WO2025034029A1 - Lipid nanoparticles for delivering gene drug containing perfluorocarbons, and method for preparing same - Google Patents

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 genetic drug delivery, and method for producing the same

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

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.

Theragnostic agents are substances that enable simultaneous diagnosis and treatment of diseases. They are usually composed of small substances, and are usually in the form of fluorescent dyes, radioactive molecules, etc. being loaded inside liposomes, polymers, nanoparticles, etc., and drugs or diagnostic markers being introduced to the outside. Recently, research has been focused on the synthesis of therapeutic contrast agents composed of lipid structures with excellent biocompatibility, and in particular, research on bioimaging analysis using microbubbles (MB), which are micrometer-sized air bubble ultrasound contrast agents composed of lipid structures, is being actively conducted, and much research is also being conducted on treatment methods using microbubbles, nanoparticle drug delivery systems, and their combined therapy.

Microbubbles repeatedly contract and expand in response to ultrasound stimulation after injection, causing cavitation outside the cell. When cavitation stimulates the cell membrane in close proximity, it generates strong streaming, creating numerous perforations in the cell membrane, and this phenomenon is called sonoporation. When perforations occur in the cell membrane, the efficiency of intracellular drug delivery can be increased, so that when the same amount is injected, the amount of drug that can penetrate the cell increases, which is suitable for the purpose of treatment and is economical. In addition, it has the advantage of being able to estimate the location of drug release using ultrasound imaging.

However, research on drug delivery using microbubbles has several limitations. One of the most important characteristics for serving as a drug delivery vehicle is the stability of the particle itself. In the case of microbubbles, even if the stability is greatly improved by the shell, the stability duration is lower than that of solid or liquid particles. In particular, since they are greatly affected by temperature and pressure changes in the body, a lot of loss occurs during the circulation process, so if the microbubbles collapse before reaching the target point, the drug is released to normal cells, causing side effects. In addition, due to their micro size, they have the disadvantage of being difficult to absorb from blood vessels to cancer tissue.

Meanwhile, gene therapy refers to a method of treating diseases by delivering nucleic acids such as DNA or RNA into cells using a gene delivery vehicle and expressing the delivered gene. For successful gene therapy, it is important to use a delivery vehicle that can effectively deliver genes. An effective gene delivery vehicle should have high delivery efficiency and low toxicity to cells. Gene delivery vehicles can be broadly divided into viral and non-viral delivery vehicles. In general, viral delivery vehicles show higher efficiency, but since the delivered genetic material can be integrated into the host chromosome, it can cause mutations and develop into cancer, and can cause various side effects such as immune responses. On the other hand, non-viral delivery vehicles have the advantage of being relatively stable, having low cytotoxicity, and having a low possibility of inducing an immune response. However, since the delivery efficiency is lower than that of viral delivery vehicles, there is a need to improve this.

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.

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, thereby 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.

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

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

Another object of the present invention is to provide a method for producing ultrasound-responsive lipid nanoparticles according to the present invention.

However, the technical problems to be achieved 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.

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

In one embodiment of the present invention, the cationic lipid may be at least one selected from the group consisting of, but is not limited to, 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).

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.

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.

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

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

(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, and 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.

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

In another embodiment of the present invention, the perfluorocarbon may be at least one selected from the group consisting of perfluoropropane (C ₃ F ₈ ), perfluorobutane (C ₄ F ₁₀ ), perfluoropentane (C ₅ F ₁₂ ), perfluorohexane (C ₆ F ₁₄ ), and perfluoromethylcyclohexane (C ₆ F ₁₁ CF ₃ ), but is not limited thereto.

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.

In another embodiment of the present invention, the perfluorocarbon may be cavitated by ultrasound, but is not limited thereto.

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.

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

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

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

[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) Step of extruding 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 prepared in step (a) and the buffer solution;

(c) a step of adding perfluorocarbon; and

(d) Step of extruding 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.

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.

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

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.

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

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

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 coupled; or a composition comprising the same.

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 maximizes sonoporation by inducing cavitation of perfluorocarbon inside the lipid nanoparticle by ultrasound, thereby improving the efficiency of gene (drug) delivery. Therefore, it is expected that the lipid nanoparticle can be usefully used in gene-based treatment.

