KR102143160B1 - Pharmaceutical composition for delivering microvesicle including cardiac targeting peptide to heart and method using the same - Google Patents

Abstract

It provides a pharmaceutical composition for delivering the active ingredient to the heart, a method for preparing a cell-derived microvesicle for delivering the active ingredient to the heart, and a method for delivering the active ingredient to the heart using the same. According to this, the active ingredient can be selectively or specifically delivered to the heart. Thereby, it can be used to diagnose or treat cardiovascular diseases.

Description

Pharmaceutical composition for delivering microvesicle including cardiac targeting peptide to heart and method using the same method for delivering an active ingredient containing a cardiac targeting protein to the heart

The present invention relates to a pharmaceutical composition for delivering an active ingredient including microvesicles containing a cardiac target protein to the heart, a method of preparing the microvesicles, and a method using the pharmaceutical composition.

Heart disease is considered the leading cause of death worldwide, and about 50% of patients with acute heart attack are known to have died due to arrhythmia as a major cause. Arrhythmia refers to a disease in which the heartbeat is abnormally fast, delayed, or irregular due to the inability to continue regular contractions when electrical stimulation is not generated or improperly transmitted from the heart. Ventricular fibrillation is an arrhythmia in which several parts of the ventricle behave disorderly and form a very fast wave at about 400 to 600 times per minute, thereby forming an irregular pulse.Ventricular tachycardia is an arrhythmia in which the ventricle is more than 100 times per minute. It is a fatal arrhythmia that runs quickly and threatens the patient's life.

Exosomes are small vesicles or microvesicles with a membrane structure secreted from various types of cells. It has been reported that the diameter of exosomes is approximately 30 nm to 100 nm. It was observed that exosomes originate in specific compartments within cells called multivesicular bodies (MVBs), and are released and secreted outside the cells, rather than directly detaching from the plasma membrane in the study by electron microscopy. In other words, when the fusion of the polycystic body and the plasma membrane occurs, such vesicles are released into the extracellular environment, which are called exosomes. It is reported that various kinds of proteins, nucleic acids, lipids, etc. derived from cells are contained in exosomes. In particular, exosomes are known to contain microRNA (miRNA), and miRNA can block various types of mRNA. Exosomes can exclude risks such as tumor induction and immune response compared to stem cells. Since various proteins exist on the surface of exosomes, they are also used as drug delivery systems to deliver to cells of various organs or tissues (EP 2788019 B1). However, no exosomes specifically delivered to the heart have been developed.

Therefore, there is a need to develop a drug delivery system that selectively or specifically delivers drugs to the heart using exosomes.

It provides a pharmaceutical composition for delivering the active ingredient to the heart.

It provides a method of preparing cell-derived microvesicles for delivering an active ingredient to the heart.

It provides a way to deliver the active ingredients to the heart.

One aspect is a fusion protein of a cardiac targeting peptide (CTP) or a fragment thereof, and an exosome transmembrane protein; And

It provides a pharmaceutical composition for delivering an active ingredient to the heart, comprising the fusion protein and cell-derived microvesicles containing the active ingredient.

The cardiac targeting peptide (CTP) is a protein that targets cardiac tissue. The CTP may be an exogenous protein. The CTP may include an amino acid sequence encoded by the amino acid sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 2. The nucleotide sequence of SEQ ID NO: 2 can be changed in consideration of codons mainly used in humans or mammals, that is, codon usage.

The CTP may be located on the surface of the microvesicle.

The fragment of the CTP contains 5 to 11, 6 to 11, 7 to 11, 8 to 11, 9 to 11, or 10 to 11 consecutive amino acid sequences as part of CTP can do.

The exosome transmembrane protein may be a protein located in a double membrane of an exosome. For example, the exosome transmembrane protein is LAMP (Lysosome-associated membrane protein)-1, LAMP-2, CD13, CD86, Flotilin, Syntaxin-3, CD2, CD36, CD40, CD40L, CD41a, CD44, CD45, ICAM((Intercellular Adhesion Molecule)-1, Integrin alpha4, L1 protein, LFA(Lymphocyte function-associated antigen)-1, macrophage-1 antigen, Vti(Vesicle transport through) interaction with t-SNAREs homolog)-1A, Vti-1B, CD3 epsilon, CD3 zeta, CD9, CD18, CD37, CD53, CD63, CD81, CD82, CXCR4, FcR, GluR(Glutamate receptor)2/3, HLA-DM (human leukocyte antigen DM), immunoglobulin, MHC-I component, MHC-II component, TCR beta, and tetraspanin may be selected from the group consisting of LAMP-2, for example LAMP-2A. , LAMP-2B and LAMP-2C.

