A nucleic acid nanomicrosphere, a preparation method and application thereof

Abstract

The application discloses a nucleic acid nanomicrosphere and a preparation method and application thereof, and belongs to the technical field of nano delivery. The preparation method comprises the following steps: mixing nucleic acid with part of water to perform pretreatment, solidification, and forming a nucleic acid semi-solid gel; melting the nucleic acid semi-solid gel at 45-80 DEG C, adjusting the pH to 4-6, and forming phase A; mixing spermidine-containing plant extract with the rest of the water to form phase B; mixing phase A, phase B and an auxiliary agent to form the nucleic acid nanomicrosphere; the nucleic acid nanomicrosphere comprises the following components in percentage by mass: nucleic acid 0.5-8%, spermidine-containing plant extract 0.1-7.5%, auxiliary agent 0-6% and water 78.5-99.4%; the mass ratio of the nucleic acid to the spermidine-containing plant extract is 1-10:1. The nucleic acid nanomicrosphere has improved transdermal effect and stability, and can be administered transdermally.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanometer delivery technology, in particular to a nucleic acid nanomicrosphere and a preparation method and application thereof. BACKGROUND

[0002] Nucleic acid is an important biological molecule in vivo, which plays a key role in storing and transmitting genetic information in cells. The commonly used nucleic acid raw materials are deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and their salts. Among them, DNA sodium as a kind of biological active molecule of growth factor is one of the raw materials for DNA synthesis in cells, which has natural biocompatibility. It has been widely used in small nucleic acid drugs, mRNA vaccines, gene therapy and other medical fields, and plays a strong anti-inflammatory repair effect in wound healing, scar repair, joint injury and other aspects, and has a positive promoting effect on vascularization, collagen synthesis, fibroblast and osteoblast cell activity.

[0003] However, most nucleic acids have a short half-life and a short shelf life, for example, the half-life of DNA sodium is only about 3 hours, the stability is poor, and the bioavailability is low; the nucleic acid surface carries a negative charge, has a high molecular weight and high hydrophilicity, and is not easy to penetrate the cell membrane to reach the intracellular to play a role; the targeting of nucleic acid in vivo is weak and easy to off-target. The above characteristics greatly limit the application of nucleic acid in transdermal drug delivery products and the like.

[0004] Microspheres are small spherical particles with a diameter of microns, which are made of natural or synthetic polymer materials, and can encapsulate drugs, biological active substances, dyes, catalysts and other substances. Microspheres have a highly uniform spherical structure, which makes them have good stability and repeatability in physical and chemical properties. The surface can be modified to change its hydrophilicity, hydrophobicity, charge properties, etc., so as to realize specific functions. The nucleic acid is made into a nucleic acid microsphere, which can improve the stability, bioavailability and targeting of the nucleic acid.

[0005] However, the stability of the nucleic acid microsphere is limited, and the drug loading capacity is insufficient. SUMMARY

[0006] Therefore, the main purpose of the present application is to provide a preparation method of a nucleic acid nanomicrosphere, so as to prepare a nucleic acid nanomicrosphere with improved transdermal effect and stability, which can effectively load nucleic acid and meet the application requirements in transdermal drug delivery products and the like.

[0007] In a first aspect, the present application provides a preparation method of a nucleic acid nanomicrosphere, comprising the following steps:

[0008] The nucleic acid is first mixed with part of water for pretreatment, solidification, and formation of a nucleic acid semi-solid gel;

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

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

[0011] A phase, B phase and an auxiliary agent are mixed to form the nucleic acid nanomicrosphere;

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

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

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

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

[0016] (2) The spermidine-containing plant extract comprises 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 pH adjusting agent comprises citric acid;

[0018] (4) The auxiliary agent comprises at least one of thickening agent and preservative;

[0019] (5) The amount of the part of water is 35-60wt% of the nucleic acid nanomicrosphere;

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

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

[0022] (1) The thickening agent comprises at least one of wrinkle chondrus extract, xanthan gum, microbacterial gum and AVC;

[0023] (2) The preservative comprises at least one of hexanediol, pentanediol and ethylhexyl glycerol;

[0024] (3) The amount of the thickening agent is 0.1-0.5wt%;

[0025] (4) the amount of the preservative is 1-5wt%.

[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℃;

[0028] (2) the pretreatment comprises homogenization treatment and / or microfluidization treatment;

[0029] (3) the solidification condition comprises: solidification temperature 10-20℃; solidification time 0.5-24h;

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

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

[0032] (6) the third mixing comprises the following steps: adding B phase into A phase, stirring at 2000-12000rpm for 5-60min; then adding the adjuvant and mixing;

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

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

[0035] (1) the homogenization treatment condition comprises: homogenization temperature 40-60℃; homogenization pressure 100-2000bar; homogenization times 2-20;

[0036] (2) the microfluidization treatment condition comprises: pressure 1400-15000Psi, treatment times 1-5.

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

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

[0039] In a third aspect of the present application, the nucleic acid nanosphere of the second aspect is used in the preparation of a transdermally administered product.

[0040] In some embodiments, the product comprises an immunity-enhancing drug or cosmetic;

[0041] Optionally, the immunity-boosting drug comprises an anti-infection drug, an immunomodulatory drug, an anti-tumor drug, or an anti-inflammatory drug.

[0042] Optionally, the cosmetic comprises at least one of a mask liquid, a skin care water, an essence, a spray, and a lotion.

