Nucleic acid-encapsulated stabilized nanoliposome carrier and method for producing same

Abstract

The present invention relates to a nucleic acid-encapsulated stabilized nanoliposome carrier and a method for producing same.

Description

Stabilized nanoliposome carrier encapsulated with nucleic acid and method for preparing the same

[0001] The present invention relates to a stabilized nanoliposome carrier encapsulating nucleic acid and a method for producing the same.

[0002]

[0003] Liposomes are spherical particles formed by the aggregation of molecules such as phospholipids that have both hydrophilic and hydrophobic groups within a single molecule through interactions between solvent molecules and phospholipid molecules in an aqueous solution or organic solvent. Liposomes are highly biocompatible drug delivery vehicles made of a phospholipid bilayer similar to cell membranes, and can encapsulate hydrophilic and lipophilic bioactive substances. They can efficiently deliver various active substances to the human body, so they can be used as delivery vehicles for bioactive substances. In addition, their small particle size of 100 to 150 nm makes them advantageous for aqueous dispersion.

[0004] However, the skin permeability of liposomes outside of internal applications has been reported to be low, and recent research into skin permeation has been focused on this topic. To improve skin permeability, a drug delivery system was developed that incorporated ethanol, a substance that aids skin permeation, into the phospholipid bilayer. This drug delivery system demonstrated approximately 100 times greater absorption compared to liposomes.

[0005] However, depending on the type of encapsulating material, these carriers may experience dissolution of the encapsulating material, which is a problem directly related to the stability of the carrier. In particular, further research and development is required for formulations that deliver nucleic acids such as negatively charged DNA or RNA as stable carriers. In particular, sodium DNA, commonly called polydeoxyribonucleotide (PDRN), has recently been used as a carrier for adenosine A. 2A receptor (adenosine A 2APDRN (polydeoxyribonucleotide) material is known to have anti-inflammation, cell proliferation, and tissue regeneration effects by stimulating receptors (Kim, YH, et al., The wound healing effect of PDRN (polydeoxyribonucleotide) material on full thickness skin defect in the mouse, J. Korean Soc. Plast. Reconstr. Surg., 37(3), 220-226, 2010.), and is used for the purpose of improving skin beauty. In this case, its carrier and its skin penetration rate are directly related to its skin improvement efficacy.

[0006]

[0007] [Prior Art Literature]

[0008] [Non-patent literature]

[0009] Kim, YH, et al., The wound healing effect of PDRN(polydeoxyribonucleotide) material on full thickness skin defect in the mouse, J. Korean Soc. Plast. Reconstr. Surg., 37(3), 220-226, 2010.

[0010] The present invention aims to provide a nanoliposome delivery vehicle in which nucleic acids are encapsulated with high encapsulation efficiency through a combination of coating agents such as phospholipids, ascorbic acid derivatives, and optionally chitosan, in order to improve the skin absorption rate of nucleic acids.

[0011] The present invention also seeks to provide a method for producing the nano liposome delivery vehicle.

[0012] Other purposes and advantages of this application will be further clarified by the detailed description below, taken in conjunction with the appended claims and drawings. Any details not described herein are readily apparent and inferable to those skilled in the technical field of this application or similar technical fields, and therefore, their description is omitted.

[0013]

[0014] The following examples and the attached drawings illustrate embodiments of the present invention in detail, so that those skilled in the art can easily implement them. The present invention may be embodied in various different forms and is not limited to the embodiments described herein. Furthermore, detailed descriptions of widely known and publicly known techniques are omitted.

[0015] Throughout the specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

[0016] The term “about” as used herein means within 10%, preferably within 5%, and more preferably within 1% of a given value or range.

[0017]

[0018] The present application is intended to solve the aforementioned conventional problems, and it has been confirmed that a core-shell nanoliposome-type carrier comprising a nucleic acid (e.g., DNA or RNA; a specific example is sodium DNA, etc.) core and a phospholipid (e.g., lecithin) bilayer shell not only exhibits high encapsulation efficiency, but also exhibits excellent stability and skin absorption ability, and the present invention has been completed based on this. In this case, the phospholipid bilayer may include internal and external hydrophilic portions, and the nucleic acid may be located at the center of the nanoliposome carrier.

[0019] A nucleic acid-encapsulated nanoliposome delivery vehicle according to one embodiment of the present invention may include an ascorbic acid derivative within the phospholipid bilayer of the liposome structure.

[0020] The nanoliposome delivery vehicle according to one embodiment of the present invention may additionally include a coating agent such as a (-) polar substance or polysaccharide on the surface to increase stability.

[0021] Accordingly, in one embodiment, the present invention comprises a core comprising a nucleic acid (such as DNA or RNA; as a specific example, sodium DNA); and

[0022] Shell of phospholipid bilayer surrounding the above core

[0023] A nanoliposome delivery vehicle comprising an ascorbic acid derivative within the phospholipid bilayer is provided.

[0024]

[0025] The term "liposome" as used herein refers to a hollow, droplet-like structure formed by a phospholipid bilayer when phospholipids, a major component of biological membranes, are added to an aqueous solution. Figure 1 illustrates one shape of a liposome composed of a phospholipid bilayer.

[0026] By incorporating a specific substance into the hollow interior ("core") of the liposome, the liposome can be used as a delivery vehicle for that substance. Because liposomes can readily penetrate biological membranes, they can safely transport substances within the liposome into the membrane, and are widely used for this purpose.

[0027] The term "core" as used herein refers to the main body located at the center of the nanoliposome delivery vehicle. The core portion may contain a physiologically active substance, i.e., a nucleic acid.

[0028] The physiologically active substance included in the core of the present invention, i.e., nucleic acid, may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a DNA-RNA hybrid mixture. The term “nucleic acid,” well known in the art, has a basic backbone of a 5-carbon sugar, a phosphate group, and a nitrogenous base. When the sugar portion is ribose, it is referred to as RNA, and when it is deoxyribose, it is defined as DNA. The base portion of DNA is composed of four bases: adenine (A), thymine (T), cytosine (C), and guanine (G), and the base portion of RNA includes uridyl (U) instead of thymine (T) of DNA. The term “nucleic acid” as used herein includes nucleic acid derivatives or analogs, and the nucleic acid derivatives or analogs may be those in which any of the components of the nucleic acids defined above are changed.

[0029] The nucleic acids according to the present invention may be naturally occurring or synthesized with different nucleotide lengths, and may be fragments of a specific length or molecular weight. In one embodiment, the nucleic acids may be generated by enzymatic cleavage or physical destruction of naturally occurring nucleic acids; chemical synthesis of nucleic acids of various sizes; or some combination thereof, and any naturally occurring nucleic acid, including nucleic acids from any species, prokaryotes, eukaryotes, fungi, etc., may be used.

