application, a preparation method of a nucleic acid nano-microsphere is provided, including the following steps:

[0008] Mix the nucleic acid with a part of water for the first time, perform pretreatment, and solidify to form a nucleic acid semi-solid gel;

[0009] The nucleic acid semi-solid gel is melted at 45 - 80 °C, and the pH is adjusted to 4 - 6 to form Phase A;

[0010] The plant extract containing spermidine is secondarily mixed with the remaining water, and the pH is adjusted to 4 - 6 to form Phase B;

[0011] Phase A, Phase B and the auxiliary agent are tertiarily mixed to form the nucleic acid nanospheres;

[0012] Among them, the nucleic acid nanospheres include the following components by mass percentage: nucleic acid 0.5 - 8%, plant extract containing spermidine 0.1 - 7.5%, auxiliary agent 0 - 6% and water 78.5 - 99.4%;

[0013] The mass ratio of the nucleic acid to the plant extract containing spermidine is 1 - 10:1.

[0014] In some embodiments, the preparation method satisfies one or more of the following conditions:

[0015] (1) The nucleic acid includes at least one of DNA and DNA derivatives;

[0016] (2) The plant extract containing spermidine includes at least one of wheat (TRITICUM VULGARE) germ extract, soybean (GLYCINE MAX) extract, pea (PISUM SATIVUM) extract and sesame (SESAMUM INDICUM) extract;

[0017] (3) The reagent for adjusting pH includes citric acid;

[0018] (4) The auxiliary agent includes at least one of a thickener and a preservative;

[0019] (5) The dosage of the partial water is 35 - 60 wt% of the nucleic acid nanospheres;

[0020] (6) The content of spermidine in the plant extract containing spermidine is ≥ 30 wt%.

[0021] In some embodiments, the preparation method satisfies one or more of the following conditions:

[0022] (1) The thickener includes at least one of chondrus crispus extract, xanthan gum, sclerotium gum and AVC;

[0023] (2) The preservative includes at least one of hexylene glycol, pentylene glycol and ethylhexyl glycerin;

[0024] (3) The dosage of the thickener is 0.1 - 0.5 wt%;

[0025] The dosage of the preservative is 1-5 wt%.

[0026] In some embodiments, the preparation method satisfies one or more of the following conditions:

[0027] (1) The temperature of the first mixing is 40-80 °C;

[0028] (2) The pretreatment includes homogenization treatment and / or microfluidization treatment;

[0029] (3) The conditions for curing include: curing temperature 10-20 °C; curing time 0.5-24 h;

[0030] (4) The temperature of the second mixing is 40-80 °C;

[0031] (5) The temperature of the third mixing is 40-80 °C;

[0032] (6) The third mixing includes the following steps: adding phase B to phase A, stirring at 2000-12000 rpm for 5-60 min; then adding an auxiliary agent and mixing;

[0033] (7) The melting time is 40-120 min.

[0034] In some embodiments, the preparation method satisfies one or more of the following conditions:

[0035] (1) The conditions for homogenization treatment include: homogenization temperature 40-60 °C; homogenization pressure 100-2000 bar; number of homogenization times 2-20 times;

[0036] (2) The conditions for microfluidization treatment include: pressure 1400-15000 Psi, number of treatment times 1-5 times.

[0037] In some embodiments, the nucleic acid nanospheres by mass percentage include the following components: nucleic acid 0.5%-8%, spermidine-containing plant extract 0.1%-7.5%, thickener 0.1-0.5%, preservative 1-5% and water 79%-98.3%.

[0038] In the second aspect of the present application, there is provided a nucleic acid nanosphere prepared by the preparation method described in the first aspect.

[0039] In the third aspect of the present application, there is provided the use of the nucleic acid nanosphere described in the second aspect in the preparation of a transdermal drug delivery product.

[0040] In some embodiments, the product includes an immune-enhancing drug or a cosmetic;

[0041] Optionally, the immunity-enhancing drug includes an anti-infective drug, an immunomodulatory drug, an anti-tumor drug or an anti-inflammatory drug;

[0042] Optionally, the cosmetic includes at least one of facial mask liquid, skin care lotion, essence, spray and emulsion.

[0043] In the fourth aspect of the present application, an immunity-enhancing drug is provided, and the raw materials include the nucleic acid nanospheres described in the second aspect.

[0044] In the fifth aspect of the present application, a cosmetic is provided, and the raw materials include the nucleic acid nanospheres described in the second aspect.

[0045] Advantages of the present application:

[0046] 1. In the present application, a nucleic acid microsphere is prepared by electrostatic adsorption self-assembly of a positively charged spermidine-containing plant extract and a negatively charged nucleic acid, and the preparation process is optimized. Nucleic acid microspheres with high nucleic acid loading, good stability and uniform nanoscale particle size can be prepared. It has better permeability and targeting, is easier to diffuse into the skin, has improved transdermal effect, can improve the bioavailability of nucleic acid, and meets the application requirements in products such as transdermal drugs.

[0047] 2. The nucleic acid nanospheres of the present application have the effect of enhancing the phagocytic ability of macrophages, can improve immunity, and can be used to prepare immunity-enhancing drugs such as anti-infective drugs, immunomodulatory drugs, anti-tumor drugs or anti-inflammatory drugs.

[0048] 3. The nucleic acid nanospheres of the present application have high uniformity and high stability, are gentle to the human body and have a sustained release effect, and show excellent stability under 7 different harsh environments of normal temperature, refrigeration, freezing, heating, freeze-thaw, light and darkness. Description of the drawings

[0049] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required to be used in the description of the embodiments or the prior art. The drawings are only for the purpose of showing the preferred embodiments and are not considered to be a limitation of the present application. Throughout the drawings, the same reference numerals are used to represent the same components. In the drawings:

[0050] Figure 1 Photographs of the bottom of the bottles after 1 day of placement for Example 11 and Comparative Example 6, where the left figure is Example 11 and the right figure is Comparative Example 6;

[0051] Figure 2 Standard curve for DNA sodium content determination;

[0052] Figure 3 Standard curve for spermidine content determination;

[0053] Figure 4 SEM electron micrograph of the nucleic acid nanospheres prepared in Example 11;

[0054] Figure 5 TEM electron micrograph of the nucleic acid nanospheres prepared in Example 11;

[0055] Figure 6 Results graph of the Turbiscan lab stability analyzer for the nucleic acid nanospheres prepared in Example 11;

[0056] Figure 7 Comparison of the in vitro cumulative permeation curves of the nucleic acid nanospheres prepared in Example 11 and the sample of Comparative Example 7;

[0057] Figure 8 Comparison of the in vitro 24-hour cumulative permeation amount and the retention amount in the skin of the nucleic acid nanospheres prepared in Example 11 and the sample of Comparative Example 7;

[0058] Figure 9 Effect of the nucleic acid nanospheres prepared in Example 11 on the phagocytic ability of macrophages. Detailed implementation manners

[0059] To make the objectives, technical solutions, and advantages of this application clearer and the understanding of the disclosed content of this application more thorough and comprehensive, the technical solutions of this application will be clearly and completely described below in conjunction with the specific embodiments and corresponding drawings of this application. The described embodiments are only a part of the embodiments of this application, rather than all of the embodiments.

