KR20140018150A - Microbubble-nanoliposome complex for diagnosis and treatment of cancer - Google Patents

Microbubble-nanoliposome complex for diagnosis and treatment of cancer Download PDF

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KR20140018150A
KR20140018150A KR1020130092029A KR20130092029A KR20140018150A KR 20140018150 A KR20140018150 A KR 20140018150A KR 1020130092029 A KR1020130092029 A KR 1020130092029A KR 20130092029 A KR20130092029 A KR 20130092029A KR 20140018150 A KR20140018150 A KR 20140018150A
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microbubble
glycero
complex
nanoliposome
cancer
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KR101488822B1 (en
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이학철
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(주)아이엠지티
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Abstract

The present invention relates to a microbubble-nanoliposome complex enabling diagnosis and treatment specific to cancer and a method for manufacturing the same and more specifically, the microbubble-nanoliposome complex, in which a hydrophobic microbubble and a hydrophilic nanoliposome are combined by stable covalence bond, carries one or more extra hydrophobic or hydrophilic fluorescent materials as well as one or more drugs and has a targeting moiety on the outside, thereby showing an excellent target specificity. The microbubble-nanoliposome complex enables the diagnosis of cancer through multiple image analysis using cancer-specific ultrasound or fluorescence contrast and the treatment of the cancer by efficiently delivering at least one of the drugs at the same time, thereby being able to be useful for the effective diagnosis and development of medicine for cancers including breast cancer, liver cancer, pancreatic cancer, brain cancer, stomach cancer, lung cancer, esophageal cancer, colon cancer, prostate cancer, kidney cancer, and ovary cancer. [Reference numerals] (AA) Lipid structure (Biocompatible material); (BB) Ultrasonography effect; (CC) Antibody (cancer-specific recognition); (DD,EE) Therapeutic effect

Description

The present invention relates to a microbubble-nanoliposome complex for diagnosis and treatment of cancer,

The present invention relates to a microbubble-nanoliposome complex capable of diagnosing and treating cancer cells and a method of preparing the same. More specifically, the present invention relates to a microbubble- Since the bubble-nanoliposome complex can carry one or more additional hydrophobic or hydrophilic fluorescent substances as well as one or more therapeutic agents in the complex structure and exhibits excellent target specificity including an external targeting moiety, Cancer, breast cancer, brain cancer, stomach cancer, lung cancer, esophageal cancer, colon cancer, and the like can be diagnosed through the multiple image analysis using specific ultrasound or fluorescence imaging, and at the same time, one or more therapeutic agents can be effectively delivered to the target cancer cells, Cancer, prostate cancer, kidney cancer, ovarian cancer May be useful to take advantage of the development of effective diagnostic and treatment of cancer.

Therapeutic agents are substances that make diagnosis and treatment of the disease possible at the same time. These are usually made of small-sized materials, and usually have a form in which fluorescent dyes and radioactive molecules are carried on the inside of liposomes, polymers and nanoparticles, and drugs or diagnostic markers are introduced to the outside. Recently, the synthesis of therapeutic contrast material composed of lipid structure with excellent biocompatibility has been mainly studied. Especially, microbubble ultrasound agent (microbubble), which is a micrometer-sized air- , MB) have been actively studied [David C., Eur J Radiol (2006), 60 (3), 324-330]. These studies have recently focused primarily on the application of properties such as non-toxic, high-water-soluble, or easy surface modification for applications in the biotechnology field of nanomaterials, which are now referred to as "nano-bio fields" It is set.

The microbubbles based on the known lipid structures can be used for various biocompatibility in vivo or in vitro studies as well as diagnosis by imaging analysis of blood vessels or cancer tissues (Katherine F. et al., Annual Review of Biomedical Engineering (2007), 9, 415-447). However, ultrasound contrast analysis through microbubbles is insufficient for accurate diagnosis of cancer due to low resolution and nonspecific properties. Therefore, by using ultrasonic imaging as well as ultrasound imaging, (Bart G. et al., Journal of Controlled Release (2011), 152, 249-49). In this study, we investigated the fluorescence and ultrasound- 256].

However, most of the known substances still remain in the target nonspecific contrasting effect, so that they can be used only for the linear diagnosis of tissues such as blood vessels and lymphatic vessels, or the therapeutic gene or drug is delivered only to lesion cells (Wang C. et al., Biomaterials (2012), 33, 1939-1947], which requires forming a hybrid body with another structure to support a therapeutic substance.

Accordingly, the inventors of the present invention have conducted studies to develop a therapeutic contrast material capable of treating cancer through target-specific delivery of one or more therapeutic agents at the same time as diagnosis of cancer through cancer-specific multi-image analysis. As a result, And at least one additional hydrophobic or hydrophilic fluorescent material, as well as at least one therapeutic agent, may be contained in the complex structure, and a targeting moiety may be included on the outside of the composite structure. The microbubble-nanoliposome complex is diagnosed with cancer through multiple image analysis using ultrasound or fluorescence contrast specifically for cancer cells, and at the same time, one or more therapeutic agents such as therapeutic genes and drugs are stably transferred to the target cells, The present invention is not limited thereto. It was.

Accordingly, an object of the present invention is to provide a theragnostic agent capable of diagnosing cancer through cancer-specific multi-image analysis and simultaneously delivering one or more therapeutic agents specifically to target cells to enable cancer treatment .

Another object of the present invention is to provide a method for producing the therapeutic contrast material.

In order to accomplish the above object, the present invention provides a complex comprising a hydrophobic microbubble and a hydrophilic nanoliposome bound by a covalent bond, wherein a targetting moiety is externally supported while supporting at least one therapeutic agent in the complex structure Microbubble-nanoliposome complex.

According to one embodiment of the present invention, the therapeutic agent may be selected from a therapeutic gene and a drug.

According to an embodiment of the present invention, one or more hydrophobic or hydrophilic fluorescent materials may be further supported inside the composite structure.

To achieve these and other objects,

1) A film is formed by reacting a film-forming substance, a phospholipid, a negative charge compound, a compound having an amine group and a compound having a disulfide group in an organic solvent and reacting the resultant with a hydrophobic gas in an organic mixed solvent, Producing a bubble;

2) preparing a film by reacting a film-forming substance, a phospholipid, a negative charge compound and a compound having an amine group in an organic solvent to prepare a film, adding the resulting film to water, and dispersing and filtering the nanoliposome by ultrasonic dispersion;

3) reacting the nanoliposome obtained in step 2) with one or more therapeutic agents to carry one or more therapeutic agents in the nanoliposome structure;

4) mixing the microbubbles obtained in the step 1) and the nanoliposome obtained in the step 3) and shaking them to prepare a microbubble-nanoliposome complex; And

5) A method for producing the microbubble-nanoliposome complex of the present invention, comprising reacting the complex prepared in step 4) with a targeting moiety to bind the targeting moiety to the outside of the complex to provide.

According to an embodiment of the present invention, the step 1) or 2) may further include a step of reacting the microbubble or nanoliposome with the fluorescent material to support the fluorescent material in the microbubble or nanoliposome structure have.

The microbubble-nanoliposome complex enabling the cancer cell-specific diagnosis and treatment according to the present invention is characterized in that a hydrophobic microbubble and a hydrophilic nanoliposome are chemically linked by a stable covalent bond so that a therapeutic gene, Of the present invention can stably support one or more additional hydrophobic or hydrophilic fluorescent substances as well as one or more therapeutic agents of the present invention and exhibits excellent target specificity due to the combination of targeting moieties on the outside, Cancer, cancer, cancer of the lung, cancer of the esophagus, cancer of the colon, prostate cancer, cancer of the breast, cancer of the liver, pancreatic cancer, lung cancer, Cancer, kidney cancer, ovarian cancer and other cancer diseases Diagnosis and can be usefully utilized in drug development.