[0030] In one embodiment, the size / length of the nucleic acid may vary depending on the specific purpose for which it is used. For example, the length of the nucleic acid may be any length within 20 nt to 10,000 nt, or 50 nt to 2,000 nt. For example, the length of the nucleic acid may have a specific size at 10 nt intervals within 20 nt to 10,000 nt (e.g., 20 nt, 30 nt, 40 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, 100 nt, etc.), or may be a length range including or between any two of the above characteristic lengths (e.g., 1,000 nt to 2,000 nt, etc.).

[0031] Likewise, the molecular weight of the nucleic acid may also vary depending on the specific purpose for which it is used. For example, the molecular weight of the nucleic acid may be 1 to 100,000 kDa, preferably 10 to 10,000 kDa, and most preferably 50 to 3,500 kDa. The molecular weight of the nucleic acid may also have a specific value in the range of 1 to 100,000 kDa at intervals of 1 kDa, 5 kDa, or 10 kDa (e.g., 1 kDa, 2 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, etc.), or may be a length range including or between any two of the above characteristic values ​​(e.g., 50 to 1,500 kDa, etc.).

[0032] In one specific example, the nucleic acid may be DNA, and the DNA may be DNA extracted and prepared from an animal or plant, preferably DNA or DNA fragment extracted from the semen or testes of fish, particularly trout or salmon, or a mixture thereof. Preferably, the DNA may be sodium DNA.

[0033] The term "sodium DNA" used herein is a physiologically active substance also called polydeoxyribonucleotide (PDRN). Sodium DNA or PDRN is an active mixture of polynucleotides with a molecular weight of 50 to 1500 kDa, and is known to have a high DNA content without active proteins or peptides. Sodium DNA or PDRN is currently approved by the Korea Food and Drug Safety as a pharmaceutical or cosmetic raw material, and is mainly used for tissue regeneration and wound treatment, and is adenosine A. 2A Receptor (A 2A It is known as an effector of R), which is involved in regulating myocardial blood flow, regulating the secretion of glutamate and dopamine, and is known to be effective in anti-aging such as collagen synthesis, anti-inflammation, and inhibition of active oxygen. Usually, sodium DNA or PDRN is commonly used in the form of those isolated from the testis or semen of fish, such as salmon or trout, but is not limited thereto, and may be extracted from other animals or plants, or may be synthetic.

[0034] The phospholipid used in the “shell” of the present invention and which is a main component of the bilayer structure of the liposome may include a natural phospholipid or a synthetic lipid. Examples of the naturally occurring phospholipid may include one or more selected from the group consisting of natural lecithin (phosphatidylcholine (PC) derivative), lecithin obtainable from soybean, sunflower or other plant or animal sources, preferably plant sources, cholesterol, egg yolk lecithin (phosphatidylcholine), soybean lecithin, lysolecithin, sphingomyelin, phosphatidylinositol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol-4,5-diphosphate, cardiolipin, and plasmalogen. In addition, the synthetic phospholipid may be at least one selected from the group consisting of dicetyl phosphate, distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylserine, eleostearoylphosphatidylcholine, eleostearoylphosphatidylethanolamine, and eleostearoylphosphatidylserine. Preferably, it may be lecithin.

[0035] According to one embodiment of the present invention, a structural stabilizer may be included to further stabilize the phospholipid bilayer. Any structural stabilizer capable of stabilizing the structure of the nanoliposome delivery vehicle of the present invention may be used without limitation. Preferably, the structural stabilizer is an ascorbic acid derivative, such as an ascorbic acid ester derivative. Examples of the above ascorbic acid derivatives include ascorbyl palmitate, ascorbyl dipalmitate, ascorbyl tetraisopalmitate, trisodium ascorbyl isopalmitate phosphate, ascorbyl stearate, ascorbyl isostearate, disodium isostearyl ascorbyl phosphate, ascorbyl linoleate, ascorbyl tocopheryl acetate, ascorbyl tocophgeryl maleate, ascorbyl asiatate. It may be at least one selected from the group consisting of ascorbyl palmitate, ascorbyl methoxycinnamate, and sodium ascorbyl / cholesteryl phosphate, and is preferably ascorbyl palmitate.

[0036] In one specific embodiment, the nanoliposome delivery system according to the present invention may further comprise a coating layer comprising a coating material on the shell for additional stability. The coating layer may form a layer on the exterior of the existing structure through electrostatic bonding, and may be distinguished from a particulate structure formed by simple electrostatic bonding. The coating material includes, but is not limited to, at least one selected from the group consisting of chitosan, hyaluronic acid, alginate, pectin, carrageenan, dextran sulfate, guar gum, gum arabic, xanthan gum, polyacrylic acid, polygalacturonic acid, carboxymethylcellulose, polyglutamic acid (PGA) carbomer, agar, collagen peptide, starch, hydrolyzed starch, cross-linked starch, modified starch, dextrin, gamma-polyglutamic acid, gelatin, alginic acid, poly-L-lysine, poly-L-lysine and grafted onto polypropylene, lactoferrin, cross-linked lactoferrin, and glycated lactoferrin. Preferably, the coating material may be chitosan.

[0037] In one specific embodiment, the nucleic acid (e.g., DNA or RNA, particularly sodium DNA) may be present in an amount of about 0.1 to about 1.5 wt %, preferably about 0.1 to about 1.2 wt %, and more preferably about 0.2 to about 1 wt %, based on the total weight of the nanoliposomal delivery vehicle. In a specific embodiment, the nucleic acid (e.g., DNA or RNA, particularly sodium DNA) may be present in an amount of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.8, about 1, or about 1.2 wt %.

[0038] In one specific embodiment, the phospholipid, such as lecithin, may be present in an amount of about 1 to about 12 wt%, preferably about 2 to about 12 wt%, and more preferably about 2 to about 4 wt%, based on the total weight of the nanoliposome carrier. In certain embodiments, the phospholipid, such as lecithin, may be present in an amount of about 2, about 4, about 6, about 8, about 10, or about 12 wt%.

[0039] Meanwhile, the amount of phospholipid (e.g., lecithin) used to encapsulate the nucleic acid (e.g., DNA or RNA, particularly sodium DNA) as a physiologically active substance may be more advantageous for the overall particle stability compared to the nucleic acid. For example, the content ratio of sodium DNA to lecithin may be 1:20 to 1:2, and preferably 1:10 to 1:2. In a specific embodiment, the content ratio may be 1:20, 1:10, 1:5, or 1:2, and preferably 1:10.

