KR102166542B1 - Conjugate of telomerase derived peptide and contrast substances for detecting stem cell and Contrast composition comprising the same - Google Patents

Abstract

Disclosed herein are a conjugate of a telomerase-derived peptide and a contrast material for detection of stem cells, and a contrast agent for detection of stem cells including the same. According to the present invention, a peptide having the sequence of SEQ ID NO: 1, a peptide that is a fragment of the sequence of SEQ ID NO: 1, or a peptide having a sequence homology of 80% or more with the peptide sequence is an excellent cell-penetrating peptide that binds to a contrast material to form stem cells. It can be used as a contrast medium for detection. In particular, the contrast agent conjugate according to the present invention has the advantage of being able to detect stem cells without affecting the basic functions of stem cells.

Description

Conjugate of telomerase derived peptide and contrast substances for detecting stem cell and Contrast composition comprising the same}

Disclosed herein are a conjugate of a telomerase-derived peptide and a contrast material for detection of stem cells, and a contrast agent for detection of stem cells including the same.

Telomere is a genetic material that is repetitively present at the end of a chromosome, and is known to prevent damage to the chromosome or binding to other chromosomes. Each time a cell divides, the length of the telomeres decreases little by little. If there are more than a certain number of cell divisions, the telomeres become very short, and the cells stop dividing and die. On the other hand, it is known that lengthening telomeres extends the lifespan of cells. For example, cancer cells secrete an enzyme called telomerase to prevent shortening of telomeres, so it is known that cancer cells can continue to proliferate without dying.

Stem cells are pluripotent cells, that is, cells that can develop into any tissue. When stem cells are injected into a living body, research reports that stem cells can move themselves to damaged or affected areas in the living body and differentiate into various cell types ranging from cardiomyocytes, nerve cells, or skeletal muscles to produce therapeutic effects. Has been announced. In research experiments related to treatment using these stem cells, it is necessary to monitor whether stem cells migrate to the damaged or affected site. In addition, in order to monitor the treatment of patients using stem cells, it is necessary to monitor the movement of stem cells in vivo. However, little is known about a means for tracking stem cells injected into a living body.

In addition, stem cell research is very active and competitive in countries around the world, and the related business market is constantly growing, and as it can be usefully applied to the treatment of numerous intractable diseases, interest in research continues to increase. The tracking of research is not universally performed, so the rapid progress of the research has not been achieved.

Korean Patent Publication No. 10-2007-0024206 (2007.03.02.) European Patent Publication No.1362597 (2003.11.19) European Patent Publication No. 0841396 (1998.05.13.)

Accordingly, the present inventors have made diligent efforts to develop a contrast agent for detecting stem cells, resulting in the completion of the present invention.

An object according to an aspect of the present invention is to provide a contrast medium that can be effectively used for the detection of stem cells.

An aspect of the present invention is to provide a contrast agent capable of penetrating into stem cells.

An aspect of the present invention is to provide a contrast medium capable of contrasting stem cells.

An aspect of the present invention is to provide a stem cell contrast agent with less side effects to the human body.

One aspect of the present invention is a conjugate of a cell penetrating peptide and a contrast material, wherein the cell penetrating peptide is a peptide having the sequence of SEQ ID NO: 1, a peptide that is a fragment of the sequence of SEQ ID NO: 1, or the peptide sequence and 80 It is a peptide having a sequence homology of% or more and provides a conjugate for detection of stem cells.

In the conjugate according to an aspect of the present invention, the peptide may be composed of 30 or less amino acids.

In the conjugate according to one aspect of the present invention, the contrast material is from the group consisting of a radiopaque contrast agent, a paramagnetic contrast material, a superparamagnetic contrast material, and a CT contrast material. It may be selected.

In the conjugate according to an aspect of the present invention, the contrast material may be an iron-based material.

In the conjugate according to an aspect of the present invention, the contrast material may be ferrocene carboxylate.

In the conjugate according to an aspect of the present invention, the peptide and the contrast material may be conjugated by a covalent bond.

In the conjugate according to an aspect of the present invention, the peptide and the contrast material may be conjugated by a non-covalent bond.

In the conjugate according to an aspect of the present invention, the stem cells for detection may be neural stem cells or mesenchymal stem cells.

Another aspect of the present invention is a contrast agent comprising a cell-penetrating peptide and a conjugate of a contrast material, wherein the cell-penetrating peptide is a peptide having the sequence of SEQ ID NO: 1, a peptide that is a fragment of the sequence of SEQ ID NO: 1, or A contrast agent for detection of stem cells comprising a conjugate, a peptide having 80% or more sequence homology with the peptide sequence, is provided.

A peptide having the sequence of SEQ ID NO: 1, a peptide that is a fragment of the sequence of SEQ ID NO: 1, or a peptide having a sequence homology of 80% or more with the peptide sequence is an excellent cell-penetrating peptide that binds to a contrast material and is Can be utilized. In particular, the contrast agent conjugate according to the present invention has the advantage of being able to detect stem cells without affecting the basic functions of stem cells.

