KR20160074883A - Microbubble-Liposome-Melanine nanoparticle complex and Contrast agent comprising the same - Google Patents

Abstract

The present invention relates to a microbubble-liposome-melanin nanoparticle complex comprising: a microbubble; a liposome covalently bound to the microbubble; melanin nanoparticles which are attached to the surface of the liposome and to which paramagnetic metal ions are bound; and a targeting ligand attached to the surface of the microbubble or liposome, and to a contrast agent and a pharmaceutical composition comprising the same. According to the present invention, an MR contrast agent and a tumor targeting ligand are attached to the microbubble-liposome-melanin nanoparticle complex, such that the complex is selectively attached to a tumor expressing a particular protein. Also, by using a principle of being selectively delivered into particular cells such as cancer cells and stem cells due to an external ultrasonic energy, the complex serves a role as a selective MR contrast agent. Further, by including a material such as a gene therapeutic agent or anticancer agent into the inside of the microbubble-liposome-melanin nanoparticle complex used in the present invention, the complex plays a role of selectively delivering a therapeutic agent to tumor cells expressing a particular protein and simultaneously exhibiting an effect as an MR contrast agent.

Description

Microbubble-liposome-melanin nanoparticle complexes and contrast agents containing same (Microbubble-Liposome-Melanine nanoparticle complex and Contrast agent comprising the same)

The present invention relates to microbubble-liposome-melanin nanoparticle complexes, and more particularly to microbubble-liposome-melanin nanoparticle complexes. Liposomes covalently bonded to the microbubbles; Melanin nanoparticles attached to the surface of the liposome and bound to paramagnetic metal ions; And a targeting ligand attached to the surface of the microbubble or liposome, a contrast agent containing the same, and a pharmaceutical composition.

One of the main advantages of theranostic materials is their ability to perform multiple functions simultaneously. Single particles can be used to diagnose disease and to imaging and targeting affected cells (X. Xie, et al. Adv . Drug Deliv . Rev. 2010, 62, 1064; HL Liu, et al. Theranostics 2014, 4, 432). To date, most of the organic, inorganic and compound therapeutic diagnostic agents developed include magnetic nanoparticles, quantum dots and new metal substrates. These therapeutic diagnostic agents have a variety of attractive properties: increased biological availability due to prolonged circulation time, selective tumor accumulation due to enhanced permeability and retention (EPR) effects (H. Xiao, et al., J. Control Release 2014, 10, 173; G. Shi, et al. Adv . Healthc Mater . 2014, 3, 142), and imaging compatibility with existing detection patterns (e.g., magnetic resonance imaging or optical excitation), their clinical interpretation remains challenging. The potential toxicity of constituent materials (especially inorganic nanoparticles) results in significant safety concerns and toxicological indications, all of which need to be handled carefully prior to clinical application (A. Gojova, et al., Environ, Health Perspect . 2007, 115, 403; A. Sarkar, et al. J. Nanosci . Nanotechnol . 2014, 14, 730).

Microbubbles (MBs) and liposomes (Lipos) are emerging as biocompatible materials for cancer diagnosis and treatment and are therefore presented as safer alternatives to traditional therapeutic diagnostic agents (S. Capece, et Chem . Commun ., 2013, 49, 5763, H. Cho, et al., Nanomedicine 2014, 10, 619). By coupling MBs and Lipos as a complex, this conceptual substance is known to achieve highly efficient target delivery of therapeutic agents as well as simultaneous ultrasound (US) imaging of cells (SP Wrenn, et al. Theranostics 2012, 2 , 1140). In addition, when introducing superparamagnetic iron oxide nanoparticles, the aggregate provided double contrast in both magnetic resonance (MR) and US imaging (F. Yang, et al. Biomaterials 2009, 30, 23). Although such formulations enable multi-model imaging, the introduction of inorganic particles, like traditional therapeutic diagnostic agents, reduces its biocompatibility and limits its clinical potential.

In the present invention, it is possible to obtain a completely US-MR imaging, including MB, Lipo, and Fe ^{3 +} ion-chelated melanin nanoparticles (MNPs), which enable enhanced gene transfer as well as dual US- Hybrid, hybrid and multi-functional hybrid. As the advantage of the high Fe ^{3 +} loading capacity (loading capacity) of MNPs, complex occurs as a result shows a MR contrast only biocompatible chemicals (chemicals) (KY Ju, et al. Biomacromolecules 2013, 14, 3491). In the present invention, the hybrid particles are also functionalized with antibodies to 1) specific targeting of agents to tumor cells, and 2) US-stimulated delivery of the linked MNPs to Lipo cells. After local application of high sound pressure (US flash), the inventors have confirmed the enhancement of the therapeutic effect of the internalized MNP and Lipo particles, together with the MRI signal.

It is therefore an object of the present invention to provide microbubble-liposome-melanin nanoparticle complexes capable of fluorescence and ultrasound (US) image analysis with improved MR imaging effect.

It is another object of the present invention to provide a multi-modal contrast agent for fluorescence, ultrasound, and magnetic resonance (MR) including the microbubble-liposome-melanin nanoparticle complex.

It is still another object of the present invention to provide a pharmaceutical composition comprising the microbubble-liposome-melanin nanoparticle complex and a therapeutic gene or drug as an active ingredient.

It is another object of the present invention to provide a method for producing a microbubble-liposome-melanin nanoparticle complex which is specific for cancer cells and capable of multimodal imaging for fluorescence, ultrasound and magnetic resonance.

It is another object of the present invention to provide a method for preparing a microbubble-liposome-melanin nanoparticle complex capable of imaging the expression of a tissue or a specific cell protein that has relied on pathological histology.

According to one aspect of the present invention, the present invention provides a microbubble; Liposomes covalently bonded to the microbubbles; Melanin nanoparticles attached to the surface of the liposome and bound to paramagnetic metal ions; And a microbubble-liposome-melanin nanoparticle complex comprising a targeting ligand attached to the surface of the microbubble or liposome.

In the present invention, the complex comprises a hydrophobic microbubble and a hydrophilic nanoliposome bound to each other through a hybrid reaction, melanin nanoparticles bonded to the outside of the liposome, targeting molecules such as an antibody to the outside of the complex, Are combined with each other.

As shown in FIG. 1, the microbubble-liposome-melanin nanoparticles of the present invention are nanoparticles of a hydrophilic lipid structure having a size of about 200 nm on the outside of a spherical hydrophobic microbubble structure having a uniform particle size of about 1.3 μm in average diameter It is a particle with a size of about 1.7 μm with a combination of doped melanin nanoparticles attached to the complex bound to the surface. The inside of the micro bubble contains a hydrophobic gas such as SF ₆ , CO ₂ or CF ₄ .