[0040] According to the present invention, since the higher the content of ascorbic acid derivatives as structural stabilizers, the lower the charge (zeta potential) on the nanoparticle surface, including a certain content of ascorbic acid derivatives may be advantageous for stabilization. However, using too much ascorbic acid derivatives may not only increase the nanoparticle size but also cause non-uniform particle properties, so it is important to appropriately control the content.

[0041] Nucleic acid delivery systems have been studied for the treatment of human genetic diseases. Nucleic acid delivery systems can be divided into viral and non-viral delivery systems. Among them, liposomes using phospholipids are non-viral delivery systems that have high biocompatibility and low immune responses. For example, when DNA is encapsulated in liposomes, the repulsion between the strongly negatively charged DNA molecules causes problems with the physical stability of the liposomes. To address this, DNA, cationic lipids, polymers, proteins, and calcium ions have been used to suppress the repulsion and enhance the stability of DNA delivery systems. However, problems such as high cost (cationic lipids), toxicity of stabilizers, and low delivery efficiency have hindered their widespread commercial application. Therefore, the present invention aims to develop an effective DNA-encapsulating nanoliposome delivery system by selecting an ascorbic acid derivative as a stabilizer with high economic efficiency and low toxicity that can be commercially utilized.

[0042] In one specific embodiment, the ascorbic acid derivative, such as ascorbyl palmitate, may be present in an amount of about 0.01 to about 1 wt %, preferably about 0.02 to about 0.5 wt %, and more preferably about 0.02 to about 0.3 wt %, based on the total weight of the nanoliposomal carrier. In a specific embodiment, the ascorbic acid derivative, such as ascorbyl palmitate, may be present in an amount of about 0.02, about 0.033, about 0.04, about 0.05, about 0.065, about 0.1, about 0.12, about 0.16, about 0.2, about 0.22, about 0.24, about 0.25, about 0.286, about 0.33, about 0.4, or about 0.5 wt %.

[0043] The present invention includes nucleic acids (e.g., DNA or RNA, particularly sodium DNA), phospholipids (e.g., lecithin), and ascorbic acid derivatives (e.g., ascorbyl palmitate) as essential components. In this case, if the total content of these three solid components increases, the total zeta potential value may increase, which may lower particle stability, and therefore, it may be necessary to limit the total content to a certain range.

[0044] In one embodiment, the total content of the three solid components can be about 2 to about 14 wt %, preferably about 2.2 to about 10 wt %, based on the total weight of the nanoliposome carrier. In a specific embodiment, the content can be about 2.14, about 2.22, about 2.233, about 2.24, about 2.25, about 2.265, about 2.3, about 2.4, about 2.42, about 2.44, about 2.45, about 2.486, about 2.53, about 2.6, about 2.7, about 3.04, about 4.04, about 6.72, about 8.96, about 13.44 wt %.

[0045] The nanoliposome delivery vehicle according to the present invention has increased particle stability when it additionally includes a coating layer containing a coating agent (e.g., chitosan) on the shell. However, if the coating agent (e.g., chitosan) is included in too large an amount, particle aggregation may occur.

[0046] In a specific embodiment, the coating agent (e.g., chitosan) may be included in an amount of about 0.001 to about 0.02 wt%, preferably about 0.0015 to about 0.015 wt%, based on the total weight of the nanoliposome carrier. In a particular embodiment, the coating agent (e.g., chitosan) may be included in an amount of about 0.015, about 0.0015 wt%.

[0047] In one embodiment, the nanoliposome delivery vehicle according to the present invention may comprise, based on the total weight of the nanoliposome delivery vehicle, a nucleic acid (e.g., DNA or RNA, particularly sodium DNA), a phospholipid (e.g., lecithin), and an ascorbic acid derivative (e.g., ascorbyl palmitate), and further a coating agent (e.g., chitosan) (which is an optional component), in any combination of the above-mentioned contents.

[0048] In one specific example, the nanoliposome delivery vehicle according to the present invention comprises, based on the total weight of the nanoliposome delivery vehicle,

[0049] About 0.1 to about 1.5 wt%, preferably about 0.1 to about 1.2 wt%, more preferably about 0.2 to about 1 wt% of a nucleic acid (e.g., DNA or RNA, particularly sodium DNA);

[0050] about 1 to about 15 wt%, preferably about 2 to about 12 wt%, more preferably about 2 to about 4 wt% of a phospholipid (e.g., lecithin); and

[0051] Ascorbic acid derivative (e.g., ascorbyl palmitate) about 0.015 to about 1 wt%, preferably about 0.02 to about 0.5 wt%, more preferably about 0.02 to about 0.3 wt%

[0052] Includes,

[0053] Optionally, when including a coating layer, the coating agent (e.g., chitosan) may be included in an amount of about 0.001 to about 0.02 wt%, preferably about 0.0015 to about 0.015 wt%.

[0054] In a more specific embodiment, the nanoliposome delivery vehicle according to the present invention comprises, based on the total weight of the nanoliposome delivery vehicle,

[0055] About 0.1 to about 1.2 wt% of a nucleic acid (e.g., DNA or RNA, particularly sodium DNA);

[0056] about 2 to about 12 wt% of a phospholipid (e.g., lecithin); and

[0057] About 0.02 to about 0.5 wt% of an ascorbic acid derivative (e.g., ascorbyl palmitate);

[0058] and optionally, when including a coating layer, may include about 0.0015 to about 0.015 wt% of a coating agent (e.g., chitosan).

[0059]

[0060] In a more specific embodiment, the nanoliposome delivery vehicle according to the present invention comprises, based on the total weight of the nanoliposome delivery vehicle,

[0061] About 0.2% by weight of a nucleic acid (e.g., DNA or RNA, particularly sodium DNA);

[0062] about 2% by weight of phospholipids (e.g., lecithin); and

[0063] About 0.02 to about 0.5 wt% of an ascorbic acid derivative (e.g., ascorbyl palmitate);

[0064] and optionally, when including a coating layer, may include about 0.0015 to about 0.015 wt% of a coating agent (e.g., chitosan).

[0065]

[0066] In a more specific embodiment, the nanoliposome delivery vehicle according to the present invention comprises, based on the total weight of the nanoliposome delivery vehicle,

[0067] Sodium DNA approximately 0.2 wt%;

[0068] About 2% by weight of lecithin; and

[0069] Ascorbyl palmitate about 0.02 to about 0.5 wt%

[0070] and optionally, when including a coating layer, may include about 0.0015 to about 0.015 wt% of chitosan.

[0071] The above numerical range is merely suggested as an embodiment for carrying out the present invention, but even if it is outside the above numerical range, if the stability is maintained while forming a “nanoparticle carrier,” it may be included in the scope of the present invention, and this can be easily confirmed by a person skilled in the art through a method commonly used in the art.