[0043] In a fourth aspect of the present application, an immunity-boosting drug is provided, and the raw material comprises the nucleic acid nanosphere of the second aspect.

[0044] In a fifth aspect of the present application, a cosmetic is provided, and the raw material comprises the nucleic acid nanosphere of the second aspect.

[0045] Advantages of the present application:

[0046] 1. The nucleic acid nanosphere of the present application is prepared by electrostatic adsorption self-assembly of positively charged spermidine-containing plant extract and negatively charged nucleic acid, and the preparation process is optimized, so that the nucleic acid nanosphere with high nucleic acid loading, good stability and uniform nanoscale particle size can be prepared, which has better permeability and targeting, is more easily diffused to the inside of the skin, has improved transdermal effect, can improve the bioavailability of nucleic acid, and meets the application requirements in transdermal drug delivery products.

[0047] 2. The nucleic acid nanosphere of the present application has the effect of enhancing the phagocytic ability of macrophages, can improve immunity, and can be used for preparing immunity-boosting drugs such as anti-infection drugs, immunomodulatory drugs, anti-tumor drugs, or anti-inflammatory drugs.

[0048] 3. The nucleic acid nanosphere of the present application has high uniformity and high stability, is mild to the human body and has a sustained-release effect, and exhibits excellent stability under 7 different harsh environments of normal temperature, refrigeration, freezing, heating, freeze-thaw, light, and darkness. BRIEF DESCRIPTION OF DRAWINGS

[0049] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiment or prior art description will be briefly introduced below, and the drawings are only used for the purpose of illustrating the preferred embodiments, and are not considered as limiting the present application. In the entire drawings, the same reference signs represent the same parts. In the drawings:

[0050] Figure 1 The bottle bottom photos of Example 11 and Comparative Example 6 after being placed for 1 day, wherein the left photo is Example 11 and the right photo is Comparative Example 6;

[0051] Figure 2 The standard curve for determination of DNA sodium content;

[0052] Figure 3 The standard curve for determination of spermidine content;

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

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

[0055] Figure 6 Turbiscan lab stability analyzer result chart of the nucleic acid nanospheres prepared in Example 11;

[0056] Figure 7 In-vitro cumulative permeation curve comparison of the nucleic acid nanospheres prepared in Example 11 and the sample of Comparative Example 7;

[0057] Figure 8 In-vitro 24h cumulative permeation amount and skin retention amount comparison 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 macrophage phagocytosis. DETAILED DESCRIPTION

[0059] In order to make the objectives, technical solutions and advantages of the present application clearer, and to make the understanding of the disclosure of the present application more thorough and comprehensive, the technical solutions of the present application will be described in detail below with reference to the specific embodiments of the present application and the corresponding drawings. The described embodiments are only some of the embodiments of the present application, not all the embodiments.

[0060] The embodiments of the present application will be described in detail below with reference to the drawings. The embodiments are implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present 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 one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application.

[0062] Terminology

[0063] Unless otherwise indicated or contradictory, the terms or phrases used herein have the following meanings:

[0064] In the present application, "a plurality of", "a plurality of kinds" and the like refer to more than two or equal to two in number, unless otherwise specified. For example, "one or more", "at least one" means one or more than or equal to two.

[0065] In the present application, "further", "particularly" and the like are used to describe purposes and indicate differences in content, but should not be understood as limiting the scope of protection of the present application.

[0066] In the present application, in the technical features described in an open form, both the closed technical solution consisting of the listed features and the open technical solution containing the listed features are included.

[0067] In the present application, with respect to a numerical interval (i.e. a numerical range), if no specific description is provided, the distribution of the optional values in the numerical interval is considered to be continuous, and includes both numerical end points (i.e. the minimum value and the maximum value) of the numerical interval and each value between the two numerical end points. If no specific description is provided, when the numerical interval only refers to integers in the numerical interval, the two end point integers of the numerical range and each integer between the two end points are equivalent to directly listing each integer. When multiple numerical ranges are provided to describe a feature or a property, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges encompassed therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. The "numerical interval" is allowed to broadly include numerical interval types such as percentage interval, ratio interval, and value interval.

[0068] In the present application, unless specifically limited, the temperature parameter allows both constant temperature treatment and treatment within a certain temperature interval. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuation within a range such as ±5°C, ±4°C, ±3°C, ±2°C and ±1°C is allowed.

[0069] In the present application, with respect to the unit of the data range, if only the unit is provided after the right end point, it means that the units of the left end point and the right end point are the same. For example, 2-5h means that the units of the left end point "2" and the right end point "5" are both h (hour).

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

[0071] In the present application, unless specifically limited, the temperature parameter allows both constant temperature treatment and treatment within a certain temperature interval. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. The room temperature described in the present application refers to 0-40°C, preferably 10°C-35°C, and further preferably 20°C-30°C.

[0072] In a first aspect of the present application, a preparation method of a nucleic acid nanomicrosphere is provided, comprising the following steps:

[0073] mixing the nucleic acid with part of the water to form a semi-solid gel of the nucleic acid by pretreatment and solidification;

[0074] melting the semi-solid gel of the nucleic acid at 45-80℃ and adjusting the pH to 4-6 to form phase A;

[0075] mixing the spermidine-containing plant extract with the rest of the water to form phase B by adjusting the pH to 4-6;

[0076] mixing phase A, phase B and the auxiliary agent to form the nucleic acid nanomicrosphere;

[0077] The nucleic acid nanomicrosphere comprises the following components by mass percentage: nucleic acid 0.5-8%, spermidine-containing plant extract 0.1-7.5%, auxiliary agent 0-6% and water 78.5-99.4%.

