Ultrasound-sensitive nanoparticles comprising perfluorocarbon and method for manufacturing same

Abstract

The present invention relates to ultrasound-sensitive nanoparticles comprising perfluorocarbon. The ultrasound-sensitive nanoparticles according to the present invention not only can improve storage stability through perfluorocarbon supported (loaded) therein, but also can enhance a drug delivery effect through the maximization of sonoporation by increasing cavitation due to volumetric expansion resulting from the vaporization of perfluorocarbon induced by ultrasound, so that the ultrasound-sensitive nanoparticles are expected to exhibit various functions in the field of drug delivery systems.

Description

Ultrasound-sensitive nanoparticles containing perfluorocarbon and method for producing the same

[0001] The present invention relates to ultrasonic-sensitive nanoparticles containing perfluorocarbon and a method for producing the same.

[0002] This invention claims the benefit of Korean Patent Application No. 10-2023-0103756, filed on August 8, 2023, and the benefit of Korean Patent Application No. 10-2024-0105671, filed on August 7, 2024, the entire contents of which are incorporated herein by reference.

[0003]

[0004] Drug Delivery Systems (DDS), which transport drugs in micro-sized capsules through the bloodstream to the affected area to ensure their effective action, are attracting attention as a dream treatment technology of the future. DDS drug delivery routes include not only through blood vessels but also through the digestive tract and the skin. The technologies that accurately deliver drugs to the affected area are collectively called drug delivery systems. The realization of DDS requires 1) a microbubble manufacturing technology applicable to the body, 2) a drug injection technology into the capsule, 3) a targeting technique to ensure that the capsule introduced into the body remains in the affected area, and 4) a drug absorption enhancement technology to deliver the drug internally through the biological membrane with low drug permeability.

[0005] Ultrasound-assisted DDS using microbubbles as drug delivery capsules is attracting significant attention because ultrasound can be used for targeting and enhancing drug absorption. Since the size, direction, and location of the acoustic radiation pressure applied to the bubbles can be controlled by ultrasound, methods for utilizing this force to capture microbubbles at a desired location are also being explored. Furthermore, the use of ultrasound is believed to enable not only targeted targeting but also simultaneous enhancement of drug absorption within biological tissues.

[0006] Meanwhile, microbubbles exposed to ultrasound cause cavitation, which temporarily forms pores in the cell membranes of surrounding cells, which increases the lymphocyte phenomenon near cancer cells, and tumor ablation activates dendritic cells, which in turn activates the immune system. Therefore, when ultrasound-based immunotherapy is used alone as an indirect treatment method, its range and therapeutic effect are weaker than those of immunotherapy drugs, but it is expected that a synergistic effect can be produced when the unique physical mechanism of ultrasound is combined with existing immunotherapy methods.

[0007]

[0008] The present inventors have conducted extensive research on methods to maximize the effectiveness of therapeutic methods using ultrasound, and have confirmed that when perfluorocarbon is loaded (supported) on nanoparticles, not only is the storage stability of the nanoparticles improved, but also the sonoporation effect is maximized by inducing cavitation when the perfluorocarbon is vaporized by ultrasound, thereby completing the present invention.

[0009] Accordingly, an object of the present invention is to provide a composition for promoting drug delivery, which comprises ultrasound-sensitive nanoparticles containing DSPC (1,2-Distearoyl-sn-glycero-3-phosphorylcholine), DSPE-mPEG2k (1,2-distearoyl-sn-glycero-3 phosphoethanolamine with conjugated methoxyl poly(ethylene glycol2000)), cholesterol, DOPE (1,2-Dioleoyl-sn-glycero 3-phosphoethanolamine), lyso-PC (lysophosphatidylcholine), and perfluorocarbon as active ingredients.

[0010] Another object of the present invention is to provide a method for producing ultrasound-sensitive nanoparticles.

[0011] Another object of the present invention is to provide a method for manufacturing ultrasound-sensitive nanoparticles.

[0012] Another object of the present invention is to provide a kit for preparing ultrasound-sensitive nanoparticles (SNPs) comprising a first composition comprising DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC; and a second composition comprising perfluorocarbon.

[0013]

[0014] 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.

[0015]

[0016] In order to achieve the above object, the present invention provides a composition for promoting drug delivery, which comprises ultrasound-sensitive nanoparticles containing DSPC (1,2-Distearoyl-sn-glycero-3-phosphorylcholine), DSPE-mPEG2k (1,2-distearoyl-sn-glycero-3 phosphoethanolamine with conjugated methoxyl poly(ethylene glycol2000)), cholesterol, DOPE (1,2-Dioleoyl-sn-glycero 3-phosphoethanolamine), lyso-PC (lysophosphatidylcholine), and perfluorocarbon as active ingredients.

[0017] In one embodiment of the present invention, the perfluorocarbon may be loaded into the nanoparticles, but is not limited thereto.

[0018] In another embodiment of the present invention, the perfluorocarbon and nanoparticles may be included in a concentration ratio of 0.01 to 10:1, but is not limited thereto.

[0019] 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).

[0020] In another embodiment of the present invention, the perfluorocarbon may be capable of cavitation induced by ultrasound, but is not limited thereto.

[0021] In another embodiment of the present invention, the nanoparticle membrane may be composed of, but is not limited to, DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC.

[0022] In another embodiment of the present invention, DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC may be included in a molar ratio (mole%) of, but not limited to, 1 to 90: 1 to 20: 1 to 80: 0.1 to 40: 1 to 20.

[0023] In another embodiment of the present invention, the nanoparticles may have a diameter of, but is not limited to, 100 to 1000 nm.

[0024] In another embodiment of the present invention, the nanoparticles may have secured storage stability, but are not limited thereto.

[0025] In addition, the present invention provides a method for producing ultrasound-sensitive nanoparticles comprising the following steps:

[0026] (a) a step of hydrating DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC by stirring with a buffer solution; and

[0027] (b) Step of adding perfluorocarbon.