[0072] The term “nanoliposome delivery vehicle” as used herein refers to a nucleic acid (e.g., DNA or RNA, particularly sodium DNA) crystallized into a liposome-shaped nanoparticle using a phospholipid bilayer, meaning that the size is in the nm order, i.e., the size is less than 1000 nm.

[0073] In one specific example, the nanoliposome carrier may have a size of less than 1000 nm, less than or equal to 900 nm, less than or equal to 800 nm, less than or equal to 700 nm, less than or equal to 600 nm, or less than or equal to 500 nm, specifically, 10 nm to 800 nm, 20 nm to 700 nm, 30 nm to 600 nm, or 40 nm to 500 nm.

[0074] If the size of the nano liposome delivery vehicle is less than the above value, a problem may arise in which the drug is not properly encapsulated, and if it exceeds the above value, it may not be dispersed in the solution, causing precipitation, and a problem in which the stability of the particles is reduced, and it may not conform to the definition of a nano particle.

[0075] In another embodiment, the present invention provides a composition comprising the nanoliposome delivery vehicle. The composition may be in a solid or liquid formulation.

[0076] The above solid or liquid formulation composition is formulated as a pharmaceutical composition or cosmetic composition, and techniques known in the art can be applied without limitation.

[0077] The term "composition" as used herein includes a product comprising the specified ingredients in the specified amounts and any product resulting directly or indirectly from the combination of the specified ingredients in the specified amounts. When referring to a pharmaceutical or cosmetic composition, the term is intended to include a product comprising the active ingredient and the inert ingredients that constitute the carrier, and any product resulting directly or indirectly from the combination, complexation, or agglomeration of any two or more ingredients, or from the dissociation, other type of reaction, or interaction of one or more of the ingredients.

[0078] In one embodiment, the composition may be a pharmaceutical composition, such as an injection.

[0079] When the composition of the present invention is a pharmaceutical composition, the composition may be formulated into solid preparations such as tablets, pills, powders, granules, and capsules for oral administration, and such solid preparations may be prepared by mixing the mixture with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. In addition, the pharmaceutical composition may be formulated into liquid preparations such as suspensions, oral solutions, emulsions, and syrups for oral administration, and may be formulated into liquid preparations using various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin.

[0080] In addition, the pharmaceutical composition of the present invention may include a sterilized aqueous solution, non-aqueous solvent, suspension, emulsion, lyophilized preparation, etc. for formulation for parenteral administration. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0081] In another embodiment, the composition may be a cosmetic composition, and the cosmetic composition may further contain additives or additives commonly used in the art in addition to the effective ingredient, and may have a basic and color cosmetic formulation such as a skin, skin toner, booster, lotion, nutritional lotion, milk lotion, mist lotion, nutritional cream, massage cream, hand cream, essence, serum, pack, mask, soap, cleansing foam, colander, cleansing cream, body cleanser, body lotion, shampoo, rinse, makeup base, etc.

[0082] The above cosmetic composition may be formulated as a topical skin preparation. Specifically, the composition may be in the form of a liquid, ointment, cream, lotion, spray, patch, gel, or aerosol.

[0083] The above cosmetic composition may appropriately mix ingredients commonly used in external skin preparations such as cosmetics or medicines, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, and various skin nutrients as needed.

[0084] The above cosmetic composition may also appropriately incorporate metal sequestrants such as sodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; caffeine, tannin, bellapamil, licorice extract, glabridin, hot water extract of the fruit of Kalin, various herbal medicines, tocopherol acetate, glycyrrhizic acid, tranexamic acid and derivatives or salts thereof; and sugars such as vitamin C, magnesium ascorbic acid phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, and trehalose.

[0085] The cosmetic composition may additionally include a cosmetically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and the lubricant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a glidant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium dihydrogen phosphate anhydrous, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

[0086] The present invention also provides a method for producing a stabilized nanoliposome carrier encapsulating the nucleic acid.

[0087] In one embodiment, the manufacturing method of the present invention comprises:

[0088] (a) a step of preparing a nucleic acid aqueous solution by dissolving a nucleic acid (e.g., DNA or RNA, particularly sodium DNA) in water;

[0089] (b) a step of preparing a phospholipid-ascorbyl derivative mixed solution by dissolving a phospholipid and an ascorbyl derivative in ethanol;

[0090] (c) a step of preparing a dispersion by adding the above nucleic acid solution to the above phospholipid-ascorbyl derivative mixed solution;

[0091] (d) a step of preparing a hydrated liquid phase by adding the dispersion to water;

[0092] (e) a step of homogenizing the above hydrated liquid phase under high pressure;

[0093] Includes.

[0094] The above (e) high pressure homogenization can be performed three times at 1000 bar.

[0095] In addition, steps (a) to (d) above may be performed at a high temperature of 70 degrees, and step (e) may be performed by cooling to room temperature.

[0096] When the nano liposome delivery system according to the present invention additionally includes a coating layer, the manufacturing method is as follows:

[0097] (f) a step of preparing a coating solution by dissolving a coating agent (e.g., chitosan) in water;

[0098] (g) a step of adding the coating solution (e.g., chitosan solution) to the aqueous solution prepared in step (e) to coat the coating solution.

[0099] may additionally include:

[0100]

[0101] A stabilized nanoliposome carrier encapsulating nucleic acid according to one embodiment of the present invention and a method for producing the same can improve the encapsulation rate of nucleic acid, improve the particle stability of the nanoliposome carrier, increase the human absorption rate of nucleic acid as a physiologically active substance, and improve the skin regeneration effect, which has recently been in the spotlight due to the effect of nucleic acid fragments, especially sodium DNA.

[0102] In addition, the stabilized nanoliposome carrier encapsulating nucleic acid and the method for manufacturing the same can easily perform mass production of nanoliposome carriers with uniform particle size and particle stability, and can be easily applied to various product groups including cosmetic compositions and pharmaceutical compositions.

[0103]

[0104] In particular, the nanoliposome delivery system of the present invention uses a method of encapsulating by improving the physical performance of the delivery system rather than a method of directly affecting existing nucleic acids, so it is easily applicable to substances with a high zeta potential value, such as DNA or RNA, and can be easily applied to commercial product groups as a delivery system designed according to the characteristics of the encapsulating material.

[0105]

[0106] Figure 1 is a schematic diagram showing the structure of a nano liposome delivery vehicle encapsulating sodium DNA according to an example of the present invention.

[0107] Figures 2 to 7 show the results for stability and particle size of drug delivery systems manufactured in examples and comparative examples of the present invention.