In the present invention, the microbubble-liposome-melanin nanoparticle complex is also expressed in the present specification or in the figures as "MB-Lipo-MNP (Fe)" or "ML-MNP (Fe)".

In the present invention, the complex may preferably exhibit a uniform size of 1 to 2 μm in average diameter, but it is not limited thereto, and may be a particle size ranging from 100 nm to 10 μm according to the concentration and the filtration method of the liposome during the preparation of the complex Can be adjusted. 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.

In the present invention, the microbubbles may be synthesized by bubbling a mixture of a phospholipid compound and cholesterol with a hydrophobic gas accompanied by film formation and mechanical vibration.

According to embodiments of the present invention, the microbubbles may be prepared using film forming materials, phospholipids, negative charge compounds, compounds having amine groups, and compounds having disulfide groups, wherein the film forming materials include DPPC (1,2 -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 the compound having an amine group include 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dierucoylsn-glycero-3-phosphoethanolamine (DLPE) 3-phosphoethanolamine, DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine); And DSPE-PEG-SPDP {1,2-distearoyl-snglycero-3-phosphoethanolamine-N- [PDP (polyethylene glycol) -2000]] may be used as the compound having a disulfide group.

In the present invention, the liposome may be prepared by film formation, ultrasonic treatment and extrusion of a mixture of a phospholipid compound and cholesterol.

According to an embodiment of the present invention, the liposome may be prepared using a film-forming substance, a phospholipid, a negative charge compound and a compound having an amine group, wherein the film forming material is 1,2-dipalmitory-snglycero-3- phosphatidylcholine, DDPC, 1,2-didecanoyl-sn-glycero-3-phosphocholine, DEPC phosphocholine, 1,2-dilauroyl-sn-glycero-3-phosphocholine, DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DOPC (1,2- dioleoyl- 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- dimyristoylsn-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-snglycero-3-phosphoethanolamine) or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine).

In the present invention, the hybrid of microbubbles and liposomes can be produced by modifying a lipid structure of a microbubble or a lipid structure of a nanoliposome by controlling pH, reaction temperature or reaction time, .

In the present invention, the microbubble or liposome may contain at least one fluorescent substance therein. As the fluorescent material, any hydrophobic or hydrophilic fluorescent material that can be used for clinical diagnosis can be used. For example, FITC, 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.

In the present invention, the term 'melanin' refers to a biological polymer present in various parts of various living things such as plants, animals, protists, etc., and generally classified into eumelanin of dark brown color and pheomelanin of yellowish red color do. In the present invention, melanin nanoparticles are used, and the diameter of the melanin nanoparticles is 30 nm to 600 nm, preferably 30 nm to 200 nm.

In the present invention, the melanin nanoparticle (MNP) may be extracted from nature or chemically synthesized. In the case of extraction in a natural environment, it can be recovered by centrifugation from squid ink. When chemically synthesized, it can be synthesized from melamine precursors of dopamine, DOPA or cysteine.

As an example, the melanin nanoparticles can be synthesized from the spontaneous oxidation and polymerization of dopamine, a melanin precursor. For example,

Which can be prepared by a process comprising the following steps:

1) dopamine · H ⁺ X ^- the step of neutralization by adding a base to the solution containing, and

2) polymerizing dopamine in the solution of step 1)

Here, the molar ratio of the dopamine · H ⁺ X ^- to the base is 1: 0.1 to 1: 1.

The step 1 is a dopamine · H ⁺ X ^- with a base and a step of reaction by neutralizing the dopamine · H ⁺ X ^- and the molar ratio of the base from 1: 0.1 to 1: Nano having Adjust to 1 constant melanin-like particle Can be prepared. Step 2 is a step of neutralizing with a base and then polymerizing to prepare melanin nanoparticles.

The X ^- may be selected from the group consisting of halide ions, HSO ₄ ^- , NO ₃ ^- , H ₂ PO ₄ ^- and CH ₃ COO ^- . The base may be selected from the group consisting of alkaline metal hydroxides, alkaline earth metal hydroxides, carbonates of alkali metals, carbonates of alkaline earth metals, bicarbonates of alkali metals, bicarbonates of alkaline earth metals, acetates of alkali metals, phosphates of alkali metals, ~ 20), ammonia, ammonia water, and amines.

In the case of the melanin nanoparticles prepared by the above-described method, the shape of the particles is more uniform and more dispersed in water than the melanin nanoparticles extracted from nature.

In the present invention, the paramagnetic metal ion refers to a material showing a nuclear magnetic resonance image. Unpaired spins existing in the inner part exhibit an irregular spin arrangement due to thermal motion in an ordinary state. However, when an external magnetic field is applied, As a result, spin alignment occurs in a certain direction, which means a material which is not magnetized normally but is magnetized in the magnetic field direction when an external magnetic field is applied. Examples thereof include a group consisting of gadolinium (Gd), iron (Fe), manganese (Mn), nickel (Ni), copper (Cu), erbium (Er), europium (Eu), holmium (Ho) May be used as the metal ion.

The paramagnetic metal ions, can be combined melanin and coordination of melanin nanoparticles and, if to be coordinated to the paramagnetic metal ion to the melanin of the melanin nanoparticles, the conventional Gd-based contrast agent and more T ₁ The damping effect is more dominant T ₁ weighted images It is possible to exert an excellent nuclear magnetic resonance imaging effect.

The paramagnetic metal ion may be combined with 1 to 10 μg, preferably 2 to 7 μg, relative to the weight of the melanin nanoparticles. Also, since the smaller the size of the melanin nanoparticles, the larger the surface area relative to the weight of the melanin nanoparticles, the smaller the size of the melanin nanoparticles is, considering the binding amount of the paramagnetic metal ions.

In the present invention, the targeting ligand can selectively recognize / bind a target substance such as a molecule, a ligand, or a receptor on the surface of a target cell Any substance can be used, for example, molecules such as antibodies, hormones, drugs, etc. that can bind to a specific receptor present on the surface of a target cell.

The targeting ligand may be incorporated into the complex surface of the present invention directly or indirectly through a common reaction in a covalent or noncovalent manner and may include, for example, an ionic bond, an electrostatic bond, a hydrophobic bond, a hydrogen bond, Or via a van der Waals bond. 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 an embodiment of the present invention, the targeting ligand may be a cancer cell-specific antibody. As an example, an antibody specifically recognizing a Her receptor expressed in breast cancer cells may be administered to ML-MNP (Fe) particles of the present invention Thereby enabling specific targeting of breast cancer cells.

