WO2019182353A1 - Method for preparing liposome comprising ultrasound reactive microbubble for drug delivery and liposome using same - Google Patents

Method for preparing liposome comprising ultrasound reactive microbubble for drug delivery and liposome using same Download PDF

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Publication number
WO2019182353A1
WO2019182353A1 PCT/KR2019/003248 KR2019003248W WO2019182353A1 WO 2019182353 A1 WO2019182353 A1 WO 2019182353A1 KR 2019003248 W KR2019003248 W KR 2019003248W WO 2019182353 A1 WO2019182353 A1 WO 2019182353A1
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Prior art keywords
glycerol
phosphocholine
drug
shell
liposomes
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PCT/KR2019/003248
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French (fr)
Korean (ko)
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김철우
박동희
원종호
Original Assignee
(주) 바이오인프라생명과학
주식회사 퍼시픽시스템
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Priority to CN201980020482.8A priority Critical patent/CN111886002A/en
Priority to US16/982,485 priority patent/US11504328B2/en
Priority claimed from KR1020190031738A external-priority patent/KR102289921B1/en
Publication of WO2019182353A1 publication Critical patent/WO2019182353A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • A—HUMAN NECESSITIES
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    • A61K41/0047—Sonopheresis, i.e. ultrasonically-enhanced transdermal delivery, electroporation of a pharmacologically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
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    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A61K47/6913—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the liposome being modified on its surface by an antibody
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    • A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
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    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
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    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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    • B01F23/80—After-treatment of the mixture
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    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01F23/40—Mixing liquids with liquids; Emulsifying
    • B01F23/41—Emulsifying
    • B01F23/414—Emulsifying characterised by the internal structure of the emulsion
    • B01F23/4143—Microemulsions

Definitions

  • the present invention relates to a method for preparing liposomes containing ultrasonically reacted microbubbles and drugs for drug delivery, and liposomes using the same.
  • a liposome comprising an ultrasonic reaction micro-bubbles for drug delivery
  • the drug delivery system is a dosage formulation that efficiently delivers the amount of drug needed to treat a disease by minimizing side effects and optimizing the efficacy and effects of the drug. Can be.
  • Such drug delivery system may be a transdermal, oral or vascular method according to the drug delivery route.
  • drug delivery systems that treat lesions by introducing micro-sized capsules into blood vessels have been in the spotlight as a dream treatment technology in the future.
  • the element technology can be said to be a technology that accurately targets the drug to the affected area and the drug control in the affected area. Therefore, the target drug delivery system using ultrasonic waves and ultrasonic reaction-type microbubbles has recently attracted more attention as a technology that can solve these problems.
  • microbubbles used as ultrasound contrast agent are cavitation by ultrasonic energy, which increases the drug delivery effect to the skin or inside the cell.
  • a method of simultaneously mounting a micro bubble containing an inert gas and a drug in a space between liposome shells has a disadvantage in that it is difficult to form a multilayer structure and does not effectively mount a drug.
  • the amount of drug to be loaded is different depending on the size of the microbubble trapped inside the liposome and the characteristics of the drug, and in severe cases, the drug cannot be mounted on the liposome or the microbubble cannot be loaded.
  • the present invention aims to solve all the above-mentioned problems.
  • Another object of the present invention is to block drug generation in normal tissues and exhibit high responsiveness to ultrasonic energy so that the drug can be delivered by mutually reacting only in a target region to which ultrasonic energy is being irradiated.
  • Another object of the present invention is to be able to quantify the amount of the drug to be loaded into the liposome by forming a constant size of the micro-bubbles and liposomes.
  • Another object of the present invention is to be able to mount a certain amount or more of the drug to exhibit a significant drug effect.
  • a liposome comprising an ultrasonic reaction micro-bubbles for drug delivery, (a) after generating an ultrasonic reaction micro-bubbles containing an inert gas inside, the first shell formed on the outer surface of the ultrasonic wave through an extruder Uniformly forming a size distribution of the reactive microbubbles; And (b) uniformly forming the size distribution of the liposomes through an extruder after generating a liposome having a second shell formed on the outer surface thereof, including the ultrasonically-responsive microbubbles and a drug having a uniform size distribution therein;
  • a method comprising a.
  • the ultrasonic reaction-type micro-bubbles containing an inert gas therein and the first shell is formed on the outer surface
  • the ultrasonic reaction-type micro bubbles having a uniform size distribution of the ultrasonic reaction-type micro bubbles through an extruder after being generated;
  • the liposome including the ultrasonic reaction-type microbubble and the drug having a uniform size distribution therein, and having a uniform size distribution of the liposomes through an extruder after a liposome having a second shell is formed on the outer surface thereof.
  • a liposome including an ultrasonic reaction microbubble for drug delivery is provided.
  • the present invention can encapsulate a drug into liposomes to protect the drug from the external environment.
  • the present invention blocks drug development in normal tissues and exhibits high responsiveness to ultrasound energy, thereby allowing the drug to be delivered by reacting with each other only in the target area where ultrasound energy is being irradiated.
  • the present invention can quantify the amount of drug to be loaded inside the liposome by forming a constant size of the micro-bubbles and liposomes.
  • the present invention can be loaded with a certain amount or more of the drug to exhibit a significant drug effect.
  • FIG. 1 schematically illustrates liposomes containing ultrasound-responsive microbubbles and drugs for drug delivery according to an embodiment of the present invention
  • Figure 2 schematically shows a state of adjusting the size of the micro-bubble according to an embodiment of the present invention
  • Figure 3 schematically shows a confocal microscope image of a microbubble according to an embodiment of the present invention
  • 4a to 4c schematically show the results of analyzing the particle size of the microbubble according to an embodiment of the present invention by intensity, volume, and number distribution,
  • Figure 5 schematically shows a state of adjusting the size of the liposomes according to an embodiment of the present invention
  • Figure 6 shows a confocal microscope analysis image for liposomes according to an embodiment of the present invention
  • Figure 7 shows a comparison of the confocal microscopic analysis of the liposomes and liposomes produced by the conventional method according to an embodiment of the present invention, respectively,
  • FIG. 1 schematically illustrates a liposome including an ultrasonic reaction-type microbubble 11 for drug delivery according to an embodiment of the present invention.
  • the liposome may have a micro bubble 11 formed therein and a second shell 22 formed on an outer surface thereof.
  • a drug may be mounted in the region 21 between the microbubbles 11 and the second shell 22.
  • a first shell 12 may be formed on an outer surface of the microbubbles 11.
  • the liposome having such a structure first generates a micro bubble 11 having a uniform size distribution according to an embodiment of the present invention, and then includes a micro bubble and a drug therein and a liposome having a second shell formed on its outer surface. And, it is made through a process of uniformly forming the size distribution of liposomes through an extruder.
  • the first mixture powder comprising the first lipid may be dissolved in the first solvent to produce a solution of the first shell material.
  • the first mixture powder containing the first lipid may further include albumin, polymers, PEG, surfactants, proteins, biodegradable polymers, and the like, and cholesterol (cholesterol) may be added to increase the durability of the ultrasonic reaction microbubbles. Can be added.
  • the first lipid is DPPC (1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine), HSPC (phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycerol-3-phosphocholine), DEPC (1,2 -Di (cis-13-docosenoyl) -sn-glycerol-3-phosphocholine), DOPC (1,2-Dioleoyl-sn-glycerol-3-phosphocholine), DMPC (1,2-Dimyristoyl-sn-glycerol-3- phosphorylcholine), DLPC (1,2-Dilauroyl-sn-glycerol-3-phosphorylcholine), DEPC (1,2-Didecanoyl-sn-glycerol-3-phosphocholine), DSPC (1,2-Distearoyl-sn-glycerol-3 -phosphocholine), MPPC (1-myristoyl-2-palmito
  • Albumin may also include serum albumin, ovalbumin, and the like.
  • the polymer may include poly ( ⁇ -benzyl-L-asparate), poly-DL-lactic acid (PBLA), or the like.
  • the surfactant may be fatty acid sodium, monoalkyl sulfate, alkylpolyoxyethylene sulfate, alkylbenzenesulfonate, monoalkyl phosphate, dialkyldimethylammonium salt, alkylbenzylmethylammonium salt, alkylsulfobetaine, alkylcarboxybetaine, polyoxyethylene Alkyl ethers, fatty acid sorbitan esters, fatty acid diethanolamines, alkyl monoglyceryl ethers, benzalkonium chloride, benzethonium chloride, and the like.
  • the protein may include albumin, globulin, collagen, and the like.
  • biodegradable polymers include PHB-based plastics, polysaccharide-based plastics, ⁇ polycaprolactone (PCL), polylactic acid (PLA), polypropylene glycol acid (PG), polylithoxybutyrate-co-valerate (PHBV), polyvinyl Alcohol (PVA), polybutylene succinate (PBS), Chitin-based plastics, Oil-based plastics and the like.
  • PCL polycaprolactone
  • PLA polylactic acid
  • PG polypropylene glycol acid
  • PHBV polylithoxybutyrate-co-valerate
  • PVA polyvinyl Alcohol
  • PBS polybutylene succinate
  • Chitin-based plastics Oil-based plastics and the like.
  • the first solvent may include at least one or more of Saline and / or tertiary distilled water, glycerin and propylene glycol.
  • lipid, albumin, polymer, cholesterol, polyethylene glycol (PEG), and the like which form a shell of powder form, may be used as saline and / or tertiary distilled water (40 to 60). %), glycerin (2-10%), propylene glycol (40-60%) mixed with a solvent containing at least one of the first 1 to 6 hours by melting at a temperature between 60 ° C ⁇ 100 ° C It is possible to create a solution of shell material.
  • micelles specialized in ultrasound for drug delivery include DPPC (Dipalmitoyl-Phos Phatidyl-Choline) and DPPA (Diphenyl-phosphoryl-Azide) as the shell material to capture inert gas and increase bubble stability.
  • DPPC Dipalmitoyl-Phos Phatidyl-Choline
  • DPPA Diphenyl-phosphoryl-Azide
  • Normal saline, glycerin, and propylene glycol can be added together.
  • cholesterol may be added to increase the durability of the micelles.
  • a solution of the first shell material and an inert gas may be mixed to increase responsiveness to ultrasonic energy.
  • the inert gas may be a perfluorocarbon-based gas, and as a perfluorocarbon-based inert gas, perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoro-n-pentane, perfluoro-n-hexane, perfluoromethylcyclopentane, perfluoro-1 , 3-dimethylcyclohexane, perfluorodecalin, perfluoromethyldecalin, perfluoroperhydrobenzyltetralin and the like can be used.
  • perfluoromethane perfluoroethane, perfluoropropane, perfluorobutane, perfluoro-n-pentane, perfluoro-n-hexane, perfluoromethylcyclopentane, perfluoro-1 , 3-dimethylcyclohexane, perfluorodecalin, perfluoromethyl
  • the shell material and the inert gas are separated through a vial mixer. Mechanical mixing is possible.
  • the mechanical mixing speed can be adjusted to 1000 ⁇ 5000 rpm to appropriately adjust the size and particle size distribution of the ultrasonic reaction-type micro bubbles (11).
  • the perfluorocarbon-based inert gas is finely broken into a nano / micro size oil / water emulsion, and the inert gas is naturally hydrophilic phospholipid of the amphiphilic phospholipid by self-assembling. And a stable state can be maintained to form a micro bubble 11 having an inert gas as a core therein as shown in FIG.
  • the fatty acid chain corresponding to the tail portion of the phospholipid is hydrophobic
  • the phosphoric acid and base portions, which are the header portion have amphiphilic properties that are hydrophilic.
  • Amphipathic phospholipids which have both hydrophilic and hydrophobic properties, play an important role in shell construction.
  • the micro bubbles 11 may be ultrasonic reactive bubbles.
  • the ultrasonic reaction by filtering the ultrasonic reaction-type micro-bubbles 11 produced in various sizes using a filter and an extruder having a pore size of any one of a predetermined pore size, for example 30nm to 1um
  • bubble microbubbles 11 can be made uniform.
  • the filter may be a membrane filter, the membrane filter may be formed of poly carbonite.
  • the temperature for the filtering of the ultrasonic reaction-type microbubble 11 can be variously adjusted from room temperature to the phase transition temperature range of each material, and the number of times of filtering may be performed at least five times or more.
  • the mixture including the ultrasonic reaction-type micro bubbles 11 having a uniform size distribution by filtering is centrifuged through a centrifuge to settle the micelles well formed, and then the supernatant present in the upper layer is removed and DW (deionized) By washing with water, it is possible to obtain an ultrasonic reaction-type microbubble 11 adjusted to a desired size.
  • DPPC + DPPA powder in a solvent mixed with Normal saline + Glycerol + Propylene glycol and heat it for 3 hours using a hot plate to prevent the solution from boiling and overflowing.
  • the solvent for dissolving Lipid is mixed with Saline, glycerol, propylene glycol in a ratio of 20: 1: 21 and put Lipid powder in a ratio of DPPC (0.1g), DPPA (0.01g) in a total 100ml mixture.
  • DSPE-mPEG (0.127g) can be further mixed.
  • the heating may use a microwave.
  • perfluorobutane 0.1ml was mixed with the mixture of shell material and core gas (v / v) in 20: 1 ratio and 2ml Dispense vials and mechanically mix for 45 seconds after sealing. At this time, the mixing sets the frequency so that vibration 4530 ⁇ 100 times per minute.
  • Example 1 DPPC (0.1 g) + DPPA (0.01 g) + Cholesterol (0.127 g) were dissolved in a solvent and mixed as an example of the preparation of the ultrasonic reaction-type micro bubbles 11, and the amount of injected gas was In the case of liquefied state (-80 °C ⁇ -20 °C) 10 ⁇ 100ul (17.5 ⁇ 175 mg in terms of mass).
  • Ultrasonic responsive microbubbles (11) fabricated in various sizes were filtered using a polycarbonite membrane filter and an extruder, the well-formed micelles were allowed to settle by centrifugation through a centrifuge, and then the supernatant in the upper layer was removed and DW Washing with deionized water to complete the ultrasonic response micro-bubbles (11).