[0108] Figure 8 illustrates the results of measuring the zeta potential of a nano liposome delivery system manufactured according to Example 21 of the present invention.

[0109] Figure 9 illustrates the results of measuring the particle size of a nano liposome delivery vehicle manufactured according to Example 21 of the present invention.

[0110] Figure 10 illustrates the results of Cryo-TEM measurement of a nano liposome delivery vehicle manufactured according to Example 21 of the present invention.

[0111] Figure 11 illustrates the results of confocal laser scanning measurement of skin absorption behavior after fluorescence treatment of a nano liposome delivery system manufactured according to Example 21 of the present invention.

[0112] Figure 12 illustrates the results of fluorescence analysis after skin penetration of a nano liposome delivery system manufactured according to Example 21 of the present invention.

[0113] Figure 13 is a microscopic photograph of the cell proliferation ability of a nano liposome delivery system manufactured according to Example 21 of the present invention.

[0114] Figure 14 shows the results of an MTT assay for cell proliferation of a nanoliposome delivery system manufactured according to Example 21 of the present invention.

[0115]

[0116] Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the following examples.

[0117]

[0118] Example 1

[0119] A sodium DNA drug delivery system was manufactured according to the composition described in Table 1.

[0120] Specifically, 0.2 wt% of sodium DNA (purchased from Japan Pharmaceutical Co., Ltd.) was mixed with 10 wt% of purified water and completely dissolved in a 70 degree water bath to prepare a sodium DNA solution.

[0121] Separately, 2 wt% of lecithin (purchased from: Lipoid GmbH) and 0.02 wt% of ascorbyl palmitate (purchased from: Shanghai Cosroma Biotech Co., Ltd) were mixed with 10 wt% of ethanol (purchased from: Samjeon Pure Chemical Industry Co., Ltd.) and completely dissolved at 70 degrees to prepare a phospholipid solution.

[0122] A DNA-phospholipid dispersion was prepared by slowly adding a sodium DNA solution to a phospholipid solution while maintaining the temperature at 70°C. The prepared DNA-phospholipid dispersion was added to 77.78 wt% purified water to prepare a hydrated liquid crystal phase solution.

[0123] The dispersed hydrated liquid crystal phase was cooled to room temperature. The cooled hydrated liquid crystal phase solution was passed through a high-pressure homogenizer at a pressure of 1000 bar and passed three times to produce a drug delivery vehicle with a uniform particle size.

[0124]

[0125] Example 2

[0126] It was manufactured in the same manner as Example 1, except that the content of ascorbyl palmitate was changed to 0.033 wt%.

[0127]

[0128] Example 3

[0129] According to the composition of Table 1, it was manufactured in the same manner as Example 1, except that the content of ascorbyl palmitate in Example 1 was changed to 0.04 wt%.

[0130]

[0131]

[0132] Example 4

[0133] According to the composition of Table 1, it was manufactured in the same manner as Example 1, except that the content of ascorbyl palmitate in Example 1 was changed to 0.05 wt%.

[0134]

[0135] Example 5

[0136] According to the composition of Table 1, it was manufactured in the same manner as Example 1, except that the content of ascorbyl palmitate in Example 1 was changed to 0.065 wt%.

[0137]

[0138] Example 6

[0139] According to the composition of Table 1, it was manufactured in the same manner as Example 1, except that the content of ascorbyl palmitate in Example 1 was changed to 0.1 wt%.

[0140]

[0141] Example 7

[0142] According to the composition of Table 1, it was manufactured in the same manner as Example 1, except that the content of ascorbyl palmitate in Example 1 was changed to 0.2 wt%.

[0143]

[0144] Composition (% by weight) of Examples 1 to 7 Example 1 2 3 4 5 6 7 Sodium DNA 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.0 3 0.0 4 0.0 5 0.0 6 5 0.1 ...

[0145]

[0146] Example 8

[0147] According to the composition of Table 2, it was manufactured in the same manner as Example 1, except that the content of ascorbyl palmitate in Example 1 was changed to 0.22 wt%.

[0148]

[0149] Example 9

[0150] According to the composition of Table 2, it was manufactured in the same manner as Example 1, except that the content of ascorbyl palmitate in Example 1 was changed to 0.25 wt%.

[0151]

[0152] Example 10

[0153] According to the composition of Table 2, it was manufactured in the same manner as Example 1, except that the content of ascorbyl palmitate in Example 1 was changed to 0.286 wt%.

[0154]

[0155] Example 11

[0156] According to the composition of Table 2, it was manufactured in the same manner as Example 1, except that the content of ascorbyl palmitate in Example 1 was changed to 0.33 wt%.

[0157]

[0158] Example 12

[0159] According to the composition of Table 2, it was manufactured in the same manner as Example 1, except that the content of ascorbyl palmitate in Example 1 was changed to 0.4 wt%.

[0160]

[0161] Example 13

[0162] According to the composition of Table 2, it was manufactured in the same manner as Example 1, except that the content of ascorbyl palmitate in Example 1 was changed to 0.5 wt%.

[0163]

[0164] Composition (% by weight) of Examples 8 to 13 Example 89 10 11 12 13 Sodium DNA 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 2 Lecithin 2 2 2 2 2 Ascorbyl palmitate 0.2 2 0.2 5 0.2 8 6 0.3 3 0.4 0.5 Ethanol 10 10 10 10 10 10 Purified water up to 100 up to 100 up to 100 up to 100 up to 100

[0165]

[0166] Example 14

[0167] According to the composition of Table 3, it was manufactured in the same manner as Example 3, except that the content of sodium DNA in Example 3 was changed to 0.1 wt%.

[0168]

[0169] Example 15

[0170] According to the composition of Table 3, it was manufactured in the same manner as Example 3, except that the content of sodium DNA in Example 3 was changed to 0.4 wt%.

[0171]

[0172] Example 16

[0173] According to the composition of Table 3, it was manufactured in the same manner as Example 3, except that the content of sodium DNA in Example 3 was changed to 1 wt%.

[0174]

[0175] Composition (weight %) of Examples 14 to 16 Example 14 15 16 Sodium DNA 0.1 0.4 1 Lecithin 2 2 Ascorbyl palmitate 0.04 0.04 0.04 Ethanol 10 10 10 Purified water up to 100 up to 100 up to 100

[0176]

[0177] Example 17

[0178] According to the composition of Table 4, it was manufactured in the same manner as Example 3, except that the sum of the solids (sodium DNA, lecithin, ascorbyl palmitate) in Example 3 was changed to 6.72 wt%.

[0179]

[0180] Example 18

[0181] According to the composition of Table 4, it was manufactured in the same manner as Example 3, except that the sum of the solids (sodium DNA, lecithin, ascorbyl palmitate) in Example 3 was changed to 8.96 wt%.