In the fusion protein, the CTP may be located in the N-terminal direction of the exosome transmembrane protein. In the fusion protein, the CTP is located in the outer surface direction of the exosome, and the exosome transmembrane protein may be arranged to pass through the lipid bilayer of the exosome.

In addition to CTP and exosome transmembrane protein, the fusion protein may further include a signal peptide, a label protein, a glycosylation site, a spacer sequence, or a combination thereof. The labeling protein may be a FLAG tag, a hemagglutinin (HA) protein, a histidine tag, or a combination thereof.

The term "microvesicle" refers to small vesicles of a membrane structure that are present in or secreted from several types of cells. Microvesicles secreted to the outside of the cell are (i) exosomes: membrane-like vesicles with a diameter of 30 to 100 nm of phagocytic origin, (ii) ectosomes (also referred to as shedding microvesicles (SMV)): flowing directly from the plasma membrane. Large membranous vesicles with a diameter of 50 to 1000 nm, (iii) apoptotic bleb: containing vesicles 50 to 5000 nm in diameter that are shed by dying cells. The microvesicle may be an exosome. Exosomes are small vesicles of a membrane structure secreted from several types of cells. The diameter of exosomes may be approximately 30 to 100 nm. It was observed that exosomes originate in a specific compartment within a cell called a multivesicular body (MVB), and are released and secreted outside the cell, rather than directly detaching from the plasma membrane in the study by electron microscopy. In other words, when the fusion of the polycystic body and the plasma membrane occurs, such vesicles are released into the extracellular environment, which are called exosomes.

The cells may be kidney cells. The kidney cells may be human embryonic kidney cells (eg, HEK293 cells).

The cell-derived microvesicle may be separated from the cell.

The active ingredient may be a drug for diagnosis, prevention, treatment, or a combination of cardiovascular diseases. The cardiovascular disease may be arrhythmia, ventricular fibrillation, ventricular tachycardia, angina, myocardial infarction, or a combination thereof. The arrhythmia may be ischemic-related arrhythmia, atrial fibrillation-related arrhythmia, or a combination thereof. The term "diagnosis" refers to the act of determining a disease name. The diagnosis may include determining the disease name, etiology, disease type, severity, detailed mode of the disease condition, the presence or absence of complications, prognosis, or a combination thereof. The term "prevention" refers to any action that suppresses the occurrence of cardiovascular disease or delays the onset of cardiovascular disease by administration of the pharmaceutical composition. The term "treatment" refers to any action in which symptoms of cardiovascular disease are improved or beneficially altered by administration of the pharmaceutical composition.

The drug may be a contrast agent, a radioactive isotope, a fluorescent substance, a luciferase, or a combination thereof. The contrast agent increases the contrast of images by artificially increasing the difference in X-ray absorption of each tissue so that tissues or blood vessels can be well seen during examinations such as magnetic resonance imaging (MRI), ultrasound, and computed tomography (CT). It could be a drug that does it. The contrast agent may include an iodine organic compound, a metal colloid, an inorganic salt (eg, barium sulfate, bismuth oxycarbonate, iodide), a metal colloid (eg, a silver-protein colloid, a silver iodide-gelatin colloid), or a combination thereof. have.