1 is a structural schematic diagram of a microbubble-nanoliposome complex according to an embodiment of the present invention.
FIG. 2 is a graph showing the results of microscopic observation using an optical microscope and a cryogenic electron microscopy (Cryo-EM) of a microbubble (a) and a nanoliposome (b) prepared according to an embodiment of the present invention. It is a photograph.
FIG. 3 is a photograph of a microbubble-nanoliposome complex prepared according to an embodiment of the present invention, using a confocal fluorescence microscope, at a low magnification and a high magnification (upper right inset).
FIG. 4 is a graph showing the results of UV-vis spectroscopy of a microbubble-nanoliposome complex prepared according to an embodiment of the present invention. As a result, the by-product SPDP (N-succinimidyl-3 - [2-pyridyldithio] -propionate).
FIG. 5 is a graph showing the results obtained by using SonoVue (TM) (a), a microbubble ultrasound contrast agent commercially available, microbubble (b) and microbubble-nanoliposome complex (c) prepared according to an embodiment of the present invention, The results of this study are as follows.
FIG. 6 is a graph showing changes in the ultrasound contrast effect in the artificial blood vessel phantom when the microbubble-nanoliposome complex manufactured according to an embodiment of the present invention was subjected to 1 to 10 times of sound stimulation 1 to 10 times) and a graph.
FIG. 7 is a graph showing the amount of antibody contained on the surface of a composite by performing BSA quantitative analysis on a microbubble-nanoliposome complex prepared according to an embodiment of the present invention.
FIG. 8 is a graph showing the results of immunohistochemical analysis of SkBr3 cells as a breast cancer cell line using a microbubble-nanoliposome complex prepared according to an embodiment of the present invention and a microbubble (MB) -nanol liposome complex prepared by omitting antibody introduction as a control group. The results of multimodal imaging analysis (a) and FACS analysis (b) using a focused fluorescence microscope are shown in the photographs and figures.
FIG. 9 is a graph showing the results of a microbubble-nanoliposome complex prepared according to an embodiment of the present invention by treating SkBr3 cells as a breast cancer cell line, applying external sound stimulation for 1 minute, and carrying out confocal fluorescence microscopy analysis It is a photograph that confirms whether the fluorescent substance which was present was transmitted to the inside of the cancer cell.
FIG. 10 is a graph showing the result of spectroscopic analysis of a drug-harvesting efficiency of a microbubble-nanoliposome complex prepared according to an embodiment of the present invention using an ultraviolet-visible spectrometer.
FIG. 11 shows the results of (1) the breast cancer cell line Skbr3 treated with the microbubble-nanoliposome complex 2 or doxorubicin 3 prepared according to one embodiment of the present invention for a certain period of time (24 hours, 48 hours, 72 hours) and MTT assay was performed to confirm cytotoxicity.
FIG. 12 is a graph showing the results obtained by treating the microbubble-nanoliposome complex prepared according to an embodiment of the present invention into a breast cancer cell line SkBr3 and culturing it for a predetermined time (48 hours, 72 hours, or 96 hours) And then the expression of tGFP was confirmed using a confocal fluorescence microscope.
Fig. 13 shows the optimization and loadings of protamine (PA) and pGFP complexes for gene transfer experiments of microbubble-nanoliposome complexes.
14 compares the pGFP delivery effect of the microbubble-nanoliposome complex of the present invention.
Figure 15 monitors the loading capacity and cellular properties of doxorubicin in microbubble-nanoliposome complexes.
16 shows the results of cell viability test by drug and therapeutic gene transfer using microbubble-nanoliposome complex. Pop-particle refers to a microbubble-nanoliposome complex.
FIG. 17 shows the expression levels of STAT3 protein and gene in cancer cells after cell death test of microbubble-nanoliposome complex containing siSTAT3.
Figure 18 shows the characteristics of VX2 tumors transplanted in the kidney of a rabbit.
Figure 19 shows cancer-specific ultrasound images and delivery of drugs and genes in vivo.
20 shows the evaluation of the effect of cancer treatment using a microbubble-nanoliposome complex in a rabbit tumor model.
21 shows the in vivo distribution of the microbubble-nanoliposome complex containing the fluorescent substance in the living body.
22 shows the measurement of the excretion of a microbubble-nanoliposome complex containing a fluorescent substance in vivo.

Hereinafter, the present invention will be described in detail.

The microbubble-nanoliposome complex according to the present invention is a complex in which a hydrophobic microbubble and a hydrophilic nanoliposome are covalently bonded to each other, and a targeting moiety such as an antibody targetting moiety. In addition, the microbubble-nanoliposome complex of the present invention may further contain at least one hydrophobic or hydrophilic fluorescent substance inside the complex structure, wherein the hydrophobic fluorescent substance is contained in the microbubble structure, the hydrophilic fluorescent substance is contained in the nanoliposome structure As shown in FIG.

As shown in FIG. 1, the microbubble-nanoliposome 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 1-2 탆 in average diameter Nano-liposome is bound to the surface, and SF 6 , CO 2 Or CF 4 And the like. At this time, the hydrophobic microbubbles and the hydrophilic nanoliposome are chemically bonded to each other through a stable covalent bond, and in addition to a therapeutic agent selected from one or more genes and drugs, hydrophobic or hydrophilic fluorescent substances And a targetting moiety such as an antibody is included in the outside of the conjugate. Therefore, it is possible to realize mutimodal imaging through ultrasound or fluorescence imaging in a target-specific manner, and at the same time, There is a characteristic that can be transmitted stably. The microbubble-nanoliposome complex is also referred to herein as a 'pop-particle' in the specification or the drawings.

According to one embodiment of the present invention, the complex of the present invention may preferably exhibit a uniform size of an average diameter of 1 to 2 탆, but it is not limited thereto, and may be selected according to the concentration and the filtration method of the nanoliposome nm to 10 [mu] m. These particle sizes can be varied according to the frequency of the currently commercialized ultrasonic devices. If the frequency of the ultrasonic devices used increases, the average diameter of the complexes can be reduced.

According to one embodiment of the present invention, in the complex of the present invention, the microbubble may include a film forming material, a phospholipid, a negative charge compound, a compound having an amine group, and a compound having a disulfide group, Glycero-3-phosphatidylcholine, 1,2-didecanoyl-sn-glycero-3-phosphocholine, DEPC (1,2-didecanoyl- Glycero-3-phosphocholine (DLOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine, DMPC (1,2-dimyristoyl- , DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) or DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine); Phospholipids include cholesterol and the like; Examples of negative charge carriers include DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl- dimyristoyl-sn-glycero-3-phosphate or DOPA (1,2-dioleoyl-sn-glycero-3-phosphate); Examples of the compound having an amine group include 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine, DLPE (1,2- glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine); As the compound having a disulfide group, DSPE-PEG-SPDP {1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [PDP (polyethylene glycol) -2000]} may be used.

According to an embodiment of the present invention, in the complex of the present invention, the nanoliposome may include a film forming material, a phospholipid, a negative charge compound, and a compound having an amine group, -dipalmitory-sn-glycero-3-phosphatidylcholine), DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPC 2-dilinoleoyl-sn-glycero-3-phosphocholine, DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl- , 2-dioleoyl-sn-glycero-3-phosphocholine, or DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine); Phospholipids include cholesterol and the like; Examples of negative charge carriers include DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl- dimyristoyl-sn-glycero-3-phosphate or DOPA (1,2-dioleoyl-sn-glycero-3-phosphate); Examples of compounds having an amine group include 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2- glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine).

According to one embodiment of the present invention, the complex of the present invention can be prepared by modifying a lipid structure of a microbubble or a lipid structure of a nanoliposome by controlling pH, reaction temperature or reaction time, .

According to one embodiment of the present invention, one or more therapeutic agents carried on the inside of the complex structure of the present invention may be any of those selected from therapeutic genes and drugs, and examples thereof include DNAs and RNAs Genes and drugs such as anticancer drugs, and the like. Specifically, when a therapeutic gene is used, for example, an expression plasmid, an siRNA, an shRNA, a microRNA or an antagonist of a microRNA capable of inhibiting the expression of a gene having known antitumor effect or a gene involved in cancer development DNA or RNA can be used. When a drug is used, for example, doxorubicin, paclitaxel or docetaxel, which are known to have anticancer effects, may be used, but the present invention is not limited thereto. These genes and drugs can be reacted with nanoliposome according to a conventional method to be carried in a complex structure. In the case of a gene, it can be supported by reacting with a nanoliposome in the form of a hybrid with a biological polymer such as protamine, Drugs can be loaded into liposomes through the usual 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 is 88.57%, and the tGFP expression plasmid gene collection efficiency is 30%.