[0060] The following will make a detailed description of the implementation of this application in conjunction with the drawings. This embodiment is implemented on the premise of the technical solution of this application, and the detailed implementation manners and specific operation processes are given, but the protection scope of this application is not limited to the following embodiments.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0062] Terms

[0063] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:

[0064] In this application, when referring to "a plurality of", "multiple", etc., unless otherwise specified, it means greater than 2 or equal to 2 in quantity. For example, "one or more", "at least one" means one or greater than or equal to two.

[0065] In this application, terms such as "further" and "especially" are used for descriptive purposes, indicating differences in content, but should not be construed as limiting the scope of protection of this application.

[0066] In the open-ended technical features described in this application, it includes closed technical solutions composed of the listed features, as well as open technical solutions containing the listed features.

[0067] In this application, regarding numerical intervals (i.e., numerical ranges), unless otherwise specified, the distribution of optional numerical values within this numerical interval is considered continuous, and it includes the two numerical endpoints of this numerical interval (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within this numerical interval, it includes the two endpoint integers of this numerical range, as well as each integer between the two endpoints, which is equivalent to directly listing each integer. When providing multiple numerical ranges to describe features or characteristics, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein. The "numerical values" in this numerical interval can be any quantitative values, such as numbers, percentages, ratios, etc. The "numerical interval" allows for a broad inclusion of numerical interval types such as percentage intervals, ratio intervals, and ratio value intervals.

[0068] In this application, unless otherwise specified, the temperature parameter allows for both constant temperature treatment and variation within a certain temperature range. It should be understood that the so-called constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0069] In this application, regarding the units of data ranges, if there is only a unit after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same. For example, 2 - 5h means that the units of the left endpoint "2" and the right endpoint "5" are both h (hours).

[0070] The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0071] In this application, unless otherwise specified, the temperature parameter allows for both constant temperature treatment and treatment within a certain temperature range. The so-called constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. The room temperature described in this application refers to 0 - 40°C, preferably 10°C - 35°C, and more preferably 20°C - 30°C.

[0072] In the first aspect of this application, a method for preparing nucleic acid nanospheres is provided, including the following steps:

[0073] Mix the nucleic acid with part of the water for the first time, perform pretreatment, and solidify to form a nucleic acid semi-solid gel;

[0074] Melt the nucleic acid semi-solid gel at 45 - 80 °C, adjust the pH to 4 - 6 to form Phase A;

[0075] Mix the plant extract containing spermidine with the remaining water for the second time, adjust the pH to 4 - 6 to form Phase B;

[0076] Perform a third mixing of Phase A, Phase B and the auxiliary agent to form the nucleic acid nanospheres;

[0077] Among them, the nucleic acid nanospheres include the following components by mass percentage: nucleic acid 0.5 - 8%, plant extract containing spermidine 0.1 - 7.5%, auxiliary agent 0 - 6% and water 78.5 - 99.4%;

[0078] The mass ratio of the nucleic acid to the plant extract containing spermidine is 1 - 10:1.

[0079] It is understandable that when calculating the above mass percentages in this application, the dosage of the pH regulator is not included, that is, the dosage of the raw materials other than the pH regulator is counted as 100%.

[0080] This application uses a positively charged plant extract containing spermidine and a negatively charged nucleic acid to prepare nucleic acid microspheres by electrostatic adsorption self-assembly, and optimizes the preparation process, and can prepare nucleic acid microspheres with high nucleic acid loading, good stability and uniform nano-scale particle size, which have better permeability and targeting, are easier to diffuse into the skin, have improved transdermal effects, can improve the bioavailability of nucleic acids, and meet the application requirements in products such as transdermal drug delivery.

[0081] In a specific example, the melting temperature is 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, etc. It is understandable that an appropriate melting temperature can extend the double helix structure of the nucleic acid and promote the interaction between DNA and spermidine, thereby preparing nano nucleic acid microspheres with small and uniform particle size.

[0082] In a specific example, the pH of Phase B is 4, 4.5, 5, 5.5, 6, etc.

[0083] In a specific example, the dosage of the nucleic acid is 0.5 - 8%, including but not limited to 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 6.5%, 7% or 8%, preferably 0.5 - 4%. It is understandable that this application directly uses a positively charged plant extract containing spermidine and a negatively charged nucleic acid to prepare nucleic acid microspheres by electrostatic adsorption self-assembly, and can achieve high loading of nucleic acids.

[0084] In a specific example, the nucleic acid includes at least one of DNA and DNA derivatives, such as sodium DNA. Among them, the content of DNA in the nucleic acid is 70% or more, and the number of base pairs of the nucleic acid is 30 - 6000 bp, such as 30 bp, 100 bp, 500 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp, 6000 bp, etc.

[0085] In a specific example, the dosage of the spermidine-containing plant extract is 0.1 - 7.5%, including but not limited to 0.1%, 0.3%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7% or 7.5%, preferably 0.1 - 3%.

[0086] In a specific example, the spermidine-containing plant extract includes at least one of wheat (TRITICUM VULGARE) germ extract, soybean (GLYCINE MAX) extract, pea (PISUM SATIVUM) extract, and sesame (SESAMUM INDICUM) extract.

[0087] Spermidine is a polyamine substance with multiple amino groups in its molecular structure. These amino groups are protonated under physiological conditions, making spermidine positively charged and tightly binding to sodium DNA. They are combined into microspheres through electrostatic attraction and hydrogen bonding and other interaction methods to achieve targeted delivery and controlled release of nucleic acids. Wheat germ is one of the sources with relatively rich spermidine content in plants. Compared with other plant tissues, its spermidine content is usually at a relatively high level. Preferably, the content of spermidine in the spermidine-containing plant extract is ≥30 wt%, such as 30 - 100 wt%, specifically it can be 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, 70 wt%, 75 wt%, 80 wt%, 85 wt%, 90 wt%, 95 wt%, 100 wt%, etc.

[0088] In a specific example, the auxiliary agent includes at least one of a thickener and a preservative; there are no special limitations on the specific types of the thickener and the preservative.

[0089] In a specific example, the thickener includes at least one of Chondrus crispus (Carrageenan) Extract (i.e., carrageenan), xanthan gum, scleroglucan, and AVC; its dosage is 0.1 - 0.5 wt%, such as 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, etc., preferably 0.3 - 0.5 wt%.

[0090] In a specific example, the preservative includes at least one of hexylene glycol, pentylene glycol, and ethylhexylglycerin; the dosage is 1-5 wt%, such as 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, etc., preferably 1-4 wt%.

