KR20200145115A - Anti-cancer hybrid exosome composition having amphiphilic doxorubicin and exosome - Google Patents

Abstract

The present invention relates to an anticancer hybrid exosome composition using amphiphilic doxorubicin and exosomes. In the present invention, a hybrid exosome formulation (Lexobrid A) was prepared by mixing lipid-conjugated doxorubicin and exosomes. The Lexobrid A can increase the residence time and half-life in cancer cells, improve bioavailability, and increase the anticancer effect by slowly releasing doxorubicin in the environment around cancer cells in the body (pH 5.5). Therefore, the hybrid exosome of the present invention can exhibit a lasting anticancer effect, and thus can be usefully used as a composition for drug delivery.

Description

Anti-cancer hybrid exosome composition having amphiphilic doxorubicin and exosome} Using amphiphilic doxorubicin and exosome

The present invention relates to an anticancer hybrid exosome composition using amphiphilic doxorubicin and exosomes.

Doxorubicin (Dox), which was approved by the FDA in 1974, is an anthracycline anticancer drug and is used to treat various cancers such as ovarian, leukemia, and myeloma cancer. However, doxorubicin exhibits severe cardiotoxicity depending on the cumulative dose, and other side effects include a short in vivo retention time and easy elution in the body due to its hydrophilic properties. In a previous study (Bioorg Med Chem Lett, 27 (2017) 723-728.), an amphiphilic doxorubicin derivative (L-Dox AD) modified with cholesterol was synthesized and tested for anticancer activity and lipophilic properties. It was confirmed that the retention time was increased compared to doxorubicin in HeLa and MDAMB 231 cell lines.

In order to avoid capture and removal by the mononuclear phagocyte system (MPS), polyethylene glycol (PEG) was coated on the liposome surface. PEG exhibits low immunogenicity, low cytotoxicity and good excretion kinetics. Pegylation not only prolongs the circulation half-life in vivo, but also plays a role in stabilizing the particles. DOXIL is a drug belonging to the anthracycline drug group and was developed as a PEGylated STEALTH liposome in which doxorubicin is trapped in a water-soluble core in order to avoid detection and elimination by the immune system, which can increase the residence time of DOXIL in the body. Liposomes allow more time to reach tumor tissue and slowly release doxorubicin from liposomes. Pegylated liposomes are protected from bodily fluids, but it is difficult to interact with target tumor cells, and the released doxorubicin can be easily released by cancer cells, resulting in poor anticancer effect. Therefore, the use of lipid-conjugated amphiphilic doxorubicin can solve the above problem by increasing the residence time in cancer cells.

Since Bangham et al. first mentioned liposomes in 1964, many studies have been conducted to solve drug delivery and its shortcomings. Liposomes are biocompatible, biodegradable, and can deliver insoluble hydrophobic drugs. Liposomes are composed of amphiphilic lipids such as phospholipids and cholesterol, and their form has a diameter of 0.03 to 10 μm. One of the advantages of liposomes is that they can modify the lipid moiety and its composition ratio. The physicochemical properties, size, zeta potential, drug encapsulation efficacy and release kinetics of liposomes can be altered by lipid modification. In addition, the presentation of a ligand or a peptide on the surface of a liposome (active-target liposome) shows a good homing effect through the affinity between the ligand and the receptor. Currently, various liposome studies are being conducted to target folate receptors (FRs), epidermal growth factor receptors (EGFR), and transferrin receptors (TfR) overexpressed in cancer cells.

On the other hand, exosomes are known to be produced in the budding of late endosomes and fused with the plasma membrane before being released into the extracellular space. Exosomes are 40-200 nm-sized follicles composed of a lipid bilayer membrane rich in phosphocholine, cholesterol and ceramide, secreted by almost all types of cells, and stably present in all types of body fluids such as blood, lymph and sweat. do. In addition, exosomes have the advantage of reaching the inside of organs and long circulation time due to their small size and weak negative charge. Exosomes can also deliver hydrophilic or hydrophobic drugs by avoiding phagocytosis and can pass through the vascular endothelium to target cells. Exosomes are known to exhibit targeted effects on specific cells due to specific surface proteins such as tetraspanin. Encapsulation of exosomes has been reported to increase the stability and bioavailability of curcumin in vitro and in vivo, and to increase anti-inflammatory activity.In other studies, exosomes cross the blood-brain barrier to the brain. It has been reported to deliver doxorubicin, deliver siRNA to cells, and effectively reduce the level of RAD 51 protein, a potent target for cancer treatment.