[0028] In one embodiment of the present invention, the buffer solution may be one or more selected from the group consisting of, but is not limited to:

[0029] a) sucrose;

[0030] b) A buffer solution containing glycol and propylene glycol;

[0031] c) a buffer solution containing sodium phosphate and sucrose; and

[0032] d) PBS.

[0033] In another embodiment of the present invention, step (a) may be performed at 30 to 80°C for 1 to 5 hours, but is not limited thereto.

[0034] In another embodiment of the present invention, step (a) may further include, but is not limited to, a step of lowering the temperature.

[0035] In addition, the present invention provides a method for preparing ultrasound-sensitive nanoparticles, comprising the following steps:

[0036] (a) a step of preparing a first solution by dissolving DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC in an organic solvent;

[0037] (b) a step of preparing a second solution by mixing perfluorocarbon into a buffer solution; and

[0038] (c) A step of mixing the first solution and the second solution, filtering them, and mixing them.

[0039] In one embodiment of the present invention, the organic solvent in step (a) may be at least one selected from the group consisting of dimethylacetamide, dimethylformamide, dimethylsulfoxide, chloroform, methanol, ethanol, and ether, but is not limited thereto.

[0040] In another embodiment of the present invention, the buffer solution may be one or more selected from the group consisting of, but is not limited to:

[0041] a) sucrose;

[0042] b) A buffer solution containing glycol and propylene glycol;

[0043] c) a buffer solution containing sodium phosphate and sucrose; and

[0044] d) PBS.

[0045] In another embodiment of the present invention, in step (c), the first solution and the second solution may be mixed in a volume ratio of 1:1 to 20, but is not limited thereto.

[0046] In another embodiment of the present invention, the filter in step (c) may have a pore size of 0.1 to 5 μm, but is not limited thereto.

[0047] In addition, the present invention provides a kit for preparing ultrasound-sensitive nanoparticles (SNPs) comprising a first composition comprising DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC; and a second composition comprising a perfluorocarbon.

[0048] In addition, the present invention provides a use of the ultrasound-sensitive nanoparticles according to the present invention for promoting drug delivery.

[0049] The present invention also provides the use of the ultrasound-sensitive nanoparticles according to the present invention for preparing a drug delivery facilitator.

[0050] The present invention also provides a method for promoting drug delivery, comprising administering ultrasound-sensitive nanoparticles according to the present invention and a drug to a subject in need thereof.

[0051]

[0052] The ultrasound-sensitive nanoparticles according to the present invention not only have improved storage stability due to the perfluorocarbon contained (loaded) therein, but also maximize sonoporation by increasing cavitation as the perfluorocarbon is vaporized by ultrasound, thereby enhancing the drug delivery effect, and are therefore expected to exhibit various functions in the field of drug delivery vehicles.

[0053]

[0054] Figures 1a and 1b show particle size graphs of a sonoporation agent loaded with perfluorobutane (hereinafter, MB: microbubble).

[0055] Figures 2a and 2b show particle size distribution graphs of a sonoporation agent loaded with perfluorobutane.

[0056] Figure 3a shows the particle size of a sonoporation agent loaded with perfluoropentane.

[0057] Figure 3b shows an electron microscope image of a sonoporation agent loaded with perfluoropentane.

[0058] Figures 4a and 4b show graphs of particle size and size change after vaporization of a sonoporation agent according to the content ratio of lipid and perfluoropentane.

[0059] Figures 5a to 5c show changes in particle size according to temperature changes of a sonoporation agent loaded with perfluoropentane.

[0060] Figures 6a and 6b show the size distribution of sonoporation agents loaded with perfluoropentane for two months depending on the type of buffer solution.

[0061] Figures 7a and 7b are C5F 12 nanoemulsion (Fig. 7a) and C8F 18 The size distribution of the nanoemulsion (Fig. 7b) is shown.

[0062] Fig. 8 is C5F 12 nanoemulsion and C8F 18 This shows the ultrasound image of nanoemulsion according to the ultrasound intensity.

[0063] Figure 9 is C5F manufactured by the method of Example 8. 12 This shows the particle size distribution of nanoemulsion.

[0064]

[0065] The present inventors have conducted extensive research on methods to maximize the effectiveness of therapeutic methods using ultrasound, and have confirmed that when perfluorocarbon is loaded (supported) on nanoparticles, not only is the stability of the nanoparticles improved, but the perfluorocarbon is vaporized by ultrasound, thereby increasing cavitation and maximizing the sonoporation effect, thereby completing the present invention.

[0066]

[0067] Hereinafter, the present invention will be described in detail.

[0068]

[0069] The present invention provides a composition for promoting drug delivery, which comprises ultrasound-sensitive nanoparticles as active ingredients, including DSPC (1,2-Distearoyl-sn-glycero-3-phosphorylcholine), DSPE-mPEG2k (1,2-distearoyl-sn-glycero-3 phosphoethanolamine with conjugated methoxyl poly(ethylene glycol 2000)), cholesterol, DOPE (1,2-Dioleoyl-sn-glycero 3-phosphoethanolamine), lyso-PC (lysophosphatidylcholine), and perfluorocarbon.

[0070] The term "DSPC" used in the present invention is an abbreviation for 1,2-Distearoyl-sn glycero-3-phosphocholine, which is a phospholipid composed of two palmitic acids attached to a phosphatidylcholine head group.

[0071] The term "DSPE-mPEG2k" used in the present invention is an abbreviation for 1,2-Distearoyl-sn-Glycero-3-Phosphoethanol amine with conjugated methoxyl poly(ethylene glycol2000) and is a PEGylated derivative of 1,2-distearoyl-sn-glycero-3-PE (DSPE).