1 is a photograph of measuring a state over time after treatment of ferrocenecarboxylic-pep1 on neural stem cells.
Figure 2 is a photograph of ferrocenecarboxylic-pep1 treatment on neural stem cells, staining the cell nucleus with DAPI, and then measuring the state of neural stem cell penetration with a confocal laser scanning system.
3 is a cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) as a result of a toxicity test of ferrocenecarboxylic-pep1 itself. It shows the change in cell viability and cytotoxicity, respectively, using an activity assay.
FIG. 4 shows the results of toxicity confirmation for NSCs through pro-apoptotic signals.
5 and 6 show experimental results confirming that the cell proliferation of stem cells was not affected by ferrocene carboxylic-pep1.
7 shows the cellular mobility of neural stem cells (NSCs) by ferrocene carboxylic-pep1.
Figure 8 shows the free radical change of stem cells by ferrocene carboxylic-pep1 in NSC.
9 is an MRI photograph of the brain of a group (Stem cells with Ferrocenecarboxylic-pep1 group) transplanted with ferrocenecarboxylic-pep1 treated neural stem cells, and FIG. 10 is a photograph of ferrocene carboxylic-pep1 treated with ferrocenecarboxylic-pep1. This is an MRI photograph of the brain of the group (Stem cells without Ferrocenecarboxylic-pep1 group) transplanted with non-neural stem cells (Stem cells without Ferrocenecarboxylic-pep1 group). It is a picture, and FIG. 12 is an MRI photograph of the brain of the group (Saline group) transplanted with physiological saline.
FIG. 13 shows the contents of different amounts of cells for each part of rat for MRI detection of ferrocenecarboxylic-pep1.
14 shows a picture taken by MRI up to 5 weeks.
FIG. 15 shows the results of Prussian blue staining by dividing the part corresponding to the control part and the part where Fe was detected in MRI tissue.
FIG. 16 is a photograph confirming the NSC with an electron microscope where ferrocene carboxylic-pep1 was observed in the cell membrane, and which organelles in the cell are located in more detail.
17 shows the results of measuring pro-apoptotic signals in mesenchymal stem cells (MSC) by Western blot and FACS.
18 and 19 show the measurement results measured by pAkt and BrdU, which are cell survival signals, whether there is a change in proliferation of stem cells by ferrocenecarboxylic-pep1 in MSC.
Figure 20 shows the cell mobility of mesenchymal stem cells (MSCs) by ferrocene carboxylic-pep1.
Figure 21 shows the free radical change of stem cells by ferrocene carboxylic-pep1 in MSC.
22 is a photograph of MSC+Fe observed and shown in Prussian blue stain in in-vitro as confirmed in In-vivo.
23 is a photograph confirming that MSCs were located in a number of cell membranes as well as cell Prussian blue stain through an electron microscope, and were observed in autophagosome.

Proteins, nucleic acids, peptides, or viruses have great potential as therapeutic substances, but their use is very limited because they cannot penetrate tissues and cell membranes because they have a molecular size. In addition, even a material having a small size cannot penetrate the lipid bilayer of a cell membrane due to its nature or structure. Accordingly, there have been attempts to move proteins, nucleic acids, peptides, or viruses into cells by electroporation or heat shock, but while maintaining the activity of the substances as they are without damaging the cell membrane. It was difficult to move these substances into the cells. Meanwhile, the Trans-Activating Transcriptional activator (TAT) protein derived from Human Immuno-deficiency Virus (HIV) can act as a Cell Penetrating Peptide that can move giant active substances into cells. As this became known, research on it was actively conducted. Specifically, unlike TAT proteins, which are known to cause intracellular toxicity, studies have been conducted on substances that can more effectively move macromolecules such as proteins, nucleic acids, peptides, or viruses into target cells without causing toxicity in vivo. . Accordingly, the inventors of the present invention have found that the peptide derived from telomerase has an excellent effect as a cell penetrating peptide while having little cytotoxicity through continuous research.

The cell penetrating peptide disclosed herein may be a peptide having the sequence of SEQ ID NO: 1, a peptide that is a fragment of the sequence of SEQ ID NO: 1, or a peptide having 80% or more sequence homology with the peptide sequence.

The peptides disclosed herein include a peptide having the sequence of SEQ ID NO: 1 or a peptide that is a fragment of the sequence of SEQ ID NO: 1, and a peptide having sequence homology of 80% or more, 85% or more, 90% or more, or 95% or more. I can.

The peptides disclosed herein can be wild-type peptides that have been identified and isolated from native sources. Meanwhile, the peptide disclosed in the present specification may be an artificial variant comprising an amino acid sequence in which one or more amino acids have been substituted, deleted, and/or inserted compared to a peptide that is SEQ ID NO: 1 or a fragment thereof. Amino acid changes in wild-type polypeptides as well as in artificial variants include conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein. Examples of conservative substitutions include basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids (phenylalanine, Tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine). In general, amino acid substitutions that do not alter a specific activity are known in the art. The most common exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly, and vice versa.