  • FIG. 3 (a) is a fluorescence microscope image, (b) is an optical microscope image, and (c) is a merging image of the fluorescence microscope image and the optical microscope image.
  • the lipid fluorescence signal forms a micelle border in the form of a shell
  • the core portion of the core is a hollow space or gas rather than lipid You can see that it consists of.
  • the general air bubbles and micelles can be divided into bubbles consisting of fluorescent lipids and bubbles consisting of fluorescent lipids, in most cases the shell as fluorescent lipids It can be seen that it is configured.
  • the principle of particle size analysis is to measure the particle size using diffraction and scattered light generated when the laser passes through the sample.
  • the equipment specifications used for the particle size analysis of micelles are as follows.
  • ELS Electrophoretic Light Scattering
  • 4A and 4B show the particle size analysis results for micelles as Intensity distribution, Volume distribution, and Number distribution.
  • Figure 4c shows the progress of the diameter and dispersion of the micelles prepared according to Example 1 and the diffusion constant, measurement environment and the like.
  • the size distribution environment of the bubble was measured in water at 25 °C, the viscosity was set to 0.8878 (cP), dispersion strength 25762 (cps), attenuator 0.72 (%).
  • the micelle produced in Example 1 had an average diameter of 257.1 nm and a diffusion constant of 1.913 e-8 (cm 2 / sec).
  • the second mixture powder including the second lipid may further include at least one or more of albumin, polymer, and polyethylene glycol (PEG), and cholesterol may be added to increase the durability of the liposome.
  • PEG polyethylene glycol
  • the second lipid is DPPC (1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine), HSPC (phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycerol-3-phosphocholine), DEPC (1,2 -Di (cis-13-docosenoyl) -sn-glycerol-3-phosphocholine), DOPC (1,2-Dioleoyl-sn-glycerol-3-phosphocholine), DMPC (1,2-Dimyristoyl-sn-glycerol-3- phosphorylcholine), DLPC (1,2-Dilauroyl-sn-glycerol-3-phosphorylcholine), DEPC (1,2-Didecanoyl-sn-glycerol-3-phosphocholine), DSPC (1,2-Distearoyl-sn-glycerol-3 -phosphocholine), MPPC (1-myristoyl-2-palmito
  • micelles for collecting drugs or genes may include DPPC (Dipalmitoyl -Phos Phatidyl-Choline) and DPPA (Diphenyl-phosphoryl-Azide) as main components to collect drugs or genes and increase the stability of micelles.
  • DPPC Dipalmitoyl -Phos Phatidyl-Choline
  • DPPA Diphenyl-phosphoryl-Azide
  • DPPC, DPPA, and cholesterol may be mixed in a ratio of 60% to 85%: 2% to 10%: 10% to 30%.
  • DSPE-mPEG can be added as a shell material.
  • DPPA DPPA
  • DMPA-Na DPPA-Na
  • DPPA-Na DPPA-Na
  • DSPG DSPS and the like can be used to charge the liposomes with phospholipids with negative or positive charges.
  • Albumin may also include serum albumin, ovalbumin, and the like.
  • the polymer may include poly ( ⁇ -benzyl-L-asparate), poly-DL-lactic acid (PBLA), or the like.
  • the organic solvent may include a mixed solvent of chloroform and methanol, chloroform and metalol may be mixed in a mixing ratio of 1: 1 to 3: 1.
  • the second mixture powder may be stirred using a magnetic stirrer to dissolve in the organic solvent, the temperature of the stirrer may be stirred for 10 to 30 minutes at about 40 ° C ⁇ 60 ° C. .
  • the organic solvent in which the shell material is dissolved can be removed by a rotary evaporation method.
  • the vacuum drying in the vacuum chamber is preferably carried out for at least 6 hours or more, more preferably 24 hours.
  • lipid films may be formed on the bottom of the flask, and the bottom of the flask may form a film cake in which the lipid material is rotated.
  • a solution of the second shell material is generated by hydrating a second solvent having an appropriate capacity in the lipid film, for example, PBS (Phosphate-buffered saline), followed by pulverization using an ultrasonic wave generator.
  • a second solvent having an appropriate capacity in the lipid film for example, PBS (Phosphate-buffered saline)
  • PBS Phosphate-buffered saline
  • the responsive microbubbles 11 and the drug and / or genes of the lipid film pulverized by the ultrasonic wave generator and the size distribution are formed uniformly are ultrasonic responsive microbubbles 11 by a self-assembling mechanism.
  • And liposomes containing drugs and / or genes are formed uniformly are ultrasonic responsive microbubbles 11 by a self-assembling mechanism.
  • liposomes produced in various sizes are filtered by using a filter and an extruder to adjust the size. That is, liposomes having a diameter larger than the pores of the filter are destroyed without passing through the pores, and the ultrasonic reaction-type microbubbles 11 located inside the destroyed liposomes, the drug, and the lipid film are recombined.
  • the mixture containing the liposomes having a uniform size distribution by filtering is cooled by centrifugation of the micelle well configured by centrifugation, followed by washing with a DW (deionized water) to remove the supernatant present in the upper layer. Liposomes adjusted to the desired size can be obtained.
  • DPPC + DPPA + Cholesterol are each mixed in a 75: 5: 20 ratio to form a lipid material, and the resulting lipid material is dissolved in an organic solvent of chloroform / methanol (2: 1, v / v).
  • the organic solvent is evaporated at 30 ° C. for 20 minutes using a rotary stirrer.
  • Ultrasonic responsive microbubbles (11) made of various sizes were filtered using a polycarbonite membrane filter and an extruder, the well-formed liposomes were settled through a centrifuge, and then the supernatant in the upper layer was removed and DW (deionized water) Wash) to complete liposomes for drug delivery
  • the liposomes that collect the drug by the same method as in Example 2 were observed through confocal microscopy.
  • the trapping of the microbubbles 11 having the gas space therein and the outside of the micelle It can be seen that the liposomes in which the hydrophilic fluorescent material (MW: 4k Dextran) was collected.
  • the ultrasonic reaction-responsive micro-bubbles (11) and (b) is a state produced according to the embodiment of the present invention as compared to the state (a) without adjusting the size of the liposomes It can be seen that each liposome effectively captures the drug.
  • the liposomes containing the ultrasonically-responsive microbubbles 11 and the drug for drug delivery produced above generate a cavitation effect when ultrasonic energy is received in a specific area so that the drug contained in the liposomes can be released at the corresponding position. Can be.
  • ultrasonic reaction-type microbubble 11 and drug-containing liposomes for drug delivery may be applied to target molecules such as proteins expressed on specific cell surfaces such as cancer cells in order to increase target delivery efficiency.
  • Targeting ligands such as antibodies or peptides that are made to react, may bind to the liposome surface.
  • the liposome according to an embodiment of the present invention may bring a target effect of passive target orientation by focused ultrasound using the collected drug and active target orientation by a specific ligand.
  • the ligand library screening for the target pathogen is confirmed to identify target targets such as antibodies, proteins, peptides, receptors, etc., and the identified target targets are bound to the liposome surface.
  • target targets such as antibodies, proteins, peptides, receptors, etc.
  • the target object bound to the liposome surface can then be induced by the pathogen.
  • the method of binding the target object to the surface of the liposome for example, to form a liposome to which PEG-COOH is bound by Example 1 and Example 2 (DNase, RNase free water).
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide
  • N-hydroxysuccinimide are added to the mixture for 15 minutes at room temperature to enable the carboxyl group (COOH) to be activated.
  • a target object containing an amino group (NH2) is added to an N-hydroxysuccinimide-activated liposome to allow carboxyl and amine to react to form an amide.

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Abstract

Provided are a method for preparing liposomes comprising ultrasound reactive microbubbles for drug delivery, comprising (a) a step of producing ultrasound reactive microbubbles comprising an inert gas therein and having a first shell formed on the outer surface thereof, followed by forming a uniform size distribution of the ultrasound reactive microbubbles through an extruder; and (b) a step of producing liposomes comprising the ultrasound reactive microbubbles distributed in a uniform size and a medicament therein and having a second shell formed on the outer surface thereof, followed by forming a uniform size distribution of the liposomes through an extruder; and a liposome using same.

Description

약물전달을 위한 초음파 반응형 미소기포를 포함하는 리포좀 제조 방법 및 이를 이용한 리포좀Method for producing liposomes containing ultrasonically reactive microbubbles for drug delivery and liposomes using the same
본 발명은 약물전달을 위한 초음파 반응형 미소기포와 약물을 포함하는 리포좀의 제조 방법 및 이를 이용한 리포좀에 관한 것이다.The present invention relates to a method for preparing liposomes containing ultrasonically reacted microbubbles and drugs for drug delivery, and liposomes using the same.
보다 상세하게는, 약물전달을 위한 초음파 반응형 미소기포를 포함하는 리포좀의 제조 방법에 있어서, (a) 내부에 불활성 가스를 포함하며 외면에 제1쉘이 형성된 초음파 반응형 미소기포를 생성한 이후 압출기를 통해 상기 초음파 반응형 미소기포의 크기 분포를 균일하게 형성하는 단계; 및 (b) 내부에 크기 분포가 균일하게 형성된 상기 초음파 반응형 미소기포와 약물을 포함하며 외면에 제2쉘이 형성된 리포좀을 생성한 이후 압출기를 통해 상기 리포좀의 크기 분포를 균일하게 형성하는 단계;를 포함하는 방법 및 이를 이용한 리포좀에 관한 것이다.More specifically, in the method for producing a liposome comprising an ultrasonic reaction micro-bubbles for drug delivery, (a) after generating the ultrasonic reaction-type micro bubbles containing an inert gas therein and the first shell is formed on the outer surface Uniformly forming a size distribution of the ultrasonic reaction micro bubbles through an extruder; And (b) uniformly forming the size distribution of the liposomes through an extruder after generating a liposome having a second shell formed on the outer surface thereof, including the ultrasonically-responsive microbubbles and a drug having a uniform size distribution therein; It relates to a method comprising and a liposome using the same.
약물전달시스템(DDS: Drug Delivery System)은 기존의 약물을 부작용을 최소화하고 약물이 가지고 있는 효능 및 효과를 최적화하여 질병치료를 위하여 필요한 양을 약물을 효율적으로 전달하기 위한 제형(dosage formulation)이라 할 수 있다.The drug delivery system (DDS) is a dosage formulation that efficiently delivers the amount of drug needed to treat a disease by minimizing side effects and optimizing the efficacy and effects of the drug. Can be.
이러한 약물전달시스템은 약물전달경로에 따라서 경피, 경구 또는 혈관을 통한 방법 등이 있다. 또한 마이크로 크기의 캡슐을 혈관에 도입하여 환부를 치료하는 약물전달시스템이 앞으로 꿈의 치료기술로써 각광을 받고 있다.Such drug delivery system may be a transdermal, oral or vascular method according to the drug delivery route. In addition, drug delivery systems that treat lesions by introducing micro-sized capsules into blood vessels have been in the spotlight as a dream treatment technology in the future.
그리고 약물전달시스템의 기술 중에서 요소기술은 약물을 목적하는 환부에 정확히 타겟팅하는 기술과 환부에서의 약물방출을 제어하는 기술이라고 할 수 있다. 따라서 초음파와 초음파 반응형 미소기포에 의한 표적약물전달 시스템은 이러한 문제점들을 해결할 수 있는 기술로써, 최근에 더욱 관심을 모으고 있다.In the technology of the drug delivery system, the element technology can be said to be a technology that accurately targets the drug to the affected area and the drug control in the affected area. Therefore, the target drug delivery system using ultrasonic waves and ultrasonic reaction-type microbubbles has recently attracted more attention as a technology that can solve these problems.
특히, 초음파 조영제로 사용되는 미소기포는 초음파 에너지에 의해 공동화 현상(cavitation)이 발생하고 이 현상은 피부나 세포 내부로의 약물전달 효과를 증가시킨다는 연구 결과에 따라 미소기포의 막에 원하는 약물이나 수용체(receptor)를 리간드 결합(ligand binding)하여 약물을 인체에 전달하고자 하였다.In particular, microbubbles used as ultrasound contrast agent are cavitation by ultrasonic energy, which increases the drug delivery effect to the skin or inside the cell. Ligand binding to the receptor (receptor) to deliver the drug to the human body.
그러나, 이러한 방법은 막 표면에 약물을 결합시키므로, 미소기포가 타겟 위치까지 이동하는 중에 약물의 유실이 발생할 수 있어 약물 전달체의 역할을 완벽하게 수행할 수 없다는 한계가 있다. 또한, 많은 양의 약물을 탑재할 수 없다는 점에서 한계가 있다.However, since this method binds the drug to the membrane surface, the drug may be lost while the microbubbles move to the target position, and thus there is a limitation in that the drug carrier cannot be completely performed. In addition, there is a limitation in that a large amount of drug cannot be loaded.
이를 개선하기 위하여, 최근에는 초음파 에너지와 반응성을 높이기 위한 미소기포와 약물을 동시에 탑재한 리포좀 제조 기술이 대두되고 있다.In order to improve this, in recent years, liposome preparation technology equipped with microbubbles and drugs simultaneously to increase the ultrasonic energy and reactivity has emerged.
하지만, 불활성 가스를 함유한 미소기포와 약물을 리포좀 막(shell) 사이 공간에 동시에 탑재하는 방식의 제조법은 다층 구조를 형성하기 어려우며 또한 효과적으로 약물을 탑재하지 못한다는 단점이 있다.However, a method of simultaneously mounting a micro bubble containing an inert gas and a drug in a space between liposome shells has a disadvantage in that it is difficult to form a multilayer structure and does not effectively mount a drug.
즉, 리포좀의 내부에 포집되는 미소기포의 크기 및 약물의 특성에 따라 탑재되는 약물의 양이 서로 다르게 되며, 심한 경우 리포좀에 약물이 탑재되지 못하거나 미소기포가 탑재되지 못하는 경우가 발생한다.That is, the amount of drug to be loaded is different depending on the size of the microbubble trapped inside the liposome and the characteristics of the drug, and in severe cases, the drug cannot be mounted on the liposome or the microbubble cannot be loaded.