Accordingly, in the present invention, an effective hybrid exosome is developed by mixing liposomes and exosomes having an amphiphilic doxorubicin derivative in liposomes.

Republic of Korea Patent Publication No. 10-2011-0085318 (published on July 27, 2011)

An object of the present invention is to provide an anticancer hybrid exosome, and a cancer disease prevention or composition comprising the same as an active ingredient.

Another object of the present invention is to provide a composition for delivery of a drug or a physiologically active substance, including the anticancer hybrid exosome, and a drug or a physiologically active substance encapsulated in the hybrid exosome.

In order to achieve the above object, the present invention provides a liposome consisting of a lipid-bound doxorubicin, a lipid-bound water-soluble polymer, and a first phospholipid; and an exosome consisting of an exosome membrane protein and a second phospholipid. , In the hybrid exosome, doxorubicin is exposed to the inner and outer aqueous phases of the first phospholipid double membrane, the water-soluble polymer is exposed to the outer aqueous phase of the first phospholipid double membrane, and the exosome membrane protein is exposed to the outer aqueous phase of the second phospholipid double membrane It provides a hybrid exosome characterized in that.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer diseases comprising the hybrid exosome as an active ingredient.

In addition, the present invention provides a composition for delivery of a drug or a physiologically active substance, including the hybrid exosome, and a drug or a physiologically active substance encapsulated in the hybrid exosome.

In the present invention, a hybrid exosome formulation (Lexobrid A) was prepared by mixing lipid-conjugated doxorubicin and exosomes. The Lexobrid A can increase the residence time and half-life in cancer cells, improve bioavailability, and increase the anticancer effect by slowly releasing doxorubicin in the environment around cancer cells in the body (pH 5.5). Therefore, the hybrid exosome of the present invention can exhibit a lasting anticancer effect, and thus can be usefully used as a composition for drug delivery.

Figure 1 shows the chemical structure of the lipid-conjugated amphiphilic doxorubicin derivative (L-Dox AD) (Chol: cholesterol structure).
2 is a result of confirming the physicochemical properties (particle size, Pdi, zeta potential) of the liposomal preparation of L-Dox.
Figures 3 (a) to 3 (c) are the results of confirming the composition, safety and physicochemical properties of Lipodoxome A (L-Dox A liposomal preparation), Figure 3 (d) is L-Dox from Lipodoxome A This is the result of confirming the release of
Figure 4 shows the configuration of a liposome-exosome hybrid (Lexobrid A).
5 is a liposome-exosome hybrid, (a) a result of performing a fluorescence resonance energy transfer (FRET) analysis using a liposome containing a total molar ratio of 1% NBD-cholesterol and 1% Rho-cholesterol fluorescent lipid. (FT 0; mixed exosomes and liposomes without freezing and thawing, FT 5; 5 freezing and thawing, FT 10; freezing and thawing 10 times), (b) dynamic laser scattering method (DLS) of particle size and image And the result of confirmation by a transmission electron microscope (TEM).
6 is a result of confirming the cytotoxicity of Dox, Lipodoxome A and Lexobrid A by MTT analysis using the A549 cell line and the MDA MB 231 cell line.
7 is a result of confirming cell uptake of Dox, Lipodoxome A and Lexobrid A by flow cytometry (Control; gray, Dox; red, Lipodoxome A; black, Lexobrid A; blue, solid line; measurement immediately after treatment, dotted line; 24 hours) Measured after incubation).
8 is a result of confirming the localization of Dox, Lipodoxome A and Lexobrid A with a confocal microscope.

The inventors of the present invention completed the present invention by confirming that a hybrid exosome preparation (Lexobrid A) prepared by mixing lipid-conjugated doxorubicin and exosomes exhibits anticancer effects by increasing the residence time and cytotoxicity in cancer cells.