[0091] In a specific example, the mass ratio of the nucleic acid to the spermidine-containing plant extract is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc., preferably 2-7:1. It can be understood that the positively charged spermidine-containing plant extract and the negatively charged nucleic acid in the present application self-assemble by electrostatic adsorption to prepare nucleic acid microspheres, and the mass ratio of the nucleic acid to the spermidine-containing plant extract has a direct impact on the preparation and morphology of the microspheres. The ratio defined in the present application is beneficial to preparing nano nucleic acid microspheres with small particle size and good uniformity.

[0092] In a specific example, the pH-adjusting reagent includes citric acid.

[0093] In a specific example, the dosage of the partial water is 35-60 wt% of the nucleic acid nanomicrospheres, such as 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, etc.

[0094] In a specific example, the temperature of the first mixing is 40-80 °C, such as 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, etc.

[0095] In a specific example, the pretreatment includes homogenization treatment and / or microfluidization treatment.

[0096] In a specific example, the conditions of the homogenization treatment include: homogenization temperature 40-60 °C, such as 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, etc.; homogenization pressure 100-2000 bar, such as 100 bar, 500 bar, 1000 bar, 1500 bar, 2000 bar, etc.; the number of homogenization times is 2-20 times, such as 2 times, 5 times, 10 times, 15 times, 20 times, etc.

[0097] In a specific example, the conditions of the microfluidization treatment include: pressure 1400-15000 Psi, such as 1400 Psi, 2500 Psi, 5000 Psi, 7500 Psi, 10000 Psi, 12500 Psi, 15000 Psi, etc., and the number of treatment times is 1-5 times, such as 1 time, 2 times, 3 times, 4 times, 5 times, etc.

[0098] In a specific example, the conditions for curing include: a curing temperature of 10-20°C, such as 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, etc.; a curing time of 0.5-24 h, such as 0.5 h, 1 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, etc. It is understandable that the curing means standing still at 10-20°C for 0.5-24 h to achieve the gelation of nucleic acid.

[0099] In a specific example, the temperature of the second mixing is 40-80°C, such as 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, etc.

[0100] In a specific example, the temperature of the third mixing is 40-80°C, such as 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, etc.

[0101] In a specific example, the third mixing includes the following steps: adding the B phase to the A phase, stirring at 2000-12000 rpm for 5-60 min; then adding an auxiliary agent and mixing; wherein, the stirring speed can specifically be 2000 rpm, 4000 rpm, 6000 rpm, 8000 rpm, 10000 rpm, 12000 rpm, etc.; the stirring time can specifically be 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, etc.

[0102] In a specific example, the melting time is 40-120 min, such as 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, 120 min, etc.

[0103] In a specific example, the nucleic acid nanospheres include the following components by mass percentage: nucleic acid 0.5%-8%, plant extract containing spermidine 0.1%-7.5%, thickener 0.1-0.5%, preservative 1-5%, and water 79%-98.3%.

[0104] In the second aspect of the present application, there is provided the nucleic acid nanospheres prepared by the preparation method described in the first aspect.

[0105] The nucleic acid nanospheres of the present application have high homogeneity and high stability, are gentle to the human body and have a sustained release effect, show excellent stability under 7 different harsh environments of normal temperature, refrigeration, freezing, heating, freeze-thaw, light and dark, and have improved transdermal effects, can improve the bioavailability of nucleic acid, and meet the application requirements in products such as transdermal drug delivery.

[0106] In a specific example, the nucleic acid nanospheres have a particle size of 100 - 700 nm, such as 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, etc.; the PDI is 0.05 - 0.55, such as 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.55, etc.; the pH is 4 - 6, such as 4, 4.5, 5, 5.5, 6, etc.

[0107] In the third aspect of the present application, there is provided an application of the nucleic acid nanospheres described in the second aspect in the preparation of a transdermal drug delivery product.

[0108] The nucleic acid nanospheres of the present application have improved transdermal effects and stability, can improve the bioavailability of nucleic acids, and meet the application requirements in products such as transdermal drug delivery drugs. The nucleic acid nanospheres of the present application have the effect of enhancing the phagocytic ability of macrophages, can improve immunity, and can be used to prepare immunity-enhancing drugs such as anti-infective drugs, immunomodulatory drugs, anti-tumor drugs, or anti-inflammatory drugs.

[0109] In a specific example, the product includes an immunity-enhancing drug or a cosmetic.

[0110] In a specific example, the immunity-enhancing drug includes an anti-infective drug, an immunomodulatory drug, an anti-tumor drug, or an anti-inflammatory drug.

[0111] In a specific example, the cosmetic includes at least one of a facial mask liquid, a skin care lotion, a serum, a spray, and a lotion.

[0112] In the fourth aspect of the present application, there is provided an immunity-enhancing drug, the raw materials of which include the nucleic acid nanospheres described in the second aspect.

[0113] The nucleic acid nanospheres of the present application have improved transdermal effects and stability, can improve the bioavailability of nucleic acids, and have the effect of enhancing the phagocytic ability of macrophages, can improve immunity, and can be used to prepare immunity-enhancing drugs such as anti-infective drugs, immunomodulatory drugs, anti-tumor drugs, or anti-inflammatory drugs.

[0114] In the fifth aspect of the present application, there is provided a cosmetic, the raw materials of which include the nucleic acid nanospheres described in the second aspect.

[0115] The nucleic acid nanospheres of the present application have improved transdermal effects and stability, can improve the bioavailability of nucleic acids, and have the effect of enhancing the phagocytic ability of macrophages, can improve skin immunity, and can be used in cosmetics.

[0116] Unless otherwise specified, the raw materials used in the following tests can all be conventionally purchased from the market.

[0117] Exemplary descriptions of the raw materials used in the examples and comparative examples are as follows:

[0118] Nucleic acid: Sodium DNA, with a DNA content of ≥70%, purchased from Beijing Weiye Innovation Technology Co., Ltd.;

[0119] Wheat (TRITICUM VULGARE) germ extract: Spermidine content ≥50%, purchased from Xi'an Zebang Biotechnology Co., Ltd.;

[0120] Soybean (GLYCINE MAX) extract: Spermidine content ≥50%, purchased from Xi'an Wisbio Biotechnology Co., Ltd.;

[0121] Pea (PISUM SATIVUM) extract: Spermidine content ≥50%, purchased from Xi'an Wisbio Biotechnology Co., Ltd.;

[0122] Sesame (SESAMUM INDICUM) extract: Spermidine content ≥50%, purchased from Xi'an Wisbio Biotechnology Co., Ltd.;

[0123] Thickener: Chondrus crispus extract, purchased from CP Kelco US Inc.;

[0124] Preservative: Hexylene glycol, commercially available;

[0125] Other raw materials are commercially available.

[0126] The following are specific examples.