본 발명은 상술한 문제점들을 모두 해결하는 것을 그 목적으로 한다.The present invention aims to solve all the above-mentioned problems.
또한, 본 발명은 약물을 리포좀 내부로 캡슐화하여 외부 환경으로부터 약물을 보호할 수 있도록 하는 것을 다른 목적으로 한다.It is another object of the present invention to encapsulate the drug into liposomes so as to protect the drug from the external environment.
또한, 본 발명은 정상 조직에서의 약효 발생을 차단하며 초음파 에너지에 높은 반응성을 나타내어 초음파 에너지가 조사되고 있는 표적 영역에서만 상호 반응하여 약물을 전달할 수 있도록 하는 것을 또 다른 목적으로 한다.In addition, another object of the present invention is to block drug generation in normal tissues and exhibit high responsiveness to ultrasonic energy so that the drug can be delivered by mutually reacting only in a target region to which ultrasonic energy is being irradiated.
또한, 본 발명은 미소기포 및 리포좀의 크기를 일정하게 형성함으로써 리포좀 내부에 탑재되는 약물의 양을 정량화할 수 있도록 하는 것을 또 다른 목적으로 한다.In addition, another object of the present invention is to be able to quantify the amount of the drug to be loaded into the liposome by forming a constant size of the micro-bubbles and liposomes.
또한, 본 발명은 유의한 약물 효과를 나타내기 위한 일정량 이상의 약물을 탑재할 수 있도록 하는 것을 또 다른 목적으로 한다.In addition, another object of the present invention is to be able to mount a certain amount or more of the drug to exhibit a significant drug effect.
상기 목적을 달성하기 위한 본 발명의 대표적인 구성은 다음과 같다.Representative configuration of the present invention for achieving the above object is as follows.
약물전달을 위한 초음파 반응형 미소기포를 포함하는 리포좀의 제조 방법에 있어서, (a) 내부에 불활성 가스를 포함하며 외면에 제1쉘이 형성된 초음파 반응형 미소기포를 생성한 이후 압출기를 통해 상기 초음파 반응형 미소기포의 크기 분포를 균일하게 형성하는 단계; 및 (b) 내부에 크기 분포가 균일하게 형성된 상기 초음파 반응형 미소기포와 약물을 포함하며 외면에 제2쉘이 형성된 리포좀을 생성한 이후 압출기를 통해 상기 리포좀의 크기 분포를 균일하게 형성하는 단계;를 포함하는 방법이 제공된다.In the method for producing a liposome comprising an ultrasonic reaction micro-bubbles for drug delivery, (a) after generating an ultrasonic reaction micro-bubbles containing an inert gas inside, the first shell formed on the outer surface of the ultrasonic wave through an extruder Uniformly forming a size distribution of the reactive microbubbles; And (b) uniformly forming the size distribution of the liposomes through an extruder after generating a liposome having a second shell formed on the outer surface thereof, including the ultrasonically-responsive microbubbles and a drug having a uniform size distribution therein; There is provided a method comprising a.
또한, 본 발명의 일 실시예에 따르면, 약물전달을 위한 초음파 반응형 미소기포를 포함하는 리포좀의 제조 방법에 있어서, 내부에 불활성 가스를 포함하며 외면에 제1쉘이 형성된 초음파 반응형 미소기포가 생성된 이후 압출기를 통해 상기 초음파 반응형 미소기포의 크기 분포가 균일하게 형성된 상기 초음파 반응형 미소기포; 및 내부에 크기 분포가 균일하게 형성된 상기 초음파 반응형 미소기포와 약물을 포함하며 외면에 제2쉘이 형성된 리포좀이 생성된 이후 압출기를 통해 상기 리포좀의 크기 분포가 균일하게 형성된 상기 리포좀;을 포함하는 약물 전달을 위한 초음파 반응형 미소기포를 포함하는 리포좀을 제공한다.In addition, according to an embodiment of the present invention, in the method for producing a liposome comprising an ultrasonic reaction-type micro-bubbles for drug delivery, the ultrasonic reaction-type micro-bubbles containing an inert gas therein and the first shell is formed on the outer surface The ultrasonic reaction-type micro bubbles having a uniform size distribution of the ultrasonic reaction-type micro bubbles through an extruder after being generated; And the liposome including the ultrasonic reaction-type microbubble and the drug having a uniform size distribution therein, and having a uniform size distribution of the liposomes through an extruder after a liposome having a second shell is formed on the outer surface thereof. Provided is a liposome including an ultrasonic reaction microbubble for drug delivery.
본 발명에 의하면, 다음과 같은 효과가 있다.According to the present invention, the following effects are obtained.
본 발명은 약물을 리포좀 내부로 캡슐화 하여 외부 환경으로부터 약물을 보호할 수 있다.The present invention can encapsulate a drug into liposomes to protect the drug from the external environment.
또한, 본 발명은 정상 조직에서의 약효 발생을 차단하며 초음파 에너지에 높은 반응성을 나타내어 초음파 에너지가 조사되고 있는 표적 영역에서만 상호 반응하여 약물을 전달할 수 있다.In addition, the present invention blocks drug development in normal tissues and exhibits high responsiveness to ultrasound energy, thereby allowing the drug to be delivered by reacting with each other only in the target area where ultrasound energy is being irradiated.
또한, 본 발명은 미소기포 및 리포좀의 크기를 일정하게 형성함으로써 리포좀 내부에 탑재되는 약물의 양을 정량화할 수 있다.In addition, the present invention can quantify the amount of drug to be loaded inside the liposome by forming a constant size of the micro-bubbles and liposomes.
또한, 본 발명은 유의한 약물 효과를 나타내기 위한 일정량 이상의 약물을 탑재할 수 있다.In addition, the present invention can be loaded with a certain amount or more of the drug to exhibit a significant drug effect.
도 1은 본 발명의 일 실시예에 따른 약물전달을 위한 초음파 반응형 미소기포와 약물을 함유한 리포좀을 개략적으로 도시한 것이고,1 schematically illustrates liposomes containing ultrasound-responsive microbubbles and drugs for drug delivery according to an embodiment of the present invention,
도 2는 본 발명의 일 실시예에 따른 미소기포의 사이즈를 조절하는 상태를 개략적으로 도시한 것이고,Figure 2 schematically shows a state of adjusting the size of the micro-bubble according to an embodiment of the present invention,
도 3은 본 발명의 일 실시예에 따른 미소기포의 공초점 현미경 영상을 개략적으로 도시한 것이고,Figure 3 schematically shows a confocal microscope image of a microbubble according to an embodiment of the present invention,
도 4a 내지 도 4c는 본 발명의 일 실시예에 따른 미소기포의 입도를 강도(intensity), 용적(volume), 개수(number) 분포로 분석한 결과를 개략적으로 도시한 것이고,4a to 4c schematically show the results of analyzing the particle size of the microbubble according to an embodiment of the present invention by intensity, volume, and number distribution,
도 5는 본 발명의 일 실시예에 따른 리포좀의 사이즈를 조절하는 상태를 개략적으로 도시한 것이고,Figure 5 schematically shows a state of adjusting the size of the liposomes according to an embodiment of the present invention,
도 6은 본 발명의 일 실시예에 따른 리포좀에 대한 공초점 현미경 분석 이미지를 도시한 것이고,Figure 6 shows a confocal microscope analysis image for liposomes according to an embodiment of the present invention,
도 7은 본 발명의 일 실시예에 따른 리포좀과 종래 방법에 의해 제작된 리포좀에 대한 공초점 현미경 분석 이미지를 각각 비교하여 도시한 것이고,Figure 7 shows a comparison of the confocal microscopic analysis of the liposomes and liposomes produced by the conventional method according to an embodiment of the present invention, respectively,
도 8은 본 발명의 일 실시예에 따른 리포좀을 이용하여 골드 나노 파티클 포집 실험을 수행한 상태를 개략적으로 도시한 것이다.8 schematically illustrates a state in which gold nanoparticle collection experiments are performed using liposomes according to an embodiment of the present invention.
후술하는 본 발명에 대한 상세한 설명은, 본 발명이 실시될 수 있는 특정 실시예를 예시로서 도시하는 첨부 도면을 참조한다. 이들 실시예는 당업자가 본 발명을 실시할 수 있기에 충분하도록 상세히 설명된다. 본 발명의 다양한 실시예는 서로 다르지만 상호 배타적일 필요는 없음이 이해되어야 한다. 예를 들어, 여기에 기재되어 있는 특정 형상, 구조 및 특성은 일 실시예에 관련하여 본 발명의 정신 및 범위를 벗어나지 않으면서 다른 실시예로 구현될 수 있다. 또한, 각각의 개시된 실시예 내의 개별 구성요소의 위치 또는 배치는 본 발명의 정신 및 범위를 벗어나지 않으면서 변경될 수 있음이 이해되어야 한다. 따라서, 후술하는 상세한 설명은 한정적인 의미로서 취하려는 것이 아니며, 본 발명의 범위는, 적절하게 설명된다면, 그 청구항들이 주장하는 것과 균등한 모든 범위와 더불어 첨부된 청구항에 의해서만 한정된다. 도면에서 유사한 참조부호는 여러 측면에 걸쳐서 동일하거나 유사한 기능을 지칭한다.DETAILED DESCRIPTION The following detailed description of the invention refers to the accompanying drawings that show, by way of illustration, specific embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. It should be understood that the various embodiments of the present invention are different but need not be mutually exclusive. For example, certain shapes, structures, and characteristics described herein may be embodied in other embodiments without departing from the spirit and scope of the invention with respect to one embodiment. In addition, it is to be understood that the location or arrangement of individual components within each disclosed embodiment may be changed without departing from the spirit and scope of the invention. The following detailed description, therefore, is not to be taken in a limiting sense, and the scope of the present invention, if properly described, is defined only by the appended claims, along with the full range of equivalents to which such claims are entitled. Like reference numerals in the drawings refer to the same or similar functions throughout the several aspects.
이하, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 본 발명을 용이하게 실시할 수 있도록 하기 위하여, 본 발명의 바람직한 실시예들에 관하여 첨부된 도면을 참조하여 상세히 설명하기로 한다.Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily implement the present invention.
도 1은 본 발명의 일 실시예에 따른 약물전달을 위한 초음파 반응형 미소기포(11)를 포함하는 리포좀을 개략적으로 도시한 것이다.1 schematically illustrates a liposome including an ultrasonic reaction-type microbubble 11 for drug delivery according to an embodiment of the present invention.
도 1을 참조하면, 리포좀은 내부에 미소기포(11)가 형성되며 외면에 제2쉘(22)이 형성될 수 있다. 그리고, 미소기포(11)와 제2쉘(22)의 사이 영역(21)에는 약물이 탑재될 수 있다. 또한, 미소기포(11)의 외면에는 제1쉘(12)이 형성될 수 있다.Referring to FIG. 1, the liposome may have a micro bubble 11 formed therein and a second shell 22 formed on an outer surface thereof. In addition, a drug may be mounted in the region 21 between the microbubbles 11 and the second shell 22. In addition, a first shell 12 may be formed on an outer surface of the microbubbles 11.
이와 같은 구조의 리포좀은 본 발명의 일 실시예에 의해 크기 분포가 균일하게 형성된 미소기포(11)를 먼저 생성한 다음, 내부에 미소기포와 약물을 포함하며 외면에 제2쉘이 형성된 리포좀을 생성하고, 압출기를 통해 리포좀의 크기 분포를 균일하게 형성하는 과정을 거쳐 만들어지게 된다.The liposome having such a structure first generates a micro bubble 11 having a uniform size distribution according to an embodiment of the present invention, and then includes a micro bubble and a drug therein and a liposome having a second shell formed on its outer surface. And, it is made through a process of uniformly forming the size distribution of liposomes through an extruder.
아래에서는 초음파 반응형 미소기포(11)를 생성하는 과정에 대해서 구체적으로 설명한다.Hereinafter, a process of generating the ultrasonic reaction-type micro bubbles 11 will be described in detail.
먼저, 미소기포(11)를 제조하기 위한 제1쉘 물질의 솔루션을 준비한다.First, a solution of the first shell material for preparing the microbubbles 11 is prepared.
이를 위해 제1 리피드를 포함하는 제1 혼합물 파우더를 제1 용매에 용해하여 제1쉘 물질의 솔루션을 생성할 수 있다. 여기서, 제1 리피드가 포함된 제1 혼합물 파우더에는 알부민, 폴리머, PEG, 계면활성제, 단백질, 생분해성 고분자 등이 더 포함될 수 있으며, 초음파 반응형 미소기포의 내구성을 높여주기 위해 콜레스테롤(cholesterol)이 첨가될 수 있다.To this end, the first mixture powder comprising the first lipid may be dissolved in the first solvent to produce a solution of the first shell material. Here, the first mixture powder containing the first lipid may further include albumin, polymers, PEG, surfactants, proteins, biodegradable polymers, and the like, and cholesterol (cholesterol) may be added to increase the durability of the ultrasonic reaction microbubbles. Can be added.