Thus, the present invention is a hybrid exosome in which a lipid-bound doxorubicin, a lipid-bound water-soluble polymer, and a liposome consisting of a first phospholipid; and an exosome consisting of an exosome membrane protein and a second phospholipid are bonded, wherein the hybrid exosome Doxorubicin is exposed to the first phospholipid double membrane inner and outer aqueous phase, the water-soluble polymer is exposed to the first phospholipid double membrane outer aqueous phase, and the exosome membrane protein is exposed to the second phospholipid double membrane outer aqueous phase. Hybrid exosomes are provided.

The lipids are cholesterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, chenodeoxycholic acid, glycoke It may be selected from the group consisting of glycochenodeoxycholic acid, taurochenodeoxycholic acid, and lithocholic acid, but is not limited thereto.

The water-soluble polymer is polyethylene glycol (poly(ethylene glycol); PEG), polylactide-co-glycolide (PLGA), polyglicolide (PGA) and polylactic acid (poly (lactic acid); PLA), but may be one or more polymers selected from the group consisting of, but is not limited thereto.

The first or second phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DPPC), 1,2-diss Thearoyl-sn-glycero-3-phosphoethanolamine (1,2-distearoyl-sn-glycero-3-phosphoethanolamine; DSPE), phosphatidylcholine, dilauroyl phosphatidylcholine (DLPC), Dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl Phosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3- Phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPC), 1-palmitoyl-2-oleoyl phosphatidylcholine (1-palmitoyl-2-oleoyl phosphatidylcholine; POPC), diethyl phosphorocyana Date (diethyl phosphorocyanidate; DEPC), phosphatidylglycerol (phosphatidylglycerol), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (dipalmitoylphosphatidylglycerol; dipalmitoylphosphatidylglycerol; DPPG sulfate proteoglycanderma; , 1-palmitoyl-2-oleoyl-phosphatidylglycerol (1-palmitoyl-2-oleoyl-phosphatidylglycerol; POPG), phosphatidylethanolamine, bis(1.2-dimethylphosphino)ethane (bis(1,2- dimethylphosphino)e thane; DMPE), 1,2-bis(diphenylphosphino)ethane (1,2-Bis(diphenylphosphino)ethane; DPPE), dioleoylphosphatidylethanolamine (DOPE), phosphatidylserine (phosphatidylserine) and dioleoyl Phosphatidylserine (dioleoyl phosphatidylserine; DOPS) may be selected from the group consisting of, but is not limited thereto.

It should be noted that the liposome and exosome may be included in a volume ratio of (20 ~ 50): (50 ~ 80), but is not limited thereto.

The liposome may contain a lipid-bound doxorubicin, a lipid-bound water-soluble polymer, and a first phospholipid in a weight ratio of (25 to 35): (10 to 20): (50 to 60), but is limited thereto. Specify not.

The lipid-bound doxorubicin may include lipids and doxorubicin in a weight ratio of (40 to 60): (40 to 60), but is not limited thereto. The lipid and doxorubicin may be bound by an ether bond. Unlike general doxorubicin, lipid-bound doxorubicin has amphiphilic properties, so it can stay in cancer cells for a longer time and increase its anticancer effect.

The water-soluble polymer to which the lipid is bound may include a lipid and a water-soluble polymer in a weight ratio of (3 to 43): (57 to 97), but is not limited thereto.

It should be noted that the hybrid exosome may have an average diameter of 80 to 150 nm, but is not limited thereto.

The exosome membrane protein may be selected from the group consisting of CD9, CD63, CD81 and CD90, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer diseases comprising the hybrid exosome as an active ingredient.

The liposome does not release doxorubicin under extracellular conditions of pH 7 to pH 7.7, and releases doxorubicin in a cancer cell environment of pH 5.2 to pH 5.8 to induce the death of cancer cells.

The cancer diseases include lung cancer, skin cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, anal muscle cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer. Tumor, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma , Bladder cancer, kidney or ureteral carcinoma, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma. However, it is stated that this is not limited thereto.