[0072] The term "cholesterol" as used herein refers to one of the steroid compounds. The cholesterol includes cholesterol derivatives. The cholesterol derivatives may be, for example, sitosterol, ergosterol, stigmasterol, 4,22-stigmasteradien-3-one, stigmasterol acetate, lanosterol, cycloartenol, or combinations thereof. Cholesterol is located in the lipid bilayer, and its amount can be adjusted to lower or increase permeability, and can be used regardless of the ratio within the nanoparticle.

[0073] The term "DOPE" used in the present invention is an abbreviation for 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, which is known to form heteroliposomes with DOTAP, which is used as a delivery vehicle for therapeutic agents.

[0074] The term "lyso-PC" used in the present invention is an abbreviation for lysophosphatidylcholine, and is a representative term for a lipid composed of one acyl chain derived from phosphocholine, where the head group is composed of choline. Various types of lyso-PC exist, and according to one embodiment of the present invention, the lyso-PC may be soy lyso-PC (S-LysoPC), but is not limited thereto.

[0075] In the present invention, the lyso-PC may be more specifically represented by the following chemical formula 1 or chemical formula 2, but is not limited thereto.

[0076] In the present invention, the lyso-PC may be 1-LPC (lysophosphatidylcholine) or 2-LPC represented by the following chemical formula 1 or 2, but is not limited thereto.

[0077] [Chemical Formula 1]

[0078]

[0079] [Chemical Formula 2]

[0080]

[0081] (In the above chemical formula 1 or 2, R is C6 to C 26 Alkyl group of C6 to C 26 Alkenyl group, C6 to C 26 Alkynyl group, substituted or unsubstituted C6 to C 26 Cycloalkyl, substituted or unsubstituted C6 to C 26 Aryl group, substituted or unsubstituted C7 to C 26 arylalkyl group, or H.)

[0082] In the present invention, the lyso-PC is lyso-PC (6:0), lyso-PC (7:0), lyso-PC (8:0), lyso-PC (9:0), lyso-PC (10:0), lyso-PC (11:0), lyso-PC (12:0), lyso-PC (13:0), lyso-PC (14:0), lyso-PC(15:0), lyso-PC(16:0), 2-lyso-PC(16:0), lyso-PC(17:0), lyso-PC(17:1), lyso-PC(18:0), lyso-PC(18:1), lyso-PC(18:2), 2-lyso-PC(18:0), 2-lyso-PC(18:1), lyso-PC(19:0), It may be at least one selected from the group consisting of lyso-PC(20:1), lyso-PC(20:4), lyso-PC(22:0), lyso-PC(22:5), lyso-PC(24:0), lyso-PC(26:0), and lyso-PC(20:5), but is not limited thereto.

[0083] The term "perfluorocarbons (PFCs)" used in the present invention 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 (C6F11 It may be one or more selected from the group consisting of, but is not limited to, CF3).

[0084] In the present invention, the perfluorocarbon is characterized in that it is loaded into the nanoparticle, and the perfluorocarbon and the nanoparticle are, that is, perfluorocarbon: nanoparticle = 0.01 to 10:1, 0.01 to 8:1, 0.01 to 5:1, 0.01 to 3:1, 0.01 to 1:1, 0.01 to 0.8:1, 0.01 to 0.4:1, 0.01 to 0.2:1, 0.01 to 0.04:1, 0.04 to 8:1, 0.04 to 5:1, 0.04 to 3:1, 0.04 to 1:1, 0.04 to 0.8:1, 0.04 to 0.4:1, It can be included or loaded into the nanoparticles at a concentration ratio (% v / v) of 0.04 to 0.2:1, 0.2 to 5:1, 0.2 to 3:1, 0.2 to 1:1, 0.2 to 0.8:1, 0.2 to 0.4:1, 0.4 to 3:1, 0.4 to 1:1, 0.4 to 0.8:1, 0.8 to 1:1, 0.04:1, 0.2:1, 0.4:1, or 0.8:1, and according to one embodiment of the present invention, it can be included at a concentration ratio (v / v%) of 0.04 to 0.8:1, but is not limited thereto.

[0085] According to one embodiment of the present invention, when perfluorocarbon is loaded (contained or supported) inside a nanoparticle composed of lipid, not only is the storage stability of the nanoparticle improved, but the effect of sonoporation is maximized as the perfluorocarbon causes cavitation by ultrasound, thereby exhibiting an improved drug delivery effect.

[0086] In the present invention, the nanoparticles 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 also perfluorocarbons inside the nanoparticles are induced to cavitation by ultrasound, so that cavitation is excessively generated and the drug penetration effect (sonoporation) is improved. Therefore, the nanoparticles of the present invention can be co-administered with other drugs as an accelerator that can promote drug delivery (absorption). By co-administering with the ultrasound-responsive nanoparticles of the present invention that include perfluorocarbons inside, the efficacy of each drug can be maximized and the dosage of each drug can be reduced, thereby minimizing side effects such as toxicity. The drug and the nanoparticles may be individually formulated and administered simultaneously or sequentially. In this case, there are no restrictions on the order of administration in the case of sequential administration, and the dosing regimen may be appropriately adjusted depending on the patient's condition, etc.

[0087] The nanoparticles of the present invention may preferably be liposomes loaded with perfluorocarbons therein. In the present invention, the nanoparticles are formed by an amphiphilic compound comprising a phospholipid. Such amphiphilic compounds are typically arranged at the interface between an aqueous medium and an essentially water-insoluble organic solvent, thereby stabilizing emulsified solvent microdroplets. The amphiphilic compounds include compounds having molecules having a hydrophilic polar head moiety (e.g., a polar or ionic group) that can react with the aqueous medium and a hydrophobic organic tail moiety (e.g., a hydrocarbon chain) that can react with the organic solvent. Amphiphilic compounds are compounds that can stabilize mixtures of substances that are not normally mixed by other methods, such as mixtures of two immiscible liquids (e.g., water and oil), mixtures of a liquid and a gas (e.g., gas microbubbles in water), or mixtures of a liquid and insoluble particles (e.g., metal nanoparticles in water).