[0182]

[0183] Example 19

[0184] According to the composition of Table 4, it was manufactured in the same manner as Example 3, except that the sum of the solids (sodium DNA, lecithin, ascorbyl palmitate) in Example 3 was changed to 13.44 wt%.

[0185]

[0186] Composition (weight %) of Examples 17 to 19 Example 17 18 19 Sodium DNA 0.6 0.8 1.2 Lecithin 68 12 Ascorbyl palmitate 0.1 2 0.1 6 0.2 4 Ethanol 10 10 10 Purified water up to 100 up to 100 up to 100

[0187]

[0188] Example 20

[0189] A sodium DNA drug delivery system was manufactured according to the composition described in Table 5.

[0190] Specifically, 0.2 wt% of sodium DNA (purchased from Japan Pharmaceutical Co., Ltd.) was mixed with 10 wt% of purified water and completely dissolved in a 70 degree water bath to prepare a sodium DNA solution.

[0191] Separately, 2 wt% of lecithin (purchased from: Lipoid GmbH) and 0.04 wt% of ascorbyl palmitate (purchased from: Shanghai Cosroma Biotech Co., Ltd) were mixed with 10 wt% of ethanol (purchased from: Samjeon Pure Chemical Industry Co., Ltd.) and completely dissolved at 70 degrees to prepare a phospholipid solution.

[0192] A DNA-phospholipid dispersion was prepared by slowly adding a sodium DNA solution to a phospholipid solution while maintaining the temperature at 70°C. The prepared DNA-phospholipid dispersion was added to 67.76 wt% purified water to prepare a hydrated liquid crystal phase solution. The hydrated liquid crystal phase, after complete dispersion, was cooled to room temperature. The cooled hydrated liquid crystal phase solution was passed through a high-pressure homogenizer at a pressure of 1000 bar and 3 passes to prepare a drug delivery vehicle with a uniform particle size.

[0193] Separately, a chitosan solution containing 0.015 wt% chitosan dissolved in 9.85 wt% purified water was mixed with the drug delivery system solution prepared above, and chitosan was coated on the surface of the drug delivery system.

[0194]

[0195] Example 21

[0196] According to the composition of Table 5, it was manufactured in the same manner as Example 21, except that the content of chitosan in Example 21 was changed to 0.0015 wt%.

[0197]

[0198] Composition (weight %) of Examples 20 to 21 Example 2021 Sodium DNA 0.20.2 Lecithin 22 Ascorbyl palmitate 0.040.04 Chitosan 0.0150.0015 Ethanol 1010 Purified water up to 100 up to 100

[0199]

[0200] Comparative Example 1

[0201] A sodium DNA drug delivery system was manufactured according to the composition described in Table 6.

[0202] Specifically, 0.2 wt% of sodium DNA (purchased from Japan Pharmaceutical Co., Ltd.) was mixed with 10 wt% of purified water and completely dissolved in a 70 degree water bath to prepare a sodium DNA solution.

[0203] Separately, 2 wt% of lecithin (purchased from: Lipoid GmbH) was mixed with 10 wt% of ethanol (purchased from: Samjeon Pure Chemical Industries, Ltd.) and completely dissolved at 70 degrees to prepare a phospholipid solution.

[0204] A DNA-phospholipid dispersion was prepared by slowly adding a sodium DNA solution to a phospholipid solution while maintaining the temperature at 70°C. The prepared DNA-phospholipid dispersion was added to 67.76 wt% purified water to prepare a hydrated liquid crystal phase solution. The hydrated liquid crystal phase, after complete dispersion, was cooled to room temperature. The cooled hydrated liquid crystal phase solution was passed through a high-pressure homogenizer at a pressure of 1000 bar and 3 passes to prepare a drug delivery vehicle with a uniform particle size.

[0205]

[0206] Comparative Example 2

[0207] According to the composition of Table 6, it was manufactured in the same manner as Example 3, except that the content of ascorbyl palmitate in Example 3 was changed to 0.01 wt%.

[0208]

[0209] Comparative Example 3

[0210] A sodium DNA drug delivery system was manufactured according to the composition described in Table 6.

[0211] Specifically, 10 wt% purified water was prepared.

[0212] Separately, 2 wt% of lecithin (purchased from: Lipoid GmbH) and 0.04 wt% of ascorbyl palmitate (purchased from: Shanghai Cosroma Biotech Co., Ltd) were mixed with 10 wt% of ethanol (purchased from: Samjeon Pure Chemical Industry Co., Ltd.) and completely dissolved at 70 degrees to prepare a phospholipid solution.

[0213] The purified water prepared above was slowly added to the phospholipid solution to prepare a phospholipid dispersion. The prepared phospholipid dispersion was added to 77.96 wt% purified water to prepare a hydrated liquid crystal phase solution. The hydrated liquid crystal phase, after complete dispersion, was cooled to room temperature. The cooled hydrated liquid crystal phase solution was passed through a high-pressure homogenizer at a pressure of 1000 bar and 3 passes to prepare a drug delivery vehicle with a uniform particle size.

[0214]

[0215] Comparative Example 4

[0216] According to the composition of Table 6, it was manufactured in the same manner as Example 3, except that the content of sodium DNA in Example 3 was changed to 2 wt%.

[0217]

[0218] Comparative examples 5 and 6

[0219] According to the composition of Table 6, the same method as Example 20 was used for manufacturing, except that the content of chitosan in Example 20 was changed to 0.15 and 0.00015 wt%, respectively.

[0220]

[0221] Composition (weight %) of Comparative Examples 1 to 6 Example (Reference) Comparative Example 3 21123456 Sodium DNA 0.20.20.20.20 20.20.2 Lecithin 2 22222222 Ascorbyl Palmitate 0.04 0.04 00.01 0.04 0.04 0.04 0.04 Chitosan-0.0015 ---- 0.15 0.000 15 Ethanol 10 10 10 10 10 10 10 10 Purified water up to 100 up to 100 up to 100 up to 100 up to 100 up to 100 up to 100

[0222]

[0223] Experimental Example 1. Comparison of particle size and zeta potential by manufacturing method

[0224] The physical properties of the DNA carriers according to Examples 1 to 21 were first evaluated.

[0225] Afterwards, the carriers that did not form sediment were measured for particle size and zeta potential using a nano particle size analyzer (Zetasizer, Malvern). As a sample pretreatment for each analysis, the particle size was analyzed by diluting the samples of Examples 1 to 21 and Comparative Examples 1 to 6 30-fold with purified water.

[0226] The results are shown in Figures 2 to 7.