[0127] Example 1

[0128] Preparation of nucleic acid nanospheres:

[0129] Based on the total amount of raw materials other than the pH regulator being 100 wt%, the formulation is as follows: Nucleic acid (sodium DNA) 0.5 wt%, wheat (TRITICUM VULGARE) germ extract 0.15 wt%, thickener (chondrus crispus extract) 0.5 wt%, preservative (hexylene glycol) 2 wt%, and the balance is water; among them, the mass ratio of nucleic acid to wheat (TRITICUM VULGARE) germ extract is 3.3:1.

[0130] The preparation process is as follows:

[0131] Dissolve the nucleic acid in 49 wt% of water (50 °C) of the final product. After complete dissolution without agglomerated particles, the pressure of high-pressure homogenization is 500 bar, and the number of times is 10 times. Then place it at 20 °C for curing for 1 h to form a nucleic acid semi-solid gel;

[0132] Heat the nucleic acid semi-solid gel to 60 °C and melt it for 40 min. Adjust the pH to 5.5 with an anhydrous citric acid solution to obtain Phase A;

[0133] Dissolve the wheat (TRITICUM VULGARE) germ extract in the remaining water (50 °C). After the solution is completely dissolved, filter it and adjust the pH to 5.5 with an anhydrous citric acid solution to obtain Phase B.

[0134] Add the said Phase B to the said Phase A, stir at 5000 rpm for 30 min, and add a thickening agent and a preservative to obtain nucleic acid nanospheres.

[0135] Example 2

[0136] Except that the dosage of nucleic acid is 2 wt% (keeping the mass ratio of nucleic acid to wheat (TRITICUM VULGARE) germ extract the same as that in Example 1), the rest is the same as in Example 1, and the specific formulation is as follows:

[0137] Nucleic acid (sodium DNA) 2 wt%, wheat (TRITICUM VULGARE) germ extract 0.6 wt%, thickening agent (chondrus crispus extract) 0.5 wt%, preservative (hexylene glycol) 2 wt% and water balance (total 100 wt%); among them, the mass ratio of nucleic acid to wheat (TRITICUM VULGARE) germ extract is 3.3:1.

[0138] Example 3

[0139] Except that the dosage of nucleic acid is 4 wt% (keeping the mass ratio of nucleic acid to wheat (TRITICUM VULGARE) germ extract the same as that in Example 1), the rest is the same as in Example 1, and the specific formulation is as follows:

[0140] Nucleic acid (sodium DNA) 4 wt%, wheat (TRITICUM VULGARE) germ extract 1.2 wt%, thickening agent (chondrus crispus extract) 0.5 wt%, preservative (hexylene glycol) 2 wt% and water balance (total 100 wt%); among them, the mass ratio of nucleic acid to wheat (TRITICUM VULGARE) germ extract is 3.3:1.

[0141] Example 4

[0142] Except that the dosage of nucleic acid is 2 wt%, the mass ratio of nucleic acid to wheat (TRITICUM VULGARE) germ extract is 4:1, and the pH is adjusted to 5, the rest is the same as in Example 1, and the specific formulation is as follows:

[0143] Nucleic acid (sodium DNA) 2 wt%, wheat (TRITICUM VULGARE) germ extract 0.5 wt%, thickener (chondrus crispus extract) 0.5 wt%, preservative (hexylene glycol) 2 wt% and the balance water (total 100 wt%); wherein, the mass ratio of nucleic acid to wheat (TRITICUM VULGARE) germ extract is 4:1.

[0144] Example 5

[0145] Except that the dosage of nucleic acid is 2 wt% and the mass ratio of nucleic acid to wheat (TRITICUM VULGARE) germ extract is 6:1, the rest is the same as Example 4, and the specific formula is as follows:

[0146] Nucleic acid (sodium DNA) 2 wt%, wheat (TRITICUM VULGARE) germ extract 0.33 wt%, thickener (chondrus crispus extract) 0.5 wt%, preservative (hexylene glycol) 2 wt% and the balance water (total 100 wt%); wherein, the mass ratio of nucleic acid to wheat (TRITICUM VULGARE) germ extract is 6:1.

[0147] Example 6

[0148] Except that the mass ratio of nucleic acid to wheat (TRITICUM VULGARE) germ extract is 2:1, the rest is the same as Example 4, and the specific formula is as follows:

[0149] Nucleic acid (sodium DNA) 2 wt%, wheat (TRITICUM VULGARE) germ extract 1 wt%, thickener (chondrus crispus extract) 0.5 wt%, preservative (hexylene glycol) 2 wt% and the balance water (total 100 wt%); wherein, the mass ratio of nucleic acid to wheat (TRITICUM VULGARE) germ extract is 2:1.

[0150] Example 7

[0151] Preparation of nucleic acid nanospheres:

[0152] The formula is as follows: Nucleic acid (sodium DNA) 4 wt%, wheat (TRITICUM VULGARE) germ extract 0.8 wt%, thickener (chondrus crispus extract) 0.5 wt%, preservative (hexylene glycol) 2 wt% and the balance water (total 100 wt%); wherein, the mass ratio of nucleic acid to wheat (TRITICUM VULGARE) germ extract is 5:1.

[0153] The preparation process is as follows:

[0154] Dissolve the nucleic acid in water (at 40 °C) accounting for 42% wt of the final product quality. After complete dissolution without agglomerated particles, the pressure for high-pressure homogenization treatment is 500 bar, and the number of times is 10. Then, cure at 20 °C for 1 h to form a nucleic acid semi-solid gel;

[0155] Heat the nucleic acid semi-solid gel to 40 °C and melt for 40 min, and adjust the pH to 5 with an anhydrous citric acid solution to obtain Phase A;

[0156] Dissolve the wheat (TRITICUM VULGARE) germ extract in the remaining water (at 40 °C). After the solution is completely dissolved, filter it, and adjust the pH to 5 with an anhydrous citric acid solution to obtain Phase B;

[0157] Add the said Phase B to the said Phase A, stir at 5000 rpm for 30 min, and add a thickening agent and a preservative to obtain nucleic acid nanospheres.

[0158] Example 8

[0159] Except for dissolving the nucleic acid in water at 60 °C and dissolving the wheat (TRITICUM VULGARE) germ extract in water at 60 °C, the rest is the same as in Example 7.

[0160] Example 9

[0161] Except for dissolving the nucleic acid in water at 80 °C and dissolving the wheat (TRITICUM VULGARE) germ extract in water at 80 °C, the rest is the same as in Example 7.

[0162] Example 10

[0163] Except for adjusting the pH to 4 and the melting temperature to 60 °C, the rest is the same as in Example 8.

[0164] Example 11

[0165] Except for adjusting the pH to 5, the rest is the same as in Example 10.

[0166] Example 12

[0167] Except for adjusting the pH to 6, the rest is the same as in Example 10.

