KR20140042747A - Conjugate of telomerase derived peptide and contrast substances for detecting stem cell and contrast composition comprising the same - Google Patents

Abstract

The present specification relates to a conjugate of a telomerase derived peptide and a contrast substance for detecting a stem cell and a contrast medium comprising the same for detecting the stem cell. A peptide having a sequence of SEQ ID NO. 1, a peptide which is a fragment of the sequence of SEQ ID NO. 1, or a peptide having 80% or more sequence homology with the peptide sequence, according to the present invention, is an excellent cell permeation peptide which can be used as a contrast medium for detecting a stem cell by combining with a contrast substance. In particular, the contrast medium conjugate according to the present invention can detect a stem cell without affecting primary actions of the stem cell.

Description

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

Disclosed herein is a conjugate of a telomerase-derived peptide and contrast agent for stem cell detection and a contrast agent for stem cell detection comprising the same.

Telomere is a genetic material that is repeatedly present at the end of a chromosome. It is known to prevent damage to the chromosome and its binding to other chromosomes. As the cell divides, the length of the telomere is getting shorter. When there is more than a certain number of cell divisions, the telomere becomes very short, and the cell stops dividing and dies. On the other hand, it is known that lengthening the telomeres prolongs the life of the cells. For example, it is known that the cancer cells secrete an enzyme called telomerase and prevent the shortening of the telomeres.

Stem cells refer to pluripotent cells, ie cells that can develop into any tissue. Injecting stem cells into living organisms continues to report that stem cells can migrate to damaged or diseased areas of the body and differentiate into various cell types, from cardiomyocytes, nerve cells, or skeletal muscle, to produce therapeutic effects. Is being announced. In research experiments related to the treatment using such stem cells, it is necessary to monitor whether the stem cells migrate to the site of injury or onset. In addition, in order to monitor the treatment of patients using stem cells, it is necessary to follow the movement of stem cells in vivo. However, little is known about tracking stem cells injected in vivo.

In addition, stem cell research is very active in various countries around the world, and the related business market is continuously growing, and it can be usefully applied to treat numerous intractable diseases. Tracing is not universally accomplished and the rapid progress of research is not achieved.

Korean Patent Publication No. 10-2007-0024206 (2007.03.02.) European Patent Publication No. 1352597 (2003.11.19) European Patent Publication No. 0841396 (1998.05.13.)

Thus, the present inventors have completed the present invention as a result of intensive efforts to develop a contrast agent for detecting stem cells.

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

One aspect of the present invention is to provide a contrast agent that can penetrate into stem cells.

One aspect of the present invention is to provide a contrast agent capable of stem cell contrast.

One aspect of the present invention is to provide a stem cell contrast medium with fewer side effects to the human body.

One aspect of the invention is a conjugate of a cell penetrating peptide and a contrast material, the cell penetrating peptide is a peptide having a 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 Peptides having at least% sequence homology and provide a conjugate for detection of stem cells.

In the conjugate according to one aspect of the invention, the peptide may be composed of up to 30 amino acids.

In the conjugate according to one aspect of the invention, the contrast material is from a 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 one aspect of the invention, the contrast material may be an iron-based material.

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

In the conjugate according to one aspect of the invention, the peptide and the contrast material may be conjugated by covalent bonds.

In the conjugate according to one aspect of the invention, the peptide and the contrast material may be conjugated by non-covalent bonds.

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

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

Peptides having the sequence of SEQ ID NO: 1, peptides which are fragments of the sequence of SEQ ID NO: 1, or peptides having more than 80% sequence homology with the peptide sequence are excellent cell penetrating peptides that bind to the contrast material and serve as contrast agents for stem cell detection. Can be utilized. In particular, the contrast agent conjugate according to the present invention has the advantage that it is possible to detect stem cells without affecting the basic action of stem cells.