[0227] From Examples 1 to 13, the content of ascorbyl palmitate increased, and from the results in Figures 2 and 3, it was confirmed that although the content of ascorbyl palmitate in the present invention increased, the color of the solution only became darker and a stable dispersion was still maintained. However, it was also suggested that excessive ascorbyl palmitate may increase the nanoparticle size and exhibit non-uniform particle properties. In addition, when ascorbyl palmitate was not used or its content was low (Comparative Examples 1 and 2; Figure 7), the dispersion was not stable, sedimentation was observed, and particle stability was very low.

[0228] In Examples 14 to 16, the sodium DNA content increased, and from the results in Fig. 4, the higher the amount of lecithin compared to the sodium DNA content in encapsulating DNA, the more stable it was. In particular, when the amount of lecithin compared to the sodium DNA content was small (Comparative Example 4; Fig. 7), the particles increased due to the environment in which DNA was encapsulated in a small amount, and it is judged that the excessively clumped DNA deteriorates particle stability due to the mutual repulsion and the relatively high zeta potential formed on the particle surface.

[0229] In Examples 17 to 19, the total solid content (sodium DNA, lecithin, ascorbyl palmitate) was increased, and according to Fig. 5 showing the results, as the total solid content increased, the concentration became darker, and the color of the dispersion was dark, but it was confirmed that all were stable. However, it was confirmed that the viscosity increased during the preparation of the above sample, and during this process, the preparation equipment was overloaded and showed low particle size.

[0230] In Examples 20 and 21, a chitosan coating process was added, and Fig. 6, which shows the results related to this, confirms that particle stability was increased through chitosan coating after the high-pressure homogenization process. However, it was confirmed that excessive chitosan can cause particle aggregation (Comparative Example 6; Fig. 7). Accordingly, it was confirmed that using 0.0015 wt% as the most optimal chitosan concentration resulted in the most stability when looking at particle properties.

[0231]

[0232]

[0233] Experimental Example 2. Stability Comparison by Manufacturing Method

[0234] In Experimental Example 1, examples in which particle formation occurred were evaluated by placing 30 g of each particle solution in a glass vial in a 45°C thermostat.

[0235] The morphological changes of the nanoliposome delivery vehicle aqueous dispersion placed in the above temperature-controlled chamber were observed with the naked eye.

[0236] The results are shown in Figure 7.

[0237] In the concentrations of ascorbyl palmitate in Examples 1 to 13, it showed a stable appearance, and the stability of the carrier was also shown at the concentration of ascorbyl palmitate higher than that of Example 13. It was observed that the stability was reduced at the concentration of ascorbyl palmitate between Comparative Example 1 and Example 1, and Comparative Example 2, which is at the boundary of this concentration, was also confirmed to be unstable.

[0238] The stability of the carrier was confirmed according to the ratio of sodium DNA and ascorbyl palmitate. According to Examples 3 to 13, it was confirmed that all carriers were stable even when the ratio of sodium DNA to ascorbyl palmitate was lowered from 5:1 to 0.4:1.

[0239] In addition, in Examples 14 to 16, the contents of lecithin and ascorbyl palmitate were kept the same, and the contents of sodium DNA were changed. In the same contents as Comparative Example 3, where sodium DNA was not used at all, unstable behavior was observed. In Examples 14 to 16, where only sodium DNA was added to the same composition as Comparative Example 3, stability was confirmed, and in particular, stability was confirmed even when the ratio of DNA and phospholipid was 1:2.

[0240] The optimal amount for chitosan coating is as confirmed from Examples 20 to 21, and chitosan in excess as in Comparative Example 5 caused aggregation between particles, and it was confirmed that the particle size was further increased even with the use of a small amount of chitosan as in Comparative Example 6.

[0241]

[0242] Experimental Example 3. Nanoliposome delivery vehicle image using Cryo-TEM

[0243] The nanoliposome carrier particles generated in Example 3 were visualized using Cryo-TEM (Talos L120C, Thermofisher scientific, Waltham, MA, USA).

[0244] Cryo-TEM was performed using a Talos L120C (Thermofisher scientific, Waltham, MA, USA). As a sample preparation method, a 200-mesh grid was mounted using Vitrobot Mark IV (Thermofisher scientific, Waltham, MA, USA) tweezers, and 20 microliters of sample was placed on the grid. After squeezing with filter paper to minimize moisture, the sample was placed in liquid ethane cooled with liquid nitrogen and cryogenically processed into an amorphous solid. The cryogenically processed sample was mounted on a cryo-TEM to visualize the nanoliposome delivery particles.

[0245] The measurement results of particles imaged by cryo-TEM are shown in Fig. 10. As can be confirmed from the image of the drug delivery system placed on the grid of Fig. 10, the drug delivery system is formed in a uniform particle shape of 30 nm.

[0246]

[0247] Experimental Example 4. Measurement of nanoliposome delivery vehicle uptake behavior using confocal laser scanning microscopy (Franzcell)

[0248] An experiment was conducted to observe the skin absorption behavior of nanoliposome delivery systems manufactured using the method of Example 3, using DNA stained with DAPI (4',6-diamidino-2-phenylindole). Sodium DNA was used as a control (indicated as “before delivery” in Figure 11 and as DNA in Figure 12).

[0249] 2.5 milligrams of DAPI were mixed with 500 microliters of purified water and sonicated for 30 minutes to completely dissolve. 0.2 grams of DNA was mixed with 2 milliliters of purified water and completely dissolved at 70°C before cooling to room temperature. The DAPI solution and DNA solution were mixed at a volume ratio of 10:1 and stained in the dark for 5 minutes. The stained DNA solution was then placed in a 10 kDa centrifugal filter and centrifuged at 10,000 g for 10 minutes to remove any remaining DAPI and then collected. The collected DNA was used to prepare a vector according to the method of Example 3.

[0250] For the absorption experiment, a MicroPig membrane (1.5 cm * 1.5 cm * 1000 um) was used, and the MicroPig membrane was mounted on a Franz cell and stabilized (32°C, 800 rpm stirring) with a receptor solution, which was a 1:1 mixture of PBS (Phosphate-buffer saline) and ethanol, and then sampled. Each sample was checked for 5, 20, 40, and 60 min.

[0251] After completing the absorption experiment, the MicroPig membrane was cut from the skin, placed in OCT (Optimal Cutting Temperature) compound, frozen in dry ice, and then sliced ​​into 20 μm pieces using a precision microtome. The sliced ​​tissue was transferred to slice glass, dried in a dry oven at 37°C, and observed using a confocal microscope (AX10, Zeiss, Baden-Wuerttemberg, Oberkochen, Germany).

