Lipid nanoparticles for delivering gene drug containing perfluorocarbons, and method for preparing same

Abstract

The present invention relates to lipid nanoparticles for delivering a gene drug containing perfluorocarbons, and a method for preparing same. The lipid nanoparticles of the present invention can not only deliver a gene (drug), as the lipid nanoparticles contain positive ionic lipids, but can also improve the efficiency of gene (drug) delivery by perfluorocarbons inside the lipid nanoparticles inducing cavitation due to ultrasonic waves, and, thus, the lipid nanoparticles are expected to be effectively utilized in therapy using genes.

Description

Lipid nanoparticles containing perfluorocarbons for gene drug delivery, and method for preparing the same

[0001] The present invention relates to lipid nanoparticles for gene drug delivery containing perfluorocarbons, and a method for producing the same.

[0002] This invention claims the benefit of Korean Patent Application No. 10-2023-0103770, filed on August 8, 2023, and the benefit of Korean Patent Application No. 10-2024-0105733, filed on August 7, 2024, the entire contents of which are incorporated herein by reference.

[0003]

[0004] Theragnostic agents are substances that enable simultaneous diagnosis and treatment of diseases. They are typically composed of small substances, typically containing fluorescent dyes, radioactive molecules, etc., encapsulated within liposomes, polymers, or nanoparticles, with drugs or diagnostic markers introduced externally. Recently, research has focused on the synthesis of therapeutic contrast agents composed of lipid structures with excellent biocompatibility. In particular, research on bioimaging analysis using microbubbles (MBs), micrometer-sized air bubble ultrasound contrast agents composed of lipid structures, is actively underway. Furthermore, extensive research is also being conducted on treatment methods utilizing microbubbles, nanoparticle drug delivery systems, and their combination therapies.

[0005] Microbubbles respond to ultrasound stimulation after injection by repeatedly contracting and expanding, causing cavitation outside the cell. When cavitation stimulates nearby cell membranes, it generates powerful streaming, creating numerous perforations in the cell membrane. This phenomenon is called sonoporation. Perforation of the cell membrane can enhance intracellular drug delivery efficiency, ultimately increasing the amount of drug that can penetrate cells with the same dose, making it both therapeutically effective and cost-effective. Furthermore, ultrasound imaging offers the advantage of allowing the location of drug release to be determined.

[0006] However, research on drug delivery using microbubbles has several limitations. One of the most important characteristics for drug delivery is particle stability. Even though microbubbles' stability is significantly enhanced by their shell, their stability persists less than that of solid or liquid particles. Furthermore, they are significantly affected by temperature and pressure changes within the body, resulting in significant loss during circulation. Therefore, if microbubbles collapse before reaching the target site, the drug may be released into normal cells, causing adverse effects. Furthermore, their microscopic size makes them difficult to absorb from blood vessels into tumor tissue.

[0007] Meanwhile, gene therapy refers to a method of treating diseases by delivering nucleic acids such as DNA or RNA into cells using gene vectors and inducing the expression of the delivered genes. For successful gene therapy, the use of a vector capable of delivering genes effectively is crucial. An effective gene vector must have high delivery efficiency and minimal cellular toxicity. Gene delivery vehicles can be broadly categorized into viral and non-viral vectors. While viral vectors generally exhibit higher efficiency, the delivered genetic material can integrate into the host chromosome, leading to mutations that can lead to cancer and other side effects, such as immune responses. Conversely, non-viral vectors offer the advantages of being relatively stable, exhibiting low cytotoxicity, and having a lower risk of triggering immune responses. However, their delivery efficiency is lower than that of viral vectors, and this needs to be improved.

[0008] Accordingly, the inventors of the present invention have completed the present invention by confirming that when perfluorocarbon is loaded (supported) on lipid nanoparticles containing cationic lipids, stability is improved, and the sonoporation effect can be maximized by inducing cavitation by vaporization by ultrasonic waves, and that since the lipid nanoparticles contain cationic lipids, they can be utilized as gene delivery vehicles.

[0009]

[0010] The present inventors have completed the present invention by confirming that when perfluorocarbon is included in lipid nanoparticles, the perfluorocarbon is vaporized by ultrasound, thereby inducing cavitation and maximizing the sonoporation effect, and that the lipid nanoparticles of the present invention can be utilized as gene delivery vehicles because they contain cationic lipids.

[0011] Accordingly, an object of the present invention is to provide ultrasound-responsive lipid nanoparticles comprising cationic lipids, non-cationic lipids, and perfluorocarbons.

[0012] Another object of the present invention is to provide a composition for gene delivery comprising the ultrasound-sensitive lipid nanoparticle according to the present invention as an active ingredient.

[0013] Another object of the present invention is to provide a method for producing ultrasound-sensitive lipid nanoparticles according to the present invention.

[0014]

[0015] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0016]

[0017] To achieve the above purpose, the present invention provides ultrasound-responsive lipid nanoparticles comprising cationic lipids, non-cationic lipids, and perfluorocarbons.

[0018] In one embodiment of the present invention, the cationic lipid may be at least one selected from the group consisting of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol), and dimethyldioctadecylammonium bromide salt (DDAB), but is not limited thereto.

[0019] In another embodiment of the present invention, the non-cationic lipid may be at least one selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), distearoylglycerophosphoethanolaminemethyloxyethylene glycol (DSPE-mPEG), and cholesterol, but is not limited thereto.

[0020] In another embodiment of the present invention, the cationic lipid may be at least one selected from the group consisting of DOTAP and DC-Chol, and the non-cationic lipid may be at least one selected from the group consisting of DSPC, DOPE, and DSPE-mPEG, but is not limited thereto.

[0021] In another embodiment of the present invention, the cationic lipid may be DODAP and DC-Chol, and the non-cationic lipid may be at least one selected from the group consisting of DOPE, DSPE-mPEG, and cholesterol, but is not limited thereto.

[0022] In another embodiment of the present invention, the cationic lipid and non-cationic lipid may be selected from the group consisting of, but not limited to:

[0023] (a) cationic lipids are DOTAP, non-cationic lipids are DSPC, DOPE, and DSPE-mPEG;

[0024] (b) cationic lipids are DOTAP and DC-Chol, non-cationic lipids are DOPE and DSPE-mPEG;

[0025] (c) cationic lipids are DODAP and DC-Chol, non-cationic lipids are DOPE;

[0026] (d) cationic lipids are DODAP and DC-Chol, non-cationic lipids are DOPE and DSPE-mPEG; and

[0027] (e) Cationic lipids include DODAP and DC-Chol, and non-cationic lipids include DOPE, DSPE-mPEG, and cholesterol.

