WO2014021678A1 - Microbubble-nanoliposome complex for cancer diagnosis and treatment - Google Patents

Microbubble-nanoliposome complex for cancer diagnosis and treatment Download PDF

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WO2014021678A1
WO2014021678A1 PCT/KR2013/006993 KR2013006993W WO2014021678A1 WO 2014021678 A1 WO2014021678 A1 WO 2014021678A1 KR 2013006993 W KR2013006993 W KR 2013006993W WO 2014021678 A1 WO2014021678 A1 WO 2014021678A1
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complex
glycero
microbubble
nanoliposomal
cancer
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French (fr)
Korean (ko)
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이학철
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(주)아이엠지티
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    • A61K31/33—Heterocyclic compounds
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    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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Definitions

  • the present invention relates to a microbubble-nanoliposomal complex and a method for preparing the same, which enables cancer cell-specific diagnosis and treatment, and specifically, a microbubble in which hydrophobic microbubbles and hydrophilic nanoliposomes are chemically stable covalently bound.
  • Bubble-nanoliposomal complexes can carry one or more additional hydrophobic or hydrophilic phosphors, as well as one or more therapeutic agents, within the complex structure and exhibit excellent target specificity, including targeting moieties to the outside.
  • Multiple imaging analysis using specific ultrasound or fluorescence contrast can diagnose cancer and effectively deliver one or more therapeutic agents to the target cancer cells to treat the cancer, so breast cancer, liver cancer, pancreatic cancer, brain cancer, gastric cancer, lung cancer, esophageal cancer and large intestine Cancer, Prostate Cancer, Kidney Cancer, Ovarian Cancer May be useful to take advantage of the development of effective diagnostic and treatment of cancer.
  • Therapeutic contrast agents refers to substances that enable the simultaneous diagnosis and treatment of the disease. They usually consist of small sized materials, usually fluorescent dyes, radioactive molecules, etc. are carried on the inside of liposomes, polymers and nanoparticles, and drugs or diagnostic markers are introduced outside. Recently, research has been focused on the synthesis of therapeutic contrast materials consisting of lipid structures with excellent biocompatibility.
  • microbubble ultrasound agent a micrometer-sized bubble ultrasound contrast medium composed of lipid structures , MB
  • MB micrometer-sized bubble ultrasound contrast medium composed of lipid structures
  • These studies focus primarily on imposing properties such as nontoxicity, high water solubility, or easy surface modification for the application of nanomaterials in the biological field, which is now called the 'nano-bio field'. It is aligned.
  • microbubbles based on known lipid structures, the excellent biocompatibility allows for treatment as well as diagnosis through imaging analysis of blood vessels or cancerous tissues in various in vivo or in vitro studies. It has been reported that cancer can be treated by delivering genes or drugs (Katherine F. et al., Annual Review of Biomedical Engineering (2007), 9, 415-447). However, microbubble ultrasonography analysis is inadequate for accurate diagnosis of cancer due to its low resolution and target nonspecific properties. At the same time, studies have been made to develop a multi-image contrast material using fluorescence and ultrasound to confirm detailed distribution through fluorescence image analysis [Bart G. et al., Journal of Controlled Release (2011), 152, 249-. 256].
  • the present inventors have studied to develop a therapeutic contrast agent capable of treating cancer by simultaneously delivering one or more therapeutic agents to cancer diagnosis through cancer cell-specific multi-image analysis, resulting in hydrophobic microbubbles.
  • the microbubble-nanoliposomal complex is used to diagnose cancer through multiple image analysis using ultrasound or fluorescence contrast, and at the same time, stably deliver one or more therapeutic agents, such as therapeutic genes and drugs, to target cells.
  • the present invention has been found to be curable. It was.
  • an object of the present invention is to provide a therapeutic contrast agent capable of treating cancer by specifically delivering one or more therapeutic agents to a target cell at the same time as diagnosing cancer through cancer cell-specific multi-image analysis.
  • the present invention is a complex in which hydrophobic microbubbles and hydrophilic nanoliposomes are covalently bonded to each other, carrying a targeting moiety to the outside while carrying at least one therapeutic agent inside the complex structure. It provides a microbubble-nanoliposomal complex containing.
  • the therapeutic agent may be selected from a therapeutic gene and a drug.
  • one or more hydrophobic or hydrophilic fluorescent materials may be additionally supported in the complex structure.
  • a film-forming substance, a phospholipid, a negatively charged compound, a compound having an amine group, and a compound having a disulfide group are added to an organic solvent and reacted to prepare a film, which is then mixed with an hydrophobic gas into an organic mixed solvent and vibrated. Preparing a bubble;
  • step 2) reacting the nanoliposomes obtained in step 2) with at least one therapeutic agent to support the at least one therapeutic agent in the nanoliposomal structure;
  • step 5) reacting the complex prepared in step 4) with a targeting moiety (targeting moiety) to bind the targeting moiety to the outside of the complex, the method of producing a microbubble-nanoliposomal complex of the present invention to provide.
  • a targeting moiety targeting moiety
  • the step 1) or 2) may further include a step of supporting the fluorescent material in the microbubble or nanoliposomal structure by reacting it with a fluorescent material after preparing the microbubble or nanoliposomes. have.
  • microbubble-nanoliposomal complex which enables cancer cell-specific diagnosis and treatment according to the present invention is hydrophobic microbubbles and hydrophilic nanoliposomes are chemically stable covalently bonded to the therapeutic genes and drugs in the complex structure, etc.
  • Ultrasonic or fluorescence specific to cancer cells as it can reliably carry one or more additional hydrophobic or hydrophilic fluorescent materials as well as a targeting moiety externally and exhibits excellent target specificity Cancer can be diagnosed through multiple imaging analysis using contrast, and at the same time, one or more therapeutic agents can be effectively delivered to the target cancer cells to treat the cancer, so breast cancer, liver cancer, pancreatic cancer, brain cancer, gastric cancer, lung cancer, esophageal cancer, colon cancer, and prostate Efficacy of cancer diseases such as cancer, kidney cancer and ovarian cancer Diagnosis and can be usefully utilized in drug development.
  • FIG. 1 is a schematic diagram of a microbubble-nanoliposomal complex according to an embodiment of the present invention.
  • FIG. 2 shows the results of microbubbles (a) and nanoliposomes (b) prepared according to one embodiment of the present invention, respectively, by optical microscope and cryogenic electron microscopy (Cryo-EM). It is a photograph.
  • FIG. 3 is a photograph of the microbubble-nanoliposomal complex prepared according to one embodiment of the present invention using a confocal fluorescence microscope (confocal fluorescence microscope) at low and high magnification (top right insertion drawing).
  • confocal fluorescence microscope confocal fluorescence microscope
  • Figure 4 is a by-product SPDP (N-succinimidyl-3) generated by covalent bonding by performing ultraviolet-vis spectroscopy (UV-vis spectroscopy) on the microbubble-nanoliposomal complex prepared according to an embodiment of the present invention -[2-pyridyldithio] -propionate).
  • SPDP N-succinimidyl-3
  • UV-vis spectroscopy ultraviolet-vis spectroscopy
  • TM microbubble ultrasound contrast agent SonoVue
  • b microbubble
  • c microbubble-nanoliposomal complex
  • FIG. 6 is a photograph showing the results of confirming the change in the ultrasound contrast effect in the artificial vascular phantom when a sonic stimulation is applied from 1 to 10 times using the microbubble-nanoliposomal complex prepared according to an embodiment of the present invention. 1 to 10 times) and graphs.
  • microbubble-nanoliposomal complex prepared according to one embodiment of the present invention and microbubbles (MB) -nanoliposomal complex prepared by omitting antibody introduction as a control using a target for SkBr3 cells, breast cancer cell line
  • MB microbubbles
  • FIG. 9 is a microbubble-nanoliposomal complex prepared according to one embodiment of the present invention treated with SkBr3 cells, a breast cancer cell line, and subjected to external sonic stimulation for 1 minute, followed by confocal fluorescence microscopy to be carried inside the complex. The result of confirming whether the fluorescent material was delivered inside the cancer cell is a photograph.
  • UV-vis spectrometer ultraviolet-vis spectrometer
  • FIG. 11 shows that breast cancer cell line Skbr3 treated with (1) microbubble-nanoliposomal complex (2) or doxorubicin (3) prepared according to one embodiment of the present invention is treated for a certain time (24 hours, 48 hours or 72 hours) and then cultured for MTT analysis to confirm the cytotoxicity is a graph.
  • microbubble-nanoliposomal complex prepared according to an embodiment of the present invention to breast cancer cell line SkBr3 and cultured for a predetermined time (48 hours, 72 hours or 96 hours) with or without external acoustic stimulation.
  • a predetermined time 48 hours, 72 hours or 96 hours
  • the result of confirming the expression of tGFP using a confocal fluorescence microscope is a photograph.
  • FIG. 13 shows the optimization and loading capacity of protamine (PA) and pGFP complexes for gene delivery experiments of micro bubble-nanoliposomal complexes.
  • PA protamine
  • Figure 14 compares the pGFP delivery effect of the micro bubble-nanoliposomal complex of the present invention.
  • Figure 15 monitors the loading capacity of doxorubicin and its properties in cells in the micro bubble-nanoliposomal complex.
  • Figure 16 shows the results of cell viability test by drug and therapeutic gene delivery using the micro bubble-nanoliposomal complex.
  • Pop-particle means micro bubble-nanoliposomal complex.
  • FIG. 17 shows the expression levels of STAT3 proteins and genes in cancer cells after apoptosis testing of the microbubble-nanoliposomal complex containing siSTAT3.
  • Figure 21 shows the distribution in vivo of the micro bubble-nanoliposomal complex containing a fluorescent material in raw vegetables.
  • FIG. 22 measures the excretion of the microbubble-nanoliposomal complex containing fluorescent material in vivo.
  • the microbubble-nanoliposomal complex according to the present invention is a complex in which a hydrophobic microbubble and a hydrophilic nanoliposome are covalently bonded, and a targeting moiety such as an antibody to the outside while carrying at least one therapeutic agent in the complex structure (targetting moiety).
  • the microbubble-nanoliposomal complex of the present invention may additionally support one or more hydrophobic or hydrophilic fluorescent materials in the complex structure, wherein the hydrophobic fluorescent material is in the microbubble structure, the hydrophilic fluorescent material in the nanoliposome structure Can be supported.
  • the microbubble-nanoliposomal complex of the present invention is a hydrophilic lipid structure having a size of 100-200 nm outside the hydrophobic microbubble structure in the form of fine air bubbles having a uniform particle size of an average diameter of 1-2 ⁇ m as shown in FIG.
  • Nanoliposomes have a form bound to the surface, the hydrophobic gas such as SF 6, CO 2 or CF 4 is contained in the complex.
  • the hydrophobic microbubbles and the hydrophilic nanoliposomes are chemically stable covalently bonded to each other, and depending on the hydrophobic or hydrophilic properties within each structure, the hydrophobic or hydrophilic fluorescent substance may be added to a therapeutic agent selected from one or more genes and drugs.
  • one or more of the complexes may include a targeting moiety such as an antibody outside the complex
  • one or more therapeutic agents may be in vivo while simultaneously implementing mutimodal imaging through ultrasound or fluorescence imaging. There is a characteristic that can be delivered stably.
  • the microbubble-nanoliposomal complex is also referred to herein as the term 'pop-particle'.
  • the complex of the present invention may preferably exhibit a uniform size of an average diameter of 1-2 ⁇ m, but is not limited thereto, depending on the concentration of the nanoliposome used in the preparation of the complex and the filtration method 100 Particle size can be adjusted to nm to 10 ⁇ m.
  • the particle size can be varied according to the frequency of the ultrasonic device that is currently commercialized, the higher the frequency of the ultrasonic device to be used, the average diameter of the composite may be smaller.
  • the microbubbles in the composite of the present invention may include a film forming material, a phospholipid, a negatively charged compound, a compound having an amine group and a compound having a disulfide group, wherein the film forming material is DPPC (1,2-dipalmitory-sn-glycero-3-phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) , DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) , DOPC (1
  • the nanoliposomes in the composite of the present invention may include a film-forming material, a phospholipid, a negatively charged compound and a compound having an amine group, wherein the film-forming material is DPPC (1, 2 -dipalmitory-sn-glycero-3-phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DLOPC (1, 2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DOPC (1 , 2-dioleoyl-sn-glycero-3-phosphocholine) or DSPC
  • the complex of the present invention is simply covalently bonded by modifying and shaking part of the lipid structure of the microbubble or the lipid structure of the nanoliposome through the control of pH, reaction temperature or reaction time, etc. It can be formed by induction.
  • any one or more therapeutic agents carried in the complex structure of the present invention may be used as long as the selected one among therapeutic genes and drugs, for example, DNA, RNA, etc., which may be used for cancer treatment.
  • therapeutic genes for example, antagonists of expression plasmids, siRNAs, shRNAs, microRNAs or microRNAs that can express genes with known anticancer effects or inhibit the expression of genes involved in cancer development. DNA or RNA, etc.
  • genes and drugs may be supported in the complex structure by reacting with the nanoliposomes according to a conventional method.
  • the genes and drugs may be supported by reacting with the nanoliposomes in a hybrid form with biopolymers such as protamine.
  • they may be supported inside liposomes through conventional ammonium sulfate gradient method (Bowen T. et al., International Journal of Pharmaceutics (2011), 416, 443-447).
  • the doxorubicin drug collection efficiency of the complex of the present invention was 88.57%
  • tGFP expression plasmid gene collection efficiency was 30%.
  • any of the hydrophobic or hydrophilic fluorescent materials that can be additionally supported in the complex structure of the present invention can be used for clinical diagnosis, for example, FITC , Texas red. RITC, Cy3, Cy5 or Cy7 may be used, but is not limited thereto.
  • fluorescent materials may be supported by microbubbles in the case of hydrophobic fluorescent materials and nanoliposomes in the case of hydrophilic fluorescent materials by stirring or sonication at 30 ° C. according to a conventional method.
  • the targeting moiety of the antibody or the like bound to the surface of the complex of the present invention may be a molecule, ligand or receptor on the surface of the target cell.
  • Any material capable of selectively recognizing / binding the target material may be used, for example, nucleic acid molecules (DNA or RNA), proteins, antibodies, antigens, aptamers (RNA, DNA and Peptide aptamers), receptors, hormones, streptavidin, avidin, biotin, lectins, ligands, agonists, antagonists, enzymes, coenzymes, inorganic ions, enzyme cofactors, sugars, lipids, enzyme substrates, hapten, neutra Neutravidin, Protein A, Protein G, Selectin, Calcium Sulfate and Gas Binders (e.g.
  • the target material means a material in a sample to be separated (or isolated and detected, or isolated, detected and quantified), and specifically, a nucleic acid molecule (DNA or RNA), a protein, a peptide, an antigen, a sugar, a lipid, a bacterium. , Viruses, cells, organic compounds, inorganic compounds, metals and inorganic ions.
  • the sample includes a biological sample, a chemical sample, and an environmental sample
  • the biological sample includes, for example, blood, plasma, serum, virus, bacteria, tissue, cells, lymph, bone marrow fluid, saliva, milk, urine, feces, eyeballs.
  • targeting moieties such as antibodies contained on the surface of the complex of the present invention are directly or indirectly covalently or indirectly shared with the surface of the complex of the present invention through a conventional reaction. It may be included in combination, for example, may be included in combination through ionic bonds, electrostatic bonds, hydrophobic bonds, hydrogen bonds, covalent bonds, hydrophilic bonds or van der Waals bonds. In addition, in the case of the indirect binding, an intervening agent such as a binder may be used.
  • sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate sulfosuccinimidyl- 4- (N-maleimidomethyl) cyclohexane-1-carboxylate
  • sulfosuccinimidyl- 4- N-maleimidomethyl
  • the therapeutic genes and drugs carried in the complex structure are destroyed by external acoustic stimulation (ultrasonic flow, UF).
  • flash exposing the composite of the present invention to high energy or high mechanical index on ultrasonic waves is referred to as flash, and is also described as flash in the following examples.
  • the FITC and Texas red as a fluorescent material, tGFP expression plasmid as a gene, doxorubicin as a drug is carried inside the complex structure, and the anti-HER2 monoclonal antibody specific for breast cancer cells on the surface
  • UF external acoustic stimulation
  • the microbubble-nanoliposomal complex of the present invention can diagnose cancer through multiple image analysis using ultrasound or fluorescence contrast specific to cancer cells while minimizing side effects through high target specificity and at least one therapeutic agent such as therapeutic genes and drugs.
  • cancer diseases such as breast cancer, liver cancer, pancreatic cancer, brain cancer, stomach cancer, lung cancer, esophageal cancer, colon cancer, prostate cancer, kidney cancer, ovarian cancer, etc.
  • a film-forming substance, a phospholipid, a negatively charged compound, a compound having an amine group, and a compound having a disulfide group are added to an organic solvent and reacted to prepare a film, which is then mixed with an hydrophobic gas into an organic mixed solvent and vibrated. Preparing a bubble;
  • step 2) reacting the nanoliposomes obtained in step 2) with at least one therapeutic agent to support the at least one therapeutic agent in the nanoliposomal structure;
  • step 5) reacting the complex prepared in step 4) with a targeting moiety (targeting moiety) to bind the targeting moiety to the outside of the complex, the method of producing a microbubble-nanoliposomal complex of the present invention to provide.
  • a targeting moiety targeting moiety
  • the hydrophobic gas used in the step 1) may be used a hydrophobic gas used in the manufacture of conventional microbubbles such as SF 6 , CO 2 or CF 4 , chloroform is an organic solvent, an organic mixed solvent As the solvent, a solvent in which glycerin, propylene glycol and water are mixed can be used.
  • the film forming material used in step 2) is DPPC (1,2-dipalmitory-sn-glycero-3-phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPC ( 1,2-didecanoyl-sn-glycero-3-phosphocholine), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) or DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) this; Phospholipids include cholesterol (cholesterol); Negative charge compounds include DCP (dicetyl phosphate
  • the step of supporting the at least one therapeutic agent in the nanoliposomal structure in step 3), if the therapeutic agent is a therapeutic gene can be carried out by reacting with the nanoliposome in the form of a hybrid with a biopolymer such as protamine (protamine) , If the therapeutic agent is a drug, it can be carried out by stirring at room temperature through a conventional ammonium sulfate gradient method, Bowen T et al., International Journal of Pharmaceutics (2011), 416, 443-447.
  • the complex formation reaction of the microbubbles and nanoliposomes of step 4) is simply by modifying a portion of the lipid structure of the nanobubble and the liposomes of the nanoliposomes by controlling the pH, the reaction temperature or the reaction time and shaking. This can be done by inducing covalent bonds.
  • the step of binding the targeting moiety, such as the antibody of step 5) outside the complex the targeting moiety binds to the surface of the complex of the present invention directly or indirectly in a covalent or non-covalent manner through a conventional reaction. It may be carried out by, for example, may be carried out by bonding through ionic bonds, electrostatic bonds, hydrophobic bonds, hydrogen bonds, covalent bonds, hydrophilic bonds, or van der Waals bonds. In this case, in the case of the indirect binding, an intervening agent such as a binder may be used.
  • sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate sulfosuccinimidyl- 4- (N-maleimidomethyl) cyclohexane-1-carboxylate
  • sulfosuccinimidyl- 4- N-maleimidomethyl
  • the step 1) or 2) further includes a step of supporting the fluorescent material in the microbubble or nanoliposomal structure by adding a fluorescent material to the prepared microbubble or nanoliposomes and reacting. can do.
  • the present invention provides a contrast agent composition for cancer cell-specific ultrasound, magnetic resonance imaging (MRI) or fluorescence analysis comprising the microbubble-nanoliposomal complex.
  • MRI magnetic resonance imaging
  • fluorescence analysis comprising the microbubble-nanoliposomal complex.
  • the contrast agent refers to a substance that is administered in vivo to contrast or image cancer cells in a powerful and specific manner in vivo, and is currently used for enhancing image of tissues and cells in the medical and diagnostic fields. Widely used.
  • the term contrast agent of the present invention is not limited to the range of conventionally known CT or MRI contrast agents, and is used in the sense of including an imaging agent for ultrasound image analysis, an imaging agent for fluorescence image analysis, and the like.
  • the contrast agent composition of the present invention may be usefully used for the targeting and diagnosis of cancer cells or cancer tissues, and may be formulated in oral or parenteral dosage forms.
  • Parenteral formulations preferably comprise sterile aqueous solutions or suspensions comprising the microbubble-nanoliposomal complex of the present invention, and various techniques for preparing pharmaceutical aqueous solutions or suspensions are known in the art.
  • the solution may also include pharmaceutically acceptable buffers, stabilizers, antioxidants and electrolytes such as sodium chloride.
  • Parenteral formulations may be injected directly or mixed with large amounts of parenteral formulations.
  • Formulations for oral administration can vary widely and are known in the art. Generally such formulations comprise an aqueous solution or suspension comprising a diagnostically effective amount of the microbubble-nanoliposomal complex according to the invention.
  • the oral formulation may optionally include buffers, surfactants, adjuvants, thixotropic agents, and the like.
  • Formulations for oral administration may also include ingredients for increasing flavor and other functionalities.
  • the contrast agent composition according to the invention is administered in an amount effective to achieve the desired contrast effect of the imaging image.
  • dosages can vary widely depending on the organ or tissue that is the subject of the imaging procedure, the imaging device used, and the like.
  • the dosage concentration for using the microbubble-nanoliposomal complex as a contrast agent may be 0.1 mM to 10 M.
  • contrast agent compositions of the present invention are used in a conventional manner of imaging diagnostic analysis.
  • the contrast agent composition may be administered systemically to the mammal or locally to an organ or tissue being imaged, in an amount sufficient to provide adequate visualization, and then the mammal may be subjected to ultrasound or MRI imaging.
  • the present invention provides a pharmaceutical composition for anticancer containing the microbubble-nanoliposomal complex and an anticancer agent or anticancer gene as an active ingredient.
  • an anticancer agent or anticancer gene into the microbubble-nanoliposomal complex of the present invention to confirm the effect of killing cancer cells or inhibiting the growth of cancer tissues, it was confirmed that it can be usefully used for cancer treatment ( See Test Examples 6 and 7).
  • the pharmaceutical composition for anticancer of the present invention may be prepared by a method known in the pharmaceutical field, in this case, the microbubble-nanoliposomal complex of the present invention carrying a known anticancer agent or anticancer gene as an active ingredient.
  • the microbubble-nanoliposomal complex of the present invention carrying a known anticancer agent or anticancer gene as an active ingredient.
  • it may include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the formulation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, phosphorus calcium, alginate, gelatin, silicic acid Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils, and the like. It is not.
  • the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
  • compositions of the present invention may be prepared in unit dosage form by being formulated with a pharmaceutically acceptable carrier or excipient according to methods which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a dose container.
  • the formulation may be in the form of a solution, suspension or emulsion in oil or medium, or may be in the form of extracts, powders, granules, tablets, capsules or injections, and may further comprise dispersants or stabilizers. They may be parenterally administered (eg, applied intravenously, subcutaneously, intraperitoneally or topically) or orally.
  • Appropriate dosages of the pharmaceutical compositions of the present invention are appropriately selected by such factors as the formulation method, mode of administration, age, weight, sex, health condition, degree of disease symptom, food, time of administration, method of administration, and reaction sensitivity of the patient.
  • 0.01 to 100 mg can be administered per day of adult.
  • DPPC (1,2-dipalmitory-sn-glycero-3-phosphatidylcholine), cholesterol (cholesterol), DCP (dicetyl phosphate), DPPE (1,2-dipalmitory-sn-glycero-3-phosphoethanolamine), DSPE-PEG-SPDP ⁇ 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [polyethylene glycol (PDP) -2000] ⁇ , sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate ( sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, Trauts Reagent (2-iminothiolane), ammonium sulfate and protamine were all purchased from Sigma-Aldrich. It was used without adding a strain.
  • SkBr3 a breast cancer cell line, was incubated under humidified 5% CO 2 atmosphere and 37 ° C. temperature in RPMI 1640 medium (Hyclone, Logan, UT) containing 10% FBS.
  • Cytotoxicity assay using MTT assay kit was performed as follows.
  • Cultured cell lines were harvested after media removal for tryout, resuspended after trypsin treatment.
  • the recovered cells were placed in 96-well plates at a concentration of 5,000 cells per well and incubated overnight under 5% CO 2 atmosphere and 37 ° C. temperature conditions. After the test solution was treated to the cultured cells at various concentrations, the treated cells were again incubated for a predetermined test time under the same conditions. After incubation, the medium was removed, and 0.1 ml of MTT solution per well was added and reacted for 3 hours at 37 ° C. and 5% CO 2. The MTT solution was removed, and 0.1 ml of MTT solubilization solution was added thereto. Formazan crystals were dissolved by addition. Cell viability was determined by measuring the absorbance at 570 nm at a well plate using an ELISA reader.
  • DCP a negative charge compound to prevent aggregation of DPPC, cholesterol, and microbubbles
  • DPPE an amine end group
  • DSPE-PEG-SPDP a compound having disulfide groups
  • Step 1-1) Supporting Hydrophobic Phosphor in Microbubble Structure
  • FITC a hydrophobic organic fluorescent dye having green fluorescence
  • DPPC a negatively charged compound to prevent aggregation of DPPC, cholesterol and nanoliposomes, and DPPE, a compound having an amine end group
  • 5 mL chloroform a compound having an amine end group
  • the obtained solution was placed in a rotary evaporator, reacted at 35 ° C. for 5 minutes to remove the solvent, and lyophilized at ⁇ 45 ° C. for about 24 hours to prepare a film. After adding 2 mL of H 2 O to the obtained film, the mixture was dispersed for 5 minutes at 60 ° C.
  • the liposome mixed solution was filtered twice at 60 ° C. with a 200 nm filter to prepare nanoliposomes having a particle size of 200 nm or less.
  • 0.1 mg of Texas-red (Texas-red), a hydrophilic organic fluorescent dye showing red fluorescence, was added to the nanoliposomes prepared in step 2), followed by stirring at room temperature to obtain nanoliposomes in which the fluorescent material was supported in the structure ( Average size: 100-200 nm).