[0088] 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 a drug, is increased.

[0089] In the present invention, the nanoparticle (liposome) membrane may be composed of DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC, but is not limited thereto.

[0090] In the present invention, DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC are 1~90 : 1~20 : 1~80 : 0.1~40 : 1~20, 10~90 : 3~20 : 10~80 : 0.5~40 : 3~20, 10~80 : 3~18 : 10~70 : 0.5~30 : 3~18, 20~80 : 5~18 : 20~70 : 1~30 : 5~18, 20~70 : 5~15 : 20~60 : 1~25 : 5~15, 30~70 : 8~15 : 30~60 : 3~25 : 8~15, 30~60 : 8~13 : 30~50 : It may be included in a molar ratio (mole%) of 3~20 : 8~13, 35~60 : 8~10 : 30~40 : 3~15 : 8~11, 35~45 : 8~10 : 30~40 : 5~7 : 9~11, or 40 : 9 : 35 : 6 : 10, but is not limited thereto.

[0091] In the present invention, the nanoparticle may have a diameter of, but is not limited to, 100 to 1000 nm, 100 to 900 nm, 100 to 800 nm, 100 to 700 nm, 100 to 600 nm, 100 to 500 nm, 100 to 400 nm, 100 to 300 nm, 100 to 200 nm, 200 to 800 nm, 200 to 700 nm, 200 to 600 nm, 200 to 500 nm, 200 to 400 nm, 200 to 300 nm, 300 to 600 nm, 300 to 500 nm, or 300 to 400 nm.

[0092] According to one embodiment of the present invention, the nanoparticles of the present invention loaded with perfluorocarbon can be stably maintained without being vaporized when stored at room temperature, and thus the nanoparticles are characterized in that the storage stability of the nanoparticles is secured by the perfluorocarbon loaded therein.

[0093] In the present invention, "ultrasound" means a sound wave exceeding the frequency of 16 Hz to 20 kHz, which is the 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.

[0094] 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 MRI-guided HIFU. The frequency of the ultrasound is, for example, 1 kHz to 100 kHz, 1 kHz to 90 kHz, 1 kHz to 80 kHz, 1 kHz to 70 kHz, 1 kHz to 60 kHz, 1 kHz to 50 kHz, 1 kHz to 40 kHz, 1 kHz to 30 kHz, 1 kHz to 20 kHz, 1 kHz to 10 kHz, 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, It may be, but is not limited to, 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, 950 kHz to 1 MHz, or 1 MHz.

[0095] Alternatively, in the present invention, the intensity of the ultrasonic waves is 10 to 3000 W / cm 2 , 50 to 3000 W / cm 2 , 90 to 3000 W / cm 2 , 90 to 2800 W / cm 2 , 95 to 2800 W / cm 2 , 95 to 2795 W / cm 2 , 95 to 2790 W / cm 2 , 95 to 2785 W / cm 2 , 95 to 2780 W / cm 2 , 95 to 2779 W / cm2 , 95 to 2778 W / cm 2 , 95 to 2777 W / cm 2 , 95 to 2776 W / cm 2 , 95 to 2775 W / cm 2 , 95 to 2774 W / cm 2 , 95 to 2773 W / cm 2 , 95.1 W / cm 2 , 273.6 W / cm 2 , 1344.6 W / cm 2 , or 2772.6 W / cm 2 It may be, but is not limited to,

[0096] The term "drug" as used herein refers to any compound possessing 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.

[0097] In the present invention, the drug may be an anticancer agent, and may be at least one selected from the group consisting of, for example, doxorubicin, paclitaxel, docetaxel, cisplatin, Gleevec, 5-fluorouracil (5-FU), tamoxifen, carboplatin, topotecan, belotecan, imatinib, irinotecan, floxuridine, vinorelbine, gemcitabine, leuprolide, flutamide, zoledronate, methotrexate, camptothecin, vincristine, hydroxyurea, streptozocin, valrubicin, retinoic acid, mechlorethamine, chlorambucil, busulfan, doxifluridine, vinblastine, mitomycin, prednisone, afinitor, and mitoxantrone, but is not limited thereto.

[0098] In the present invention, the ultrasound-sensitive nanoparticles or the pharmaceutical composition containing them may be administered sequentially or simultaneously with the ultrasound treatment, but is not limited thereto.

[0099]

[0100] In addition, the present invention provides a method for producing ultrasound-sensitive nanoparticles comprising the following steps:

[0101] (a) a step of hydrating DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC by stirring with a buffer solution; and

[0102] (b) Step of adding perfluorocarbon.

[0103] According to one embodiment of the present invention, the perfluorocarbon added in step (b) can be loaded onto ultrasonic-sensitive nanoparticles using an ultrasonic homogenizer.

[0104] In the present invention, the buffer solution may be one or more selected from the group consisting of, but is not limited to:

[0105] a) sucrose;

[0106] b) A buffer solution containing glycol and propylene glycol;

[0107] c) a buffer solution containing sodium phosphate and sucrose; and

[0108] d) PBS.

[0109] The above a) sucrose is preferably 33%-Sucrose (w / w%);

[0110] The buffer solution containing the above b) glycol and propylene glycol is preferably an Emulsifier (5%-glycol & 10%-propylene glycol mixture, w / w%);

[0111] c) The buffer solution containing sodium phosphate and sucrose is preferably 0.01M sodium phosphate & 30%-sucrose buffer; and

[0112] The above d) PBS is preferably PBS (Phosphate buffered saline (0.01M, pH 7.4)).

[0113] According to one embodiment of the present invention, the buffer solution can be stably maintained regardless of the type of buffer solution at refrigerated temperature, but can be stably stored at room temperature only when the buffer solution is PBS. Therefore, those skilled in the art can appropriately select a buffer solution by considering the manufacturing and storage environment (temperature) of the nanoparticles of the present invention.