When the composition of the present invention is a pharmaceutical composition, for administration, it may include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the above-described active ingredients. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

The pharmaceutical compositions of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injectable solutions according to a conventional method. . Specifically, when formulated, it may be prepared using diluents or excipients such as fillers, weight agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such a solid preparation may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. in addition to the active ingredient. Further, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. It can be prepared by adding various excipients, such as wetting agents, sweetening agents, fragrances, preservatives, and the like, in addition to oral liquids and liquid paraffin. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and tasks. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyloleate, and the like may be used. As a base for suppositories, witepsol, macrosol, Tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, the form of the drug, and the time, but may be appropriately selected by a person skilled in the art, and the daily dosage of the composition is preferably It is 0.001 mg/kg to 50 mg/kg, and it can be administered once to several times a day as needed.

In addition, the present invention provides a composition for delivery of a drug or a physiologically active substance, including the hybrid exosome, and a drug or a physiologically active substance encapsulated in the hybrid exosome.

The physiologically active substance may be selected from the group consisting of peptides, proteins, anticancer agents, anti-inflammatory analgesics, antibiotics, antibacterial agents, hormones, genes, and vaccines, but is not limited thereto.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Cell line and material

The A549 cell line (human lung cancer cell line) and the MDA MB 231 cell line (human breast cancer cell line) were provided by the Korean Cell Line Bank, and the adipose derived stem cell (ADSC) was provided by Remybio Inc (Dongtan, Korea). Cells were cultured in a medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin in an incubator at 37° C. and 5% CO ₂ . ADSC cells were DMEM/F12 medium, and RPMI 1640 medium was used for other cell lines. Cell culture reagents including fetal calf serum were purchased from WelGENE (Korea), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTT) and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Example 2: Lipid-conjugated doxorubicin (L-Dox) synthesis

Referring to the previous paper (Bioorg Med Chem Lett, 27 (2017) 723-728.), as shown in Fig. 1, as doxorubicin conjugated with cholesterol having an ether (A and C) or amide (B and D) linker The L-Dox derivative (L-Dox AD) was synthesized.

Example 3: Preparation of amphiphilic doxorubicin liposome and analysis of its physicochemical properties

To prepare the Lipodoxome A liposome preparation, L-Dox derivatives were mixed with phospholipid (DPPC) and Chol-PEG ₂₀₀₀ in an eggplant-shaped flask using an anhydrous organic solvent (dichloromethane/methanol=9:1 mixed solvent). Thereafter, the organic solvent was removed using an evaporator, and residual traces were completely removed overnight under vacuum to prepare a mixed lipid film. The mixed lipid film was resuspended in phosphate buffered saline (PBS, pH 7.4) so that the concentration of the L-Dox derivative was 1 mg/mL. The mixture was sonicated at 60° C. for 20 minutes to form liposomes, and the prepared Lipodoxome A liposomes were stored at 4° C. until used in experiments.

To measure the size and zeta potential of Lipodoxome A liposomes, after diluting Lipodoxome A liposomes at a concentration of 100 μg/mL using distilled water, they were measured using Nano-ZS Zetasizer (Malvern Instruments Ltd., Worcestershire, UK). .

As a result, when DPPC, L-Dox A and Chol-PEG ₂₀₀₀ were in a weight ratio of 54:31:15, Lipodoxome A liposomes were well formed, and as shown in FIG. 2, L-Dox B to L-Dox D liposomes and In contrast, it was confirmed that L-Dox A liposomes form particles with a low Pdi value and a size of 100 nm. Therefore, unless otherwise stated, Lipodoxome A liposomes having the above weight ratio were used in all experiments.

Example 4: exosome extraction

Exosomes were extracted using the exoEasy Maxi Kit (Quiagen, Germany). After incubation until ADSC has a density of about 80-90%, it was replaced with a fresh medium containing no fetal calf serum and cultured for an additional 3 days. The extraction process of exosomes was performed according to the protocol provided. Before using the exoEasy Maxi Kit, the medium was purified with a 0.8 μm syringe filter. The extracted exosomes were stored at 4° C. until use in the test.

Example 5: Preparation of liposome-exosome hybrid

For the preparation of liposome-exosomal hybrid, exosomes extracted from ADSC and Lipodoxome A were mixed at room temperature for 5 minutes at a volume ratio of 50:50. The mixture was quick frozen at -80°C and thawed slowly at 4°C. This process was repeated 10 times to prepare a hybrid particle, Lexobrid A.