In the present invention, the targeting ligand is attached to the surfaces of microbubbles or liposomes to facilitate the targeting of ML-MNP (Fe) particles. The targeting ligands are attached to microbubbles of ML-MNP (Fe) The efficiency can be further improved.

In the present invention, when the complex approaches the target cell by the targeting ligand on the surface, it can be destroyed through the external sound stimulus to transfer the therapeutic agent such as the therapeutic gene and the drug and the fluorescent substance carried in the complex structure simultaneously into the target cell At this time, the intensity of the sound wave stimulus is not particularly limited, but the mechanical index 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 another aspect of the present invention, the present invention provides a contrast agent for multi-modal analysis for fluorescence, US and magnetic resonance imaging (MR) comprising said microbubble-liposome-melanin nanoparticle complex do.

In the present invention, the contrast agent refers to a substance that is administered in the body to contrast or image cancer cells in a strong and specific manner in vivo. Is widely used. The term contrast agent of the present invention is not limited to the range of conventional CT or MRI contrast agents, and is used to include an imaging agent for ultrasound (US) image analysis, an imaging agent for fluorescence image analysis, and the like.

According to the embodiment of the present invention, the imaging pattern of ML-MNP (Fe) particles included in the contrast agent of the present invention was compared with SonoVue, a commercially available clinical ultrasound (US) contrast agent, and Gadovist, Showing similar echo occurrence (FIG. 8), showing a T ₁ -w MR imaging signal twice as high as Gadovist (FIG. 9). Thus, the contrast agent comprising the complex of the present invention can be used in multiple modes of ultrasound and MR imaging, and is superior in contrast to conventional contrast agents.

In addition, the complexes of the present invention exhibit remarkably enhanced MR imaging images with the formation of microbubbles, liposomes, and complex formation of melanin nanoparticles. MNP (Fe) -HER2 particles conjugated to melanin nanoparticles without microbubble-liposomes (ML) exhibited low T ₁ -w MR signals, while ML-MNPs of the present invention Fe) -HER2 particles displayed a remarkably bright image (c in Fig. 3). This indicates that it is not enough to increase the MR imaging efficiency only with MNP (Fe) without the ML complex.

In the contrast agent of the present invention, the complex may further contain a therapeutic gene or drug inside the liposome. The step of carrying a therapeutic agent such as the therapeutic gene or drug in the liposome structure can be carried out 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 gene, In the case of 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 contrast agent 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. Parenteral formulations preferably comprise sterile aqueous solutions or suspensions comprising the microbubble-liposome-melanin nanoparticle complexes 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 comprising a diagnostically effective amount of a microbubble-liposome-melanin nanoparticle 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.

Contrast agents according to the present invention are 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-liposome-melanin nanoparticle complex as the contrast agent may be 0.1 mM to 10 M.

The contrast agents of the present invention are used in a conventional manner for imaging analysis. For example, the contrast agent may be administered to a mammal in an amount sufficient to provide appropriate visualization locally to the organ or tissue to be systemically or imaged, and then the mammal may be fluoroscopically, sonographically, or MRI-scored.

The contrast agent of the present invention can be useful not only as a general imaging effect through specific cell-specific targeting as described above, but also as a means for providing pathological or pharmacological information necessary for a patient prior to diagnosis. For example, targeting ligands included in the contrast agent can target a specific protein, that is, a marker protein expressed in a specific tissue or cell, so that the pathological or pharmacological Information on marker proteins that are differentially expressed depending on the type of information, such as the type of cancer, progression, and metastasis range, can be known through imaging before the biopsy. Therefore, it is possible to know the effect of the specific drug on the patient in advance, which may be helpful in selecting the treatment plan and the therapeutic drug of the patient.

According to another aspect of the present invention, there is provided an anticancer pharmaceutical composition comprising the microbubble-liposome-melanin nanoparticle complex, an anticancer gene or an anticancer agent as an active ingredient.

In the present invention, the anticancer gene carried on the inside of the complex structure may be an expression plasmid, an siRNA, an shRNA, an antagonist of microRNA or microRNA capable of inhibiting the expression of a gene having known antitumor effect, Of DNA or RNA can be used. For example, doxorubicin, paclitaxel or docetaxel may be used as the above-mentioned drugs, but the present invention is not limited thereto. These genes and drugs can be reacted with liposomes according to a conventional method to be carried in a complex structure. In the case of a gene, the gene and the drug can be supported by reacting with a nanoliposome in the form of a hybrid with a biopolymer such as protamine, , It can be carried in the liposome through the usual ammonium sulfate concentration gradient method.

According to embodiments of the present invention, as an example of the anti-cancer gene, siRNA against a cell suicide-suppressing gene (Survivin) is carried in the complex, and the complex of the present invention is targeted to cancer cells to measure cancer cell viability. As a result, the siRNA gene was effectively internalized into cells due to the ultrasound-mediated uptake process, and this absorption effectively down-regulated survivin expression in the cells (Fig. 4c). Cell viability after siRNA delivery was significantly decreased. The concept of theranostics using the microbubble-liposome-melanin nanoparticle complex of the present invention is shown in FIG.

Therefore, the microbubble-liposome-melanin nanoparticle complex of the present invention diagnoses cancer through multiple image analysis using fluorescence, ultrasound, or fluorescence imaging specific to cancer cells while minimizing adverse effects through high target specificity, Can effectively treat cancer by effectively delivering one or more therapeutic agents to the target cancer cells so that effective diagnosis 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 and ovarian cancer And can be usefully used for the development of therapeutic agents.

In order to use the pharmaceutical composition for anti-cancer medicine of the present invention as a medicine, it may be prepared by a method known in the pharmaceutical field. In this case, microbubble-liposome-melanin of the present invention, which contains a known anti- In addition to nanoparticle complexes, pharmaceutically acceptable carriers may be included. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, phosphorus calcium, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, no. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, 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.

According to another aspect of the present invention, the present invention provides a microbubble-liposome-melanin nanoparticle complex capable of multimodal imaging for fluorescence, ultrasound, and magnetic resonance (MR) Lt; RTI ID = 0.0 > of:

a) forming a phospholipid film from a phospholipid compound to which a fluorescent substance is added, adding the phospholipid film to an organic solvent, and synthesizing a microbubble through bubbling and mechanical vibrating of a hydrophobic gas;

b) adding a phospholipid film formed from a phospholipid-containing phospholipid compound to an organic solvent, and ultrasonically dispersing the liposomes, and synthesizing liposomes by extrusion using a nanofilter;

c) adding a solution containing paramagnetic metal ions to a solution containing melanin nanoparticles to bind paramagnetic metal ions to the melanin nanoparticles;

d) synthesizing a microbubble-liposome complex by introducing a thiol group into the ziazospores synthesized in the step b) and then shaking the microbubble with the microbubble synthesized in the step a);

e) preparing a microbubble-liposome-melanin nanoparticle complex by mixing the thiolated microbubble-liposome complex of d) with melanin nanoparticles bound to paramagnetic metal ions of step c); And

f) introducing a maleimide group into the microbubble-liposome-melanin nanoparticle complex, and then binding the maleimide group to the cancer cell-specific antibody.