  • a mixed solution containing 26.6 ⁇ g of a green fluorescent protein (tGFP) expression plasmid gene and 18.8 ⁇ M of protamine was added to a powdered nanoliposome (21 mg). And shaken at 25 ° C. for 5 minutes. The resulting reaction mixture was centrifuged twice at 13000 rpm for 5 minutes to remove residual genes and protamine not trapped in liposomes to obtain nanoliposomes carrying the therapeutic genes inside the structure.
  • tGFP green fluorescent protein
  • doxorubicin a solution of 0.5-20 mM doxorubicin, which is known to be effective in treating cancer, is added to the nanoliposomal solution at a concentration of 1-120 mM, and then stirred at room temperature, so that ammonium sulfate in the nanoliposomes is osmotic by the osmotic phenomenon.
  • the nanoliposomes containing the drug (doxorubicin) were prepared in the structure by replacing the doxorubicin with the outside to support the doxorubicin inside the nanoliposomes.
  • microbubble-nanoliposome complex 0.2 mL of the microbubbles obtained in step 1) or 1-1); And 1.8 mL of the nanoliposome obtained in step 3) was put in a sealed bottle (hermetic vial) was adjusted to pH 8 and shaken at 25 °C for 2 hours to prepare a microbubble-nanoliposome complex.
  • sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate was added to the complex prepared in step 4), and the pH was adjusted to 8, followed by shaking at 25 ° C. for 3 hours.
  • 0.1 mL of herceptin, an anti-HER2 monoclonal antibody specific for breast cancer cells was added, and the pH was adjusted to 7. After shaking for 24 hours at 4 °C bound the antibody outside the complex.
  • the resultant mixed solution was centrifuged twice at 13000 rpm for 5 minutes to remove unreacted material, thereby preparing a microbubble-nanoliposomal complex according to the present invention.
  • Test Example 1 Checking the physical properties of the microbubble (MB) -nanoliposomal complex of the present invention
  • An optical microscope and a cryogenic electron microscope were used for the microbubbles obtained in step 1) or 1-1) of the preparation example and the nanoliposomes obtained in step 2), 2-1) or 3) of the preparation example.
  • Cryogenic electron microscopy (Cryo-EM) and dynamic light scattering (DLS) analyzes were performed to confirm each molecular shape and size distribution.
  • the microbubbles (MB) prepared according to an embodiment of the present invention has a spherical shape and the average size was 1.713 ⁇ m, nanoliposomes also the average size in the shape of a sphere 197 nm.
  • both microbubbles and nanoliposomes were found to exhibit uniform particle size distribution.
  • the microbubble-nanoliposomal complex according to the present invention is a microbubble and nanoliposomes are covalently bonded with excellent chemical stability, the gene, drug or fluorescent material carried in the particles to target organs, tissues or cells in vivo Excellent stability in transport and delivery. Therefore, in order to confirm whether the complex prepared in the above preparation is covalently bonded, SPDP (N-succinimidyl-3- [2-) which is a by-product generated during covalent bonding through UV-vis spectroscopy. pyridyldithio] -propionate) was confirmed.
  • microbubble-nanoliposomal complex according to the present invention is covalently bonded to microbubbles and nanoliposomes with excellent chemical stability, so that genes, drugs, or fluorescent substances supported in the particle structure are transferred to target organs, tissues, or cells in vivo. It can be expected to show good stability in transportation and delivery.
  • microbubble ultrasound contrast agent SonoVue TM
  • the microbubbles obtained in the US were used to analyze the effect of ultrasound imaging on the artificial vascular phantom through the ultrasound imaging apparatus (IU22, Philips) used in the clinic.
  • the microbubble-nanoliposomal complex (c) of the present invention finally prepared in the microbubbles (b) and step 5) obtained in step 1) or 1-1) of the preparation example ) was confirmed to exhibit a sufficiently high level of contrast effect compared to the existing commercial product (a).
  • microbubble-nanoliposomal complex of the present invention having such excellent stability reaches the target organ, tissue, or cell, one or more therapeutic agents or fluorescent substances contained in the structure are destroyed by stimulation of external sound waves to the target cell.
  • a total of 10 sonic stimuli were performed by using an ultrasonic imaging device (IU22, Philips) to see how the effect of ultrasound contrast on the artificial vascular phantom was changed. The results are shown in the photograph and graph of FIG. Exposing the composite of the present invention to high energy or high mechanical index on ultrasound is called flash.
  • microbubble-nanoliposomal complex of the present invention can effectively deliver fluorescent materials as well as one or more therapeutic agents simultaneously to target sites such as cancer cells.
  • the BSA assay which is a conventional protein analysis technique, is performed on the MB-nanoliposomal complex prepared in Preparation Example In this case, spectroscopic analysis was performed using an UV-vis spectrometer.
  • the antibody introduced to the surface was detected at a wavelength of 560 nm and the amount was confirmed to be 1.62 ⁇ M per mg complex by quantitative analysis.
  • Test Example 2 Multiple Image Analysis Test Using Microbubble (MB) -Nanoliposomal Complex of the Present Invention
  • the microbubble (MB) -nano containing the antibody obtained in step 4) of the preparation as a control and the complex prepared in the preparation Liposome complexes were used to compare SkBr3 cells, breast cancer cell lines, by multimodal imaging analysis and FACS analysis using confocal fluorescence microscopy.
  • sonic stimulation mechanical index: 0.7
  • fluorescence contrast was confirmed by confocal fluorescence microscopy.
  • the microbubble-nanoliposomal complex of the present invention enables effective diagnosis through multiple image analysis specific to a specific cancer tissue or cell target.
  • Test Example 3 Drug delivery test using the microbubble (MB) -nanoliposomal complex of the present invention
  • the complex prepared in the preparation example was subjected to spectroscopic analysis using an ultraviolet-visible spectrometer to collect the drug (doxorubicin) supported on the complex. The efficiency was confirmed.
  • the capture efficiency of the representative anti-cancer doxorubicin (88%) contained in the complex of the present invention was 88.57%, and thus the complex of the present invention is a complex of various drugs involved in the treatment of cancer It can be seen that it can be effectively carried and delivered to target cells.
  • cytotoxicity test was performed using the MB-nanoliposomal complex prepared in Preparation Example to confirm the efficacy of cancer cell therapy using the complex of the present invention.
  • Skbr3 a breast cancer cell line
  • 10 mg of the microbubble-nanoliposomal complex having the doxorubicin prepared in the above preparation or 0.2 ⁇ g of doxorubicin itself as a control followed by incubation for 3 hours.
  • a strong ultrasonic wave was added for 5 minutes and incubated for another 3 hours, and then cultured for a predetermined time (24 hours, 48 hours or 72 hours) after removing the treatment medium.
  • MTT assay was then performed to confirm cytotoxicity.
  • the anticancer drug-containing microbubble-nanoliposomal complex of the present invention exhibits excellent cytotoxicity compared to the cell line treated with only the anticancer agent itself as well as the cell line not treated with the drug. (85% cell survival after 72 hours of treatment with drug alone, 63% after 24 hours, 61% after 48 hours, and 35% after 72 hours with complexes of the present invention) .
  • the complex of the present invention it can be seen that due to the cell specificity caused by the antibody bound to the complex, the drug is effectively delivered to the cell specific to the cancer cells can bring excellent therapeutic effect.
  • Test Example 4 Gene delivery test using the microbubble (MB) -nanoliposomal complex of the present invention
  • the collection efficiency of the tGFP expression plasmid supported in the complex prepared in Preparation Example was about 30% using a spectrophotometer (Nano drop).
  • 10 mg of the complexes were treated with SkBr3, incubated for 3 hours, the complexes which did not bind to the cells were removed, and then subjected to a strong ultrasonic wave (mechanical index: 0.07) for 5 minutes. Sonication was omitted.
  • the cells were further incubated for 3 hours, and then all complexes were removed. After incubation for a period of time (48 hours, 72 hours, or 96 hours), the expression of tGFP was confirmed using confocal fluorescence microscopy.
  • the microbubble-nanoliposomal complex according to the present invention is a microbubble and nanoliposomes are bonded through a chemically stable covalent bond, one of a variety of therapeutic genes and drugs in the hydrophobic microbubble and hydrophilic nanoliposome structure
  • one or more additional hydrophobic or hydrophilic fluorescent materials can be loaded, and a targeting moiety is coupled to the exterior of the complex. Therefore, multimodal analysis using cancer cell-specific ultrasound or fluorescence contrast is performed. Through imaging analysis, cancer can be accurately diagnosed and at the same time, one or more therapeutic agents can be stably supported and delivered to target cells specifically until they approach the target cancer cells, thereby effectively treating the cancer. Can be widely used have.
  • Test Example 5 Application of the microbubble-nanoliposomal complex as a gene transfer material
  • Green fluorescent protein plasmid DNA (p GFP, 10 kbp) was placed inside the hydrophilic region of the nanoliposome (Lipo).
  • p GFP Green fluorescent protein plasmid DNA
  • PA protamine
  • pGFP was combined with PA at different concentration ratios to optimize the PA-pGFP complex ratio to 20 ⁇ M PA and 34 nM pGFP representing 600 PAs per pGFP.
  • the weakly positive complex migrated to negatively charged Lipo and showed a loading capacity of 95% or more (see FIG. 13).
  • the microbubble-nanoliposomal complex is also expressed as a pop-particle.
  • Figure 13 measures the optimization and loading capacity of the PA and pGFP complex.
  • the complex of protamine (PA) and plasmid-GFP (pGFP) was optimized at molar ratios and the electrophoretic test could specify the exact values of PA and pGFP. As a result, 600 PAs of 7.5 kD are linked to one 10 kbp pGFP.
  • the PA-pGFP complex can be delivered into the nanoliposomes of the microbubble-nanoliposomal complex because the complex has a sustained positive charge.
  • Nanoliposomes containing pGFP were presented in the microbubble-nanoliposomal complex system of the present invention in the same ligation process.
  • MBB-Lipo (R + pGFP) -Her2 Ab which contains red fluorescence and pGFP and attached Her2 receptor antibody to the surface, for 1 hour to SKBR3, a breast cancer cell line High ultrasonic energy (Mechanical index: 0.61) was exposed for 1 minute (called 'flash'). The cells were then incubated for 48 hours.
  • Figure 14 compares the pGFP transfer effect of the microbubble-nanoliposomal complex system of the present invention. Plasmid gene transfer effect of the microbubble-nanoliposomal complex was compared with Lipofectamine TM , a representative transfection material that is commercially available. Green fluorescent protein expressed in SKBR3 cells was observed as green dots in single cells observed with low magnification confocal laser fluorescence microscopy (CLSM). It was characterized by quantitative analysis using flow cytometry (FACS) studies. The microbubble-nanoliposomal complex system has about three times better plasmid gene transfer compared to Lipofetamine TM . When observed at high magnification, most cancer cells contained green protein in the cell substrate (b right-hand corner). In addition, the microbubble-nanoliposomal complex system has a pGFP transition to primary cells (CFt) extracted from cardiac fibroblasts, which has been shown to have a high effect of about 60% by CLSM and FACS analysis.
  • microbubbles than that representatively cell experiments transformants of Lipofectamine TM (a) injecting material is used in a control nano-liposome complex is highly superior transfer efficiency than Lipofectamine TM showed bright green fluorescence in the cytosol of most cells (3 times).
  • microbubble-nanoliposomal complexes with CD29 antibodies specific for cardiac fibroblast primary cell lines (CFf) were synthesized and used for pGFP transfer. This is known as a gene that is difficult to transfer to the cell line but showed good efficiency. From these results, the microbubble-nanoliposomal complex was easy to target gene delivery for any cell based on targeting and ultrasound energy stimulation experiments (Test Examples 3, 4, 5).
  • Test Example 6 Therapeutic Application of Microbubble-Nanoliposomal Complex as Drug or siRNA Carrier
  • Doxorubicin is a drug used to treat cancer and works by interfering with DNA in the nucleus. Dox can be easily incorporated into the hydrophilic core of a nano liposome (Lipo), according to the ammonium sulfate gradient method (Haran, G., et al., Biochim. Biophys. Acta 1151, 201-215 (1993)).
  • Figure 15 monitors the loading capacity of doxorubicin in pop-particles and the properties in the cells.
  • Dox was inserted into nanoliposomes of pop-particles (microbubble-nanoliposomal complexes) via an ammonium sulfate gradient method, and the delivery effect was quantitatively calculated through an ultraviolet-visible absorption test.
  • the loading efficiency of Dox was confirmed to be 90% or more using UV-Vis spectroscopy (FIG. 15A).
  • MB-Lipo (Dox) -Her2 Ab with Herb receptor antibody attached and microbubble-nanoliposomes (DPPC 2.1 mM and Dox 19 ⁇ M) containing Dox has advanced breast cancer cell therapy.
  • the loaded Dox can simply be monitored for translocations exhibiting self-red fluorescence in CLSM measurements (FIG. 15B). It appeared on the cell membrane at the time of targeting and disappeared in the cytoplasm after flash application of exposure to ultrasonic energy such as the above conditions. As the culture continued for 24 hours, Dox penetrated the cell nucleus.
  • FIG. 16 shows cell viability test results for therapeutic microbubble-nanoliposomal complexes such as drug and therapeutic gene delivery.
  • the microbubble-nanoliposomal complex containing Dox proceeded to apoptosis of cancer cells (survival rate of 40% or less), but when only ultrasound was exposed without the microbubble-nanoliposomal complex or the cells treated with Dox alone were cells. There was no significant decrease in survival.
  • analysis of cells treated with microbubble-nanoliposomal complexes containing Dox induced higher cell death (survival rate ⁇ 35%) than 72-hour treatment of free Dox (survival rate ⁇ 90%). (A in FIG. 16).
  • FIG. 16 shows cell viability test results for therapeutic microbubble-nanoliposomal complexes such as drug and therapeutic gene delivery.
  • STAT3 Signal transducer and activator of transcription 3
  • siRNAs for STAT3 have been reported to have pathways associated with death receptors DR4 and DR5 (Kang, Y. et al. Biochim. Biophys. Acta 1830, 2638-2648 (2013)).
  • siRNA for STAT3 siSTST3 was simply inserted into liposomes after PA complex formation, showing a similar loading capacity of about 95%.
  • the target genes and proteins were measured for expression levels using PCR and Western blotting (see FIGS. 17 a and b).
  • FIG. 17 measures the expression levels of STAT3 proteins and genes in cancer cells after microbubble-nanoliposomal complex (siSTAT3) including siSTAT3 treatment and apoptosis test.
  • siSTAT3 microbubble-nanoliposomal complex
  • MB-Lipo siSTAT3
  • FIG. 17 measures the expression levels of STAT3 proteins and genes in cancer cells after microbubble-nanoliposomal complex (siSTAT3) including siSTAT3 treatment and apoptosis test.
  • target protein expression levels of cells treated with microbubble-nanoliposomal complex (siSTAT3) containing siSTAT3 were significantly reduced in their target genes and protein intensity.
  • As a control cells exposed only to ultrasound or cells treated with only siSTAT3 showed no change in STAT3 gene expression.
  • the microbubble-nanoliposomal complex (siSTAT3) and MB-Lipo (siSTAT3) -Her2 Ab containing siSTAT3 were exposed to ultrasound only as a control or compared to naked siSTAT3 treated cells. Inhibition levels of pear target STAT3 genes and proteins were shown.
  • cells treated with microbubble-nanoliposomal complex (siSTAT3) containing siSTAT3 and exposed to ultrasound had a cell viability of ⁇ 30% at 120 hours.
  • the present inventors treated cancer cells with the microbubble-nanoliposomal complex containing Dox and siSTAT3 simultaneously.
  • the cell survival rate was ⁇ 20% at 72 hours and below 10% at 120 hours. Observation of the cells during the treatment under a microscope revealed virtually no cells to survive at 120 hours (FIG. 16 d).
  • apoptotic cell morphology was confirmed during treatment of microbubble-nanoliposomal complex (Dox + siSTAT3) including Dox and siSTAT3.
  • FIG. 18 shows the characteristics of VX2 cancer transplanted in rabbit kidneys.
  • FIG. 18A to confirm Her2 receptor expression of VX2 tumors in the kidney, hematoxylin and eosin (embedded tumor (positive) and rabbit muscle tissue (negative control) after histological cleavage H & E) staining was performed.
  • FIG. 18B dark brown reflecting Her2 expression appeared in the tumor area.
  • Her2 receptor expression levels were analyzed by Western blot. Transplanted VX2 cancers showed relatively high Her2 expression levels.
  • VX2 cancer expressed specific Her2 receptors and their levels were determined using hematoxylin and eosin (H & E) staining and western blotting of tissues.
  • H & E hematoxylin and eosin
  • the microbubble-nanoliposomal complex, MB-Lipo (R) -Her2 Ab which contained red fluorescent substance and bound to Her2 antibody, was able to recognize VX2 cancer tissue in vitro.
  • microbubble-nanoliposomal complex which has the dual ability of ultrasound (US) imaging and therapeutic effect, directly implanted tumors into the kidney via trans-catheter intra-articular (IA) injection in rabbits.
  • US ultrasound
  • IA intra-articular
  • FIG. 19 after IA injection of a microbubble-nanoliposomal complex including herx antibody and Dox and siSTAT3, tumors in rabbit kidney were examined by ultrasound imaging effect of microbubble in the microbubble-nanoliposomal complex.
  • the image of the echo was bright and imaged brightly and after 10 ultrasonic high energy exposures (Mechanical index: 0.61) the echo gradually faded.
  • the ultrasonic signal indicating the strong targeting ability of the antibody-bound pop-particle in vivo did not show high intensity (data not shown). .
  • Microbubble-nanoliposomal complexes containing an anticancer agent (Dox) and a therapeutic gene (siSTAT3) were injected into rabbits and tumor tissues were extracted after ultrasound high energy exposure to VX2 tumors grown in rabbits.
  • the extracted tumor tissues were confirmed by CLSM (Confocal Laser Scanning Microscope) and PCR to deliver the anticancer agent and therapeutic genes to the tumor tissues.
  • CLSM Confocal Laser Scanning Microscope
  • the Dox transferred to the VX2 cancer tissues clearly showed red fluorescence by themselves, and the target STAT3 gene level was suppressed by the delivered siSTAT3.
  • Tissues that were not exposed to ultrasound high energy did not detect any effects of Dox and siSTAT3, and controls that were not treated with microbubble-nanoliposomal complexes with her2 antibody bound and containing Dox and siSTAT3 also showed no effects of Dox and siSTAT3.
  • the target-directed therapeutic agent-containing microbubble-nanoliposomal complex of the present invention can be delivered only to specific cancer sites so that the release of the therapeutic agent can be controlled by external ultrasonic high energy stimulation.
  • H & E staining cancer cell density of cancer tissues treated with microbubble-nanoliposomal complexes containing Dox and siSTAT3 combined with her2 antibody compared to the control group was sparsely distributed compared to the control group. This is because the complex was partially destroyed by delivery of the carrier to tumor cells.
  • the therapeutic effect was confirmed in vivo in an environment similar to the actual clinic.
  • FIG. 20 evaluates the treatment of cancer in rabbit tumors using a target-directed therapeutic agent containing microbubble-nanoliposomal complex.
  • a target-directed therapeutic agent containing microbubble-nanoliposomal complex To monitor the progress of tumor growth inhibition by MB2Lipo (Dox + siSTAT3) -Her2 Ab , the herb antibody bound in vivo and microbubble-nanoliposomal complexes including Dox and siSTAT3, magnetic resonance imaging was performed. (MRI, cross section) and ultrasound imaging (a: white arrows indicate transplanted cancer tumors).
  • microbubble-nanoliposomal complexes bound to her2 antibody and containing Dox and siSTAT3 were repeatedly administered on days 1 and 5 of the start of the experiment, and tumors were obtained by MRI and ultrasound on days 0, 4, and 10 of the experiment. The exact size of was measured.
  • the her2 antibody was bound to a microbubble-nanoliposomal complex containing Dox and siSTAT3, and the mass treated with high energy of the ultrasonic mass was not treated with ultra high energy or a microbubble-nanoliposomal complex. The growth rate was reduced compared to the mass not administered.
  • microbubble-nanoliposomal conjugates with her2 antibody in rabbit animal tumor models was confirmed in liver, lung, kidney and cancer.
  • Figure 21 shows the in vivo distribution of the micro bubble-nanoliposomal complex containing fluorescence in raw vegetables.
  • green and red fluorescent micro bubble-nanoliposomal complexes MB (G) -Lipo (R) -Her2 Ab , were injected intravenously and the organic dye intensity was characterized by CLSM analysis outside the living tissue.
  • Organic dyes were found to be mainly distributed in liver and lung organs regardless of flash application. However, in tumor tissues, the dye increased in intensity by about three times after flash.
  • FIG. 22 measures the release of micro bubble-nanoliposomal complexes in vivo.
  • FIG. In order to test imaging agents or therapeutic applications using the micro bubble-nanoliposomal complex system, an exclusion study is required. Here, we used a mouse animal model because it is a faster metabolic biological model than a rabbit. After injecting the microbubble-nanoliposomal complex containing the fluorescent material into the tail vein, fluorescence was measured in various organs of the mouse at 48 hours. As a result, the fluorescent substance-containing micro bubble-nanoliposomal complex completely exited the mouse body without any abnormal tissue morphology. That is, 48 hours after injection of the fluorescent substance-containing micro bubble-nanoliposomal complex, clear release of the fluorescent substance-containing micro bubble-nanoliposomal complex was confirmed in various organs of the mouse.
  • microbubble-nanoliposomal complex which enables cancer cell-specific diagnosis and treatment according to the present invention is hydrophobic microbubbles and hydrophilic nanoliposomes are chemically stable covalently bonded to the therapeutic genes and drugs in the complex structure, etc. It is possible to stably support one or more additional hydrophobic or hydrophilic fluorescent materials as well as one or more therapeutic agents of the externally targeting targeting moiety (targeting moiety) coupled to exhibit excellent target specificity. Therefore, it is possible to diagnose cancer through multiple image analysis and at the same time, it can be usefully used for effective diagnosis and treatment of various cancer diseases.

Abstract

The present invention relates to a microbubble-nanoliposome complex allowing cancer-cell-specific diagnosis and treatment. More specifically the microbubble-nanoliposome complex, in which a hydrophobic microbubble and a hydrophilic nanoliposome are bonded by means of chemically stable covalent bonding, is able to carry not only one or more therapeutic agent but also one or more additional hydrophobic or hydrophilic fluorescent substance on the inside of the composite structure while comprising a targeting moiety on the outside thereof so as to exhibit outstanding target specificity, and thus said complex allows cancer to be diagnosed by means of multiple image analysis using ultrasound or fluorescence imaging specific to cancer cells and at the same time allows cancer to be treated as one or more therapeutic agent is effectively delivered to target cancer cells, and thus the invention can be used to advantage in the development of effective diagnostic and therapeutic agents for cancers such as breast cancer, liver cancer, pancreatic cancer, brain cancer, stomach cancer, lung cancer, oesophageal cancer, colon cancer, prostate cancer, kidney cancer and ovarian cancer.

Description

암의 진단 및 치료를 위한 마이크로버블-나노리포좀 복합체Microbubble-nanoliposomal complexes for the diagnosis and treatment of cancer
본 발명은 암 세포 특이적인 진단 및 치료를 가능하게 하는 마이크로버블-나노리포좀 복합체 및 그 제조방법에 관한 것으로, 구체적으로 소수성인 마이크로버블과 친수성인 나노리포좀이 화학적으로 안정적인 공유결합으로 결합되어 있는 마이크로버블-나노리포좀 복합체는 복합체 구조 내부에 하나 이상의 치료제 뿐 아니라 추가의 소수성 또는 친수성 형광물질을 하나 이상 담지할 수 있으면서 외부에 타켓팅 모이어티(targetting moiety)를 포함하여 우수한 표적 특이성을 나타내므로, 암세포에 특이적인 초음파 또는 형광 조영을 이용한 다중 영상 분석을 통해 암을 진단함과 동시에 하나 이상의 치료제를 표적 암세포에 효과적으로 전달하여 암을 치료할 수 있으므로, 유방암, 간암, 췌장암, 뇌암, 위암, 폐암, 식도암, 대장암, 전립선암, 신장암, 난소암 등의 암 질환의 효과적인 진단 및 치료제 개발에 유용하게 활용될 수 있다.The present invention relates to a microbubble-nanoliposomal complex and a method for preparing the same, which enables cancer cell-specific diagnosis and treatment, and specifically, a microbubble in which hydrophobic microbubbles and hydrophilic nanoliposomes are chemically stable covalently bound. Bubble-nanoliposomal complexes can carry one or more additional hydrophobic or hydrophilic phosphors, as well as one or more therapeutic agents, within the complex structure and exhibit excellent target specificity, including targeting moieties to the outside. Multiple imaging analysis using specific ultrasound or fluorescence contrast can diagnose cancer and effectively deliver one or more therapeutic agents to the target cancer cells to treat the cancer, so breast cancer, liver cancer, pancreatic cancer, brain cancer, gastric cancer, lung cancer, esophageal cancer and large intestine Cancer, Prostate Cancer, Kidney Cancer, Ovarian Cancer May be useful to take advantage of the development of effective diagnostic and treatment of cancer.
치료용 조영 물질(theragnostic agent)은 병의 진단과 치료를 동시에 가능하게 하는 물질들을 일컫는다. 이들은 보통 작은 크기의 물질로 이뤄져 있으며, 대개 형광 염료, 방사성 분자 등이 리포좀, 고분자 및 나노 입자 등의 내부에 담지되고 약물이나 진단용 마커가 외부에 도입된 형태를 갖는다. 최근에는 생체 적합성(biocompatibility)이 우수한 지질구조체(lipid structure)로 이루어진 치료용 조영 물질의 합성 연구가 주를 이루고 있으며, 특히 지질구조체로 이뤄진 마이크로미터 크기의 공기방울 초음파 조영제인 마이크로버블(Microbubble ultrasound agent, MB)을 이용한 생체 영상 분석 연구가 활발히 진행되고 있다[David C., Eur J Radiol (2006), 60(3), 324-330]. 이러한 연구들은 최근 '나노-바이오 분야(nano-bio field)'로 불리고 있는 나노물질의 생물관련 분야로의 응용을 위해 비독성, 고-수용성 또는 용이한 표면 변형 등의 특성을 부과하는데 주로 초점이 맞춰져 있다.Therapeutic contrast agents (theragnostic agents) refers to substances that enable the simultaneous diagnosis and treatment of the disease. They usually consist of small sized materials, usually fluorescent dyes, radioactive molecules, etc. are carried on the inside of liposomes, polymers and nanoparticles, and drugs or diagnostic markers are introduced outside. Recently, research has been focused on the synthesis of therapeutic contrast materials consisting of lipid structures with excellent biocompatibility. In particular, microbubble ultrasound agent, a micrometer-sized bubble ultrasound contrast medium composed of lipid structures , MB) has been actively studied in vivo image analysis (David C., Eur J Radiol (2006), 60 (3), 324-330). These studies focus primarily on imposing properties such as nontoxicity, high water solubility, or easy surface modification for the application of nanomaterials in the biological field, which is now called the 'nano-bio field'. It is aligned.