In addition, according to one embodiment of the present invention, the fluorescent substance that can be additionally carried in the complex structure of the present invention can be any hydrophobic or hydrophilic fluorescent substance that can be used for clinical diagnosis. Examples of the fluorescent substance include FITC , Texas Red (Texas-red). RITC, Cy3, Cy5, or Cy7 may be used, but the present invention is not limited thereto. Such fluorescent materials can be supported by micro bubbles in the case of a fluorescent substance having hydrophobicity and nanoriposomes in the case of a fluorescent substance having hydrophilic property by stirring or ultrasonic dispersion (sonication) at 30 캜 according to a conventional method.

According to one embodiment of the present invention, a targeting moiety of an antibody or the like bound to the surface of the complex of the present invention may be a target molecule such as a molecule, a ligand, or a receptor (DNA or RNA), a protein, an antibody, an antigen, an aptamer (such as RNA, DNA, or DNA), or any other substance capable of selectively recognizing / binding the target substance. Peptide, aptamer), receptor, hormone, streptavidin, avidin, biotin, lectin, ligand, agonist, antagonist, enzyme, coenzyme, inorganic ion, enzyme cofactor, sugar, lipid, enzyme substrate, hapten, (Such as Pt, Pd, Au, Ag, Nb, Ir, Rh and Ru), preferably biotin, streptavidin Deamin, avidin, antibodies, aptamers, polypeptides, Available Tide, ligand, receptor, lectin, sugar, lipid, glycolipid or nucleic acid, such as, but not limited thereto. The target substance refers to a substance in a sample to be separated (or separated and detected or separated, detected and quantified), and specifically includes nucleic acid molecules (DNA or RNA), proteins, peptides, antigens, sugars, lipids, , 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 is, for example, blood, plasma, serum, virus, bacteria, tissue, cell, lymph, bone marrow, saliva, milk, urine, But are not limited to, liquid, semen, brain extract, spinal fluid, joint fluid, thymus fluid, ascites fluid, amniotic fluid, cell tissue fluid and cell culture fluid.

According to one embodiment of the present invention, a targeting moiety, such as an antibody, included on the surface of the complex of the invention can be attached to the surface of the composite of the invention directly or indirectly in a shared or non- And may be included in combination via, for example, 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 coupling, an intervening agent such as a binder may be used, wherein the intermediate mediator is sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane- 4- (N-maleimidomethyl) cyclohexane-1-carboxylate).

According to one embodiment of the present invention, when the complex of the present invention approaches the target cell by the targeting moiety of the surface, it is destroyed through an external ultrasonic flow (UF), so that the therapeutic gene and the drug The intensity of the sound wave stimulation is not particularly limited, but its mechanical index (mechanical index = PNP /

Figure pat00001
: PNP; Peak negative pressure of the ultrasound wave (MPa), Fc; center frequency of the ultrasound wave (MHz)) is preferably 0.01 to 2.0. Thus, exposing the composite of the present invention to high energy or high mechanical index in ultrasound is referred to as flash, and is also referred to in the following examples as the term flash.

According to one embodiment of the present invention, FITC and Texas Red as a fluorescent substance, a tGFP expression plasmid as a gene, and doxorubicin as a drug are carried in the complex structure, and an anti-HER2 monoclonal antibody specific for breast cancer cells anti-HER2 monoclonal antibody (Herceptin) was used to treat SkBr3, which is a breast cancer cell line, and that the fluorescent materials FITC and Texas Red were delivered into the cells under external stimulation (UF) After 3 days, more than 65% cell necrosis was confirmed by doxorubicin. From 2 days later, more than 90% of the cells were transduced with the gene, thus confirming that tGFP was expressed.

Therefore, the microbubble-nanoliposome complex of the present invention can diagnose cancer through multiple image analysis using ultrasound or fluorescence imaging specific to cancer cells while minimizing adverse effects through high target specificity, and at the same time, Can be effectively used for the development of effective diagnostic and therapeutic agents for cancer diseases such as breast cancer, liver cancer, pancreatic cancer, brain cancer, stomach cancer, lung cancer, esophageal cancer, colon cancer, prostate cancer, kidney cancer and ovarian cancer. .

In addition,

1) A film is formed by reacting a film-forming substance, a phospholipid, a negative charge compound, a compound having an amine group and a compound having a disulfide group in an organic solvent and reacting the resultant with a hydrophobic gas in an organic mixed solvent, Producing a bubble;

2) preparing a film by reacting a film-forming substance, a phospholipid, a negative charge compound and a compound having an amine group in an organic solvent to prepare a film, adding the resulting film to water, and dispersing and filtering the nanoliposome by ultrasonic dispersion;

3) reacting the nanoliposome obtained in step 2) with one or more therapeutic agents to carry one or more therapeutic agents in the nanoliposome structure;

4) mixing the microbubbles obtained in the step 1) and the nanoliposome obtained in the step 3) and shaking the microbubbles to prepare a microbubble-nanoliposome complex; And

5) A method for producing the microbubble-nanoliposome complex of the present invention, comprising reacting the complex prepared in step 4) with a targeting moiety to bind the targeting moiety to the outside of the complex to provide.

Examples of the film-forming material used in step 1) include 1,2-dipalmitory-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-didecanoyl-sn- glycero-3-phosphocholine , DEPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC ), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl- phosphocholine and the like; Phospholipids include cholesterol and the like; Examples of negative charge carriers include DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl- dimyristoyl-sn-glycero-3-phosphate or DOPA (1,2-dioleoyl-sn-glycero-3-phosphate); Examples of the compound having an amine group include 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine, DLPE (1,2- glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine); As the compound having a disulfide group, DSPE-PEG-SPDP {1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [PDP (polyethylene glycol) -2000]} may be used.

The hydrophobic gas used in the step 1) may be a hydrophobic gas used in the production of conventional micro bubbles such as SF 6 , CO 2 or CF 4. As the organic solvent, chloroform may be used as an organic solvent A solvent in which glycerin, propylene glycol and water are mixed can be used.

Examples of the film-forming material used in the step 2) include 1,2-dipalmitory-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-didecanoyl-sn- glycero-3-phosphocholine Glycero-3-phosphocholine, DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl- (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) or DSPC (1,2-distearoyl- this; Phospholipids include cholesterol and the like; Examples of negative charge carriers include DCP (dicetyl phosphate), DEPA (1,2-dierucoyl-sn-glycero-3-phosphate), DLPA (1,2-dilauroyl- dimyristoyl-sn-glycero-3-phosphate or DOPA (1,2-dioleoyl-sn-glycero-3-phosphate); Examples of compounds having an amine group include 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2- glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine).

In addition, the step of supporting one or more therapeutic agents in the nanoliposome structure in the step 3) may be performed by reacting the therapeutic agent with a nanoliposome in the form of a hybrid body with a biological polymer such as protamine when the therapeutic agent is a therapeutic gene , And when 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).

In addition, the complex formation reaction of the microbubble and the nanoliposome in the step 4) may be performed simply by modifying a lipid structure of the microbubble and a lipid structure of the nanoliposome by shaking to regulate the pH, the reaction temperature or the reaction time, Or by inducing covalent bonding.

In addition, the step of bonding a targeting moiety such as the antibody of step 5) to the outside of the conjugate may be carried out by a conventional reaction in which the targeting moiety is directly or indirectly bonded to the surface of the conjugate of the present invention in a covalent or non- And can be carried out, for example, 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 coupling, an intervening agent such as a binder may be used. As the intermediate agent, sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane- 4- (N-maleimidomethyl) cyclohexane-1-carboxylate).