In the method of the present invention, before the phospholipid film formed from the phospholipid compound in the step b) is homogenized with the mixed solution of the biopolymer and the therapeutic gene using an electrophoresis protocol, You can do more.

The present inventors have produced hybrid multiplexable particles composed of MB, Lipo and MNP (Fe). The prepared composite particles showed high biocompatibility and selectivity and demonstrated multi-mode imaging performance for fluorescence, US and MR. Interestingly, the associated therapeutic Lipos and MNP (Fe) s rapidly penetrated into cancer cells after exposure to the US flash and ultimately improved treatment effects and MR imaging due to MB cavitation and sonoporation . As a non-toxic therapeutic diagnostic agent, the hybrid complexes of the present invention can easily lead to US and MR-guided cancer treatment in clinical applications.

Figure 1 shows the properties of microbubbles (MB), nanoliposomes (Lipo), melanin nanoparticles (MNP (Fe)), and their complexes.
Spherical shapes of MB and Lipo particles prepared from the phospholipid compounds were confirmed with a microscope (a, left) and a frozen-electron microscope (a, right), respectively. In addition, the Fe ^{3 +} ion-chelated MNP and MNP (Fe) also showed a spherical shape as evidenced by the TEM analysis (b), and the intensity of the ion metal bound to the MNP surface was mapped to the scanning TEM (b, . Quantitative analysis was also performed with EDX equipped TEM and several energy-dispersion peaks indicated the presence of ionic metal (b, lower right, corresponding peaks indicated by black arrows). After MNP (Fe) was anchored on the ML, the hybrid particle solution was loaded onto a silicon wafer plate and then slowly dried. The sunken particles are marked with a red dotted circle circle and the black arrows indicate connected MNP (Fe) particles on the ML. The size distribution of all particles produced was measured by DLS analysis (d).
Figure 2 shows specific targeting, US flash effect, and cytotoxicity assays.
green and red fluorescence M _G L _R -MNP (Fe) -HER2 particles specifically recognized Her2 receptors expressed in breast cancer cells (SKBR3) as compared to negative cells (MCF7) . HER2 conjugated particles appeared on the cell membrane (a, stomach-right, blue represents DAPI-stained nuclei). SKBR3 cells treated with antibody-unbound particles showed little fluorescence after US flash (MI = 0.61) (a, bottom-left). However, SKBR3 cells treated with M _G L _R -MNP (Fe) -HER2 particles with US Flash showed fluorescent dyes in the cell cytosol (a, bottom-right). The released MNP (Fe) s was found to be located in the cytoplasm of SKBR3 cells using bio-TEM analysis (b), and there was no change in the size and shape of MNP (b, inserted drawing). M _G L _R -MNP (Fe) -HER2 and US flushed cells exhibited a survival rate of 90% or more (c) for various concentrations and incubation times. For cytotoxicity analysis measured at the protein level, 0.66 mg / ml of particles were added to the cells and then measured for protein level changes associated with cell organelle function using Western blotting (d). These cells exhibited similar expression levels as untreated control cells (all experiments were performed three times in c and d). Data show mean +/- SD.
Figure 3 shows the effect of US and MR imaging of the targeted cells, and flash stimulation.
In phantom US analysis (iU22, Philips), the treated SKBR3 cell solution showed significantly higher echogenicity compared to the untreated cell solution (a, left and middle, MI = 0.08). The signal disappeared completely after 6 hours of flash (a, right, see Figure 8b). HER positive SKBR3 and negative MCF7 cells were characterized for expression levels of HER2 receptor by Western blotting (b). After US flash, treated cells showed a significant T ₁ -w MR imaging signal for SKBR3 as compared to control cells (c, I: flash, ii: MNP (Fe) -HER2, iii: ML-MNP ) -HER2)
4 is in RTI ID = 0.0 &gt; siRNA < / RTI &gt; in vitro .
To introduce the therapeutic gene, siRNA against Survivin was blended with protamine (PA) via electrostatic interaction between the two. Optimal complexity ratios (50 μM siSurv and 40 μM PA) were measured using electrophoresis (a). The loading capacity of the siSurv- PA complex in Lipo was calculated by monitoring the absorption spectrum generated by the UV-Vis spectrophotometer (b). The target protein showed an 8-fold reduced level of expression, as evidenced by Western blotting, compared to siSurv alone cells (c). After treatment, cell viability was observed as a function of time. ML _siSurv -MNP (Fe) -HER2 and the flashed cells showed a significant reduction in viability (survival less than 60%) at day 3, while other controls showed sustained viability up to 90% (d). (All experiments in c and d were performed three times, with data representing mean +/- SD * P <0.05, ** P <0.01)
Figure 5 shows the chelating capacity of Fe ^{3 +} ions for MNP.
To test the chelating capacity of Fe ^{3 +} ions for melanin nanoparticles (MNP), Fe (NO ₃ ) ₃ .6H ₂ O was dissolved in distilled at various concentrations (210 μmol) And mixed with 1 ml of solution containing dispersed MNPs (1 mg / ml). After completion, it was washed and then precipitated at 13,000 rpm for 10 minutes to remove excess Fe ^{3 +} ions. The amount of Fe ^{3 +} ions loaded was directly measured by ICP-AES analysis (a). During the treatment of 8 μmol Fe ^{3 +} ion solution, the Fe ^{3 +} ions increased to a maximum of 3 μmol loading per mg MNP. More importantly, the chelated Fe ^{3 +} ions should not be released from the MNP particles at various pH levels, indicating retention of the contrast agent. According to ICP-AES analysis, very small amounts of Fe ^{3 +} ions (<5%) were detected at various pH levels (4, 7 and 10) after 72 hours of stirring (b).
FIG. 6 shows the production process and characteristics of ML-MNP (Fe) -HER2 hybrid particles.
The red and green fluorescent M _G L _R -MNP (Fe) -HER 2 particle complexes were synthesized by a three-step chemical reaction; MB-Lipo hybrid reaction, MNP (Fe) anchoring, and antibody conjugation (a). For ML fabrication (a, i), DSPE-PEG-SPDP in MB after thiolation of Lipo-DPPE was cross-linked with a sulfhydryl group and the complete hybrid reaction was carried out using pyridine-2 as a chemical leaving group -Thione (b) was confirmed by UV-Vis spectrophotometer. MNP (Fe) was attached to the thiolated Lipo (a, ii), which was further treated with HS-PEG chemistry to increase solubility in an aqueous environment. Finally, in order to impart specificity to the particles, the HER2 antibody (HER2) was bound to the maleimide functional group on MB and Lipo after reaction of DPPE and Sulfo-SMCC (a, iii). All processes are fully described in the Examples. The red and green organic dyes introduced into the Lipo and MB complexes were characterized by a confocal laser scanning microscope (c). The MB appeared as a hollow spherical shape due to the core SF ₆ gas, and the Lipos around the green MB appeared as a small point of red (c, inset drawing).
Figure 7 shows an improvement in absorption efficiency through specific targeting and US flash stimulation.
ML-MNP (Fe) -HER2 particles can be attached to the cells with specific targeting to HER2-positive SKBR3 cells. After 1 minute of US flash (MI = 0.61), the MB of the particles quickly disintegrated and released the Lipo- and MNP (Fe) -conjugated materials into the cells.
In addition, the destructive force of MB induces a pore (about 220 nm in diameter) in the cell membrane, thereby absorbing the connected substances (a). As a control, MCF7 cells (FIG. 3 b) expressing very low levels of HER2 receptor were treated with the particles under the same conditions. Rare MNP (Fe) particles were identified in cell cytosol due to non-specific inclusion (b). Red and green fluorescent organic dyes from MB and Lipo were quantitatively analyzed using FACS (c).
8 is a US phantom analysis result for monitoring comparison of contrast agents echogenicity and MB cavitation under flash processing.
After loading the US Phantom plastic tube, the synthesized M _G L _R -MNP (Fe) -HER2 particle solution was used clinically in the same concentration of phospholipid (DPPC, 10.5 mM) under US imaging mode (MI = 0.08) Similar to SonoVue US contrast agent, echogenicity and intensity were shown (a). After flash application (MI = 0.61), the echogenicity gradually disappeared (b). The particles were found to be destroyed within 6 flashes.
Fig. 9 shows measurement results of longitudinal axis relaxation ( r ₁ ) and T ₁ -w MR in phantom analysis.
The present inventors measured the longitudinal relaxivity value of the ML-MNP (Fe) -HER2 particles and the clinically used T ₁ MR agent (Gadovist) using a 0.47-T magnetic relaxometer (mq20, Bruker) Respectively. The longitudinal relaxation time was measured at various concentrations of metal ions and also accurately calculated using ICP-AES analysis. The particle solution showed a r ₁ that was two times higher than Gadovist (a). According to the Solomon-Bloembergen-Morgan (SBM) hypothesis, the second sphere of water molecules and oxygen atoms in Fe ^{3 +} chelated catechol complexes increases r _1, and the nanoparticle properties of MNPs Shows increased r ₁ and limited rotational mobility. In the MR phantom analysis using a clinically used MRI device (3.0-T, Philips), the ML-MNP (Fe) -HER2 particle solution showed a roughly two-fold higher white intensity at the same metal ion concentration (b ).
Figure 10 illustrates the concept of diagnostic-therapeutic application using the microbubble-liposome-nanocomposite according to the present invention.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of MB, Lipo, MNP (Fe) and composite thereof