[0028] In another embodiment of the present invention, the perfluorocarbon may be loaded into nanoparticles, but is not limited thereto.

[0029] In another embodiment of the present invention, the perfluorocarbon is perfluoropropane (C3F8), perfluorobutane (C4F 10 ), perfluoropentane (perfluoropentane: C5F 12 ), perfluorohexane (perfluorohexane: C6F 14 ), and perfluoromethylcyclohexane (C6F 11 It may be one or more selected from the group consisting of, but is not limited to, CF3).

[0030] In another embodiment of the present invention, the perfluorocarbon may be included in a dosage of 1 to 50 uL per 1 mg of nanoparticles, but is not limited thereto.

[0031] In another embodiment of the present invention, the perfluorocarbon may be cavitized by ultrasound, but is not limited thereto.

[0032] In addition, the present invention provides a composition for gene delivery comprising the ultrasound-sensitive lipid nanoparticle according to the present invention as an active ingredient.

[0033] In one embodiment of the present invention, the ultrasound-sensitive lipid nanoparticle and the gene may be combined at an N / P ratio of 1:1 to 20, but is not limited thereto.

[0034] In another embodiment of the present invention, the gene may be selected from the group consisting of, but not limited to, gDNA, cDNA, pDNA, mRNA, tRNA, rRNA, siRNA, miRNA, and antisense nucleotides.

[0035] In addition, the present invention provides a method for producing ultrasound-sensitive lipid nanoparticles according to the present invention, comprising one method selected from the group consisting of the following methods 1 to 3:

[0036] [Method 1]

[0037] (a) a step of dispersing cationic lipids and non-cationic lipids in a buffer solution;

[0038] (b) a step of adding perfluorocarbon; and

[0039] (c) Extruding step through an extruder.

[0040] [Method 2]

[0041] (a) a step of dissolving cationic lipids and non-cationic lipids in ethanol;

[0042] (b) a step of mixing the ethanol solution and buffer solution prepared in step (a);

[0043] (c) a step of adding perfluorocarbon; and

[0044] (d) Extruding step through an extruder.

[0045] [Method 3]

[0046] (a) a step of dissolving cationic lipids and non-cationic lipids in ethanol;

[0047] (b) a step of adding perfluorocarbon to the ethanol solution prepared in step (a); and

[0048] (c) Ultrasonic processing step.

[0049] In one embodiment of the present invention, step (a) of the above method 1 may be, but is not limited to, forming a lipid film with cationic lipids and non-cationic lipids and then dispersing them by stirring and ultrasonication in a buffer solution.

[0050] In another embodiment of the present invention, the stirring may be performed at, but is not limited to, 30 to 80°C.

[0051] In another embodiment of the present invention, step (a) of the above method 3 may further include a step of lowering the temperature of the ethanol solution prepared in step (a), but is not limited thereto.

[0052] The present invention also provides a use of the ultrasound-responsive lipid nanoparticle according to the present invention; or a composition comprising the same, for gene delivery.

[0053] The present invention also provides a use of the ultrasound-sensitive lipid nanoparticle according to the present invention for the manufacture of a gene delivery vehicle; or a composition comprising the same.

[0054] In addition, the present invention provides a gene delivery method comprising a step of administering to a subject in need thereof an ultrasound-sensitive lipid nanoparticle according to the present invention to which a gene is linked; or a composition comprising the same.

[0055]

[0056] The present invention relates to a lipid nanoparticle containing perfluorocarbon for gene delivery. The lipid nanoparticle of the present invention contains a cationic lipid, so that it can not only deliver a gene (drug), but also maximize sonoporation by causing cavitation by ultrasonic waves in the perfluorocarbon inside the lipid nanoparticle, thereby improving the efficiency of gene (drug) delivery. Therefore, it is expected that the lipid nanoparticle can be usefully used in gene-based treatment.

[0057]

[0058] Figure 1 shows ultrasound-sensitive images of the compositions of DSPC, DOPE, and DSPE-mPEG in Table 1.

[0059] Figure 2 shows cyro-TEM data according to the manufacturing method of a cationic lipid-based sonoporation agent for gene delivery containing perfluoropentane.

[0060] Figure 3 shows the results of a gel retardation experiment for setting the N / P ratio of a cationic lipid-based sonoporation agent for gene delivery.

[0061] Figure 4 shows the results of a gel retardation experiment for setting the N / P ratio of a sonoporation agent for gene delivery according to the cationic lipid ratio.

[0062] Figure 5 shows data on the enhancement of contrast after ultrasound irradiation of a cationic lipid-based sonoporation agent for gene delivery containing perfluoropentane.

[0063] Figure 6 shows the results of confirming the vaporization of a cationic lipid-based sonoporation agent for gene delivery containing perfluoropentane using a portable ultrasonic device.

[0064] Figure 7 shows the results of a pDNA gel retardation experiment of a sonoporation agent for gene delivery according to the manufacturing method.

[0065] Figure 8 shows the in vitro cell gene expression results of a sonoporation agent for gene delivery according to the manufacturing method.

[0066] Figure 9 shows the results of confirming the gene expression rate of a sonoporation agent for gene transfer.

[0067]

[0068] The present inventors have completed the present invention by confirming that when perfluorocarbon is included in lipid nanoparticles, the perfluorocarbon is vaporized by ultrasound, thereby inducing cavitation and maximizing the sonoporation effect, and that the lipid nanoparticles of the present invention can be utilized as gene delivery vehicles because they contain cationic lipids.

[0069]

[0070] Hereinafter, the present invention will be described in detail.

[0071]

[0072] The present invention provides ultrasound-responsive lipid nanoparticles comprising cationic lipids, non-cationic lipids, and perfluorocarbons.

[0073] In the present invention, the cationic lipid may be at least one selected from the group consisting of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol), and dimethyldioctadecylammonium bromide salt (DDAB), but is not limited thereto.

[0074] In the present invention, the non-cationic lipid refers to a neutral lipid or anionic lipid, and may be at least one selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), distearoylglycerophosphoethanolaminemethyloxyethylene glycol (DSPE-mPEG), and cholesterol, but is not limited thereto.