또한, 제1 리피드는 DPPC(1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine), HSPC(phosphatidylcholine), DDPC(1,2-didecanoyl-sn-glycerol-3-phosphocholine), DEPC(1,2-Di(cis-13-docosenoyl)-sn-glycerol-3-phosphocholine), DOPC(1,2-Dioleoyl-sn-glycerol-3-phosphocholine), DMPC(1,2-Dimyristoyl-sn-glycerol-3-phosphorylcholine), DLPC(1,2-Dilauroyl-sn-glycerol-3-phosphorylcholine), DEPC(1,2-Didecanoyl-sn-glycerol-3-phosphocholine), DSPC(1,2-Distearoyl-sn-glycerol-3-phosphocholine), MPPC(1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphocholine), MSPC(1-myristoyl-2-stearoyl-sn-glycerol-3-phosphocholine), egg PC(phosphocholine), DPPA(Diphenylphosphoryl azide), DMPA-Na(1,2-Dimyristoyl-sn-glycerol-3-phosphate), DPPA-Na(1,2-Dipalmitoyl-sn-glycerol-3-phosphate), DOPA-Na(1,2-Dioleoyl-sn-glycerol-3-phosphate), DSPE(Distearoylphosphatidylethanolamine), DMPE(Dimyristoyl phosphatidylethanolamine), DOPE(Dioleoyl phosphatidylethanolamine), DPPE(Dipalmitoyl phosphatidylethanolamine), DOPE-Glutaryl-(Na)2(1,2-Dioleoyl-sn-glycerol-3-phosphoethanolamine), egg PE(phosphatidylethanolamine), DSPG(Distearoyl phosphatidylglycerol), DMPG-Na(1,2-Dimyristoyl-sn-glycerol-3-Phosphoglycerol), DPPG-Na(1,2-Dipalmitoyl-sn-glycerol-3-Phosphoglycerol), DOPG-Na(1,2-Dioleoyl-sn-glycerol-3-Phosphoglycerol), DOPS(ioleoyl phosphatidylserine), DMPS(Dimyristoyl phosphatidylserine), DMPS- Na(1,2-Dimyristoyl-sn-glycerol-3-phosphoserine), DPPS-Na(1,2-Dipalmitoyl-sn-glycerol-3-phosphoserine), DOPS-Na(1,2-Dioleoyl-sn-glycerol-3-phosphoserine), DSPS(Distearoylphosphatidylserine), DSPE-mPEG(1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), DSPE-mPEG-2000-Na(1,2-Distearoyl-sn-glycerol-3-phosphoethanolamine), DSPE-mPEG-5000-Na, DSPE-Maleimide PEG-2000-Na, Surfactant: Tween 80, Span 80, dipotassium glycyrrhizinate 중 적어도 하나 이상을 포함할 수 있다.In addition, the first lipid is DPPC (1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine), HSPC (phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycerol-3-phosphocholine), DEPC (1,2 -Di (cis-13-docosenoyl) -sn-glycerol-3-phosphocholine), DOPC (1,2-Dioleoyl-sn-glycerol-3-phosphocholine), DMPC (1,2-Dimyristoyl-sn-glycerol-3- phosphorylcholine), DLPC (1,2-Dilauroyl-sn-glycerol-3-phosphorylcholine), DEPC (1,2-Didecanoyl-sn-glycerol-3-phosphocholine), DSPC (1,2-Distearoyl-sn-glycerol-3 -phosphocholine), MPPC (1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphocholine), MSPC (1-myristoyl-2-stearoyl-sn-glycerol-3-phosphocholine), egg PC (phosphocholine), DPPA ( Diphenylphosphoryl azide), DMPA-Na (1,2-Dimyristoyl-sn-glycerol-3-phosphate), DPPA-Na (1,2-Dipalmitoyl-sn-glycerol-3-phosphate), DOPA-Na (1,2- Dioleoyl-sn-glycerol-3-phosphate (DPE), Distearoylphosphatidylethanolamine (DSPE), Dimyristoyl phosphatidylethanolamine (DMPE), Dioleoyl phosphatidylethanolamine (DOPE), Dipalmitoyl phosphatidylethanolamine (DPPE), DOPE-Glutleo- (Na) 2 (1) yl-sn-glycerol-3-phosphoethanolamine, egg PE (phosphatidylethanolamine), DSPG (Distearoyl phosphatidylglycerol), DMPG-Na (1,2-Dimyristoyl-sn-glycerol-3-Phosphoglycerol), DPPG-Na (1,2- Dipalmitoyl-sn-glycerol-3-Phosphoglycerol, DOPG-Na Dimyristoyl-sn-glycerol-3-phosphoserine), DPPS-Na (1,2-Dipalmitoyl-sn-glycerol-3-phosphoserine), DOPS-Na (1,2-Dioleoyl-sn-glycerol-3-phosphoserine), DSPS (Distearoylphosphatidylserine), DSPE-mPEG (1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000]), DSPE-mPEG-2000-Na (1,2-Distearoyl-sn -glycerol-3-phosphoethanolamine), DSPE-mPEG-5000-Na, DSPE-Maleimide PEG-2000-Na, Surfactant: Tween 80, Span 80, dipotassium glycyrrhizinate may include at least one or more.
또한, 알부민은 세럼 알부민 (serum albumin), 오발부민 (ovalbumin) 등을 포함할 수 있다.Albumin may also include serum albumin, ovalbumin, and the like.
또한, 폴리머는 Poly(β-benzyl-L-asparate), PBLA(poly-DL-lactic acid) 등을 포함할 수 있다.In addition, the polymer may include poly (β-benzyl-L-asparate), poly-DL-lactic acid (PBLA), or the like.
또한, 계면활성제는 지방산 나트륨, 모노알킬 황산염, 알킬폴리옥시에틸렌 황산염, 알킬벤젠술폰산염, 모노알킬인산염, 디알킬디메틸암모늄염, 알킬벤질메틸암모늄염, 알킬설포베타인, 알킬카르복시베타인, 폴리옥시에틸렌알킬에테르, 지방산 솔비탄에스테르, 지방산 디에탄올아민, 알킬모노글리세릴에테르, 벤잘코늄 염화물 (benzalkonium chloride), 벤제토늄 염화물 (benzethonium chloride) 등을 포함할 수 있다.In addition, the surfactant may be fatty acid sodium, monoalkyl sulfate, alkylpolyoxyethylene sulfate, alkylbenzenesulfonate, monoalkyl phosphate, dialkyldimethylammonium salt, alkylbenzylmethylammonium salt, alkylsulfobetaine, alkylcarboxybetaine, polyoxyethylene Alkyl ethers, fatty acid sorbitan esters, fatty acid diethanolamines, alkyl monoglyceryl ethers, benzalkonium chloride, benzethonium chloride, and the like.
또한, 단백질은 알부민, 글로불린, 콜라겐 등을 포함할 수 있다.In addition, the protein may include albumin, globulin, collagen, and the like.
또한, 생분해성 고분자는 PHB계 플라스틱, 다당류계 플라스틱,　폴리카프로락톤(PCL),　폴리유산(PLA),　폴리필렌 글리콜산(PG), 폴리리드록시부티레이트-코-발레레이트(PHBV), 폴리비닐알콜(PVA), 폴리부틸렌 숙시네이트(PBS), Chitin계 플라스틱, Oil계 플라스틱 등을 포함할 수 있다.In addition, biodegradable polymers include PHB-based plastics, polysaccharide-based plastics, 락 polycaprolactone (PCL), polylactic acid (PLA), polypropylene glycol acid (PG), polylithoxybutyrate-co-valerate (PHBV), polyvinyl Alcohol (PVA), polybutylene succinate (PBS), Chitin-based plastics, Oil-based plastics and the like.
또한, 제1 용매에는 Saline 및/또는 3차 증류수, glycerin 및 propylene glycol 중 적어도 하나 이상이 포함될 수 있다.In addition, the first solvent may include at least one or more of Saline and / or tertiary distilled water, glycerin and propylene glycol.
일 예로, 파우더 상태의 쉘을 구성하는 리피드(lipid), 알부민(albumin), 폴리머(polymer), 콜레스테롤(cholesterol) 및 PEG(polyethylene glycol) 등의 물질을 Saline 및/또는 3차 증류수(40~60%), glycerin(2~10%), propylene glycol(40~60%) 중 하나 이상을 포함하는 용매와 혼합한 뒤 60°C~100°C 사이의 온도에서 1~6시간 동안 녹여줌으로써 제1쉘 물질의 솔루션을 생성할 수 있다 .For example, lipid, albumin, polymer, cholesterol, polyethylene glycol (PEG), and the like, which form a shell of powder form, may be used as saline and / or tertiary distilled water (40 to 60). %), glycerin (2-10%), propylene glycol (40-60%) mixed with a solvent containing at least one of the first 1 to 6 hours by melting at a temperature between 60 ° C ~ 100 ° C It is possible to create a solution of shell material.
일 예로, 약물 전달을 위한 초음파에 특화된 미셀(micelle)은 불활성 가스를 포집하고 기포의 안정성을 높이기 위해 쉘 물질로 DPPC (Dipalmitoyl -Phos Phatidyl - Choline) 및 DPPA(Diphenyl-phosphoryl-Azide)를 주요 구성물로 하고 Normal saline, Glycerol, Propylene glycol이 함께 첨가될 수 있다.As an example, micelles specialized in ultrasound for drug delivery include DPPC (Dipalmitoyl-Phos Phatidyl-Choline) and DPPA (Diphenyl-phosphoryl-Azide) as the shell material to capture inert gas and increase bubble stability. Normal saline, glycerin, and propylene glycol can be added together.
그리고, 주요 구성물인 DPPC와 DPPA가 쉘을 구성할 때 미셀의 내구성을 높여주기 위해 콜레스테롤(cholesterol)이 첨가될 수 있다.In addition, when the main components DPPC and DPPA form a shell, cholesterol may be added to increase the durability of the micelles.
다음으로, 초음파 에너지에 반응성을 높이기 위해 제1쉘 물질의 솔루션과 불활성 가스를 혼합할 수 있다.Next, a solution of the first shell material and an inert gas may be mixed to increase responsiveness to ultrasonic energy.
이때, 불활성 가스는 퍼플루오로카본 계열의 가스일 수 있으며, 퍼플루오로카본 계열의 불활성 가스로서 Perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoro-n-pentane, perfluoro-n-hexane, perfluoromethylcyclopentane, perfluoro-1,3-dimethylcyclohexane, perfluorodecalin, perfluoromethyldecalin, perfluoroperhydrobenzyltetralin 등을 사용할 수 있다.In this case, the inert gas may be a perfluorocarbon-based gas, and as a perfluorocarbon-based inert gas, perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoro-n-pentane, perfluoro-n-hexane, perfluoromethylcyclopentane, perfluoro-1 , 3-dimethylcyclohexane, perfluorodecalin, perfluoromethyldecalin, perfluoroperhydrobenzyltetralin and the like can be used.
일 예로, 제1쉘 물질의 솔루션과 불활성 가스의 혼합비율(v/v)을 1:1 내지 20:1의 비율로 바이얼에 분주하여 밀봉 후, 바이얼믹서를 통해 쉘 물질과 불활성 가스를 기계적 믹싱할 수 있다.For example, after dispensing and mixing the mixing ratio (v / v) of the solution of the first shell material and the inert gas to the vial at a ratio of 1: 1 to 20: 1, the shell material and the inert gas are separated through a vial mixer. Mechanical mixing is possible.
이때, 기계적 믹싱 속도는 1000 ~ 5000 rpm으로 조절하여 초음파 반응형 미소기포(11)의 크기 및 입도 분포를 적절하게 조절할 수 있다.At this time, the mechanical mixing speed can be adjusted to 1000 ~ 5000 rpm to appropriately adjust the size and particle size distribution of the ultrasonic reaction-type micro bubbles (11).
그러면, 기계적 믹싱을 통해 퍼플루오로카본 계열의 불활성 가스가 nano 내지 micro 사이즈의 oil/water 에멀젼 형태로 잘게 부수어지며, 불활성 가스는 자기 정합(self-assembling)에 의해 자연스럽게 양친매성 인지질의 소수성 꼬리 부분과 결합되어 안정된 상태를 유지할 수 있게 되어 도 1에서와 같이 내부에 코어로서의 불활성 가스를 가지는 미소기포(11)를 형성하게 된다. 구체적으로는, 인지질 중 꼬리 부분에 해당하는 지방산 사슬은 소수성이고, 헤더 부분인 인산과 염기 부분은 친수성인 양친매성 특성을 가진다. 이런 친수성 특성과 소수성 특성을 동시에 지니는 양친매성(amphipathic) 인지질은 쉘을 구성하는데 있어 중요한 역할을 한다. 그리고, 미소기포(11)는 초음파 반응성 기포일 수 있다.Then, through mechanical mixing, the perfluorocarbon-based inert gas is finely broken into a nano / micro size oil / water emulsion, and the inert gas is naturally hydrophilic phospholipid of the amphiphilic phospholipid by self-assembling. And a stable state can be maintained to form a micro bubble 11 having an inert gas as a core therein as shown in FIG. Specifically, the fatty acid chain corresponding to the tail portion of the phospholipid is hydrophobic, and the phosphoric acid and base portions, which are the header portion, have amphiphilic properties that are hydrophilic. Amphipathic phospholipids, which have both hydrophilic and hydrophobic properties, play an important role in shell construction. The micro bubbles 11 may be ultrasonic reactive bubbles.
다음으로, 도 2에서와 같이, 다양한 크기로 제작된 초음파 반응형 미소기포(11)를 일정한 공극 사이즈, 일 예로 30nm 내지 1um 중 어느 하나의 공극 사이즈를 갖는 필터와 압출기를 사용하여 필터링함으로써 초음파 반응형 미소기포(11)의 크기 분포를 균일하게 할 수 있다. 이때, 필터는 멤브레인 필터일 수 있으며, 멤브레인 필터는 폴리 카보나이트로 형성될 수 있다.Next, as shown in FIG. 2, the ultrasonic reaction by filtering the ultrasonic reaction-type micro-bubbles 11 produced in various sizes using a filter and an extruder having a pore size of any one of a predetermined pore size, for example 30nm to 1um The size distribution of the mold | bubble microbubbles 11 can be made uniform. In this case, the filter may be a membrane filter, the membrane filter may be formed of poly carbonite.
또한, 초음파 반응형 미소기포(11)의 필터링을 위한 온도는 상온에서 각 물질의 상전이 온도 범위까지 다양하게 조절할 수 있으며, 필터링의 횟수는 최소 5회에서 그 이상으로 수행할 수 있다.In addition, the temperature for the filtering of the ultrasonic reaction-type microbubble 11 can be variously adjusted from room temperature to the phase transition temperature range of each material, and the number of times of filtering may be performed at least five times or more.