In one aspect of the present invention, amino acid changes belong to properties that cause the physicochemical properties of the peptide to be altered. For example, amino acid changes such as improving the thermal stability of the peptide, altering the substrate specificity, changing the optimal pH, and the like can be performed.

In one aspect of the present invention, the cell penetrating peptide may be composed of 30 or less amino acids.

In one aspect of the present invention, the cell penetrating peptide includes a peptide derived from telomerase, specifically human ( Homo sapiens ) telomerase.

The peptides listed in SEQ ID NO: 1 are shown in Table 1 below. The "name" in Table 1 below is named to distinguish the peptide. In one aspect of the invention, the peptide set forth in SEQ ID NO: 1 represents the entire peptide of human telomerase. In another aspect of the present invention, a peptide having the sequence of SEQ ID NO: 1, a peptide that is a fragment of the sequence of SEQ ID NO: 1, or a peptide having a sequence homology of 80% or more with the peptide sequence is a corresponding among the peptides included in telomerase. It includes "synthetic peptides" obtained by selecting and synthesizing a peptide at a position. SEQ ID NO: 2 shows the amino acid sequence of all telomerase.

[Table 1]

In the present specification, "Cell Penetrating Peptide (CPP)" is in vitro and/or in It refers to a peptide capable of moving a carriage into cells in vivo .

In the present specification, the term "contrast material" is a broad concept including all materials used for the imaging of structures or fluids in vivo in medical imaging. Suitable contrast materials include, but are not limited to, radiopaque contrast agents, paramagnetic contrast agents, superparamagnetic contrast agents, computed tomography (CT) contrast materials, and other contrast materials. Does not. For example, radiopaque contrast materials (for X-ray imaging) include inorganic iodine compounds and organic iodine compounds (eg, diatrizoat), radiopaque metals and salts thereof (eg, silver, gold, platinum). Etc.) and other radiopaque compounds (e.g., calcium salts, barium salts such as barium sulfate, tantalum and tantalum oxide). Suitable paramagnetic contrast materials (for MR imaging) are gadolinium diethylene triaminepentaacetic acid (Gd-DTPA) and derivatives thereof, and other gadolinium, manganese, iron, dysprosium, copper, uro Europium, erbium, chromium, nickel and cobalt complexes, for example 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N,-N',N"-triacetic acid (D03A), 1,4,7-triazacyclo Nonane-N,N',N"-triacetic acid (NOTA), 1,4,8,10-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid (TETA), hydroxy Benzylethylene-diamine diacetic acid (HBED), and the like. Suitable superparamagnetic contrast materials (for MR imaging) include magnetite, super-paramagnetic iron oxide (SPIO), ultrasmall superparamagnetic iron oxide (USPIO) and monocrystalline iron oxide. do. Other suitable contrast media are iodized and non-iodinated, ionic and nonionic CT contrast media, and contrast media such as spin-labels, or other diagnostically effective agents.

Other examples of the contrast material include, but are not limited to, β-galactosidase, green fluorescent protein, blue fluorescent protein, luciferase, etc. When expressed in cells, a marker gene encoding an easily detectable protein Include. Various labels such as radionuclides, fluores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (especially hapten) may be used.

In one embodiment of the present invention, the contrast material may be ferrocenecarboxylic acid represented by the following formula (2). The structure of ferrocene is shown in Formula 1.

[Formula 1]

[Formula 2]

In one embodiment of the present invention, the conjugate of the cell penetrating peptide and the contrast material may be ferrocenecarboxylic-pep1 of the following formula (3).

[Formula 3]

In one aspect of the present invention, a transport target, that is, a contrast material may be directly bound to the peptide. In another aspect of the present invention, a moving object may be bound to the peptide through several binding methods, including covalent bonding or non-covalent bonding. For example, a disulfide bond, or a covalent bond that binds a migration object to the peptide N-terminus or peptide C-terminus, specifically, the alpha amine or C- of the peptide N-terminal glutamate (E) The migration target can be bound to the peptide through a covalent bond that binds the migration target to the amine of the terminal lysine (K) residue. Alternatively, one of the peptide and the moving object may bind the peptide and the moving object through a non-covalent bond surrounding the other in a form such as a capsule form.

In another aspect of the present invention, it is possible to bind the peptide and the target to be moved through a linker. For example, in the amine of the peptide N-terminus or the peptide C-terminus, specifically the alpha amine of the peptide N-terminal glutamate or the C-terminal lysine residue, Hynic(6-hydrazinopyridine-3-carboxylic acid , 6-hydrazinopyridine-3-carboxylic acid) After introducing a linker such as a linker, the transfer target can be bonded to the peptide by binding the transfer target to the linker.