[0075] According to one embodiment of the present invention, the cationic lipid is at least one selected from the group consisting of DOTAP and DC-Chol,

[0076] The non-cationic lipid is at least one selected from the group consisting of DSPC, DOPE, and DSPE-mPEG; or

[0077] The cationic lipids are DODAP and DC-Chol,

[0078] The non-cationic lipid may be at least one selected from the group consisting of DOPE, DSPE-mPEG, and cholesterol, and specifically, the cationic lipid and the non-cationic lipid may be selected from the group consisting of, but not limited to:

[0079] (a) cationic lipids are DOTAP, non-cationic lipids are DSPC, DOPE, and DSPE-mPEG;

[0080] (b) cationic lipids are DOTAP and DC-Chol, non-cationic lipids are DOPE and DSPE-mPEG;

[0081] (c) cationic lipids are DODAP and DC-Chol, non-cationic lipids are DOPE;

[0082] (d) cationic lipids are DODAP and DC-Chol, non-cationic lipids are DOPE and DSPE-mPEG; and

[0083] (e) Cationic lipids include DODAP and DC-Chol, and non-cationic lipids include DOPE, DSPE-mPEG, and cholesterol.

[0084] According to one embodiment of the present invention, the cationic lipid and non-cationic lipid of (a) are DOTAP: DSPC: DOPE: DSPE-mPEG=10~50:30~80:1~20:1~10, 10~40:30~70:1~15:1~8, 10~30:40~70:5~15:1~5, 20~40:50~70:5~10:3~10, 20~30:60~70:10~15:3~8, 10~20:60~65:5~15:3~5, 10~20:65~70:5~15:5~8, 15~25:60~70:5~15:1~10, or It may be included in a molar ratio (mole%) of 20:65:10:5, but is not limited thereto.

[0085] According to one embodiment of the present invention, the cationic lipid and non-cationic lipid of (b) are DOTAP: DC-Chol: DOPE: DSPE-mPEG=30~80:10~50:10~50:0.1~8, 30~80:10~40:10~40:0.1~5, 30~70:10~30:10~30:0.1~3, 30~60:20~30:20~30:0.1~1, 40~70:20~26:20~25:0.1~0.5, 40~60:25~30:25~30:0.5~1, 45~55:20~30:20~30:0.1~1, or 50: It may be included in a molar ratio (mole%) of 25.4:25:0.5, but is not limited thereto.

[0086] According to one embodiment of the present invention, the cationic lipid and non-cationic lipid of (c) may be included in a molar ratio (mole%) of DODAP: DC-Chol: DOPE=30~80:10~50:10~50, 30~80:10~40:10~40, 30~70:10~30:10~30, 30~60:20~30:20~30, 40~70:20~26:20~25, 40~60:25~30:25~30, 45~55:20~30:20~30, or 50:25:25, but is not limited thereto.

[0087] According to one embodiment of the present invention, the cationic lipid and non-cationic lipid of (d) are DODAP: DC-Chol: DOPE: DSPE-mPEG=30~80:10~50:10~50:0.1~8, 30~80:10~40:10~40:0.1~5, 30~70:10~30:10~30:0.1~3, 30~60:20~30:20~30:0.1~1, 40~70:20~25:20~25:0.1~0.5, 40~60:24~30:25~30:0.5~1, 45~55:20~30:20~30:0.1~1, or 50: It may be included in a molar ratio (mole%) of 24.5:24:0.5, but is not limited thereto.

[0088] According to one embodiment of the present invention, the cationic lipid and non-cationic lipid of (e) are DODAP: DC-Chol: Chol: DOPE: DSPE-mPEG=30~80: 10~50: 10~50: 1~20: 0.1~8, 30~80: 10~40: 10~40: 1~15: 0.1~5, 30~70: 10~30: 10~30: 1~10: 0.1~3, 30~60: 20~30: 20~30: 5~20: 0.1~1, 40~70: 20~25: 20~25: 5~15: 0.1~0.5, 40~60: 24~30: 24~30: 5~10: It may be included in a molar ratio (mole%) of 0.5~1, 40~60 : 24~30 : 24~30 : 10~15 : 0.5~1, 45~55 : 20~30 : 20~30 : 5~15 : 0.1~1, or 50 : 24.5 : 24 : 10 : 0.5, but is not limited thereto.

[0089] In the present invention, the membrane of the nanoparticle is composed of a cationic lipid and a non-cationic lipid, and the perfluorocarbon may be loaded (supported) on the lipid nanoparticle. Specifically, the perfluorocarbon may be supported (loaded, contained) on the inside of the nanoparticle, but is not limited thereto.

[0090] The term "perfluorocarbons (PFCs)" in this specification refers to compounds in which all CH in the chain are replaced with CF, and may include PFC precursors in which a perfluoroalkyl moiety is bonded to a non-perfluorinated atom but which may eventually be converted into a PFC. The perfluorocarbons include artificial compounds composed only of carbon and fluorine, such as CF4, C2F6, and C4F8, and are known to be chemically very stable and almost non-toxic substances. In the present invention, the perfluorocarbons include perfluoropropane (C3F8), perfluorobutane (C4F 10 ), perfluoropentane (perfluoropentane: C5F 12 ), perfluorohexane (perfluorohexane: C6F 14 ), and perfluoromethylcyclohexane (C6F 11 It may be one or more selected from the group consisting of, but is not limited to, CF3).

[0091] According to one embodiment of the present invention, the perfluorocarbon may be included in a volume of 1 to 50 uL per 1 mg of nanoparticles, 10 to 50 uL per 1 mg, 20 to 50 uL per 1 mg, 30 to 50 uL per 1 mg, 40 to 50 uL per 1 mg, 20 to 50 uL per 1 mg, 20 to 40 uL per 1 mg, 20 to 30 uL per 1 mg, 10 to 40 uL per 1 mg, 10 to 30 uL per 1 mg, 15 to 25 uL per 1 mg, 10 uL per 1 mg, 20 uL per 1 mg, 30 uL per 1 mg, 40 uL per 1 mg, or 50 uL per 1 mg, and preferably, may be included in a volume of 20 uL per 1 mg. However, it is not limited to this.

[0092] According to one embodiment of the present invention, when perfluorocarbon is loaded (contained, supported) inside a nanoparticle composed of lipids, the perfluorocarbon causes cavitation by ultrasound, thereby maximizing the effect of sonoporation, thereby exhibiting an improved drug delivery effect.