Figure 1 is a photograph measuring the state over time after ferrocene carboxylic-pep1 (Ferrocenecarboxylic-pep1) treated with neural stem cells.
FIG. 2 is a photograph of measuring neural stem cell transmission by confocal laser scanning system after conferring ferrocenecarboxylic-pep1 (Ferrocenecarboxylic-pep1) to neural stem cells and staining cell nuclei with DAPI.
Figure 3 is a cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) as a result of toxicity test of Ferrocenecarboxylic-pep1 (Ferrocenecarboxylic-pep1) itself Activity assays were used to show changes in cell viability and cytotoxicity, respectively.
Figure 4 shows the results of the toxicity confirmation through the pro-apoptotic signal for NSC.
5 and 6 show experimental results confirming that cell proliferation of stem cells was not affected by ferrocene carboxylic-pep1.
Figure 7 shows the cell mobility of neural stem cells (NSC) by ferrocene carboxy-pep1.
Figure 8 shows the free radical change of stem cells by ferrocene carboxy-pep1 in NSC.
9 is an MRI photograph of the brain of a group of transplanted neural stem cells treated with Ferrocenecarboxylic-pep1 (Stem cells with Ferrocenecarboxylic-pep1 group), and FIG. 10 is a ferrocene carboxylic-pep1 treated group. MRI photograph of the brain of the stem cells without (Stem cells without Ferrocenecarboxylic-pep1 group) transplanted, Figure 11 is a MRI of the brain of the group (Ferrocenecarboxylic-pep1 group) implanted with ferrocenecarboxylic-pep1 It is a photograph, and FIG. 12 is the MRI photograph of the brain of the group which implanted the saline solution (Saline group).
Figure 13 shows the contents of different cell amounts for each region of the rat for MRI detection of ferrocene carboxylic-pep1.
14 shows MRI photographs taken up to 5 weeks.
FIG. 15 shows the result of Prussian blue staining by dividing a portion corresponding to a control portion and a portion where Fe is detected in the MRI tissue. FIG.
Figure 16 is a photograph of the NSC confirmed by ferrocene carboxylic-pep1 on the cell membrane in which organelles in the cell in more detail it is located.
Figure 17 shows the result of measuring the pro-apoptotic signal by Western blot and FACS in mesenchymal stem cells (MSC).
FIG. 18 and FIG. 19 show measurement results of pAkt and BrdU, which are cell survival signals, indicating whether there is a change in proliferation of stem cells by ferrocene carboxy-pep1 in MSC.
Figure 20 shows the cell mobility of mesenchymal stem cells (MSC) by ferrocene carboxylic-pep1.
Figure 21 shows the change of free radicals of stem cells by ferrocene carboxylic-pep1 in MSC.
FIG. 22 is a photograph of MSC + Fe observed with Prussian blue stain in in-vitro as shown in In-vivo.
FIG. 23 is a photograph confirming that MSCs were located in a large number of cell membranes and were observed in an autophagosome as in the case of cell Prussian blue stain through electron microscopy.

Proteins, nucleic acids, peptides, or viruses have great potential as therapeutic substances, but because of their molecular size, they have not been able to penetrate tissue and cell membranes, and their use has been very limited. In addition, even small-sized substances do not penetrate the lipid bilayer of the cell membrane due to their properties and structure. Thus, there have been attempts to move proteins, nucleic acids, peptides or viruses into cells by electroporation or heatshock, but do not damage cell membranes and at the same time maintain their activity. It was difficult to move these substances into cells. Meanwhile, TAT (Trans-Activating Transcriptional activator) protein derived from Human Immuno-deficiency Virus (HIV) can act as a Cell Penetrating Peptide that can transfer large active substances into cells. As it became known, research on this was actively conducted. In particular, unlike TAT proteins, which are known to cause intracellular toxicity, studies have been conducted on substances capable of more effectively transferring large molecules 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 peptides derived from telomerase have excellent effects as cell penetrating peptides with little cytotoxicity.

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

Peptides disclosed herein can include peptides having a sequence of SEQ ID NO: 1 or a peptide that is a fragment of sequence of SEQ ID NO: 1 and a peptide having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology. Can be.

Peptides disclosed herein can be wild-type peptides identified and isolated from a natural source. On the other hand, a peptide disclosed herein may be an artificial variant, comprising an amino acid sequence in which one or more amino acids are substituted, deleted, and / or inserted as compared to a peptide that is SEQ ID NO: 1 or a fragment thereof. Amino acid changes in the wild type polypeptide 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). Amino acid substitutions that generally do not alter 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 invention, amino acid changes belong to the property that allows the physicochemical properties of the peptide to be altered. For example, amino acid changes can be made, such as improving the thermal stability of the peptide, altering substrate specificity, changing the optimal pH, and the like.

In one aspect of the invention, the cell penetrating peptide may consist of up to 30 amino acids.

In one aspect of the invention, cell penetrating peptides include telomerases, specifically peptides derived from human ( Homo sapiens ) telomerase.

The peptide described in SEQ ID NO: 1 is shown in Table 1 below. The "name" in Table 1 below is to distinguish peptides. 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 a sequence of SEQ ID NO: 1, a peptide of a sequence of SEQ ID NO: 1, or a peptide having a sequence homology of 80% or more with the above peptide sequence, And "synthetic peptide" synthesized by selecting peptides at positions. SEQ ID 2 shows the amino acid sequence of the entire telomerase.