Example 6: Fluorescence Resonance Energy Transfer (FRET) Analysis

FRET analysis was performed using Chol-NBD and Chol-Rho pairs synthesized according to the previous paper (Bioorg Med Chem Lett 2015, 25 (18), 3893-3896). Lipodoxome A containing 1% Chol-NBD and 1% Chol-Rho was mixed with ADSC exosomes to analyze the difference in spectrum. Before and after several freeze and thaw (FT) processes, the fluorescence spectrum of the mixture was analyzed using a fluorescence photometer (Germini EM, Molecular Devices, USA). It was confirmed that the formed particles were excited at 450 nm and emitted at 500 nm to 650 nm.

Example 7: Measurement of particle size by dynamic laser scattering (DLS)

In order to analyze the size of the particles, the sample was diluted 10-fold with distilled water, and then measured using Nano-ZS Zetasizer (Malvern Inc, UK). Particle size was measured 3 times in 15 replicates using a disposable cuvette (DTS1070, Malvern Inc, UK).

Example 8: Transmission electron microscopy (TEM)

The particles were stirred overnight at 4° C. with 2% formaldehyde. A drop of 5 μL of particle suspension was placed on the Para film, and a Formva-carbon coated grid (200 mesh, TED PELLA, USA) was placed on the drop and adsorbed at room temperature. After 20 minutes, the grid was washed with PBS and dried under lighting. The cross section of the particles was observed with an electron microscope (Hitachi Ltd. H-7000B, Japan) operating at 70 kV.

Example 9: Cell viability analysis

After treatment with Lipodoxome A and Lexobrid A on the A549 cell line and the MDA MB 231 cell line, MTT analysis was performed to confirm the cytotoxicity of the drug. Briefly, cells were seeded in 96-well plates at a density of 4,000 cells per well and incubated overnight. Thereafter, the cells were treated with various concentrations of drugs and cultured for 2 hours, then replaced with fresh medium and further cultured for 72 hours. For MTT analysis, 80 μL of fresh medium and 20 μL of MTT solution (5 mg/ml) were added to each well and incubated for 3 hours. Formazan crystals were completely dissolved in 100 μL of DMSO. Absorbance was measured at 550 nm using a microplate reader (Tecan, Tecan AG, Switzerland), and cytotoxicity was normalized to the absorbance of untreated control cells.

Example 10: Observation of cell uptake using flow cytometry and confocal microscopy

To compare the transcytosis of doxorubicin, liposomes and liposome-exosomal hybrids, 24-well plates were seeded with A549 and MDA MB 231 cell lines at a density of 50,000 cells per well. After incubation overnight, the cells were recovered, fixed with 1% paraformaldehyde, and then placed on ice until analysis and stored in a dark room. Fixed cells were analyzed by BD FACSAriaTM III (BD Biosciences, Germany), and a total of 10,000 gated cells were analyzed using FlowJo V10 (Ashland, OR).

For confocal microscopy analysis, 24-well plates were seeded with the A549 and MDA MB 231 cell lines at a density of 50,000 cells per well. After incubation overnight, the cells were treated with Lipodoxome A and Lexobrid A for 2 hours. Lysosomes were stained using lyso-tracker green (Invitrogen, USA), and nuclei were stained using TO-PRO III (Invitrogen, USA) to compare drug localization. Each dye reagent was performed according to the protocol provided, and the prepared sample was observed with a confocal microscope (TCS SP2 AOBS, Leika, Germany) equipped with an objective lens at 40X magnification. All images with merged photos were acquired using LAS AF lite software (Leica, Germany).