[0114] In the present invention, the step (a) may be performed at 30 to 80°C, 30 to 70°C, 30 to 60°C, 30 to 50°C, 30 to 40°C, 40 to 70°C, 40 to 60°C, 40 to 50°C, or 50 to 60°C for 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, 1 to 2 hours, or 1 hour, and may be performed without limitation as long as it is a temperature and time range in which DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC can be completely hydrated in a buffer solution.

[0115] In the present invention, the step (a) may further include a step of lowering the temperature to prevent vaporization of perfluorocarbon, which may be, but is not limited to, lowering the temperature to 1 to 10°C, 1 to 8°C, 1 to 6°C, 1 to 5°C, 1 to 4°C, 1 to 2°C, 2 to 8°C, 2 to 6°C, 2 to 5°C, 2 to 4°C, 2 to 3°C, 3 to 6°C, 3 to 5°C, 3 to 4°C, 4 to 5°C, 1°C, 2°C, 3°C, 4°C, or 5°C by stirring and hydrating DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC in a buffer solution in the step (a) and storing the lipid solution in a refrigerator.

[0116]

[0117] In addition, the present invention provides a method for preparing ultrasound-sensitive nanoparticles, comprising the following steps:

[0118] (a) a step of preparing a first solution by dissolving DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC in an organic solvent;

[0119] (b) a step of preparing a second solution by mixing perfluorocarbon into a buffer solution; and

[0120] (c) A step of mixing the first solution and the second solution, filtering them, and mixing them.

[0121] According to one embodiment of the present invention, the method for preparing the ultrasonic sensitive nanoparticles may be performed at a temperature of 1°C, 2°C, 3°C, 4°C, or 5°C to prevent vaporization of perfluorocarbon.

[0122] According to one embodiment of the present invention, the step (c) is characterized in that the mixed solution of the first solution and the second solution is repeatedly filtered through a filter to mix the first solution and the second solution.

[0123] 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.

[0124] In the present invention, in step (a), the organic solvent may be at least one selected from the group consisting of dimethylacetamide, dimethylformamide, dimethylsulfoxide, chloroform, methanol, ethanol, and ether, and according to one embodiment of the present invention, the organic solvent may be ethanol, but is not limited thereto.

[0125] In the present invention, the buffer solution may be one or more selected from the group consisting of, but is not limited to:

[0126] a) sucrose;

[0127] b) A buffer solution containing glycol and propylene glycol;

[0128] c) a buffer solution containing sodium phosphate and sucrose; and

[0129] d) PBS.

[0130] The above a) sucrose is preferably 33%-Sucrose (w / w%);

[0131] The buffer solution containing the above b) glycol and propylene glycol is preferably an Emulsifier (5%-glycol & 10%-propylene glycol mixture, w / w%);

[0132] c) The buffer solution containing sodium phosphate and sucrose is preferably 0.01M sodium phosphate & 30%-sucrose buffer; and

[0133] The above d) PBS is preferably PBS (Phosphate buffered saline (0.01 M, pH 7.4)). According to one embodiment of the present invention, the buffer solution may be PBS, but is not limited thereto.

[0134] In the present invention, in step (c), the first solution and the second solution may be mixed in a volume ratio of 1:1 to 20, 1:1 to 18, 1:1 to 15, 1:1 to 13, 1:1 to 12, 1:1 to 11, 1:1 to 10, 1:1 to 9, 1:1 to 8, 1:1 to 5, 1:1 to 3, 1:1 to 2, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, or 1:9, but is not limited thereto.

[0135] In the present invention, in the step (c), the filter may have a pore size of 0.1 to 5, 0.1 to 4, 0.1 to 3, 0.1 to 2, 0.1 to 1, 0.1 to 0.5, 0.2 to 4, 0.2 to 2, 0.2 to 1, 0.2 to 0.8, or 0.2 to 0.5 μm, but is not limited thereto.

[0136] In addition, the present invention provides a kit for preparing ultrasound-sensitive nanoparticles (solubilized) comprising a first composition comprising DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC; and a second composition comprising a perfluorocarbon, wherein the kit may further include an instruction manual describing a method for preparing ultrasound-sensitive nanoparticles of the present invention.

[0137] In the present invention, the first composition may be a solution in which DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC are dissolved in an organic solvent (ethanol) as a first solution of the method for preparing ultrasound-sensitive nanoparticles of the present invention, and the second composition may be a solution in which perfluorocarbon and a buffer solution (PBS) are mixed as a second solution of the method for preparing ultrasound-sensitive nanoparticles of the present invention.

[0138] The kit of the present invention comprises two or more compositions with different compositions, and the end user can easily produce ultrasonic-sensitive nanoparticles containing perfluorocarbons by simply mixing the compositions. The term "simple mixing" can encompass any act of "mixing" and means that no specific conditions are imposed on the mixing act. The mixing can be accomplished by various methods, including, but not limited to, dripping, vortexing, and decanting.

[0139] In the present invention, the kit may include a container; instructions; a first composition; and a second composition. The container may serve to package the substance, and may also serve to store and secure the substance. The material of the container may be, for example, plastic, glass, or the like, but is not limited thereto.

[0140]

[0141] The terms used in this invention have been selected from widely used, current terms, taking into account the functions of the invention. 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.

[0142] 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.

[0143] 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.

[0144]

[0145]

[0146] 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.

[0147]

[0148] [Example]

[0149] Example 1. Preparation and property analysis of lipid-based sonoporation agent loaded with perfluorobutane

[0150] To prepare a sonoporation agent loaded with perfluorobutane consisting of a lipid shell, the lipid was dispersed in 0.01 M phosphate buffered saline (PBS, pH 7.4) at a concentration of 0.5 mg / mL. The dispersion was stirred for more than 1 hour at a temperature of 50-60°C, which is higher than the phase transition temperature, to ensure complete dispersion. 1 mL was then added to a glass vial, and the vial was completely sealed by filling the head space with perfluorobutane gas. The mixture was then vigorously mixed for 45 seconds using VialMIX.