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

[0079] It is understood that the above mass percentage is calculated without the amount of the pH adjuster, i.e., the amount of the raw material other than the pH adjuster is 100%.

[0080] The positively charged spermidine-containing plant extract and the negatively charged nucleic acid are self-assembled by electrostatic adsorption to prepare the nucleic acid microsphere, and the preparation process is optimized, so that the nucleic acid microsphere with high nucleic acid loading, good stability and uniform nanoscale particle size is prepared, which has better permeability and targeting, is easier to diffuse into the skin, has improved transdermal effect, can improve the bioavailability of the nucleic acid, and meets the application requirements of the nucleic acid in transdermal drug delivery products.

[0081] In a specific example, the melting temperature is 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, etc. It is understood that the appropriate melting temperature can extend the double helix structure of the nucleic acid, promote the interaction between DNA and spermidine, and thus prepare the nanometer nucleic acid microsphere with small particle size and good uniformity.

[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 amount 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 understood that the nucleic acid microsphere is prepared by directly using the positively charged spermidine-containing plant extract and the negatively charged nucleic acid through electrostatic adsorption self-assembly, which can achieve high loading of the nucleic acid.

[0084] In one specific example, the nucleic acid comprises at least one of DNA and DNA derivatives, such as DNA sodium. In this case, 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 one specific example, the spermidine-containing plant extract is used in an amount of 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 one specific example, the spermidine-containing plant extract comprises 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 kind of polyamine substance, and its molecular structure contains multiple amino groups. These amino groups are protonated under physiological conditions, making spermidine positively charged, tightly combined with DNA sodium, and combined into microspheres through electrostatic attraction and hydrogen bonding, etc. to achieve targeted delivery and controlled release of nucleic acids. Wheat germ is one of the sources with relatively rich content of spermidine in plants, and its content of spermidine is usually at a high level compared with other plant tissues. Preferably, the content of spermidine in the spermidine-containing plant extract is ≥30wt%, such as 30-100wt%, and specifically can be 30wt%, 35wt%, 40wt%, 45wt%, 50wt%, 55wt%, 60wt%, 70wt%, 75wt%, 80wt%, 85wt%, 90wt%, 95wt%, 100wt%, etc.

[0088] In one specific example, the adjuvant comprises at least one of thickening agents and preservatives; and the specific types of thickening agents and preservatives are not particularly limited.

[0089] In one specific example, the thickening agent comprises at least one of Chondrus crispus (Carrageenan) extract, xanthan gum, small nucleus fungus gum and AVC; and its amount is 0.1-0.5wt%, such as 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, etc., preferably 0.3-0.5wt%.

[0090] In one specific example, the preservative includes at least one of hexylene glycol, pentylene glycol, and ethylhexylglycerin; and the amount 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 one 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 are self-assembled to prepare the nucleic acid microspheres by electrostatic adsorption, 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 conducive to the preparation of nanometer nucleic acid microspheres with small particle size and good uniformity.

[0092] In one specific example, the pH adjusting agent includes citric acid.

[0093] In one specific example, the amount of the part of water is 35-60 wt% of the nucleic acid nanometer microspheres, such as 35 wt%, 40 wt%, 45 wt%, 50 wt%, 55 wt%, 60 wt%, etc.

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

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

[0096] In one specific example, the homogenization treatment includes a homogenization temperature of 40-60℃, such as 40℃, 45℃, 50℃, 55℃, 60℃, etc.; a homogenization pressure of 100-2000 bar, such as 100 bar, 500 bar, 1000 bar, 1500 bar, 2000 bar, etc.; and a homogenization frequency of 2-20 times, such as 2 times, 5 times, 10 times, 15 times, 20 times, etc.

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

[0098] In one specific example, the conditions for the solidification include: a solidification temperature of 10-20℃, such as 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, etc.; a solidification time of 0.5-24h, such as 0.5h, 1h, 4h, 8h, 12h, 16h, 20h, 24h, etc. It can be understood that the solidification refers to standing at 10-20℃ for 0.5-24h to achieve gelation of the nucleic acid.

[0099] In one specific example, the temperature for the second mixing is 40-80℃, such as 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, etc.

[0100] In one specific example, the temperature for the third mixing is 40-80℃, such as 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, etc.

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

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

[0103] In one specific example, the nucleic acid nanomicrosphere includes the following components in terms of mass percentage: nucleic acid 0.5%-8%, spermidine-containing plant extract 0.1%-7.5%, thickening agent 0.1-0.5%, preservative 1-5%, and water 79%-98.3%.

[0104] In a second aspect of the present application, the nucleic acid nanomicrosphere prepared by the preparation method of the first aspect is provided.

[0105] The nucleic acid nanomicrosphere of the present application has high uniformity and high stability, is mild to the human body, has a sustained-release effect, exhibits excellent stability under 7 different harsh environments of normal temperature, refrigeration, freezing, heating, freeze-thawing, light, and dark, has improved transdermal effect, can improve the bioavailability of nucleic acid, and meets the application requirements in transdermal drug products and the like.