In one aspect of the present invention, the cell penetrating peptide may be combined with a dyeing material or a fluorescent material, specifically fluorescein isothiocyanate (FITC). In one aspect of the present invention, fluorescein isothiocyanate may bind to the amino group (NH ₃ ⁺ ) of the N-terminal or C-terminal Lys of the cell penetrating peptide. In the case of a peptide having no Lys at the end, the peptide and FITC may be bonded through a linker containing Lys.

In one aspect of the present invention, cells include all cells used herein as a cell therapy agent. For example, it may be a stem cell, an islet cell, a dendritic cell, or an immune cell, but is not limited thereto. In particular, the cells may be stem cells.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to experimental examples. However, the experimental examples below are provided for illustrative purposes only to aid understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

[Example 1] Ferrocenecarboxylic (Ferrocenecarboxylic)-pep1 Conjugate Permeation Test in Neural Stem Cells

(One) Ferrocene carboxylic ( Ferrocenecarboxylic )- pep1 Conjugated Manufacturing and Experiment Method

A. Ferrocene carboxylic ( Ferrocenecarboxylic )- pep1 Conjugated Produce

For coupling, the amino acid protected in NH ₂ -Lys(Dde)-2-chloro-trityl resin (8 equivalents) and the coupling reagent HBTU (8 equivalents)/HOBt (8 equivalents)/NMM (16 equivalents) were added to DMF. After dissolving and adding, it was reacted at room temperature for 2 hours and washed in the order of DMF, MeOH, and DMF. Thereafter, for Fmoc deprotection, piperidine in 20% DMF was added, reacted twice for 5 minutes at room temperature, and washed in the order of DMF, MeOH, and DMF.

By repeatedly performing the above reaction, the peptide base skeleton (NH2-E(OtBu)-AR(Pbf)-PALLT(tBu)-S(tBu)-R(Pbf)LR(Pbf)-FIPK(Dde)-2-chloro -Trityl Resin) was made. Here, hydrazine in 2% DMF was treated to remove Dde, which is a residue protecting group of C-terminal Lys. Then, ferrocenecarboxylic acid (Sigma Aldrich cat.#46264, 16 equivalents) and coupling reagent HBTU (16 equivalents)/HOBt (16 equivalents)/NMM (32 equivalents) were dissolved in DMF and added, followed by room temperature. After reacting for 2 hours, DMF, MeOH, and DMF were washed in that order. The peptide was separated from the resin by adding TFA/TIS/H2O=95/2.5/2.5 to the synthesized peptide resin. After cooling diethyl ether was added to the obtained mixture, the peptide obtained by centrifugation was precipitated. This was purified by HPLC, confirmed by MS, and freeze-dried.

B. Western Blot

The levels of Ki67, Bax, cytosolic cytochrome c, cleaved caspase-9, COX-2 and cleaved caspase-3 (Asp175) were analyzed by Western blot. Western blot analysis of various signals in cells was performed immediately after 24 hours cell treatment. After washing 5 X 10 ⁶ cells twice with cold PBS solution, lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.2% SDS, 100 μg/ml phenyl methyl sulfonyl fluoride ( PMSF), 50 μl/ml aprotinin, 1% Igepal 630, 100 mM NaF, 0.5% sodium deoxy choate, 0.5 mM EDTA, 0.1 mM EGTA]; Incubated on ice, unbroken cells and nuclei were removed using centrifugation (2000 xg) for 10 minutes, and lysates were removed using centrifugation (10,000 xg). To evaluate cytoplasmic HMGB-1 and cytosolic cytochrome c levels, mitochondrial and cytosolic fractions were isolated using a mitochondrial/cytoplasmic nucleation kit (Abcam, UK) according to the manufacturer's instructions. For 48 hours, after 20 μM Aβ25-35 with various concentrations of PEP1, neural stem cells were obtained. After washing with cold PBS solution, 1.0 mL of 1× cytoplasmic extraction buffer mixed solution containing dithiothreitol (DTT) and protease inhibitors Resuspended in After incubation on ice for 10 minutes, the cell suspension was homogenized with a syringe 30-50 times. The sample was centrifuged at 3,000 rpm at 4° C. for 10 minutes. The supernatant was centrifuged again at 13,000 rpm for 30 minutes to separate the mitochondrial fraction (pellets) and the cytoplasmic fraction (supernatants). The mitochondrial pellet was washed once with an isolation buffer, and then lysed using a mitochondrial extraction buffer containing DTT and a protease inhibitor. Samples containing the same amount (20 μg) of protein were digested by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Nitrocellulose membranes were blocked with 5% skim milk, and cultured with specific basic antibodies.

Anti-Ki67 (1:200, Abcam, UK), anti-BAX (1:1000, Cell Signaling), anti-cytochrome c (1:200, Cell Signaling), anti-cleaved caspase-9 (1: 1000, Cell signaling), COX-2 (1:1000, Cell signaling) and anti-cleaved caspase-3 (Asp 175) (1:1000, Cell signaling, Beverly, MA, USA).