[0151] The size distribution and particle number of the manufactured sonoporation agent were analyzed using a Coulter counter. The particle size distribution and particle number were measured by diluting the sonoporation agent approximately 500-1000 times and passing it through the membrane pores. At this time, the particles showed an average size of 1-3 μm and an average particle size of approximately 0.5-3 × 10 9 / mL particle count was shown.

[0152]

[0153] Example 2. Derivation of the optimal composition of a lipid-based sonoporation agent loaded with perfluorobutane.

[0154] In order to derive the optimal composition of a sonoporation agent loaded with perfluorobutane, the composition of lipids was adjusted as shown in Table 1 to manufacture a sonoporation agent.

[0155] According to the composition in Table 1, each lipid was weighed into a vial and hydrated with 0.01 M phosphate buffered saline (PBS, pH 7.4) at a final concentration of 0.5 mg / mL by stirring at 50-60°C for more than 1 hour. After that, 1 mL was placed into a vial, the head space of the vial was filled with perfluorobutane gas, and the vial was completely sealed. Then, vialMIX was used to vigorously agitrate to prepare a sonoporation agent. The particle size distribution and particle number of the prepared sonoporation agent were analyzed using a Coulter counter, and the analysis results are shown in Table 1. Specifically, the particle size distribution of all compositions showed an average value of 0.5-3.0 μm in size and a D90 value of less than 5.0 μm. In addition, most of them had a particle size of 1.0 × 10 8 / mL or more of particles were shown. The particle size distribution graphs of the five representative species are shown in Figures 1a and 1b.

[0156]

[0157]

[0158] Afterwards, to derive the optimal composition ratio, the molar ratios of DSPC and DOPE were fixed at 40 and 35%, respectively, and other compositions were varied to manufacture sonoporation agents loaded with perfluorobutane, and the particle size distribution and particle number were compared and analyzed. The composition molar ratios of each sonoporation agent are as shown in Table 2.

[0159] After weighing the lipids in a vial with the composition shown in Table 2, they were completely hydrated by stirring at the phase transition temperature of 50-60℃ for more than 1 hour with 0.01 M phosphate buffered saline (PBS, pH 7.4). Then, 1 mL was transferred to a glass vial and vigorously agitized to prepare a sonoporation agent. The particle distribution and particle number of the sonoporation agent were measured using a Coulter counter. As a result, as shown in Table 2, all compositions showed a particle size distribution of 1-3 μm and 0.5-3 × 10 9 / mL of particles was shown. The particle size distribution is shown in Fig. 2a and Fig. 2b.

[0160]

[0161]

[0162] Example 3. Development of a sonoporation agent loaded with perfluoropentane

[0163] To prepare a perfluoropentane-loaded sonoporation agent, based on the results of Examples 1 and 2, one composition that produced a large number of particles and maintained stability was selected from Table 1 to prepare a perfluoropentane-loaded sonoporation agent. At this time, the stability of the composition was compared visually by observing that the solution became clear as the sonoporation agent was destroyed. The selected composition is shown in Table 3. After weighing the lipids in a vial at the molar ratios shown in Table 3, they were hydrated using 0.01 M phosphate buffered saline (PBS, pH 7.4) at a concentration of 5 mg / mL. The hydration was performed by stirring at the phase transition temperature of 50-60°C for more than 1 hour until complete hydration. The fully hydrated lipid solution was stored in a refrigerator to lower the temperature to approximately 4°C, and then perfluoropentane was added in an amount corresponding to 2% (v / v%) of the total solution. The perfluoropentane was loaded into the lipid using an ultrasonic homogenizer at 4°C to prepare a sonoporation agent. Afterwards, the sonoporation agent was purified by centrifugation at 4°C and 6000 rpm for 5 minutes. The particle size of the prepared sonoporation agent was measured using a zetasizer, and the particle number was analyzed using a nanosight.

[0164] As a result of the analysis, as shown in Fig. 3a, the particle size of the manufactured sonoporation agent showed an average particle size value of about 300-400 nm, and the number of particles was about 5-15×10 11 / mL of particles were observed. In addition, the morphology of the particles was photographed using an ultra-low temperature transmission electron microscope, and the results are shown in Fig. 3b.

[0165]

[0166]

[0167] Example 4. Analysis of physical properties according to the concentration of sonoporation agent loaded with perfluoropentane

[0168] To analyze the physical properties of sonoporation agents loaded with perfluoropentane according to their concentration, sonoporation agents were prepared with the lipid and perfluoropentane volume % (v / v%) shown in Table 4. The lipid composition was prepared using the lipid molar ratio performed in Example 3, and the manufacturing process and physical property analysis were also performed using the same process as in Example 3. To determine the function of each prepared sonoporation agent, the agent was stored at 70°C for 24 hours, and the vaporization of the perfluoropentane inside was observed, and the particle size distribution before and after vaporization was analyzed. The analysis results are shown in Table 4, Figs. 4a and 4b.

[0169]

[0170]

[0171] As a result of the analysis, all of the manufactured perfluoropentane-loaded sonoporation agents possessed the function of sonoporation agents by possessing vaporization ability, and this could be confirmed by changes in particle size.

[0172]

[0173] Example 5. Temperature-dependent stability of sonoporation agent loaded with perfluoropentane

[0174] To analyze the temperature-dependent stability of the sonoporation agent loaded with perfluoropentane, changes in particle size were analyzed at refrigerated, room temperature, 37, 50, and 70°C. The sonoporation agent loaded with perfluoropentane was prepared using the same method as in Example 3, and the particle distribution was measured after 1 hour, 3 hours, and 24 hours, respectively.