[0093] In the present invention, "sonoporation" refers to a phenomenon in which the permeability of a cell membrane increases due to ultrasound. When strong ultrasound is applied to a cell or molecule, the outer membrane surrounding it is broken in a very short moment, and the movement of a substance through the membrane, i.e. the penetration of genes (drugs), is increased.

[0094] The nanoparticles of the present invention are ultrasound-responsive (ultrasound-sensitive) nanoparticles that react to ultrasound. Ultrasound-responsive nanoparticles refer to nanoparticles whose permeability increases or whose structure collapses when exposed to ultrasound. That is, when the ultrasound-responsive nanoparticles of the present invention are exposed to ultrasound, not only does cavitation occur due to ultrasound, but perfluorocarbons inside the nanoparticles also undergo cavitation due to ultrasound, so that cavitation is excessively generated and the sonoporation effect of genes (drugs) is enhanced.

[0095] Therefore, the nanoparticles of the present invention can be administered in combination with other drugs as a facilitator capable of promoting drug delivery (absorption). By co-administering with the ultrasound-sensitive nanoparticles containing the perfluorocarbon of the present invention, the penetration effect of the drugs can be increased, thereby maximizing the efficacy of each drug and minimizing side effects such as toxicity by reducing the dosage of each drug. The drugs and the nanoparticles can be formulated separately and administered simultaneously or sequentially. In this case, in the case of sequential administration, the administration order is not limited, and the administration regimen can be appropriately adjusted depending on the patient's condition, etc.

[0096] In the present invention, "ultrasound" means a sound wave having a frequency exceeding 16 Hz to 20 kHz, which is a frequency of sound waves that can generally be heard by the human ear, and high-intensity focused ultrasound introduces focused ultrasound that provides continuous, high-intensity ultrasonic energy to a focus, and can produce instantaneous thermal effects (65-100℃), cavitation effects, mechanical effects, and sonochemical effects depending on the energy and frequency. Ultrasound is not harmful when passing through human tissue, but high-intensity ultrasound that forms a focus generates enough energy to cause coagulation necrosis and thermocautery effects regardless of the type of tissue.

[0097] In the present invention, the ultrasound refers to sound waves having a frequency higher than the audible frequency range of 16 Hz to 20 kHz. The ultrasound may be, but is not limited to, high-intensity focused ultrasound (HIFU), high-intensity unfocused ultrasound, or a combination of the two. HIFU refers to ultrasound that focuses high-intensity ultrasound energy in one place to create a concentrated focus. Depending on which image is viewed while performing the HIFU treatment, there are ultrasound-guided HIFU and magnetic resonance imaging-guided HIFU. The frequency of ultrasound is, for example, 20 kHz to 3.0 MHz, 40 kHz to 2.0 MHz, 60 kHz to 2.0 MHz, 80 kHz to 2.0 MHz, 100 kHz to 2.0 MHz, 150 kHz to 2.0 MHz, 200 kHz to 2.0 MHz, 250 kHz to 2.0 MHz, 300 kHz to 2.0 MHz, 350 kHz to 2.0 MHz, 400 kHz to 2.0 MHz, 450 kHz to 2.0 MHz, 500 kHz to 2.0 MHz, 550 kHz to 2.0 MHz, 600 kHz to 2.0 MHz, 650 kHz to 2.0 MHz, 700 kHz to 2.0 MHz, 750 kHz to 2.0 MHz, 800 kHz to 2.0 MHz, 850 kHz to 2.0 MHz, 900 kHz to 2.0 MHz, 950 kHz to 2.0 MHz, 600 kHz to 1.5 MHz, 650 kHz to 1.5 MHz, 700 kHz to 1.5 MHz, 750 kHz to 1.5 MHz, 800 kHz to 1.5 MHz, 850 kHz to 1.5 MHz, 900 kHz to 1.5 MHz, 950 kHz to 1.5 MHz, 1 MHz to 1.5 MHz, 600 kHz to 1.3 MHz, 650 kHz to 1.3 MHz, 700 kHz to 1.3 MHz, 750 kHz to 1.3 MHz, 800 kHz to 1.3 MHz, 850 kHz to 1.3 MHz, 900 kHz to 1.3 MHz, 950 kHz to 1.3 MHz, 600 kHz to 1.1 MHz, 650 kHz to 1.1 MHz, 700 kHz to 1.1 MHz, 750 kHz to 1.1 MHz, 800 kHz to 1.1 MHz, 850 kHz to 1.1 MHz, 900 kHz to 1.1 MHz, 950 kHz to 1.1 MHz, 600 kHz to 1 MHz, 650 kHz to 1 MHz, 700 It may be, but is not limited to, kHz to 1 MHz, 750 kHz to 1 MHz, 800 kHz to 1 MHz, 850 kHz to 1 MHz, 900 kHz to 1 MHz, or 950 kHz to 1 MHz.

[0098]

[0099] In addition, since the ultrasound-sensitive lipid nanoparticles of the present invention contain cationic lipids, genes can be bound to the inside / outside of the nanoparticles, the present invention provides a composition for gene delivery containing the ultrasound-sensitive nanoparticles of the present invention as an active ingredient.

[0100] In the present invention, the ultrasound-sensitive lipid nanoparticle and the gene may be combined at an N / P ratio of 1:1 to 20, 1:1 to 15, 1:1 to 10, 1:1 to 5, 1:1 to 3, 1:3 to 20, 1:3 to 15, 1:3 to 10, 1:3 to 5, 1:5 to 20, 1:5 to 15, 1:5 to 10, or 1:5 to 8, and according to one embodiment of the present invention, may be combined at an N / P ratio of 1:3 to 10, but is not limited thereto.

[0101] In the present invention, the gene may be selected from the group consisting of gDNA, cDNA, pDNA, mRNA, tRNA, rRNA, siRNA, miRNA, and antisense nucleotides, but is not limited thereto. The genes in the present invention may exist in nature or be synthesized, and may exist in various sizes, from oligonucleotides to chromosomes. These genes originate from humans, animals, plants, bacteria, viruses, and the like. They may be obtained using methods known in the art.

[0102]

[0103] Furthermore, since the lipid nanoparticles of the present invention contain cationic lipids, they can be used as carriers for anionic substances requiring intracellular delivery, in addition to genes. Accordingly, the present invention provides a drug delivery composition comprising the ultrasound-sensitive lipid nanoparticles of the present invention as an active ingredient.