Cell membranes were washed with Tris-buffered saline containing 0.05% Tween-20 (TBST), treated with HRP-conjugated rabbit antibody or anti-mouse antibody (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and then ECL detection (Amersham Pharmacia Biotech). The blot was quantified with an image analyzer (GE Healthcare, ImageQuant LAS 4000).

C. BrdU cell proliferation assay

Immediately after exposure to ferrocenecarboxylic-PEP1 for 24 hours, MSCs were collected. Thereafter, MSCs were cultured in BrdU-labeling medium (10 μM BrdU) for 5 hours, and cell proliferation was measured using BrdU Labeling and Detection Kit (Roche Boehringer??Mannheim, IN, USA) according to the manufacturer's instructions.

Cell proliferation was evaluated using an ELISA plate reader at a reference wavelength of 492 nm and a wavelength of 370 nm, and all results were normalized to the OD of the same well without cells.

D. Colony forming unit (CFU) analysis

Neural stem cells were dispensed at 0.25 × 10 ⁵ cells per 6 well, and then the cells were exposed to 10 μM ferrocenecarboxylic-PEP1 for 24 hours. After washing the cells with DPBS, the medium was replaced. After 14 days, the cells were washed again with DPBS and stained with .5% crystal violet (Sigma) in methanol for 30 minutes at room temperature. After cell staining, the plate was washed with DPBS and dried. Colony count was performed using a dissecting microscope, and cells with faint staining or less than 2 nm in diameter were excluded.

E. Move( Migration ) Measure

Cell migration was measured according to the manufacturer's instructions using a QCM 24-well colorimetric cell migration assay kit (Chemicon, Temecula, CA). 10 μM ferrocenecarboxylic-PEP1 was added to the lower chamber. NSCs (3 × 10 ⁵ cells) and MSCs (1 × 10 ⁵ ) were dispensed in the upper chamber and incubated at 37°C for 24 hours. NSCs (3 × 10 ⁵ cells) and MSCs (1 × 10 ⁵ ) that migrated through the cell membrane were stained, and the migrated cells were determined by measuring absorbance at 560 nm.

Cell migration was measured according to the manufacturer's instructions using a QCM 24-well colorimetric cell migration assay kit (Chemicon, Temecula, CA).

F. Test to determine the production of free radicals

To measure the production of free radicals, NSCs and MSCs were exposed to Ferrocenecarboxylic-GV1001 for 24 hours, followed by fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) for 15 minutes (Molecular Probes Inc., Eugene, OR, USA). ) And incubated. Thereafter, NSCs and MSCs were treated with 10 μM Ferrocenecarboxylic-GV1001, and the control group was incubated for 24 hours. After incubation with 10 μM DCFH-DA at 37° C. for 15 minutes, the cells were washed three times with PBS. DCFH-DA freely passes through the cell membrane and is hydrolyzed to 2',7'-dichlorodihydrofluorescein (DCFH2) by cellular esterases, and then oxidized in the presence of peroxides as fluorescent 2'7'-dichlorofluorescein (DCF). This means that DCF fluorescence mainly indicates the level of hydrogen peroxide in the cell, not peroxide. The accumulation of DCF in the cells was observed using an Olympus fluorescence microscope with an excitation wavelength suitable for ROS staining, which is the subject of flow cytometry analysis (FACS C6, BD).

(2) Neural stem cell penetration experiment

In the experiment, rat (Sprague-Dawley, SD) (Orient bio, Kyungki, Korea) separating the cerebral cortex from the head of the embryo on the 13th day of pregnancy, poly-L-ornithine/fibronectin in Ca ²⁺ /Mg ²⁺ -free PBS ( GIBCO, Grand Island, NY, USA) coated with 2 x 10 ⁴ cells/cm ² and N2 medium (DMEM/F12, 4.4 IM insulin, 100 mg/l transferrin, 30 nM selenite, 0.6 IM pu). Basic fibroblast growth factor daily in putrescine, 20 nM progesterone, 0.2 mM ascorbic acid, 2 nM L-glutamine, 8.6 mM D(+) glucose, 20 nM NaHCO ₃ (Sigma, St. Louis, MO)). (basic fibroblast growth factor, BFGF) (R&D Systems, Minneapolis, MN) 10 ng/ml was treated to obtain more than 95% of neural stem cells after 4-6 days at 37°C and 5% CO ₂ .

The obtained neural stem cells were dispensed into a chamber well plate at 1×10 ⁵ . Ferrocene carboxylic-pep1 was treated with 10 μM and incubated for 0 to 24 hours in a 37°C, 5% CO ₂ incubator. After washing twice with PBS and fixing with 4% paraformaldehyde for 20 minutes at room temperature, 4',6-diamidino-2-phenylindole (DAPI) mount medium (mount medium with DAPI) (Vetor Laboratories, CA, USA). After that, a confocal laser scanning system was used to compare and analyze the blue wavelength of 430-500nm and the red wavelength of 620-700nm, respectively, and the combined wavelength of the two.