Experimental Example 1: Structure of lipid-conjugated amphiphilic doxorubicin

As shown in Figure 1, the newly synthesized L-Dox derivatives of cholesterol-conjugated doxorubicin having ether (A and C) or amide (B and D) linkers exhibit amphiphilic properties due to conjugated lipids, unlike water-soluble general doxorubicin. Have. L-Dox A and B derivatives have similar in vitro cytotoxicity to doxorubicin, and it has been reported by the present inventors that they stay in cancer cells for a longer time than water-soluble doxorubicin because of the lipid properties of conjugated cholesterol (Bioorg Med Chem Lett , 27 (2017) 723-728). The amphiphilic property can solve the problem that conventional water-soluble doxorubicin is easily released by multi-drug resistance (MDR) cells and has a short half-life in the biological circulation. However, since these amphiphilic properties are soluble only in a specific organic solvent, they exhibit low bioavailability in vivo due to the problem of having low solubility in aqueous conditions in vivo. Therefore, the liposomal preparation of amphiphilic doxorubicin can improve low bioavailability by enabling in vivo injection.

Experimental Example 2: Physicochemical properties of amphiphilic doxorubicin liposomes

DOXIL is a STEALTH liposome in which doxorubicin is captured in a water-soluble core, and was developed to avoid detection and destruction from the immune system, thereby increasing the residence time of DOXIL in vivo.

As a result, these liposomal dispersions allow more time to reach cancer tissues, and then doxorubicin is slowly released from the liposomes to act effectively on cancer tissues. Doxorubicin can effectively prevent its disappearance in vivo by the pegylated liposomal dispersion, but due to the nature of STEALTH liposomes, it can also interfere with the action of target cancer cells, and the released doxorubicin can be easily released by cancer cells. Eventually the effect can be diminished. Therefore, the use of lipid-conjugated amphiphilic doxorubicin can overcome the disadvantages of conventional DOXIL by increasing the residence time in cancer cells while maximizing the advantages of the conventional liposomal dispersion.

Accordingly, in order to increase the bioavailability and retention time of doxorubicin in cancer cells, various concentrations of phospholipids such as DPPC and Chol-PEG ₂₀₀₀ were used to make L-Dox derivatives into liposome preparations. When resuspended in PBS so that the concentration of each L-Dox derivative is 1 mg/mL and dispersed by sonicating at 60° C. for 20 minutes, as shown in FIG. 2, the L-Dox A derivative has the smallest and even particle size. It was easy to generate liposomes, and in the case of L-Dox C and L-Dox D derivatives, it was confirmed that a precipitate was generated without liposomeization. To prepare amphiphilic liposomal doxorubicin for cancer target treatment, L-Dox A, which is the most easily liposomed, was used, and liposomes containing 31% L-Dox A were named Lipodoxome A and further studies were conducted on this.

As shown in Figure 4, the new Lipodoxome A liposome contains more doxorubicin than doxorubicin encapsulated in conventional commercial DOXIL liposomes, and doxorubicin is organically bound to cholesterol, so there is no fear of spontaneous release of doxorubicin in the liposome. . The size and zeta potential of Lipodoxome A liposome particles were measured to be 93.9±1.4 nm and -12.7±0.7 mV, and it was confirmed that the particle shape was stably maintained for 5 weeks at 4°C in PBS.

Lipodoxome A The release experiment of L-Dox A from liposomes was performed by mechanical agitation under two conditions using pH 7.4 and pH 5.5 solutions. The conditions of the two solutions are similar to those of the extracellular and intracellular pH conditions, so that the stability of the particles can be more accurate than the particle size and zeta potential data. As a result of conducting an experiment for the release of L-Dox A from Lipodoxome A liposomes for a total of 7 days, the release of L-Dox A was not observed under the condition of pH 7.4, and when compared to the initial concentration, 44.1% of L-Dox A was observed at pH 5.5. The emission of Dox A was observed by fluorescence analysis. Therefore, it was confirmed that Lipodoxome A liposomes are stable at pH 7.4, which is an extracellular pH condition, and can effectively kill cancer cells without loss of doxorubicin by slowly releasing L-Dox A from Lipodoxome A liposomes at pH 5.5, which is an intracellular pH condition.

Experimental Example 3: Physicochemical properties of hybrid exosomes

ADSC was cultured to a density of about 90%, replaced with a fresh medium containing no fetal calf serum, and cultured for an additional 3 days. Then, the culture medium was recovered to extract exosomes. After mixing the exosomes containing 1% Chol-NBD and 1% Chol-Rho and Lipodoxome A, several freezing and thawing processes were performed to prepare hybrid exosomes. The particle properties of the prepared Lexobrid A were measured by FRET analysis.