[0104] The term "drug" as used herein refers to any compound having a desired biological activity. The desired biological activity includes an activity useful for diagnosing, curing, alleviating, treating, or preventing a disease in humans or other animals, and may be selected from the group consisting of, but not limited to, immune cell activators, anticancer agents, therapeutic antibodies, antibiotics, antibacterial agents, antiviral agents, anti-inflammatory agents, contrast media, protein drugs, growth factors, cytokines, peptide drugs, hair tonics, and anesthetics, and may include any anionic substance.

[0105] The above anionic substance refers to a substance having an anionic group in its molecule, and any substance that can form a complex with the lipid nanoparticle according to the present invention containing a cationic lipid through electrostatic interaction may be included.

[0106] In the present invention, the ultrasound-sensitive nanoparticles or the composition containing them may be administered sequentially or simultaneously with the ultrasound treatment, but is not limited thereto.

[0107]

[0108] In addition, the present invention provides a method for producing ultrasound-responsive lipid nanoparticles, comprising one method selected from the group consisting of the following methods 1 to 3, wherein the following methods 1 and 2 may be methods for producing ultrasound-responsive lipid nanoparticles:

[0109] [Method 1]

[0110] (a) a step of dispersing cationic lipids and non-cationic lipids in a buffer solution;

[0111] (b) a step of adding perfluorocarbon; and

[0112] (c) Extruding step through an extruder.

[0113]

[0114] [Method 2]

[0115] (a) a step of dissolving cationic lipids and non-cationic lipids in ethanol;

[0116] (b) a step of mixing the ethanol solution and buffer solution prepared in step (a);

[0117] (c) a step of adding perfluorocarbon; and

[0118] (d) Extruding step through an extruder.

[0119]

[0120] [Method 3]

[0121] (a) a step of dissolving cationic lipids and non-cationic lipids in ethanol;

[0122] (b) a step of adding perfluorocarbon to the ethanol solution prepared in step (a); and

[0123] (c) Ultrasonic processing step.

[0124] In the present invention, the term “preparation (manufacturing) at the time of use” means preparation at the time of use, meaning use immediately after preparation.

[0125] In the present invention, step (a) of the above method 1 may be performed by forming a lipid film with cationic lipids and non-cationic lipids and then dispersing the lipids by stirring and ultrasonication in a buffer solution, but is not limited thereto.

[0126] In step (a) of the above method 1, stirring may be performed at, but is not limited to, 30 to 80°C, 30 to 70°C, 30 to 60°C, 30 to 50°C, 40 to 70°C, 40 to 60°C, 40 to 50°C, or 50 to 60°C.

[0127] In the present invention, the buffer solution may be a buffer solution containing 10% sucrose, 0.9% NaCl, and / or nuclease free water, but is not limited thereto.

[0128] In the present invention, step (a) of the above method 3 may further include a step of lowering the temperature of the ethanol solution prepared in step (a) by placing it in an ice bath to prevent vaporization of perfluorocarbon, and the steps after step (a) may be performed while maintaining the lowered temperature.

[0129] Since the ultrasound-responsive lipid nanoparticles of the present invention contain cationic lipids and can deliver genes, the above-described preparation methods 1 to 3 may further include a step of adding a gene. Specifically, the preparation method 1 may further include a step of adding a genetic material after the step (c) of extruding through an extruder, the preparation method 2 may mix the ethanol solution prepared in the step (a) with the buffer solution, and the genetic material, and the preparation method 3 may further include a step of adding a genetic material after the step (a) of dissolving the cationic lipid and non-cationic lipid in ethanol.

[0130]

[0131] Unless otherwise specified, all numbers, values, and / or expressions expressing ingredients, reaction conditions, and quantities of ingredients used herein are approximations that inherently reflect, among other things, the various uncertainties of measurement that arise in obtaining such values, and therefore should be understood as being modified in all instances by the term "about." Furthermore, whenever a numerical range is disclosed herein, such range is continuous and includes every value from the minimum value to the maximum value inclusive, unless otherwise indicated. Furthermore, whenever such a range refers to integers, every integer from the minimum value to the maximum value inclusive, unless otherwise indicated, is included.

[0132] In this specification, when a range is described for a variable, the variable will be understood to include all values ​​within the described range including the described endpoints of the range. For example, the range "5 to 10" will be understood to include the values ​​5, 6, 7, 8, 9, and 10, as well as any subranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc., and any value between integers that fall within the described range, such as 5.5, 6.5, 7.5, 5.5 to 8.5, and 6.5 to 9. Also, for example, a range of "10% to 30%" would be understood to include all integers up to and including 30%, as well as any subranges such as 10% to 15%, 12% to 18%, 20% to 30%, etc., and any value between reasonable integers within the stated range, such as 10.5%, 15.5%, 25.5%, etc.

[0133] The terms used in this invention have been selected from widely used, current terms, taking into account their functions. However, these terms may vary depending on the intentions of those skilled in the art, precedents, the emergence of new technologies, etc. Furthermore, in certain cases, terms may be arbitrarily selected by the applicant, in which case their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should not be defined simply as names, but rather based on their inherent meanings and the overall content of the invention.

[0134] Throughout the specification of the present invention, when a part is said to "include" a certain component, unless specifically stated otherwise, this does not exclude other components, but rather means that other components may be included. The terms "about," "substantially," and the like, as used throughout the specification of the present invention, are used in a meaning at or near the numerical value when manufacturing and material tolerances inherent to the meaning stated are presented, and are used to prevent unscrupulous infringers from unfairly exploiting disclosures that state precise or absolute values ​​to aid understanding of the present invention.

[0135] Throughout the specification of the present invention, the term "combination thereof" included in the expressions in the Makushi format means a mixture or combination of one or more selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of the components.

[0136]

[0137] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0138]

[0139] [Example]

[0140] Example 1. Preparation of lipid-based ultrasound-responsive particles containing perfluorocarbon

[0141] Ultrasound-responsive lipid-based particles containing perfluorocarbons were developed by adjusting the compositions of DSPC, DOPE, and DSPE-mPEG as shown in Table 1. First, the lipids weighed for each composition were dissolved in ethanol at a high concentration and then mixed with 0.9% NaCl to a concentration of 1 mg / mL. Then, perfluoropentane was added to the mixture at a total volume% of 2%, and the mixture was extruded using an Avanti extruder mini to produce ultrasonic-responsive lipid particles. The particle size, particle number, and ultrasonic sensitivity of the produced particles were evaluated using a zetasizer, nanosight, and IMD10R, respectively, and the results are shown in Table 1 and Fig. 1.