The results are shown in FIGS. 1 and 2.

1 is a photograph of measuring a state over time after treatment of ferrocenecarboxylic-pep1 on neural stem cells.

As shown in FIG. 1, after treatment with ferrocene carboxylic-pep1 on neural stem cells, a comparative analysis result confirmed that ferrocene carboxylic-pep1 at a concentration of 10 μM penetrated into the cell from 2 hours. It was confirmed that a large amount penetrated into the cells.

Figure 2 is a photograph of ferrocenecarboxylic-pep1 treatment on neural stem cells, staining the cell nucleus with DAPI, and then measuring the state of neural stem cell penetration with a confocal laser scanning system. In FIG. 4, a portion that appears blue at a wavelength of 430-500 nm is a nucleus stained with DAPI, and a portion that appears red at a wavelength of 620-700 nm is ferrocenecarboxylic-pep1. As shown in the combined photo of the two wavelengths, ferrocenecarboxylic-pep1 is aggregated around the nucleus of the cell, indicating that ferrocenecarboxylic-pep1 penetrated into the cell and migrated into the cytoplasm.

(3) Toxicity test

A. Confirmation of toxicity of ferrocenecarboxylic-pep1 itself

For the toxicity test of pep1 itself conjugated with ferrocenecarboxylic acid, neural stem cells were dispensed into 96-well and 24-well plates at 4x10 ⁴ and 2.5x10 ⁵ , respectively, and incubated at 37℃, 5% CO _{2 for} at least 12 hours. Cultured. Ferrocenecarboxylic-pep1 was treated at concentrations of 0, 0.01, 0.1, 1, 10, and 100 μM, respectively, and cultured in an incubator for up to 24 hours, followed by cell counting kit-8 (CCK- 8) Using the assay and lactate dehydrogenase (LDH) activity assay, changes in cell viability and cytotoxicity were confirmed, respectively. The results are shown in FIG. 3.

As shown in FIG. 3, it was confirmed that there was no effect on cell viability and cytotoxicity up to 100 μM in neural stem cells.

B. Toxicity confirmation through pro-apoptotic signal

PEP1 has already been confirmed that there is no toxicity in neurons and other cells through various experiments, but it is necessary to evaluate whether a new cytotoxicity occurs due to conjugation of Ferrocenecarboxy, so pro-apoptotic signal is measured by western blot and FACS. I did.

The results of confirming toxicity through a pro-apoptotic signal for NSC are shown in FIG. 4. As shown in FIG. 4, it was confirmed that no specific change occurred in pro-apoptotic signals, and through this, it was confirmed that Fe-PEP1 did not show toxicity.

(4) Ferrocene carboxylic - pep1 of NSC The impact on

A. Ferrocene carboxylic - pep1 Cell proliferation of stem cells by

The change in proliferation of stem cells by ferrocenecarboxylic-pep1 was measured by Ki67, a cell proliferation marker, pAkt, a cell survival signal, colony forming unit assay, and BrdU. The results measured by each are shown in FIGS. 5 and 6. As can be seen through FIGS. 5 and 6, it can be confirmed that cell proliferation of stem cells was not affected by ferrocenecarboxylic-pep1.

B. Ferrocene carboxylic - pep1 Cell migration of stem cells by

Due to the nature of stem cells, cell mobility is a very important part. As can be seen from FIG. 7, it can be seen that there was no change in stem cell migration by ferrocenecarboxylic-pep1.

C. Ferrocene carboxylic - pep1 Of stem cells by Free radical change

Oxidative stress can damage all cells. If ROS increases by that amount, it will be problematic to use as a stem cell contrast agent. After treatment with ferrocenecarboxylic-pep1 using DCF-DA staining samples, changes in the generation of active oxygen were observed. As can be seen through FIG. 8, no change in active oxygen of NSC was observed.

(5) MRI Through filming NSC of Detectability Confirm

A. Ferrocene carboxylic - pep1 Labeled Preparation of neural stem cells

The neural stem cells obtained in (2) above were dispensed into a 100 mm dish at 5x10 ⁶ . Ferrocene carboxylic-pep1 was treated with 10 μM and incubated for 24 hours in a 37° C., 5% CO ₂ incubator. Cells were separated from the plate with TrypLE (GIBCO), washed with free media, and counted, and ferrocene carboxyl per animal Rig-pep1 labeled neural stem cells were prepared 3×10 ⁵ 10 μl.