As a result, it was confirmed that excitation of Chol-NBD at 460 nm induces fluorescence emission at 530 nm and 588 nm corresponding to Chol-NBD and Chol-Rho, respectively. In addition, as a result of performing the freezing and thawing processes 5 times, it was confirmed that NBD fluorescence increased (530 nm) and Rho fluorescence decreased (580 nm). That is, as shown in Figure 5a, it was confirmed that the liposome and the exosome are well fused to form Lexobrid A.

To compare the physicochemical properties of Lexobrid A before and after hybrid formation, particle size distribution and morphology were analyzed by three methods. As a result, as shown in Table 1 below, the size of Lexobrid A measured by DLS was 141.9 ± 3.2 nm, which was confirmed to represent a value between liposomes (93.9 ± 1.4 nm) and exosomes (227.7 ± 2.7 nm). I did. As a result of measuring the particles by TEM, it was confirmed that liposomes exhibited a particle size of 87.0 ± 21.8 nm, exosomes 110.9 ± 19.2 nm, and liposome-exosome hybrids 99.6 ± 17.5 nm. In addition, as shown in Figure 5b, the shape of the exosome showed a spherical shape when dried in a TEM grid.

Experimental Example 4: Cytotoxicity and cell uptake of liposome-exosome hybrid

The amounts of doxorubicin contained in a total of three drugs, Dox, Lipodoxome A, and Lexobrid A, were 0.5, 1, and 2 μM, and the cytotoxicity in A549 and MDA MB 231 cell lines was analyzed using this.

As a result, as shown in Figure 6, Loxobrid A showed improved cytotoxicity at all concentrations compared to Dox and Lipodoxome A. In the A549 cell line, Lexobrid A inhibited the growth of cancer cell lines by about 30% or more than Dox and Lipodoxome A at all concentrations. In MDA MB 231 cell line, Lexobrid A containing 0.5 μM of doxorubicin showed more than about 25% cytotoxicity than Dox and Lipodoxome A. Lexobrid A also inhibited the growth of cancer cell lines by more than 10% in MDA MB 231 cells than Dox and Lipodoxome A.

In order to analyze the cause of cytotoxicity, the cell line was cultured for 0 or 24 hours after 2 hours of drug treatment, and then cell uptake was measured by flow cytometry. As a result, as shown in FIG. 7, it was confirmed that the fluorescence signals of Dox and Lipodoxome A decreased after 24 hours in both A549 and MDA MB 231 cell lines, whereas the fluorescence signals of Lexobrid A were increased under all conditions. In both cells, when cultured with Lexobrid A for 24 hours showed higher fluorescence than immediately after treatment. From the above results, it was confirmed that Lexobrid A can maintain the effect longer than Dox and Lipodoxome A and can continuously act as an anticancer agent.

Experimental Example 5: Microscopic observation of doxorubicin, liposome, and liposome-exosome hybrid

As shown in FIG. 8, Dox was localized and concentrated in the nucleus 2 hours after treatment with the A549 cell line, and Lipodoxome A and Lexobrid A were found in the cytoplasm and nucleus after 2 hours. In the cytoplasm, the red signals of Lipodoxome A and Lexobrid A were mainly found as particles, and Lexobrid A showed a higher signal than Lipodoxome A. The results were consistent with the results of flow cytometry. That is, it was confirmed that doxorubicin in the nucleus was released from Lipodoxome A and Lexobrid A particles.

As the specific parts of the present invention have been described in detail above, it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

The scope of the present invention is indicated by the claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (16)