[0142]

[0143]

[0144] As shown in Table 1, the average particle size was approximately 300 nm or larger, and the particle number was approximately 1 to 2 x 10 11 / mL. In addition, as a result of evaluating the ultrasonic sensitivity of the particles, it was confirmed that all the particles in Table 1 responded to ultrasonic waves and induced cavitation, as can be seen in the ultrasonic image of Fig. 1.

[0145]

[0146] Example 2. Preparation and physical property analysis of a cationic lipid-based sonoporation agent for gene delivery containing perfluoropentane.

[0147] There are two main methods for manufacturing a sonoporation agent for gene delivery containing perfluoropentane, which is composed of a shell of lipids of various compositions, including cationic lipids. (1) In the case of manufacturing method 1, the lipid is dispersed in a buffer at a concentration of 1 mg / mL (buffer such as 10% sucrose, 0.9% NaCl, nuclease-free water, etc.). Depending on the composition, the lipid is dried into a lipid film, and then completely dispersed using the buffer above at a temperature above the phase transition temperature, by stirring and sonication. Afterwards, 1 mL of the stock solution and 2% of the total volume of perfluoropentane are added, and the solution is filled into a glass syringe. Avanti ® Extrusion is performed using a Mini-Extruder. Afterwards, genetic material such as pDNA is added to the manufactured particles according to the specified N / P ratio, so that anionic pDNA is attached to the surface of the cationic lipid particles.

[0148] (2) In the case of manufacturing method 2, after dissolving the lipid of the same composition in ethanol at a high concentration, mix the ethanol solution with a buffer such as 10% sucrose, 0.9% NaCl, nuclease-free water, etc. At this time, mix so that ethanol is less than 10 v / v % of the total buffer solution. In addition, genetic material such as pDNA is mixed with the buffer according to the set N / P ratio so that pDNA can be encapsulated inside the particles. In the same way as manufacturing method 1, make the total volume of the above materials 1 mL (lipid concentration 1 mg / mL) and add perfluoropentane in an amount that is 2% of the total volume%, and then proceed in the same way as manufacturing method 1 to obtain Avanti ®Extrusion is performed using a Mini-Extruder.

[0149] To analyze the size distribution and zeta potential of the manufactured sonoporation agent, a zetasizer was used. The manufactured particles were diluted approximately 50-100 times, and the particle size distribution and zeta potential were measured. The morphology of the particles according to Manufacturing Methods 1 and 2 was analyzed using cryo-TEM. The TEM image is shown in Fig. 2.

[0150] TEM images of the particles showed that both particles manufactured by methods 1 and 2 had a spherical particle shape, and particle size analysis using a zetasizer showed a particle size of approximately 300 nm, showing similar patterns to the particles manufactured previously. Since the particle properties did not change depending on the manufacturing method, it was determined that the particle properties were determined by the particle constituents and composition.

[0151]

[0152] Example 3. Derivation of the range of cationic lipids for cationic lipid-based sonoporation agents for gene delivery.

[0153] To derive the approximate range of cationic lipids in a cationic lipid-based gene delivery sonoporation agent containing perfluoropentane, the composition ratio of lipids was adjusted as shown in Table 2 to manufacture a sonoporation agent.

[0154] Each lipid was weighed into a vial according to the composition in Table 2, and the final concentration was 1 mg / mL in 10% sucrose 10 mM histidine buffer, and then a sonoporation agent was prepared using the same method as in Preparation 1. The particle size distribution and zeta potential of the completed sonoporation agent were analyzed using a zetasizer, and the results are shown in Table 2.

[0155] As shown in Table 2, it was confirmed that particles were stably produced at a ratio of 20% or more of DOTAP. The particle size distribution at 20% or more of DOTAP showed an average value of 0.1 - 1.5 μm, and a z-average value of around 300 nm.

[0156] For DOTAP 10%, sediment was observed during extrusion and then released, requiring excessive pressure.

[0157]

[0158]

[0159] Example 4. Derivation of the range of N / P ratios of cationic lipid-based sonoporation agents for gene delivery containing perfluoropentane.

[0160] To establish the range of the N / P ratio of a cationic lipid-based gene delivery sonoporation agent containing perfluoropentane, the ratio of cationic lipid was fixed at 40% and the experiment was conducted. For the gel retardation assay, the manufactured particles were mixed with a DNA staining solution, and the sample was loaded onto a 1.0% agarose gel. Electrophoresis was performed using an electrophoresis machine and the results were confirmed using a Gel Doc system. The particle size distribution and zeta potential were measured using a zetasizer.

[0161] A sonoporation agent for gene delivery was produced using two types of cationic lipids, DOTMA and DOTAP. After extrusion using a Mini-Extruder in the same manner as in Manufacturing Method 1, pDNA was added to the produced particles according to the N / P ratio listed in Table 3 to conjugate the genetic material to the surface of the cationic lipid-based particles.

[0162] As shown in Table 3 and Fig. 3, when the N / P ratio was 1, the amount of pDNA was large, so the particles clumped together, and both types of cationic lipid particles had large sizes. When the N / P ratio was 5, it was confirmed that the particles were properly manufactured without clumping together. It was also confirmed that the zeta potential was approximately 10 mV lower than that of particles without pDNA due to the influence of negatively charged pDNA.

[0163]

[0164]

[0165] Additionally, to determine whether pDNA loading is affected by the cationic lipid ratio, pDNA was conjugated to the particle surface while adjusting the N / P ratio at 20% and 40% DOTAP, as shown in Table 4. The formulation was also manufactured in the same manner as in Manufacturing Method 1.

[0166] As shown in Table 4 and Fig. 4, the gel retardation assay results confirmed that pDNA was completely conjugated to the particle surface at all N / P ratios for both 20% and 40% DOTAP. In addition, when the N / P ratio was 3 or higher, the z-average value was around 300-400 nm, but when the N / P ratio was 1, the particles were confirmed to have clumped together and become somewhat larger.

[0167] DOTAP 20%DOTAP 40%N / P ratioSize (nm)PDIZeta Potential (mV)Size (nm)PDIZeta Potential (mV)Bare279.170.18054.9295.830.20750.431:17340.670.823-25.17816.400.582-1 .253:1358.900.44151.57391.370.47644.835:1282.030.25951.57319.270.30245.43

[0168]

[0169] From the above results, it was determined that when the N / P ratio in the manufactured ultrasonic-sensitive nanoparticles was 1:1, genes were bound while inhibiting the existing particle size, but when the N / P ratio was 3:1 or higher, genes could be stably loaded while maintaining the particle size.