[0106] In one specific example, the nucleic acid nanosphere has a particle size of 100-700 nm, such as 100 nm, 2000 nm, 3000 nm, 4000 nm, 5000 nm, 6000 nm, 7000 nm, etc.; a PDI of 0.05-0.55, such as 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.55, etc.; and a pH of 4-6, such as 4, 4.5, 5, 5.5, 6, etc.

[0107] In a third aspect of the present application, the nucleic acid nanosphere of the second aspect is used in the preparation of a transdermally administered product.

[0108] The nucleic acid nanosphere of the present application has improved transdermal effect and stability, can improve the bioavailability of nucleic acids, and meets the application requirements in transdermally administered drugs and the like. The nucleic acid nanosphere of the present application has the effect of enhancing the phagocytic ability of macrophages, can improve immunity, and can be used in the preparation of anti-infective drugs, immunomodulatory drugs, anti-tumor drugs, or anti-inflammatory drugs and the like to improve immunity drugs.

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

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

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

[0112] In a fourth aspect of the present application, an immunity improving drug is provided, and the raw material includes the nucleic acid nanosphere of the second aspect.

[0113] The nucleic acid nanosphere of the present application has improved transdermal effect and stability, can improve the bioavailability of nucleic acids, and has the effect of enhancing the phagocytic ability of macrophages, can improve immunity, and can be used in the preparation of anti-infective drugs, immunomodulatory drugs, anti-tumor drugs, or anti-inflammatory drugs and the like to improve immunity drugs.

[0114] In a fifth aspect of the present application, a cosmetic is provided, and the raw material includes the nucleic acid nanosphere of the second aspect.

[0115] The nucleic acid nanosphere of the present application has improved transdermal effect and stability, can improve the bioavailability of nucleic acids, and has 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 be purchased from the market.

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

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

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

[0120] Soybean (GLYCINE MAX) extract: spermidine content ≥ 50%, purchased from Xi'an Weisibo Biological Technology Co., Ltd.;

[0121] Pea (PISUM SATIVUM) extract: spermidine content ≥ 50%, purchased from Xi'an Weisibo Biological Technology Co., Ltd.;

[0122] Sesame (SESAMUM INDICUM) extract: spermidine content ≥ 50%, purchased from Xi'an Weisibo Biological Technology Co., Ltd.;

[0123] Thickening agent: Chondrus crispus extract, purchased from SPATHEWARE, USA;

[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] The formulation is as follows: nucleic acid (DNA sodium) 0.5wt%, wheat (TRITICUM VULGARE) germ extract 0.15wt%, thickening agent (Chondrus crispus extract) 0.5wt%, preservative (hexylene glycol) 2wt%, and water in the balance, based on 100wt% of the total amount of raw materials excluding the pH regulator; wherein 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 49wt% water (50℃) of the final product mass, and after complete dissolution without agglomeration particles, high-pressure homogenization treatment at a pressure of 500bar for 10 times, solidify at 20℃ for 1h, form a nucleic acid semi-solid gel;

[0132] Warm the nucleic acid semi-solid gel to 60℃ for 40min, adjust the pH to 5.5 with anhydrous citric acid solution, and obtain phase A;

[0133] Dissolve wheat (TRITICUM VULGARE) germ extract in the remaining water (50°C), after the solution is completely dissolved, filter, adjust pH to 5.5 with anhydrous citric acid solution, to obtain phase B;

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

[0135] Example 2

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

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

[0138] Example 3

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

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

[0141] Example 4

[0142] Except that the amount of nucleic acid is 2wt%, 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 example 1, the specific formulation is as follows:

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

[0144] Example 5

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

[0146] Nucleic acid (DNA sodium) 2wt%, wheat (TRITICUM VULGARE) germ extract 0.33wt%, thickening agent (Chondrus crispus extract) 0.5wt%, preservative (hexylene glycol) 2wt% and water balance (total 100wt%); 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, the specific formula is as follows:

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

[0150] Example 7

[0151] Preparation of nucleic acid nanosphere:

[0152] The formula is as follows: nucleic acid (DNA sodium) 4wt%, wheat (TRITICUM VULGARE) germ extract 0.8wt%, thickening agent (Chondrus crispus extract) 0.5wt%, preservative (hexylene glycol) 2wt% and water balance (total 100wt%); 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 nucleic acid in the remaining water (40°C), after the solution is completely dissolved, filter, adjust pH to 5 with anhydrous citric acid solution, to obtain phase B;

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

[0156] Dissolve wheat (TRITICUM VULGARE) germ extract in the remaining water (40°C), after the solution is completely dissolved, filter, adjust pH to 5 with anhydrous citric acid solution, to obtain phase B;

[0157] Add the B phase to the A phase, stir at 5000 rpm for 30 min, add thickening agent and preservative, to obtain nucleic acid nanoscale microspheres.

[0158] Example 8

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

[0160] Example 9

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

[0162] Example 10

[0163] Except that the pH is adjusted to 4 and the melting temperature is 60°C, the rest is the same as example 8.

[0164] Example 11

[0165] Except that the pH is adjusted to 5, the rest is the same as example 10.

[0166] Example 12

[0167] Except that the pH is adjusted to 6, the rest is the same as example 10.