According to an embodiment of the present invention, the step 1) or 2) further includes a step of adding a fluorescent substance to the prepared microbubble or nanoliposome and then reacting the fluorescent substance to support the fluorescent substance in the microbubble or nanoliposome structure can do. At this time, the step of supporting the fluorescent substance in the microbubble or nanoliposome structure may be carried out by using a microbubble in the case where the fluorescent substance is hydrophobic or a nano liposome in the case where the fluorescent substance is hydrophilic, Ultrasonic dispersion (sonication).

According to another aspect of the present invention, there is provided a cancer cell-specific ultrasound, magnetic resonance imaging (MRI) or contrast agent composition for fluorescence analysis comprising the microbubble-nanoliposome complex.

In the present invention, the contrast agent refers to a substance administered intracorporeally to contrastively or imaging cancer cells in a strong and specific manner in vivo. Currently, the contrast agent is used for enhancing images of tissues and cells in the medical field, It is 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.

In one embodiment of the present invention, it was confirmed that ultrasonic analysis, MRI analysis and fluorescence analysis were easily performed using a microbubble-nanoliposome complex carrying a fluorescent substance, a target gene, an antibody, etc. (see Test Example 7).

The contrast agent composition of the present invention can be useful for targeting and diagnosis of cancer cells or cancer tissues and can be formulated into oral or parenteral administration forms. The parenteral formulation preferably comprises a sterile aqueous solution or suspension comprising the microbubble-nanoliposome complex of the present invention, and various techniques for the preparation of pharmaceutical aqueous solutions or suspensions are known in the art. The solution may also contain 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 may vary widely and are known in the art. Such formulations generally comprise an aqueous solution or suspension containing a diagnostically effective amount of a microbubble-nanoliposome complex according to the present invention. The oral formulations may optionally include buffers, surfactants, adjuvants, thixotropic agents, and the like. Formulations for oral administration may also contain ingredients for increasing spices and other functionalities.

The contrast agent composition according to the present invention is administered in an amount effective to achieve the desired imaging effect of the imaging image. Such dosage can vary widely depending on the organ or tissue to be imaged, the imaging device used, and the like. The administration concentration for using the microbubble-nanoliposome complex as a contrast agent may be 0.1 mM to 10 M.

The contrast agent compositions of the present invention are used in a conventional manner for imaging analysis. For example, the contrast agent composition may be administered to a mammal systemically or to an organ or tissue being imaged locally, in an amount sufficient to provide adequate visualization, and then the mammal may be sonicated or MRI.

According to still another aspect of the present invention, there is provided a pharmaceutical composition for an anticancer comprising the microbubble-nanoliposome complex, an anticancer agent or an anti-cancer gene as an active ingredient.

In one embodiment of the present invention, an anticancer agent or an anti-cancer gene is introduced into the microbubble-nanoliposome complex of the present invention to confirm the effect of inhibiting cancer cell death or cancer tissue growth, See Test Examples 6 and 7).

In order to use the pharmaceutical composition for anti-cancer medicine of the present invention as a medicine, it may be prepared by a known method in the pharmaceutical field. In this case, the microbubble-nanoliposome complex of the present invention In addition, a pharmaceutically acceptable carrier may be included. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, phosphorus calcium, alginate, gelatin, But are not limited to, calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. 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).

The pharmaceutical composition of the present invention may be prepared in a unit dose form by being formulated using a pharmaceutically acceptable carrier or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. Into a capacity container. The formulations may be in the form of solutions, suspensions or emulsions in oils or milks, or they may be in the form of excipients, powders, granules, tablets, capsules or injectables, and may additionally contain dispersing or stabilizing agents. They may be administered parenterally (e. G., Intravenously, subcutaneously, intraperitoneally or topically) or orally.

The appropriate dosage of the pharmaceutical composition of the present invention may be appropriately selected depending on factors such as the formulation method, administration method, age, body weight, sex, health condition, disease symptom, food, administration time, administration method, And preferably 0.01 to 100 mg per adult standard daily may be administered.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Materials and methods

1) Experimental material

DPP (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine), cholesterol, DCP (dicetyl phosphate), DPPE 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [PDP (polyethylene glycol) -2000]}, sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, Trauts Reagent, 2-iminothiolane, ammonium sulfate and protamine were purchased from Sigma-Aldrich Was used without any modification.

2) Cell culture

SkBr3, a breast cancer cell line, was cultured in RPMI 1640 medium (Hyclone, Logan, UT) containing 10% FBS under a humidified atmosphere of 5% CO 2 and 37 ° C.

3) MTT analysis

The cytotoxicity assay using an MTT assay kit (Sigma-Aldrich) was performed as follows.

The cultured cell line was washed after removal of the medium for use in the test, and recovered by resuspending after treatment with trypsin. Recovered cells were plated in 96-well plates at a concentration of 5,000 cells per well and incubated overnight at 37 ° C under 5% CO 2 atmosphere. The cultured cells were treated with various concentrations of the test solution, and the treated cells were again cultured under the same conditions for a certain test time. After incubation, the medium was removed, and 0.1 ml of MTT solution per well was added. The mixture was incubated at 37 ° C under 5% CO 2 for 3 hours. The MTT solution was removed, and 0.1 ml of MTT solubilization solution Was added to dissolve the formazan crystals. Cell viability was determined by measuring absorbance at 570 nm using an ELISA reader in the well plate.

Preparation Example: Preparation of microbubble (MB) -nanolithosome complex according to the present invention

Step 1) Preparation of micro bubble (MB)

DPPE, which is a compound having an amine end group, and DSPE-PEG-SPDP, which is a compound having a disulfide group, were used as a negative charge compound to prevent DPPC, cholesterol, mg, 1.0 mg, 1.2 mg and 5 mg each in 5 mL of chloroform. The resulting solution was placed in a rotary evaporator and reacted at 35 ° C for 5 minutes to remove the solvent chloroform. The solution was lyophilized at -45 ° C for about 24 hours to prepare a film. 2 mL of glycerin, propylene glycol and water (glycerin: propylene glycol: H 2 O = 1: 2: 7) was added to the mixture, and the resultant mixed solution was placed in a hermetic vial. The microbubbles (MB) were prepared by filling SF 6 gas and then vibrating for 15 seconds with a vibrator.

Step 1-1) Carrying a hydrophobic fluorescent substance in the micro bubble structure

0.1 g of FITC, which is a hydrophobic organic fluorescent dye having green fluorescence, was added to the microbubbles prepared in the above step 1) and stirred at room temperature to obtain a microbubble having an average diameter of 1 to 2 占 퐉 ).

Step 2) Preparation of nanoliposome

DPP, a cholesterol, a negative charge compound DCP for preventing nanoliposome clumping, and DPPE, a compound having an amine end group, were dissolved in 5 mL of chloroform in amounts of 15.4 mg, 3.5 mg, 1.0 mg and 1.2 mg, respectively. The obtained solution was put into a rotary evaporator and reacted at 35 ° C for 5 minutes to remove the solvent. The solution was freeze-dried 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 at 60 ° C for 5 minutes using an ultrasonic disperser, and then the liposome mixed solution was prepared by repeating the freezing and thawing five times using liquid nitrogen. This liposome mixed solution was filtered twice with a 200 nm filter at 60 ° C to prepare a nanoliposome having a particle size of 200 nm or less.

Step 2-1) Carrying a hydrophilic fluorescent substance in the nanoliposome structure

0.1 mg of Texas-red, a hydrophilic organic fluorescent dye exhibiting red fluorescence, was added to the nanoliposome prepared in step 2), and the mixture was stirred at room temperature to obtain a nanoliposome in which the fluorescent material was supported inside the structure Average size: 100 to 200 nm).

Step 3) Loading the drug and gene in the nanoliposome structure

The following reaction was carried out on the nanoliposome obtained in the above step 2) or 2-1) to carry the gene and / or the drug inside the nanoliposome structure.