B. Ferrocene carboxylic - pep1 Treated neural stem cells ( NSC ) Of the transplant

All animal experiments described in this specification were conducted after obtaining the approval of IACUC from Hanyang University, Korea. Eight-week-old SD rats were purchased, and after an adaptation period for 1 week, rats weighing about 290 g +/- 15 g were used in this experiment. The animals were divided into 4 groups, and the experiment was conducted with NSCs with Ferrocenecarboxylic-pep1 group, NSCs without Ferrocenecarboxylic-pep1 group, Ferrocenecarboxylic-pep1 group, and physiological saline group (Saline group). When cells, ferrocenecarboxylic-pep1 or saline were transplanted into each group, they were transplanted into the brain of SD rats using stereotaxic surgery. Anesthesia was performed using ketamine and rompun before transplantation, and the skull was partially ground and removed after shaving the scalp. After that, it was implanted stereotaxic at coordinates AP = +0.7, R = +2, V = -5.5.

C. MRI shooting

The implanted NSCs and ferrocenecarboxylic-pep1 were confirmed by MRI for in vivo observation. MRI was performed after 3 periods of implantation, and images were taken using Philips' Best 3T MRI equipment. Anesthesia was also performed using ketamine and rompun, and a multiplanar gradient-recalled (MPGR) pulse sequence (TR = 596 ms, TE = 16 ms, section thickness = 0.7 mm, phosphorus -In-plane resolution: 292 x 290 μm (pixel size 0.0593 mm ³ ), and number of acquisitions = 1) was used.

The results are shown in Figs. 9 to 12. 9 is an MRI photograph of the brain of a group (Stem cells with Ferrocenecarboxylic-pep1 group) transplanted with ferrocene carboxylic-pep1-treated neural stem cells, and FIG. 10 is a diagram of a neural stem cell not treated with ferrocene carboxylic-pep1 This is an MRI photograph of the brain of the transplanted group (Stem cells without Ferrocenecarboxylic-pep1 group), and FIG. 61 is an MRI photograph of the brain of the ferrocenecarboxylic-pep1 transplanted group (Ferrocenecarboxylic-pep1 group), and FIG. 12 Is an MRI photograph of the brain of the group transplanted with physiological saline (Saline group). As shown in FIGS. 9 to 12, it was found that the in vivo detection of neural stem cells treated with ferrocenecarboxylic-pep1 was very excellent. Comparing FIGS. 9 and 10, neural stem cells not treated with ferrocenecarboxylic-pep1 are not detected, whereas neural stem cells treated with ferrocenecarboxylic-pep1 have excellent detection. On the other hand, compared to the group transplanted with only physiological saline (FIG. 12), it can be seen that the group transplanted with ferrocenecarboxylic-pep1 (FIG. 11) had much better detection.

D. MRI Through filming Ferrocene carboxylic - pep1 In vivo observation of

The implanted NSCs and ferrocenecarboxylic-pep1 were confirmed by MRI for in vivo observation. MRI was performed three periods after transplantation, and images were taken using Philips' Best 3T MRI equipment. Ketamin and rompun were also used for anesthesia, and a multiplanar gradient-recalled (MPGR) pulse sequence (TR= 596 ms, TE = 16 ms, section thickness = 0.7 mm, in-plane resolution: 292 x 290 μm (voxel size of 0.0593). mm3), and number of acquisitions = 1). 13 shows the contents of different amounts of cells for each part of rats for MRI detection of ferrocenecarboxylic-pep1. 14 shows a picture taken by MRI up to 5 weeks. For confirmation, the detected point was sectioned and the correct Fe was detected by Prussian blue stain. FIG. 15 shows a photograph of reconfirming fe detection as a result of Prussian blue staining by dividing a part corresponding to a control part and a part where Fe was detected in MRI tissue, and seeing that the part corresponding to Fe is blue. As confirmed in in-vivo, in-vitro was also observed with Prussian blue stain.

In FIG. 16, ferrocene carboxylic-pep1 was observed on the cell membrane as shown in the picture, and it was confirmed with an electron microscope in which organelles in the cell are located in more detail. As shown in the electron micrograph of NSCs, it was confirmed that a number of cells were located on the cell membrane as in the cell Prussian blue stain and were also observed in the autophagosome.

[Example 2] Ferrocenecarboxylic (Ferrocenecarboxylic)-pep1 Conjugate Permeation Test in Mesenchymal Stem Cells

(One) Ferrocene carboxylic ( Ferrocenecarboxylic )- pep1 Conjugated Produce

A ferrocenecarboxylic (Ferrocenecarboxylic)-pep1 conjugate was prepared and prepared according to the method described in Example 2.

(2) Toxicity test

The results of measuring pro-apoptotic signals in mesenchymal stem cells (MSCs) by Western blot and FACS are shown in FIG. 17. As shown in FIG. 17, it was confirmed that no specific change occurred in pro-apoptotic signals, and through this, it was confirmed that Fe-PEP1 did not show toxicity.