The drug may be an antiarrhythmic agent, an antiplatelet agent, an anticoagulant, or a combination thereof. The antiarrhythmic agent may be a sodium channel blocker, a beta-blocker, a potassium channel blocker, a calcium channel blocker, digoxin, adenosine, or magnesium sulfate. The sodium channel blockers are, for example, quinidine, azmaline, procainamide, disopyramide, lidocaine, phenytoin, and mexiletine. ), Tocainide, Encainide, Flecainide, Propafenone, and Moricizine. The beta-blockers are, for example, Carvedilol, Propanolol, Esmolol, Timolol, Metoprolol, Atenolol, Atenolol, Bisoprolol ( Bisoprolol), and Nebivolol. The potassium channel blockers are, for example, Amiodarone, Sotalol, Ibutilide, Dofetilide, Dronedarone, and E-4031. The calcium channel blockers are, for example, Verapamil and Diltiazem. Anticoagulants are, for example, warfarin and heparin. Antiplatelet agents are, for example, aspirin and Clopidogrel.

The drug may contain inhibitory RNA. The inhibitory RNA may be a short-length RNA that inhibits transcription, translation, or protein function of a nucleic acid in a cell. The inhibitory RNA may be microRNA (miRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), or fragments thereof.

The active ingredient may be located in the lipid bilayer or the inner aqueous layer of the exosome. When the active ingredient is hydrophobic, the active ingredient may be located in the lipid bilayer of the exosome. When the active ingredient is hydrophilic, the active ingredient may be located in the inner aqueous portion of the exosome.

The active ingredient may be introduced into the microvesicle by transformation, transfection, electroporation, or a combination thereof.

The active ingredient can be delivered specifically or selectively to the heart. The rate at which the active ingredient is delivered to the heart is about 1.2 times or more, about 1.5 times or more, about 2 times or more, or about 3 times or more for other tissues such as liver, spleen, kidney, lung, skeletal muscle or brain. Can increase. The rate of delivery of the active ingredient to the heart may increase by about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 75% or more, or about 100% compared to the negative control. I can.

The heart may be a cardiac muscle cell. The cardiac muscle cells are, for example, H9C2 cells or HL-1 cardiac muscle cell lines.

The pharmaceutical composition may contain a pharmaceutically acceptable carrier. The carrier is used in the sense of including an excipient, diluent or adjuvant. The carrier is, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl blood. It may be selected from the group consisting of rolidone, water, physiological saline, a buffer such as PBS, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil. The composition may contain a filler, an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, or a combination thereof.

The pharmaceutical composition may be prepared in any formulation according to a conventional method. The composition may be formulated as a parenteral formulation (eg, injection) or an oral dosage form (eg, powder, tablet, capsule, syrup, pill, or granule). In addition, the composition may be prepared in a systemic formulation or a topical formulation.

The pharmaceutical composition comprises a fusion protein of the CTP or a fragment thereof and an exosome transmembrane protein; And it may contain a cell-derived microvesicle containing the same in an effective amount. The term “effective amount” refers to an amount sufficient to exhibit the effect of prophylaxis or treatment when administered to an individual in need thereof. The effective amount can be appropriately selected by a person skilled in the art according to the individual. The severity of the disease, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors, including drugs used in combination or concurrent with the composition used, and other medical fields It can be determined according to factors well known in the. The effective amount may be about 0.5 μg to about 2 g, about 1 μg to about 1 g, about 10 μg to about 500 mg, about 100 μg to about 100 mg, or about 1 mg to about 50 mg per pharmaceutical composition. have.

The pharmaceutical composition may be administered in a conventional manner through oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, or intradermal routes. The pharmaceutical composition is administered, for example, by intravascular injection. The dosage of the pharmaceutical composition is, for example, about 0.001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 10 mg/kg, or about 0.1 mg/kg to about 1 mg/kg on an adult basis. It can be in the range of kg. The administration may be administered once a day, multiple times a day, or once a week, once every two weeks, once every three weeks, or once every four weeks to once a year.

Another aspect is the step of transforming the cell with a vector expressing a fusion protein of CTP or a fragment thereof and an exosome transmembrane protein;

Culturing the transformed cells to obtain a cell culture solution; And

Including the step of separating the microvesicles from the cell culture solution,

It provides a method for producing a cell-derived microvesicle comprising a fusion protein of CTP or a fragment thereof and an exosome transmembrane protein.

CTP, CTP fragment, exosome transmembrane protein, fusion protein, cell, and microvesicle are as described above.

The method includes transforming the cell with a vector expressing a fusion protein of CTP or a fragment thereof and an exosome transmembrane protein.

The vector may include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, or a nucleotide sequence of SEQ ID NO: 2.