[0175] As a result of the measurement, as shown in Figures 5a to 5c, in the case of the sonoporation agent loaded with perfluoropentane, the particle size was maintained at most temperatures for up to 3 hours, but thereafter, the particle size changed at temperatures above 37°C, and in particular, at 70°C, it was confirmed that sufficient vaporization occurred and the particle size average peak shifted.

[0176] From the above results, it was confirmed that the sonoporation agent loaded with perfluoropentane was stable when stored at room temperature, and when administered into the body, although some vaporization may occur at body temperature of 37℃, it could be primarily vaporized by heat generation by ultrasound.

[0177]

[0178] Example 6. Stability of perfluoropentane-loaded sonoporation agent in different buffers

[0179] To conduct a test on the storage stability of a perfluoropentane-loaded sonoporation agent, a perfluoropentane-loaded sonoporation agent was prepared using the following four buffers.

[0180] 1) 33%-Sucrose (w / w%),

[0181] 2) Emulsifier (5%-glycol & 10%-propylene glycol mixture, w / w%),

[0182] 3) 0.01M Sodium phosphate & 30%-Sucrose buffer,

[0183] 4) Phosphate buffered saline (0.01M, pH 7.4)

[0184] The ratio and preparation of lipids and perfluoropentane were identical to those in Example 3. The final buffer was dispersed in each of the four buffers, and stability was tested by analyzing changes in size distribution. The buffer was stored for two months at refrigerated (4°C) and room temperature (RT, 25°C). Particle size was analyzed using dynamic light scattering (DLS).

[0185] As shown in Figures 6a and 6b, the analysis results confirmed that all four buffers were stably stored for two months without changes in particle size when stored under refrigeration. However, at room temperature (RT), the particle size distribution range changed polydispersely in all buffers except PBS buffer, which was believed to be due to particle instability.

[0186] From the above results, it was determined that PBS was the buffer that could most stably maintain the particles.

[0187]

[0188] Example 7. Confirmation of vaporization of sonoporation agent loaded with perfluoropentane under ultrasonic conditions.

[0189] To confirm the functionality of the sonoporation agent loaded with perfluoropentane, vaporization was analyzed according to ultrasonic conditions. The sonoporation agent loaded with perfluoropentane was prepared using phosphate buffered saline (0.01 M-PBS, pH 7.4), and the lipid composition, ratio of perfluorooctane, and preparation were prepared in the same manner as in Example 3. To compare and analyze the presence or absence of vaporization, a nanoemulsion loaded with perfluorooctane was prepared with the same lipid composition and ratio using perfluorooctane, a non-vaporization oil. The prepared perfluoropentane (C5F 12 ) nanoemulsion and perfluoroocatane (C8F 18 ) The size distribution and particle number of nanoemulsion were analyzed using nanosight, a nanoparticle tracking method, and the analysis results are shown in Fig. 7a, Fig. 7b, and Table 5.

[0190] Perfluorocarbon type particle number (1 x 10 11 / mL)Perfluoropentane(C5F 12 )216.5 nm6.11 ± 0.32Perfluorooctane(C8F 18 )248.6 nm7.23 ± 0.22

[0191] Also, manufactured C5F 12 nanoemulsion and C8F 18Each nanoemulsion was diluted 10-fold with phosphate buffered saline, 1 mL was placed on a dialysis membrane, and vaporization by ultrasonic emission was evaluated at 37°C. The ultrasonic equipment used for the evaluation was VIFU2000, and the ultrasonic conditions emitted were as shown in Table 6. Vaporization by ultrasonic emission was confirmed through ultrasonic imaging, and C5F 12 nanoemulsion and C8F 18 The results of the vaporization of the nanoemulsion are shown in Fig. 8. As shown in Fig. 8, the vaporizable C5F 12 In the case of nanoemulsion, cavitation occurred while vaporizing under all ultrasonic conditions in Table 6. As a result, echogenicity increased, allowing for white imaging in ultrasound images. Vaporization occurred strongly in proportion to the intensity of the ultrasound, making it appear very bright in the ultrasound image. In contrast, C8F, which cannot be vaporized, 18 In the case of nanoemulsion, echogenicity in ultrasound images due to cavitation was not observed in all images. From the above results, C5F by ultrasound 12 It was confirmed that nanoemulsion vaporized and cavitation occurred.

[0192] Ultrasonic emission conditions Frequency 1 MHz Duty cycle 5% Pulse repetition frequency (PRF) 10 Hz Time / spot 5 sec / spot Watt (intensity) 1) 1 W (non-detectable) 2) 5 W (95.1 W / cm 2 )3) 10W (273.6 W / cm 2 )4) 40W (1344.6 W / cm 2 )5) 80W (2772.6 W / cm 2 )

[0193] Example 8. Preparation of sonoporation agent loaded with perfluoropentane by the pre-mixing method

[0194] To prepare a sonoporation agent loaded with perfluoropentane, each solvent was prepared. Specifically, the first solvent was prepared by dissolving the same ratio of lipids as in Example 3 in ethanol to prepare a 10 mg / mL lipid solution. The second solvent was prepared by mixing perfluoropentane in PBS (0.01 M, pH 7.4) to make a 2% (v / v%) solution. The first and second solvents were each collected using a syringe at a mixing ratio of 1:9. The two collected solvents were mixed by repeatedly passing them through a filter with pores of 0.2 to 0.5 μm to prepare a sonoporation agent loaded with perfluoropentane. At this time, the temperature was maintained in a refrigerated state (4°C) to prevent perfluoropentane from vaporizing during preparation.

[0195] The manufactured sonoporation agent has an average particle size of 300-400 nm and a particle number of 5-10 x 10 11 / mL was.

[0196]

[0197] 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.