지금까지 공지된 지질구조체를 기반으로 하는 마이크로버블들을 살펴보면, 우수한 생체적합성을 통해 여러 생체 내(in vivo) 또는 시험관 내(in vitro) 연구에서 혈관 또는 암조직의 영상 분석을 통한 진단 뿐 아니라 치료용 유전자나 약물을 전달하여 암을 치료할 수 있는 것으로 보고되고 있다[Katherine F. 등, Annual Review of Biomedical Engineering (2007), 9, 415-447]. 그러나 마이크로버블을 통한 초음파 조영 분석은 낮은 해상도와 표적 비특이성(nonspecific property)으로 인해 암의 정확한 진단에는 역부족이며, 따라서 초음파 조영 뿐 아니라 형광 조영을 동시에 사용하여 초음파 분석을 통한 생체 조직의 형태 확인과 동시에 형광 영상 분석을 통한 세부적인 분포도를 확인할 수 있도록 하는 형광 및 초음파를 통한 다중 영상 조영 물질을 개발하기 위한 연구들이 이루어지고 있다[Bart G. 등, Journal of Controlled Release (2011), 152, 249-256].Looking at microbubbles based on known lipid structures, the excellent biocompatibility allows for treatment as well as diagnosis through imaging analysis of blood vessels or cancerous tissues in various in vivo or in vitro studies. It has been reported that cancer can be treated by delivering genes or drugs (Katherine F. et al., Annual Review of Biomedical Engineering (2007), 9, 415-447). However, microbubble ultrasonography analysis is inadequate for accurate diagnosis of cancer due to its low resolution and target nonspecific properties. At the same time, studies have been made to develop a multi-image contrast material using fluorescence and ultrasound to confirm detailed distribution through fluorescence image analysis [Bart G. et al., Journal of Controlled Release (2011), 152, 249-. 256].
그러나 공지된 물질들 대부분이 여전히 표적 비특이적인 조영 효과에 그치고 있어 실질적으로 혈관, 림프관 등의 선형적인 조직 진단에만 활용가능하거나, 병변 세포에만 표적 특이적으로 치료 유전자나 약물을 전달하여 치료하는데 한계를 나타내고 있어 치료용 물질을 담지하기 위해 또 다른 구조체와의 혼성체를 형성시켜야 하는 구조적 어려움이 있어왔다[Wang C. 등, Biomaterials (2012), 33, 1939-1947].However, most of the known substances are still only targeted non-specific contrast effects, which can be used only for the diagnosis of linear tissues such as blood vessels and lymphatic vessels, or to limit the treatment of delivering specific therapeutic genes or drugs to lesion cells. There have been structural difficulties in forming hybrids with other structures to support therapeutic materials (Wang C. et al., Biomaterials (2012), 33, 1939-1947).
이에 본 발명자들은 암세포 특이적인 다중 영상 분석을 통한 암 진단과 동시에 하나 이상의 치료제를 표적 특이적으로 전달하여 암 치료를 가능하게 할 수 있는 치료용 조영 물질을 개발하기 위해 연구한 결과, 소수성인 마이크로버블과 친수성인 나노리포좀이 공유결합으로 결합되어 있는 복합체로서, 상기 복합체 구조 내부에 하나 이상의 치료제 뿐 아니라 추가의 소수성 또는 친수성 형광물질을 하나 이상 담지할 수 있으면서 외부에 타겟팅 모이어티(targetting moiety)를 포함하고 있는 마이크로버블-나노리포좀 복합체가 암세포 특이적으로 초음파 또는 형광 조영을 이용한 다중 영상 분석을 통해 암을 진단함과 동시에 치료용 유전자 및 약물 등의 하나 이상의 치료제를 표적 세포에 안정적으로 전달하여 암을 치료할 수 있음을 발견하여 본 발명을 완성하였다.Accordingly, the present inventors have studied to develop a therapeutic contrast agent capable of treating cancer by simultaneously delivering one or more therapeutic agents to cancer diagnosis through cancer cell-specific multi-image analysis, resulting in hydrophobic microbubbles. A complex in which a hydrophilic nanoliposome and a covalent bond are bonded to each other, and include a targeting moiety externally while carrying at least one additional hydrophobic or hydrophilic fluorescent substance as well as one or more therapeutic agents in the complex structure. The microbubble-nanoliposomal complex is used to diagnose cancer through multiple image analysis using ultrasound or fluorescence contrast, and at the same time, stably deliver one or more therapeutic agents, such as therapeutic genes and drugs, to target cells. The present invention has been found to be curable. It was.
따라서 본 발명의 목적은 암세포 특이적인 다중 영상 분석을 통한 암 진단과 동시에 하나 이상의 치료제를 표적 세포에 특이적으로 전달하여 암 치료를 가능하게 할 수 있는 치료용 조영 물질(theragnostic agent)를 제공하는 것이다.Accordingly, an object of the present invention is to provide a therapeutic contrast agent capable of treating cancer by specifically delivering one or more therapeutic agents to a target cell at the same time as diagnosing cancer through cancer cell-specific multi-image analysis. .
또한 본 발명의 다른 목적은 상기 치료용 조영 물질의 제조방법을 제공하는 것이다.It is another object of the present invention to provide a method for preparing the therapeutic contrast material.
상기 목적을 달성하기 위하여, 본 발명은 소수성인 마이크로버블과 친수성인 나노리포좀이 공유결합으로 결합되어 있는 복합체로서, 상기 복합체 구조 내부에 하나 이상의 치료제를 담지하면서 외부에 타겟팅 모이어티(targetting moiety)를 포함하고 있는 마이크로버블-나노리포좀 복합체를 제공한다.In order to achieve the above object, the present invention is a complex in which hydrophobic microbubbles and hydrophilic nanoliposomes are covalently bonded to each other, carrying a targeting moiety to the outside while carrying at least one therapeutic agent inside the complex structure. It provides a microbubble-nanoliposomal complex containing.
본 발명의 일실시예에 따르면, 상기 치료제는 치료유전자 및 약물 중에서 선택된 것일 수 있다.According to one embodiment of the invention, the therapeutic agent may be selected from a therapeutic gene and a drug.
본 발명의 일실시예에 따르면, 상기 복합체 구조 내부에 소수성 또는 친수성 형광물질을 하나 이상 추가로 담지할 수 있다.According to an embodiment of the present invention, one or more hydrophobic or hydrophilic fluorescent materials may be additionally supported in the complex structure.
상기 다른 목적을 달성하기 위하여, 본 발명은 In order to achieve the above another object, the present invention
1) 필름형성물질, 인지질, 음전하 화합물, 아민기를 갖는 화합물 및 다이설파이드기를 갖는 화합물을 유기용매에 넣고 반응시켜 필름을 제조한 후 이를 소수성 가스와 함께 유기 혼합용매에 넣고 진동반응(vibrating)시켜 마이크로버블을 제조하는 단계;1) A film-forming substance, a phospholipid, a negatively charged compound, a compound having an amine group, and a compound having a disulfide group are added to an organic solvent and reacted to prepare a film, which is then mixed with an hydrophobic gas into an organic mixed solvent and vibrated. Preparing a bubble;
2) 필름형성물질, 인지질, 음전하 화합물 및 아민기를 갖는 화합물을 유기용매에 넣고 반응시켜 필름을 제조한 후 이를 물에 넣고 초음파분산 및 여과하여 나노리포좀을 제조하는 단계;2) preparing a film by adding a film-forming substance, a phospholipid, a negatively charged compound, and a compound having an amine group into an organic solvent to prepare a film, and then putting the same into water and dispersing ultrasonically and filtering to prepare nanoliposomes;
3) 상기 단계 2)에서 얻어진 나노리포좀을 하나 이상의 치료제와 반응시켜 하나 이상의 치료제를 나노리포좀 구조 내에 담지시키는 단계;3) reacting the nanoliposomes obtained in step 2) with at least one therapeutic agent to support the at least one therapeutic agent in the nanoliposomal structure;
4) 상기 단계 1)에서 얻어진 마이크로버블과 상기 단계 3)에서 얻어진 나노리포좀을 혼합하고 진탕(shaking)시켜 마이크로버블-나노리포좀 복합체를 제조하는 단계; 및4) preparing a microbubble-nanoliposomal complex by mixing and shaking the microbubbles obtained in step 1) and the nanoliposomes obtained in step 3); And
5) 상기 단계 4)에서 제조된 복합체를 타겟팅 모이어티(targetting moiety)와 반응시켜 타겟팅 모이어티를 복합체 외부에 결합시키는 단계를 포함하는, 상기 본 발명의 마이크로버블-나노리포좀 복합체를 제조하는 방법을 제공한다.5) reacting the complex prepared in step 4) with a targeting moiety (targeting moiety) to bind the targeting moiety to the outside of the complex, the method of producing a microbubble-nanoliposomal complex of the present invention to provide.
본 발명의 일실시예에 따르면, 상기 단계 1) 또는 2)는 마이크로버블 또는 나노리포좀 제조 후 이를 형광물질과 반응시켜 형광물질을 상기 마이크로버블 또는 나노리포좀 구조 내에 담지시키는 공정을 추가로 포함할 수 있다.According to an embodiment of the present invention, the step 1) or 2) may further include a step of supporting the fluorescent material in the microbubble or nanoliposomal structure by reacting it with a fluorescent material after preparing the microbubble or nanoliposomes. have.
본 발명에 따른 암 세포 특이적인 진단 및 치료를 가능하게 하는 마이크로버블-나노리포좀 복합체는 소수성인 마이크로버블과 친수성인 나노리포좀이 화학적으로 안정적인 공유결합으로 결합되어 있어 복합체 구조 내부에 치료 유전자 및 약물 등의 하나 이상의 치료제 뿐 아니라 추가의 소수성 또는 친수성의 형광물질을 하나 이상 안정적으로 담지할 수 있으면서 외부에 타켓팅 모이어티(targetting moiety)가 결합되어 있어 우수한 표적 특이성을 나타내므로, 암세포에 특이적인 초음파 또는 형광 조영을 이용한 다중 영상 분석을 통해 암을 진단할 수 있음과 동시에 하나 이상의 치료제를 표적 암세포에 효과적으로 전달하여 암을 치료할 수 있으므로, 유방암, 간암, 췌장암, 뇌암, 위암, 폐암, 식도암, 대장암, 전립선암, 신장암, 난소암 등의 암 질환의 효과적인 진단 및 치료제 개발에 유용하게 활용될 수 있다.The microbubble-nanoliposomal complex which enables cancer cell-specific diagnosis and treatment according to the present invention is hydrophobic microbubbles and hydrophilic nanoliposomes are chemically stable covalently bonded to the therapeutic genes and drugs in the complex structure, etc. Ultrasonic or fluorescence specific to cancer cells as it can reliably carry one or more additional hydrophobic or hydrophilic fluorescent materials as well as a targeting moiety externally and exhibits excellent target specificity Cancer can be diagnosed through multiple imaging analysis using contrast, and at the same time, one or more therapeutic agents can be effectively delivered to the target cancer cells to treat the cancer, so breast cancer, liver cancer, pancreatic cancer, brain cancer, gastric cancer, lung cancer, esophageal cancer, colon cancer, and prostate Efficacy of cancer diseases such as cancer, kidney cancer and ovarian cancer Diagnosis and can be usefully utilized in drug development.
도 1은 본 발명의 일실시예에 따른 마이크로버블-나노리포좀 복합체의 구조 모식도이다.1 is a schematic diagram of a microbubble-nanoliposomal complex according to an embodiment of the present invention.
도 2는 본 발명의 일실시예에 따라 제조된 마이크로버블(a) 및 나노리포좀(b)을 대상으로 각각 광학현미경(optical microscope) 및 극저온 전자 현미경(cryogenic electron microscopy, Cryo-EM)으로 확인한 결과 사진이다.FIG. 2 shows the results of microbubbles (a) and nanoliposomes (b) prepared according to one embodiment of the present invention, respectively, by optical microscope and cryogenic electron microscopy (Cryo-EM). It is a photograph.
도 3은 본 발명의 일실시예에 따라 제조된 마이크로버블-나노리포좀 복합체를 공초점 형광 현미경(confocal fluorescence microscope)을 사용하여 저배율 및 고배율(상단 우측 삽입도면)로 확인한 결과 사진이다. FIG. 3 is a photograph of the microbubble-nanoliposomal complex prepared according to one embodiment of the present invention using a confocal fluorescence microscope (confocal fluorescence microscope) at low and high magnification (top right insertion drawing).
도 4는 본 발명의 일실시예에 따라 제조된 마이크로버블-나노리포좀 복합체를 대상으로 자외-가시선 분광분석(UV-vis spectroscopy)을 수행하여 공유결합시 발생되는 부산물인 SPDP(N-succinimidyl-3-[2-pyridyldithio]-propionate)의 존재 여부를 확인한 결과이다.Figure 4 is a by-product SPDP (N-succinimidyl-3) generated by covalent bonding by performing ultraviolet-vis spectroscopy (UV-vis spectroscopy) on the microbubble-nanoliposomal complex prepared according to an embodiment of the present invention -[2-pyridyldithio] -propionate).
도 5는 시판중인 마이크로버블 초음파 조영제인 SonoVue(TM)(a), 본 발명의 일실시예에 따라 제조된 마이크로버블(b) 및 마이크로버블-나노리포좀 복합체(c)를 사용하여 각각 초음파 영상 장치를 통한 인공혈관 팬텀에서의 초음파 조영 효과를 비교 분석한 결과 사진이다.5 is an ultrasonic imaging apparatus using a commercially available microbubble ultrasound contrast agent SonoVue (TM) (a), a microbubble (b) and a microbubble-nanoliposomal complex (c) prepared according to an embodiment of the present invention. The result of the comparative analysis of the ultrasound contrast effect on the artificial vascular phantom through
도 6은 본 발명의 일실시예에 따라 제조된 마이크로버블-나노리포좀 복합체를 사용하여 1회에서 10회까지의 음파 자극을 가하였을 때 인공혈관 팬텀에서의 초음파 조영 효과의 변화를 확인한 결과 사진(1회~10회) 및 그래프이다.6 is a photograph showing the results of confirming the change in the ultrasound contrast effect in the artificial vascular phantom when a sonic stimulation is applied from 1 to 10 times using the microbubble-nanoliposomal complex prepared according to an embodiment of the present invention. 1 to 10 times) and graphs.
도 7은 본 발명의 일실시예에 따라 제조된 마이크로버블-나노리포좀 복합체를 대상으로 BSA 정량분석을 수행하여 복합체 표면에 포함된 항체의 양을 확인한 결과 그림이다. 7 is a result of confirming the amount of the antibody contained in the surface of the complex by performing the BSA quantitative analysis on the microbubble-nanoliposomal complex prepared according to an embodiment of the present invention.
도 8은 본 발명의 일실시예에 따라 제조된 마이크로버블-나노리포좀 복합체 및 대조군으로써 항체 도입을 생략하여 제조한 마이크로버블(MB)-나노리포좀 복합체를 사용하여 유방암 세포주인 SkBr3 세포를 대상으로 공초점 형광 현미경을 통한 다중 영상 분석(multimodal imaging analysis)(a) 및 FACS 분석(b)을 수행한 결과 사진 및 그림이다.8 is a microbubble-nanoliposomal complex prepared according to one embodiment of the present invention and microbubbles (MB) -nanoliposomal complex prepared by omitting antibody introduction as a control using a target for SkBr3 cells, breast cancer cell line The results of the multimodal imaging analysis (a) and FACS analysis (b) through a focus fluorescence microscope are photographs and pictures.
도 9는 본 발명의 일실시예에 따라 제조된 마이크로버블-나노리포좀 복합체를 유방암 세포주인 SkBr3 세포에 처리하고 외부 음파 자극을 1분 동안 가한 후 공초점 형광 현미경 분석을 수행하여 복합체 내부에 담지되어 있던 형광 물질이 암세포 내부에 전달되었는지를 확인한 결과 사진이다. FIG. 9 is a microbubble-nanoliposomal complex prepared according to one embodiment of the present invention treated with SkBr3 cells, a breast cancer cell line, and subjected to external sonic stimulation for 1 minute, followed by confocal fluorescence microscopy to be carried inside the complex. The result of confirming whether the fluorescent material was delivered inside the cancer cell is a photograph.
도 10은 본 발명의 일실시예에 따라 제조된 마이크로버블-나노리포좀 복합체의 약물 포집 효율을 자외-가시선 분광기(UV-vis spectrometer)를 이용한 분광분석을 통해 확인한 결과 그림이다.10 is a result of confirming the drug collection efficiency of the microbubble-nanoliposomal complex prepared according to an embodiment of the present invention by spectroscopic analysis using an ultraviolet-vis spectrometer (UV-vis spectrometer).
도 11은 아무것도 처리하지 않거나(1) 본 발명의 일실시예에 따라 제조된 마이크로버블-나노리포좀 복합체(2) 또는 독소루비신(3)을 처리한 유방암 세포주 Skbr3를 일정 시간(24시간, 48시간 또는 72시간) 동안 배양한 후 MTT 분석을 수행하여 세포독성을 확인한 결과 그래프이다. FIG. 11 shows that breast cancer cell line Skbr3 treated with (1) microbubble-nanoliposomal complex (2) or doxorubicin (3) prepared according to one embodiment of the present invention is treated for a certain time (24 hours, 48 hours or 72 hours) and then cultured for MTT analysis to confirm the cytotoxicity is a graph.
도 12는 본 발명의 일실시예에 따라 제조된 마이크로버블-나노리포좀 복합체를 유방암 세포주 SkBr3에 처리한 후 외부 음파 자극을 가하거나 가하지 않고 일정 시간(48시간, 72시간 또는 96시간) 동안 배양한 후 공초점 형광 현미경을 사용하여 tGFP의 발현을 확인한 결과 사진이다.12 is treated with a microbubble-nanoliposomal complex prepared according to an embodiment of the present invention to breast cancer cell line SkBr3 and cultured for a predetermined time (48 hours, 72 hours or 96 hours) with or without external acoustic stimulation. The result of confirming the expression of tGFP using a confocal fluorescence microscope is a photograph.
도 13은 마이크로 버블-나노리포좀 복합체의 유전자 전달 실험을 위한 프로타민(PA)과 pGFP 복합체의 최적화 및 로딩용량을 측정한 것이다.FIG. 13 shows the optimization and loading capacity of protamine (PA) and pGFP complexes for gene delivery experiments of micro bubble-nanoliposomal complexes.
도 14는 본 발명의 마이크로 버블-나노리포좀 복합체의 pGFP 전달 효과를 비교한 것이다. Figure 14 compares the pGFP delivery effect of the micro bubble-nanoliposomal complex of the present invention.
도 15는 마이크로 버블-나노리포좀 복합체에서 독소루비신의 로딩용량 및 세포에서의 특성을 모니터한 것이다.Figure 15 monitors the loading capacity of doxorubicin and its properties in cells in the micro bubble-nanoliposomal complex.
도 16은 마이크로 버블-나노리포좀 복합체를 이용한 약물 및 치료 유전자 전달에 의한 세포 생존율 시험 결과이다. Pop-particle은 마이크로 버블-나노리포좀 복합체를 의미한다.Figure 16 shows the results of cell viability test by drug and therapeutic gene delivery using the micro bubble-nanoliposomal complex. Pop-particle means micro bubble-nanoliposomal complex.
도 17은 siSTAT3를 포함하고 있는 마이크로 버블-나노리포좀 복합체의 세포사멸 시험 후 암세포에서 STAT3 단백질 및 유전자에 대한 발현 수준을 측정한 것이다.FIG. 17 shows the expression levels of STAT3 proteins and genes in cancer cells after apoptosis testing of the microbubble-nanoliposomal complex containing siSTAT3.
도 18은 토끼의 신장에서 이식된 VX2 종양의 특성을 나타낸 것이다.18 shows the characteristics of VX2 tumors transplanted in rabbit kidneys.
도 19는 생체 내에서 약물 및 유전자의 암 특이적 초음파 이미지 및 전달을 나타낸 것이다.19 shows cancer specific ultrasound images and delivery of drugs and genes in vivo.
도 20은 토끼 종양 모델에서 마이크로 버블-나노리포좀 복합체를 이용한 암치료 효과에 대해 평가한 것이다.20 is an evaluation of the cancer treatment effect using the micro bubble-nanoliposomal complex in the rabbit tumor model.
도 21은 생채 내에서 형광물질을 포함한 마이크로 버블-나노리포좀 복합체의 생체 내 분포를 나타낸 것이다.Figure 21 shows the distribution in vivo of the micro bubble-nanoliposomal complex containing a fluorescent material in raw vegetables.
도 22는 생체 내에서 형광물질을 포함한 마이크로 버블-나노리포좀 복합체의 방출(excretion)을 측정한 것이다.FIG. 22 measures the excretion of the microbubble-nanoliposomal complex containing fluorescent material in vivo.
이하, 본 발명을 구체적으로 설명한다.Hereinafter, the present invention will be described in detail.
본 발명에 따른 마이크로버블-나노리포좀 복합체는 소수성인 마이크로버블과 친수성인 나노리포좀이 공유결합으로 결합되어 있는 복합체로서, 상기 복합체 구조 내부에 하나 이상의 치료제를 담지하면서 외부에 항체 등의 타겟팅 모이어티(targetting moiety)를 포함하고 있는 것을 특징으로 한다. 또한, 본 발명의 마이크로버블-나노리포좀 복합체는 복합체 구조 내부에 소수성 또는 친수성 형광물질을 하나 이상 추가로 담지할 수 있으며, 이때 소수성 형광물질은 마이크로버블 구조 내부에, 친수성 형광물질은 나노리포좀 구조 내부에 담지될 수 있다.The microbubble-nanoliposomal complex according to the present invention is a complex in which a hydrophobic microbubble and a hydrophilic nanoliposome are covalently bonded, and a targeting moiety such as an antibody to the outside while carrying at least one therapeutic agent in the complex structure ( targetting moiety). In addition, the microbubble-nanoliposomal complex of the present invention may additionally support one or more hydrophobic or hydrophilic fluorescent materials in the complex structure, wherein the hydrophobic fluorescent material is in the microbubble structure, the hydrophilic fluorescent material in the nanoliposome structure Can be supported.
본 발명의 마이크로버블-나노리포좀 복합체는 도 1에 나타낸 바와 같이 평균 직경 1-2 ㎛의 균일한 입자 크기를 갖는 미세 공기방울 형태의 소수성 마이크로버블 구조 외부에 100-200 nm 크기의 친수성 지질 구조체인 나노리포좀이 표면에 결합된 형태를 가지며, 복합체 내부에는 SF6, CO2 또는 CF4 등의 소수성의 기체가 내포되어 있다. 이때, 소수성 마이크로버블과 친수성 나노리포좀은 서로 화학적으로 안정적인 공유결합으로 결합되어 있고, 각각의 구조내에 소수성 또는 친수성의 성질에 따라 하나 이상의 유전자 및 약물 중에서 선택된 치료제 뿐 아니라 추가의 소수성 또는 친수성 형광물질을 하나 이상 담지할 수 있으며 복합체 외부에는 항체 등의 타겟팅 모이어티(targetting moiety)를 포함하므로, 표적 특이적으로 초음파 또는 형광 조영을 통한 다중 영상화(mutimodal imaging)를 구현함과 동시에 하나 이상의 치료제를 생체 내에 안정적으로 전달할 수 있는 특징이 있다. 상기 마이크로버블-나노리포좀 복합체는 본 명세서 또는 도면에서 '팝-파티클(pop-particle)'이라는 용어로도 표현된다.The microbubble-nanoliposomal complex of the present invention is a hydrophilic lipid structure having a size of 100-200 nm outside the hydrophobic microbubble structure in the form of fine air bubbles having a uniform particle size of an average diameter of 1-2 μm as shown in FIG. Nanoliposomes have a form bound to the surface, the hydrophobic gas such as SF 6, CO 2 or CF 4 is contained in the complex. At this time, the hydrophobic microbubbles and the hydrophilic nanoliposomes are chemically stable covalently bonded to each other, and depending on the hydrophobic or hydrophilic properties within each structure, the hydrophobic or hydrophilic fluorescent substance may be added to a therapeutic agent selected from one or more genes and drugs. Since one or more of the complexes may include a targeting moiety such as an antibody outside the complex, one or more therapeutic agents may be in vivo while simultaneously implementing mutimodal imaging through ultrasound or fluorescence imaging. There is a characteristic that can be delivered stably. The microbubble-nanoliposomal complex is also referred to herein as the term 'pop-particle'.
본 발명의 일실시예에 따르면, 본 발명의 복합체는 바람직하게 평균 직경 1-2 ㎛의 균일한 크기를 나타낼 수 있으나 이에 제한되는 것은 아니며, 복합체 제조시 나노리포좀의 사용 농도 및 여과 방법에 따라 100 nm 내지 10 ㎛로 입자크기를 조절할 수 있다. 또한, 이러한 입자크기는 현재 상용화 되어 있는 초음파 기기의 주파수에 맞춰 다양화시킬 수 있는데, 사용되는 초음파 기기의 주파수가 높아지면 복합체의 평균 직경은 작아질 수 있다.According to one embodiment of the invention, the complex of the present invention may preferably exhibit a uniform size of an average diameter of 1-2 ㎛, but is not limited thereto, depending on the concentration of the nanoliposome used in the preparation of the complex and the filtration method 100 Particle size can be adjusted to nm to 10 ㎛. In addition, the particle size can be varied according to the frequency of the ultrasonic device that is currently commercialized, the higher the frequency of the ultrasonic device to be used, the average diameter of the composite may be smaller.