이후, 필터링에 의해 크기 분포가 균일하게 형성된 초음파 반응형 미소기포(11)를 포함하는 혼합물을 원심분리기를 통해 원심분리로 잘 구성된 미셀을 가라앉힌 다음 상층에 존재하는 상층액을 제거하고 DW(deionized water)를 이용하여 워싱함으로써 원하는 크기로 조정된 초음파 반응형 미소기포(11)를 획득할 수 있다.Subsequently, the mixture including the ultrasonic reaction-type micro bubbles 11 having a uniform size distribution by filtering is centrifuged through a centrifuge to settle the micelles well formed, and then the supernatant present in the upper layer is removed and DW (deionized) By washing with water, it is possible to obtain an ultrasonic reaction-type microbubble 11 adjusted to a desired size.
실시예1. 초음파 반응형 미소기포 제작Example 1 Ultrasonic Responsive Microbubble Fabrication
1. 리피드 쉘 물질 준비1. Ready shell material preparation
Normal saline + Glycerol + Propylene glycol이 혼합된 용매에 DPPC + DPPA 파우더를 넣고 핫 플레이트(hot plate)를 이용하여 용액이 끓어서 넘치지 않도록 주의하여 3시간 동안 가열한다. 이때, Lipid를 용해할 용매는 Saline, glycerol, propylene glycol을 20:1:21의 비율로 혼합하여 total 100ml의 혼합물에 DPPC(0.1g), DPPA(0.01g)의 비율로 Lipid 파우더를 넣어준다. 이때, DSPE-mPEG(0.127g)를 더 혼합할 수 있다. 그리고, 가열은 microwave를 이용할 수도 있다.Put DPPC + DPPA powder in a solvent mixed with Normal saline + Glycerol + Propylene glycol and heat it for 3 hours using a hot plate to prevent the solution from boiling and overflowing. At this time, the solvent for dissolving Lipid is mixed with Saline, glycerol, propylene glycol in a ratio of 20: 1: 21 and put Lipid powder in a ratio of DPPC (0.1g), DPPA (0.01g) in a total 100ml mixture. At this time, DSPE-mPEG (0.127g) can be further mixed. In addition, the heating may use a microwave.
2. 콜레스테롤 쉘 물질 준비2. Cholesterol Shell Substance Preparation
Normal saline + Propylene glycol (+ Glycerol, 생략가능)을 유리 비커에 넣고 전체 100ml의 혼합물에 콜레스테롤(0.127g)을 넣은 뒤 핫 플레이트를 이용하여 용액의 온도를 80°C로 유지하며 3시간 동안 가열한다.Put normal saline + propylene glycol (+ Glycerol, optional) in a glass beaker, put cholesterol (0.127g) in a total 100ml mixture and heat the solution for 3 hours while maintaining the temperature of the solution at 80 ° C using a hot plate .
3. 초음파 반응형 미소기포 제작3. Ultrasonic Response Micro Bubble Fabrication
준비된 DPPC+DPPA 혼합액(1.5ml)과 Cholesterol 혼합액(0.5ml)을 더해준 뒤, perfluorobutane(0.1ml)을 쉘 물질의 솔루션과 코어 가스의 혼합비율(v/v)을 20:1 비율로 혼합하여 2ml 바이얼에 분주하여 밀봉 후 45초 동안 기계적 믹싱한다. 이때, 믹싱은 1분에 4530±100번 진동이 이루어지도록 주파수를 설정한다.After adding the prepared DPPC + DPPA mixture (1.5ml) and Cholesterol mixture (0.5ml), perfluorobutane (0.1ml) was mixed with the mixture of shell material and core gas (v / v) in 20: 1 ratio and 2ml Dispense vials and mechanically mix for 45 seconds after sealing. At this time, the mixing sets the frequency so that vibration 4530 ± 100 times per minute.
즉, 실시예1에서는 초음파 반응형 미소기포(11) 제조의 일례로 DPPC(0.1g) + DPPA(0.01g) + Cholesterol(0.127g)를 각각 용매에 녹여 혼합하였으며, 주입된 가스의 양은 perfluorobutane의 경우 액화상태(-80℃~-20℃)에서 10~100ul (질량으로 환산 시 17.5~175 mg) 이다.That is, in Example 1, DPPC (0.1 g) + DPPA (0.01 g) + Cholesterol (0.127 g) were dissolved in a solvent and mixed as an example of the preparation of the ultrasonic reaction-type micro bubbles 11, and the amount of injected gas was In the case of liquefied state (-80 ℃ ~ -20 ℃) 10 ~ 100ul (17.5 ~ 175 mg in terms of mass).
4. 초음파 반응형 미소기포의 사이즈 조절 및 분리4. Size Control and Separation of Ultrasonic Responsive Microbubbles
다양한 크기로 제작된 초음파 반응형 미소기포(11)를 폴리카보나이트 멤브레인 필터와 압출기를 사용하여 필터링하고, 원심분리기를 통해 원심분리로 잘 구성된 미셀을 가라앉힌 다음 상층에 존재하는 상층액을 제거하고 DW(deionized water)로 워싱하여 초음파 반응형 미소기포(11)를 완성한다.Ultrasonic responsive microbubbles (11) fabricated in various sizes were filtered using a polycarbonite membrane filter and an extruder, the well-formed micelles were allowed to settle by centrifugation through a centrifuge, and then the supernatant in the upper layer was removed and DW Washing with deionized water to complete the ultrasonic response micro-bubbles (11).
5. 공초점 현미경 분석5. Confocal Microscopy Analysis
상기 실시예1에서와 같은 방법에 의해 NBD PC로 제작된 미셀을 이용하여 미셀의 형성 여부와 형태 분석 및 형광 지질의 위치와 물리적 기포의 상관관계를 확인하기 위하여 도 3에서와 같은 공초점 현미경 영상을 획득하였다.Confocal microscopy image as shown in FIG. 3 in order to determine the formation and morphology of micelles and the correlation of the location and physical bubbles of the fluorescent lipids using micelles made of NBD PC by the same method as in Example 1 Obtained.
도 3에서 (a)는 형광 현미경 이미지이며, (b)는 광학 현미경 이미지이며, (c)는 형광 현미경 이미지와 광학 현미경 이미지를 합한(merging) 이미지이다.In FIG. 3, (a) is a fluorescence microscope image, (b) is an optical microscope image, and (c) is a merging image of the fluorescence microscope image and the optical microscope image.
도 3의 (a)와 (b)를 통해 알 수 있는 바와 같이, 형광 신호를 띄는 지질이 쉘 형태로 미셀 테두리를 형성하고 있는 것을 확인할 수 있으며, 가운데 코어 부분은 지질이 아닌 빈 공간 혹은 가스로 이루어져 있는 것을 확인할 수 있다.As can be seen from (a) and (b) of Figure 3, it can be seen that the lipid fluorescence signal forms a micelle border in the form of a shell, the core portion of the core is a hollow space or gas rather than lipid You can see that it consists of.
그리고, 도 3의 (c)를 통해 알 수 있는 바와 같이, 일반 공기방울과 미셀의 구분은 형광지질로 이루어져 있지 않은 기포와 형광 지질로 구성된 기포로 구분할 수 있으며, 대부분의 경우 형광 지질로 쉘이 구성되어 있음을 알 수 있다.And, as can be seen through (c) of Figure 3, the general air bubbles and micelles can be divided into bubbles consisting of fluorescent lipids and bubbles consisting of fluorescent lipids, in most cases the shell as fluorescent lipids It can be seen that it is configured.
6. 입도분석6. Particle size analysis
입도분석의 원리는 레이저가 샘플을 통과할 때 발생하는 회절, 산란광을 이용하여 입자의 크기를 측정하는 것으로, 미셀의 입도분석에 사용된 장비 스펙은 다음과 같다.The principle of particle size analysis is to measure the particle size using diffraction and scattered light generated when the laser passes through the sample. The equipment specifications used for the particle size analysis of micelles are as follows.
가. 모델명: ELS-2000ZSend. Model Name: ELS-2000ZS
나. 입도분석: DLS (Dynamic Light Scattering)I. Particle size analysis: DLS (Dynamic Light Scattering)
다. 제타전위: ELS (Electrophoretic Light Scattering)All. Zeta Potential: ELS (Electrophoretic Light Scattering)
라. 모든 용매에 분산된　Particle의 입도분포 및　Zeta Potential　측정la. Particle size distribution and Zeta Potential measurement of particles dispersed in all solvents
마. Flat Sample의 표면　Zeta Potential　측정 가능hemp. Surface of flat sample SampleZeta Potential can be measured
바. 온도　Control &　경시변화 측정 가능bar. Possible to measure changes in temperature and control
사. 미량　Sample　측정 가능four. Trace amount can be measured
아. Size: 0.1nm~10000nm / Zeta Potential:　1nm~50000nm　대응 가능Ah. Size: 0.1nm ~ 10000nm / Zeta Potential: 1nm ~ 50000nm
자. 시료 농도: 0.001~40% 대응 가능character. Sample concentration: 0.001 to 40% available
도 4a 및 도 4b는 미셀에 대한 입도분석 결과를 Intensity distribution, Volume distribution, Number distribution으로 나타낸 것이다.4A and 4B show the particle size analysis results for micelles as Intensity distribution, Volume distribution, and Number distribution.
그 결과 Intensity distribution: 308 nm, Volume distribution: 184 nm, Number Distribution: 139nm로 최종 결과치가 나타난다.The final result is shown as Intensity distribution: 308 nm, Volume distribution: 184 nm, Number Distribution: 139 nm.
그리고, 도 4c는 실시예1에 따라 제작된 미셀의 직경과 분산도 및 확산 상수, 측정환경 등에 대한 경과를 나타낸 것이다. 이때, 기포의 크기 분포 측정 환경은 25℃의 물에서 측정을 하였으며 점도 0.8878(cP), 분산 강도 25762(cps), 감쇠기 0.72(%)로 설정하였다.And, Figure 4c shows the progress of the diameter and dispersion of the micelles prepared according to Example 1 and the diffusion constant, measurement environment and the like. At this time, the size distribution environment of the bubble was measured in water at 25 ℃, the viscosity was set to 0.8878 (cP), dispersion strength 25762 (cps), attenuator 0.72 (%).
측정 결과, 실시예1에 의해 제작된 미셀의 평균 직경은 257.1 nm이며, 확산상수는 1.913 e-8(cm2/sec)로 나타났다.As a result, the micelle produced in Example 1 had an average diameter of 257.1 nm and a diffusion constant of 1.913 e-8 (cm 2 / sec).
다음으로는, 상기에서와 같은 방법에 의해 만들어진 크기 분포가 균일하게 형성된 초음파 반응형 미소기포(11)를 이용하여 초음파 반응형 미소기포(11)와 약물을 포함하는 리포좀을 제조하는 방법을 설명하면 다음과 같다.Next, a method of manufacturing a liposome including the ultrasonic reaction-type microbubble 11 and the drug using the ultrasonic reaction-type microbubble 11 having a uniform size distribution made by the method described above will be described. As follows.
먼저, 리포좀을 제조하기 위한 제2쉘 물질의 솔루션을 준비한다.First, a solution of the second shell material for preparing liposomes is prepared.
이를 위해 제2 리피드를 포함하는 제2 혼합물 파우더를 유기 용매에 용해한 후 유기 용매를 제거하여 리피드 필름을 획득하는 과정을 수행할 수 있다. 여기서 제2 리피드가 포함된 제2 혼합물 파우더에는 알부민(albumin), 폴리머(polymer) 및 PEG(polyethylene glycol) 중 적어도 하나 이상이 더 포함될 수 있으며, 리포좀의 내구성을 높여주기 위해 콜레스테롤이 첨가될 수 있다.To this end, a process of obtaining a lipid film by dissolving the second mixture powder including the second lipid in an organic solvent and then removing the organic solvent may be performed. Here, the second mixture powder including the second lipid may further include at least one or more of albumin, polymer, and polyethylene glycol (PEG), and cholesterol may be added to increase the durability of the liposome. .