The vector may be an expression vector. Expression vectors refer to constructs designed for gene expression in cells. The expression vector may be a plasmid vector or a viral vector. The expression vector includes a promoter, a terminator, an enhancer, an origin of replication, a selectable marker, a multiple cloning site, or a combination thereof. Can include.

The transformation means that an exogenous substance (eg, compound, protein, or nucleic acid) is introduced into a microvesicle. The transformation may include transfection, lipofection, electroporation, or a combination thereof.

The method includes culturing the transformed cells to obtain a cell culture solution.

The method includes separating microvesicles from the cell culture solution. The microvesicle may be separated by performing centrifugation, density gradient method, filtration, precipitation, dialysis, immunoaffinity chromatography, or a combination of the cell culture solution.

The method may further include introducing an active ingredient into the separated microvesicles. Introducing the active ingredient into the isolated microvesicles may be performed by transformation, transduction, electroporation, or a combination thereof.

Another aspect provides a method of delivering an active ingredient to the heart, comprising administering to a cell or individual a pharmaceutical composition according to one aspect.

The pharmaceutical composition, cell, active ingredient, heart, and delivery to the heart are as described above.

The subject can be a mammal, for example, a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be suffering from or at risk of suffering from a cardiovascular disease.

The pharmaceutical composition can be administered to cells in vitro.

The pharmaceutical composition may be administered to an individual in vivo. The method of administration may be oral or parenteral administration. The method of administration may be oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, intranasal, intratracheal, or intradermal route. For example, the pharmaceutical composition is administered to a subject by intravascular injection. The pharmaceutical composition may be administered systemically or locally, and may be administered alone or in combination with other pharmaceutically active compounds.

The preferred dosage of the pharmaceutical composition varies depending on the condition and weight of the patient, the degree of disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art.

The dosage is, for example, in the range of about 0.001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 10 mg/kg, or about 0.1 mg/kg to about 1 mg/kg on an adult basis. Can be The administration may be administered once a day, multiple times a day, or once a week, once every two weeks, once every three weeks, or once every four weeks to once a year.

According to a pharmaceutical composition for delivering the active ingredient to the heart according to an aspect, a method for preparing a cell-derived microvesicle for delivering the active ingredient to the heart, and a method for delivering the active ingredient to the heart using the same, the heart It is possible to selectively or specifically deliver the active ingredient to. Thereby, it can be used to diagnose or treat cardiovascular diseases.

1 is a simple schematic diagram of a method according to an aspect, showing a method of specifically delivering exosomes containing cardiac target protein (CTP) to cardiac cells.
Fig. 2 is a schematic diagram of a LAMP2B plasmid containing a CTP coding nucleotide sequence.
3 is a schematic diagram showing a process of separating exosomes from a cell culture solution.
4 is an image confirming the proteins of exosomes isolated from cells and cell culture media by immunoblotting.
Figure 5 shows the expression pattern of exosomes isolated from cotransformed cells of CTP-LAMP2 plasmid and CD81-mcherry by fluorescence microscopy (A), immunoblotting (B), and nanoparticle tracking analysis (C). to be.
6 is a fluorescence microscope image showing exosome delivery over time in a kidney cell line or a heart cell line in vitro (arrow: intracellular exosome delivery).
7 is a graph calculated by confirming the expression level of exosome proteins in the heart, liver, and spleen tissues of mice after injection of exosomes into the tail vein of mice (CTL: control exosome injection, CTP : CTP exosome injection, *: P<0.1, NS: not significant).
8A is a fluorescence image of a rat heart after injection of a CTP polypeptide or CTP exosome into a rat, and FIG. 8B is a graph showing the average radiant efficiency calculated from the fluorescence intensity measured in the rat heart tissue (ns : Not significant).

It will be described in more detail through the following examples. However, these examples are intended to illustrate one or more embodiments by way of example, and the scope of the present invention is not limited to these examples.

Example 1. Containing cardiac target protein Exosome Heart cell line Confirmation of specific delivery

One. CTP - LAMP2B Plasmid construction

To prepare the expression vector, pcDNA GNSTM-3-Flag-10-Lamp2b-HA (Addgene, Plasmid #71293) ('LAMP2B plasmid') was purchased.