[0168] Example 13

[0169] Preparation of nucleic acid nanospheres:

[0170] The formula is as follows: nucleic acid (sodium DNA) 4 wt%, soybean (GLYCINE MAX) extract 1.2 wt%, thickener (chondrus crispus extract) 0.5 wt%, preservative (hexylene glycol) 4 wt%, and the balance is water (total 100 wt%); among them, the mass ratio of nucleic acid to soybean (GLYCINE MAX) extract is 3.3:1.

[0171] The preparation process is as follows:

[0172] Dissolve the nucleic acid in 42% wt of water (60 °C) of the final product. After complete dissolution without agglomerated particles, the pressure for high-pressure homogenization is 500 bar, and the number of times is 10 times. Then place it at 20 °C for curing for 1 h to form a nucleic acid semi-solid gel.

[0173] Heat the nucleic acid semi-solid gel to 60 °C and melt it for 60 min, and adjust the pH to 5.5 with an anhydrous citric acid solution to obtain Phase A.

[0174] Dissolve the soybean (GLYCINE MAX) extract in the remaining water (60 °C). After the solution is completely dissolved, filter it, and adjust the pH to 5.5 with an anhydrous citric acid solution to obtain Phase B.

[0175] Add the said Phase B to the said Phase A, stir at 5000 rpm for 30 min, and add the thickener and the preservative to obtain nucleic acid nanospheres.

[0176] Example 14

[0177] Preparation of nucleic acid nanospheres:

[0178] The formula is as follows: nucleic acid (sodium DNA) 4 wt%, pea (PISUM SATIVUM) extract 1 wt%, thickener (chondrus crispus extract) 0.5 wt%, preservative (hexylene glycol) 4 wt%, and the balance is water (total 100 wt%); among them, the mass ratio of nucleic acid to pea (PISUM SATIVUM) extract is 4:1.

[0179] The preparation process is as follows:

[0180] Dissolve the nucleic acid in 42% wt of water (60 °C) of the final product. After the solution is clear and transparent, the pressure for high-pressure homogenization is 500 bar, and the number of times is 10 times. Then place it at 20 °C for curing for 1 h to form a nucleic acid semi-solid gel.

[0181] Heat the nucleic acid semi-solid gel to 60 °C and melt it for 60 min, and adjust the pH to 4.5 with an anhydrous citric acid solution to obtain Phase A.

[0182] Dissolve the pea (PISUM SATIVUM) extract in the remaining water (60 °C). After the solution is completely dissolved, filter it and adjust the pH to 4.5 with an anhydrous citric acid solution to obtain Phase B.

[0183] Add the obtained Phase B to Phase A, stir at 5000 rpm for 30 min, and add a thickener and a preservative to obtain nucleic acid nanospheres.

[0184] Example 15

[0185] Preparation of nucleic acid nanospheres:

[0186] The formula is as follows: nucleic acid (sodium DNA) 4 wt%, sesame (SESAMUM INDICUM) extract 0.6 wt%, thickener (chondrus crispus extract) 0.5 wt%, preservative (hexylene glycol) 4 wt% and the balance is water (total 100 wt%); among them, the mass ratio of nucleic acid to sesame (SESAMUM INDICUM) extract is 6.67:1.

[0187] The preparation process is as follows:

[0188] Dissolve the nucleic acid in 42% wt of water (60 °C) of the final product mass. After the solution is clear and transparent, the pressure for high-pressure homogenization is 500 bar and the number of times is 10 times. Then place it at 20 °C for curing for 1 h to form a nucleic acid semi-solid gel.

[0189] Heat the nucleic acid semi-solid gel to 60 °C and melt it for 60 min, and adjust the pH to 4 with an anhydrous citric acid solution to obtain Phase A.

[0190] Dissolve the sesame (SESAMUM INDICUM) extract in the remaining water (60 °C). After the solution is completely dissolved, filter it and adjust the pH to 4 with an anhydrous citric acid solution to obtain Phase B.

[0191] Add the obtained Phase B to Phase A, stir at 5000 rpm for 30 min, and add a thickener and a preservative to obtain nucleic acid nanospheres.

[0192] Comparative Example 1

[0193] The formula is as follows: nucleic acid (sodium DNA) 10 wt%, wheat (TRITICUM VULGARE) germ extract 2 wt%, thickener (chondrus crispus extract) 0.5 wt%, preservative (hexylene glycol) 2 wt% and the balance is water (total 100 wt%); among them, the mass ratio of nucleic acid to wheat (TRITICUM VULGARE) germ extract is 5:1;

[0194] The preparation process is the same as that of Example 11.

[0195] Comparative Example 2

[0196] The formulation is as follows: nucleic acid (sodium DNA) 2 wt%, wheat (TRITICUM VULGARE) germ extract 4 wt%, thickener (chondrus crispus extract) 0.5 wt%, preservative (hexylene glycol) 2 wt%, and the balance is water (total 100 wt%); among them, the mass ratio of nucleic acid to wheat (TRITICUM VULGARE) germ extract is 1:2;

[0197] The preparation process is the same as that of Example 11.

[0198] Comparative Example 3

[0199] Except that the melting temperature is 20 °C, the rest is the same as that of Example 11.

[0200] Comparative Example 4

[0201] Except that pH adjustment is not carried out, the rest is the same as that of Example 11.

[0202] Comparative Example 5

[0203] Except that high-pressure homogenization is not carried out, the rest is the same as that of Example 11. The particle size and PDI of this microsphere are 1250 nm and 0.87 respectively, both higher than those of Example 11, and there will be precipitation at the bottom after standing for 7 days.

[0204] Comparative Example 6

[0205] Except that curing is not carried out, the rest is the same as that of Example 11. When it is just prepared, its particle size and PDI are similar to those of Example 11, but after standing for one day, the particle size and PDI will increase, and precipitation will occur. Photographs of the bottom of the bottle after standing for one day in Example 11 and Comparative Example 6 are shown in Figure 1 .

[0206] Comparative Example 7

[0207] Prepare a free sodium DNA solution: Dissolve 2 wt% of sodium DNA in 96 wt% of deionized water, and add 2 wt% of hexylene glycol to obtain a 2 wt% free sodium DNA solution. The particle size of this solution is 1320 nm, much higher than that of the nucleic acid nanospheres in Examples 1-15.

[0208] Comparative Example 8

[0209] Cationic liposomes are used to encapsulate nucleic acids, specifically as follows: Weigh raw materials according to the ratio of 19490 parts of phosphate buffer solution, 160 parts of soybean lecithin, 20 parts of cholesterol, 160 parts of solubilizer - polyethylene glycol, 160 parts of glycerol, and 10 parts of nucleic acid, mix at 30 °C for 20 min, and obtain a suspension after ultrasonication for 3 min. The suspension is filtered through a microporous membrane with a diameter of 0.45 μm to obtain cationic liposomes;

[0210] The nucleic acid encapsulated by cationic liposomes through electrostatic adsorption is 0.5%, which is lower than the drug loading of nucleic acid nanospheres.