본 발명의 일실시예에 따르면, 본 발명의 복합체에서 상기 마이크로버블은 필름형성물질, 인지질, 음전하 화합물, 아민기를 갖는 화합물 및 다이설파이드기를 갖는 화합물을 포함할 수 있으며, 이때 필름형성물질로는 DPPC (1,2-dipalmitory-sn-glycero-3-phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) 또는 DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) 등이; 인지질로는 콜레스테롤(cholesterol) 등이; 음전하 화합물로는 DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl-sn-glycero-3-phosphate), DMPA (1,2-dimyristoyl-sn-glycero-3-phosphate) 또는 DOPA (1,2-dioleoyl-sn-glycero-3-phosphate) 등이; 아민기를 갖는 화합물로는 DPPE (1,2-dipalmitory-sn-glycero-3-phosphoethanolamine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) 또는 DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) 등이; 그리고 다이설파이드기를 갖는 화합물로는 DSPE-PEG-SPDP {1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000]} 등이 사용될 수 있다.According to an embodiment of the present invention, the microbubbles in the composite of the present invention may include a film forming material, a phospholipid, a negatively charged compound, a compound having an amine group and a compound having a disulfide group, wherein the film forming material is DPPC (1,2-dipalmitory-sn-glycero-3-phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) , DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) or DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine); Phospholipids include cholesterol (cholesterol); Negative charge compounds include DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl-sn-glycero-3-phosphate), DMPA (1,2- dimyristoyl-sn-glycero-3-phosphate) or DOPA (1,2-dioleoyl-sn-glycero-3-phosphate); Compounds having an amine group include DPPE (1,2-dipalmitory-sn-glycero-3-phosphoethanolamine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), and DLPE (1,2-dilauroyl-sn- glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine); And a compound having a disulfide group may be used DSPE-PEG-SPDP {1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [polyethylene glycol (2000)}}.
또한, 본 발명의 일실시예에 따르면, 본 발명의 복합체에서 상기 나노리포좀은 필름형성물질, 인지질, 음전하 화합물 및 아민기를 갖는 화합물을 포함할 수 있으며, 이때 필름형성물질로는 DPPC (1,2-dipalmitory-sn-glycero-3-phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) 또는 DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) 등이; 인지질로는 콜레스테롤(cholesterol) 등이; 음전하 화합물로는 DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl-sn-glycero-3-phosphate), DMPA (1,2-dimyristoyl-sn-glycero-3-phosphate) 또는 DOPA (1,2-dioleoyl-sn-glycero-3-phosphate) 등이; 그리고 아민기를 갖는 화합물로는 DPPE (1,2-dipalmitory-sn-glycero-3-phosphoethanolamine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) 또는 DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) 등이 사용될 수 있다.In addition, according to an embodiment of the present invention, the nanoliposomes in the composite of the present invention may include a film-forming material, a phospholipid, a negatively charged compound and a compound having an amine group, wherein the film-forming material is DPPC (1, 2 -dipalmitory-sn-glycero-3-phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DLOPC (1, 2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DOPC (1 , 2-dioleoyl-sn-glycero-3-phosphocholine) or DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine); Phospholipids include cholesterol (cholesterol); Negative charge compounds include DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl-sn-glycero-3-phosphate), DMPA (1,2- dimyristoyl-sn-glycero-3-phosphate) or DOPA (1,2-dioleoyl-sn-glycero-3-phosphate); Compounds having an amine group include DPPE (1,2-dipalmitory-sn-glycero-3-phosphoethanolamine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), and DLPE (1,2-dilauroyl-sn -glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) may be used.
본 발명의 일실시예에 따르면, 본 발명의 복합체는 pH, 반응온도 또는 반응시간 등의 조절을 통해 마이크로버블의 지질구조체 또는 나노리포좀의 지질구조체의 일부분을 변형하여 진탕반응시킴으로써 간단하게 공유결합을 유도하여 형성될 수 있다.According to one embodiment of the present invention, the complex of the present invention is simply covalently bonded by modifying and shaking part of the lipid structure of the microbubble or the lipid structure of the nanoliposome through the control of pH, reaction temperature or reaction time, etc. It can be formed by induction.
본 발명의 일실시예에 따르면, 본 발명의 복합체 구조 내부에 담지되는 하나 이상의 치료제는 치료용 유전자 및 약물 중에서 선택된 것이라면 어느 것이나 사용될 수 있으며, 예를 들면 암 치료에 사용될 수 있는 DNA, RNA 등의 유전자 및 항암제 등의 약물 등을 포함하는 군에서 선택된 것을 사용될 수 있다. 구체적으로, 치료용 유전자를 사용하는 경우에는 예를 들면 항암 효과가 공지된 유전자를 발현시키거나 암 발생에 관련된 유전자의 발현을 저해할 수 있는 발현 플라스미드, siRNA, shRNA, 마이크로 RNA 또는 마이크로 RNA의 길항제 등의 DNA 또는 RNA가 사용될 수 있으며, 약물을 사용하는 경우에는 예를 들면 항암 효과가 알려진 독소루비신(doxorubicin), 파클리탁셀(paclitaxel) 또는 도세탁셀(docetaxel) 등이 사용될 수 있으나 이에 제한되는 것은 아니다. 이러한 유전자 및 약물은 통상적인 방법에 따라 나노리포좀과 반응시켜 복합체 구조 내에 담지시킬 수 있는데, 유전자의 경우 프로타민(protamine) 등의 생체고분자와의 혼성체 형태로 나노리포좀과 반응시켜 담지시킬 수 있으며, 약물의 경우 통상적인 황산암모늄 농도구배법[ammonium sulfate gradient method, Bowen T. 등, International Journal of Pharmaceutics (2011), 416, 443-447]을 통해 리포좀 내부에 담지시킬 수 있다. 본 발명의 일실시예에 따르면, 본 발명의 복합체의 독소루비신(doxorubicin) 약물 포집 효율은 88.57%이며, tGFP 발현 플라스미드 유전자 포집 효율은 30%를 나타내었다.According to one embodiment of the present invention, any one or more therapeutic agents carried in the complex structure of the present invention may be used as long as the selected one among therapeutic genes and drugs, for example, DNA, RNA, etc., which may be used for cancer treatment. Those selected from the group containing drugs such as genes and anticancer agents can be used. Specifically, when using therapeutic genes, for example, antagonists of expression plasmids, siRNAs, shRNAs, microRNAs or microRNAs that can express genes with known anticancer effects or inhibit the expression of genes involved in cancer development. DNA or RNA, etc. may be used, and in the case of using a drug, for example, doxorubicin, paclitaxel, docetaxel, or the like, which are known to have anticancer effects, may be used, but is not limited thereto. Such genes and drugs may be supported in the complex structure by reacting with the nanoliposomes according to a conventional method.In the case of genes, the genes and drugs may be supported by reacting with the nanoliposomes in a hybrid form with biopolymers such as protamine. In the case of drugs, they may be supported inside liposomes through conventional ammonium sulfate gradient method (Bowen T. et al., International Journal of Pharmaceutics (2011), 416, 443-447). According to one embodiment of the present invention, the doxorubicin drug collection efficiency of the complex of the present invention was 88.57%, tGFP expression plasmid gene collection efficiency was 30%.
또한 본 발명의 일실시예에 따르면, 본 발명의 복합체 구조 내부에 추가로 담지될 수 있는 상기 형광물질은 임상 진단에 사용될 수 있는 소수성 또는 친수성 형광물질이라면 어느 것이든 사용할 수 있으며, 예를 들면 FITC, 텍사스 레드(Texas-red). RITC, Cy3, Cy5 또는 Cy7 등이 사용될 수 있으나 이에 제한되는 것은 아니다. 이러한 형광물질들은 통상적인 방법에 따라 소수성을 갖는 형광물질인 경우 마이크로버블과, 친수성을 갖는 형광물질인 경우 나노리포좀과 30℃에서 교반 또는 초음파분산(sonication)시켜 담지시킬 수 있다.In addition, according to an embodiment of the present invention, any of the hydrophobic or hydrophilic fluorescent materials that can be additionally supported in the complex structure of the present invention can be used for clinical diagnosis, for example, FITC , Texas red. RITC, Cy3, Cy5 or Cy7 may be used, but is not limited thereto. Such fluorescent materials may be supported by microbubbles in the case of hydrophobic fluorescent materials and nanoliposomes in the case of hydrophilic fluorescent materials by stirring or sonication at 30 ° C. according to a conventional method.
본 발명의 일실시예에 따르면, 본 발명의 복합체의 표면에 결합되어 있는 항체 등의 타겟팅 모이어티(targetting moiety)는 표적세포 표면의 분자(molecule), 리간드(ligand) 또는 수용체(receptor) 등의 표적물질을 선택적으로 인식(recognition)/결합(binding)할 수 있는 물질이라면 어느 것이든 사용가능하며, 예를 들면 핵산분자(DNA 또는 RNA), 단백질, 항체, 항원, 앱타머(RNA, DNA 및 펩타이드 앱타머), 수용체, 호르몬, 스트렙타비딘, 아비딘, 바이오틴, 렉틴, 리간드, 아고니스트, 안타고니스트, 효소, 조효소, 무기이온, 효소보조인자, 당, 지질, 효소기질, 합텐(hapten), 뉴트라비딘(neutravidin), 프로테인 A, 프로테인 G, 셀렉틴 (selectin), 칼슘 설페이트 및 기체 결합제(예: Pt, Pd, Au, Ag, Nb, Ir, Rh 및 Ru) 등을, 바람직하게는 바이오틴, 스트렙타비딘, 아비딘, 항체, 앱타머, 폴리펩타이드, 펩타이드, 리간드, 수용체, 렉틴, 당, 지질, 당지질 또는 핵산 등을 사용할 수 있으나 이에 제한되는 것은 아니다. 또한, 상기 표적물질은 분리(또는 분리와 검출, 또는 분리, 검출 및 정량)하고자 하는 시료 내 물질을 의미하며, 구체적으로 핵산분자(DNA 또는 RNA), 단백질, 펩타이드, 항원, 당, 지질, 세균, 바이러스, 세포, 유기 화합물, 무기화합물, 금속 및 무기이온을 포함하나, 이에 한정되는 것은 아니다. 또한, 상기 시료는 생물학적 시료, 화학적 시료 및 환경 시료를 포함하며, 상기 생물학적 시료는 예컨대 혈액, 혈장, 혈청, 바이러스, 세균, 조직, 세포, 림프, 골수액, 타액, 우유, 소변, 분변, 안구액, 정액, 뇌 추출물, 척수액, 관절액, 흉선액, 복수, 양막액, 세포 조직액 및 세포 배양액을 포함하나, 이에 제한되는 것은 아니다.According to an embodiment of the present invention, the targeting moiety of the antibody or the like bound to the surface of the complex of the present invention may be a molecule, ligand or receptor on the surface of the target cell. Any material capable of selectively recognizing / binding the target material may be used, for example, nucleic acid molecules (DNA or RNA), proteins, antibodies, antigens, aptamers (RNA, DNA and Peptide aptamers), receptors, hormones, streptavidin, avidin, biotin, lectins, ligands, agonists, antagonists, enzymes, coenzymes, inorganic ions, enzyme cofactors, sugars, lipids, enzyme substrates, hapten, neutra Neutravidin, Protein A, Protein G, Selectin, Calcium Sulfate and Gas Binders (e.g. Pt, Pd, Au, Ag, Nb, Ir, Rh and Ru), preferably biotin, streptabi Dean, avidin, antibodies, aptamers, polypeptides, Available Tide, ligand, receptor, lectin, sugar, lipid, glycolipid or nucleic acid, such as, but not limited thereto. In addition, the target material means a material in a sample to be separated (or isolated and detected, or isolated, detected and quantified), and specifically, a nucleic acid molecule (DNA or RNA), a protein, a peptide, an antigen, a sugar, a lipid, a bacterium. , Viruses, cells, organic compounds, inorganic compounds, metals and inorganic ions. In addition, the sample includes a biological sample, a chemical sample, and an environmental sample, and the biological sample includes, for example, blood, plasma, serum, virus, bacteria, tissue, cells, lymph, bone marrow fluid, saliva, milk, urine, feces, eyeballs. Fluids, semen, brain extracts, spinal fluid, joint fluids, thymic fluids, ascites, amniotic fluids, cell tissue fluids and cell culture fluids.
본 발명의 일실시예에 따르면, 본 발명의 복합체의 표면에 포함되어 있는 항체 등의 타겟팅 모이어티(targetting moiety)는 통상적인 반응을 통해 본 발명의 복합체 표면에 직접 또는 간접적으로 공유 또는 비공유 방식으로 결합되어 포함될 수 있으며, 예를 들어 이온결합, 정전기적 결합, 소수성 결합, 수소 결합, 공유결합, 친수성 결합 또는 반데르 발스 결합을 통해 결합되어 포함될 수 있다. 또한, 상기 간접 결합의 경우 결합제 등의 중간 매개체(intervening agent)가 사용될 수 있는데, 이때 중간 매개체로는 설포숙신이미딜-4-(N-말레이미도메틸)사이클로헥산-1-카복실레이트(sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) 등을 사용할 수 있다.According to one embodiment of the present invention, targeting moieties such as antibodies contained on the surface of the complex of the present invention are directly or indirectly covalently or indirectly shared with the surface of the complex of the present invention through a conventional reaction. It may be included in combination, for example, may be included in combination through ionic bonds, electrostatic bonds, hydrophobic bonds, hydrogen bonds, covalent bonds, hydrophilic bonds or van der Waals bonds. In addition, in the case of the indirect binding, an intervening agent such as a binder may be used. In this case, as an intermediate medium, sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfosuccinimidyl- 4- (N-maleimidomethyl) cyclohexane-1-carboxylate) can be used.
본 발명의 일실시예에 따르면, 본 발명의 복합체가 표면의 타겟팅 모이어티에 의해 표적 세포에 접근하면 외부 음파 자극(ultrasonic flow, UF)을 통해 파괴시켜 복합체 구조 내부에 담지되어 있던 치료 유전자 및 약물 등의 치료제 및 형광물질을 동시에 표적 세포내로 전달시킬 수 있으며, 이때 음파 자극의 세기는 특별히 제한되는 것은 아니나 그 기계지수(mechanical index) (mechanical index = PNP/
Figure PCTKR2013006993-appb-I000001
: PNP; Peak negative pressure of the ultrasound wave (MPa), Fc; center frequency of the ultrasound wave (MHz))가 0.01 내지 2.0인 것이 바람직하다. 이와 같이, 본 발명의 복합체를 초음파에서 고에너지 또는 높은 기계지수에 노출시키는 것을 플래쉬(flash)라고 하며, 이하 실시예에서 플래쉬라는 용어로도 설명한다.According to an embodiment of the present invention, when the complex of the present invention approaches the target cell by a targeting moiety on the surface, the therapeutic genes and drugs carried in the complex structure are destroyed by external acoustic stimulation (ultrasonic flow, UF). The therapeutic agent and the fluorescent substance of can be delivered to the target cell at the same time, the intensity of the sonic stimulation is not particularly limited, but the mechanical index (mechanical index = PNP /
Figure PCTKR2013006993-appb-I000001
: PNP; Peak negative pressure of the ultrasound wave (MPa), Fc; Center frequency of the ultrasound wave (MHz) is preferably 0.01 to 2.0. As such, exposing the composite of the present invention to high energy or high mechanical index on ultrasonic waves is referred to as flash, and is also described as flash in the following examples.
본 발명의 일실시예에 따르면, 형광물질로 FITC 및 텍사스 레드를, 유전자로 tGFP 발현 플라스미드를, 약물로 독소루비신을 복합체 구조 내부에 담지시키고, 표면에 유방암 세포에 특이적인 항-HER2 단일클론 항체(anti-HER2 monoclonal antibody)인 헤르셉틴(Herceptin)을 결합시킨 복합체를 사용하여 유방암 세포주인 SkBr3에 처리한 후 외부 음파 자극(UF)을 준 조건에서 형광물질인 FITC 및 텍사스 레드가 세포내로 전달된 것을 확인하였으며, 3일 후에는 독소루비신에 의한 65% 이상의 세포괴사를 확인하였으며, 또한 2일 후부터 90% 이상의 세포에 유전자가 전달되어 tGFP가 발현되는 것을 확인하였다.According to an embodiment of the present invention, the FITC and Texas red as a fluorescent material, tGFP expression plasmid as a gene, doxorubicin as a drug is carried inside the complex structure, and the anti-HER2 monoclonal antibody specific for breast cancer cells on the surface ( Treatment of breast cancer cell line SkBr3 using a complex conjugated with herceptin, an anti-HER2 monoclonal antibody, and the delivery of fluorescent substances FITC and Texas red into cells under external acoustic stimulation (UF) After 3 days, more than 65% cell necrosis by doxorubicin was confirmed, and the gene was delivered to 90% or more cells from 2 days later, and it was confirmed that tGFP was expressed.
따라서 본 발명의 마이크로버블-나노리포좀 복합체는 높은 표적 특이성을 통해 부작용을 최소화하면서 암세포에 특이적인 초음파 또는 형광 조영을 이용한 다중 영상 분석을 통해 암을 진단함과 동시에 치료 유전자 및 약물 등의 하나 이상의 치료제를 표적 암세포에 효과적으로 전달하여 암을 치료할 수 있으므로, 유방암, 간암, 췌장암, 뇌암, 위암, 폐암, 식도암, 대장암, 전립선암, 신장암, 난소암 등의 암 질환의 효과적인 진단 및 치료제 개발에 유용하게 활용될 수 있다. Therefore, the microbubble-nanoliposomal complex of the present invention can diagnose cancer through multiple image analysis using ultrasound or fluorescence contrast specific to cancer cells while minimizing side effects through high target specificity and at least one therapeutic agent such as therapeutic genes and drugs. Can be effectively treated to target cancer cells to treat cancer, which is useful for the effective diagnosis and treatment of cancer diseases such as breast cancer, liver cancer, pancreatic cancer, brain cancer, stomach cancer, lung cancer, esophageal cancer, colon cancer, prostate cancer, kidney cancer, ovarian cancer, etc. Can be utilized.
또한, 본 발명은In addition, the present invention
1) 필름형성물질, 인지질, 음전하 화합물, 아민기를 갖는 화합물 및 다이설파이드기를 갖는 화합물을 유기용매에 넣고 반응시켜 필름을 제조한 후 이를 소수성 가스와 함께 유기 혼합용매에 넣고 진동반응(vibrating)시켜 마이크로버블을 제조하는 단계;1) A film-forming substance, a phospholipid, a negatively charged compound, a compound having an amine group, and a compound having a disulfide group are added to an organic solvent and reacted to prepare a film, which is then mixed with an hydrophobic gas into an organic mixed solvent and vibrated. Preparing a bubble;
2) 필름형성물질, 인지질, 음전하 화합물 및 아민기를 갖는 화합물을 유기용매에 넣고 반응시켜 필름을 제조한 후 이를 물에 넣고 초음파분산 및 여과하여 나노리포좀을 제조하는 단계;2) preparing a film by adding a film-forming substance, a phospholipid, a negatively charged compound, and a compound having an amine group into an organic solvent to prepare a film, and then putting the same into water and dispersing ultrasonically and filtering to prepare nanoliposomes;
3) 상기 단계 2)에서 얻어진 나노리포좀을 하나 이상의 치료제와 반응시켜 하나 이상의 치료제를 나노리포좀 구조 내에 담지시키는 단계;3) reacting the nanoliposomes obtained in step 2) with at least one therapeutic agent to support the at least one therapeutic agent in the nanoliposomal structure;
4) 상기 단계 1)에서 얻어진 마이크로버블과 상기 단계 3)에서 얻어진 나노리포좀을 혼합하고 진탕시켜 마이크로버블-나노리포좀 복합체를 제조하는 단계; 및4) preparing a microbubble-nanoliposomal complex by mixing and shaking the microbubbles obtained in step 1) and the nanoliposomes obtained in step 3); And
5) 상기 단계 4)에서 제조된 복합체를 타겟팅 모이어티(targetting moiety)와 반응시켜 타겟팅 모이어티를 복합체 외부에 결합시키는 단계를 포함하는, 상기 본 발명의 마이크로버블-나노리포좀 복합체를 제조하는 방법을 제공한다.5) reacting the complex prepared in step 4) with a targeting moiety (targeting moiety) to bind the targeting moiety to the outside of the complex, the method of producing a microbubble-nanoliposomal complex of the present invention to provide.
상기 제조방법에서, 상기 단계 1)에서 사용되는 필름형성물질로는 DPPC (1,2-dipalmitory-sn-glycero-3-phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) 또는 DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) 등이; 인지질로는 콜레스테롤(cholesterol) 등이; 음전하 화합물로는 DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl-sn-glycero-3-phosphate), DMPA (1,2-dimyristoyl-sn-glycero-3-phosphate) 또는 DOPA (1,2-dioleoyl-sn-glycero-3-phosphate) 등이; 아민기를 갖는 화합물로는 DPPE (1,2-dipalmitory-sn-glycero-3-phosphoethanolamine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) 또는 DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) 등이; 그리고 다이설파이드기를 갖는 화합물로는 DSPE-PEG-SPDP {1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000]} 등이 사용될 수 있다.In the manufacturing method, the film forming material used in step 1) is DPPC (1,2-dipalmitory-sn-glycero-3-phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine) , DEPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine ), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) or DSPC (1,2-distearoyl-sn-glycero-3- phosphocholine); Phospholipids include cholesterol (cholesterol); Negative charge compounds include DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl-sn-glycero-3-phosphate), DMPA (1,2- dimyristoyl-sn-glycero-3-phosphate) or DOPA (1,2-dioleoyl-sn-glycero-3-phosphate); Compounds having an amine group include DPPE (1,2-dipalmitory-sn-glycero-3-phosphoethanolamine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), and DLPE (1,2-dilauroyl-sn- glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine); And a compound having a disulfide group may be used DSPE-PEG-SPDP {1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [polyethylene glycol (2000)}}.
또한, 상기 단계 1)에서 사용되는 소수성 가스는 SF6, CO2 또는 CF4 등의 통상적인 마이크로버블 제조시 사용되는 소수성 가스를 사용할 수 있으며, 유기용매로는 클로로포름(chloroform)이, 유기 혼합용매로는 글리세린, 프로필렌글리콜 및 물을 혼합한 용매를 사용할 수 있다. In addition, the hydrophobic gas used in the step 1) may be used a hydrophobic gas used in the manufacture of conventional microbubbles such as SF 6 , CO 2 or CF 4 , chloroform is an organic solvent, an organic mixed solvent As the solvent, a solvent in which glycerin, propylene glycol and water are mixed can be used.
또한, 상기 단계 2)에서 사용되는 필름형성물질로는 DPPC (1,2-dipalmitory-sn-glycero-3-phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) 또는 DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) 등이; 인지질로는 콜레스테롤(cholesterol) 등이; 음전하 화합물로는 DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl-sn-glycero-3-phosphate), DMPA (1,2-dimyristoyl-sn-glycero-3-phosphate) 또는 DOPA (1,2-dioleoyl-sn-glycero-3-phosphate) 등이; 그리고 아민기를 갖는 화합물로는 DPPE (1,2-dipalmitory-sn-glycero-3-phosphoethanolamine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) 또는 DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) 등이 사용될 수 있다.In addition, the film forming material used in step 2) is DPPC (1,2-dipalmitory-sn-glycero-3-phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPC ( 1,2-didecanoyl-sn-glycero-3-phosphocholine), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) or DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) this; Phospholipids include cholesterol (cholesterol); Negative charge compounds include DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl-sn-glycero-3-phosphate), DMPA (1,2- dimyristoyl-sn-glycero-3-phosphate) or DOPA (1,2-dioleoyl-sn-glycero-3-phosphate); Compounds having an amine group include DPPE (1,2-dipalmitory-sn-glycero-3-phosphoethanolamine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), and DLPE (1,2-dilauroyl-sn -glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) may be used.
또한, 상기 단계 3)에서 하나 이상의 치료제를 나노리포좀 구조 내에 담지시키는 공정은, 상기 치료제가 치료 유전자인 경우 프로타민(protamine) 등의 생체고분자와의 혼성체 형태로 나노리포좀과 반응시켜 수행할 수 있고, 상기 치료제가 약물인 경우에는 통상적인 황산암모늄 농도구배법[ammonium sulfate gradient method, Bowen T 등, International Journal of Pharmaceutics (2011), 416, 443-447]을 통해 상온에서 교반시켜 수행할 수 있다.In addition, the step of supporting the at least one therapeutic agent in the nanoliposomal structure in step 3), if the therapeutic agent is a therapeutic gene can be carried out by reacting with the nanoliposome in the form of a hybrid with a biopolymer such as protamine (protamine) , If the therapeutic agent is a drug, it can be carried out by stirring at room temperature through a conventional ammonium sulfate gradient method, Bowen T et al., International Journal of Pharmaceutics (2011), 416, 443-447.
또한, 상기 단계 4)의 마이크로버블과 나노리포좀의 복합체 형성 반응은 pH, 반응온도 또는 반응시간 등의 조절을 통해 마이크로버블의 지질구조체와 나노리포좀의 지질구조체의 일부분을 변형하여 진탕반응시킴으로써 간단하게 공유결합을 유도하여 수행할 수 있다.In addition, the complex formation reaction of the microbubbles and nanoliposomes of step 4) is simply by modifying a portion of the lipid structure of the nanobubble and the liposomes of the nanoliposomes by controlling the pH, the reaction temperature or the reaction time and shaking. This can be done by inducing covalent bonds.
또한, 상기 단계 5)의 항체 등의 타겟팅 모이어티(targetting moiety)를 복합체 외부에 결합시키는 공정은 통상적인 반응을 통해 타겟팅 모이어티를 본 발명의 복합체 표면에 직접 또는 간접적으로 공유 또는 비공유 방식으로 결합시켜 수행할 수 있으며, 예를 들어 이온결합, 정전기적 결합, 소수성 결합, 수소 결합, 공유결합, 친수성 결합, 또는 반데르 발스 결합을 통해 결합시켜 수행할 수 있다. 이때, 상기 간접 결합의 경우 결합제 등의 중간 매개체(intervening agent)가 사용될 수 있는데, 이때 중간 매개체로는 설포숙신이미딜-4-(N-말레이미도메틸)사이클로헥산-1-카복실레이트(sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) 등을 사용할 수 있다.In addition, the step of binding the targeting moiety, such as the antibody of step 5) outside the complex, the targeting moiety binds to the surface of the complex of the present invention directly or indirectly in a covalent or non-covalent manner through a conventional reaction. It may be carried out by, for example, may be carried out by bonding through ionic bonds, electrostatic bonds, hydrophobic bonds, hydrogen bonds, covalent bonds, hydrophilic bonds, or van der Waals bonds. In this case, in the case of the indirect binding, an intervening agent such as a binder may be used. In this case, as an intermediate medium, sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfosuccinimidyl- 4- (N-maleimidomethyl) cyclohexane-1-carboxylate) can be used.