[0168] Example 13

[0169] Preparation of nucleic acid nanoscale microspheres:

[0170] The formula is as follows: nucleic acid (DNA sodium) 4wt%, soybean (GLYCINE MAX) extract 1.2wt%, thickening agent (Chondrus crispus extract) 0.5wt%, preservative (hexylene glycol) 4wt% and water balance (total 100wt%); wherein 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 the final product mass 42%wt water (60°C), after complete dissolution without agglomeration particles, high pressure homogenization treatment pressure is 500bar, 10 times, placed at 20°C for 1h, forming a nucleic acid semi-solid gel;

[0173] Warm the nucleic acid semi-solid gel to 60°C for 60min, adjust the pH to 5.5 with anhydrous citric acid solution, to obtain phase A;

[0174] Dissolve the soybean (GLYCINE MAX) extract in the remaining water (60°C), after complete dissolution of the solution, filter, adjust the pH to 5.5 with anhydrous citric acid solution, to obtain phase B;

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

[0176] Example 14

[0177] Preparation of nucleic acid nanospheres:

[0178] The formula is as follows: nucleic acid (DNA sodium) 4wt%, pea (PISUM SATIVUM) extract 1wt%, thickening agent (Chondrus crispus extract) 0.5wt%, preservative (hexylene glycol) 4wt% and water balance (total 100wt%); wherein 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 the final product mass 42%wt water (60°C), after complete dissolution without agglomeration particles, high pressure homogenization treatment pressure is 500bar, 10 times, placed at 20°C for 1h, forming a nucleic acid semi-solid gel;

[0181] Warm the nucleic acid semi-solid gel to 60°C for 60min, adjust the pH to 4.5 with anhydrous citric acid solution, to obtain phase A;

[0182] Pisum sativum extract was dissolved in the remaining water (60°C), after the solution was completely dissolved, filtered, and the pH was adjusted to 4.5 with anhydrous citric acid solution to obtain phase B;

[0183] The phase B was added to the phase A, stirred at 5000 rpm for 30 min, and thickening agent and preservative were added to obtain nucleic acid nanomicrospheres.

[0184] Example 15

[0185] Preparation of nucleic acid nanomicrospheres:

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

[0187] The preparation process is as follows:

[0188] The nucleic acid was dissolved in 42%wt water (60°C) of the final product, after the solution was clear and transparent, the pressure of high pressure homogenization was 500bar, the number of times was 10, and it was placed at 20°C for 1h to form a nucleic acid semi-solid gel;

[0189] The nucleic acid semi-solid gel was heated to 60°C and melted for 60min, and the pH was adjusted to 4 with anhydrous citric acid solution to obtain phase A;

[0190] Sesamum indicum extract was dissolved in the remaining water (60°C), after the solution was completely dissolved, filtered, and the pH was adjusted to 4 with anhydrous citric acid solution to obtain phase B;

[0191] The phase B was added to the phase A, stirred at 5000 rpm for 30 min, and thickening agent and preservative were added to obtain nucleic acid nanomicrospheres.

[0192] Comparative example 1

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

[0194] The preparation process is the same as example 11.

[0195] Comparative Example 2

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

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

[0198] Comparative Example 3

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

[0200] Comparative Example 4

[0201] Except that no pH adjustment is performed, the rest is the same as Example 11.

[0202] Comparative Example 5

[0203] Except that no high pressure homogenization is performed, the rest is the same as Example 11. The particle size and PDI of the microspheres are 1250nm and 0.87, respectively, which are higher than those of Example 11, and there is a precipitate at the bottom after 7 days of storage.

[0204] Comparative Example 6

[0205] Except that no solidification is performed, the rest is the same as Example 11. Just after preparation, the particle size and PDI are similar to those of Example 11, but after one day of storage, the particle size and PDI increase and a precipitate is generated. The bottle bottom photos of Example 11 and Comparative Example 6 after one day of storage are shown in Figure 1 .

[0206] Comparative Example 7

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

[0208] Comparative Example 8

[0209] The nucleic acid is loaded into the cationic liposome as follows: the raw materials are weighed according to the ratio of phosphate buffer 19490 parts, soybean lecithin 160 parts, cholesterol 20 parts, solubilizer-polyethylene glycol 160 parts, glycerol 160 parts and nucleic acid 10 parts, mixed at 30℃ for 20min, and the suspension is obtained after ultrasonic treatment for 3min. The suspension is passed through a microporous filter membrane with a diameter of 0.45μm to obtain the cationic liposome;

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

[0211] Test Example 1

[0212] The particle size, PDI and Zeta potential of the nucleic acid microspheres of the examples and the comparative examples were determined by 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 Performance summary of nucleic acid microspheres of examples and comparative examples

[0214]

[0215] As can be seen from Table 1, the nucleic acid microspheres prepared in Examples 1-15 have small diameters, uniform particle size distribution and high stability, while the diameters of the nucleic acid microspheres prepared in Comparative Examples 1-4 significantly increase, and the uniformity significantly deteriorates.

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

[0217] The standard curve for determining the DNA sodium content was prepared according to the following steps:

[0218] 0.05 g of nucleic acid (DNA sodium) powder was taken in a 50 mL volumetric flask and diluted to the mark to prepare a 1 mg / mL solution;

[0219] The above solution was diluted to prepare solutions of 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04 mg / mL;

[0220] The absorption at 260 nm (baseline with pure water) was detected by ultraviolet spectrophotometer;

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

[0222] The detected DNA content of the nucleic acid raw material was 80%.