A liposome consisting of a lipid-bound doxorubicin, a lipid-bound water-soluble polymer, and a first phospholipid; and an exosome consisting of an exosome membrane protein and a second phospholipid are bonded to each other, wherein in the hybrid exosome, doxorubicin is the first A hybrid exosome, characterized in that the phospholipid double membrane is exposed to the inner aqueous phase and the external aqueous phase, the water-soluble polymer is exposed to the first phospholipid double membrane outer aqueous phase, and the exosome membrane protein is exposed to the second phospholipid double membrane external aqueous phase. The method of claim 1, wherein the lipid is cholesterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and kenodeoxycholic acid ( chenodeoxycholic acid), glycochenodeoxycholic acid (glycochenodeoxycholic acid), taurochenodeoxycholic acid (taurochenodeoxycholic acid) and lithocholic acid (lithocholic acid) hybrid exosome, characterized in that selected from the group consisting of. According to claim 1, wherein the water-soluble polymer is polyethylene glycol (poly(ethylene glycol); PEG), polylactide-co-glycolide (PLGA), polyglicolide; PGA ) And polylactic acid (poly(lactic acid); PLA) hybrid exosome, characterized in that at least one polymer selected from the group consisting of. The method of claim 1, wherein the first or second phospholipid is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DPPC) , 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (1,2-distearoyl-sn-glycero-3-phosphoethanolamine; DSPE), phosphatidylcholine, dilauroyl phosphatidylcholine ( dilauroyl phosphatidylcholine; DLPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (distearoyl Phosphatidylcholine), 1,2-DSPC -Glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPC), 1-palmitoyl-2-oleoyl phosphatidylcholine (1-palmitoyl-2-oleoyl phosphatidylcholine; POPC), Diethyl phosphorocyanidate (DEPC), phosphatidylglycerol (phosphatidylglycerol), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (dipalmitoylphosphatidylglycerol), dermaglycerol dermatan sulfate proteoglycan; DSPG), 1-palmitoyl-2-oleoyl-phosphatidylglycerol (1-palmitoyl-2-oleoyl-phosphatidylglycerol; POPG), phosphatidylethanolamine (phosphatidylethanolamine), bis(1.2-dimethylphosphino)ethane ( bis(1,2-dimethyl phosphino)ethane; DMPE), 1,2-bis(diphenylphosphino)ethane (1,2-Bis(diphenylphosphino)ethane; DPPE), dioleoylphosphatidylethanolamine (DOPE), phosphatidylserine (phosphatidylserine) and dioleoyl Hybrid exosomes, characterized in that selected from the group consisting of phosphatidylserine (dioleoyl phosphatidylserine; DOPS). The hybrid exosome according to claim 1, wherein the liposome and the exosome are included in a volume ratio of (20 ~ 50): (50 ~ 80). The method of claim 1, wherein the liposome comprises doxorubicin to which lipid is bound, water-soluble polymer to which lipid is bound, and the first phospholipid in a weight ratio of (25 to 35): (10 to 20): (50 to 60). Hybrid exosome, characterized in that. The hybrid exosome according to claim 1, wherein the lipid-bound doxorubicin contains lipids and doxorubicin in a weight ratio of (40 to 60): (40 to 60). The hybrid exosome according to claim 1, wherein the lipid-bound doxorubicin is characterized in that the lipid and doxorubicin are bound by an ether bond. The hybrid exosome according to claim 1, wherein the water-soluble polymer to which the lipid is bound contains the lipid and the water-soluble polymer in a weight ratio of (3 to 43): (57 to 97). The hybrid exosome according to claim 1, wherein the average diameter of the hybrid exosome is 80 to 150 nm. The hybrid exosome according to claim 1, wherein the exosome membrane protein is selected from the group consisting of CD9, CD63, CD81 and CD90. A pharmaceutical composition for preventing or treating cancer diseases comprising the hybrid exosome of any one of claims 1 to 11 as an active ingredient. The pharmaceutical composition for preventing or treating cancer according to claim 12, wherein the hybrid exosome releases doxorubicin in a cancer cell environment of pH 5.2 to pH 5.8. The method of claim 12, wherein the cancer disease is lung cancer, skin cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, muscle cancer of the anus, colon cancer, breast cancer, fallopian tube carcinoma, Endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic Or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. A pharmaceutical composition for preventing or treating cancer diseases, characterized in that selected from the group. A composition for delivery of a drug or a physiologically active substance comprising the hybrid exosome of claim 1, and a drug or a physiologically active substance encapsulated in the hybrid exosome. [16] The composition of claim 15, wherein the physiologically active substance is selected from the group consisting of peptides, proteins, anticancer agents, anti-inflammatory analgesics, antibiotics, antibacterial agents, hormones, genes and vaccines.

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