(1) Preparation of micro bubble (MB)

DPPE (1.2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine), 1.0 mg of DCP (diacetyl phosphate), 1.2 mg of DPPE (1,2-dipalmitoyl- 5.0 mg of PDP (1,2-distearoyl-sn-phosphoethanolamine-N- [PDP (polyethylene glycol) -2000) and 3.5 mg of cholesterol were dissolved in 5 ml of chloroform (99.9%, Sigma-Aldrich, , USA). The mixed solution was evaporated at room temperature for 5 minutes and lyophilized at -45 ° C for 24 hours to induce the formation of a phospholipid film. Subsequently, 2 ml of a solution containing glycerin, propylene, and H ₂ O (volume ratio: 1: 2: 7) was added to the film and the solution was gently shaken. After that, it was transferred to a hermetic vial (Wheaton, NJ, USA), and a high-speed shaking-device (KIMS, South Korea) was used for bubbling with SF ₆ gas. Microbubbles (MB) were prepared. The prepared MB particle solution was stored in a refrigerator.

(2) Production of nanoliposome (Lipo)

Lipo was prepared from a chloroform solution containing DPPC, DCP, DPPE and cholesterol (the amount of each ingredient used is the same as in (1) above). The solution was evaporated and lyophilized as described above. After addition of 2 ml H ₂ O, the lipid film was sonicated at 60 ° C for 5 minutes. The liquid was subjected to a freeze-thaw cycle five times using liquid nitrogen and a constant temperature bath. The prepared Lipo dispersion solution was extruded at 60 ° C through a 200 nm filter (mini-extruder; Avanti polar lipids) to prepare a nanoliposome (Lipo).

(3) Carrying of fluorescent substance

1 mg of fluorescein isothiocyanate (Sigma-Aldrich) and 1 mg of Texas-red (Sigma-Aldrich) were added prior to forming the MB and Lipo films, respectively, 1 mg was uniformly added to the lipid mixed solution. Excess organic dyes were continuously removed by centrifugation (5 min at 13,000 rpm) and washing with H ₂ O until the color of the supernatant completely disappeared.

(4) Introduction of Sulphor drill machine

After adjusting the pH to 8.2 with 1 M NaHCO ₃ to make the sulfhydryl functional group on the lipo particles, 2 ml of amine active Lipo (21 mg) derived from DPPE was added to 5 mg of 2-iminothiorene HCl (2-iminothiolane · HCl; Traut's reagent, Pierce) at room temperature for 2 hours. The 2 ml thiolated Lipo (10.5 mg / ml) and 1.5 ml MB (13.1 mg / ml) solution was gently shaken for 2 hours at room temperature and the complex reaction was monitored by UV-Vis spectroscopy, Was monitored by calculating the amount of 2-thione (pyridine-2-thione).

(5) Preparation of melanin nanoparticles (MNP)

To prepare MNP particles, 180 mg of dopamine hydrochloride was dissolved in distilled water at 50 캜 and 780 μl of NaOH solution (1 M) was vigorously stirred. After 6 hours, when it turned black, the solution was centrifuged (20,000 rpm for 10 minutes) and washed several times with distilled water. As measured by TEM and DLS, the MNP particles exhibited a characteristic size (diameter = 100 nm) and exhibited a uniform size distribution.