또한, 제2 리피드는 DPPC(1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine), HSPC(phosphatidylcholine), DDPC(1,2-didecanoyl-sn-glycerol-3-phosphocholine), DEPC(1,2-Di(cis-13-docosenoyl)-sn-glycerol-3-phosphocholine), DOPC(1,2-Dioleoyl-sn-glycerol-3-phosphocholine), DMPC(1,2-Dimyristoyl-sn-glycerol-3-phosphorylcholine), DLPC(1,2-Dilauroyl-sn-glycerol-3-phosphorylcholine), DEPC(1,2-Didecanoyl-sn-glycerol-3-phosphocholine), DSPC(1,2-Distearoyl-sn-glycerol-3-phosphocholine), MPPC(1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphocholine), MSPC(1-myristoyl-2-stearoyl-sn-glycerol-3-phosphocholine), egg PC(phosphocholine), DPPA(Diphenylphosphoryl azide), DMPA-Na(1,2-Dimyristoyl-sn-glycerol-3-phosphate), DPPA-Na(1,2-Dipalmitoyl-sn-glycerol-3-phosphate), DOPA-Na(1,2-Dioleoyl-sn-glycerol-3-phosphate), DSPE(Distearoylphosphatidylethanolamine), DMPE(Dimyristoyl phosphatidylethanolamine), DOPE(Dioleoyl phosphatidylethanolamine), DPPE(Dipalmitoyl phosphatidylethanolamine), DOPE-Glutaryl-(Na)2(1,2-Dioleoyl-sn-glycerol-3-phosphoethanolamine), egg PE(phosphatidylethanolamine), DSPG(Distearoyl phosphatidylglycerol), DMPG-Na(1,2-Dimyristoyl-sn-glycerol-3-Phosphoglycerol), DPPG-Na(1,2-Dipalmitoyl-sn-glycerol-3-Phosphoglycerol), DOPG-Na(1,2-Dioleoyl-sn-glycerol-3-Phosphoglycerol), DOPS(ioleoyl phosphatidylserine), DMPS(Dimyristoyl phosphatidylserine), DMPS- Na(1,2-Dimyristoyl-sn-glycerol-3-phosphoserine), DPPS-Na(1,2-Dipalmitoyl-sn-glycerol-3-phosphoserine), DOPS-Na(1,2-Dioleoyl-sn-glycerol-3-phosphoserine), DSPS(Distearoylphosphatidylserine), DSPE-mPEG(1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), DSPE-mPEG-2000-Na(1,2-Distearoyl-sn-glycerol-3-phosphoethanolamine), DSPE-mPEG-5000-Na, DSPE-Maleimide PEG-2000-Na, Surfactant: Tween 80, Span 80, dipotassium glycyrrhizinate 중 적어도 하나 이상을 포함할 수 있다.In addition, the second lipid is DPPC (1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine), HSPC (phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycerol-3-phosphocholine), DEPC (1,2 -Di (cis-13-docosenoyl) -sn-glycerol-3-phosphocholine), DOPC (1,2-Dioleoyl-sn-glycerol-3-phosphocholine), DMPC (1,2-Dimyristoyl-sn-glycerol-3- phosphorylcholine), DLPC (1,2-Dilauroyl-sn-glycerol-3-phosphorylcholine), DEPC (1,2-Didecanoyl-sn-glycerol-3-phosphocholine), DSPC (1,2-Distearoyl-sn-glycerol-3 -phosphocholine), MPPC (1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphocholine), MSPC (1-myristoyl-2-stearoyl-sn-glycerol-3-phosphocholine), egg PC (phosphocholine), DPPA ( Diphenylphosphoryl azide), DMPA-Na (1,2-Dimyristoyl-sn-glycerol-3-phosphate), DPPA-Na (1,2-Dipalmitoyl-sn-glycerol-3-phosphate), DOPA-Na (1,2- Dioleoyl-sn-glycerol-3-phosphate (DPE), Distearoylphosphatidylethanolamine (DSPE), Dimyristoyl phosphatidylethanolamine (DMPE), Dioleoyl phosphatidylethanolamine (DOPE), Dipalmitoyl phosphatidylethanolamine (DPPE), DOPE-Glutleo- (Na) 2 (1) yl-sn-glycerol-3-phosphoethanolamine, egg PE (phosphatidylethanolamine), DSPG (Distearoyl phosphatidylglycerol), DMPG-Na (1,2-Dimyristoyl-sn-glycerol-3-Phosphoglycerol), DPPG-Na (1,2- Dipalmitoyl-sn-glycerol-3-Phosphoglycerol, DOPG-Na Dimyristoyl-sn-glycerol-3-phosphoserine), DPPS-Na (1,2-Dipalmitoyl-sn-glycerol-3-phosphoserine), DOPS-Na (1,2-Dioleoyl-sn-glycerol-3-phosphoserine), DSPS (Distearoylphosphatidylserine), DSPE-mPEG (1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000]), DSPE-mPEG-2000-Na (1,2-Distearoyl-sn -glycerol-3-phosphoethanolamine), DSPE-mPEG-5000-Na, DSPE-Maleimide PEG-2000-Na, Surfactant: Tween 80, Span 80, dipotassium glycyrrhizinate may include at least one or more.
일 예로, 약물이나 유전자를 포집하기 위한 미셀은 약물이나 유전자를 채집하고 미셀의 안정성을 높이기 위해 쉘 물질로 DPPC (Dipalmitoyl -Phos Phatidyl - Choline), DPPA(Diphenyl-phosphoryl-Azide)를 주요 구성물로 할 수 있다. 이때, DPPC, DPPA, 및 콜레스테롤은 60%~85%:2%~10%:10%~30%의 비율로 혼합될 수 있다. 그리고, 쉘 물질로 DSPE-mPEG가 추가될 수 있다.For example, micelles for collecting drugs or genes may include DPPC (Dipalmitoyl -Phos Phatidyl-Choline) and DPPA (Diphenyl-phosphoryl-Azide) as main components to collect drugs or genes and increase the stability of micelles. Can be. At this time, DPPC, DPPA, and cholesterol may be mixed in a ratio of 60% to 85%: 2% to 10%: 10% to 30%. And, DSPE-mPEG can be added as a shell material.
특히, DPPA, DMPA-Na, DPPA-Na, DSPG, DSPS 등은 음전하 또는 양전하를 가지는 인지질로 리포좀이 전하를 띄게 하기 위하여 사용되어질 수 있다.In particular, DPPA, DMPA-Na, DPPA-Na, DSPG, DSPS and the like can be used to charge the liposomes with phospholipids with negative or positive charges.
또한, 알부민은 세럼 알부민 (serum albumin), 오발부민 (ovalbumin) 등을 포함할 수 있다.Albumin may also include serum albumin, ovalbumin, and the like.
또한, 폴리머는 Poly(β-benzyl-L-asparate), PBLA(poly-DL-lactic acid) 등을 포함할 수 있다.In addition, the polymer may include poly (β-benzyl-L-asparate), poly-DL-lactic acid (PBLA), or the like.
여기서, 유기용매는 클로로포름과 메탄올의 혼합 용매를 포함할 수 있으며, 클로로포름과 메탈올은 1:1 내지 3:1의 혼합비로 혼합될 수 있다.Here, the organic solvent may include a mixed solvent of chloroform and methanol, chloroform and metalol may be mixed in a mixing ratio of 1: 1 to 3: 1.
또한, 제2 혼합물 파우더를 유기 용매에 녹이기 위하여 자석 교반기(magnetic stirrer)를 사용하여 교반할 수 있으며, 교반기의 온도는 약 40°C~60°C로 하여 10분 내지 30분간 교반을 진행할 수 있다.In addition, the second mixture powder may be stirred using a magnetic stirrer to dissolve in the organic solvent, the temperature of the stirrer may be stirred for 10 to 30 minutes at about 40 ° C ~ 60 ° C. .
또한, 회전 증발 방식에 의해 쉘 물질이 녹아있는 유기 용매를 제거할 수 있다.In addition, the organic solvent in which the shell material is dissolved can be removed by a rotary evaporation method.
일 예로, 회전증발기(rotary evaporator)를 이용하여 20°C 내지 40°C의 온도에서 10분 내지 30분간 유기 용매를 증발시키며, 잔류된 유기 용매를 완전히 제거하기 위하여 진공 챔버에 넣은 후 감압 건조하여 준다. 이때 진공 챔버에서의 감압 건조는 적어도 6시간 이상 진행하는 것이 바람직하며, 보다 상세하게는 24시간 진행하는 것이 바람직하다.For example, using a rotary evaporator to evaporate the organic solvent for 10 to 30 minutes at a temperature of 20 ° C to 40 ° C, put in a vacuum chamber to completely remove the remaining organic solvent and dried under reduced pressure give. At this time, the vacuum drying in the vacuum chamber is preferably carried out for at least 6 hours or more, more preferably 24 hours.
그러면, 유기 용매의 클로로포름과 메탄올 등이 증발된 뒤 플라스크 바닥에는 리피드 필름이 다층으로 형성될 수 있으며, 플라스크 바닥에는 뿌옇게 리피드 물질이 회전이 적용된 형태로 필름 케이크(film cake)를 형성하게 된다.Then, after the chloroform and methanol of the organic solvent is evaporated, a plurality of lipid films may be formed on the bottom of the flask, and the bottom of the flask may form a film cake in which the lipid material is rotated.
다음으로, 리피드 필름에 적절한 용량의 제2 용매, 일 예로, PBS(Phosphate-buffered saline)를 넣어 수화함으로써 제2쉘 물질의 솔루션을 생성한 다음, 초음파발생장치를 이용하여 분쇄하여 준다. 이때, 약물의 조건 및 미셀의 특성에 따라 크기 분포가 균일하게 형성된 초음파 반응형 미소기포(11)와 약물 및/또는 유전자를 함께 넣고 초음파 분해를 할 수 있으나 이에 한정되는 것은 아니며 리피드 필름을 분쇄한 이후에 초음파 반응형 미소기포(11)와 약물 및/또는 유전자를 넣을 수 있다.Next, a solution of the second shell material is generated by hydrating a second solvent having an appropriate capacity in the lipid film, for example, PBS (Phosphate-buffered saline), followed by pulverization using an ultrasonic wave generator. At this time, the ultrasonic reaction-type micro bubbles 11 having a uniform size distribution according to the condition of the drug and the characteristics of the micelle and the drug and / or gene can be put together and ultrasonic decomposition, but is not limited thereto. Thereafter, the ultrasonic reaction microbubbles 11 and drugs and / or genes may be put.
그러면, 초음파발생장치에 의해 분쇄된 리피드 필름과 크기 분포가 균일하게 형성된 초음파 반응형 미소기포(11)와 약물 및/또는 유전자는 자기정합(self-assembling) 기전에 의해 초음파 반응형 미소기포(11)와 약물 및/또는 유전자를 포함하는 리포좀의 형태로 제작된다.Then, the responsive microbubbles 11 and the drug and / or genes of the lipid film pulverized by the ultrasonic wave generator and the size distribution are formed uniformly are ultrasonic responsive microbubbles 11 by a self-assembling mechanism. ) And liposomes containing drugs and / or genes.
다음으로, 도 5에서와 같이, 다양한 크기로 제작된 리포좀을 필터와 압출기를 사용하여 필터링하여 사이즈를 조절한다. 즉, 필터의 공극보다 큰 직경을 가지는 리포좀은 공극을 통과하지 못하고 파괴되며, 파괴된 리포좀 내부에 위치하는 초음파 반응형 미소기포(11)와 약물, 리피드 필름은 재결합하게 된다.Next, as shown in Figure 5, liposomes produced in various sizes are filtered by using a filter and an extruder to adjust the size. That is, liposomes having a diameter larger than the pores of the filter are destroyed without passing through the pores, and the ultrasonic reaction-type microbubbles 11 located inside the destroyed liposomes, the drug, and the lipid film are recombined.
이후, 필터링에 의해 크기 분포가 균일하게 형성된 리포좀을 포함하는 혼합물을 원심분리기를 통해 원심분리로 잘 구성된 미셀을 가라앉힌 다음 상층에 존재하는 상층액을 제거하고 DW(deionized water)를 이용하여 워싱함으로써 원하는 크기로 조정된 리포좀을 획득할 수 있다.Subsequently, the mixture containing the liposomes having a uniform size distribution by filtering is cooled by centrifugation of the micelle well configured by centrifugation, followed by washing with a DW (deionized water) to remove the supernatant present in the upper layer. Liposomes adjusted to the desired size can be obtained.
실시예2. 리포좀 제작Example 2. Liposome production
1. 쉘 물질 준비1. Shell material preparation
DPPC + DPPA + Cholesterol을 각각 75:5:20 비율로 혼합하여 리피드 물질을 생성하고, 생성된 리피드 물질을 클로로포름/메탄올(2:1, v/v)의 유기 용매에 녹인다.DPPC + DPPA + Cholesterol are each mixed in a 75: 5: 20 ratio to form a lipid material, and the resulting lipid material is dissolved in an organic solvent of chloroform / methanol (2: 1, v / v).
그리고, 회전교반기를 이용하여 30℃에서 20분간 유기 용매를 증발시킨다.Then, the organic solvent is evaporated at 30 ° C. for 20 minutes using a rotary stirrer.
이후, 진공 챔버에 넣은 뒤 감압하여 24시간 감압 건조하여 리피드 필름을 제작한다.Thereafter, the resultant was placed in a vacuum chamber and depressurized to dry for 24 hours to produce a lipid film.
2. 리포좀 제작2. Liposome Production
리피드 필름에 2ml의 PBS를 넣어 수화한 다음, 상온에서 1분 동안 100W의 에너지로 초음파 분해를 진행한다. 이때, 실시예1에서 제작된 사이즈가 조절된 미소기포(11)와 약물 및/또는 유전자를 함께 넣고 초음파 분해를 진행하여 다양한 크기를 가지는 리포좀을 제작할 수 있다.2 ml of PBS was hydrated in the lipid film, and then ultrasonically decomposed at 100W for 1 minute at room temperature. At this time, the micro-bubbles (11) and the drug and / or gene with the adjusted size produced in Example 1 are put together and ultrasonic decomposition can be produced to produce liposomes having various sizes.
3. 리포좀의 사이즈 조절 및 재결합3. Size Control and Recombination of Liposomes
다양한 크기로 제작된 초음파 반응형 미소기포(11)를 폴리카보나이트 멤브레인 필터와 압출기를 사용하여 필터링하고, 원심분리기를 통해 잘 구성된 리포좀을 가라앉힌 다음 상층에 존재하는 상층액을 제거하고 DW(deionized water)로 워싱하여 약물전달을 위한 리포좀을 완성한다Ultrasonic responsive microbubbles (11) made of various sizes were filtered using a polycarbonite membrane filter and an extruder, the well-formed liposomes were settled through a centrifuge, and then the supernatant in the upper layer was removed and DW (deionized water) Wash) to complete liposomes for drug delivery
4. 공초점 현미경 분석4. Confocal Microscopy Analysis
상기 실시예2에서와 같은 방법에 의해 약물을 포집하는 리포좀을 공초점 현미경을 통해 관찰한 결과 도 6에서 알 수 있는 바와 같이, 내부에 가스 공간이 존재하는 미소기포(11)의 포집 및 미셀 외부에 친수성 형광 물질(MW: 4k의 Dextran)이 포집된 리포좀이 확인됨을 알 수 있다.As shown in FIG. 6, the liposomes that collect the drug by the same method as in Example 2 were observed through confocal microscopy. As shown in FIG. 6, the trapping of the microbubbles 11 having the gas space therein and the outside of the micelle It can be seen that the liposomes in which the hydrophilic fluorescent material (MW: 4k Dextran) was collected.