본 발명의 일실시예에 따르면, 상기 단계 1) 또는 2)는 제조된 마이크로버블 또는 나노리포좀에 형광물질을 첨가 후 반응시켜 형광물질을 상기 마이크로버블 또는 나노리포좀 구조 내에 담지시키는 공정을 추가로 포함할 수 있다. 이때, 상기 형광물질을 마이크로버블 또는 나노리포좀 구조 내에 담지시키는 공정은, 상기 형광물질이 소수성인 경우 마이크로버블과, 또는 상기 형광물질이 친수성인 경우 나노리포좀과 통상적인 방법에 따라 30℃에서 교반 또는 초음파분산(sonication)시켜 수행될 수 있다.According to an embodiment of the present invention, the step 1) or 2) further includes a step of supporting the fluorescent material in the microbubble or nanoliposomal structure by adding a fluorescent material to the prepared microbubble or nanoliposomes and reacting. can do. At this time, the step of supporting the fluorescent material in the microbubble or nanoliposomal structure, the microbubble when the fluorescent material is hydrophobic, or when the fluorescent material is hydrophilic or nanoliposomes and the hydrophilic at 30 ℃ according to a conventional method or It may be performed by sonication.
본 발명의 다른 양태에 따르면, 본 발명은 상기 마이크로버블-나노리포좀 복합체를 포함하는 암세포 특이적 초음파, 자기공명영상(MRI) 또는 형광 분석용 조영제 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a contrast agent composition for cancer cell-specific ultrasound, magnetic resonance imaging (MRI) or fluorescence analysis comprising the microbubble-nanoliposomal complex.
본 발명에서, 상기 조영제는 생체 내에서 강력하고 특이적으로 암세포를 조영(contrast) 또는 영상화(imaging)하기 위해 체내 투여되는 물질을 말하는 것으로, 현재 의료, 진단 분야에서 조직 및 세포의 이미지 강화를 위해 광범위하게 사용되고 있다. 본 발명의 조영제라는 용어는 종래 알려진 CT 또는 MRI 조영제의 범위로 한정되지 않으며 초음파 이미지 분석용 영상제, 형광 이미지 분석용 영상제 등을 포함하는 의미로 사용된다. In the present invention, the contrast agent refers to a substance that is administered in vivo to contrast or image cancer cells in a powerful and specific manner in vivo, and is currently used for enhancing image of tissues and cells in the medical and diagnostic fields. Widely used. The term contrast agent of the present invention is not limited to the range of conventionally known CT or MRI contrast agents, and is used in the sense of including an imaging agent for ultrasound image analysis, an imaging agent for fluorescence image analysis, and the like.
본 발명의 일실시예에서는 형광물질, 타겟 유전자, 항체 등을 담지시킨 마이크로버블-나노리포좀 복합체를 이용하여 초음파 분석, MRI 분석, 형광 분석이 용이하게 이루어짐을 확인하였다 (시험예 7 참조).In one embodiment of the present invention, it was confirmed that ultrasonic analysis, MRI analysis, fluorescence analysis was easily performed using a microbubble-nanoliposomal complex carrying a fluorescent substance, a target gene, an antibody, and the like (see Test Example 7).
본 발명의 조영제 조성물은 암세포 또는 암조직의 표적 및 진단에 유용하게 사용될 수 있으며, 경구 또는 비경구 투여 형태로 제형화될 수 있다. 비경구 제형은 바람직하게는 본 발명의 마이크로버블-나노리포좀 복합체를 포함하는 멸균 수용액 또는 현탁액을 포함하며, 약학적 수용액 또는 현탁액의 다양한 제조기술이 당업계에 공지되어 있다. 상기 용액은 또한 약학적으로 허용되는 완충제, 안정제, 항산화제 및 염화나트륨과 같은 전해질을 포함할 수 있다. 비경구 제형은 직접 주사되거나 대용량의 비경구 제형과 혼합될 수 있다.The contrast agent composition of the present invention may be usefully used for the targeting and diagnosis of cancer cells or cancer tissues, and may be formulated in oral or parenteral dosage forms. Parenteral formulations preferably comprise sterile aqueous solutions or suspensions comprising the microbubble-nanoliposomal complex of the present invention, and various techniques for preparing pharmaceutical aqueous solutions or suspensions are known in the art. The solution may also include pharmaceutically acceptable buffers, stabilizers, antioxidants and electrolytes such as sodium chloride. Parenteral formulations may be injected directly or mixed with large amounts of parenteral formulations.
경구 투여용 제형은 매우 다양할 수 있으며, 이는 당업계에 공지되어 있다. 일반적으로 그러한 제형은 진단적 유효량의 본 발명에 따른 마이크로버블-나노리포좀 복합체를 포함하는 수용액 또는 현탁액을 포함한다. 상기 경구 제형은 선택적으로 완충제, 계면활성제, 보조제(adjuvant), 요변성제(thixotropic agent) 등을 포함할 수 있다. 또한 경구 투여용 제형은 향미료 및 기타 관능성을 증가시키기 위한 성분을 포함할 수 있다.Formulations for oral administration can vary widely and are known in the art. Generally such formulations comprise an aqueous solution or suspension comprising a diagnostically effective amount of the microbubble-nanoliposomal complex according to the invention. The oral formulation may optionally include buffers, surfactants, adjuvants, thixotropic agents, and the like. Formulations for oral administration may also include ingredients for increasing flavor and other functionalities.
본 발명에 따른 조영제 조성물은 이미징 영상의 목적하는 조영 효과를 달성하는데 유효한 양으로 투여된다. 그러한 투여량은 영상 절차의 대상인 기관 또는 조직, 사용되는 영상 장치 등에 따라 광범위하게 변할 수 있다. 상기 마이크로버블-나노리포좀 복합체를 조영제로서 사용하기 위한 투여 농도는 0.1 mM 내지 10 M이 될 수 있다. The contrast agent composition according to the invention is administered in an amount effective to achieve the desired contrast effect of the imaging image. Such dosages can vary widely depending on the organ or tissue that is the subject of the imaging procedure, the imaging device used, and the like. The dosage concentration for using the microbubble-nanoliposomal complex as a contrast agent may be 0.1 mM to 10 M.
본 발명의 조영제 조성물은 영상 진단 분석의 통상적인 방식으로 사용된다. 예를 들어, 상기 조영제 조성물을 포유동물에 전신적으로 또는 영상화되는 기관 또는 조직에 국부적으로, 적절한 시각화를 제공하는데 충분한 양으로 투여한 다음, 포유동물을 초음파 촬영 또는 MRI 촬영할 수 있다.The contrast agent compositions of the present invention are used in a conventional manner of imaging diagnostic analysis. For example, the contrast agent composition may be administered systemically to the mammal or locally to an organ or tissue being imaged, in an amount sufficient to provide adequate visualization, and then the mammal may be subjected to ultrasound or MRI imaging.
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 마이크로버블-나노리포좀 복합체와, 항암제 또는 항암 유전자를 유효성분으로 함유하는 항암용 약학 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a pharmaceutical composition for anticancer containing the microbubble-nanoliposomal complex and an anticancer agent or anticancer gene as an active ingredient.
본 발명의 일실시예에서 본 발명의 마이크로버블-나노리포좀 복합체에 항암제 또는 항암 유전자를 도입시켜 암세포의 사멸 또는 암조직의 성장 억제 효과를 확인함으로써, 암 치료에 유용하게 사용될 수 있음을 확인하였다 (시험예 6 및 7 참조).In an embodiment of the present invention, by introducing an anticancer agent or anticancer gene into the microbubble-nanoliposomal complex of the present invention to confirm the effect of killing cancer cells or inhibiting the growth of cancer tissues, it was confirmed that it can be usefully used for cancer treatment ( See Test Examples 6 and 7).
본 발명의 항암용 약학 조성물이 약제로 이용되기 위해서는 약제학적 분야에서 공지된 방법에 의해 제조될 수 있으며, 이 경우 유효성분으로서 공지의 항암제 또는 항암 유전자를 담지시킨 본 발명의 마이크로버블-나노리포좀 복합체 이외에 약제학적으로 허용되는 담체를 포함할 수 있다. 본 발명의 약학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제화 시에 통상적으로 이용되는 것으로서 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인살 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약학 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences(19th ed., 1995)에 상세히 기재되어 있다.In order to use the pharmaceutical composition for anticancer of the present invention may be prepared by a method known in the pharmaceutical field, in this case, the microbubble-nanoliposomal complex of the present invention carrying a known anticancer agent or anticancer gene as an active ingredient. In addition, it may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the formulation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, phosphorus calcium, alginate, gelatin, silicic acid Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils, and the like. It is not. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명의 약학 조성물은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 또는 부형제를 이용하여 제제화 됨으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제, 캅셀제 또는 주사제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다. 이들은 비경구 투여(예컨대, 정맥 내, 피하, 복강 내 또는 국소에 적용)하거나 경구 투여될 수 있다.The pharmaceutical compositions of the present invention may be prepared in unit dosage form by being formulated with a pharmaceutically acceptable carrier or excipient according to methods which can be easily carried out by those skilled in the art. It can be prepared by incorporation into a dose container. The formulation may be in the form of a solution, suspension or emulsion in oil or medium, or may be in the form of extracts, powders, granules, tablets, capsules or injections, and may further comprise dispersants or stabilizers. They may be parenterally administered (eg, applied intravenously, subcutaneously, intraperitoneally or topically) or orally.
본 발명의 약학 조성물의 적합한 투여량은 제제화 방법, 투여방식, 환자의 연령, 체중, 성, 건강상태, 질병 증상의 정도, 음식, 투여시간, 투여방법 및 반응 감응성과 같은 요인들에 의해 적절히 선택될 수 있으며, 바람직하게는 성인기준 1일 당 0.01 ~ 100 mg이 투여될 수 있다.Appropriate dosages of the pharmaceutical compositions of the present invention are appropriately selected by such factors as the formulation method, mode of administration, age, weight, sex, health condition, degree of disease symptom, food, time of administration, method of administration, and reaction sensitivity of the patient. Preferably, 0.01 to 100 mg can be administered per day of adult.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당 업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
재료 및 방법Materials and methods
1) 실험재료1) Experimental material
DPPC (1,2-dipalmitory-sn-glycero-3-phosphatidylcholine), 콜레스테롤(cholesterol), DCP (dicetyl phosphate), DPPE (1,2-dipalmitory-sn-glycero-3-phosphoethanolamine), DSPE-PEG-SPDP {1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000]}, 설포숙신이미딜-4-(N-말레이미도메틸)사이클로헥산-1-카복실레이트(sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), 트라우츠 시약(Trauts Reagent, 2-iminothiolane), 암모늄 설페이트(ammonium sulfate) 및 프로타민(protamine)은 모두 Sigma-Aldrich 사에서 구입하였으며, 별도의 변형을 가하지 않고 사용하였다. DPPC (1,2-dipalmitory-sn-glycero-3-phosphatidylcholine), cholesterol (cholesterol), DCP (dicetyl phosphate), DPPE (1,2-dipalmitory-sn-glycero-3-phosphoethanolamine), DSPE-PEG-SPDP {1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [polyethylene glycol (PDP) -2000]}, sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate ( sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, Trauts Reagent (2-iminothiolane), ammonium sulfate and protamine were all purchased from Sigma-Aldrich. It was used without adding a strain.
2) 세포 배양2) cell culture
유방암 세포주인 SkBr3를 10% FBS 함유 RPMI 1640 배지(Hyclone, Logan, UT)에서 습기를 가한 5% CO2 대기 및 37℃ 온도 조건 하에 배양하였다.SkBr3, a breast cancer cell line, was incubated under humidified 5% CO 2 atmosphere and 37 ° C. temperature in RPMI 1640 medium (Hyclone, Logan, UT) containing 10% FBS.
3) MTT 분석 3) MTT analysis
MTT 분석키트(Sigma-Aldrich 사)를 사용한 세포독성 시험(cytotoxicity assay)을 다음과 같이 수행하였다. Cytotoxicity assay using MTT assay kit (Sigma-Aldrich) was performed as follows.
배양된 세포주를 시험에 사용하기 위해 배지제거 후 세척하고 트립신 처리 후 재부유(resuspend)시켜 회수하였다. 회수된 세포들을 96-웰 플레이트(96-well plate)에 웰 당 5,000 세포의 농도로 넣어 5% CO2 대기 및 37℃ 온도 조건하에 밤새 배양하였다. 배양된 세포에 시험 용액을 다양한 농도로 처리한 후, 처리된 세포들을 다시 동일 조건하에 일정시험시간 동안 배양하였다. 배양 후 배지를 제거하고, 웰 당 MTT 용액 0.1 ml씩을 첨가하여 37℃, 5% CO2 조건 하에 3시간 동안 반응시킨 후 MTT 용액을 제거하였으며, 여기에 MTT 가용화 용액(MTT solubilization solution) 0.1 ml 씩을 첨가하여 포르마잔 크리스탈(formazan crystals)을 용해시켰다. 웰 플레이트를 ELISA 리더(reader)를 사용해 570 nm 파장에서 흡광도를 측정하여 세포생존성을 결정하였다. Cultured cell lines were harvested after media removal for tryout, resuspended after trypsin treatment. The recovered cells were placed in 96-well plates at a concentration of 5,000 cells per well and incubated overnight under 5% CO 2 atmosphere and 37 ° C. temperature conditions. After the test solution was treated to the cultured cells at various concentrations, the treated cells were again incubated for a predetermined test time under the same conditions. After incubation, the medium was removed, and 0.1 ml of MTT solution per well was added and reacted for 3 hours at 37 ° C. and 5% CO 2. The MTT solution was removed, and 0.1 ml of MTT solubilization solution was added thereto. Formazan crystals were dissolved by addition. Cell viability was determined by measuring the absorbance at 570 nm at a well plate using an ELISA reader.
제조예: 본 발명에 따른 마이크로버블(MB)-나노리포좀 복합체의 제조Preparation Example: Preparation of microbubble (MB) -nanoliposomal complex according to the present invention
단계 1) 마이크로버블(MB) 제조Step 1) Manufacturing microbubbles (MB)
DPPC, 콜레스테롤, 마이크로버블의 뭉침을 막기 위한 음전하(negative charge) 화합물인 DCP, 아민 말단기를 갖는 화합물인 DPPE 및 다이설파이드기(disulfide)를 갖는 화합물인 DSPE-PEG-SPDP를 각각 15.4 mg, 3.5 mg, 1.0 mg, 1.2 mg 및 5 mg 씩 5 mL 클로로포름(chloroform)에 용해시켰다. 얻어진 용액을 회전식 증발농축기(Rotary evaporator)에 넣어 35℃에서 5분 동안 반응시켜 용매인 클로로포름을 제거한 후, -45℃에서 약 24시간 동결건조시켜 필름을 제조하였다. 여기에 글리세린, 프로필렌글리콜 및 물을 혼합한 용매(glycerin:propylene glycol:H2O=1:2:7) 2 mL를 가하여 혼합한 후 수득한 혼합용액을 밀봉된 병(hermetic vial)에 담았다. 여기에 SF6 가스(gas)를 충진한 후 진동기(vibrator)로 15초 동안 진동시켜 마이크로버블(MB)을 제조하였다.DCP, a negative charge compound to prevent aggregation of DPPC, cholesterol, and microbubbles, DPPE, an amine end group, and DSPE-PEG-SPDP, a compound having disulfide groups, were respectively 15.4 mg, 3.5 mg, 1.0 mg, 1.2 mg and 5 mg were dissolved in 5 mL chloroform. The obtained solution was placed in a rotary evaporator and reacted at 35 ° C. for 5 minutes to remove chloroform as a solvent, and then freeze-dried at −45 ° C. for about 24 hours to prepare a film. 2 mL of a mixture of glycerin, propylene glycol, and water (glycerin: propylene glycol: H 2 O = 1: 2: 7) was added thereto, and the mixed solution obtained after mixing was put in a hermetic vial. After filling the SF 6 gas (gas) and vibrated for 15 seconds with a vibrator (vibrator) to prepare a microbubble (MB).
단계 1-1) 마이크로버블 구조 내에 소수성 형광물질 담지Step 1-1) Supporting Hydrophobic Phosphor in Microbubble Structure
상기 단계 1)에서 제조된 마이크로버블에 녹색 형광을 갖는 소수성 유기 형광 염료인 FITC 0.1 mg을 가하고 상온에서 교반 반응시켜 형광물질을 구조 내부에 담지시킨 마이크로버블을 수득하였다(평균직경: 1 ~ 2 ㎛). 0.1 mg of FITC, a hydrophobic organic fluorescent dye having green fluorescence, was added to the microbubbles prepared in step 1), followed by stirring at room temperature to obtain a microbubble in which a fluorescent material was loaded in the structure (average diameter: 1 to 2 μm). ).
단계 2) 나노리포좀 제조Step 2) Nanoliposomal Preparation
DPPC, 콜레스테롤, 나노리포좀의 뭉침을 막기 위한 음전하 화합물인 DCP 및 아민 말단기를 갖는 화합물인 DPPE를 각각 15.4 mg, 3.5 mg, 1.0 mg 및 1.2 mg 씩 5 mL 클로로포름에 넣어 용해시켰다. 얻어진 용액을 회전 증발농축기에 넣어 35℃에서 5분 동안 반응시켜 용매를 제거한 후 -45℃에서 약 24시간 동안 동결건조시켜 필름을 제조하였다. 수득한 필름에 H2O 2 mL을 가한 후 초음파 분산기(sonicator)를 이용하여 60℃에서 5분 동안 분산시킨 후, 액체 질소를 이용하여 동결 및 해동을 5회 반복하여 리포좀 혼합용액을 제조하였다. 이 리포좀 혼합용액을 200 nm 필터로 60℃에서 2회 여과하여 입자크기가 200 nm 이하인 나노리포좀을 제조하였다. DPPC, a negatively charged compound to prevent aggregation of DPPC, cholesterol and nanoliposomes, and DPPE, a compound having an amine end group, were dissolved in 5 mL chloroform at 15.4 mg, 3.5 mg, 1.0 mg, and 1.2 mg, respectively. The obtained solution was placed in a rotary evaporator, reacted at 35 ° C. for 5 minutes to remove the solvent, and lyophilized at −45 ° C. for about 24 hours to prepare a film. After adding 2 mL of H 2 O to the obtained film, the mixture was dispersed for 5 minutes at 60 ° C. using an ultrasonic disperser, and then freeze and thaw was repeated 5 times with liquid nitrogen to prepare a liposome mixed solution. The liposome mixed solution was filtered twice at 60 ° C. with a 200 nm filter to prepare nanoliposomes having a particle size of 200 nm or less.
단계 2-1) 나노리포좀 구조 내에 친수성 형광물질 담지Step 2-1) Supporting Hydrophilic Phosphors in the Nanoliposomal Structure
상기 단계 2)에서 제조한 나노리포좀에 붉은색 형광을 나타내는 친수성 유기 형광 염료인 텍사스-레드(Texas-red) 0.1 mg을 가하고 상온에서 교반시켜 형광물질을 구조 내부에 담지시킨 나노리포좀을 수득하였다(평균크기: 100~200 nm). 0.1 mg of Texas-red (Texas-red), a hydrophilic organic fluorescent dye showing red fluorescence, was added to the nanoliposomes prepared in step 2), followed by stirring at room temperature to obtain nanoliposomes in which the fluorescent material was supported in the structure ( Average size: 100-200 nm).
단계 3) 나노리포좀 구조 내에 약물 및 유전자 담지Step 3) Supporting Drugs and Genes in the Nanoliposomal Structure
상기 단계 2) 또는 2-1)에서 수득한 나노리포좀을 대상으로 다음과 같은 반응을 수행하여 나노리포좀 구조 내부에 유전자 및/또는 약물을 담지시켰다.Genes and / or drugs were carried in the nanoliposomal structure by performing the following reaction on the nanoliposomes obtained in step 2) or 2-1).
상기 나노리포좀을 동결건조한 후 수득한 파우더(powder) 형태의 나노리포좀(21 mg)에 녹색형광단백질(green fluorescent protein, tGFP) 발현 플라스미드 유전자 26.6 ㎍ 및 프로타민(protamine) 18.8 μM 함유 혼합용액 0.1 ml을 가하여 25℃에서 5분 동안 진탕(shaking)시켰다. 생성된 반응 혼합물을 13000 rpm에서 5분 동안 2회 원심분리하여 리포좀에 포집되지 않은 잔여 유전자 및 프로타민을 제거하여 치료 유전자가 구조 내부에 담지된 나노리포좀을 수득하였다. After lyophilizing the nanoliposomes, 0.1 ml of a mixed solution containing 26.6 μg of a green fluorescent protein (tGFP) expression plasmid gene and 18.8 μM of protamine was added to a powdered nanoliposome (21 mg). And shaken at 25 ° C. for 5 minutes. The resulting reaction mixture was centrifuged twice at 13000 rpm for 5 minutes to remove residual genes and protamine not trapped in liposomes to obtain nanoliposomes carrying the therapeutic genes inside the structure.
또한, 암 치료에 효과적인 것으로 알려진 0.5 ~ 20 mM의 독소루비신(doxorubicin) 용액을 1 ~ 120 mM 농도의 상기 나노리포좀 용액에 첨가한 후, 상온에서 교반하여 나노리포좀 내부에 있던 황산암모늄이 삼투압 현상에 의해서 외부에 있는 독소루비신과 교체되어 독소루비신을 나노리포좀 내부에 담지시킴으로써 구조 내부에 약물(독소루비신)을 함유하는 나노리포좀을 제조하였다.In addition, a solution of 0.5-20 mM doxorubicin, which is known to be effective in treating cancer, is added to the nanoliposomal solution at a concentration of 1-120 mM, and then stirred at room temperature, so that ammonium sulfate in the nanoliposomes is osmotic by the osmotic phenomenon. The nanoliposomes containing the drug (doxorubicin) were prepared in the structure by replacing the doxorubicin with the outside to support the doxorubicin inside the nanoliposomes.
단계 4) 마이크로버블-나노리포좀 복합체 제조Step 4) Preparation of the microbubble-nanoliposomal complex
상기 단계 1) 또는 1-1)에서 수득한 마이크로버블 0.2 mL; 및 상기 단계 3)에서 수득한 나노리포좀 1.8 mL를 밀봉 병(hermetic vial)에 넣고 pH를 8로 조정 후 25℃에서 2시간 동안 진탕반응시켜 마이크로버블-나노리포좀 복합체를 제조하였다.0.2 mL of the microbubbles obtained in step 1) or 1-1); And 1.8 mL of the nanoliposome obtained in step 3) was put in a sealed bottle (hermetic vial) was adjusted to pH 8 and shaken at 25 ℃ for 2 hours to prepare a microbubble-nanoliposome complex.
단계 5) 마이크로버블-나노리포좀 복합체 외부에 항체 결합Step 5) Binding Antibodies Outside the Microbubble-Nanoliposomal Complex
상기 단계 4)에서 제조된 복합체에 설포숙신이미딜-4-(N-말레이미도메틸)사이클로헥산-1-카복실레이트 5 mg을 가한 후 pH를 8로 조정하여 25℃에서 3시간 동안 진탕시켰으며, 얻어진 말레이미드(maleimide)-마이크로버블-나노리포좀 복합체 용액에 유방암 세포에 특이적인 항-HER2 단일클론 항체(anti-HER2 monoclonal antibody)인 헤르셉틴(Herceptin) 0.1 mL를 가하고 pH를 7로 조정한 후 4℃에서 24시간동안 진탕반응시켜 복합체 외부에 항체를 결합시켰다. 얻어진 혼합용액을 13000 rpm에서 5분 동안 2 회 원심분리하여 미반응 물질을 제거함으로써 본 발명에 따른 마이크로버블-나노리포좀 복합체를 제조하였다. 5 mg of sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate was added to the complex prepared in step 4), and the pH was adjusted to 8, followed by shaking at 25 ° C. for 3 hours. To the obtained maleimide-microbubble-nanoliposomal complex solution, 0.1 mL of herceptin, an anti-HER2 monoclonal antibody specific for breast cancer cells, was added, and the pH was adjusted to 7. After shaking for 24 hours at 4 ℃ bound the antibody outside the complex. The resultant mixed solution was centrifuged twice at 13000 rpm for 5 minutes to remove unreacted material, thereby preparing a microbubble-nanoliposomal complex according to the present invention.
시험예 1: 본 발명의 마이크로버블(MB)-나노리포좀 복합체의 물성 확인Test Example 1: Checking the physical properties of the microbubble (MB) -nanoliposomal complex of the present invention
1) 광학현미경, 극저온 전자 현미경 및 동적광산란 분석 1) Optical microscope, cryogenic electron microscopy and dynamic light scattering analysis
상기 제조예의 단계 1) 또는 1-1)에서 수득한 마이크로버블 및 상기 제조예의 단계 2), 2-1) 또는 3)에서 수득한 나노리포좀을 대상으로 광학현미경(optical microscope), 극저온 전자 현미경(cryogenic electron microscopy, Cryo-EM) 및 동적광산란(dynamic light scattering, DLS) 분석을 수행하여 각 분자 모양 및 크기 분포를 확인하였다. An optical microscope and a cryogenic electron microscope were used for the microbubbles obtained in step 1) or 1-1) of the preparation example and the nanoliposomes obtained in step 2), 2-1) or 3) of the preparation example. Cryogenic electron microscopy (Cryo-EM) and dynamic light scattering (DLS) analyzes were performed to confirm each molecular shape and size distribution.