The amino acid sequence of the known cardiac target protein (CTP) is as follows.

CTP: N-APWHLSSQYSRT-C (SEQ ID NO: 1)

As a nucleotide encoding the amino acid sequence of SEQ ID NO: 1, the nucleotide sequence reverse-translated with a codon frequently used in humans and mice is as follows.

CTP coding sequence: 5'-GCCCCCTGGCACCTGTCCTCCCAGTACTCCCGGACC-3' (SEQ ID NO: 2)

A polynucleotide having the nucleotide sequence of SEQ ID NO: 2 was synthesized by requesting Bioneer (Korea). The synthesized polynucleotide was inserted into the LAMP2B plasmid through a polymerase chain reaction (PCR) (FIG. 2). In FIG. 2,'SP' denotes a signal peptide,'NST' of'GNSTM' denotes a standard N-linked glycosylation sequon, and'G' and'M' Represents an amino acid that increases the glycosylation rate in mammals, and'CTP' represents a cardiac targeting peptide (CTP).

The CTP nucleotide sequence inserted into the plasmid was confirmed through sequencing. The LAMP2B plasmid into which the CTP coding sequence was inserted was named'CTP-LAMP2B plasmid'.

2. CTP - LAMP2B Proteins in cells and In exosomes Confirmation of expression

(One) CTP - LAMP2B Manifestation Exosome Preparations

HEK293 cell line (Korean Cell Line Bank, Korea) was inoculated into a 150 mm cell culture dish. The control LAMP2B plasmid or the CTP-LAMP2 plasmid prepared as described in Example 1.1 was added to the prepared HEK293 cells, and transformed using Lipofectamine 3000 reagent (Thermo Fischer Scientific).

After about 8 hours thereafter, it was replaced with a fresh medium containing no serum and containing GlutaMax supplement. After about 36 hours, the cell culture medium was collected and centrifuged at 300x g for 10 minutes to remove the cells. Then, the supernatant was centrifuged at 2,000x g for 10 minutes to remove cell debris, and then centrifuged at 10,000x g for 30 minutes to remove microvesicles. The supernatant was ultracentrifuged at a speed of 36,900 rpm for 2 hours to precipitate exosomes or the precipitated exosomes were separated using tangential flow filtration. The separated exosomes were washed with phosphate buffered saline (PBS) until they became transparent, and then resuspended (FIG. 3). After collecting the medium, the remaining cell lines and the separated exosomes were dissolved in RIPA buffer and then quantified using a 660 nm Protein Assay reagent (Pierce).

(2) Exosome medium CTP - LAMP2B of Immunoblotting

Expression of CTP-LAMP2B was confirmed in exosomes isolated from cells and cell culture solutions prepared in Example 1.2(1).

Specifically, immunoblotting was performed using an anti-FLAG antibody (Sigma) and an anti-HA antibody (Santacruz Biotechnology) for the same amount of protein. The image obtained by immunoblotting is shown in FIG. 4.

As shown in Figure 4, it was confirmed that the fusion protein of CTP-LAMP2B was expressed in both cells and exosomes. The exosome containing the fusion protein of CTP-LAMP2B was named'CTP exosome'. Meanwhile, exosomes isolated from cells transformed with LAMP2B plasmid without CTP were named as'control exosomes'.

3. CTP - Exosome Confirm

In order to be used as a reporter of CTP exosomes prepared as described in Example 1.2(1), mCherry-CD81 plasmid (Addgene, Flismid #55012) was prepared. HEK293 cells were transformed with herry-CD81 plasmid (8 µg), control (8 µg) or CTP-LAMP2 plasmid (8 µg) together. Exosomes were isolated from cells as described in Example 1.2(1).