(3) Ferrocene carboxylic - pep1 of MSC The impact on

A. Ferrocene carboxylic - pep1 Cell proliferation of stem cells by

The changes in the proliferation of stem cells by ferrocenecarboxylic-pep1 were measured by cell survival signals, pAkt and BrdU. The measurement results are shown in FIGS. 18 and 19.

B. Ferrocene carboxylic - pep1 Cell migration of stem cells by

Due to the nature of stem cells, cell mobility is a very important part. There was no change in stem cell migration by ferrocenecarboxylic-pep1. The results for the mobility confirmation are shown in FIG. 20.

C. Ferrocene carboxylic - pep1 Of stem cells by Free radical change

Oxidative stress can damage all cells. If ROS increases by that amount, it will be problematic to use as a stem cell contrast agent. After treatment with ferrocenecarboxylic-pep1 using DCF-DA staining samples, changes in the generation of active oxygen were observed. As can be seen through FIG. 21, no change in active oxygen was observed for MSC.

D. MRI Through filming Ferrocene carboxylic - pep1 In vivo observation of

MSCs were prepared in the same manner as described in Example 1, and confirmed using MRI for in vivo observation of the implanted MSCs and ferrocenecarboxylic-pep1. For MSC+Fe, as confirmed in In-vivo, it was observed with Prussian blue stain in in-vitro as well as shown in FIG. 22. In Figure 23, as shown in the picture, ferrocene carboxylic-pep1 was observed on the cell membrane, and it was confirmed with an electron microscope in which organelles in the cell are located in more detail. As shown in the electron micrograph of MSC, it was confirmed that many were located on the cell membrane as in the cell Prussian blue stain and were also observed in the autophagosome.

<110> KAEL-GEMVAX CO., LTD. KIM, Sangjae <120> Conjugate of telomerase derived peptide and contrast substances for detecting stem cell and Contrast composition comprising the same <130> 13P552/IND <150> KR10-2012-109207 <151> 2012-09-28 <160> 2 <170> PatentIn version 3.2 <210> 1 <211> 16 <212> PRT <213> Homo sapiens <400> 1 Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys 1 5 10 15 <210> 2 <211> 1132 <212> PRT <213> Homo sapiens <400> 2 Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser 1 5 10 15 His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly 20 25 30 Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg 35 40 45 Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro 50 55 60 Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu 65 70 75 80 Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val 85 90 95 Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro 100 105 110 Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr 115 120 125 Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 130 135 140 Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val 145 150 155 160 Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr 165 170 175 Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly 180 185 190 Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg 195 200 205 Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg 210 215 220 Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg 225 230 235 240 Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp 245 250 255 Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val 260 265 270 Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala 275 280 285 Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His 290 295 300 Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro 305 310 315 320 Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly 325 330 335 Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro 340 345 350 Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser 355 360 365 Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln 370 375 380 Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His 385 390 395 400 Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg 405 410 415 Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln 420 425 430 Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu 435 440 445 Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe 450 455 460 Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser 465 470 475 480 Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser 485 490 495 Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met 500 505 510 Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys 515 520 525 Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe 530 535 540 Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe 545 550 555 560 Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr 565 570 575 Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His 580 585 590 Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln 595 600 605 His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile 610 615 620 Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val 625 630 635 640 Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser 645 650 655 Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg 660 665 670 Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg 675 680 685 Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro 690 695 700 Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile 705 710 715 720 Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln 725 730 735 Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His 740 745 750 Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp 755 760 765 Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser 770 775 780 Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu 785 790 795 800 Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His 805 810 815 Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro 820 825 830 Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp 835 840 845 Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu 850 855 860 Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala 865 870 875 880 Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys 885 890 895 Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu 900 905 910 Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe 915 920 925 Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser 930 935 940 Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe 945 950 955 960 Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly 965 970 975 Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn 980 985 990 Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln 995 1000 1005 Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln 1010 1015 1020 Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala 1025 1030 1035 1040 Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu 1045 1050 1055 Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gln Trp 1060 1065 1070 Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr 1075 1080 1085 Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser 1090 1095 1100 Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala Ala Ala Asn 1105 1110 1115 1120 Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp 1125 1130

Claims (9)

As a conjugate of a cell penetrating peptide and a contrast material,
The cell penetrating peptide,
It is a peptide consisting of SEQ ID NO: 1,
Conjugate for detection of stem cells.
delete The method of claim 1,
The conjugate is selected from the group consisting of a radiopaque contrast agent, a paramagnetic contrast material, a superparamagnetic contrast material, and a CT contrast material.
The conjugate of claim 1, wherein the contrast material is an iron-based material.
The conjugate of claim 4, wherein the contrast material is ferrocene carboxylate.
The conjugate for detection of stem cells according to claim 1, wherein the peptide and the contrast material are conjugated by covalent bonds.
The conjugate of claim 1, wherein the peptide and the contrast material are conjugated by a non-covalent bond.
The conjugate for detection of stem cells according to claim 1, wherein the stem cells for detection are neural stem cells or mesenchymal stem cells.
A contrast agent for detection of stem cells comprising the conjugate according to any one of claims 1 and 3 to 8.

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