그 결과, 도 2에서 볼 수 있는 바와 같이, 본 발명의 일실시예에 따라 제조된 마이크로버블(MB)은 구형의 모양을 갖고 있으며 평균 크기는 1.713 ㎛였고, 나노리포좀 역시 구형의 모양에 평균 크기 197 nm를 나타내었다. 또한, 마이크로버블 및 나노리포좀은 모두 균일한 입자 크기 분포를 나타내는 것으로 확인되었다.As a result, as can be seen in Figure 2, the microbubbles (MB) prepared according to an embodiment of the present invention has a spherical shape and the average size was 1.713 ㎛, nanoliposomes also the average size in the shape of a sphere 197 nm. In addition, both microbubbles and nanoliposomes were found to exhibit uniform particle size distribution.
2) 공초점 형광 현미경 분석 및 자외-가시선 분광분석2) Confocal Fluorescence Microscopy and Ultraviolet-Vis Spectroscopy
본 발명에 따른 마이크로버블-나노리포좀 복합체의 조영 효과 및 안정성을 확인하기 위해, 상기 제조예에서 제조된 복합체를 대상으로 공초점 형광 현미경(confocal fluorescence microscope) 분석을 수행하였다.In order to confirm the contrast effect and stability of the microbubble-nanoliposomal complex according to the present invention, a confocal fluorescence microscope analysis was performed on the complex prepared in Preparation Example.
그 결과, 도 3에서 볼 수 있는 바와 같이, 저배율 분석에서는 대부분 입자 들이 붉은색 형광과 녹색 형광을 동시에 갖고 있음을 확인하였으며, 이로부터 녹색 형광의 FITC를 함유하는 마이크로버블과 붉은색 형광의 텍사스 레드를 함유하는 나노리포좀이 한 입자 내에 잘 결합되어 있음을 알 수 있었다. 또한, 도 3의 삽입 도면에서 볼 수 있는 바와 같이, 고배율 분석에서는 입자 내부의 녹색 형광과 외부의 붉은색 형광이 뚜렷하게 관찰되었으며, 이로써 본 발명에 따른 마이크로버블-나노리포좀 복합체가 도 1에서와 같이 마이크로버블의 표면에 나노리포좀이 결합되어 있는 구형의 구조로 이루어졌음을 육안으로 확인할 수 있었다. 또한, 본 발명의 마이크로버블-나노리포좀 복합체는 평균 약 1-2 ㎛ 직경의 균일한 크기 분포를 나타내는 것을 확인하였다.As a result, as can be seen in Figure 3, in the low magnification analysis, it was confirmed that most of the particles have red fluorescence and green fluorescence at the same time, from this microbubble containing FITC of green fluorescence and Texas red of red fluorescence It was found that the nanoliposome containing was well bound in one particle. In addition, as can be seen in the inset of FIG. 3, in the high magnification analysis, the green fluorescence inside the particle and the red fluorescence outside were clearly observed, whereby the microbubble-nanoliposomal complex according to the present invention is as shown in FIG. It was visually confirmed that the spherical structure in which nanoliposomes are bound to the surface of the microbubble was formed. In addition, it was confirmed that the microbubble-nanoliposomal complex of the present invention exhibited a uniform size distribution with an average diameter of about 1-2 μm.
한편, 본 발명의 따른 마이크로버블-나노리포좀 복합체는 마이크로버블과 나노리포좀이 화학적 안정성이 우수한 공유결합으로 결합되어 있어, 입자 내에 담지된 유전자, 약물 또는 형광물질을 생체 내 표적 기관, 조직 또는 세포로 운송 및 전달하는 데 우수한 안정성을 나타낸다. 따라서 상기 제조예에서 제조된 복합체가 공유결합으로 결합되어 있는지 확인하기 위해, 자외-가시선 분광분석(UV-vis spectroscopy)을 통해 공유결합시 발생되는 부산물인 SPDP(N-succinimidyl-3-[2-pyridyldithio]-propionate)의 존재 여부를 확인하였다.On the other hand, the microbubble-nanoliposomal complex according to the present invention is a microbubble and nanoliposomes are covalently bonded with excellent chemical stability, the gene, drug or fluorescent material carried in the particles to target organs, tissues or cells in vivo Excellent stability in transport and delivery. Therefore, in order to confirm whether the complex prepared in the above preparation is covalently bonded, SPDP (N-succinimidyl-3- [2-) which is a by-product generated during covalent bonding through UV-vis spectroscopy. pyridyldithio] -propionate) was confirmed.
그 결과, 도 4에서 볼 수 있는 바와 같이, 340 nm 영역에서 SPDP를 확인할 수 있었으며, 텍사스 레드와 FITC 형광도 확인되었다. 이로써 본 발명에 따른 마이크로버블-나노리포좀 복합체는 마이크로버블과 나노리포좀이 화학적 안정성이 우수한 공유결합으로 결합되어 있어, 입자 구조 내에 담지된 유전자, 약물 또는 형광물질을 생체 내 표적 기관, 조직 또는 세포로 운송 및 전달하는 데 우수한 안정성을 나타낼 것을 기대할 수 있다.As a result, as shown in Figure 4, it was possible to confirm the SPDP in the 340 nm region, Texas red and FITC fluorescence was also confirmed. As a result, the microbubble-nanoliposomal complex according to the present invention is covalently bonded to microbubbles and nanoliposomes with excellent chemical stability, so that genes, drugs, or fluorescent substances supported in the particle structure are transferred to target organs, tissues, or cells in vivo. It can be expected to show good stability in transportation and delivery.
3) 초음파 조영 분석3) Ultrasound Contrast Analysis
본 발명에 따른 마이크로버블-나노리포좀 복합체의 초음파 조영 효과를 확인하기 위해, 시판중인 마이크로버블 초음파 조영제인 SonoVue(TM)와 상기 제조예에서 제조된 복합체 및 상기 제조예의 단계 1) 또는 1-1)에서 수득한 마이크로버블을 각각 사용하여 임상에서 사용되고 있는 초음파 영상 장치(IU22, Philips사)를 통한 인공혈관 팬텀에서의 초음파 조영 효과를 비교 분석하였다. In order to confirm the ultrasonic contrast effect of the microbubble-nanoliposomal complex according to the present invention, a commercially available microbubble ultrasound contrast agent SonoVue (TM) and the composite prepared in the above preparation and the preparation example 1) or 1-1) The microbubbles obtained in the US were used to analyze the effect of ultrasound imaging on the artificial vascular phantom through the ultrasound imaging apparatus (IU22, Philips) used in the clinic.
그 결과, 도 5에서 볼 수 있는 바와 같이, 상기 제조예의 단계 1) 또는 1-1)에서 수득한 마이크로버블(b) 및 단계 5)에서 최종 제조된 본 발명의 마이크로버블-나노리포좀 복합체(c)는 기존 시판 제품(a)과 비교하여도 충분히 높은 수준의 조영 효과를 나타냄을 확인하였다. As a result, as shown in Figure 5, the microbubble-nanoliposomal complex (c) of the present invention finally prepared in the microbubbles (b) and step 5) obtained in step 1) or 1-1) of the preparation example ) Was confirmed to exhibit a sufficiently high level of contrast effect compared to the existing commercial product (a).
또한, 이렇게 우수한 안정성을 갖는 본 발명의 마이크로버블-나노리포좀 복합체가 표적 기관, 조직 또는 세포에 도달했을 때 외부 음파의 자극으로 파괴되어 구조 내부에 담지되어 있던 하나 이상의 치료제 또는 형광물질을 표적 세포에 효과적으로 전달할 수 있는지를 확인하기 위해, 초음파 영상 장치(IU22, Philips사)를 통해 총 10회의 음파 자극(기계지수: 0.7)을 수행하여 인공혈관 팬텀에서의 초음파 조영 효과가 어떻게 변화하는지를 확인하였으며, 그 결과를 도 6의 사진 및 그래프로 나타내었다. 본 발명의 복합체를 초음파에서 고에너지 또는 높은 기계지수에 노출시키는 것을 플래쉬(flash)라고 한다.In addition, when the microbubble-nanoliposomal complex of the present invention having such excellent stability reaches the target organ, tissue, or cell, one or more therapeutic agents or fluorescent substances contained in the structure are destroyed by stimulation of external sound waves to the target cell. In order to confirm the effective delivery, a total of 10 sonic stimuli (mechanical index: 0.7) were performed by using an ultrasonic imaging device (IU22, Philips) to see how the effect of ultrasound contrast on the artificial vascular phantom was changed. The results are shown in the photograph and graph of FIG. Exposing the composite of the present invention to high energy or high mechanical index on ultrasound is called flash.
그 결과, 도 6에서 볼 수 있는 바와 같이, 음파 자극의 횟수가 증가함에 따라 복합체의 구조가 파괴되어 초음파 조영 효과가 크게 감소하는 것을 확인하였으며, 특히 5회 이상에서는 대부분의 복합체의 구조가 파괴되어 조영 효과가 거의 사라지는 것을 확인하였다. 이로써 본 발명의 마이크로버블-나노리포좀 복합체가 암세포와 같은 타겟 부위에 하나 이상의 치료제 뿐 아니라 형광물질을 동시에 효과적으로 전달할 수 있음을 알 수 있다.As a result, as can be seen in Figure 6, as the number of sonic stimulation increases the structure of the complex was confirmed that the ultrasound contrast effect is greatly reduced, especially in more than five times the structure of most of the complex is destroyed It was confirmed that the contrast effect disappeared almost. As a result, it can be seen that the microbubble-nanoliposomal complex of the present invention can effectively deliver fluorescent materials as well as one or more therapeutic agents simultaneously to target sites such as cancer cells.
4) 단백질 정량 분석(BSA 분석)4) Protein Quantitation (BSA Analysis)
본 발명에 따른 마이크로버블-나노리포좀 복합체의 표면에 결합된 항체의 양을 확인하기 위해, 상기 제조예에서 제조된 MB-나노리포좀 복합체를 대상으로 당분야에 통상적인 단백질 분석 기법인 BSA 분석을 실시하였으며, 이때 분광분석은 자외-가시선 분광분석기(UV-vis spectrometer)를 사용하여 수행되었다.In order to confirm the amount of antibody bound to the surface of the microbubble-nanoliposomal complex according to the present invention, the BSA assay, which is a conventional protein analysis technique, is performed on the MB-nanoliposomal complex prepared in Preparation Example In this case, spectroscopic analysis was performed using an UV-vis spectrometer.
그 결과, 도 7에서 볼 수 있는 바와 같이, 파장 560 nm에서 표면에 도입된 항체가 검출되었으며 그 양은 정량분석을 통해 복합체 1 mg 당 1.62 μM임을 확인하였다. As a result, as can be seen in Figure 7, the antibody introduced to the surface was detected at a wavelength of 560 nm and the amount was confirmed to be 1.62 μM per mg complex by quantitative analysis.
시험예 2: 본 발명의 마이크로버블(MB)-나노리포좀 복합체를 이용한 다중 영상 분석 시험Test Example 2: Multiple Image Analysis Test Using Microbubble (MB) -Nanoliposomal Complex of the Present Invention
본 발명에 따른 마이크로버블-나노리포좀 복합체의 암세포 표적 특이성을 확인하기 위해, 상기 제조예에서 제조된 복합체와 대조군으로써 상기 제조예의 단계 4)에서 수득한 항체를 함유하지 않는 마이크로버블(MB)-나노리포좀 복합체를 각각 사용하여 유방암 세포주인 SkBr3 세포를 대상으로 공초점 형광 현미경을 통한 다중 영상 분석(multimodal imaging analysis) 및 FACS 분석을 실시하여 비교하였다.In order to confirm the cancer cell target specificity of the microbubble-nanoliposomal complex according to the present invention, the microbubble (MB) -nano containing the antibody obtained in step 4) of the preparation as a control and the complex prepared in the preparation Liposome complexes were used to compare SkBr3 cells, breast cancer cell lines, by multimodal imaging analysis and FACS analysis using confocal fluorescence microscopy.
그 결과, 도 8의 a에서 볼 수 있는 바와 같이, 아무것도 처리하지 않은 유방암 세포 및 항체를 함유하지 않는 MB-나노리포좀 복합체에서는 형광조영이 거의 나타나지 않은 반면, 본 발명에 따른 항체 함유 마이크로버블-나노리포좀 복합체의 경우 표적 세포에서 매우 우수한 형광 조영을 나타냄을 확인하였다. 또한 FACS를 사용한 정량 분석을 통해 녹색 및 붉은색 형광의 세포 함유량이 94% 이상으로 우수한 것을 확인하였다(도 8의 b). 이로써 본 발명의 마이크로버블-나노리포좀 복합체가 항체결합을 통해 우수한 표적 특이성을 나타냄을 알 수 있다. As a result, as can be seen in Figure 8a, in the MB-nanoliposomal complex containing no antibody and breast cancer cells that did not treat anything, the fluorescence was hardly observed, whereas the antibody-containing microbubble-nano according to the present invention It was confirmed that the liposome complexes showed very good fluorescence contrast in target cells. In addition, the quantitative analysis using FACS confirmed that the cell content of the green and red fluorescence was superior to 94% or more (FIG. 8B). It can be seen that the microbubble-nanoliposomal complex of the present invention exhibits excellent target specificity through antibody binding.
또한, 외부 음파 자극에 의해서 본 발명의 마이크로버블-나노리포좀 내부에 담지되어 있는 형광물질이 암세포 특이적으로 세포내부에 전달될 수 있는지를 확인하기 위해, 초음파 영상 장치를 통해 음파 자극(기계지수: 0.7)을 1분 동안 가한 후 공초점 형광 현미경으로 형광 조영을 확인하였다.In addition, in order to check whether the fluorescent material supported inside the microbubble-nanoliposomes of the present invention can be delivered specifically to cancer cells by external sonic stimulation, sonic stimulation (mechanical index: 0.7) was added for 1 minute, and fluorescence contrast was confirmed by confocal fluorescence microscopy.
그 결과, 도 9에서 볼 수 있는 바와 같이, 본 발명의 마이크로버블-나노리포좀 복합체 내부에 담지되어 있던 녹색 형광 염료인 FITC 및 붉은색 형광 염료인 텍사스 레드가 SkBr3 특이적으로 세포 내에 침투되어 암세포의 형광 조영을 가능하게 함을 확인하였다. 이로써 본 발명의 마이크로버블-나노리포좀 복합체를 이용하여 특정 암 조직 또는 세포 표적에 특이적인 다중 영상 분석을 통한 효과적인 진단이 가능함을 알 수 있다.As a result, as shown in Figure 9, the green fluorescent dye FITC and the red fluorescent dye Texas Red, which was carried inside the microbubble-nanoliposomal complex of the present invention specifically penetrated into the SkBr3 cells of the cancer cells It was confirmed that fluorescence contrast was enabled. As a result, it can be seen that the microbubble-nanoliposomal complex of the present invention enables effective diagnosis through multiple image analysis specific to a specific cancer tissue or cell target.
시험예 3: 본 발명의 마이크로버블(MB)-나노리포좀 복합체를 이용한 약물 전달 시험Test Example 3: Drug delivery test using the microbubble (MB) -nanoliposomal complex of the present invention
본 발명의 따른 마이크로버블-나노리포좀 복합체의 치료제 전달 효과를 확인하기 위해, 상기 제조예에서 제조된 복합체를 대상으로 자외-가시선 분광기를 이용한 분광분석을 수행하여 복합체에 담지된 약물(독소루비신)의 포집 효율을 확인하였다.In order to confirm the drug delivery effect of the microbubble-nanoliposomal complex according to the present invention, the complex prepared in the preparation example was subjected to spectroscopic analysis using an ultraviolet-visible spectrometer to collect the drug (doxorubicin) supported on the complex. The efficiency was confirmed.
그 결과, 도 10에서 볼 수 있는 바와 같이, 본 발명의 복합체에 함유된 대표 항암제인 독소루비신(doxorubicin)의 포집 효율은 88.57%였으며, 따라서 본 발명의 복합체는 내부에 암 치료에 관여하는 다양한 약물을 효과적으로 담지하여 표적 세포에 전달할 수 있음을 알 수 있다. As a result, as can be seen in Figure 10, the capture efficiency of the representative anti-cancer doxorubicin (88%) contained in the complex of the present invention was 88.57%, and thus the complex of the present invention is a complex of various drugs involved in the treatment of cancer It can be seen that it can be effectively carried and delivered to target cells.
또한, 본 발명의 복합체를 이용한 암세포 치료의 효용성을 확인하기 위해 상기 제조예에서 제조된 MB-나노리포좀 복합체를 이용하여 다음과 같은 세포독성(cytotoxicity) 시험을 수행하였다. 먼저, 유방암 세포주인 Skbr3에 상기 제조예에서 제조된 독소루비신이 내부에 담지된 마이크로버블-나노리포좀 복합체 10 mg 또는 대조예로 독소루비신 자체 0.2 ㎍을 각각 처리한 후 3시간 동안 배양하였다. 이후 세포에 결합하지 않은 복합체들을 제거하고 강한 초음파(기계지수: 0.7)를 5분 동안 가한 후 또다시 3시간 동안 배양하였으며, 처리 배지 제거 후 일정 시간(24시간, 48시간 또는 72시간) 동안 배양한 후 MTT 분석을 수행하여 세포독성을 확인하였다.In addition, the following cytotoxicity test was performed using the MB-nanoliposomal complex prepared in Preparation Example to confirm the efficacy of cancer cell therapy using the complex of the present invention. First, Skbr3, a breast cancer cell line, was treated with 10 mg of the microbubble-nanoliposomal complex having the doxorubicin prepared in the above preparation or 0.2 μg of doxorubicin itself as a control, followed by incubation for 3 hours. After removing the complexes that did not bind to the cells, a strong ultrasonic wave (mechanical index: 0.7) was added for 5 minutes and incubated for another 3 hours, and then cultured for a predetermined time (24 hours, 48 hours or 72 hours) after removing the treatment medium. MTT assay was then performed to confirm cytotoxicity.
그 결과, 도 11에서 볼 수 있는 바와 같이, 본 발명의 항암제 함유 마이크로버블-나노리포좀 복합체는 약물을 처리하지 않은 세포주 뿐 아니라 항암제 자체만 처리한 세포주와 비교하여도 우수한 세포독성을 나타냄을 확인하였다(약물만 처리한 경우 72시간이 지나도 85%의 세포 생존율을 나타낸 반면, 본 발명의 복합체를 처리한 경우 24시간 후 63%, 48시간 후 61%, 그리고 72시간 후 35%의 생존율을 나타냄). 이로써 본 발명의 복합체를 사용하는 경우 복합체에 결합되어 있는 항체로 인한 세포 특이성으로 인해 약물이 암세포 특이적으로 세포 내부로 효과적으로 전달되어 우수한 치료효과를 가져올 수 있음을 알 수 있다.As a result, as shown in Figure 11, it was confirmed that the anticancer drug-containing microbubble-nanoliposomal complex of the present invention exhibits excellent cytotoxicity compared to the cell line treated with only the anticancer agent itself as well as the cell line not treated with the drug. (85% cell survival after 72 hours of treatment with drug alone, 63% after 24 hours, 61% after 48 hours, and 35% after 72 hours with complexes of the present invention) . Thus, when using the complex of the present invention it can be seen that due to the cell specificity caused by the antibody bound to the complex, the drug is effectively delivered to the cell specific to the cancer cells can bring excellent therapeutic effect.
시험예 4: 본 발명의 마이크로버블(MB)-나노리포좀 복합체를 이용한 유전자 전달 시험Test Example 4: Gene delivery test using the microbubble (MB) -nanoliposomal complex of the present invention
본 발명에 따른 마이크로버블-나노리포좀 복합체의 유전자 전달 효과를 확인하기 위해, 상기 제조예에서 제조된 녹색 형광 단백질인 tGFP(turbo green fluorescent protein, tGFP) 발현 플라스미드가 내부에 담지된 복합체를 사용하여 유방암 세포주인 SkBr3를 대상으로 다음과 같이 유전자 전달 효과를 확인하였다.To confirm the gene transfer effect of the microbubble-nanoliposomal complex according to the present invention, breast cancer using a complex in which a green fluorescent protein (tGFP) expression plasmid, which is a green fluorescent protein prepared in Preparation Example, was carried therein The gene transfer effect of the cell line SkBr3 was confirmed as follows.
먼저, 분광광도기(spectrophotometer, Nano drop)를 이용하여 상기 제조예에서 제조된 복합체 내 담지된 tGFP 발현 플라스미드의 포집 효율이 약 30%임을 확인하였다. 또한, 복합체 10 mg을 SkBr3에 처리하여 3시간 동안 배양한 후 세포에 결합하지 않은 복합체들을 제거하고 강한 초음파(기계지수(mechanical index): 0.07)를 5분 동안 처리하였으며, 이때 대조군으로 일부 세포들은 초음파 처리를 생략하였다. 대상 세포들을 3시간 동안 추가 배양한 후 복합체를 모두 제거하였으며, 일정 시간(48시간, 72시간 또는 96시간) 동안 배양 후 공초점 형광 현미경을 사용하여 tGFP의 발현을 확인하였다.First, it was confirmed that the collection efficiency of the tGFP expression plasmid supported in the complex prepared in Preparation Example was about 30% using a spectrophotometer (Nano drop). In addition, 10 mg of the complexes were treated with SkBr3, incubated for 3 hours, the complexes which did not bind to the cells were removed, and then subjected to a strong ultrasonic wave (mechanical index: 0.07) for 5 minutes. Sonication was omitted. The cells were further incubated for 3 hours, and then all complexes were removed. After incubation for a period of time (48 hours, 72 hours, or 96 hours), the expression of tGFP was confirmed using confocal fluorescence microscopy.
그 결과, 도 12에서 볼 수 있는 바와 같이, 초음파 처리를 생략한 대조군과 비교하여 본 발명의 복합체를 처리하고 초음파 처리한 세포군에서 시간이 지남에 따라 녹색 형광이 강해진 것을 확인하였다. 이는 tGFP가 세포 내부에서 발현되고 있음을 의미하는 것으로, 따라서 강한 초음파의 처리를 통해 본 발명의 복합체가 파괴되어 내부에 담지되어 있던 플라스미드가 표적 세포 내부로 효과적으로 전달되어 tGFP가 발현되었음을 알 수 있다. As a result, as shown in Figure 12, it was confirmed that the green fluorescence intensified over time in the cell group treated with the complex of the present invention and compared with the control group, the ultrasonic treatment was omitted, compared with the ultrasonic treatment. This means that tGFP is expressed inside the cell, and thus, the plasmid contained therein is effectively delivered to the target cell by destroying the complex of the present invention through the treatment of strong ultrasonic waves, indicating that tGFP is expressed.
결과적으로, 본 발명의 따른 마이크로버블-나노리포좀 복합체는 마이크로버블과 나노리포좀이 화학적으로 안정적인 공유결합을 통해 결합되어 있으며, 소수성인 마이크로버블과 친수성인 나노리포좀 구조 내에 다양한 치료 유전자 및 약물 등의 하나 이상의 치료제 뿐 아니라 추가의 소수성 또는 친수성 형광물질을 하나 이상 담지할 수 있으면서 복합체 외부에 타겟팅 모이어티(targetting moiety)가 결합되어 있으므로, 암 세포 특이적인 초음파 조영 또는 형광 조영을 이용하여 다중 영상 분석(multimodal imaging analysis)을 통해 암을 정확히 진단함과 동시에 하나 이상의 치료제를 표적 암 세포에 접근할 때까지 안정적으로 담지하고 표적 세포 특이적으로 전달할 수 있어 암을 효과적으로 치료할 수 있으므로, 암의 진단 및 치료제 개발에 광범위하게 활용될 수 있다.As a result, the microbubble-nanoliposomal complex according to the present invention is a microbubble and nanoliposomes are bonded through a chemically stable covalent bond, one of a variety of therapeutic genes and drugs in the hydrophobic microbubble and hydrophilic nanoliposome structure In addition to the above therapeutic agents, one or more additional hydrophobic or hydrophilic fluorescent materials can be loaded, and a targeting moiety is coupled to the exterior of the complex. Therefore, multimodal analysis using cancer cell-specific ultrasound or fluorescence contrast is performed. Through imaging analysis, cancer can be accurately diagnosed and at the same time, one or more therapeutic agents can be stably supported and delivered to target cells specifically until they approach the target cancer cells, thereby effectively treating the cancer. Can be widely used have.
시험예 5: 유전자 전달 물질로서 마이크로버블-나노리포좀 복합체의 적용Test Example 5: Application of the microbubble-nanoliposomal complex as a gene transfer material
녹색형광단백질 플라스미드 DNA (pGFP, 10 kbp)를 나노리포좀(Lipo)의 친수성 영역 내부에 위치시켰다. pGFP 로딩 효율을 증가시키기 위해, 프로타민(protamine; PA, 7.5 kD)을 이용한 복합 방법을 사용하였다. pGFP는 서로 다른 농도 비율로 PA와 결합시켜 pGFP 당 600 PAs를 나타내는 20 μM PA 및 34 nM pGFP가 되도록 PA-pGFP 복합체 비를 최적화하였다. 약한 양전하를 띄는 상기 복합체는 음전하를 띄는 Lipo로 이동하여 95% 이상의 로딩용량(loading capacity)을 나타내었다 (도 13 참조). 이하 도면의 설명에서 마이크로버블-나노리포좀 복합체는 팝-파티클로도 표현된다. Green fluorescent protein plasmid DNA ( p GFP, 10 kbp) was placed inside the hydrophilic region of the nanoliposome (Lipo). In order to increase pGFP loading efficiency, a complex method using protamine (PA, 7.5 kD) was used. pGFP was combined with PA at different concentration ratios to optimize the PA-pGFP complex ratio to 20 μM PA and 34 nM pGFP representing 600 PAs per pGFP. The weakly positive complex migrated to negatively charged Lipo and showed a loading capacity of 95% or more (see FIG. 13). In the following description, the microbubble-nanoliposomal complex is also expressed as a pop-particle.