[Table 1]

As used herein, "Cell Penetrating Peptide (CPP)" is in vitro and / or in It means a peptide capable of moving a cargo into cells in vivo .

As used herein, the term "contrast material" is a broad concept encompassing all materials used for the imaging of structures or fluids in vivo in medical imaging. Suitable contrast agents include, but are not limited to, radiopaque contrast agents, paramagnetic contrast agents, superparamagnetic contrast agents, computed tomography (CT) contrast agents, and other contrast agents. Does not. For example, radiopaque contrast materials (for X-ray imaging) include inorganic iodine compounds and organic iodine compounds (e.g., diatrizoat), radiopaque metals and salts thereof (e.g., silver, gold, platinum Etc.) and other radiopaque compounds (eg, calcium salts, barium salts such as barium sulfate, tantalum and tantalum oxide). Suitable paramagnetic contrasting materials (for MR imaging) are gadolinium diethylene triaminepentaacetic acid (Gd-DTPA) and derivatives thereof, and other gadolinium, manganese, iron, dysprosium, copper, euros 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 materials (for MR imaging) include magnetite, super-paramagnetic iron oxide (SPIO), ultrasmall superparamagnetic iron oxide (USPIO), and monocrystalline iron oxide. do. Other suitable contrast agents are iodinated and non-iodinated, ionic and nonionic CT contrast agents, and contrast agents such as spin-labels, or other diagnostically effective agents.

Other examples of contrast agents include marker genes encoding proteins that are readily detectable when expressed in cells, including but not limited to β-galactosidase, green fluorescent protein, blue fluorescent protein, luciferase, and the like. Include. Various labels may be used, such as radionuclides, fluorescents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (especially hapten), and the like.

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

[Chemical Formula 1]

(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 formula (3).

(3)

In one aspect of the invention, it is possible to directly bind a cargo, ie, contrast material, to the peptide. In another aspect of the present invention, a moving object can be bound to a peptide through various binding methods, such as covalent or non-covalent bonds. For example, disulfide bonds or covalent bonds that bind the target of movement to the peptide N-terminus or peptide C-terminus, specifically the alpha amine or C- of the peptide N-terminus glutamate (E). The migration target can be linked to the peptide via a covalent bond that binds the migration target to the amine of the terminal lysine (K) residue. Alternatively, one of the peptides and the moving object may be bound to the peptide and the moving object through a non-covalent bond that encloses the other in a form such as a capsule form.

In another aspect of the invention, the linker can be linked to the peptide and the target of movement through a linker. For example, Hynic (6-hydrazinopyridine-3-carboxylic acid) is applied to the peptide N-terminus or peptide C-terminus, specifically to the amine of the alpha-amine or C-terminal lysine residue of peptide N-terminal glutamate. After the introduction of a linker such as a 6-hydrazinopyridine-3-carboxylic acid) linker, the linker may be bound to the peptide by binding the linker to the linker.

In one aspect of the invention, the cell penetrating peptide may bind to a dyeing or fluorescent material, in particular fluorescein isothiocyanate (FITC). One of fluorescein isothiocyanate in terms of the present invention can be combined with the N- terminus or the amino group (NH ₃ ⁺⁾ in the C- terminal Lys of the cell permeable peptide. In the case of a peptide having no Lys at the terminal, the peptide and the FITC may be coupled through a linker including Lys.

In one aspect of the invention, the cells include all cells used herein as cell therapy. For example, stem cells, islet cells, dendritic cells, immune cells, and the like, but are not limited thereto. In particular, the cell may be a stem cell.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to experimental examples. However, the following experimental examples are provided only for the purpose of illustration in order to help the understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

EXAMPLE 1 Permeation Experiment of Neural Stem Cells of Ferrocenecarboxylic-pep1 Conjugate

(One) Ferrocene carboxylic ( Ferrocenecarboxylic ) - pep1 Conjugate  Manufacture and Experiment Method

A. Ferrocene carboxylic ( Ferrocenecarboxylic ) - pep1 Conjugate  Produce

Amino acids (8 equivalents) and coupling reagents HBTU (8 equivalents) / HOBt (8 equivalents) / NMM (16 equivalents) protected in NH ₂ -Lys (Dde) -2-chloro-trityl Resin for coupling were DMF. After dissolving in and adding, the mixture was reacted at room temperature for 2 hours, and washed in the order of DMF, MeOH, and DMF. Then, piperidine in DMF in 20% DMF was added for Fmoc deprotection, reacted twice at room temperature for 5 minutes, and washed in the order of DMF, MeOH, and DMF.