Figure 1 shows ultrasound response images of the compositions of DSPC, DOPE, and DSPE-mPEG in Table 1.

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

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.

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.

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

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.

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

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

Figure 9 shows the results of confirming the gene expression rate of a sonoporation agent for gene delivery.

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, thereby 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.

Hereinafter, the present invention will be described in detail.

The present invention provides ultrasound-responsive lipid nanoparticles comprising a cationic lipid, a non-cationic lipid, and a perfluorocarbon.

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.

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.

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,

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

The cationic lipids are DODAP and DC-Chol,

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:

(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, and 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.

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 of 20:65:10:5, but is not limited thereto.

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.

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.

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.

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 can 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.

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) into the lipid nanoparticle. Specifically, the perfluorocarbon may be supported (loaded, contained) inside the nanoparticle, but is not limited thereto.

The term "perfluorocarbons (PFCs)" as used herein refers to compounds in which all CH in the chain are replaced with CF, and may include PFC precursors in which the 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 CF ₄ , C ₂ F ₆ , and C ₄ F ₈ , and are known to be chemically very stable and virtually non-toxic substances. In the present invention, the perfluorocarbon may be at least one selected from the group consisting of perfluoropropane (C ₃ F ₈ ), perfluorobutane (C ₄ F ₁₀ ), perfluoropentane (C ₅ F ₁₂ ), perfluorohexane (C ₆ F ₁₄ ), and perfluoromethylcyclohexane (C ₆ F ₁₁ CF ₃ ), but is not limited thereto.

According to one embodiment of the present invention, the perfluorocarbon may be included in a dose 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 dose of 20 uL per 1 mg. However, it is not limited to this.

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

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 ruptured in a very short moment, and the movement of a substance through the membrane, i.e. the penetration of genes (drugs), is increased.

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 occurs excessively and the penetration effect (sonoporation) of genes (drugs) is enhanced.

Therefore, the nanoparticles of the present invention can be administered in combination with other drugs as an accelerator capable of promoting drug delivery (absorption). By co-administering with the ultrasound-sensitive nanoparticles containing the perfluorocarbon of the present invention therein, the penetration effect of the drugs can be increased, thereby maximizing the efficacy of each drug and reducing the dosage of each drug, thereby minimizing side effects such as toxicity. The drugs and the nanoparticles can be each formulated and administered simultaneously or sequentially. In this case, in the case of sequential administration, there is no limitation on the administration order, and the administration regimen can be appropriately adjusted depending on the patient's condition, etc.

In the present invention, "ultrasound" means a sound wave exceeding a frequency of 16 Hz to 20 kHz, which is a sound wave that can generally be heard by human ears, 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 sufficient energy to cause coagulation necrosis and thermocautery effects regardless of the type of tissue.

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 to perform the HIFU treatment, there are ultrasound-guided high intensity focused ultrasound (Ultrasound-guided HIFU) and MRI-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 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, but is not limited thereto.

In addition, since the ultrasound-responsive 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-responsive nanoparticles of the present invention as an active ingredient.

In the present invention, the ultrasound-responsive lipid nanoparticle and the gene may be combined in 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 in an N/P ratio of 1:3 to 10, but is not limited thereto.

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 may be synthesized, and may exist in various sizes from oligonucleotides to chromosomes. These genes are derived from humans, animals, plants, bacteria, viruses, etc. They may be obtained using methods known in the art.

In addition, since the lipid nanoparticle of the present invention contains cationic lipids, it can be used as a carrier for anionic substances that require delivery into the body in addition to genes. Accordingly, the present invention provides a drug delivery composition containing the ultrasound-sensitive lipid nanoparticle according to the present invention as an active ingredient.

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 agents, protein drugs, growth factors, cytokines, peptide drugs, hair tonics, and anesthetics, and may include any anionic substance.

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.

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.

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:

[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) Step of extruding 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 prepared in step (a) and the buffer solution;

(c) a step of adding perfluorocarbon; and

(d) Step of extruding 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.

The term "preparation (manufacturing) at the time of use" in the present invention means preparation at the time of use, meaning use immediately after preparation.