도 13은 PA와 pGFP 복합체의 최적화 및 로딩용량을 측정한 것이다. 프로타민(PA)과 플라스미드-GFP(pGFP)의 복합체는 몰 비율로 최적화되었고 전기영동 시험에 의해 PA와 pGFP의 정확한 수치를 특정할 수 있다. 그 결과, 7.5 kD의 600 PAs는 하나의 10 kbp pGFP와 연결된다. PA-pGFP 복합체는 마이크로버블-나노리포좀 복합체의 나노리포좀 안으로 전달될 수 있는데, 이는 상기 복합체가 지속적인 양전하를 갖고 있기 때문이다. Figure 13 measures the optimization and loading capacity of the PA and pGFP complex. The complex of protamine (PA) and plasmid-GFP (pGFP) was optimized at molar ratios and the electrophoretic test could specify the exact values of PA and pGFP. As a result, 600 PAs of 7.5 kD are linked to one 10 kbp pGFP. The PA-pGFP complex can be delivered into the nanoliposomes of the microbubble-nanoliposomal complex because the complex has a sustained positive charge.
pGFP가 포함된 나노리포좀을 동일한 연결 과정으로 본 발명의 마이크로버블-나노리포좀 복합체 시스템에 제시하였다. 붉은색 형광(red dye) 및 pGFP를 포함하고 Her2 수용체 항체를 표면에 부착한 마이크로 버블-나노리포좀 복합체, MB-Lipo(R+pGFP)-Her2Ab를 유방암 세포주인 SKBR3에 1시간 동안 처리한 후 1분간 고 초음파 에너지 (Mechanical index: 0.61)를 노출 (이를 ‘flash’라 한다) 시켰다. 이후 세포들을 48시간 동안 계속 배양하였다. Nanoliposomes containing pGFP were presented in the microbubble-nanoliposomal complex system of the present invention in the same ligation process. After treatment with MBB-Lipo (R + pGFP) -Her2 Ab , which contains red fluorescence and pGFP and attached Her2 receptor antibody to the surface, for 1 hour to SKBR3, a breast cancer cell line High ultrasonic energy (Mechanical index: 0.61) was exposed for 1 minute (called 'flash'). The cells were then incubated for 48 hours.
도 14는 본 발명의 마이크로버블-나노리포좀 복합체 시스템의 pGFP 전이 효과를 비교한 것이다. 마이크로버블-나노리포좀 복합체의 플라스미드 유전자 전달 효과를 상업적으로 구할 수 있는 대표적인 형질주입 물질인 LipofectamineTM과 비교하였다. SKBR3 세포에 발현된 녹색 형광 단백질은 저배율의 공초점레이저형광현미경(CLSM)으로 관찰된 단일 세포에서 녹색 점으로 관찰되었다. 유세포분석기(FACS) 연구를 이용한 정량분석으로 특정하였다. 마이크로버블-나노리포좀 복합체 시스템은 LipofetamineTM과 비교하여 3배 정도 플라스미드 유전자 전이가 우수하다. 고배율로 관찰시, 대부분의 암 세포는 세포기질에서 녹색 단백질을 포함하였다(b right-hand corner). 게다가, 마이크로버블-나노리포좀 복합체 시스템은 심장 섬유아세포에서 추출된 1차세포(CFt)로 pGFP 전이가 되며, 상기 시스템은 CLSM 및 FACS 분석에 의해 약 60%의 높은 효과가 있음이 나타났다.Figure 14 compares the pGFP transfer effect of the microbubble-nanoliposomal complex system of the present invention. Plasmid gene transfer effect of the microbubble-nanoliposomal complex was compared with Lipofectamine ™ , a representative transfection material that is commercially available. Green fluorescent protein expressed in SKBR3 cells was observed as green dots in single cells observed with low magnification confocal laser fluorescence microscopy (CLSM). It was characterized by quantitative analysis using flow cytometry (FACS) studies. The microbubble-nanoliposomal complex system has about three times better plasmid gene transfer compared to Lipofetamine ™ . When observed at high magnification, most cancer cells contained green protein in the cell substrate (b right-hand corner). In addition, the microbubble-nanoliposomal complex system has a pGFP transition to primary cells (CFt) extracted from cardiac fibroblasts, which has been shown to have a high effect of about 60% by CLSM and FACS analysis.
도 14에서, 대조군으로서 대표적으로 세포 실험에 사용되는 형질 주입 물질인 LipofectamineTM (a)에 비하여 마이크로버블-나노리포좀 복합체는 대부분 세포의 세포기질에서 밝은 녹색 형광을 나타내었고 LipofectamineTM 보다 높은 우수한 전달 효율(3배)을 나타내었다. 게다가, 심장 섬유아세포 일차세포주(CFf)에 특이적인 CD29 항체를 가진 마이크로버블-나노리포좀 복합체 합성하여 pGFP 전이에 사용하였다. 이는 세포주에 전이시키기 어려운 유전자로 알려져 있으나 좋은 효율을 나타내었다. 이러한 결과로 볼 때, 상기 마이크로버블-나노리포좀 복합체는 타겟팅 및 초음파 에너지 자극 실험(시험예 3, 4, 5)에 기초한 임의의 세포에 대한 목적 유전자 전달체로 용이하였다.14, the microbubbles than that representatively cell experiments transformants of Lipofectamine TM (a) injecting material is used in a control nano-liposome complex is highly superior transfer efficiency than Lipofectamine TM showed bright green fluorescence in the cytosol of most cells (3 times). In addition, microbubble-nanoliposomal complexes with CD29 antibodies specific for cardiac fibroblast primary cell lines (CFf) were synthesized and used for pGFP transfer. This is known as a gene that is difficult to transfer to the cell line but showed good efficiency. From these results, the microbubble-nanoliposomal complex was easy to target gene delivery for any cell based on targeting and ultrasound energy stimulation experiments (Test Examples 3, 4, 5).
시험예 6: 약물 또는 siRNA 전달체로서 마이크로버블-나노리포좀 복합체의 치료적 적용Test Example 6: Therapeutic Application of Microbubble-Nanoliposomal Complex as Drug or siRNA Carrier
독소루비신(Doxorubicin, Dox)은 암 치료에 사용되는 약물이며 핵에서 DNA에 끼어들어 작동한다. Dox는 황산암모늄 구배 방법에 따라 (Haran, G., et al., Biochim. Biophys. Acta 1151, 201-215 (1993)), 나노리포좀(Lipo)의 친수성 코어 내부에 쉽게 포함될 수 있다. Doxorubicin (Dox) is a drug used to treat cancer and works by interfering with DNA in the nucleus. Dox can be easily incorporated into the hydrophilic core of a nano liposome (Lipo), according to the ammonium sulfate gradient method (Haran, G., et al., Biochim. Biophys. Acta 1151, 201-215 (1993)).
도 15는 팝-파티클에서 독소루비신의 로딩용량 및 세포에서의 특성을 모니터한 것이다. Dox는 황산 암모늄 구배법을 통하여 팝-파티클(마이크로버블-나노리포좀 복합체)의 나노리포좀으로 삽입되었고, 전달 효과는 자외-가시광 흡수 시험을 통해 정량적으로 산출되었다. Figure 15 monitors the loading capacity of doxorubicin in pop-particles and the properties in the cells. Dox was inserted into nanoliposomes of pop-particles (microbubble-nanoliposomal complexes) via an ammonium sulfate gradient method, and the delivery effect was quantitatively calculated through an ultraviolet-visible absorption test.
도 15에서, Dox의 로딩 효율은 UV-Vis 분광법을 이용하여 90% 이상으로 확인되었다 (도 15의 a). Her2 수용체 항체가 부착되고 Dox를 포함한 마이크로버블- 나노리포좀(DPPC 2.1 mM 및 Dox 19 μM)인 MB-Lipo(Dox)-Her2Ab는 유방암 세포 치료를 진척시켰다. 로딩된 Dox는 CLSM 측정에서 자가-붉은색 형광을 나타내는 전좌(translocation)를 간단하게 모니터될 수 있다 (도 15의 b). 그것은 타겟팅 시점에 세포막 상에서 나타났으며 상기 조건과 같은 초음파 에너지에 노출시키는 플래쉬(flash) 적용 후 세포질에서 사라졌다. 24시간 동안 계속 배양함에 따라, Dox는 세포핵에 침투되었다. 세포 사멸로 인하여 48시간 후에는 건강한 세포 형태를 관찰할 수 없었다. 초음파 노출의 대조 실험군으로서, 초음파에 노출 되지 않은 세포는 48시간 배양에서 살아있었다 (푸른색: DAPI 염색). 이는 마이크로버블-나노리포좀 복합체 시스템이 적절한 에너지의 초음파가 노출 된 경우에만 선택적으로 치료제를 전달하는 것을 의미한다. In FIG. 15, the loading efficiency of Dox was confirmed to be 90% or more using UV-Vis spectroscopy (FIG. 15A). MB-Lipo (Dox) -Her2 Ab with Herb receptor antibody attached and microbubble-nanoliposomes (DPPC 2.1 mM and Dox 19 μM) containing Dox has advanced breast cancer cell therapy. The loaded Dox can simply be monitored for translocations exhibiting self-red fluorescence in CLSM measurements (FIG. 15B). It appeared on the cell membrane at the time of targeting and disappeared in the cytoplasm after flash application of exposure to ultrasonic energy such as the above conditions. As the culture continued for 24 hours, Dox penetrated the cell nucleus. Healthy cell morphology could not be observed after 48 hours due to cell death. As a control group of ultrasound exposure, cells not exposed to ultrasound were alive in 48 hours of culture (blue: DAPI staining). This means that the microbubble-nanoliposomal complex system selectively delivers therapeutic agents only when the appropriate energy ultrasound is exposed.
도 16은 약물 및 치료 유전자 전달과 같은 치료적 마이크로버블-나노리포좀 복합체에 대한 세포 생존율 시험 결과이다. 도 16의 a에서, Dox를 포함하는 마이크로버블-나노리포좀 복합체는 암세포의 세포사멸을 진행시켰으나 (생존율 40% 이하) 마이크로버블-나노리포좀 복합체 없이 초음파만 노출시킨 경우 또는 Dox만 처리된 세포는 세포생존율의 유의한 감소가 없었다. 즉, Dox를 포함하는 마이크로버블-나노리포좀 복합체로 치료한 세포에 대한 분석은 72시간에서 동일한 농도의 유리 Dox 처리 시(생존율 ~90%) 보다 세포 사멸을 더 높게(생존율 ~35%) 유도함을 나타내었다 (도 16의 a). 도 16의 b에서, 치료 유전자 전달에 대한 실험에서는 항암제의 실험과 마찬가지 결과로서 마이크로버블-나노리포좀 복합체 없이 초음파만 노출시킨 경우 또는 siSTAT3만 투여한 세포들은 세포생존율에 영향을 미치지 않았다. 또한, siSTAT3를 포함하는 마이크로버블-나노리포좀 복합체만 처치하고 초음파를 노출 시키지 않은 경우 72시간에서 세포생존율이 약간 감소되었으나 120시간에서 세포수는 오히려 증가되었는데, 이는 비-효과적인 siSTAT3 유전자 전달로 인하여 세포 증식 과정이 회복된 것으로 판단된다. 그러나 siSTAT3를 포함하는 마이크로버블-나노리포좀 복합체를 처치하고 초음파를 노출시킨 후의 세포는 120시간 후에서 약 ~30%의 낮은 생존율을 나타내었다. 도 16의 c에서, 이중 치료적 팝-파티클(Dox+siSTAT3)로 암세포를 처리하는 경우에는 동일한 120 시간에서 약 10%만의 세포 생존율을 보여서 현저한 치료 효과를 나타내었다. 대조군으로서 Dox와 siSTAT3 (Dox+siSTAT3)만을 처리하거나 초음파의 노출이 없는 마이크로버블-나노리포좀 복합체는 세포사멸을 유도하는데 있어서 높은 효과를 나타내지 못했다. 또한 도 16의 d에서, 치료 시험 동안 현미경으로 세포 밀도를 모니터하였다. 실제로, 72시간 및 120시간에서 Dox와 siSTAT3를 포함하고 있는 마이크로버블-나노리포좀 복합체(Dox+siSTAT3)에 의하여 처리된 세포는 밀도가 낮게 나타났으며 48시간에서 세포자살 세포에 대한 전형적인 형태를 명확하게 관찰할 수 있었다.FIG. 16 shows cell viability test results for therapeutic microbubble-nanoliposomal complexes such as drug and therapeutic gene delivery. In FIG. 16A, the microbubble-nanoliposomal complex containing Dox proceeded to apoptosis of cancer cells (survival rate of 40% or less), but when only ultrasound was exposed without the microbubble-nanoliposomal complex or the cells treated with Dox alone were cells. There was no significant decrease in survival. In other words, analysis of cells treated with microbubble-nanoliposomal complexes containing Dox induced higher cell death (survival rate ~ 35%) than 72-hour treatment of free Dox (survival rate ~ 90%). (A in FIG. 16). In FIG. 16B, in the experiments on the delivery of therapeutic genes, as the result of the anticancer drug experiment, the cells exposed only to the ultrasound without the microbubble-nanoliposomal complex or the siSTAT3 alone did not affect the cell viability. In addition, when only microbubble-nanoliposomal complexes containing siSTAT3 were treated and no ultrasound exposure was observed, cell viability was slightly decreased at 72 hours, but rather increased at 120 hours, due to inefficient siSTAT3 gene delivery. The growth process seems to have recovered. However, cells treated with the microbubble-nanoliposomal complex containing siSTAT3 and exposed to ultrasound showed a low survival rate of ˜30% after 120 hours. In FIG. 16C, the treatment of cancer cells with dual therapeutic pop-particles (Dox + siSTAT3) showed only about 10% cell viability at the same 120 hours, indicating a significant therapeutic effect. The microbubble-nanoliposomal complex treated with only Dox and siSTAT3 (Dox + siSTAT3) or without ultrasound exposure did not show a high effect on inducing cell death. Also in FIG. 16 d, the cell density was monitored under the microscope during the treatment test. Indeed, cells treated with the microbubble-nanoliposomal complex (Dox + siSTAT3) containing Dox and siSTAT3 at 72 and 120 hours appeared to be less dense and clarified typical morphology for apoptotic cells at 48 hours. Could observe.
STAT3(Signal transducer and activator of transcription 3)은 전사인자이고 STAT3 유전자에 의해 암호화되는 그 단백질은 세포 증식에 관여하는 STAT 단백질 패밀리에 속한다. 최근의 STAT3에 대한 종양 억제 siRNA는 경로가 사멸 수용체 DR4 및 DR5와 관련되어 있는 것으로 보고되어 있다 (Kang, Y. et al. Biochim. Biophys. Acta 1830, 2638-2648 (2013)). 유전자 도입 과정과 같이, STAT3에 대한 siRNA(siSTST3)는 PA 복합체 형성 후 리포좀에 간단히 삽입되어 유사한 로딩 용량 약 95%를 나타내었다. SKBR3에 siSTAT3의 전달 후, 타겟 유전자 및 단백질은 PCR 및 웨스턴 블롯팅을 이용하여 발현 수준을 측정하였다 (도 17의 a와 b 참조). Signal transducer and activator of transcription 3 (STAT3) is a transcription factor and the protein encoded by the STAT3 gene belongs to the family of STAT proteins involved in cell proliferation. Recent tumor suppressor siRNAs for STAT3 have been reported to have pathways associated with death receptors DR4 and DR5 (Kang, Y. et al. Biochim. Biophys. Acta 1830, 2638-2648 (2013)). As with the transduction process, siRNA for STAT3 (siSTST3) was simply inserted into liposomes after PA complex formation, showing a similar loading capacity of about 95%. After delivery of siSTAT3 to SKBR3, the target genes and proteins were measured for expression levels using PCR and Western blotting (see FIGS. 17 a and b).
도 17은 siSTAT3를 포함한 마이크로버블-나노리포좀 복합체(siSTAT3) 처리 및 세포사멸 시험 후 암세포에서 STAT3 단백질 및 유전자에 대한 발현 수준을 측정한 것이다. siSTAT3를 포함하고 항체가 있는 마이크로버블-나노리포좀 복합체, 즉 MB-Lipo(siSTAT3)-Her2Ab로 처리한 후 암세포는 PCR 및 웨스턴 블롯으로 타겟 STAT3 유전자 및 단백질 수준을 측정하였다. FIG. 17 measures the expression levels of STAT3 proteins and genes in cancer cells after microbubble-nanoliposomal complex (siSTAT3) including siSTAT3 treatment and apoptosis test. After treatment with the microbubble-nanoliposomal complex containing siSTAT3 and with antibody, MB-Lipo (siSTAT3) -Her2 Ab , the cancer cells were subjected to PCR and western blot to determine target STAT3 gene and protein levels.
도 17의 a와 b에서, siSTAT3를 포함한 마이크로버블-나노리포좀 복합체(siSTAT3)로 처리된 세포의 타겟 단백질 발현 수준은 그 타겟 유전자 및 단백질 강도가 현저하게 감소되었다. 대조군으로서 초음파만 노출시킨 세포나 siSTAT3만 투여한 세포는 STAT3 유전자의 발현에서 별다른 변화를 보이지 않았다. 정량적인 분석을 하였을 때 상기 siSTAT3를 포함한 마이크로버블-나노리포좀 복합체(siSTAT3), MB-Lipo(siSTAT3)-Her2Ab는 대조군으로서 초음파만 노출하였거나 또는 네이키드(naked) siSTAT3 처리한 세포들와 비교하여 5배의 타겟 STAT3 유전자 및 단백질의 억제 수준을 나타내었다.In a and b of FIG. 17, target protein expression levels of cells treated with microbubble-nanoliposomal complex (siSTAT3) containing siSTAT3 were significantly reduced in their target genes and protein intensity. As a control, cells exposed only to ultrasound or cells treated with only siSTAT3 showed no change in STAT3 gene expression. In quantitative analysis, the microbubble-nanoliposomal complex (siSTAT3) and MB-Lipo (siSTAT3) -Her2 Ab containing siSTAT3 were exposed to ultrasound only as a control or compared to naked siSTAT3 treated cells. Inhibition levels of pear target STAT3 genes and proteins were shown.
또한 도 17의 c와 d에서, siSTAT3 전달을 통해 세포 사멸 메커니즘을 규명하기 위해, 본 발명자들은 세포 증식 및 세포자살성 세포사멸과 관련되어 있는 cyclin-D1, c-Myc, cyclin-D2 및 caspase3에 대한 유전자와 단백질들을 조사하였다. 이들은 대조군과 비교하여 STAT3와 cyclin-D1, c-Myc 및 cyclin-D2의 억제 수준이 유의하게 나타났다. 또한, siSTAT3를 포함한 마이크로버블-나노리포좀 복합체(siSTAT3)에 처리된 세포는 세포자살(apoptosis)에 대한 지시 단백질인, caspase3이 활성화되었으며 이는 세포자살성 세포사멸 과정이 이어짐을 의미한다. 대조군으로서 네이키드 siSTAT3로 처리되거나 siSTAT3를 포함한 마이크로버블-나노리포좀 복합체(siSTAT3)에 처리되었다 하더라도 초음파에 노출 되지 않은 세포는 세포사멸 과정을 나타내지 않았다. In addition, in Figures c and d of the present invention, in order to elucidate the mechanism of cell death through siSTAT3 delivery, the present inventors are directed to cyclin-D1, c-Myc, cyclin-D2 and caspase3, which are involved in cell proliferation and apoptosis. Genes and proteins were examined. Compared with the control group, they showed significantly higher levels of inhibition of STAT3, cyclin-D1, c-Myc and cyclin-D2. In addition, cells treated with the microbubble-nanoliposomal complex (siSTAT3) containing siSTAT3 were activated caspase3, an indicator protein for apoptosis, which means that the apoptotic apoptosis process is followed. Cells that were not exposed to ultrasound did not show apoptosis, even if treated with naked siSTAT3 as a control or microbubble-nanoliposomal complex containing siSTAT3 (siSTAT3).
도 16에서, siSTAT3를 포함한 마이크로버블-나노리포좀 복합체(siSTAT3)에 처리되고 초음파에 노출 된 세포는 120시간에서 세포 생존율이 ~ 30%였다. 또한, 마이크로버블-나노리포좀 복합체 시스템의 상승적 치료 효과를 강화하기 위해, 본 발명자들은 Dox와 siSTAT3를 동시에 포함하는 마이크로버블-나노리포좀 복합체로 암세포를 처리를 진행하였다. 72시간에서 세포생존율이 ~20%, 120시간에서 10% 이하로 나타났다. 현미경으로 처리 동안의 세포를 관찰하였을 때, 120시간에서 생존할 세포를 사실상 찾지 못했다 (도 16의 d). 또한 48시간에서 Dox와 siSTAT3를 포함한 마이크로버블-나노리포좀 복합체(Dox+siSTAT3) 치료 동안 세포자살성 세포 형태를 확인하였다.In FIG. 16, cells treated with microbubble-nanoliposomal complex (siSTAT3) containing siSTAT3 and exposed to ultrasound had a cell viability of ˜30% at 120 hours. In addition, in order to enhance the synergistic therapeutic effect of the microbubble-nanoliposomal complex system, the present inventors treated cancer cells with the microbubble-nanoliposomal complex containing Dox and siSTAT3 simultaneously. The cell survival rate was ~ 20% at 72 hours and below 10% at 120 hours. Observation of the cells during the treatment under a microscope revealed virtually no cells to survive at 120 hours (FIG. 16 d). At 48 hours, apoptotic cell morphology was confirmed during treatment of microbubble-nanoliposomal complex (Dox + siSTAT3) including Dox and siSTAT3.
시험예 7: 동물모델의 생체 내에서 초음파 기반 마이크로버블-나노리포좀 복합체의 치료진단 적용Experimental Example 7 Application of Therapeutic Diagnosis of Ultrasound-Based Microbubble-Nanoliposomal Complexes in Animal Models
초음파(ultrasound, US)에 기초한 마이크로버블-나노리포좀 복합체의 생체내 진단 및 자극-반응성 치료를 확인하기 위해, 신장에 VX2 간암세포의 이식을 통해 암 발생 토끼 동물모델을 제조하였다. 지난 수십년간, 토끼는 VX2 암종(carcinoma)을 지닌 악성종양의 진단 및 치료의 실험 연구에 사용되어 왔다 (Virmani, S. et al. J. Vasc. Interv. Radiol. 18, 1280-1286 (2007)). 본 발명자가 개발한 마이크로버블-나노리포좀을 이용한 타겟 특이적인 약물 및 유전자 전달을 확인하기 위해, 이식된 VX2 암 조직이 세포-타겟팅 마커를 적절하게 발현하는지를 확인하였다. To confirm the in vivo diagnosis and stimulus-responsive treatment of the microbubble-nanoliposomal complex based on ultrasound (US), a cancer-causing rabbit animal model was prepared through transplantation of VX2 liver cancer cells into the kidney. In the last decades, rabbits have been used in experimental studies of the diagnosis and treatment of malignancies with VX2 carcinoma (Virmani, S. et al. J. Vasc. Interv . Radiol . 18, 1280-1286 (2007) ). In order to confirm target specific drug and gene delivery using the microbubble-nanoliposomes developed by the present inventors, it was confirmed that the transplanted VX2 cancer tissues properly express the cell-targeting markers.
도 18은 토끼의 신장에서 이식된 VX2 암의 특성을 나타낸 것이다. 도 18의 a에서, 신장에서 생긴 VX2 종양의 Her2 수용체 발현을 확인하기 위해, 포매된 종양(embedded tumor)(양성) 및 토끼 근육 조직(음성 대조군)의 조직학적 절단한 후 헤마톡실린과 에오신(H&E) 염색을 수행하였다. 도 18의 b에서, Her2 발현을 반영하는 어두운 갈색은 종양 영역에서 나타났다. 도 18의 c에서, Her2 수용체 발현 수준은 웨스턴 블롯으로 분석하였다. 이식된 VX2 암은 상대적으로 높은 Her2 발현 수준을 나타내었다. 도 18의 d에서, 붉은색 형광물질을 함유하고 Her2 항체가 결합된 마이크로버블-나노리포좀 복합체(MB-Lipo(R)-Her2Ab)로 특이적 타겟팅에 대한 능력을 확인한 결과, CLSM에 의해 정상 신장 조직과 비교하여 생체외 시험에서 이식된 암 조직에 대해 충분한 선택성을 나타내었다 (d; D+R; merged DAPI and red fluorescence images).18 shows the characteristics of VX2 cancer transplanted in rabbit kidneys. In FIG. 18A, to confirm Her2 receptor expression of VX2 tumors in the kidney, hematoxylin and eosin (embedded tumor (positive) and rabbit muscle tissue (negative control) after histological cleavage H & E) staining was performed. In FIG. 18B, dark brown reflecting Her2 expression appeared in the tumor area. In c of FIG. 18, Her2 receptor expression levels were analyzed by Western blot. Transplanted VX2 cancers showed relatively high Her2 expression levels. In FIG. 18 d, the ability of specific targeting to the microbubble-nanoliposomal complex (MB-Lipo (R) -Her2 Ab ) containing a red fluorescent substance and to which the Her2 antibody was bound was confirmed, and was confirmed by CLSM. In vitro tests showed sufficient selectivity for transplanted cancer tissues compared to kidney tissues (d; D + R; merged DAPI and red fluorescence images).
즉, 포매된 VX2 암은 특정 Her2 수용체를 발현하였고 조직의 헤마톡실린과 에오신(H&E) 염색 및 웨스턴 블로팅을 이용하여 그 수준을 결정하였다. 또한 붉은색 형광물질을 함유하고 Her2 항체가 결합된 마이크로버블-나노리포좀 복합체, MB-Lipo(R)-Her2Ab은 생체외 실험에서 VX2 암 조직을 인식할 수 있었다. That is, embedded VX2 cancer expressed specific Her2 receptors and their levels were determined using hematoxylin and eosin (H & E) staining and western blotting of tissues. In addition, the microbubble-nanoliposomal complex, MB-Lipo (R) -Her2 Ab , which contained red fluorescent substance and bound to Her2 antibody, was able to recognize VX2 cancer tissue in vitro.
또한, 초음파(US) 이미징과 치료 효과의 이중 능력이 있는 상기 마이크로버블-나노리포좀 복합체는 토끼의 trans-catheter intra-articular (IA) 주사를 통해 신장에 종양을 직접적으로 이식하였다. In addition, the microbubble-nanoliposomal complex, which has the dual ability of ultrasound (US) imaging and therapeutic effect, directly implanted tumors into the kidney via trans-catheter intra-articular (IA) injection in rabbits.