(6) Preparation of boxed metal ion-bound MNP

To bind Fe ³⁺ ions, 4 ml of Fe (NO ₃ ) ₃ .6H ₂ O with a concentration range of 210 μmol was mixed with 10 ml of MNP dispersion (1 mg / ml). After stirring for 3 hours, the solution was centrifuged (20,000 rpm for 10 minutes) and unbound Fe ³⁺ ions in the supernatant solution were quantitatively evaluated to determine the total loading capacity. This was done by ICP-AES (inductively coupled plasma atomic emission spectrometer).

(7) Preparation of ML-MNP (Fe) complex

(30 mg, 15 mg / ml) was mixed homogeneously with 1 ml of MNP (Fe) solution (1 mg / ml) for 0.5 hour at room temperature Respectively. Excess MNP (Fe) particles were removed by washing the ML-MNP (Fe) complexes three times with centrifugation (5 min at 13,000 rpm) and distilled water. To increase stability in aqueous environments, the resulting composite particle solution was treated with 2 ml of PEG-SH (methoxy-poly (ethylene glycol), 2 kDa, 0.5 mM) for 1 hour at room temperature, Of PEG-SH was removed by centrifugation (2 min at 13,000 rpm). The size and shape of the complexes were characterized using FE-SEM, TEM and DLS measurements. In the present invention, all phospholipid chemicals were purchased from Avanti Polar Lipids, and all other chemical reagents were purchased from Sigma-Aldrich. All chemicals were used without further purification.

Example 2. HER2 antibody conjugation

To make an active maleimide functional group, the pH was adjusted to 8.2 with 1 M NaHCO ₃ solution and then 2 ml of ML-MNP (Fe) particle solution (14.9 mg / ml ) Was reacted with Sulfo-SMCC (sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate; 5 mg, Sigma-Aldrich) for 3 hours. Next, the maleimide moiety was conjugated to the appropriate half-antibody HER2 using a conventional bio-conjugation method. The antibody-containing particles were quantitatively analyzed using a typical protein assay assay.

Example 3. Cytotoxicity assay

MTT assay kit (Invitrogen) was used to assess cell viability in the presence of ML-MNP (Fe) particles. SKBR3 cells (ATCC, VA, USA) were cultured in RPMI supplemented with 10% FBS, penicillin and 1% streptomycin. Cells were maintained in a 5% CO ₂ incubator at 37 ° C. After the cells reached 80% confluence, the cells were washed, trypsinized and resuspended in the culture medium. Next, cells were inoculated into 96-well tissue culture plates at a concentration of 5,000 cells / well and then cultured overnight in a CO ₂ incubator. Various concentrations of ML-MNP (Fe) particles in 1 ml PBS buffer solution were added to the cell culture solution, and the cells were cultured for 24, 48, and 72 hours, respectively. To measure cell viability, the culture medium was replaced with MTT solution. After 3 hours of incubation in a CO ₂ incubator, the formazan crystals obtained by adding MTT solution were dissolved. Cell viability was measured by spectrophotometry at 570 nm, subtracting background at 690 nm.

Example 4. Ultrasonic phantom analysis

The echogenicity of ML-MNP (Fe) -HER2 was evaluated using phantom samples. This solution was prepared using a plastic connecting tube with an internal diameter of 2 mm, placed in a chamber filled with water. 8.7 mg of particles or Sonovue solution dissolved in 2 ml of PBS was passed through the plastic tube at 0.1 ml / s. (Mechanical Index, MI = 0.08) was used to image an ultrasound scanner (iU22, Philips, Bothell, WA, USA) equipped with a 512 MHz broadband linear probe.

Example  5. Phantom and in vitro MR  analysis

T ₁ emphasized (T ₁ -w) for MR phantom analysis, various concentrations (0 1000 nM, [Fe] or [Gd]) ML-MNP ( Fe) particles or Gadovist 0.5% agarose gel (agarose gel of ) (1: 1 by volume). in To test the in vitro MR imaging, particles at a concentration of 3 μM [Fe] were reacted with HER2-positive SKBR3 and HER2-negative MCF7 cells in a 5% CO ₂ incubator for 1 hour. The US probe was then placed on the back of the well plate and six flash pulses (MI = 0.61) were applied for one minute. Excess particles were removed by washing the cell culture with the culture medium, the treated cells were separated by trypsin treatment, and then centrifuged at 3,000 rpm. All T ₁ -w MR images were acquired on a 3.0-T clinical MR scanner (Philips medical system, Netherland, archieva Release 3.2.1.0 version). The axial and coronal T ₁ -w images have a TR 400 ms, a TE 10 ms, a 240 × 240 matrix, a flip angle of 90 degrees, a slice thickness of 3 mm, a number of averages with 4 , A bandwidth of 115 Hz / pixel and a 120 × 120 mm FOV.

Example  6. siSurv  Introduction and cell treatment

To load the siRNA ( siSurv , 19mer) to Lipo against Survivin, siSurv (50 μM) and PA (protamine, 7.5 kDa, 40) were homogenized in lipid film (21 mg, 2 ml) for 5 minutes at room temperature using electrophoresis. Lipo containing siSurv -PA was washed twice by centrifugation (5 min at 13,000 rpm) to remove the unloaded gene complex and the loading capacity was measured by UV-Vis spectrophotometer. The gene-PA complexes were applied to the ML-MNP (Fe) -HER2 preparation process. Subsequently, 3 x 10 ⁴ cells were inoculated on a 48-well plate (BD Falcon) and ML _siSurv- MNP (Fe) -HER2 particles (1 mg) dissolved in 0.5 ml PBS were added to a culture medium Lt; / RTI &gt; cells. After 1 hour of incubation, the excess particles were washed with PBS solution. For flash application, a clinical US scanner probe was placed on the back of a 48-well plate and a flash pulse (MI = 0.61) was applied for 1 minute. The treated cells were also washed twice with culture medium and subcultured for 2 days.

Test Example  One. Micro bubble ( MB ) And Nanoliposomes (Lipo)  Identify characteristics

MBs was synthesized by homogeneous mixing of various phospholipids and cholesterol, followed by bubbling of hydrophobic SF ₆ gas with film formation and mechanical vibrations. Similarly, Lipos was prepared through film formation, sonication, and without gas. The shape and size distributions of MBs and Lipos were analyzed by microscopy, cryo-electron microscopy and dynamic light scattering (DLS) (Fig. 1 (a) and (d)). The synthesized MBs and Lipos were spherical with mean diameters of 1.3 μm and 200 nm, respectively. The MB and Lipo cores are composed of a hydrophilic gas phase and a hydrophilic aqueous phase, respectively. In addition, during the preparation, a hydrophilic dye (fluorescein isothiocyanate, G) for positioning the alkyl chain of the MB shell and a red hydrophilic dye (Texas Red, R) for introduction into the aqueous Lipo core were added .