To the powdery nanoliposome (21 mg) obtained after freeze-drying the nanoliposome was added 26.6 ㎍ of a green fluorescent protein (tGFP) expression plasmid gene and 0.1 ml of a mixed solution containing 18.8 M M protamine Was added and shaken at 25 DEG C for 5 minutes. The resulting reaction mixture was centrifuged twice at 13000 rpm for 5 minutes to remove residual genes and protamine not captured in the liposome to obtain a nanoliposome carrying the therapeutic gene inside the structure.

In addition, a 0.5-20 mM doxorubicin solution known to be effective for cancer treatment is added to the nanoliposome solution at a concentration of 1 to 120 mM, and then stirred at room temperature to remove ammonium sulfate contained in the nanoliposome by osmotic pressure Nano liposomes containing the drug (doxorubicin) in the structure were prepared by replacing the external doxorubicin with doxorubicin inside the nanoliposome.

Step 4) Preparation of microbubble-nanoliposome complex

0.2 mL of the microbubbles obtained in the above step 1) or 1-1); And 1.8 mL of the nanoliposome obtained in the above step 3) were placed in a hermetic vial and the pH was adjusted to 8, followed by shaking reaction at 25 ° C for 2 hours to prepare a microbubble-nanoliposome complex.

Step 5) Antibody binding to the outside of microbubble-nanoliposome complex

5 mg of sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate was added to the complex prepared in step 4), the pH was adjusted to 8, and the mixture was shaken at 25 ° C for 3 hours , 0.1 mL of Herceptin, an anti-HER2 monoclonal antibody specific to breast cancer cells, was added to the resulting maleimide-microbubble-nanoliposome complex solution, and the pH was adjusted to 7 After 24 hours of shaking at 4 ° C, the antibody was bound to the outside of the complex. The obtained mixed solution was centrifuged twice at 13000 rpm for 5 minutes to remove unreacted material, thereby preparing a microbubble-nanoliposome complex according to the present invention.

Test Example  1: Micro bubble ( MB ) - Nanoliposome  Confirmation of physical properties of composites

1) Optical microscope, cryo electron microscope and dynamic light scattering analysis

The microbubbles obtained in step 1) or 1-1) and the nanoliposomes obtained in step 2), 2-1) or 3) of the above preparation example were subjected to optical microscopy, cryo-electron microscopy cryogenic electron microscopy, and cryo-EM) and dynamic light scattering (DLS) analyzes were performed to confirm the molecular shape and size distribution.

As a result, as shown in FIG. 2, the microbubble MB prepared according to an embodiment of the present invention had a spherical shape and an average size of 1.713 mu m, and the nanoliposome also had a spherical shape with an average size 197 nm. In addition, it has been found that both microbubbles and nanoliposomes exhibit a uniform particle size distribution.

2) Confocal fluorescence microscopy and ultraviolet-visible spectroscopy

In order to confirm the imaging effect and stability of the microbubble-nanoliposome complex according to the present invention, a confocal fluorescence microscope analysis was performed on the complex prepared in the above Preparation Example.

As a result, as can be seen from FIG. 3, in the low-power analysis, it was confirmed that most of the particles had red fluorescence and green fluorescence at the same time, from which microbubbles containing green fluorescence FITC and red Was found to be well bound in one particle. As can be seen from the inset of FIG. 3, in the high-power analysis, the green fluorescence inside the particle and the external red fluorescence were distinctly observed. As a result, the microbubble-nanoliposome complex according to the present invention It was visually confirmed that the microbubble was formed of a spherical structure in which a nanoliposome was bonded to the surface of the microbubble. In addition, it was confirmed that the microbubble-nanoliposome complex of the present invention exhibited a uniform size distribution with an average diameter of about 1-2 탆.

Meanwhile, the microbubble-nanoliposome complex according to the present invention is a microbubble-nanoliposome complex in which microbubbles and nanoliposomes are bonded with a covalent bond excellent in chemical stability, so that a gene, a drug or a fluorescent substance carried in the particle can be introduced into a target organ, Exhibits excellent stability in transportation and delivery. Therefore, in order to confirm whether the complex prepared in the above-mentioned preparation example is covalently bonded, UV-vis spectroscopy was used to determine the amount of SPDP (N-succinimidyl-3- [2- pyridyldithio] -propionate).

As a result, as can be seen from FIG. 4, SPDP was confirmed in the region of 340 nm, and Texas red and FITC fluorescence were also confirmed. Thus, the microbubble-nanoliposome complex according to the present invention is a microbubble-nanoliposome complex in which microbubbles and nanoliposomes are covalently bonded with each other with excellent chemical stability, so that a gene, a drug or a fluorescent substance carried in the particle structure is introduced into a target organ, It can be expected to exhibit excellent stability in transportation and delivery.

3) Ultrasound contrast analysis

In order to confirm the ultrasound imaging effect of the microbubble-nanoliposome complex according to the present invention, SonoVue (TM), a commercially available microbubble ultrasound contrast agent, the complex prepared in the above production example, and step 1) or 1-1) (IU22, Philips) using the microbubbles obtained from the micro - bubbles obtained from the micro - bubble.

As a result, as shown in Fig. 5, the microbubble-nanoliposome complex (c) of the present invention finally prepared in the microbubble (b) obtained in step 1) or 1-1) ) Showed a sufficiently high contrast effect compared to the existing commercial product (a).

In addition, when the microbubble-nanoliposome complex of the present invention having such excellent stability is destroyed by stimulation of external sound waves when it reaches a target organ, tissue or cell, one or more therapeutic agents or fluorescent substances carried in the structure are immobilized on the target cells In order to confirm whether the artificial vascular phantom can be effectively delivered, 10 sonic stimulations (mechanical index: 0.7) were performed through an ultrasound imaging device (IU22, Philips) The results are shown in the photograph and graph of FIG. Exposure of the inventive composite to high energy or high mechanical index in ultrasound is referred to as flash.

As a result, as shown in FIG. 6, it was confirmed that the structure of the complex was destroyed as the frequency of the sound wave stimulation increased, and the ultrasound contrast effect was greatly reduced. Especially, It was confirmed that the contrast effect was almost lost. Thus, it can be seen that the microbubble-nanoliposome complex of the present invention can effectively deliver not only one therapeutic agent but also a fluorescent substance to a target site such as cancer cells at the same time.

4) Protein quantitative analysis (BSA analysis)

In order to confirm the amount of the antibody bound to the surface of the microbubble-nanoliposome complex according to the present invention, the MB-nanoliposome complex prepared in the above Preparation Example was subjected to BSA analysis, which is a conventional protein analysis technique in the art Spectroscopic analysis was performed using an ultraviolet-visible spectrometer (UV-vis spectrometer).

As a result, as can be seen from FIG. 7, the antibody introduced on the surface at a wavelength of 560 nm was detected, and the amount thereof was confirmed to be 1.62 μM per 1 mg of the complex by quantitative analysis.

Test Example  2: Micro bubble ( MB ) - Nanoliposome  Multiple image analysis test using complex

In order to confirm the target specificity of the cancer cells of the microbubble-nanoliposome complex according to the present invention, the microbubble (MB) -nano-nano-liposome complex containing no antibody obtained in the step 4) Multimodal imaging analysis and FACS analysis using confocal fluorescence microscopy were performed on SkBr3 cells, a breast cancer cell line, using liposome complexes.

As a result, as can be seen from FIG. 8 (a), fluorescence contrast was hardly observed in the MB-nanoliposome complex containing no treatment of the breast cancer cells and the antibody, while the antibody-containing microbubble- It was confirmed that the liposome complex showed excellent fluorescence contrast in the target cells. Also, quantitative analysis using FACS confirmed that the cell content of green and red fluorescence was superior to that of 94% or more (FIG. 8B). It can be seen that the microbubble-nanoliposome complex of the present invention exhibits excellent target specificity through antibody binding.

In order to confirm whether the fluorescent substance carried in the microbubble-nanoliposome of the present invention by the external sound wave stimulation can be transmitted to the inside of the cells specifically for the cancer cells, a sonic stimulation (mechanical index: 0.7) was added for 1 minute, and fluorescence contrast was confirmed by confocal fluorescence microscopy.