Expression of the mCherry-CD81 protein in the transformed cells was confirmed with a fluorescence microscope, and the image is shown in FIG. 5A. 5A is an image confirming CD81-mcherry expression with a microscope (CTL: control, CTP: CTP-LAMP2 plasmid, BF: bright field, TR: red wavelength (Texas Red))

Transformed cells or proteins of exosomes isolated therefrom were confirmed by immunoblotting. For immunoblotting, anti-LAMP2 antibody (Santacruz Biotechnology), anti-CD81 antibody (Santacruz Biotechnology), anti-FLAG antibody (Sigma), anti-Alix antibody (Santacruz Biotechnology), and anti-GAPDH antibody (Santacruz Biotechnology) Was used. The immunoblotting results are shown in FIG. 5B. As shown in FIG. 5B, since CD81 and Alix are exosome-labeled proteins, it was confirmed that the CTP-LAMP2 fusion protein was present in exosomes isolated from transformed cells.

The separated exosomes were measured for the size and concentration of exosomes in a 10 second video frame using a Nanoparticle Tracking Analysis (NTA) using a NanoSight LM10 (Malvern Instruments Ltd, UK) instrument. The analyzed images and results are shown in C of FIG. 5 (X axis: nm, Y axis: concentration (X 10 ⁶ ), left: control exosomes, right: CTP exosomes). As shown in C of FIG. 5, it was confirmed that the exosomes were well separated by looking at the size and concentration of the particles.

4. In vitro Cardiac cell line Exosome relay

Two days before the exosome treatment, the HEK293 human embryonic kidney cell line and the H9C2D murine cardiomyocytes cell line were inoculated into a 6-well culture dish. 20 μg of control or CTP-exosomes were added to the cells. At this time, CD81-mcherry protein was used as a reporter. After 5, 24, and 48 hours, exosome absorption of the cell line was confirmed using a fluorescence microscope. A fluorescence microscope image is shown in FIG. 6 (CTL: control exosome, CTP: CTP exosome).

As shown in FIG. 6, in the HEK293 cell line, there was no difference in intracellular absorption rate between the control and CTP exosomes, but in the H92C cell line, CTP exosomes were better absorbed into the cells. Therefore, it was confirmed that the exosomes spiked with CTP in vitro were specifically delivered to heart cells.

5. CTP - Exosome Mouse heart cells Confirmation of specific delivery

The separated 150 μg of the control and CTP-exosomes were resuspended in 100 μl of 5% (w/v) glucose solution and injected into the tail vein of mice (n=4). After 24 hours, the rats were sacrificed, and the rat heart, liver, and spleen tissue were obtained. The obtained tissue was dissolved in RIPA buffer, and then quantified using a 660 nm Protein Assay reagent (Pierce). Equal amounts of protein were immunoblotted using anti-FLAG antibody (Sigma), anti-tubulin antibody (Santacruz Biotechnology), and anti-HA antibody (Santacruz Biotechnology). The band intensity of immunoblotting was measured, the ratio of the intensity of FLAG and the intensity of tubulin, and the ratio of the intensity of FLAG and the HA intensity in heart, liver, and spleen tissues were calculated to calculate the relative expression level of FLAG. A graph showing the relative expression level of FLAG in each tissue is shown in FIG. 7 (CTL: control exosome injection, CTP: CTP exosome injection, *: p<0.1, N.S.: not significant).

As shown in FIG. 7, CTP exosomes were better delivered to the heart than control exosomes. Therefore, it was confirmed that exosomes containing CTP are useful as cardiac specific delivery materials.

6. Confirmation of specific delivery of CTP-exosomes to mouse heart cells in vivo

Cardiac target protein (CTP) was prepared as described in Example 1.1, and a CTP-TAMRA polypeptide ('CTP polypeptide') was prepared by attaching a TAMRA (5-carboxytetramethylrhodamine, abcam) dye to CTP. As a protein control, a control polypeptide having TAMRA attached to the polypeptide of SEQ ID NO: 3 (5'-ARPLEHGSDKAT-3') was used. The polypeptide of SEQ ID NO: 3 has the same length as CTP, but differs in amino acid sequence, and is known as a polypeptide that is not specifically delivered to cardiac cells (Zahid et al., PLOS ONE, Aug 2010, vol. 5, issue 8, e12252).

As described in Example 1.3, cells expressing mCherry-CD81 protein were prepared, and exosomes were separated from the prepared cells to prepare exosomes labeled with mcherry fluorescence.

CTP polypeptides were transformed into exosomes labeled with mcherry fluorescence to prepare exosomes ('CTP exosomes') containing CTP polypeptides. On the other hand, the control polypeptide was transformed into an exosome labeled with mcherry fluorescence to prepare an exosome ('control exosome') containing the control polypeptide.