[0170] In addition, it was confirmed that particle size was dependent on the N / P ratio regardless of the ratio of cationic lipids in the particles.

[0171]

[0172] Example 5. Establishment of lipid composition of cationic lipid-based sonoporation agent for gene delivery containing perfluoropentane

[0173] To derive the lipid composition of a cationic lipid-based sonoporation agent for gene delivery containing perfluoropentane, the lipid composition was set as shown in Table 5 and manufactured. The manufacturing process was performed using the same process as Manufacturing Method 1 of Example 2. To set the ratio of DSPE-mPEG, particle formation was confirmed at 1% and 5% DSPE-mPEG, and particles were manufactured by changing the ratio of DSPC and DOPE from 10 to 69%. Particles were manufactured according to Manufacturing Method 1, and the particle size distribution of the completed sonoporation agent was measured using a zetasizer. The analysis results are shown in Table 5. As a result, it was confirmed that the size of the manufactured particles was around 300 nm regardless of the ratio of DSPE-mPEG, DSPC, and DOPE.

[0174]

[0175] In addition, after fixing the ratio of DSPE-mPEG to 5%, the ratios of DSPC and DOPE were set as shown in Table 6. At this time, the experiment was conducted after fixing DOTAP to 20%. The analysis results are shown in Table 6. As shown in Table 6, the z-average value of the particles was produced at around 300 nm in all compositions. In addition, to confirm the stability of the sonoporation agent of each composition manufactured, the particles were stored at RT and 4℃ for 24 hours and the change in particle size was observed. As a result of the observation, it was confirmed that the particles were maintained stably for 24 hours without significant change. Subsequent experiments were conducted with the ratios fixed at 20 mol % of DOTAP, 65 mol % of DSPC, 10 mol % of DOPE, and 5 mol % of DSPE-mPEG.

[0176]

[0177]

[0178] Example 6. Confirmation of the ultrasound sensitivity of a cationic lipid-based sonoporation agent for gene delivery containing perfluoropentane.

[0179] To explore the functionality of a sonoporation agent containing perfluoropentane, the enhancement of contrast by ultrasound response was confirmed. The manufactured particles were diluted 10-fold with distilled water, 1 mL was placed on a dialysis membrane, and the contrast by ultrasound emission was evaluated at 37°C. The ultrasound equipment used was IMGT's IMD10, and the conditions for the emitted ultrasound were an intensity of 2 kW / cm. 2 , PRF 10 Hz, Duty 2%, 10 s / spot. As shown in Fig. 5, there was no ultrasound contrast before ultrasound irradiation, but it was confirmed that the ultrasound contrast ability of the sonoporation agent was improved after ultrasound irradiation.

[0180] Additionally, the presence or absence of vaporization due to ultrasonic irradiation of the manufactured sonoporation agent was evaluated using a portable ultrasonic device used in in vitro experiments. Ultrasonic irradiation was performed under the same conditions as in the in vitro experiment, and as shown in Figure 6, the generation of bubbles due to vaporization in the solution after ultrasonic irradiation was visually confirmed.

[0181]

[0182] Example 7. Confirmation of gene loading and delivery efficiency of a cationic lipid-based sonoporation agent containing perfluoropentane for gene delivery.

[0183] To confirm the gene loading ability and delivery efficiency of the perfluoropentane-loaded sonoporation agent, plasmid DNA (pDNA) was loaded onto the particles and the pDNA loading efficiency was confirmed through a gel retardation assay. The N / P ratio was 5:1 and particles according to Preparation Methods 1 and 2 of Example 2 were prepared, respectively, and pDNA loading was confirmed under the same electrophoresis conditions as in Example 3. As shown in Fig. 7, the results of the gel retardation assay showed that both particles prepared according to Preparation Methods 1 and 2 had pDNA loaded onto the particles, and no free pDNA band was observed in the gel, confirming that all pDNA was conjugated to the particles.

[0184] In addition, to confirm that pDNA was loaded inside / outside the particles, the encapsulation efficiency of pDNA was confirmed using Promega's Qunatifluoro dsDNA kit according to the manufacturer's protocol. The pDNA concentrations of the particles themselves loaded with pDNA and the samples in which the particle shape was destroyed by treating the particles with 3% Triton-X (surfactant) were calculated, and the encapsulation efficiency was derived using the formula ((Triton-X-treated particles - particles themselves) / Triton-X-treated particles) * 100 (%). The pDNA encapsulation efficiency derived through the formula was confirmed to have an encapsulation efficiency of 19.8% for Preparation Method 1 and 68.6% for Preparation Method 2, as shown in Table 7.

[0185] Encapsulation Efficiency (%) Manufacturing Method 119.8 Manufacturing Method 268.6

[0186] In addition, the in vitro cell gene expression results of the sonoporation agents for gene delivery manufactured according to Manufacturing Methods 1 and 2 were confirmed using the Huh-7 cell line. In the case of plasmid DNA, plasmid DNA expressing tdtomato was used, and the results were confirmed using a fluorescence microscope. The particles manufactured according to Manufacturing Methods 1 and 2 were treated to the cell line, and then portable ultrasound was irradiated immediately after treating the cells with the particles. After irradiating with portable ultrasound, the cells were cultured for 24 hours, and the expression of tdtomato fluorescence in the cells with or without ultrasound irradiation was confirmed using fluorescence images. As a result, as shown in Fig. 8, it was confirmed that the fluorescence expression rate of the cells treated with ultrasound was further enhanced in both particles manufactured according to Manufacturing Methods 1 and 2.

[0187]

[0188] Example 8. Preparation of a cationic lipid-based sonoporation agent containing perfluoropentane for gene delivery and confirmation of gene expression effect.

[0189] Lipids were mixed in the molar ratios shown in Table 8 to prepare a lipid stock. Each lipid stock was prepared by dissolving it in EtOH, and the EtOH concentration was adjusted to 5% of the total volume. 0.9% saline was used to adjust the volume to 1 mL, and plasmid DNA was added so that the N / P ratio was 5. The mixed solution of lipids and plasmid DNA was kept cold in an ice bath, and 20 μL of perfluoropentane (2% perfluoropentane), which is 2% of the total volume, was added. Ultrasonication was performed under the conditions of 70% amp, pulse on for 3 s, pulse off for 2 s, and 2 min 30 s to load perfluoropentane into the interior of nanoparticles, thereby preparing a sonoporation agent. The particle size and gene loading rate were confirmed by performing dynamic light scattering (DLS) and electrophoresis.