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

[0224] The standard curve determination of spermidine content is prepared as follows:

[0225] Preparation of spermidine standard stock solution: accurately weigh 0.01 g of spermidine standard, place it in a 10 mL beaker, dissolve it with 0.1 mol / L hydrochloric acid solution, and then transfer it to a 10 mL volumetric flask, dilute to the mark, mix well, and prepare a standard stock solution with a concentration of 1000 mg / L (calculated based on various biological amine monomers).

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

[0227] Preparation of spermidine standard series solution: take 0.10 mL, 0.25 mL, 0.50 mL, 1.0 mL, 1.50 mL, 2.50 mL, and 5.0 mL of spermidine standard working solution (100 mg / L) respectively, place them in a 10 mL volumetric flask, dilute to the mark with 0.1 mol / L hydrochloric acid solution, mix well, and 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, and 50.0 mg / L respectively.

[0228] Dansyl chloride derivatization agent solution: accurately weigh an appropriate amount of dansyl chloride, prepare a derivatization agent working solution with a concentration of 10 mg / mL using acetone as the solvent, and store it in a 4 ℃ refrigerator away from light.

[0229] 0.1 mol / L hydrochloric acid solution: accurately weigh 8.6 mL of hydrochloric acid into a 100 mL volumetric flask, and dilute to the mark with water.

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

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

[0232] 50 mg / mL glutamate sodium solution: accurately weigh 5 g of glutamate sodium, dissolve it with saturated sodium bicarbonate solution, and dilute 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 dilute to the mark with water.

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

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

[0236] Preparation of internal standard stock solution: An appropriate amount of internal standard 1,7-diaminoheptane was accurately weighed into a 10 mL volumetric flask, dissolved with 0.1 mol / L hydrochloric acid solution, and diluted to the mark after dilution, mixed well, and prepared into an internal standard stock solution with a concentration of 10 mg / mL, stored in a-20 ℃ refrigerator, and the shelf life was 6 months.

[0237] Preparation of internal standard intermediate use solution: 1.0 mL of internal standard stock solution was taken into a 10 mL volumetric flask, diluted to the mark with 0.1 mol / L hydrochloric acid, mixed well, and used as an internal standard solution (1.0 mg / mL), with a shelf life of 3 months.

[0238] Preparation of internal standard use solution: 1 mL of internal standard stock solution was taken into a 10 mL volumetric flask, diluted to the mark with 0.1 mol / L hydrochloric acid, mixed well, and used as an internal standard solution (100 mg / L), and prepared immediately before use.

[0239] Derivation of standard:

[0240] 1 mL of spermidine standard series solution was taken into a 10 mL volumetric flask, 250 μL of internal standard use 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 were added in turn, vortexed for 1 min, and then placed in a 60 ℃ constant temperature water bath for derivation for 15 min. After taking out, 100 μL of sodium glutamate solution was added, vortexed and mixed well, and reacted at 60 ℃ for 15 min. After taking out and cooling to room temperature, 1 mL of water was added to each centrifuge tube, vortexed for 1 min, and the acetone (about 1 mL) was removed under nitrogen blowing at 40 ℃ water bath. 0.5 g of sodium chloride was vortexed and shaken until the sodium chloride was completely dissolved, then 5 mL of diethyl ether was added, vortexed and shaken for 2 min, and after standing and layering, the upper organic phase (diethyl ether layer) was aspirated and extracted again. The diethyl ether extract was combined and dried under nitrogen blowing at 40 ℃ water bath. 1 mL of acetonitrile was added and vortexed to dissolve the residue completely, filtered through a 0.22 μm filter needle filter into a sample vial, and determined.

[0241] Chromatographic conditions:

[0242] The chromatographic column is C18 column (column length 250 mm, column inner diameter 4.6 mm, column packing particle size 5 μm), UV detection wavelength 254 nm, sample injection amount 20 μL, column temperature 35 ℃, mobile phase A 90% acetonitrile / 10% (0.01 mol / L ammonium acetate solution containing 0.1% acetic acid), mobile phase B 10% acetonitrile / 90% (0.01 mol / L ammonium acetate solution containing 0.1% acetic acid), flow rate 0.8 mL / min. The phase ratio of the mobile phase is shown in Table 2:

[0243] Table 2 Mobile phase ratio

[0244]

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

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

[0247] Test Example 4 SEM electron microscope observation of nucleic acid nanometer microspheres

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

[0249] The nucleic acid nanometer microspheres sample was uniformly ultrasonically dispersed, dropped on a silicon wafer, dried, and gold sprayed for about 120 s, and then tested by field emission scanning electron microscope (Thermo Quattro S), wherein the acceleration voltage was 10 kv, the shooting magnification range was 3-30k, and the test mode was secondary electron mode. The results are shown in Figure 4 , and the SEM image obtained by diluting the solution by 20 times shows that the particles are spherical and uniformly distributed.

[0250] Test Example 5 TEM electron microscope observation of nucleic acid nanometer microspheres

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

[0252] A sample of the nucleic acid nanospheres prepared in Example 11 was dropped on a copper mesh, and after a few seconds, the copper mesh sample was gently picked up with tweezers, and the excess liquid was absorbed with filter paper along one side. After drying slightly, the copper mesh was placed on a drop of 2% phosphotungstic acid staining solution and floated for 60 s. After picking up with tweezers, the excess liquid was again absorbed with filter paper along one side, and the membrane was dried with the face up on filter paper. The sample was observed and photographed under a transmission electron microscope. The results are shown in Figure 6, which is a TEM image of the sample diluted 10 times, and the particles are approximately spherical. According to the scale, the particle size is approximately the same as that measured by the particle size analyzer. Figure 5 The results are shown in Figure 6, which is a TEM image of the sample diluted 10 times, and the particles are approximately spherical. According to the scale, the particle size is approximately the same as that measured by the particle size analyzer.