[0198]

[0199] The ultrasonic-sensitive nanoparticles according to the present invention not only have improved storage stability due to the perfluorocarbon contained (loaded) therein, but also maximize sonoporation by increasing cavitation as the perfluorocarbon is vaporized by ultrasonic waves, thereby enhancing the drug delivery effect, and are therefore expected to exhibit various functions in the field of drug delivery vehicles, and thus have industrial applicability.

Claims

1. A composition for promoting drug delivery comprising ultrasound-sensitive nanoparticles containing DSPC (1,2-Distearoyl-sn-glycero-3-phosphorylcholine), DSPE-mPEG2k (1,2-distearoyl-sn-glycero-3 phosphoethanolamine with conjugated methoxyl poly(ethylene glycol2000)), cholesterol, DOPE (1,2-Dioleoyl-sn-glycero 3-phosphoethanolamine), lyso-PC (lysophosphatidylcholine), and perfluorocarbon as active ingredients.

2. In paragraph 1, A composition for promoting drug delivery, characterized in that the perfluorocarbon is loaded into the nanoparticles.

3. In paragraph 1, A composition for promoting drug delivery, characterized in that the perfluorocarbon and nanoparticles are included in a concentration ratio of 0.01 to 10:

1.

4. In paragraph 1, A composition for promoting drug delivery, characterized in that the above perfluorocarbon causes cavitation by ultrasound.

5. In paragraph 1, The above perfluorocarbons are perfluoropropane (C3F8), perfluorobutane (C4F) 10 ), perfluoropentane (perfluoropentane: C5F 12 ), perfluorohexane (perfluorohexane: C6F 14 ), and perfluoromethylcyclohexane (C6F 11 A composition for promoting drug delivery, characterized in that it comprises at least one selected from the group consisting of (CF3).

6. In paragraph 1, A composition for promoting drug delivery, characterized in that the membrane of the above nanoparticles is composed of DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC.

7. In paragraph 1, A composition for promoting drug delivery, characterized in that DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC are contained in a molar ratio (mole%) of 1~90 : 1~20 : 1~80 : 0.1~40 : 1~20.

8. In paragraph 1, A composition for promoting drug delivery, characterized in that the nanoparticles have a diameter of 100 to 1000 nm.

9. In paragraph 1, A composition for promoting drug delivery, characterized in that the ultrasound-sensitive nanoparticles have secured storage stability.

10. A method for producing ultrasound-sensitive nanoparticles comprising the following steps: (a) a step of hydrating DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC by stirring with a buffer solution; and (b) Step of adding perfluorocarbon.

11. In paragraph 10, The above buffer solution is characterized in that it is at least one selected from the group consisting of: a) sucrose; b) A buffer solution containing glycol and propylene glycol; c) a buffer solution containing sodium phosphate and sucrose; and d) PBS.

12. In paragraph 10, A manufacturing method, characterized in that the above step (a) is performed at 30 to 80°C for 1 to 5 hours.

13. In paragraph 10, A manufacturing method, characterized in that the step (a) further includes a step of lowering the temperature.

14. A method for manufacturing a solution of ultrasound-sensitive nanoparticles comprising the following steps: (a) a step of preparing a first solution by dissolving DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC in an organic solvent; (b) a step of preparing a second solution by mixing perfluorocarbon into a buffer solution; and (c) A step of mixing the first solution and the second solution, filtering them, and mixing them.

15. In paragraph 14, A manufacturing method, characterized in that in the above step (a), the organic solvent is at least one selected from the group consisting of dimethylacetamide, dimethylformamide, dimethylsulfoxide, chloroform, methanol, ethanol, and ether.

16. In paragraph 14, A manufacturing method characterized in that the buffer solution in the above step (b) is at least one selected from the group consisting of: a) sucrose; b) A buffer solution containing glycol and propylene glycol; c) a buffer solution containing sodium phosphate and sucrose; and d) PBS.

17. In paragraph 14, A manufacturing method characterized in that in the above step (c), the first solution and the second solution are mixed in a volume ratio of 1:1 to 20.

18. In paragraph 14, A manufacturing method, characterized in that in the above step (c), the filter has a pore size of 0.1 to 5 μm.

19. A kit for producing ultrasound-sensitive nanoparticles, comprising a first composition comprising DSPC, DSPE-mPEG2k, DOPE, cholesterol, and lyso-PC; and a second composition comprising perfluorocarbon. 20.Usage of ultrasound-responsive nanoparticles comprising DSPC (1,2-Distearoyl-sn-glycero-3-phosphorylcholine), DSPE-mPEG2k (1,2-distearoyl-sn-glycero-3 phosphoethanolamine with conjugated methoxyl poly(ethylene glycol2000)), cholesterol, DOPE (1,2-Dioleoyl-sn-glycero 3-phosphoethanolamine), lyso-PC (lysophosphatidylcholine), and perfluorocarbon for promoting drug delivery.

21. Use of ultrasound-responsive nanoparticles comprising DSPC (1,2-Distearoyl-sn-glycero-3-phosphorylcholine), DSPE-mPEG2k (1,2-distearoyl-sn-glycero-3 phosphoethanolamine with conjugated methoxyl poly(ethylene glycol2000)), cholesterol, DOPE (1,2-Dioleoyl-sn-glycero 3-phosphoethanolamine), lyso-PC (lysophosphatidylcholine), and perfluorocarbon for manufacturing a drug delivery facilitator.

22. Ultrasound-responsive nanoparticles comprising DSPC (1,2-Distearoyl-sn-glycero-3-phosphorylcholine), DSPE-mPEG2k (1,2-distearoyl-sn-glycero-3 phosphoethanolamine with conjugated methoxyl poly(ethylene glycol2000)), cholesterol, DOPE (1,2-Dioleoyl-sn-glycero 3-phosphoethanolamine), lyso-PC (lysophosphatidylcholine), and a perfluorocarbon; and a method for promoting drug delivery comprising a step of administering the drug to a subject in need thereof.

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