[0190] No.Charged lipidDC-cholesterolcholesterolDOPEDSPE-mPEG20001DOTAP 50%25.4-250.52DODAP 50%24.5-250.53DODAP 50%25-25-4DODAP 50%24.524100.5

[0191] As shown in Table 9, the particles had a size of 200-500 nm, and it was confirmed that the gene loading was also effective.

[0192] No.Size(nm)PDIZeta potential (mV)1441.50.38025.92243.30.10228.943266.70.10537.454251.50.15232.85

[0193] In addition, as a result of quantitative analysis of the gene transfer and expression rate by ultrasound sensitivity at the cellular level of the manufactured particles, it was confirmed that the gene expression rate generally tends to increase by more than two times when ultrasound is emitted, as shown in Fig. 9.

[0194]

[0195] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0196]

[0197] The present invention relates to a lipid nanoparticle containing perfluorocarbon for gene delivery. The lipid nanoparticle of the present invention contains a cationic lipid, so that it can not only deliver a gene (drug), but also improve the efficiency of gene (drug) delivery by inducing cavitation by ultrasound due to perfluorocarbon inside the lipid nanoparticle, so that it can be usefully used for gene-based treatment, and thus has industrial applicability.

Claims

1. Ultrasound-responsive lipid nanoparticles comprising cationic lipids, non-cationic lipids, and perfluorocarbons.

2. In paragraph 1, Ultrasound-responsive lipid nanoparticles, characterized in that the cationic lipid is at least one selected from the group consisting of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol), and dimethyldioctadecylammonium bromide salt (DDAB).

3. In paragraph 1, Ultrasound-responsive lipid nanoparticles, characterized in that the non-cationic lipid is at least one selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), distearoylglycerophosphoethanolaminemethyloxyethylene glycol (DSPE-mPEG), and cholesterol.

4. In paragraph 1, The cationic lipid is at least one selected from the group consisting of DOTAP and DC-Chol, Ultrasound-responsive lipid nanoparticles, characterized in that the non-cationic lipid is at least one selected from the group consisting of DSPC, DOPE, and DSPE-mPEG.

5. In paragraph 1, The cationic lipids are DODAP and DC-Chol, An ultrasound-responsive lipid nanoparticle, characterized in that the non-cationic lipid is at least one selected from the group consisting of DOPE, DSPE-mPEG, and cholesterol.

6. In paragraph 1, Ultrasound-responsive lipid nanoparticles, characterized in that the cationic lipid and non-cationic lipid are selected from the group consisting of: (a) cationic lipids are DOTAP, non-cationic lipids are DSPC, DOPE, and DSPE-mPEG; (b) cationic lipids are DOTAP and DC-Chol, non-cationic lipids are DOPE and DSPE-mPEG; (c) cationic lipids are DODAP and DC-Chol, non-cationic lipids are DOPE; (d) cationic lipids are DODAP and DC-Chol, non-cationic lipids are DOPE and DSPE-mPEG; and (e) Cationic lipids include DODAP and DC-Chol, and non-cationic lipids include DOPE, DSPE-mPEG, and cholesterol.

7. In paragraph 1, Ultrasound-responsive lipid nanoparticles, characterized in that the above perfluorocarbon is loaded into the nanoparticles.

8. In paragraph 1, The above perfluorocarbons are perfluoropropane (C3F8), perfluorobutane (C4F) 10 ), perfluoropentane (perfluoropentane: C5F 12 ), perfluorohexane (perfluorohexane: C6F 14 ), and perfluoromethylcyclohexane (C6F 11 Ultrasound-responsive lipid nanoparticles characterized by at least one selected from the group consisting of CF3).

9. In paragraph 1, Ultrasound-responsive lipid nanoparticles, characterized in that the perfluorocarbon is contained in a volume of 1 to 50 uL per 1 mg of nanoparticles.

10. In paragraph 1, The above perfluorocarbon is an ultrasound-sensitive lipid nanoparticle characterized by cavitation induced by ultrasound.

11. A composition for gene delivery comprising an ultrasound-sensitive lipid nanoparticle according to any one of claims 1 to 10 as an active ingredient.

12. In paragraph 11, A composition for gene delivery, characterized in that the ultrasound-sensitive lipid nanoparticles and the gene are combined at an N / P ratio of 1:1 to 20.

13. In paragraph 11, A composition for gene delivery, characterized in that the gene is selected from the group consisting of gDNA, cDNA, pDNA, mRNA, tRNA, rRNA, siRNA, miRNA, and antisense nucleotides.

14. A method for producing ultrasound-sensitive lipid nanoparticles of claim 1, comprising one method selected from the group consisting of methods 1 to 3 below: [Method 1] (a) a step of dispersing cationic lipids and non-cationic lipids in a buffer solution; (b) a step of adding perfluorocarbon; and (c) Extruding step through an extruder. [Method 2] (a) a step of dissolving cationic lipids and non-cationic lipids in ethanol; (b) a step of mixing the ethanol solution and buffer solution prepared in step (a); (c) a step of adding perfluorocarbon; and (d) Extruding step through an extruder. [Method 3] (a) a step of dissolving cationic lipids and non-cationic lipids in ethanol; (b) a step of adding perfluorocarbon to the ethanol solution prepared in step (a); and (c) Ultrasonic processing step.

15. In paragraph 14, A method for producing ultrasound-sensitive lipid nanoparticles, characterized in that step (a) of the above method 1 comprises forming a lipid film with cationic lipids and non-cationic lipids and then dispersing the lipids by stirring and ultrasonic treatment in a buffer solution.

16. In paragraph 15, A method for producing ultrasound-sensitive lipid nanoparticles, characterized in that the above stirring is performed at 30 to 80°C.

17. In paragraph 14, A method for producing ultrasound-sensitive lipid nanoparticles, characterized in that step (a) of the above method 3 further includes a step of lowering the temperature of the ethanol solution produced in step (a).

18. Gene delivery use of the ultrasound-sensitive lipid nanoparticle of paragraph 1 or a composition containing the same 19. Use of the ultrasound-sensitive lipid nanoparticle of claim 1 for manufacturing a gene delivery system; or a composition containing the same 20. A gene delivery method comprising a step of administering the ultrasound-sensitive lipid nanoparticle of claim 1 carrying a gene; or a composition containing the same, to a subject in need thereof.

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