[0211] Test Example 1

[0212] The particle size, PDI, and Zeta potential of the nucleic acid microspheres in the examples and comparative examples were measured using a ZETASIZER PRO particle size analyzer. The particle size and PDI were tested according to the method specified in "GB / T 19077-2016 Particle Size Distribution - Laser Diffraction Method". The Zeta potential was tested according to "GB / Z 42353-2023 Zeta Potential Measurement Operation Guide". The results are shown in Table 1.

[0213] Table 1 Summary of the performance of nucleic acid microspheres in examples and comparative examples

[0214]

[0215] As can be seen from Table 1, the nucleic acid microspheres prepared in Examples 1-15 of this application have a smaller diameter, a uniform particle size distribution, and high stability. The nucleic acid microspheres prepared in Comparative Examples 1-4 outside the defined range have a significantly increased diameter and significantly deteriorated uniformity.

[0216] Test Example 2 Determination of DNA sodium content by ultraviolet spectrophotometer

[0217] The standard curve of DNA sodium content was determined as follows:

[0218] Take 0.05 g of nucleic acid (DNA sodium) powder and make it up to volume in a 50 mL volumetric flask to prepare a 1 mg / mL solution;

[0219] Dilute the above solution to prepare solutions with concentrations of 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, and 0.04 mg / mL;

[0220] Use an ultraviolet spectrophotometer to detect its absorption at 260 nm (the baseline is pure water);

[0221] Perform linear regression on the absorbance (Y) against the sample mass concentration (X) for calculating the DNA sodium content in the transdermal absorption experiment. The standard curve is as Figure 2 shown. The linear regression equation of the DNA sodium standard curve is Y = 19.58X + 0.05, R 2 = 0.9908.

[0222] After detection, the measured value of the DNA content in the nucleic acid raw material is 80%.

[0223] Test Example 3 Determination of spermidine content by high performance liquid chromatography

[0224] Determination of the standard curve for spermidine content, and the preparation steps are as follows:

[0225] Preparation of spermidine standard stock solution: Accurately weigh 0.01 g of spermidine standard, place it in a 10 mL small beaker, dissolve it with 0.1 mol / L hydrochloric acid solution, transfer it to a 10 mL volumetric flask, make up the volume to the mark, mix well, and prepare a standard stock solution with a concentration of 1000 mg / L (calculated based on each biogenic amine monomer).

[0226] Pipette 1 mL of the spermidine standard stock solution into a 10 mL volumetric flask, dilute it to the mark with 0.1 mol / L hydrochloric acid, and mix well to prepare a spermidine standard working solution (100 mg / L).

[0227] Preparation of spermidine standard series solutions: Pipette 0.10 mL, 0.25 mL, 0.50 mL, 1.0 mL, 1.50 mL, 2.50 mL, 5.0 mL of the spermidine standard working solution (100 mg / L) into 10 mL volumetric flasks respectively, dilute them to the mark with 0.1 mol / L hydrochloric acid solution, and mix well to make the concentrations 1.0 mg / L, 2.5 mg / L, 5.0 mg / L, 10.0 mg / L, 15.0 mg / L, 25.0 mg / L, 50.0 mg / L respectively.

[0228] Dansyl chloride derivatizing agent solution: Accurately weigh an appropriate amount of dansyl chloride, prepare a derivatizing agent working solution with a concentration of 10 mg / mL using acetone as the solvent, and store it in a refrigerator at 4 °C in the dark.

[0229] 0.1 mol / L hydrochloric acid solution: Accurately measure 8.6 mL of hydrochloric acid into a 100 mL volumetric flask, and make up the volume to the mark with water.

[0230] 1 mol / L sodium hydroxide solution: Weigh 4 g of sodium hydroxide, add 100 mL of water and dissolve it completely.

[0231] Saturated sodium bicarbonate solution: Weigh 15 g of sodium bicarbonate, add 100 mL of water to dissolve it, and take the supernatant as the saturated solution.

[0232] 50 mg / mL sodium glutamate solution: Accurately weigh 5 g of sodium glutamate, dissolve it with saturated sodium bicarbonate solution, and make up the volume to 100 mL.

[0233] 0.01 mol / L ammonium acetate solution containing 1% acetic acid: Weigh 0.77 g of ammonium acetate, dissolve it in water, transfer it to a 1000 mL volumetric flask, add 10 mL of formic acid, and make up the volume to the mark with water.

[0234] Mobile phase A: Measure 100 mL of 0.01 mol / L ammonium acetate solution containing 1% acetic acid and add 900 mL of acetonitrile.

[0235] Mobile phase B: Measure 900 mL of 0.01 mol / L ammonium acetate solution containing 1% acetic acid and add 100 mL of acetonitrile.

[0236] Preparation of internal standard standard stock solution: Accurately weigh an appropriate amount of the internal standard standard 1,7-diaminoheptane, place it in a 10 mL volumetric flask, dissolve it with 0.1 mol / L hydrochloric acid solution and dilute to the mark, mix well, and prepare an internal standard standard stock solution with a concentration of 10 mg / mL. Store it in a refrigerator at -20 °C, and the shelf life is 6 months.

[0237] Preparation of internal standard standard intermediate working solution: Pipette 1.0 mL of the internal standard standard stock solution into a 10 mL volumetric flask, dilute it to the mark with 0.1 mol / L hydrochloric acid, mix well, and use it as the internal standard working solution (1.0 mg / mL). The storage period is 3 months.

[0238] Preparation of internal standard standard working solution: Pipette 1 mL of the internal standard standard stock solution into a 10 mL volumetric flask, dilute it to the mark with 0.1 mol / L hydrochloric acid, mix well, and use it as the internal standard working solution (100 mg / L). Prepare it freshly before use.

[0239] Derivatization of standards:

[0240] Respectively pipette 1 mL of the spermidine standard series solution into 10 mL volumetric flasks, successively add 250 μL of the internal standard working solution (100 mg / L), 1 mL of saturated sodium bicarbonate solution, 100 μL of sodium hydroxide solution (1 mol / L), and 1 mL of dansyl chloride derivatization reagent. Vortex and mix for 1 min, then place it in a constant temperature water bath at 60 °C for derivatization for 15 min. Take it out, add 100 μL of sodium glutamate solution respectively, shake and mix well, and react at 60 °C for 15 min. Take it out, cool to room temperature, add 1 mL of water to each centrifuge tube, vortex and mix for 1 min, blow off acetone (about 1 mL) under nitrogen at 40 °C in a water bath, add 0.5 g of sodium chloride, vortex and shake until the sodium chloride is completely dissolved, then add 5 mL of ether, vortex and shake for 2 min. After standing and separating layers, suck out the upper organic phase (ether layer), extract it once again, combine the ether extraction solutions, and dry it under nitrogen at 40 °C in a water bath. Add 1 mL of acetonitrile, vortex and shake to completely dissolve the residue, filter it through a 0.22 μm syringe filter into an injection vial for determination.