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.

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.

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.

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 in order to prevent vaporization of perfluorocarbon, and the steps after step (a) may be performed while maintaining the lowered temperature.

Since the ultrasound-responsive lipid nanoparticles of the present invention contain cationic lipids and can deliver genes, the 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 the non-cationic lipid in ethanol.

Unless otherwise specified, all numbers, values, and/or expressions expressing ingredients, reaction conditions, and quantities of ingredients used herein are to be understood as being modified in all instances by the term "about" because these numbers are approximations that inherently reflect, among other things, the various uncertainties of measurement that arise in obtaining such values. 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 an integer, every integer from the minimum value to the maximum value inclusive, unless otherwise indicated, is included.

In this specification, when a range is described for a variable, it will be understood that the variable includes all values within the described range including the described endpoints of the range. For example, a range of "5 to 10" will be understood to include the values 5, 6, 7, 8, 9, and 10, as well as any subranges of 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc., and also any value between the 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 not only the values 10%, 11%, 12%, 13%, etc., and all integers up to and including 30%, but also any subranges such as 10% to 15%, 12% to 18%, 20% to 30%, and also any value between reasonable integers within the stated range, such as 10.5%, 15.5%, 25.5%, etc.

The terms used in the present invention are selected from the most widely used general terms possible while considering the functions of the present invention, but they may vary depending on the intention of engineers working in the field, precedents, the emergence of new technologies, etc. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meanings thereof will be described in detail in the description of the relevant invention. Therefore, the terms used in the present invention should be defined based on the meanings of the terms and the overall contents of the present invention, rather than simply the names of the terms.

Throughout the specification of the present invention, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components can be included, unless otherwise specifically stated. The terms "about," "substantially," and the like, used throughout the specification of the present invention, are used in a meaning at or close to the numerical value when manufacturing and material tolerances inherent in the stated meaning are presented, and are used to prevent unscrupulous infringers from unfairly utilizing the disclosure in which exact or absolute values are mentioned to aid the understanding of the present invention.

Throughout the specification of the present invention, the term "a combination of these" 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 said components.

Hereinafter, preferred examples are presented to help understand the present invention. However, the following examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following examples.

[Example]

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

Ultrasonic-responsive lipid-based particles containing perfluorocarbons were developed by controlling 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 in an amount of 2% of the total volume was added to the mixture, and then 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 by zetasizer, nanosight, and IMD10R, respectively, and the results are shown in Table 1 and Fig. 1.

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 of the particles in Table 1 responded to ultrasonic waves and induced cavitation, as can be seen in the ultrasonic image of Fig. 1.

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

There are two main manufacturing methods for producing a sonoporation agent for gene delivery containing perfluoropentane composed of a shell of various lipids 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.). In the case of the lipid, after drying in the form of a lipid film depending on the composition, it is completely dispersed by stirring and sonication at a temperature of 50-60℃ above the phase transition temperature using the above buffer. After that, 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. Extrusion is performed using an Avanti ^® Mini-Extruder. After that, genetic material such as pDNA is added to the manufactured particles according to the determined N/P ratio so that anionic pDNA is attached to the surface of the cationic lipid particles.

(2) For Manufacturing Method 2, after dissolving the lipid of the same composition in ethanol at a high concentration, the ethanol solution is mixed with a buffer such as 10% sucrose, 0.9% NaCl, nuclease free water, etc. At this time, ethanol is mixed so that it is 10 v/v % or less of the total buffer solution. In addition, genetic material such as pDNA is mixed with the buffer at a set N/P ratio so that the pDNA can be encapsulated inside the particles. In the same way as Manufacturing Method 1, the total volume of the above materials is adjusted to 1 mL (lipid concentration 1 mg/mL), and perfluoropentane is added in an amount that becomes 2% of the total volume%, and the subsequent steps are the same as in Manufacturing Method 1 to perform extrusion using an Avanti ^® Mini-Extruder.

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, and the morphology of the particles according to Manufacturing Method 1 and Manufacturing Method 2 was analyzed using cryo-TEM. The TEM image is shown in Fig. 2.