또한, 도 7에서 알 수 있는 바와 같이, 초음파 반응형 미소기포(11)와 리포좀의 사이즈를 조절하지 않은 상태인 (a)에 비하여, 본 발명에서의 실시예에 따라 제작된 상태인 (b)에서 각각의 리포좀들이 효율적으로 약물을 포집하고 있음을 확인할 수 있다.In addition, as can be seen in Figure 7, the ultrasonic reaction-responsive micro-bubbles (11) and (b) is a state produced according to the embodiment of the present invention as compared to the state (a) without adjusting the size of the liposomes It can be seen that each liposome effectively captures the drug.
5. 골드 나노 파티클 포집 실험5. Gold Nanoparticle Collection Experiment
TEM 영상을 이용하여 초음파 반응형 미소기포(11)와 친수성 약물을 함유한 리포좀을 분석하기 위한 것으로, 도 8의 (a)와 같이 골드 나노 파티클이 위치하는 영역에 본 발명의 일 실시예에 따라 제작된 초음파 반응형 미소기포(11)와 약물을 함유한 리포좀을 위치시켰다.To analyze liposomes containing the ultrasonic reaction-type microbubbles 11 and the hydrophilic drug using a TEM image, according to an embodiment of the present invention in a region where gold nanoparticles are located as shown in FIG. The prepared ultrasonic response microbubbles 11 and the liposomes containing the drug were placed.
그리고, TEM 영상을 통해 상태를 확인한 결과, 도 8의 (b)에서와 같이 골드 나노 파티클이 초음파 반응형 미소기포(11)와 약물을 함유한 리포좀에 포획되는 것을 알 수 있으며, 이를 통해 초음파 반응형 미소기포(11)와 약물을 함유한 리포좀에 유의한 약물 효과를 나타내기 위한 일정량 이상의 약물이 탑재 되었음을 확인할 수 있다.As a result of confirming the state through the TEM image, it can be seen that the gold nanoparticles are captured in the liposomes containing the ultrasonically-responsive microbubbles 11 and the drug as shown in FIG. It can be confirmed that a certain amount of drug is loaded to show a significant drug effect on the liposomes containing the type microbubbles 11 and the drug.
상기에서 제작된 약물전달을 위한 초음파 반응형 미소기포(11)와 약물을 함유한 리포좀은 특정 영역에서 초음파 에너지를 받게 되면 캐비테이션 효과를 발생시켜 리포좀에 함유된 약물이 해당 위치에서 방출될 수 있도록 할 수 있다.The liposomes containing the ultrasonically-responsive microbubbles 11 and the drug for drug delivery produced above generate a cavitation effect when ultrasonic energy is received in a specific area so that the drug contained in the liposomes can be released at the corresponding position. Can be.
또한, 본 발명의 일 실시예에 따른 약물전달을 위한 초음파 반응형 미소기포(11)와 약물을 함유한 리포좀은 표적 전달 효율을 높이기 위해 암세포와 같은 특정 세포 표면에서 발현하는 단백질 등의 표적 분자에 반응하도록 만들어진 항체나 펩타이드와 같은 타게팅 리간드(targeting ligand)를 리포좀 표면에 결합할 수도 있다.In addition, the ultrasonic reaction-type microbubble 11 and drug-containing liposomes for drug delivery according to an embodiment of the present invention may be applied to target molecules such as proteins expressed on specific cell surfaces such as cancer cells in order to increase target delivery efficiency. Targeting ligands, such as antibodies or peptides that are made to react, may bind to the liposome surface.
즉, 본 발명의 일 실시예에 따른 리포좀은 포집된 약물을 이용하는 집속형 초음파에 의한 수동형 표적 지향과 특정 리간드에 의한 능동적 표적 지향의 표적 효과를 동시에 가져올 수 있다.That is, the liposome according to an embodiment of the present invention may bring a target effect of passive target orientation by focused ultrasound using the collected drug and active target orientation by a specific ligand.
이때, 표적하는 병원체에 대한 리간드 라이브러리 스크리닝을 통해 항체, 단백질, 펩타이드, 수용체 등의 표적 대상을 확인하고, 확인된 표적 대상을 리포좀 표면에 결합한다. 그러면, 리포좀 표면에 결합된 표적 대상이 병원체에 유도될 수 있게 된다.At this time, the ligand library screening for the target pathogen is confirmed to identify target targets such as antibodies, proteins, peptides, receptors, etc., and the identified target targets are bound to the liposome surface. The target object bound to the liposome surface can then be induced by the pathogen.
또한, 리포좀 표면에 표적 대상을 결합하는 방법은, 예를 들어, 상기 실시예1 및 실시예2에 의해 PEG-COOH가 결합되어있는 리포좀을 만든다(DNase, RNase free water).In addition, the method of binding the target object to the surface of the liposome, for example, to form a liposome to which PEG-COOH is bound by Example 1 and Example 2 (DNase, RNase free water).
그리고, EDC(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide), N-hydroxysuccinimide를 첨가하여 15분간 실온에 교반시켜 카르복실기(COOH)가 활성화되도록 해준다.Then, EDC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide) and N-hydroxysuccinimide are added to the mixture for 15 minutes at room temperature to enable the carboxyl group (COOH) to be activated.
다음으로 N-hydroxysuccinimide가 활성화된 리포좀에 아미노기(NH2)가 결합되어있는 표적 대상을 넣어 카르복실과 아민이 반응하여 아마이드가 형성되어 결합되도록 해준다.Next, a target object containing an amino group (NH2) is added to an N-hydroxysuccinimide-activated liposome to allow carboxyl and amine to react to form an amide.
이상에서 본 발명이 구체적인 구성요소 등과 같은 특정 사항들과 한정된 실시예 및 도면에 의해 설명되었으나, 이는 본 발명의 보다 전반적인 이해를 돕기 위해서 제공된 것일 뿐, 본 발명이 상기 실시예들에 한정되는 것은 아니며, 본 발명이 속하는 기술분야에서 통상적인 지식을 가진 자라면 이러한 기재로부터 다양한 수정 및 변형을 꾀할 수 있다.Although the present invention has been described by specific embodiments such as specific components and the like, but the embodiments and the drawings are provided to assist in a more general understanding of the present invention, the present invention is not limited to the above embodiments. For those skilled in the art, various modifications and variations can be made from these descriptions.
따라서, 본 발명의 사상은 상기 설명된 실시예에 국한되어 정해져서는 아니 되며, 후술하는 특허청구범위뿐만 아니라 이 특허청구범위와 균등하게 또는 등가적으로 변형된 모든 것들은 본 발명의 사상의 범주에 속한다고 할 것이다.Accordingly, the spirit of the present invention should not be limited to the above-described embodiments, and all of the equivalents or equivalents of the claims, as well as the appended claims, fall within the scope of the spirit of the present invention. I will say.

Claims (25)

  1. 약물전달을 위한 초음파 반응형 미소기포를 포함하는 리포좀의 제조 방법에 있어서,In the manufacturing method of liposomes containing ultrasonic response micro-bubbles for drug delivery,
    (a) 내부에 불활성 가스를 포함하며 외면에 제1쉘이 형성된 초음파 반응형 미소기포를 생성한 이후 압출기를 통해 상기 초음파 반응형 미소기포의 크기 분포를 균일하게 형성하는 단계; 및(a) uniformly forming a size distribution of the ultrasonically reacted microbubbles through an extruder after generating an ultrasonically reacted microbubble containing an inert gas therein and having a first shell formed on its outer surface; And
    (b) 내부에 크기 분포가 균일하게 형성된 상기 초음파 반응형 미소기포와 약물을 포함하며 외면에 제2쉘이 형성된 리포좀을 생성한 이후 압출기를 통해 상기 리포좀의 크기 분포를 균일하게 형성하는 단계;(b) uniformly forming a size distribution of the liposomes through an extruder after generating a liposome having a second shell formed on the outer surface thereof, including the ultrasonically-responsive microbubbles and a drug having a uniform size distribution therein;
    를 포함하는 방법.How to include.
  2. 제1항에 있어서,The method of claim 1,
    (c) 상기 약물을 전달하고자 하는 병원체에 반응하는 타게팅 리간드(targeting ligand)를 상기 제2쉘에 결합하는 단계;(c) binding a targeting ligand to the second shell in response to a pathogen to which the drug is to be delivered;
    를 더 포함하는 방법.How to include more.
  3. 제2항에 있어서,The method of claim 2,
    상기 (c) 단계는,In step (c),
    상기 병원체에 대한 리간드 라이브러리 스크리닝을 통해 상기 병원체에 반응하는 항체, 단백질, 펩타이드 및 수용체 중 적어도 하나를 상기 타게팅 리간드로서 확인하고, 확인된 상기 타게팅 리간드를 상기 제2쉘에 결합하는 것을 특징으로 하는 방법.Ligand library screening for the pathogen identifies at least one of the antibodies, proteins, peptides and receptors that respond to the pathogen as the targeting ligand and binds the identified targeting ligand to the second shell. .
  4. 제3항에 있어서,The method of claim 3,
    상기 제2쉘에 카르복실기(COOH)를 도입시킨 후, 상기 카르복실기를 활성화시킨 다음 아미노기(NH2)가 결합된 상기 타게팅 리간드를 혼합하고 상기 카르복실기와 상기 아미노기가 반응하도록 하여 아마이드를 형성함으로써 상기 타게팅 리간드를 상기 제2쉘에 결합하는 것을 특징으로 하는 방법.After introducing the carboxyl group (COOH) into the second shell, the activating the carboxyl group and then mixing the targeting ligand with the amino group (NH2) is bonded and the carboxyl group and the amino group reacts to form an amide by forming an amide Coupling to the second shell.
  5. 제4항에 있어서,The method of claim 4, wherein
    상기 제2쉘에 상기 카르복실기를 도입시킨 후, EDC(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) 및 N-hydroxysuccinimide를 첨가하여 (i) 상기 카르복실기를 활성화시키고, (ii) 상기 아미노기가 결합된 상기 타게팅 리간드를 혼합하여 상기 카르복실기와 상기 아미노기가 반응하도록 하여 아마이드를 형성함으로써 상기 타게팅 리간드를 상기 제2쉘에 결합하는 것을 특징으로 하는 방법.After introducing the carboxyl group into the second shell, EDC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide) and N-hydroxysuccinimide were added to (i) activate the carboxyl group, and (ii) the amino group Adhering the targeting ligand to the second shell by mixing the targeting ligand to react the carboxyl group with the amino group to form an amide.
  6. 제1항에 있어서,The method of claim 1,
    상기 (a) 단계에서,In the step (a),
    제1 리피드를 포함하는 제1 혼합물 파우더를 제1 용매에 용해하여 제1쉘 물질의 솔루션을 생성하고, 상기 제1쉘 물질의 솔루션과 상기 불활성 가스를 혼합한 다음 기계적 믹싱을 하여 상기 초음파 반응형 미소기포를 생성하는 것을 특징으로 하는 방법.The first mixture powder including the first lipid is dissolved in a first solvent to produce a solution of the first shell material, the solution of the first shell material and the inert gas are mixed, and then mechanically mixed to perform the ultrasonic reaction type. A method for producing a microbubble.
  7. 제6항에 있어서,The method of claim 6,
    상기 제1 리피드는 DPPC(1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine), HSPC(phosphatidylcholine), DDPC(1,2-didecanoyl-sn-glycerol-3-phosphocholine), DEPC(1,2-Di(cis-13-docosenoyl)-sn-glycerol-3-phosphocholine), DOPC(1,2-Dioleoyl-sn-glycerol-3-phosphocholine), DMPC(1,2-Dimyristoyl-sn-glycerol-3-phosphorylcholine), DLPC(1,2-Dilauroyl-sn-glycerol-3-phosphorylcholine), DEPC(1,2-Didecanoyl-sn-glycerol-3-phosphocholine), DSPC(1,2-Distearoyl-sn-glycerol-3-phosphocholine), MPPC(1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphocholine), MSPC(1-myristoyl-2-stearoyl-sn-glycerol-3-phosphocholine), egg PC(phosphocholine), DPPA(Diphenylphosphoryl azide), DMPA-Na(1,2-Dimyristoyl-sn-glycerol-3-phosphate), DPPA-Na(1,2-Dipalmitoyl-sn-glycerol-3-phosphate), DOPA-Na(1,2-Dioleoyl-sn-glycerol-3-phosphate), DSPE(Distearoylphosphatidylethanolamine), DMPE(Dimyristoyl phosphatidylethanolamine), DOPE(Dioleoyl phosphatidylethanolamine), DPPE(Dipalmitoyl phosphatidylethanolamine), DOPE-Glutaryl-(Na)2(1,2-Dioleoyl-sn-glycerol-3-phosphoethanolamine), egg PE(phosphatidylethanolamine), DSPG(Distearoyl phosphatidylglycerol), DMPG-Na(1,2-Dimyristoyl-sn-glycerol-3-Phosphoglycerol), DPPG-Na(1,2-Dipalmitoyl-sn-glycerol-3-Phosphoglycerol), DOPG-Na(1,2-Dioleoyl-sn-glycerol-3-Phosphoglycerol), DOPS(ioleoyl phosphatidylserine), DMPS(Dimyristoyl phosphatidylserine), DMPS- Na(1,2-Dimyristoyl-sn-glycerol-3-phosphoserine), DPPS-Na(1,2-Dipalmitoyl-sn-glycerol-3-phosphoserine), DOPS-Na(1,2-Dioleoyl-sn-glycerol-3-phosphoserine), DSPS(Distearoylphosphatidylserine), DSPE-mPEG(1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), DSPE-mPEG-2000-Na(1,2-Distearoyl-sn-glycerol-3-phosphoethanolamine), DSPE-mPEG-5000-Na, DSPE-Maleimide PEG-2000-Na, Surfactant: Tween 80, Span 80, dipotassium glycyrrhizinate 중 적어도 하나 이상을 포함하는 것을 특징으로 하는 방법.The first lipid is DPPC (1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine), HSPC (phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycerol-3-phosphocholine), DEPC (1,2- Di (cis-13-docosenoyl) -sn-glycerol-3-phosphocholine), DOPC (1,2-Dioleoyl-sn-glycerol-3-phosphocholine), DMPC (1,2-Dimyristoyl-sn-glycerol-3-phosphorylcholine ), DLPC (1,2-Dilauroyl-sn-glycerol-3-phosphorylcholine), DEPC (1,2-Didecanoyl-sn-glycerol-3-phosphocholine), DSPC (1,2-Distearoyl-sn-glycerol-3- phosphocholine), MPPC (1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphocholine), MSPC (1-myristoyl-2-stearoyl-sn-glycerol-3-phosphocholine), egg PC (phosphocholine), DPPA (Diphenylphosphoryl azide), DMPA-Na (1,2-Dimyristoyl-sn-glycerol-3-phosphate), DPPA-Na (1,2-Dipalmitoyl-sn-glycerol-3-phosphate), DOPA-Na (1,2-Dioleoyl -sn-glycerol-3-phosphate), Distearoylphosphatidylethanolamine (DPE), Dimyristoyl phosphatidylethanolamine (DMPE), Dioleoyl phosphatidylethanolamine (DOPE), Dipalmitoyl phosphatidylethanolamine (DPPE), DOPE-Glutaryl- (Na) 2 (1,2-Dioleoy l-sn-glycerol-3-phosphoethanolamine, egg PE (phosphatidylethanolamine), DSPG (Distearoyl phosphatidylglycerol), DMPG-Na (1,2-Dimyristoyl-sn-glycerol-3-Phosphoglycerol), DPPG-Na (1,2- Dipalmitoyl-sn-glycerol-3-Phosphoglycerol, DOPG-Na Dimyristoyl-sn-glycerol-3-phosphoserine), DPPS-Na (1,2-Dipalmitoyl-sn-glycerol-3-phosphoserine), DOPS-Na (1,2-Dioleoyl-sn-glycerol-3-phosphoserine), DSPS (Distearoylphosphatidylserine), DSPE-mPEG (1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000]), DSPE-mPEG-2000-Na (1,2-Distearoyl-sn -glycerol-3-phosphoethanolamine), DSPE-mPEG-5000-Na, DSPE-Maleimide PEG-2000-Na, Surfactant: Tween 80, Span 80, dipotassium glycyrrhizinate.