[0253] Test Example 6: Stability of nucleic acid nanospheres

[0254] The nucleic acid nanospheres of Examples 1-15 were placed under the following conditions: daily, light, dark, 4°C, 45°C, -15°C, and freeze-thaw cycles (24 h at -15°C followed by 24 h at 45°C is one freeze-thaw cycle). The particle size stability was measured, and the results for Example 11 are shown in Table 3. The results for the other examples were similar to those of Example 11. Thus, the nucleic acid nanospheres of the present application have good stability.

[0255] Table 3: Stability data for Example 11

[0256]

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

[0258] The nucleic acid nanospheres prepared in Example 11 were tested for stability.

[0259] A certain amount of sample was taken in a Turbiscan Lab stability analyzer sample bottle, and the dynamic changes in the stability of the serum were measured. The parameters were set to scan every 30 min, and the scanning time was 24 h. The experimental results for the nanoemulsion of Example 11 are shown in Figure 5, which is a Turbiscan Lab stability analyzer chart. Generally, an average back scattered light intensity of less than 0.2 indicates that the system is in an absolutely stable state. The average back scattered light intensity of this system was ΔBS = 0.14, indicating that it has good stability at room temperature. Figure 6

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

[0261] Franz diffusion cells were used for in vitro transdermal experiments of the present application Example 11 and Comparative Example 7. The skin of guinea pigs weighing 200-250 g was used as the barrier layer for the transdermal experiment, and the intact skin was fixed between the receiving cell and the supply cell. The effective diffusion area was 1.77 cm 2 ​, receiving pool volume about 12ml, magnetic stirring speed 300 r / min. In the receiving pool filled with release medium (normal saline), exclude air bubbles, open the stirring, and constant temperature (32.0±0.5) °C. The skin surface is evenly coated with equal amounts of DNA sodium sample, respectively, at set time 2h, 4h, 6h, 8h, 12h, 24h time with long head sampling needle 1ml of receiving liquid, the sample solution is placed in the EP tube, first to the receiving pool using a non-porous puncture needle for traction air bubbles, then to the receiving pool 1ml of receiving liquid. Respectively, the concentration of DNA sodium in the receiving liquid filtered by 0.22μm filter membrane is determined by ultraviolet spectrophotometer, and the cumulative permeation of the drug at different times is calculated. The cumulative permeation of DNA sodium is calculated according to the following formula:

[0262]

[0263] In the formula, Q n is the cumulative permeation per unit area at the nth time point (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 drug mass concentration measured at the nth time point (μg / ml); C n-1 is the drug mass concentration measured at the (n-1)th sampling point (μg / ml); S is the effective area.

[0264] After 24h, the mouse skin is cut and placed in a 1.5ml EP tube, 1ml of ethanol is added, and ultrasonic is performed for 30min. After ultrasonic, centrifugation is performed at 5000 r / min for 10min, and the supernatant is collected into the EP tube. Filtration is performed by using a 0.22μm microporous filter membrane, the filtrate is used to determine the concentration of DNA sodium in the receiving liquid filtered by 0.22μm filter membrane by ultraviolet spectrophotometer, and the skin retention of the drug is calculated. The skin retention of DNA sodium is calculated according to the following formula:

[0265] Q s =VC / A

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

[0267] The transdermal test is performed on the example 11 and the comparative example 7 of the present application. The test results are shown in Figure 7 、 Figure 8 ,

[0268] From Figure 8The 24h cumulative permeation and skin retention of Example 11 nucleic acid nanospheres and the 24h cumulative permeation of sodium free DNA solution of Comparative Example 7 can be seen from the following table: the 24h cumulative permeation of Example 11 nucleic acid nanospheres and the 24h cumulative permeation of sodium free DNA solution of Comparative Example 7 are 1.52mg / cm 2 , 0.26mg / cm 2 , respectively, and the 24h skin retention of Example 11 nucleic acid nanospheres and the 24h skin retention of sodium free DNA solution of Comparative Example 7 are 0.28mg / cm 2 , 0.21mg / cm 2 , respectively. It can be seen that the permeation and retention of nucleic acid nanospheres are higher than those of free nucleic acid solution, and are increased by 5.85 times and 1.33 times, respectively, compared with free nucleic acid solution. The experimental results are consistent with the 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 the active substance, and thus can improve the bioavailability of transdermal drug delivery products, promote the absorption and use efficiency of pharmaceutical products, and improve the skin care effect of cosmetic products.