The reaction was performed repeatedly to peptide base skeleton (NH2-E (OtBu) -AR (Pbf) -PALLT (tBu) -S (tBu) -R (Pbf) LR (Pbf) -FIPK (Dde) -2-chloro -Trityl Resin). It was treated with hydrazine (hydrazine in DMF) in 2% DMF to remove Dde, the residue protecting group of C-terminal Lys. The coupling reagent HBTU (16 equiv.) / HOBt (16 equiv.) / NMM (32 equiv.) Was dissolved in DMF and added to the reaction mixture, For 2 hours and washed with DMF, MeOH and DMF in that order. Peptide was isolated from Resin by adding TFA / TIS / H 2 O = 95 / 2.5 / 2.5 to the synthesized peptide Resin. Cooling diethyl ether was added to the obtained mixture, followed by centrifugation to precipitate the obtained peptide. It was purified by HPLC, then confirmed by MS and lyophilized.

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 of cell treatment. 5 × 10 ⁶ cells were washed twice with cold PBS solution and then 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; Cultured on ice, unbroken cells and nuclei were removed by centrifugation (2000 xg) for 10 minutes, and lysates were removed by centrifugation (10,000 xg). To assess cytoplasmic HMGB-1 and cytosolic cytochrome c levels, mitochondrial and cytosolic fractions were isolated using mitochondrial / cytoplasmic kits (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, washed with cold PBS solution, and then mixed with 1.0 mL of 1 × cytoplasmic extraction buffer containing dithiothreitol (DTT) and protease inhibitors. Resuspended in. After incubation on ice for 10 minutes, the cell suspension was homogenized with a syringe 30 to 50 times. Samples were 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 fractions (pellets) and cytoplasmic fractions (supernatants). Mitochondrial pellets were washed once with isolation buffer and then dissolved using mitochondrial extraction buffer containing DTT and protease inhibitors. 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 incubated with specific basic antibodies.

Antibodies used were 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 bound rabbit antibody or anti-mouse antibody (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and then ECL detection (Amersham). Pharmacia Biotech). Blots were quantified with an image analyzer (GE Healthcare, ImageQuant LAS 4000).

C. BrdU Cell Proliferation Assay

Immediately upon exposure to ferrocenecarboxylic-PEP1 for 24 hours, MSCs were collected. MSCs were then incubated 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 492 nm reference wavelength and 370 nm wavelength, and all results were normalized to OD of the same well without cells.

D. Colony Forming Unit (CFU) Analysis

Neural stem cells were divided to 0.25 × 10 ⁵ cells per 6 wells, and then cells were exposed to 10 μM ferrocenecarboxylic-PEP1 for 24 hours. Cells were washed with DPBS and then medium replaced. After 14 days, the cells were washed again with DPBS and stained with .5% crystal violet (Sigma) in methanol at room temperature for 30 minutes. After cell staining, the plates were washed with DPBS and allowed to dry. Colony counts were performed using an anatomical microscope. Cells less faintly stained or less than 2 nm in diameter were excluded.

E. Move Migration ) Measure

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

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

F. Production Determination Test of Free Radicals

To measure the production of free radicals, 24-hour NSCs and MSCs were exposed to fluorescent probe 2 ', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) for 15 minutes after exposure to Ferrocenecarboxylic-GV1001 (Molecular Probes Inc., Eugene, OR, USA). ) And incubation. Subsequently, 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 crosses the cell membrane, hydrolyzed to 2 ', 7'-dichlorodihydrofluorescein (DCFH2) by cellular esterases, and then oxidized in the presence of peroxides with fluorescent 2'7'-dichlorofluorescein (DCF). This means that DCF fluorescence mainly represents the level of hydrogen peroxide in the cell rather than peroxide. Accumulation of intracellular DCF was observed using an Olympus fluorescence microscope at an excitation wavelength appropriate for ROS staining, which is the subject of flow cytometry analysis (FACS C6, BD).

(2) Neural Stem Cell Permeation Experiment

In the experiment, rat (Sprague-Dawley, SD) (Orient bio, Kyungki, Korea) cerebral cortex was isolated from embryonic head on the 13th day of pregnancy and poly-L-ornithine / fibronectin in Ca ²⁺ / Mg ²⁺ -free PBS ( Dispense 2 x 10 ⁴ cells / cm ² onto a plate coated with GIBCO, Grand Island, NY, USA, N2 medium (DMEM / F12, 4.4 IM insulin, 100 mg / l transferrin, 30 nM selenite, 0.6 IM fu) 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) was treated with 10 ng / ml to obtain more than 95% neural stem cells after 4-6 days at 37 ℃, 5% CO ₂ environment.