[0241] Chromatographic conditions:

[0242] The chromatographic column was a C18 column (column length 250 mm, column inner diameter 4.6 mm, column packing particle size 5 μm), the ultraviolet detection wavelength was 254 nm, the injection volume was 20 μL, the column temperature was 35 °C, mobile phase A was 90% acetonitrile / 10% (0.01 mol / L ammonium acetate solution containing 0.1% acetic acid), mobile phase B was 10% acetonitrile / 90% (0.01 mol / L ammonium acetate solution containing 0.1% acetic acid), and the flow rate was 0.8 mL / min. The proportions of the mobile phases are shown in Table 2:

[0243] Table 2 Proportions of the mobile phases

[0244]

[0245] Linear regression was performed with the peak area (Y) against the sample mass concentration (X) for the calculation of the spermidine content. The standard curve is as Figure 3 shown. The linear regression equation of the spermidine standard curve was Y = 329.25X + 29.39, and R 2 = 0.9996.

[0246] Upon detection, the measured values of the spermidine content in wheat (TRITICUM VULGARE) germ extract, soybean (GLYCINE MAX) extract, pea (PISUM SATIVUM) extract, and sesame (SESAMUM INDICUM) extract were 54.1%, 51.8%, 54.6%, and 53.5% respectively.

[0247] Test Example 4 SEM electron microscopy observation of the morphology of nucleic acid nanospheres

[0248] The nucleic acid nanospheres prepared in Example 11 were subjected to SEM detection.

[0249] Take the nucleic acid nanosphere sample. After ultrasonic dispersion, it was dropped on a silicon wafer. After drying, it was sputtered with gold for about 120 s, and tested using a field emission scanning electron microscope (Thermo Fisher Quattro S). The acceleration voltage was 10 kv, the shooting magnification range was 3 - 30k, and the test mode was the secondary electron mode. The results are as Figure 4 shown. The SEM image obtained by diluting the solution 20 times shows that the particles are spherical-like and are relatively evenly distributed.

[0250] Test Example 5 TEM electron microscopy observation of the morphology of nucleic acid nanospheres

[0251] The nucleic acid nanospheres prepared in Example 11 were subjected to TEM detection.

[0252] Weigh the nucleic acid nanospheres sample prepared in Example 11, drop it on a copper grid. After a few seconds, gently pick up the copper grid sample with forceps, and absorb the excess liquid along one side with filter paper. After it dries slightly, place the copper grid 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 5 shown. The TEM image obtained by diluting the solution 10 times shows that the particles are spherical-like. According to the scale bar, it can be shown that the particle size is roughly the same as that measured by the particle size analyzer.

[0253] Test Example 6 Investigation of the stability of nucleic acid nanospheres

[0254] The nucleic acid nanospheres of Examples 1-15 were respectively placed under the conditions of daily, light, dark, 4 °C, 45 °C, -15 °C, and freeze-thaw cycle (one freeze-thaw cycle is placing at -15 °C for 24 h and then at 45 °C for 24 h), and their particle size stability was measured. The test results of Example 11 are shown in Table 3. The test results of other examples are equivalent to those of Example 11. It can be seen that the nucleic acid nanospheres of the present application have good stability.

[0255] Table 3 Stability data of Example 11

[0256]

[0257] Test Example 7 Comparative investigation of the stability of nucleic acid nanospheres

[0258] The nucleic acid nanospheres prepared in Example 11 were subjected to a stability test.

[0259] Absorb a certain amount of the sample into the sample bottle of the Turbiscan Lab stability analyzer to measure the dynamic changes in the stability of the essence. The parameter settings are to scan once every 30 min for 24 h. The experimental results of the nanoemulsion of Example 11 are shown in Figure 6 . Generally, it is considered that when the average value of the backscattered light intensity is less than 0.2, the system is in an absolutely stable state. The average value of the backscattered light intensity △BS of this system is 0.14, indicating good stability at room temperature.

[0260] Test Example 8 Comparative analysis of in vitro skin permeability and retention

[0261] The in vitro transdermal experiment was carried out on Example 11 of the present application and Comparative Example 7 using a Franz diffusion cell. The abdominal skin of guinea pigs weighing 200 - 250 g was used as the transdermal test barrier layer, and the intact and undamaged skin was fixed between the receiving cell and the supply cell. The effective diffusion area was 1.77 cm 2, the volume of the receiving pool is about 12 ml, and the magnetic stirring speed is 300 r / min. The receiving pool is filled with the release medium (normal saline), air bubbles are removed, stirring is started, and the temperature is kept constant at (32.0 ± 0.5) °C. Samples containing an equal amount of sodium DNA are evenly coated on the skin surface. At the set times of 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h, 1 ml of the receiving solution is aspirated with a long-headed sampling needle, and the sample solution is placed in an EP tube. First, the receiving pool is evacuated of air bubbles by traction using a non-porous puncture needle, and then 1 ml of the receiving solution is added to the receiving pool. The concentration of sodium DNA in the receiving solution filtered through a 0.22 μm filter membrane is measured using an ultraviolet spectrophotometer, and the cumulative permeation amount of the drug at different times is calculated. The cumulative permeation amount of sodium DNA is calculated according to the following formula:

[0262]

[0263] In the formula, Q n is the cumulative permeation amount at the nth time point per unit area (μg / cm 2 ); V o is the volume of the liquid in the receiving pool (ml); V is the sampling volume (ml); C n is the mass concentration of the drug in the receiving solution measured at the nth time point (μg / ml); C n-1 is the mass concentration of the drug in the receiving solution measured at the (n - 1)th sampling point (μg / ml); S is the effective area.

[0264] After 24 h, the mouse skin is cut into pieces and placed in a 1.5 ml EP tube. 1 ml of ethanol is added and ultrasonicated for 30 min. After ultrasonication, it is centrifuged at 5000 r / min for 10 min, and the supernatant is collected into the EP tube. It is filtered through a 0.22 μm microporous filter membrane, and the concentration of sodium DNA in the receiving solution filtered through a 0.22 μm filter membrane is measured using an ultraviolet spectrophotometer, and the retention amount of the drug in the skin is calculated. The retention amount of sodium DNA in the skin is calculated according to the following formula:

[0265] Q s = VC / A

[0266] In the formula, Qs is the retention amount of sodium DNA in the skin per unit area (μg / cm 2 ); V is the total volume of the skin extract (ml); A is the effective diffusion area (cm 2 ); C is the concentration of the drug in the skin extract (μg / ml).