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 appearances to the particles manufactured previously. Since the properties of the particles did not change depending on the manufacturing method, it was determined that the properties of the particles were determined by the particle constituents and composition.

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

To derive the approximate range of cationic lipids in a cationic lipid-based gene delivery sonoporation agent containing perfluoropentane, sonoporation agents were manufactured by adjusting the lipid composition ratio as shown in Table 2.

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 the sonoporation agent was produced using the same method as in Manufacturing Method 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.

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

In the case of DOTAP 10%, it was found that sediment was formed and then released during extrusion, requiring excessive pressure.

Example 4. Derivation of the range of N/P ratio of cationic lipid-based sonoporation agent for gene delivery containing perfluoropentane

In order to set the range of N/P ratio of cationic lipid-based gene delivery sonoporation agent containing perfluoropentane, the ratio of cationic lipid was fixed at 40% and the experiment was conducted. In the case of gel retardation assay, DNA staining solution was mixed with the manufactured particles, and then 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.

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

As shown in Table 3 and Fig. 3, when the N/P ratio was 1, it was confirmed that the particles clumped together due to the large amount of pDNA, so both types of cationic lipid particles had large sizes, and 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.

In addition, 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% of DOTAP, as shown in Table 4. The formulation was also produced in the same manner as in Manufacturing Method 1.

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.

DOTAP 20% DOTAP 40% N/P ratio Size (nm) PDI Zeta Potential (mV) Size (nm) PDI Zeta Potential (mV) Bare 279.17 0.180 54.9 295.83 0.207 50.43 1:1 7340.67 0.823 -25.17 816.40 0.582 -1.25 3:1 358.90 0.441 51.57 391.37 0.476 44.83 5:1 282.03 0.259 51.57 319.27 0.302 45.43

From the above results, it was determined that when the N/P ratio in the manufactured ultrasound-responsive 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.

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.

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

In order 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 was performed using the same process as Manufacturing Method 1 of Example 2. In order 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.

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, in the case of DOTAP, the experiment was conducted after fixing it to 20%. The analysis results are shown in Table 6. As shown in Table 6, the z-average values of the particles were produced at around 300 nm in all compositions. In addition, in order to confirm the stability of the sonoporation agent of each composition, the particles were stored at RT and 4℃ for 24 hours and the change in the size of the particles was observed. As a result of the observation, it was confirmed that the particles were stably maintained for 24 hours without significant change. The subsequent experiments were conducted by fixing the ratios at DOTAP 20, DSPC 65, DOPE 10, and DSPE-mPEG 5 mol %.

Example 6. Confirmation of ultrasound sensitivity of cationic lipid-based sonoporation agent for gene delivery containing perfluoropentane

In order to explore the functionality of sonoporation agent containing perfluoropentane, the enhancement of contrast ability by ultrasound response was confirmed. The manufactured particles were diluted 10-fold using distilled water, 1 mL was placed in a dialysis membrane, and the ultrasound contrast ability by ultrasound emission was evaluated at 37℃. The ultrasound equipment used here was IMGT's IMD10, and the conditions of the emitted ultrasound were Intensity 2kW/ ^cm2 , PRF 10Hz, Duty 2%, and 10s/spot. As shown in Fig. 5, ultrasound contrast was not obtained before ultrasound irradiation, but it was confirmed that the ultrasound contrast ability of the sonoporation agent was enhanced after ultrasound irradiation.

In addition, 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 Fig. 6, it was possible to visually confirm the occurrence of bubbles due to vaporization in the solution phase after ultrasonic irradiation.

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

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 Manufacturing Methods 1 and 2 of Example 2 were respectively manufactured, and pDNA loading was confirmed under the same electrophoresis conditions as in Example 3. As shown in Fig. 7, the particles manufactured according to Manufacturing Methods 1 and 2 both 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.

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 calculation formula of ((Triton-X-treated particles - particle itself) / Triton-X-treated particles) * 100 (%). The pDNA encapsulation efficiency derived through the calculation 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.