[0269] Effect of nucleic acid nanospheres on the phagocytic ability of macrophages

[0270] RAW264.7 macrophages (static or undifferentiated RAW264.7) of M0 type were carefully inoculated on the well plate at a cell density of 5x10 4 cells / well, and the well plate was placed in a 37°C, 5% CO2 incubator for a total of 12h. After removing the well plate culture medium, the medium of Example 11 was added for 24h. 0.03μm latex beads (fluorescent microspheres) of carboxylate modified polystyrene with yellow-green autofluorescence were treated with FBS for 1h, and then resuspended in the medium and added to the macrophage well plate, incubated for 6h, and phagocytosis was completed. The 12-well plate was taken out of the cell incubator and placed on ice for 20-30s to stop phagocytosis. The culture medium was discarded, and the cells were washed twice with ice PBS to wash away the unphagocytosed fluorescent microspheres. 0.04% trypan blue solution was added to quench the fluorescence of extracellular microspheres for 30 min, and then washed with PBS. Finally, 4% paraformaldehyde was used to fix the cells at room temperature for 10 min, and then washed twice with PBS. DAPI dye was used to stain the cell nuclei, and then washed with PBS for 3 times. The phagocytosis of fluorescent microspheres by macrophages was observed under a laser confocal microscope (blue excitation light channel: DragonGreen (λex-480, λem-520)).

[0271] The results are shown in the following table: Figure 9As shown, in the blank control group, a small amount of macrophages had weak fluorescence, representing their basic phagocytic capacity. The fluorescence in the macrophages of the model control group was significantly stronger and more widely distributed, indicating that more cells successfully phagocytosed the fluorescent markers and the phagocytosis amount was larger. The fluorescence intensity of the experimental group using 0.025% of the nucleic acid nanospheres of Example 11 was higher than that of the blank control group, and even close to or exceeded that of the model control group, indicating that the nucleic acid nanospheres prepared in the application can effectively promote the phagocytic capacity of macrophages and significantly improve the immune function. Macrophages play an important role in anti-infection, immune regulation, anti-tumor and anti-inflammatory, and the like, and therefore the nucleic acid nanospheres of the 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 in any manner. To make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, but as long as the combinations of the technical features do not contradict each other, they should be considered within the scope of the present disclosure.

[0273] The above-described embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for preparing a nucleic acid nanomicrosphere, characterized in that, The method comprises the following steps: a first mixing of the nucleic acid with part of the water, a pretreatment, a solidification, and a formation of a semi-solid gel of the nucleic acid; a melting of the semi-solid gel of the nucleic acid at 45-80℃, a pH adjustment to 4-6, and a formation of phase A; a second mixing of the plant extract containing spermidine with the rest of the water, a pH adjustment to 4-6, and a formation of phase B; a third mixing of phase A, phase B, and an auxiliary agent, and a formation of the nucleic acid nanosphere; wherein the nucleic acid nanosphere comprises the following components in percentage by mass: 0.5-8% of the nucleic acid, 0.1-7.5% of the plant extract containing spermidine, 78.5-99.4% of the auxiliary agent and water; a mass ratio of the nucleic acid to the plant extract containing spermidine is 1-10:1; the plant extract containing spermidine comprises at least one of wheat germ extract, soybean extract, pea extract, and sesame extract; the auxiliary agent comprises at least one of a thickening agent and a preservative, wherein the thickening agent is used in an amount of 0.1-0.5wt%, and the preservative is used in an amount of 1-5wt%; the pretreatment comprises a homogenization treatment and / or a microfluidization treatment; a content of spermidine in the plant extract containing spermidine is ≥30wt%.

2. The production method according to claim 1, wherein One or more of the following conditions are met: (1) the nucleic acid comprises at least one of DNA and a salt of DNA; (2) the pH adjustment agent comprises citric acid; (3) the part of the water is used in an amount of 35-60wt% of the nucleic acid nanosphere.

3. The production method according to claim 2, wherein One or more of the following conditions are met: (1) the thickening agent comprises at least one of wrinkle chondrus extract, xanthan gum, microbacterial gum, and AVC; (2) the preservative comprises at least one of hexylene glycol, pentylene glycol, and ethylhexylglycerin.

4. The production method according to any one of claims 1 to 3, wherein One or more of the following conditions are met: (1) a temperature of the first mixing is 40-80℃; (2) a condition of the solidification comprises a solidification temperature of 10-20℃ and a solidification time of 0.5-24h; (3) a temperature of the second mixing is 40-80℃; (4) a temperature of the third mixing is 40-80℃; (5) the third mixing comprises the following steps: adding phase B into phase A, stirring at 2000-12000rpm for 5-60min, and then adding the auxiliary agent and mixing; (6) a time of the melting is 40-120min.

5. The production method according to claim 4, wherein One or more of the following conditions are met: (1) a condition of the homogenization treatment comprises a homogenization temperature of 40-60℃, a homogenization pressure of 100-2000bar, and a homogenization times of 2-20 times; (2) a condition of the microfluidization treatment comprises a pressure of 1400-15000Psi and a treatment times of 1-5 times.

6. The production method according to claim 4, wherein The nucleic acid nanosphere comprises the following components in percentage by mass: 0.5-8% of the nucleic acid, 0.1-7.5% of the plant extract containing spermidine, 0.1-0.5% of the thickening agent, 1-5% of the preservative, and 79-98.3% of water.

7. The nucleic acid nanosphere prepared by the method of any one of claims 1-6.

8. The nucleic acid nanosphere of claim 7 for use in the preparation of a transdermally administrable product.

9. Use according to claim 8, wherein the compound is ###0002### The product includes an immunity-enhancing medicine or cosmetic.

10. An anti-infective, immunomodulatory or anti-inflammatory drug, characterized in that, The raw material includes the nucleic acid nanosphere prepared by the preparation method in any one of claims 1-6.

11. A medicine for improving immunity, characterized by comprising the extract of claim 1. The raw material includes the nucleic acid nanosphere prepared by the preparation method in any one of claims 1-6.

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