The prepared control polypeptide, CTP polypeptide, control exosome, and CTP exosome were administered through the tail vein of mice (in each group, n=3). In the case of the polypeptide, the rat felt pain even while under anesthesia when injected into the rat, and one died during the injection.

Thereafter, the mice were sacrificed, and the heart was excised to obtain a fluorescence image. The obtained image is shown in Fig. 8A. Average radiant efficiency was calculated from the fluorescence intensity measured in the rat heart tissue, and the results are shown in FIG. 8B (n.s: not significant). The average radiative efficiency is the total number of photons per second per square centimeter per steradian over the irradiation range (microwatts per square centimeter): [(p/s/cm2/sr)/(μW/cm2)].

8A and 8B, the control polypeptide and control exosomes were not delivered to the heart. The average copy efficiency of CTP polypeptide increased by about 5%, and the average copy efficiency of CTP exosomes increased by about 11%, so that CTP polypeptide and CTP exosome were significantly delivered to the heart. In particular, it was confirmed that CTP exosomes were significantly increased compared to CTP polypeptides, and could be specifically and effectively delivered to heart tissue by a combination of exosomes and CTP.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Pharmaceutical composition for delivering microvesicle including cardiac targeting protein to heart and method using the same <130> PN125583KR <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Cardiac targeting protein <400> 1 Ala Pro Trp His Leu Ser Ser Gln Tyr Ser Arg Thr 1 5 10 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acids encoding cardiac targeting protein <400> 2 gccccctggc acctgtcctc ccagtactcc cggacc 36 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Control polypeptide <400> 3 Ala Arg Pro Leu Glu His Gly Ser Asp Lys Ala Thr 1 5 10

Claims (18)

As a fusion protein of a cardiac targeting peptide (CTP) or a fragment thereof, and an exosome transmembrane protein,
The CTP is a fusion protein comprising an amino acid sequence encoded by the amino acid sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 2; And
A pharmaceutical composition for delivering the active ingredient to the heart, comprising the fusion protein and cell-derived microvesicles containing the active ingredient. delete The pharmaceutical composition of claim 1, wherein the CTP is located on the surface of the microvesicle. The pharmaceutical composition of claim 1, wherein the exosome transmembrane protein is selected from the group consisting of LAMP-1, LAMP-2, CD13, CD86, Flotilin, and Syntaxin-3. The pharmaceutical composition of claim 1, wherein the cells are kidney cells. The pharmaceutical composition of claim 5, wherein the kidney cells are human embryonic kidney cells. The pharmaceutical composition of claim 1, wherein the microvesicle is an exosome. The pharmaceutical composition of claim 1, wherein the heart is a cardiac muscle cell. The pharmaceutical composition of claim 1, wherein the active ingredient is a drug for diagnosis, prevention, treatment, or a combination of cardiovascular diseases. The pharmaceutical composition of claim 9, wherein the drug is a contrast agent, a radioactive isotope, a fluorescent substance, a luciferase, or a combination thereof. The pharmaceutical composition of claim 9, wherein the drug is an antiarrhythmic agent, an antiplatelet agent, an anticoagulant, or a combination thereof. The pharmaceutical composition of claim 1, wherein the active ingredient is located in the lipid bilayer or the inner aqueous layer of the exosome. Transforming the cell with a vector expressing a fusion protein of CTP or a fragment thereof and an exosome transmembrane protein;
Culturing the transformed cells to obtain a cell culture solution; And
Including the step of separating the microvesicles from the cell culture,
As a method for producing a cell-derived microvesicle comprising a fusion protein of CTP or a fragment thereof and an exosome transmembrane protein,
Wherein the CTP comprises an amino acid sequence encoded by the amino acid sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 2. delete A method of delivering the active ingredient to heart cells or heart, comprising administering the pharmaceutical composition of claim 1 to heart cells or mammals other than humans in vitro. delete The method of claim 15, wherein the pharmaceutical composition is administered to a mammal other than a human in vivo. The method of claim 17, wherein the pharmaceutical composition is administered by intravascular injection of a mammal other than a human.

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