Encapsulation Efficiency (%) Recipe 1 19.8 Recipe 2 68.6

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 Huh-7 cell lines. In the case of plasmid DNA, plasmid DNA expressing tdtomato was used, and the results were confirmed through a fluorescence microscope. The particles manufactured according to Manufacturing Methods 1 and 2 were treated to the cell lines, 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 cells with or without ultrasound irradiation was confirmed through fluorescence images. As a result, as shown in Fig. 8, it was confirmed that the fluorescence expression rate of cells treated with ultrasound was further enhanced for both particles manufactured according to Manufacturing Methods 1 and 2.

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

Lipids were mixed in the molar ratios shown in Table 8 to prepare a lipid stock. Each lipid stock was prepared by dissolving in EtOH, and the EtOH concentration was made 5% of the total volume. 0.9% saline solution 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 placed in an ice bath to keep the solution cold, and 20 μL of perfluoropentane (2% perfluoropentane), which is 2% of the total volume, was added. Ultrasonication was performed under the conditions of Amp 70%, Pulse on 3 s, off 2 s, and 2 min 30 s to load perfluoropentane into the interior of nanoparticles, thereby preparing a sonoporation agent. The physical properties of the particles were confirmed by dynamic light scattering (DLS) and electrophoresis to confirm the particle size and gene loading rate.

No. Charged lipid DC-cholesterol cholesterol DOPE DSPE-mPEG2000 1 DOTAP 50% 25.4 - 25 0.5 2 DODAP 50% 24.5 - 25 0.5 3 DODAP 50% 25 - 25 - 4 DODAP 50% 24.5 24 10 0.5

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.

No. Size(nm) PDI Zeta potential (mV) 1 441.5 0.380 25.9 2 243.3 0.102 28.94 3 266.7 0.105 37.45 4 251.5 0.152 32.85

In addition, as a result of quantitative analysis of the gene transfer and expression rate by ultrasound response 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.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

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. Therefore, it can be usefully used for treatment using genes, and thus has industrial applicability.

Claims (20)

Ultrasound-responsive lipid nanoparticles comprising cationic lipids, non-cationic lipids, and perfluorocarbons. In the first paragraph, 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). In the first paragraph, An ultrasound-responsive lipid nanoparticle, 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. In the first paragraph, 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. In the first paragraph, 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. In the first paragraph, 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, and 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. In the first paragraph, Ultrasound-responsive lipid nanoparticles, characterized in that the above perfluorocarbon is loaded into the nanoparticles. In the first paragraph, Ultrasound-responsive lipid nanoparticles, characterized in that the perfluorocarbon is at least one selected from the group consisting of perfluoropropane (C ₃ F ₈ ), perfluorobutane (C ₄ F ₁₀ ), perfluoropentane (C ₅ F ₁₂ ), perfluorohexane (C ₆ F ₁₄ ), and perfluoromethylcyclohexane (C ₆ F ₁₁ CF ₃ ). In the first paragraph, Ultrasound-responsive lipid nanoparticles, characterized in that the perfluorocarbon is contained in a volume of 1 to 50 uL per 1 mg of nanoparticles. In the first paragraph, The above perfluorocarbon is an ultrasound-responsive lipid nanoparticle characterized by cavitation induced by ultrasound. A composition for gene delivery comprising an ultrasound-responsive lipid nanoparticle according to any one of claims 1 to 10 as an active ingredient. In Article 11, A composition for gene delivery, characterized in that the ultrasound-responsive lipid nanoparticles and the gene are combined at an N/P ratio of 1:1 to 20. In Article 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. A method for producing ultrasound-responsive 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) Step of extruding 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 prepared in step (a) and the buffer solution; (c) a step of adding perfluorocarbon; and (d) Step of extruding 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. In Article 14, A method for producing ultrasound-responsive 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 ultrasonication in a buffer solution. In Article 15, A method for producing ultrasound-sensitive lipid nanoparticles, characterized in that the stirring is performed at 30 to 80°C. In Article 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). Ultrasound-responsive lipid nanoparticles of claim 1; or a composition comprising the same for use in gene delivery Ultrasound-responsive lipid nanoparticles according to claim 1 for the manufacture of gene delivery vehicles; or use of a composition comprising the same A method for gene delivery comprising the step of administering the ultrasound-sensitive lipid nanoparticle of claim 1 carrying a gene; or a composition comprising the same, to a subject in need thereof.

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