도 19는 생체 내에서 약물 및 유전자의 암 특이적 초음파 이미지 및 수송을 나타낸 것이다. (a) Her2 항체가 결합되고 대표적인 항암제인 Dox와 대표적인 siRNA 유전자 치료제인 siSTAT3를 포함한 마이크로버블-나노리포좀 복합체, MB-Lipo(Dox+siSTAT3)-Her2Ab를 IA(intra-articular) 주사한 후, 초음파 영상으로 토끼 신장에서 이식된 종양 영역을 시각화하였고 초음파 에너지를 노출시킨 후 마이크로버블의 파괴로 인해 초음파 영상 신호가 사라짐을 볼 수 있다. (b) 추출된 암 조직에서, CLSM을 이용하여 양성대조군으로서 초음파 에너지에 노출시키지 않은 종양 조직 및 Her2 항체가 결합되고 Dox와 siSTAT3를 포함한 마이크로버블-나노리포좀 복합체를 처리하지 않은 종양조직들과 비교할 때 Her2 항체가 결합되고 Dox와 siSTAT3를 포함한 마이크로버블-나노리포좀 복합체를 처리한 종양 조직은 Dox(붉은색 형광)는 높은 농도를 나타내었다. (c) 또한, Her2 항체가 결합되고 Dox와 siSTAT3를 포함한 마이크로버블-나노리포좀 복합체에 의해 전달된 siSTAT3의 효율은 PCR로 STAT3 타겟 유전자 수준을 직접적으로 측정하였으며 세포 실험에서와 마찬가지로 현저히 감소된 수준을 나타내었다. (d) 종양 조직을 H&E 염색으로 조직학적 변화를 분석하였을 때에도 Her2 항체가 결합되고 Dox와 siSTAT3를 포함한 마이크로버블-나노리포좀 복합체로 처리한 조직이 그렇지 않은 조직들에 비하여 그 세포 밀도가 감소함을 알 수 있었다. 19 shows cancer specific ultrasound images and transport of drugs and genes in vivo. (a) IA (intra-articular) injection of MB-Lipo (Dox + siSTAT3) -Her2 Ab with Her2 antibody bound and a microbubble-nanoliposomal complex, including Dox, a representative anti-cancer agent, and siSTAT3, a representative siRNA gene therapy agent, Ultrasonographic images of the implanted tumor area of the rabbit kidney were visualized and the ultrasound image signal disappeared due to destruction of the microbubble after exposure to ultrasound energy. (b) In extracted cancer tissues, compared to tumor tissues that were not exposed to ultrasound energy as a positive control using CLSM and tumor tissues to which Her2 antibody was bound and not treated with the microbubble-nanoliposomal complex containing Dox and siSTAT3 Tumor tissues treated with the microbubble-nanoliposomal complex containing Dox and siSTAT3 bound to the Her2 antibody showed high concentrations of Dox (red fluorescence). (c) In addition, the efficiency of siSTAT3 bound to the Her2 antibody and delivered by the microbubble-nanoliposomal complex including Dox and siSTAT3 directly measured STAT3 target gene levels by PCR and markedly decreased levels as in cell experiments. Indicated. (d) When tumor tissues were analyzed for histological changes by H & E staining, tissues treated with Her2 antibody and treated with microbubble-nanoliposomal complexes containing Dox and siSTAT3 decreased their cell density compared to those without. Could know.
도 19에서, her2 항체가 결합되고 Dox와 siSTAT3를 포함한 마이크로버블-나노리포좀 복합체를 IA 주사 후, 토끼의 신장에서 발생한 종양은 마이크로버블-나노리포좀의 복합체 중 마이크로버블의 초음파 조영효과에 의하여 초음파 영상에서 고에코를 보여 밝게 이미지되었고 10회의 초음파 고에너지 노출 (Mechanical index: 0.61)후 그 에코는 점차적으로 약해졌다. 반면에 her2 항체가 결합되지 않은 마이크로버블-나노리포좀 복합체를 대조군 종양에 주입하였을 때, 생체 내에서 항체 결합된 팝-파티클의 강력한 타겟팅 능력을 나타내는 초음파 신호는 높은 강도를 나타내지 않았다 (데이터 미도시). 항암제(Dox) 및 치료 유전자(siSTAT3)를 포함하는 마이크로버블-나노리포좀 복합체를 토끼에 주사하고 토끼에서 자란 VX2 종양에 초음파 고에너지에 노출 후 종양 조직을 추출하였다. 추출한 종양 조직은 CLSM(Confocal Laser Scanning Microscope) 및 PCR을 통해 항암제와 치료 유전자의 종양조직으로의 전달을 확인하였다. 상기 VX2 암 조직에 전이된 Dox는 스스로 붉은색 형광을 뚜렷하게 나타내었으며 타겟 STAT3 유전자 수준은 전달된 siSTAT3에 의해 해당 유전자의 발현이 억제되었다. 초음파 고에너지에 노출되지 않은 조직은 Dox와 siSTAT3의 효과가 전혀 검출되지 않았고 her2 항체가 결합되고 Dox와 siSTAT3를 포함한 마이크로버블-나노리포좀 복합체로 처리하지 않은 대조군도 역시 Dox와 siSTAT3의 효과가 나타나지 않았다. 이는 본 발명의 타겟 지향성 치료제 함유 마이크로버블-나노리포좀 복합체가 단지 특이적 암 사이트에만 전달되어 외부 초음파 고에너지 자극에 의해 치료제의 방출 여부가 조절될 수 있음을 의미한다. H&E 염색에서, 대조군에 비하여 her2 항체가 결합되고 Dox와 siSTAT3를 포함한 마이크로버블-나노리포좀 복합체로 치료한 암 조직의 암세포 밀도가 대조군과 비교하여 드문드문 분포하였는데 이는 VX2 종양조직이 마이크로 버블-나노리포좀 복합체를 이용한 전달체의 종양세포로의 전달에 의하여 부분적으로 파괴되었기 때문이다. In FIG. 19, after IA injection of a microbubble-nanoliposomal complex including herx antibody and Dox and siSTAT3, tumors in rabbit kidney were examined by ultrasound imaging effect of microbubble in the microbubble-nanoliposomal complex. The image of the echo was bright and imaged brightly and after 10 ultrasonic high energy exposures (Mechanical index: 0.61) the echo gradually faded. On the other hand, when the microbubble-nanoliposomal complex without her2 antibody was injected into the control tumor, the ultrasonic signal indicating the strong targeting ability of the antibody-bound pop-particle in vivo did not show high intensity (data not shown). . Microbubble-nanoliposomal complexes containing an anticancer agent (Dox) and a therapeutic gene (siSTAT3) were injected into rabbits and tumor tissues were extracted after ultrasound high energy exposure to VX2 tumors grown in rabbits. The extracted tumor tissues were confirmed by CLSM (Confocal Laser Scanning Microscope) and PCR to deliver the anticancer agent and therapeutic genes to the tumor tissues. The Dox transferred to the VX2 cancer tissues clearly showed red fluorescence by themselves, and the target STAT3 gene level was suppressed by the delivered siSTAT3. Tissues that were not exposed to ultrasound high energy did not detect any effects of Dox and siSTAT3, and controls that were not treated with microbubble-nanoliposomal complexes with her2 antibody bound and containing Dox and siSTAT3 also showed no effects of Dox and siSTAT3. . This means that the target-directed therapeutic agent-containing microbubble-nanoliposomal complex of the present invention can be delivered only to specific cancer sites so that the release of the therapeutic agent can be controlled by external ultrasonic high energy stimulation. In H & E staining, cancer cell density of cancer tissues treated with microbubble-nanoliposomal complexes containing Dox and siSTAT3 combined with her2 antibody compared to the control group was sparsely distributed compared to the control group. This is because the complex was partially destroyed by delivery of the carrier to tumor cells.
또한, 토끼 종양 모델에서 타겟 지향성 치료제 함유 마이크로버블-나노리포좀 복합체를 반복하여 투여함으로써 실제 임상과 비슷한 환경에서의 생체 내에서 치료 효과를 확인하였다. In addition, by repeatedly administering the target-directed therapeutic agent-containing microbubble-nanoliposomal complex in the rabbit tumor model, the therapeutic effect was confirmed in vivo in an environment similar to the actual clinic.
도 20은 타겟 지향성 치료제 함유 마이크로버블-나노리포좀 복합체를 이용하여 토끼 종양에서의 암치료에 대해 평가한 것이다. 생체 내에서 her2 항체가 결합되고 Dox와 siSTAT3를 포함한 마이크로버블-나노리포좀 복합체, MB-Lipo(Dox+siSTAT3)-Her2Ab에 의한 종양 성장 저해의 진척을 모니터하기 위해, 정확한 종양 크기를 자기공명영상 (MRI, 횡단면) 및 초음파 영상기기로 측정하였다 (a: 흰색 화살표는 이식된 암 종양을 나타낸다). FIG. 20 evaluates the treatment of cancer in rabbit tumors using a target-directed therapeutic agent containing microbubble-nanoliposomal complex. To monitor the progress of tumor growth inhibition by MB2Lipo (Dox + siSTAT3) -Her2 Ab , the herb antibody bound in vivo and microbubble-nanoliposomal complexes including Dox and siSTAT3, magnetic resonance imaging was performed. (MRI, cross section) and ultrasound imaging (a: white arrows indicate transplanted cancer tumors).
도 20에서, her2 항체가 결합되고 Dox와 siSTAT3를 포함한 마이크로버블-나노리포좀 복합체를 실험 시작 1일 및 5일에 반복적으로 투여 하였고 실험 0일, 4일, 10일에 MRI 및 초음파를 이용하여 종양의 정확한 크기를 측정하였다. 상기 her2 항체가 결합되고 Dox와 siSTAT3를 포함한 마이크로버블-나노리포좀 복합체를 투여하고 초음파의 고에너지를 이용하여 치료한 종괴는 초음파 고에너지를 이용하여 치료하지 않은 종괴나 아예 마이크로버블-나노리포좀 복합체를 투여하지 않은 종괴와 비교하여 그 성장 속도가 감소되었다. In FIG. 20, microbubble-nanoliposomal complexes bound to her2 antibody and containing Dox and siSTAT3 were repeatedly administered on days 1 and 5 of the start of the experiment, and tumors were obtained by MRI and ultrasound on days 0, 4, and 10 of the experiment. The exact size of was measured. The her2 antibody was bound to a microbubble-nanoliposomal complex containing Dox and siSTAT3, and the mass treated with high energy of the ultrasonic mass was not treated with ultra high energy or a microbubble-nanoliposomal complex. The growth rate was reduced compared to the mass not administered.
또한, 토끼 동물 종양 모델에서 her2 항체가 결합된 마이크로 버블- 나노리포좀 복합체의 생체 내 분포(bio-distribution)를 간, 폐, 신장 및 암에서 확인하였다. In addition, bio-distribution of the microbubble-nanoliposomal conjugates with her2 antibody in rabbit animal tumor models was confirmed in liver, lung, kidney and cancer.
도 21은 생채 내에서 형광을 포함한 마이크로 버블-나노리포좀 복합체의 생체 내 분포를 나타낸 것이다. 검출을 위해, 녹색 및 붉은색 형광 마이크로 버블-나노리포좀 복합체, MB(G)-Lipo(R)-Her2Ab를 정맥으로 주사하고 생체 조직 밖의 CLSM 분석에 의해 유기염료 강도를 특성화하였다. 유기염료는 플래쉬 적용에 관계없이 간과 폐 기관에 주로 분포하는 것으로 나타났다. 그러나 종양 조직에서, 상기 염료는 플래쉬 후 약 3배 정도 강도가 증가하였다. Figure 21 shows the in vivo distribution of the micro bubble-nanoliposomal complex containing fluorescence in raw vegetables. For detection, green and red fluorescent micro bubble-nanoliposomal complexes, MB (G) -Lipo (R) -Her2 Ab , were injected intravenously and the organic dye intensity was characterized by CLSM analysis outside the living tissue. Organic dyes were found to be mainly distributed in liver and lung organs regardless of flash application. However, in tumor tissues, the dye increased in intensity by about three times after flash.
또한, 마이크로 버블-나노리포좀 복합체의 방출(excretion issue)은 임상적용 가능성을 이해하는데 중요하므로 다양한 조직에서 조사되었다. In addition, the release issue of the micro bubble-nanoliposomal complex has been investigated in various tissues as it is important for understanding the clinical applicability.
도 22는 생체 내에서 마이크로 버블-나노리포좀 복합체의 방출을 측정한 것이다. 마이크로 버블-나노리포좀 복합체 시스템을 이용한 조영제(imaging agent) 또는 치료 적용을 시험하기 위해, 방출 시험(excretion study)이 필요하다. 여기서는, 본 발명자들이 마우스 동물모델을 사용하였는데 토끼 보다 빠른 대사 생체 모델이기 때문이다. 형광물질을 포함하고 있는 마이크로 버블-나노리포좀 복합체를 꼬리 정맥으로 주사한 후, 48시간째에 마우스의 여러 장기에서 형광을 측정하였다. 그 결과, 상기 형광물질 함유 마이크로 버블-나노리포좀 복합체는 어떠한 이상 조직 형태가 없이 마우스 체내에서 완전하게 빠져 나갔다. 즉, 형광물질 함유 마이크로 버블-나노리포좀 복합체의 주사 후, 48시간 후 마우스의 여러 장기에서 형광물질 함유 마이크로 버블-나노리포좀 복합체의 분명한 방출을 확인하였다.FIG. 22 measures the release of micro bubble-nanoliposomal complexes in vivo. FIG. In order to test imaging agents or therapeutic applications using the micro bubble-nanoliposomal complex system, an exclusion study is required. Here, we used a mouse animal model because it is a faster metabolic biological model than a rabbit. After injecting the microbubble-nanoliposomal complex containing the fluorescent material into the tail vein, fluorescence was measured in various organs of the mouse at 48 hours. As a result, the fluorescent substance-containing micro bubble-nanoliposomal complex completely exited the mouse body without any abnormal tissue morphology. That is, 48 hours after injection of the fluorescent substance-containing micro bubble-nanoliposomal complex, clear release of the fluorescent substance-containing micro bubble-nanoliposomal complex was confirmed in various organs of the mouse.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.
본 발명에 따른 암 세포 특이적인 진단 및 치료를 가능하게 하는 마이크로버블-나노리포좀 복합체는 소수성인 마이크로버블과 친수성인 나노리포좀이 화학적으로 안정적인 공유결합으로 결합되어 있어 복합체 구조 내부에 치료 유전자 및 약물 등의 하나 이상의 치료제 뿐 아니라 추가의 소수성 또는 친수성의 형광물질을 하나 이상 안정적으로 담지할 수 있으면서 외부에 타켓팅 모이어티(targetting moiety)가 결합되어 있어 우수한 표적 특이성을 나타낸다. 따라서 다중 영상 분석을 통해 암을 진단할 수 있음과 동시에 다양한 암 질환의 효과적인 진단 및 치료제 개발에 유용하게 활용될 수 있다.The microbubble-nanoliposomal complex which enables cancer cell-specific diagnosis and treatment according to the present invention is hydrophobic microbubbles and hydrophilic nanoliposomes are chemically stable covalently bonded to the therapeutic genes and drugs in the complex structure, etc. It is possible to stably support one or more additional hydrophobic or hydrophilic fluorescent materials as well as one or more therapeutic agents of the externally targeting targeting moiety (targeting moiety) coupled to exhibit excellent target specificity. Therefore, it is possible to diagnose cancer through multiple image analysis and at the same time, it can be usefully used for effective diagnosis and treatment of various cancer diseases.

Claims (15)

  1. 소수성인 마이크로버블과 친수성인 나노리포좀이 공유결합으로 결합되어 있는 복합체로서, 상기 복합체 구조 내부에 하나 이상의 치료제를 담지하면서 외부에 타겟팅 모이어티(targetting moiety)를 포함하고 있는 마이크로버블-나노리포좀 복합체.A microbubble-nanoliposomal complex comprising a hydrophobic microbubble and a hydrophilic nanoliposome covalently bonded to each other, wherein the microbubble-nanoliposomal complex contains a targeting moiety externally while carrying at least one therapeutic agent inside the complex structure.
  2. 제 1 항에 있어서,The method of claim 1,
    상기 치료제는 치료 유전자 및 약물 중에서 선택된 것임을 특징으로 하는 마이크로버블-나노리포좀 복합체.The therapeutic agent is a microbubble-nanoliposomal complex, characterized in that selected from therapeutic genes and drugs.
  3. 제 2 항에 있어서,The method of claim 2,
    상기 치료 유전자는 발현 플라스미드, siRNA, shRNA, 마이크로RNA 및 마이크로RNA의 길항제를 포함하는 DNA 및 RNA로부터 하나 이상 선택된 것임을 특징으로 하는 마이크로버블-나노리포좀 복합체.The therapeutic gene is a microbubble-nanoliposomal complex, characterized in that at least one selected from DNA and RNA including an antagonist of expression plasmid, siRNA, shRNA, microRNA and microRNA.
  4. 제 2 항에 있어서,The method of claim 2,
    상기 약물은 독소루비신(doxorubicin), 파클리탁셀(paclitaxel), 도세탁셀(docetaxel)을 포함하는 항암제들로부터 하나 이상 선택된 것임을 특징으로 하는 마이크로버블-나노리포좀 복합체.The drug is a microbubble-nanoliposomal complex, characterized in that at least one selected from anticancer agents, including doxorubicin, paclitaxel (paclitaxel), docetaxel (docetaxel).
  5. 제 1 항에 있어서,The method of claim 1,
    상기 타겟팅 모이어티는 바이오틴, 스트렙타비딘, 아비딘, 항체, 앱타머, 폴리펩타이드, 펩타이드, 리간드, 수용체, 렉틴, 당, 지질, 당지질 및 핵산을 포함하는 군으로부터 하나 이상 선택된 것임을 특징으로 하는 마이크로버블-나노리포좀 복합체.The targeting moiety is a microbubble, characterized in that at least one selected from the group consisting of biotin, streptavidin, avidin, antibodies, aptamers, polypeptides, peptides, ligands, receptors, lectins, sugars, lipids, glycolipids and nucleic acids Nanoliposome complexes.
  6. 제 1 항에 있어서,The method of claim 1,
    상기 복합체 구조 내부에 소수성 또는 친수성 형광물질을 하나 이상 추가로 담지하는 것을 특징으로 하는 마이크로버블-나노리포좀 복합체.Microbubble-nanoliposomal complex, characterized in that for carrying one or more additional hydrophobic or hydrophilic fluorescent material in the complex structure.
  7. 제 6 항에 있어서,The method of claim 6,
    상기 소수성 또는 친수성 형광물질은 FITC, 텍사스 레드(Texas-red), RITC, Cy3, Cy5 및 Cy7을 포함하는 군으로부터 하나 이상 선택된 것임을 특징으로 하는 마이크로버블-나노리포좀 복합체.The hydrophobic or hydrophilic fluorescent substance is a microbubble-nanoliposomal complex, characterized in that at least one selected from the group comprising FITC, Texas-red, RITC, Cy3, Cy5 and Cy7.
  8. 제 1 항에 있어서,The method of claim 1,
    상기 마이크로버블은 필름형성물질, 인지질, 음전하 화합물, 아민기를 갖는 화합물 및 다이설파이드기를 갖는 화합물을 포함하는 것을 특징으로 하는 마이크로버블-나노리포좀 복합체.The microbubble is a microbubble-nanoliposomal complex, characterized in that it comprises a film-forming material, a phospholipid, a negatively charged compound, a compound having an amine group and a compound having a disulfide group.
  9. 제 8 항에 있어서,The method of claim 8,
    상기 필름형성물질은 DPPC (1,2-dipalmitory-sn-glycero-3-phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) 및 DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine)을 포함하는 군으로부터 선택된 것이고; 상기 인지질은 콜레스테롤(cholesterol)이고; 상기 음전하 화합물은 DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl-sn-glycero-3-phosphate), DMPA (1,2-dimyristoyl-sn-glycero-3-phosphate) 및 DOPA (1,2-dioleoyl-sn-glycero-3-phosphate)을 포함하는 군으로부터 선택된 것이고; 상기 아민기를 갖는 화합물은 DPPE (1,2-dipalmitory-sn-glycero-3-phosphoethanolamine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) 및 DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine)을 포함하는 군으로부터 선택된 것이고; 그리고 상기 다이설파이드기를 갖는 화합물은 DSPE-PEG-SPDP 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000]인 것을 특징으로 하는 마이크로버블-나노리포좀 복합체.The film forming material is DPPC (1,2-dipalmitory-sn-glycero-3-phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPC (1,2-didecanoyl-sn-glycero -3-phosphocholine), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn- glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine); The phospholipid is cholesterol; The negatively charged compound is DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl-sn-glycero-3-phosphate), DMPA (1,2- dimyristoyl-sn-glycero-3-phosphate) and DOPA (1,2-dioleoyl-sn-glycero-3-phosphate); Compounds having an amine group include DPPE (1,2-dipalmitory-sn-glycero-3-phosphoethanolamine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), and DLPE (1,2-dilauroyl-sn- glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine); And the compound having a disulfide group is a DSP-PEG-SPDP 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [PDP (polyethylene glycol) -2000].
  10. 제 1 항에 있어서,The method of claim 1,
    상기 나노리포좀은 필름형성물질, 인지질, 음전하 화합물 및 아민기를 갖는 화합물을 포함하는 것을 특징으로 하는 마이크로버블-나노리포좀 복합체.The nano liposome is a microbubble-nanoliposomal complex, characterized in that it comprises a film-forming substance, a phospholipid, a negatively charged compound and a compound having an amine group.
  11. 제 10 항에 있어서,The method of claim 10,
    상기 필름형성물질은 DPPC (1,2-dipalmitory-sn-glycero-3-phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) 및 DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine)을 포함하는 군으로부터 선택된 것이고; 상기 인지질은 콜레스테롤(cholesterol)이고; 상기 음전하 화합물은 DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl-sn-glycero-3-phosphate), DMPA (1,2-dimyristoyl-sn-glycero-3-phosphate) 및 DOPA (1,2-dioleoyl-sn-glycero-3-phosphate)을 포함하는 군으로부터 선택된 것이고; 그리고 상기 아민기를 갖는 화합물은 DPPE (1,2-dipalmitory-sn-glycero-3-phosphoethanolamine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) 및 DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine)을 포함하는 군으로부터 선택된 것임을 특징으로 하는 마이크로버블-나노리포좀 복합체.The film forming material is DPPC (1,2-dipalmitory-sn-glycero-3-phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPC (1,2-didecanoyl-sn-glycero -3-phosphocholine), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn- glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine); The phospholipid is cholesterol; The negatively charged compound is DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl-sn-glycero-3-phosphate), DMPA (1,2- dimyristoyl-sn-glycero-3-phosphate) and DOPA (1,2-dioleoyl-sn-glycero-3-phosphate); And the compound having an amine group is DPPE (1,2-dipalmitory-sn-glycero-3-phosphoethanolamine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), DLPE (1,2-dilauroyl-sn -glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) Microbubble-nanoliposomal complex.
  12. 1) 필름형성물질, 인지질, 음전하 화합물, 아민기를 갖는 화합물 및 다이설파이드기를 갖는 화합물을 유기용매에 넣고 반응시켜 필름을 제조한 후 이를 소수성 가스와 함께 유기 혼합용매에 넣고 진동반응(vibrating)시켜 마이크로버블을 제조하는 단계;1) A film-forming substance, a phospholipid, a negatively charged compound, a compound having an amine group, and a compound having a disulfide group are added to an organic solvent and reacted to prepare a film, which is then mixed with an hydrophobic gas into an organic mixed solvent and vibrated. Preparing a bubble;
    2) 필름형성물질, 인지질, 음전하 화합물 및 아민기를 갖는 화합물을 유기용매에 넣고 반응시켜 필름을 제조한 후 이를 물에 넣고 초음파분산 및 여과하여 나노리포좀을 제조하는 단계;2) preparing a film by adding a film-forming substance, a phospholipid, a negatively charged compound, and a compound having an amine group into an organic solvent to prepare a film, and then putting the same into water and dispersing ultrasonically and filtering to prepare nanoliposomes;
    3) 상기 단계 2)에서 얻어진 나노리포좀을 치료제와 반응시켜 치료제를 나노리포좀 구조 내에 담지시키는 단계;3) reacting the nanoliposomes obtained in step 2) with a therapeutic agent to support the therapeutic agent in the nanoliposomal structure;
    4) 상기 단계 1)에서 얻어진 마이크로버블과 상기 단계 3)에서 얻어진 나노리포좀을 혼합하고 진탕(shaking)시켜 마이크로버블-나노리포좀 복합체를 제조하는 단계; 및4) preparing a microbubble-nanoliposomal complex by mixing and shaking the microbubbles obtained in step 1) and the nanoliposomes obtained in step 3); And
    5) 상기 단계 4)에서 제조된 복합체를 타겟팅 모이어티(targetting moiety)와 반응시켜 타겟팅 모이어티를 복합체 외부에 결합시키는 단계를 포함하는, 제 1 항의 마이크로버블-나노리포좀 복합체를 제조하는 방법.5) reacting the complex prepared in step 4) with a targeting moiety to bind the targeting moiety to the outside of the complex, the method of preparing the microbubble-nanoliposomal complex of claim 1.
  13. 제 12 항에 있어서,The method of claim 12,
    상기 단계 1) 또는 2)는 마이크로버블 또는 나노리포좀 제조 후 이를 형광물질과 반응시켜 형광물질을 상기 마이크로버블 또는 나노리포좀 구조 내에 담지시키는 공정을 추가로 포함하는 것을 특징으로 하는, 마이크로버블-나노리포좀 복합체를 제조하는 방법.Step 1) or 2) further comprises the step of preparing a microbubble or nanoliposomes and reacting it with a fluorescent material to support the fluorescent material in the microbubble or nanoliposome structure, microbubble-nanoliposomes How to prepare a complex.
  14. 제 1항에 따른 마이크로버블-나노리포좀 복합체를 포함하는 암세포 특이적 초음파, 자기공명영상 또는 형광 분석용 조영제 조성물.A contrast agent composition for cancer cell-specific ultrasound, magnetic resonance imaging, or fluorescence analysis comprising the microbubble-nanoliposomal complex according to claim 1.
  15. 제 1항에 따른 마이크로버블-나노리포좀 복합체와, 항암제 또는 항암 유전자를 유효성분으로 함유하는 항암용 약학 조성물.A microbubble-nanoliposomal complex according to claim 1, and an anticancer pharmaceutical composition comprising an anticancer agent or anticancer gene as an active ingredient.
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