Test Example  2. Melanin nanoparticles ( MNP )

MNPs were prepared as follows. Briefly, 780 μl NaOH solution (1 M) was added to 90 ml of dopamine hydrochloride solution (2 mg / ml) at 50 ° C under vigorous agitation to facilitate spontaneous oxidation and polymerization of dopamine to form MNPs . After 6 hours, the particles were collected by centrifugation at 20,000 rpm (10 min) and washed several times with distilled water. The obtained spherical MNP particles showed an average diameter of 100 nm and a regular size distribution. To introduce the magnetic properties, 1 mg of MNPs was dissolved in 2 ml of Fe (NO ₃ ) ₃ .6H ₂ O (1.85 mM) solution and Fe ^{3 +} ions were added to the catechol 0.0 &gt; o-dihydroxyl &lt; / RTI &gt; group. This procedure loaded 3 μmol of Fe ³⁺ ions per mg of MNP, which was measured by ICP-AES (inductively coupled plasma atomic emission spectrometer). The chelation of the Fe ^{3 +} ions was strongly maintained on the MNP surface at various pH levels (Figure 5). Fe ^{3 +} -doped particles, MNP (Fe), were analyzed by STEM (scanning-transmission electron microscopy) (FIG. Loaded (loaded) Fe ^{3 +} ions are analyzed qualitatively by using a homogenizer located on the particle surface is mounted STEM- EDX (energy-dispersive x-ray spectrometer). Therefore, Fe ^{3 +} ion T ₁ - it is possible to effectively induce highlighted (T ₁ -w) and the vertical axis (longitudinal) (T ₁₎ reduction of the water protons (water protons) as an MR contrast agent.

Test Example  3. ML - MNP  Structural Analysis of Composites

After processing the Traut's reagent with DPPE in Lipo to produce a hybrid of MB and Lipo (see FIG. 6), the DSPE-PEG-SPDP in MB has a sulfhydryl chemical functional group (-SH And cross-linked. Ellman's reagent was used to determine the number of sulfhydryl groups present on Lipos. The connection was confirmed by measuring the pyridine-2-thione (leaving group) using a UV-Vis spectrophotometer (Fig. 6 (b)). The mean diameter of ML was about 1.6 μm. The synthesized MNP (Fe) was then purified via Schiff's base or Michael addition reaction (H. Lee, et al. Science 2007, 318, 426) with an excess of sulfhydryl moiety moiety. ML-MNP (Fe) composite particles were treated with methoxy-poly (ethylene glycol) (PEG-SH, 2 kDa) to increase solubility in aqueous buffer.

For cancer cell-specific targeting by ML-MNP (Fe) particles, antibodies against human epidermal growth factor receptor 2 (HER2) were introduced into the particles by a typical half-antibody conjugation process, Anchored antibodies were quantitatively analyzed by protein assay method (70 nM HER2 antibody per DPPC of particle) (SC Owen, et al., J. Control Release 2013, 172, 395). Green and red fluorescent dye-introduced particles (M _G L _R -MNP (Fe) -HER 2) were confirmed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) c). In CLSM, MBs clearly showed a shell structure when observed with a green fluorescent dye, and was mainly associated with red Lipos. MNP (Fe) s was confirmed to be a very small point on the surface of ML by SEM analysis.

Test Example  4. ML - MNP  Complex specific Targeting  And Absorption capacity

To investigate the specific targeting ability and uptake enhancement by US stimulation, the prepared particles were cultured with breast cancer cells. The translocation of fluorescent dyes and MNPs was analyzed using CLSM and bio-TEM (FIGS. 2 a and b). HER2 positive cells (SKBR3) were treated with a solution of M _G L _R -MNP (Fe) -HER2 (1 ml, 0.33 mg / ml PBS buffer) in a 5% CO ₂ incubator at 37 ° C for 1 hour. During the treatment, the particles adhered to the cell membrane due to the large-sized MBs. One minute of US stimulation (mechanical index, MI = 0.61, flash technique used) induced MB cavitation and increased penetration of Lipos and MNP (Fe) particles bound into the cell cytosol.

As a result, HER2-positive cells showed green and red fluorescence in cytosol, and their intensities were analyzed using flow cytometry (FIG. 7C). In comparison, HER2 negative cells (MCF7) that underwent the same treatment with M _G L _R -MNP (Fe) -HER2 particles showed a small amount of particle inclusion in the cytosol due to their nonspecific uptake (A and b in Fig. 7). The particle system thus exhibited significantly enhanced absorption efficiency with cancer cell specific selective delivery and external US flash.

Test Example  5. ML - MNP  Toxicity assessment of complex

Cell viability after targeting and flash treatment was measured using a typical cell proliferation assay (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, MTT) The effect of time was measured. The cells showed a survival rate of 90% or more (Fig. 2 (c)). Also, the present inventors have found that the present invention provides a method of screening for a cell organelle function, such as a voltage-dependent anion channel (VDAC), a nucleoporin p62 (nucleoporin p62 protein complex), and an anti- Bcl-2-related death promoter)], the biocompatibility of the particles was investigated (FIG. 2 d). All markers showed similar expression levels in treated and untreated control cells, and US flash stimulation did not cause any cytotoxic or functional abnormalities.

Test Example  6. ML - MNP ( Fe ) Complex Imaging  effect

Next, the present inventors compared the imaging aspect of the produced M _G L _R -MNP (Fe) -HER 2 particle with that of commercially available clinical US (SonoVue, Bracco) or T ₁ -w MR (Gadovist, Bayer Schering Pharma) Respectively.

As US image contrast agents, the particles produced were similar to SonoVue echogenicity as measured by clinical US scanners (iU22, Philips, Bothell, WA, USA) (Fig. 8). The particles also had a T ₁ -w MR imaging signal twice as high as Gadovist, as shown on a 0.47 T magnetic relaxometer ( r ₁ = 6.7 mM ^-1 s ^-1 , mq-20, Bruker) Respectively. A comparative study of MR phantoms confirmed the superiority of the particles as a T ₁ -w MR contrast agent and showed the same signal intensity at approximately two-fold lower capacity (FIG. 9).