As a result, as shown in FIG. 9, the green fluorescent dye FITC and the red fluorescent dye, Texas Red, which were carried in the microbubble-nanoliposome complex of the present invention, specifically penetrated into the cells to form SkBr3, It was confirmed that fluorescence contrast was possible. Thus, it can be seen that the microbubble-nanoliposome complex of the present invention can be used for effective diagnosis through multiple image analysis specific to a specific cancer tissue or cell target.

Test Example  3: Micro bubble ( MB ) - Nanoliposome  Drug delivery test using complex

In order to confirm the therapeutic delivery effect of the microbubble-nanoliposome complex according to the present invention, the complex prepared in the above Preparation Example was subjected to spectroscopic analysis using an ultraviolet-visible spectroscope to collect the drug (doxorubicin) Efficiency was confirmed.

As a result, as shown in FIG. 10, the collection efficiency of doxorubicin, which is a representative anticancer drug contained in the complex of the present invention, was 88.57%, and thus the complex of the present invention can inhibit various drugs It can be understood that it can be effectively carried and delivered to the target cells.

In order to confirm the efficacy of the cancer cell therapy using the complex of the present invention, the following cytotoxicity test was carried out using the MB-nanoliposome complex prepared in the above Preparation Example. First, 10 mg of the microbubble-nanoliposome complex in which the doxorubicin prepared in the above preparation example was carried, or 0.2 μg of doxorubicin itself was treated with Skbr3, a breast cancer cell line, and cultured for 3 hours. After incubation for 24 hours, 48 hours, or 72 hours after removal of the treated medium, the cells were incubated for 24 hours, 48 hours, or 72 hours. And MTT assay was performed to confirm cytotoxicity.

As a result, as shown in FIG. 11, it was confirmed that the anticancer drug-containing microbubble-nanoliposome complex of the present invention exhibited excellent cytotoxicity not only in the cell line not treated with the drug but also in the cell line treated with the anticancer drug alone (The cell survival rate was 85% even after 72 hours of treatment with the drug only, whereas it was 63% after 24 hours, 61% after 48 hours, and 35% after 72 hours after the complex treatment of the present invention) . As a result, when the complex of the present invention is used, it can be understood that the drug is effectively transferred to the inside of the cell by the cancer cell specifically due to the cell specificity due to the antibody bound to the complex, and thus the therapeutic effect can be obtained.

Test Example  4: Micro bubble ( MB ) - Nanoliposome  Gene delivery test using complex

In order to confirm the gene transfer effect of the microbubble-nanoliposome complex according to the present invention, a complex in which a green fluorescent protein tGFP (turbo green fluorescent protein, tGFP) expression plasmid produced in the above Preparation Example was internally supported, The effect of gene transfer on SkBr3 cell line was confirmed as follows.

First, using the spectrophotometer (Nano drop), it was confirmed that the collection efficiency of the tGFP expression plasmid carried in the complex prepared in the above example was about 30%. In addition, 10 mg of the complex was treated with SkBr3 and cultured for 3 hours. Unbound complexes were removed and treated with strong ultrasonic (mechanical index: 0.07) for 5 minutes. As a control, Ultrasonic treatment was omitted. The cells were further incubated for 3 hours and then the complex was removed. After incubation for 48 hours, 72 hours or 96 hours, the expression of tGFP was confirmed by confocal fluorescence microscopy.

As a result, as can be seen from FIG. 12, it was confirmed that green fluorescence became stronger over time in the group treated with the complex of the present invention and treated with ultrasonication as compared with the control group in which the ultrasonic treatment was omitted. This means that tGFP is expressed inside the cell. Therefore, the complex of the present invention is destroyed through strong ultrasonic treatment, and the plasmid which was carried in the inside is effectively transferred into the target cell, and tGFP is expressed.

As a result, the microbubble-nanoliposome complex according to the present invention has microbubbles and nanoliposome bound through a chemically stable covalent bond, and a hydrophobic microbubble and a hydrophilic nanoliposome structure containing various therapeutic genes and drugs Since the targetting moiety is bound to the outside of the complex while one or more additional hydrophobic or hydrophilic fluorescent substances can be supported as well as the therapeutic agent described above, multimodal analysis using a cancer cell-specific ultrasound imaging or fluorescence imaging imaging analysis to accurately diagnose cancer and simultaneously deliver one or more therapeutic agents stably to target cancer cells and deliver them specifically to target cells, thereby effectively treating cancer, Can be used extensively have.

Test Example 5: Application of microbubble-nanoliposome complex as a gene transfer substance

The green fluorescent protein plasmid DNA (p GFP, 10 kbp) was placed inside the hydrophilic regions of the nano-liposome (Lipo). To increase pGFP loading efficiency, a combined 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 be 20 μM PA and 34 nM pGFP representing 600 PAs per pGFP. The complex with a weak positive charge migrated to the negatively charged Lipo and showed a loading capacity of 95% or more (see FIG. 13). In the following description, microbubble-nanoliposome complexes are also represented as pop-particles.

Figure 13 shows the optimization and loading capacity of PA and pGFP complexes. The complexes of protamine (PA) and plasmid-GFP (pGFP) were optimized in molar proportions and the exact values of PA and pGFP can be determined by electrophoresis. As a result, 600 PAs at 7.5 kD are associated with one 10 kbp pGFP. The PA-pGFP complex can be delivered into the nanoliposome of the microbubble-nanoliposome complex because the complex has a sustained positive charge.

Nano liposomes containing pGFP were presented in the microbubble-nanoliposome complex system of the present invention in the same connection procedure. After treatment with MBBR-Lipo (R + pGFP) -Her2 Ab for 1 hour in breast cancer cell line SKBR3, the microbubble-nanoliposome complex containing the red dye and pGFP and the Her2 receptor antibody attached to the surface was treated The high-frequency ultrasound energy (mechanical index: 0.61) was exposed for 1 minute (referred to as 'flash'). The cells were then continuously cultured for 48 hours.

14 compares the pGFP transfection effect of the microbubble-nanoliposome complex system of the present invention. The plasmid gene delivery effect of the microbubble-nanoliposome complex was compared with Lipofectamine TM , which is a commercially available transfusion material. The green fluorescent protein expressed in SKBR3 cells was observed as a green spot in single cells observed with a low-power confocal laser fluorescence microscope (CLSM). And quantitative analysis using flow cytometry (FACS) studies. The microbubble-nanoliposome complex system is superior to the Lipofetamine ™ plasmid by 3-fold. When observed at high magnification, most cancer cells contained green protein in the cell matrix (b right-hand corner). In addition, the microbubble-nanoliposome complex system showed pGFP transfer to primary cells (CFt) extracted from cardiac fibroblasts, and the system showed a high effect of about 60% by CLSM and FACS analysis.

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, a microbubble-nanoliposome complex with a CD29 antibody specific for the cardiac fibroblast primary cell line (CFf) was synthesized and used for pGFP transfer. This is known as a gene which is difficult to transfer to cell line, but showed good efficiency. From these results, the microbubble-nanoliposome complex was easy to target gene carrier for any cell based on targeting and ultrasound energy stimulation experiments (Test Examples 3, 4 and 5).

Test Example 6: Therapeutic application of microbubble-nanoliposome complex as drug or siRNA carrier

Doxorubicin (Dox) is a drug used to treat cancer and works by interrupting DNA in the nucleus. Dox was prepared by ammonium sulfate precipitation method (Haran, G., et al., Biochim. Biophys . Acta 1151, 201-215 (1993)) and can be easily incorporated into the hydrophilic core of a nanoliposome (Lipo).

Figure 15 monitors the loading capacity and cellular properties of doxorubicin in pop-particles. Dox was inserted into the nanoliposome of the pop-particle (microbubble-nanoliposome complex) through the ammonium sulfate gradient method, and the transfer effect was quantitatively calculated by the ultraviolet-visible absorption test.