[0252] Figure 11 is an image of the shape of the nano liposome delivery system treated with DAPI being absorbed into the sectioned tissue. It can be confirmed that the nano liposome delivery system is absorbed into the skin overall, and that the nano liposome delivery system is gradually absorbed into the dermis.

[0253] Figure 12 shows the fluorescence analysis results of the nanoliposome delivery system confirmed after skin penetration. While there was no trend in fluorescence values ​​in the sample (control group) prior to drug delivery, a trend of increasing permeation over time was observed after delivery, confirming the absorption behavior of the delivery system.

[0254]

[0255] Experimental Example 5. Cell Proliferation Evaluation

[0256] To investigate the cell proliferation potential of the nanoliposome carrier prepared in Example 3, a cell proliferation test was conducted. First, to investigate the cell proliferation potential, Hs68 cells, a fibroblast cell line, were used and obtained from the American Type Culture Collection (Manassas, VA, USA).

[0257] To evaluate the cell proliferation ability of the nanoliposome delivery system, a cell proliferation assay was performed. First, Hs68 cells cultured in DMEM containing 10% FBS (Fetal bovine serum) and 1% Penicillin / Streptomycin were seeded at 5 x 10^4 cells / mL in a 6-well plate, and each sample was treated with different DNA concentrations (5 and 10 ppm) and incubated for 72 hours in a 5% CO2, 37°C environment. After incubation, MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl Tetrazolium Bromide) reagent was added to each well and incubated for 4 hours to allow formazan formation. After 4 hours, DMSO (Dimethyl Sulfoxide) solution was treated, and the absorbance at a wavelength of 420 nm was measured using a microplate reader. Sodium DNA was used as a control.

[0258] Figure 13 shows cell proliferation after 72 hours of culture. While cells treated with purified water and DNA itself (control) had large intercellular gaps, cells treated with nanoliposome carriers had narrow intercellular gaps. Visually, it was confirmed that cell proliferation was more active in cells treated with nanoliposome carriers.

[0259] Figure 14 shows a graph measuring the absorbance at a wavelength of 420 nm after treatment with MTT reagent, showing a higher cell proliferation rate compared to the control group treated with DNA as is. This confirms the superior intracellular delivery capability of the nanoliposome carrier.

Claims

1. A core containing nucleic acid; and Shell of phospholipid bilayer surrounding the above core It is a nano liposome delivery vehicle containing, Containing an ascorbic acid derivative within the phospholipid bilayer, Nano liposome delivery vehicle.

2. A nano liposome delivery vehicle according to claim 1, wherein the nucleic acid is a fragment of DNA, RNA, or a mixture thereof.

3. A nano liposome delivery vehicle according to claim 1, wherein the nucleic acid is sodium DNA.

4. In the first paragraph, the ascorbic acid derivative is ascorbyl palmitate, ascorbyl dipalmitate, ascorbyl tetraisopalmitate, trisodium ascorbyl isopalmitate phosphate, ascorbyl stearate, ascorbyl isostearate, disodium isostearyl ascorbyl phosphate, ascorbyl linoleate, ascorbyl tocophgeryl acetate, ascorbyl tocophgeryl maleate, ascorbyl At least one selected from the group consisting of ascorbyl asiatate, ascorbyl methoxycinnamate, and sodium ascorbyl / cholesteryl phosphate. Nano liposome delivery vehicle.

5. In the first paragraph, the nano liposome delivery vehicle, based on the total weight of the nano liposome delivery vehicle, 0.1 to 1.5 wt% nucleic acid; 1 to 15 wt% of phospholipids; and 0.015 to 1 wt% of ascorbyl derivative It includes, A nanoliposome delivery vehicle wherein the nucleic acid is sodium DNA, the phospholipid is lecithin, and the ascorbyl derivative is ascorbyl palmitate.

6. In the first paragraph, the nano liposome delivery vehicle, based on the total weight of the nano liposome delivery vehicle, 0.1 to 1.2 wt% nucleic acid; 2 to 12 wt% of phospholipids; and 0.02 to 0.5 wt% of ascorbyl derivative It includes, A nanoliposome delivery vehicle wherein the nucleic acid is sodium DNA, the phospholipid is lecithin, and the ascorbyl derivative is ascorbyl palmitate.

7. A nanoliposome carrier according to claim 1, further comprising a coating layer comprising at least one coating agent selected from the group consisting of chitosan, hyaluronic acid, alginate, pectin, carrageenan, dextran sulfate, guar gum, gum arabic, xanthan gum, polyacrylic acid, polygalacturonic acid, carboxymethylcellulose, polyglutamic acid (PGA) carbomer, agar, collagen peptide, starch, hydrolyzed starch, cross-linked starch, modified starch, dextrin, gamma-polyglutamic acid, gelatin, alginic acid, poly-L-lysine, poly-L-lysine grafted onto polypropylene, lactoferrin, cross-linked lactoferrin, and glycated lactoferrin.

8. In paragraph 7, The above coating agent is chitosan, A nanoliposome delivery system comprising 0.001 to 0.02 wt% of the coating agent based on the total weight of the nanoliposome delivery system.

9. In paragraph 7, The above coating agent is chitosan, A nanoliposome delivery system comprising 0.0015 to 0.015 wt% of the coating agent based on the total weight of the nanoliposome delivery system.

10. A nanoliposome delivery vehicle according to claim 1, wherein the diameter of the nanoliposome delivery vehicle is 10 nm or more and less than 800 nm.

11. A composition comprising a nano liposome delivery vehicle according to any one of claims 1 to 10.

12. A pharmaceutical composition comprising a nanoliposome delivery vehicle according to any one of claims 1 to 10.

13. A cosmetic composition comprising a nano liposome delivery vehicle according to any one of claims 1 to 10. 14.(a) A step of preparing a nucleic acid solution by dissolving nucleic acid in water; (b) a step of preparing a phospholipid-ascorbyl derivative mixed solution by dissolving a phospholipid and an ascorbyl derivative in ethanol; (c) a step of preparing a dispersion by adding the above nucleic acid solution to the above phospholipid-ascorbyl derivative mixed solution; (d) a step of preparing a hydrated liquid phase by adding the dispersion to water; (e) a step of homogenizing the above hydrated liquid phase under high pressure; A method for producing a nanoliposome delivery system according to any one of claims 1 to 10, comprising:

15. In paragraph 14, (f) a step of preparing a coating solution by dissolving the coating agent in water; and (g) a step of adding the coating solution to the aqueous solution prepared in step (e) to coat it with the coating solution. A method for producing a nanoliposome delivery system, further comprising:

16. A method for producing a nano liposome carrier, wherein in paragraph 14, (e) the high-pressure homogenization is performed three times at 1000 bar.

Images