Test Example  7. Cancer cells Targeting  And MR Imaging  Improving

To understand cancer cell specific US imaging capabilities, SKBR3 cells were targeted to the particles and collected in PBS buffer. The solution was then transferred to a 5 mm plastic tube and exhibited significant US echo generation under standard imaging-mode illumination (MI = 0.08) (FIG. 3 a). After applying the US Flash (MI = 0.61), the MB was completely destroyed (cavitated), thereby reducing the echo occurrence within the 6 flashes (FIG. 8b).

T ₁ -w US flash of MR Imaging - in order to check the parameters increase, HER2-positive (SKBR3) and negative (MCF7) cells to target particles (Fig. 3 b), a flash exposure (1 min) and a residue removal process . Treated cells were used for MR imaging (3.0-T, Philips, Fig. 3c). (In the absence of particle treatment) the flash-treated cells exhibited the lowest T ₁ -w MR signal regardless of HER expression. MNP (Fe) -HER2 (without ML complex) showed a similarly low signal, indicating that only MNP (Fe) particles antibody-conjugated to increase MR imaging efficiency were not sufficient. However, M _G L _R -MNP (Fe) -HER2- and flash-treated SKBR3 cells displayed a significantly bright image. The present inventors confirmed the increase of the T ₁ -w MR signal even through an external flash stimulus.

Test Example  8. Therapeutic gene transfer and Therapeutic  apply

Finally, the present invention confirmed the efficiency of the composite particles for gene delivery. The gene for survivin ( siSurv ) is well known as a therapeutic gene that triggers apoptosis; SiRNA ( siSurv , 19-mer, Thermo-scientific) for survivin can be introduced into Lipo particles (A. Gabizon, et al., Eur. J. Pharm . Sci .

Briefly, the lipid film was lyophilized and then mixed with a complex solution of siSurv (50 μM) and protamine (PA; 7.5 kDa, 40 μM) using an electrophoresis protocol at an optimized ratio of 1.25 genes per PA And homogenized (Fig. 4 (a)). The loading capacity was about 82% as measured by the UV-Vis spectrophotometer (Figure 4b). Lipos containing siSurv- PA were attached to MB (ML _siSurv ) and then anchored and conjugated to MNP (Fe) and HER2 antibodies. This gene transfer did not alter the particle system, as evidenced by the comparison of ML-MNP (Fe) -HER2, which serves as a reference, with morphology and particle size. The target cells (SKBR3) were incubated with the therapeutic particles for 1 hour and then presented in US flash (1 minute). The delivery gene was effectively internalized into the cells due to the US-mediated uptake of the proteins, and this absorption effectively down-regulated survivin expression in these cells as compared to the control (Fig. 4c).

Cell viability after siRNA delivery decreased to less than 60% after 72 hours of flash but control cells showed cell viability above 90% (FIG. 4 d). These results demonstrate that the MB-based particle system can enhance the selective delivery efficiency of bio-molecules introduced into linked Lipo.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. 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 (14)

Microbubbles;
Liposomes covalently bonded to the microbubbles;
Melanin nanoparticles attached to the surfaces of the liposome and microbubbles and bound to paramagnetic metal ions; And
A microbubble-liposome-melanin nanoparticle complex comprising a targeting ligand attached to the surface of the microbubble or liposome.
The microbubble-liposome-melanin nanoparticle complex according to claim 1, wherein the microbubble is synthesized by bubbling a mixture of a phospholipid compound and cholesterol with a hydrophobic gas accompanied by film formation and mechanical vibration.
The microbubble-liposome-melanin nanoparticle complex according to claim 1, wherein the liposome is prepared by film formation, ultrasonic treatment and extrusion of a mixture of a phospholipid compound and cholesterol.
The microbubble-liposome-melanin nanoparticle composite according to claim 1, wherein the microbubble or liposome comprises at least one fluorescent substance therein.
The microbubble-liposome-melanin nanoparticle complex according to claim 1, wherein the melanin nanoparticles are synthesized from spontaneous oxidation and polymerization of dopamine as a melanin precursor.
The method of claim 1, wherein the paramagnetic metal ions are selected from the group consisting of Gd, Fe, Mn, Ni, Cu, Er, Eu, And chromium (Cr). 10. The microbubble-liposome-melanin nanoparticle composite according to claim 1, wherein the microbubble-liposome-melanin nanoparticle complex is an ion of one or more metals selected from the group consisting of chromium (Cr)
The microbubble-liposome-melanin nanoparticle complex of claim 1, wherein the targeting ligand is a cancer cell-specific antibody.
2. The microbubble-liposome-melanin nanoparticle complex of claim 1, wherein the targeting ligand is attached to both the microbubble and the liposome surface.
A contrast agent for multi-modal analysis of fluorescence, ultrasound and magnetic resonance (MR) comprising the microbubble-liposome-melanin nanoparticle complex according to claim 1.
10. The contrast agent of claim 9, wherein the complex further comprises a therapeutic gene or drug within the liposome.
A pharmaceutical composition for anti-cancer comprising the microbubble-liposome-melanin nanoparticle complex according to claim 1 and an anti-cancer gene or an anti-cancer agent as an active ingredient.
[Claim 13] The anticancer drug composition according to claim 11, wherein the anti-cancer gene is an siRNA against a cell suicide-suppressing gene.
A method of preparing microbubble-liposome-melanin nanoparticle complexes capable of multi-modal imaging for cancer, cell specific, fluorescence, ultrasound and magnetic resonance (MR), comprising the steps of:
a) forming a phospholipid film from a phospholipid compound to which a fluorescent substance is added, adding the phospholipid film to an organic solvent, and synthesizing a microbubble through bubbling and mechanical vibrating of a hydrophobic gas;
b) adding a phospholipid film formed from a phospholipid-containing phospholipid compound to an organic solvent, and ultrasonically dispersing the liposomes, and synthesizing liposomes by extrusion using a nanofilter;
c) adding a solution containing paramagnetic metal ions to a solution containing melanin nanoparticles to bind paramagnetic metal ions to the melanin nanoparticles;
d) synthesizing a microbubble-liposome complex by introducing a thiol group into the ziazospores synthesized in the step b) and then shaking the microbubble with the microbubble synthesized in the step a);
e) preparing a microbubble-liposome-melanin nanoparticle complex by mixing the thiolated microbubble-liposome complex of d) with melanin nanoparticles bound to paramagnetic metal ions of step c); And
f) introducing a maleimide group into the microbubble-liposome-melanin nanoparticle complex, and then binding the maleimide group to the cancer cell-specific ligand.
[14] The method of claim 13, further comprising the step of homogenizing the phospholipid film formed from the phospholipid compound in the organic solvent with a hybrid solution of the biopolymer and the therapeutic gene to support the therapeutic gene, Way.

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