In Fig. 15, the loading efficiency of Dox was confirmed to be 90% or more by UV-Vis spectroscopy (Fig. 15 (a)). MB-Lipo (Dox) -Her2 Ab, a microbubble-nanoliposome (DPPC 2.1 mM and Dox 19 μM) with a Her2 receptor antibody attached and Dox, advanced breast cancer cell therapy. The loaded Dox can be simply monitored for translocation showing self-red fluorescence in the CLSM measurement (Fig. 15 (b)). It appeared on the cell membrane at the time of targeting and disappeared from the cytoplasm after flash application which exposed to ultrasonic energy such as the above conditions. By continuing to incubate for 24 hours, Dox penetrated into the nucleus. The cell morphology was not observed after 48 hours due to apoptosis. As a control group of ultrasound exposure, cells not exposed to ultrasound were alive in culture for 48 hours (blue: DAPI staining). This means that the microbubble-nanoliposome complex system selectively delivers the therapeutic agent only when the appropriate energy of ultrasound is exposed.

Figure 16 shows the cell viability test results for therapeutic microbubble-nanoliposome complexes such as drug and therapeutic gene delivery. In Fig. 16a, the microbubble-nanoliposome complex containing Dox promoted the apoptosis of cancer cells (survival rate: 40% or less), but only the supersonic wave was exposed without the microbubble-nanoliposome complex, There was no significant decrease in survival rate. In other words, the analysis of cells treated with the microbubble-nanoliposome complex containing Dox induces a higher cell death (survival rate ~ 35%) than that at the same concentration of free Dox treatment (survival rate ~ 90%) at 72 hours (Fig. 16 (a)). In Fig. 16 (b), as in the case of the anticancer drug test, only cells exposed to ultrasound without microbubble-nanoliposome complex or siSTAT3 alone did not affect cell viability. In addition, when the microbubble-nanoliposome complex containing siSTAT3 was treated and the ultrasound was not exposed, the cell viability was slightly decreased at 72 hours, but the cell number was increased at 120 hours because of the non-effective siSTAT3 gene transfer It is considered that the proliferation process has been restored. However, cells treated with microbubble-nanoliposome complex containing siSTAT3 and exposed to ultrasound showed a low survival rate of about 30% after 120 hours. In FIG. 16 (c), when the cancer cells were treated with the dual therapeutic pop-particle (Dox + siSTAT3), the cell survival rate was only about 10% at the same 120 hours, showing a remarkable therapeutic effect. As a control, the microbubble-nanoliposome complex treated only with Dox and siSTAT3 (Dox + siSTAT3) or without exposure to ultrasound did not show a high effect in inducing apoptosis. Also in FIG. 16 d, the cell density was monitored microscopically during the treatment test. In fact, the cells treated with the microbubble-nanoliposome complex (Dox + siSTAT3) containing Dox and siSTAT3 at 72 and 120 hours showed a low density and the typical morphology for apoptotic cells at 48 hours .

STAT3 (signal transducer and activator of transcription 3) is a transcription factor and the protein encoded by the STAT3 gene belongs to the STAT family of proteins involved in cell proliferation. Recently, tumor suppressor siRNAs against STAT3 have been reported to be associated with the death receptors DR4 and DR5 (Kang, Y. et al., Biochim. Biophys. Acta 1830, 2638-2648 (2013)). Like the transfection process, the siRNA for STAT3 (siSTST3) was simply inserted into the liposome after PA complex formation and exhibited a similar loading capacity of about 95%. Following delivery of siSTAT3 to SKBR3, the target gene and protein were measured for expression levels using PCR and Western blotting (see a and b in Figure 17).

FIG. 17 shows the expression levels of STAT3 protein and gene in cancer cells after treatment with microbubble-nanoliposome complex (siSTAT3) containing siSTAT3 and cell death test. After treating with MB-Lipo (siSTAT3) -Her2 Ab , which contains siSTAT3 and an antibody, the cancer cells were assayed for target STAT3 gene and protein levels by PCR and Western blotting.

17A and 17B, the target protein expression level of the cells treated with the microbubble-nanoliposome complex (siSTAT3) containing siSTAT3 significantly decreased the target gene and protein intensity. As a control, cells exposed to ultrasound alone or cells treated with siSTAT3 alone did not show any change in the expression of STAT3 gene. When quantitative analysis was performed, the microbubble-nanoliposome complex (siSTAT3) containing siSTAT3 and MB-Lipo (siSTAT3) -Her2 Ab were compared with cells exposed to ultrasound alone or naked siSTAT3 as control The inhibitory levels of the target STAT3 gene and protein in the embryo were shown.

In addition, in Fig. 17C and Fig. 17D, in order to identify the apoptosis mechanism through the siSTAT3 transfer, we have found that cyclin D1, c-Myc, cyclin-D2 and caspase 3, which are involved in cell proliferation and apoptosis, Genes and proteins were investigated. The inhibition levels of STAT3, cyclin-D1, c-Myc and cyclin-D2 were significantly higher than those of the control group. In addition, the cells treated with siSTAT3-containing microbubble-nanoliposome complex (siSTAT3) activated caspase3, an indicating protein for apoptosis, which means that apoptotic cell death process is continued. As a control, even if treated with naked siSTAT3 or treated with microbubble-nanoliposome complex (siSTAT3) containing siSTAT3, cells not exposed to ultrasound did not show apoptosis.

In FIG. 16, the cells treated with microbubble-nanoliposome complex (siSTAT3) containing siSTAT3 and exposed to ultrasonic waves had a cell survival rate of ~ 30% at 120 hours. Further, in order to enhance the synergistic therapeutic effect of the microbubble-nanoliposome complex system, the present inventors treated cancer cells with a microbubble-nanoliposome complex containing Dox and siSTAT3 at the same time. Cell viability was ~ 20% at 72 hours and less than 10% at 120 hours. When cells were observed under the microscope during the treatment, virtually no cells were found to be viable at 120 hours (Fig. 16 (d)). At 48 h, apoptotic cell morphology was also observed during the treatment of microbubble-nanoliposome complex (Dox + siSTAT3) containing Dox and siSTAT3.

Test Example 7: Therapeutic application of ultrasound-based microbubble-nanoliposome complex in vivo in animal models

In order to confirm the in vivo diagnosis and stimulation-responsive treatment of the microbubble-nanoliposome complex based on ultrasound (US), a cancer-rabbit animal model was prepared through transplantation of VX2 liver cancer cells into the kidney. Over the past few years, the rabbit has been used in the experimental study of the diagnosis and treatment of malignant tumors with a VX2 carcinoma (carcinoma) (Virmani, S. et al. J. Vasc. Interv. Radiol. 18, 1280-1286 (2007) ). In order to confirm the target specific drug and gene transfer using the microbubble-nanoliposome developed by the present inventor, it was confirmed whether the transplanted VX2 cancer tissue adequately expresses the cell-targeting marker.

Figure 18 shows the characteristics of VX2 cancer transplanted from the kidney of a rabbit. In Fig. 18a, to confirm Her2 receptor expression of VX2 tumors in the kidney, hematoxylin and eosin (Fig. 18a) were obtained after histological cleavage of embedded tumor (positive) and rabbit muscle tissue (negative control) H & E) staining. In Figure 18b, dark brown reflecting Her2 expression appeared in the tumor area. In Figure 18c, Her2 receptor expression levels were analyzed by western blot. The transplanted VX2 cancer exhibited relatively high levels of Her2 expression. In Figure 18 (d), the ability to specifically target with a microbubble-nanoliposome complex (MB-Lipo (R) -Her2 Ab ) containing a red fluorescent substance and conjugated with Her2 antibody was confirmed by CLSM (D; D + R; merged DAPI and red fluorescence images) compared to kidney tissue.

That is, embedded VX2 cancers expressed specific Her2 receptors and their levels were determined using tissue hematoxylin and eosin (H & E) staining and Western blotting. In addition, MB-Lipo (R) -Her2 Ab , a microbubble-nanoliposome complex containing a red fluorescent substance and conjugated with Her2 antibody, was able to recognize VX2 cancer tissues in vitro.