[0267] The transdermal tests of Example 11 and Comparative Example 7 of this application are carried out. The test results are as Figure 7 , Figure 8 shown,

[0268] From Figure 8It can be seen from the 24-hour cumulative permeation amount and skin retention amount that the 24-hour cumulative permeation amounts of the nucleic acid nanospheres of Example 11 and the free DNA sodium solution of Comparative Example 7 are respectively: 1.52 mg / cm 2 , 0.26 mg / cm 2 , and their 24-hour skin retention amounts are respectively: 0.28 mg / cm 2 , 0.21 mg / cm 2 . It can be seen that both the permeation amount and retention amount of the nucleic acid nanospheres are higher than those of the free nucleic acid solution, and are increased to 5.85 times and 1.33 times respectively compared with the free nucleic acid solution. The experimental results are all in line with the action path of sodium DNA in the skin layer. Obviously, the nucleic acid nanospheres of the present application have the effect of improving the transdermal penetration and skin retention of active substances, thus can improve the bioavailability of transdermal drug products, promote the absorption and use efficiency of drug products, and improve the skin care effect of cosmetic products.

[0269] Effect of nucleic acid nanospheres of Test Example 9 on macrophage phagocytosis ability

[0270] The M0 type RAW264.7 macrophages (resting or undifferentiated RAW264.7) were carefully inoculated on the well plate at a cell density of 5×10 4 cell / well. After the well plate was placed in an incubator at 37 °C and 5% CO2 for co-culture for 12 h, the culture medium in the well plate was removed, and the culture medium with different concentrations of Example 11 was added and allowed to act for 24 h; 0.03 μm latex beads (referred to as fluorescent microspheres) of carboxylate-modified polystyrene with spontaneous fluorescence of yellow-green were treated with FBS for 1 h, and then resuspended in the culture medium and added to the macrophage well plate, incubated for 6 h. After phagocytosis was completed, the 12-well plate was taken out of the cell culture incubator and placed on ice for 20 - 30 s to stop phagocytosis; the culture medium was discarded, and the cells were washed twice with ice-cold PBS to wash away the unphagocytosed fluorescent microspheres, and 0.04% trypan blue solution was added and treated for 30 min to quench the fluorescence of the extracellular microspheres, and then washed with PBS. Finally, it was fixed with 4% paraformaldehyde at room temperature for 10 min, washed twice with PBS, the cell nuclei were stained with DAPI dye, and then washed 3 times with PBS and observed under a laser confocal microscope (blue excitation light channel: DragonGreen (λex - 480, λem - 520)) for the situation of macrophages phagocytosing fluorescent microspheres.

[0271] The results are as Figure 9As shown, in the blank control group, weak fluorescence was visible in a small number of macrophages, representing their basal phagocytic ability. The fluorescence in macrophages of the model control group should be significantly stronger and widely distributed, indicating that more cells successfully phagocytosed the fluorescent label and the phagocytic amount was large. The fluorescence intensity of the experimental group using the nucleic acid nanospheres of Example 11 at 0.025% was higher than that of the blank control group and even close to or exceeding that of the model control group, indicating that the nucleic acid nanospheres prepared in this application can effectively promote the phagocytic ability of macrophages and significantly improve immune function. Macrophages play an important role in aspects such as anti-infection, immune regulation, anti-tumor, and anti-inflammation. Therefore, the nucleic acid nanospheres of this application can be used to prepare anti-infection drugs, immune regulation drugs, anti-tumor drugs, or anti-inflammatory drugs.

[0272] 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-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0273] The above-described embodiments only represent several implementation manners of this application. Their descriptions are relatively specific and detailed, but they should not be construed as limiting 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 this application, several modifications and improvements can still be made, and these all belong to the protection scope of this application. Therefore, the protection scope of the patent of this application should be subject to the appended claims.

Claims

1. A method for preparing nucleic acid nanoparticles, characterized in that: The steps include: The nucleic acid is first mixed with a portion of water, pretreated, and solidified to form a nucleic acid semi-solid gel; The nucleic acid semi-solid gel is melted at 45-80°C and the pH is adjusted to 4-6 to form phase A; The plant extract containing spermidine is mixed with the remaining water for a second time and the pH is adjusted to 4-6 to form phase B; The phase A, the phase B and the auxiliary agent are mixed for the third time to form the nucleic acid nanoparticles; The nucleic acid nanoparticles include the following components by mass percentage: 0.5-8% nucleic acid, 0.1-7.5% plant extract containing spermidine, 0-6% auxiliary agent and 78.5-99.4% water; The mass ratio of the nucleic acid to the plant extract containing spermidine is 1-10:

1.

2. The preparation method according to claim 1, characterized in that One or more of the following conditions are met: (1) The nucleic acid comprises at least one of DNA and a DNA derivative; (2) the plant extract containing spermidine includes at least one of wheat germ extract, soybean extract, pea extract and sesame extract; (3) The pH adjusting agent includes citric acid; (4) The auxiliary agent includes at least one of a thickener and a preservative; (5) The amount of the water is 35-60wt% of the nucleic acid nanoparticles; (6) The content of spermidine in the spermidine-containing plant extract is ≥ 30 wt %.

3. The preparation method according to claim 2, characterized in that: One or more of the following conditions are met: (1) The thickener comprises at least one of Chondrus crispus extract, xanthan gum, Sclerotium gum and AVC; (2) the preservative comprises at least one of hexylene glycol, pentylene glycol and ethylhexylglycerin; (3) The amount of the thickener is 0.1-0.5wt%; (4) The amount of the preservative is 1-5wt%.

4. The preparation method according to any one of claims 1 to 3, characterized in that: One or more of the following conditions are met: (1) The temperature of the first mixing is 40-80°C; (2) The pretreatment includes homogenization and / or microfluidization; (3) The curing conditions include: curing temperature 10-20°C; curing time 0.5-24h; (4) The temperature of the second mixing is 40-80°C; (5) The temperature of the third mixing is 40-80°C; (6) The third mixing comprises the following steps: adding phase B to phase A, stirring at 2000-12000 rpm for 5-60 min; then adding an auxiliary agent and mixing; (7) The melting time is 40-120 minutes.

5. The preparation method according to claim 4, characterized in that: One or more of the following conditions are met: (1) The homogenization conditions include: homogenization temperature 40-60°C; homogenization pressure 100-2000 bar; homogenization times 2-20 times; (2) The conditions of the microfluidic treatment include: pressure 1400-15000Psi, and treatment times 1-5 times.

6. The preparation method according to claim 4, characterized in that: The nucleic acid nano-microspheres include the following components by mass percentage: 0.5%-8% nucleic acid, 0.1%-7.5% plant extract containing spermidine, 0.1%-0.5% thickener, 1%-5% preservative and 79%-98.3% water.

7. The nucleic acid nanoparticles prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the nucleic acid nanoparticles according to claim 7 in preparing products for transdermal administration.

9. The use according to claim 8, characterized in that The products include immunity-enhancing drugs or cosmetics; Optionally, the immunity enhancing drug includes an anti-infective drug, an immunomodulatory drug, an anti-tumor drug or an anti-inflammatory drug.

10. A drug for improving immunity, characterized in that: The raw materials include nucleic acid nanoparticles prepared by the preparation method according to any one of claims 1 to 6.

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