  8. 제7항에 있어서,The method of claim 7, wherein
    상기 제1 리피드는 상기 DPPC 및 상기 DPPA를 포함하는 것을 특징으로 하는 방법.And wherein the first lipid comprises the DPPC and the DPPA.
  9. 제6항에 있어서,The method of claim 6,
    상기 제1 혼합물 파우더는 알부민(albumin), 폴리머(polymer), 콜레스테롤(cholesterol), PEG(polyethylene glycol), 계면활성제, 단백질 및 생분해성 고분자 중 적어도 하나 이상을 더 포함하는 것을 특징으로 하는 방법.The first mixture powder further comprises at least one of albumin (albumin), polymer (polymer), cholesterol (cholesterol), PEG (polyethylene glycol), surfactants, proteins and biodegradable polymers.
  10. 제6항에 있어서,The method of claim 6,
    상기 제1 용매는 Saline 또는 3차 증류수, glycerin 및 propylene glycol 중 적어도 하나 이상을 포함하는 것을 특징으로 하는 방법.The first solvent is a method comprising at least one of Saline or tertiary distilled water, glycerin and propylene glycol.
  11. 제6항에 있어서,The method of claim 6,
    상기 제1쉘 물질의 솔루션과 상기 불활성 가스를 1:1 내지 20:1의 부피비로 혼합하는 것을 특징으로 하는 방법.Mixing the solution of the first shell material and the inert gas in a volume ratio of 1: 1 to 20: 1.
  12. 제1항에 있어서,The method of claim 1,
    상기 (a) 단계에서,In the step (a),
    30nm 내지 1um 중 어느 하나의 공극 사이즈를 갖는 필터와 상기 압출기를 통해 상기 초음파 반응형 미소기포를 필터링함으로써, 상기 초음파 반응형 미소기포의 크기 분포를 균일하게 형성하는 것을 특징으로 하는 방법.The ultrasonically reactive microbubble is filtered through the extruder and the filter having a pore size of any one of 30 nm to 1 μm, thereby uniformly forming a size distribution of the ultrasonically reactive microbubbles.
  13. 제1항에 있어서,The method of claim 1,
    상기 불활성 가스는 퍼플루오로카본(perfluorocarbon) 계열의 가스인 것을 특징으로 하는 방법.The inert gas is a method characterized in that the gas of the perfluorocarbon (perfluorocarbon) series.
  14. 제13항에 있어서,The method of claim 13,
    상기 퍼플루오로카본 계열의 가스로서, Perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoro-n-pentane, perfluoro-n-hexane, perfluoromethylcyclopentane, perfluoro-1,3-dimethylcyclohexane, perfluorodecalin, perfluoromethyldecalin 및 perfluoroperhydrobenzyltetralin 중 적어도 하나가 사용되는 것을 특징으로 하는 방법.As the perfluorocarbon-based gas, at least one of perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoro-n-pentane, perfluoro-n-hexane, perfluoromethylcyclopentane, perfluoro-1,3-dimethylcyclohexane, perfluorodecalin, perfluoromethyldecalin and perfluoroperhydrobenzyltetralin are used. Characterized in that the method.
  15. 제1항에 있어서,The method of claim 1,
    상기 (b) 단계에서,In step (b),
    제2 리피드를 포함하는 제2 혼합물 파우더를 유기 용매에 용해한 후 상기 유기 용매를 제거하여 리피드 필름을 획득하고, 상기 리피드 필름을 제2 용매에 용해하여 제2쉘 물질의 솔루션을 생성하고, 상기 제2쉘 물질의 솔루션과 상기 초음파 반응형 미소기포 및 상기 약물을 혼합한 다음 초음파를 조사하여 상기 리포좀을 생성하는 것을 특징으로 하는 방법.Dissolving a second mixture powder comprising a second lipid in an organic solvent and then removing the organic solvent to obtain a lipid film, dissolving the lipid film in a second solvent to produce a solution of a second shell material, and A method of producing a liposome by mixing a solution of a two-shell material and the ultrasonic reaction micro-bubbles and the drug and then irradiated with ultrasonic waves.
  16. 제15항에 있어서,The method of claim 15,
    상기 제2 리피드는 DPPC(1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine), HSPC(phosphatidylcholine), DDPC(1,2-didecanoyl-sn-glycerol-3-phosphocholine), DEPC(1,2-Di(cis-13-docosenoyl)-sn-glycerol-3-phosphocholine), DOPC(1,2-Dioleoyl-sn-glycerol-3-phosphocholine), DMPC(1,2-Dimyristoyl-sn-glycerol-3-phosphorylcholine), DLPC(1,2-Dilauroyl-sn-glycerol-3-phosphorylcholine), DEPC(1,2-Didecanoyl-sn-glycerol-3-phosphocholine), DSPC(1,2-Distearoyl-sn-glycerol-3-phosphocholine), MPPC(1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphocholine), MSPC(1-myristoyl-2-stearoyl-sn-glycerol-3-phosphocholine), egg PC(phosphocholine), DPPA(Diphenylphosphoryl azide), DMPA-Na(1,2-Dimyristoyl-sn-glycerol-3-phosphate), DPPA-Na(1,2-Dipalmitoyl-sn-glycerol-3-phosphate), DOPA-Na(1,2-Dioleoyl-sn-glycerol-3-phosphate), DSPE(Distearoylphosphatidylethanolamine), DMPE(Dimyristoyl phosphatidylethanolamine), DOPE(Dioleoyl phosphatidylethanolamine), DPPE(Dipalmitoyl phosphatidylethanolamine), DOPE-Glutaryl-(Na)2(1,2-Dioleoyl-sn-glycerol-3-phosphoethanolamine), egg PE(phosphatidylethanolamine), DSPG(Distearoyl phosphatidylglycerol), DMPG-Na(1,2-Dimyristoyl-sn-glycerol-3-Phosphoglycerol), DPPG-Na(1,2-Dipalmitoyl-sn-glycerol-3-Phosphoglycerol), DOPG-Na(1,2-Dioleoyl-sn-glycerol-3-Phosphoglycerol), DOPS(ioleoyl phosphatidylserine), DMPS(Dimyristoyl phosphatidylserine), DMPS- Na(1,2-Dimyristoyl-sn-glycerol-3-phosphoserine), DPPS-Na(1,2-Dipalmitoyl-sn-glycerol-3-phosphoserine), DOPS-Na(1,2-Dioleoyl-sn-glycerol-3-phosphoserine), DSPS(Distearoylphosphatidylserine), DSPE-mPEG(1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), DSPE-mPEG-2000-Na(1,2-Distearoyl-sn-glycerol-3-phosphoethanolamine), DSPE-mPEG-5000-Na, DSPE-Maleimide PEG-2000-Na, Surfactant: Tween 80, Span 80, dipotassium glycyrrhizinate 중 적어도 하나 이상을 포함하는 것을 특징으로 하는 방법.The second lipid is DPPC (1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine), HSPC (phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycerol-3-phosphocholine), DEPC (1,2- Di (cis-13-docosenoyl) -sn-glycerol-3-phosphocholine), DOPC (1,2-Dioleoyl-sn-glycerol-3-phosphocholine), DMPC (1,2-Dimyristoyl-sn-glycerol-3-phosphorylcholine ), DLPC (1,2-Dilauroyl-sn-glycerol-3-phosphorylcholine), DEPC (1,2-Didecanoyl-sn-glycerol-3-phosphocholine), DSPC (1,2-Distearoyl-sn-glycerol-3- phosphocholine), MPPC (1-myristoyl-2-palmitoyl-sn-glycerol-3-phosphocholine), MSPC (1-myristoyl-2-stearoyl-sn-glycerol-3-phosphocholine), egg PC (phosphocholine), DPPA (Diphenylphosphoryl azide), DMPA-Na (1,2-Dimyristoyl-sn-glycerol-3-phosphate), DPPA-Na (1,2-Dipalmitoyl-sn-glycerol-3-phosphate), DOPA-Na (1,2-Dioleoyl -sn-glycerol-3-phosphate), Distearoylphosphatidylethanolamine (DPE), Dimyristoyl phosphatidylethanolamine (DMPE), Dioleoyl phosphatidylethanolamine (DOPE), Dipalmitoyl phosphatidylethanolamine (DPPE), DOPE-Glutaryl- (Na) 2 (1,2-Dioleoy l-sn-glycerol-3-phosphoethanolamine, egg PE (phosphatidylethanolamine), DSPG (Distearoyl phosphatidylglycerol), DMPG-Na (1,2-Dimyristoyl-sn-glycerol-3-Phosphoglycerol), DPPG-Na (1,2- Dipalmitoyl-sn-glycerol-3-Phosphoglycerol, DOPG-Na Dimyristoyl-sn-glycerol-3-phosphoserine), DPPS-Na (1,2-Dipalmitoyl-sn-glycerol-3-phosphoserine), DOPS-Na (1,2-Dioleoyl-sn-glycerol-3-phosphoserine), DSPS (Distearoylphosphatidylserine), DSPE-mPEG (1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000]), DSPE-mPEG-2000-Na (1,2-Distearoyl-sn -glycerol-3-phosphoethanolamine), DSPE-mPEG-5000-Na, DSPE-Maleimide PEG-2000-Na, Surfactant: Tween 80, Span 80, dipotassium glycyrrhizinate.
  17. 제16항에 있어서,The method of claim 16,
    상기 제2 리피드는 상기 DPPC 및 DPPA를 포함하는 것을 특징으로 하는 방법.And said second lipid comprises said DPPC and DPPA.
  18. 제16항에 있어서,The method of claim 16,
    상기 제2 리피드는 상기 DPPA, 상기 DMPA-Na, 상기 DPPA-Na, 상기 DSPG 및 상기 DSPS 중 적어도 하나 이상을 포함함으로써, 상기 리포좀이 전하를 띄도록 하는 것을 특징으로 하는 방법.And wherein the second lipid comprises at least one of the DPPA, the DMPA-Na, the DPPA-Na, the DSPG, and the DSPS, thereby allowing the liposomes to be charged.
  19. 제15항에 있어서,The method of claim 15,
    상기 제2 혼합물 파우더는 알부민(albumin), 폴리머(polymer), 콜레스테롤(cholesterol) 및 PEG(polyethylene glycol) 중 적어도 하나 이상을 더 포함하는 것을 특징으로 하는 방법.The second mixture powder further comprises at least one of albumin (albumin), polymer (polymer), cholesterol (cholesterol) and PEG (polyethylene glycol).
  20. 제15항에 있어서,The method of claim 15,
    상기 제2 용매는 PBS(Phosphate-buffered saline)인 것을 특징으로 하는 방법.Wherein said second solvent is a Phosphate-buffered saline (PBS).
  21. 제15항에 있어서,The method of claim 15,
    상기 유기 용매는 클로로포름과 메탄올의 혼합 용매인 것을 특징으로 하는 방법.The organic solvent is a mixed solvent of chloroform and methanol.
  22. 제1항에 있어서,The method of claim 1,
    상기 (b) 단계에서,In step (b),
    필터와 상기 압출기를 통해 상기 리포좀을 필터링함으로써, 상기 리포좀의 크기 분포를 균일하게 형성하는 것을 특징으로 하는 방법.Filtering the liposomes through a filter and the extruder, thereby uniformly forming a size distribution of the liposomes.
  23. 제15항에 있어서,The method of claim 15,
    상기 제2쉘 물질의 솔루션에 초음파를 조사한 다음, 상기 미소기포와 상기 약물을 혼합하여 상기 리포좀을 생성하는 것을 특징으로 하는 방법.Irradiating the solution of the second shell material with ultrasonic waves, and then mixing the microbubbles with the drug to generate the liposomes.
  24. 제1항에 있어서,The method of claim 1,
    상기 리포좀은 내부에 유전자를 추가로 포함하는 것을 특징으로 하는 방법.The liposome is characterized in that it further comprises a gene therein.
  25. 제1항 내지 제24항 중 어느 한 항의 방법에 의해 생성된 리포좀.A liposome produced by the method of any one of claims 1-24.
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