In addition, the microbubble-nanoliposome complex with dual capability of US imaging and therapeutic effects directly implanted tumors into the kidney through trans-catheter intra-articular (IA) injection of rabbits.

Figure 19 shows cancer-specific ultrasound images and transport of drugs and genes in vivo. (a) Intra-articular injections of MB-Lipo (Dox + siSTAT3) -Her2 Ab containing Her2 antibody and Dox, a typical anti-cancer drug, and siSTAT3, a typical siRNA gene therapy agent, The ultrasound image was visualized in the kidney of the rabbit by ultrasound imaging, and the ultrasound image signal disappeared due to microbubble destruction after exposure to ultrasound energy. (b) In extracted cancer tissues, tumor tissue not exposed to ultrasonic energy as a positive control using CLSM, and microbubble-nanoliposome complex containing Her2 antibody bound and Dox and siSTAT3 were compared with untreated tumor tissues In the tumor tissue treated with microbubble - nanoliposome complex containing Dox and siSTAT3, Dox (red fluorescence) showed high concentration when Her2 antibody was bound. (c) In addition, the efficiency of siSTAT3 conjugated with Her2 antibody and delivered by microbubble-nanoliposome complex containing Dox and siSTAT3 was directly measured by STAT3 target gene by PCR and was significantly reduced as in cell experiments Respectively. (d) Histologic analysis of tumor tissue by H & E staining revealed that the Her2 antibody binds and the tissue treated with microbubble-nanoliposome complex containing Dox and siSTAT3 has a decreased cell density Could know.

In Fig. 19, after the herb antibody-bound microbubble-nanoliposome complex containing Dox and siSTAT3 was injected IA, the tumor developed in the kidney of the rabbit was ultrasonically imaged by the ultrasonic imaging effect of the microbubble in the microbubble- And the echo gradually weakened after 10 times of ultrasonic high energy exposure (Mechanical index: 0.61). On the other hand, when a microbubble-nanoliposome complex without her2 antibody conjugated thereto was injected into a control tumor, ultrasonic signals showing strong targeting ability of antibody-bound pop-particles in vivo did not show high intensity (data not shown) . The microbubble-nanoliposome complex containing the anticancer drug (Dox) and the therapeutic gene (siSTAT3) was injected into the rabbit and the tumor tissue was extracted from the rabbit-grown VX2 tumor after exposure to ultrasound high energy. The extracted tumor tissues were confirmed by confocal laser scanning microscope (CLSM) and PCR. The Dox transferred to the VX2 cancer tissue clearly showed red fluorescence by itself, and the target STAT3 gene level was suppressed by the transferred siSTAT3. No effect of Dox and siSTAT3 on tissues not exposed to ultrasound high energy, no effect of Dox and siSTAT3 was observed in the control group which was not treated with microbubble-nanoliposome complex containing Dox and siSTAT3 . This means that the microbubble-nanoliposome complex containing the target-directed therapeutic of the present invention is delivered only to the specific cancer site, and the release of the therapeutic agent can be controlled by external ultrasound high-energy stimulation. In H & E staining, cancer cell density of cancer tissue treated with microbubble-nanoliposome complex containing Dox and siSTAT3 bound to her2 antibody as compared with the control group was infrequent compared to the control group, because the VX2 tumor tissue was microbubble-nano liposome Because they were partially destroyed by the delivery of the conjugate to the tumor cells.

In addition, in a rabbit tumor model, the microbubble-nanoliposome complex containing a target-directed therapeutic agent was repeatedly administered to confirm the therapeutic effect in vivo in a similar clinical environment.

20 shows evaluation of cancer treatment in a rabbit tumor using a microbubble-nanoliposome complex containing a target-directed therapeutic agent. In order to monitor the progress of inhibition of tumor growth by MB-Lipo (Dox + siSTAT3) -Her2 Ab , in which the herb antibody is bound in vivo and the microbubble-nanoliposome complex containing Dox and siSTAT3, (MRI, cross-sectional) and ultrasound imaging equipment (a: white arrows indicate implanted cancer tumors).

In FIG. 20, a microbubble-nanoliposome complex containing her2 antibody and Dox and siSTAT3 was repeatedly administered on the 1st and 5th days of the experiment, and on the 0th, 4th, and 10th days of the experiment, Were measured. The hernia treated with high energy of ultrasound and administered with microbubble-nanoliposome complex containing Dox and siSTAT3 and her2 antibody were treated with ultrasound high energy to treat untreated mass or microbubble-nanoliposome complex The growth rate was reduced compared to untreated tumors.

In addition, the bio-distribution of the herb-bound microbubble-nanoliposome complex in the rabbit animal tumor model was confirmed in liver, lung, kidney and cancer.

21 shows the in vivo distribution of microbubble-nanoliposome complexes containing fluorescence in living beings. For detection, organic dye intensities were characterized by intravenous injection of green and red fluorescent microbubble-nanoliposome complex, MB (G) -Lipo (R) -Her2 Ab and CLSM analysis outside the living tissue. Organic dyes were found to be mainly distributed in liver and lung irrespective of flash application. However, in tumor tissue, the dye increased in intensity by about 3-fold after flashing.

In addition, the excretion issue of the microbubble-nanoliposome complex has been investigated in various tissues since it is important to understand the possibility of clinical application.

Figure 22 shows the release of microbubble-nanoliposome complexes in vivo. An excretion study is required to test the imaging agent or therapeutic application with the microbubble-nanoliposome complex system. Here, the present inventors used a mouse animal model because it is a metabolism bio-model faster than rabbits. The microbubble-nanoliposome complex containing the fluorescent substance was injected into the tail vein, and fluorescence was measured in several organs of the mouse at 48 hours. As a result, the fluorescent substance-containing microbubble-nanoliposome complex completely disappeared in the mouse body without any abnormal tissue morphology. Namely, after 48 hours from the injection of the fluorescent substance-containing microbubble-nanoliposome complex, clear release of the fluorescent substance-containing microbubble-nanoliposome complex was observed in various organs of the mouse.

The present invention has been described with reference to the preferred embodiments. 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 an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (15)

A microbubble-nanoliposomal complex comprising a hydrophobic microbubble and a hydrophilic nanoliposome covalently bonded to each other. The method of claim 1,
The therapeutic agent is a microbubble-nanoliposomal complex, characterized in that selected from therapeutic genes and drugs. 3. The method of claim 2,
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. 3. The method of claim 2,
The drug is a microbubble-nanoliposomal complex, characterized in that at least one selected from anticancer agents, including doxorubicin, paclitaxel (paclitaxel), docetaxel (docetaxel). The method of claim 1,
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. The method of claim 1,
Microbubble-nanoliposomal complex, characterized in that for carrying one or more additional hydrophobic or hydrophilic fluorescent material in the complex structure. The method according to claim 6,
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. The method of claim 1,
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. The method of claim 8,
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]. The method of claim 1,
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. The method of claim 10,
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. 1) A film is formed by reacting a film-forming substance, a phospholipid, a negative charge compound, a compound having an amine group and a compound having a disulfide group in an organic solvent and reacting the resultant with a hydrophobic gas in an organic mixed solvent, Producing a bubble;
2) preparing a film by reacting a film-forming substance, a phospholipid, a negative charge compound and a compound having an amine group in an organic solvent to prepare a film, adding the resulting film to water, and dispersing and filtering the nanoliposome by ultrasonic dispersion;
3) reacting the nanoliposome obtained in the step 2) with a therapeutic agent to support the therapeutic agent in the nanoliposome structure;
4) mixing the microbubbles obtained in the step 1) and the nanoliposome obtained in the step 3) and shaking them to prepare a microbubble-nanoliposome complex; And
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. The method of claim 12,
Wherein the step (1) or (2) further comprises a step of reacting the microbubble or nanoliposome with a fluorescent substance to form a microbubble or nanoliposome structure, followed by supporting the fluorescent substance in the microbubble or nanoliposome structure. ≪ / RTI > A contrast agent composition for cancer cell-specific ultrasound, magnetic resonance imaging or fluorescence analysis comprising the microbubble-nanoliposomal complex according to claim 1. 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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