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

The results are shown in FIGS. 1 and 2.

Figure 1 is a photograph measuring the state over time after ferrocene carboxylic-pep1 (Ferrocenecarboxylic-pep1) treated with neural stem cells.

As shown in Figure 1, after the treatment of the ferrocene carboxylic-pep1 to the neural stem cells comparative analysis was confirmed that the ferrocene carboxylic-pep1 at a concentration of 10μM penetrated into the cell from 2 hours and more over time It was confirmed that a large amount penetrated into the cells.

FIG. 2 is a photograph of measuring neural stem cell transmission by confocal laser scanning system after conferring ferrocenecarboxylic-pep1 (Ferrocenecarboxylic-pep1) to neural stem cells and staining cell nuclei with DAPI. In FIG. 4, the portion that appears blue at the wavelength of 430-500 nm is the nucleus stained with DAPI, and the portion that appears red at the wavelength of 620-700 nm is ferrocene carboxylic-pep1. As shown in the integrated picture of the two wavelengths, the ferrocenecarboxylic-pep1 gathers around the nucleus of the cell, indicating that the ferrocenecarboxylic-pep1 has penetrated into the cell and migrated into the cytoplasm.

(3) Toxicity Test

A. Toxicity of ferrocenecarboxylic-pep1 itself

For toxicological experiments of pep1 itself conjugated with ferrocenecarboxylic acid, neural stem cells were dispensed at 4x10 ⁴ and 2.5x10 ⁵ into 96-well and 24-well plates, respectively, for at least 12 hours in a 37 ° C, 5% CO ₂ incubator. Incubated. Ferrocenecarboxylic-pep1 (Ferrocenecarboxylic-pep1) was treated at concentrations of 0, 0.01, 0.1, 1, 10, and 100 μM, respectively, and incubated in the incubator for up to 24 hours, followed by cell counting kit-8 (CCK-). 8) Assays and lactate dehydrogenase (LDH) activity assays were used to determine cell viability and cytotoxicity, respectively. The results are shown in FIG.

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

B. Toxicity Identification by Pro-apoptotic Signal

PEP1 has already been confirmed to be nontoxic in neurons and other cells through various experiments.However, it is necessary to evaluate whether new cytotoxicity is caused by the ferrocenecarboxy conjugation.The pro-apoptotic signal is measured by western blot and FACS. It was.

The results of the toxicity confirmation through pro-apoptotic signal for NSC are shown in FIG. 4. As shown in FIG. 4, it was confirmed that no special change occurred in pro-apoptotic signals, and it was confirmed that Fe-PEP1 did not exhibit toxicity.

(4) Ferrocene carboxylic - pep1 of NSC Check for impact on

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

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

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 through Figure 7, it can be seen that there was no change in stem cell migration by ferrocene carboxylic-pep1.

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

Oxidative stress can damage all cells. If the ROS increases, there will be a problem to use as a stem cell contrast medium. DCF-DA staining samples were used to observe the change in the generation of free radicals after ferrocene-carboxyl-pep1 treatment. As can be seen from FIG. 8, no change in free radicals of NSC was observed.

(5) MRI  Through shooting NSC of Detectability  Confirm

A. Ferrocene carboxylic - pep1 Labeled  Preparation of Neural Stem Cells

The neural stem cells obtained in (2) above were dispensed at 5 × 10 ⁶ into 100 mm dishes. Ferrocenecarboxylic-pep1 was treated with 10 μM for 24 hours in a 37 ° C., 5% CO ₂ incubator, and then cells were separated from the plate with TrypLE (GIBCO), washed with free media, and counted. Rick-pep1 labeled neural stem cells were prepared 10 l 3x10 ⁵ .

B. Ferrocene carboxylic - pep1  Treated neural stem cells ( NSC Transplantation

All animal experiments described in this specification were conducted after obtaining the approval of Hanyang University IACUC. 8 weeks old SD rats were purchased for 1 week, and then rats weighing about 290 g +/- 15 g were used for this experiment. Animals were divided into 4 groups and were tested with NSCs with Ferrocenecarboxylic-pep1 group, NSCs without Ferrocenecarboxylic-pep1 group, Ferrocenecarboxylic-pep1 group, and saline group (Saline group). When cells, ferrocene-carboxyl-pep1 or saline were implanted in each group, they were implanted into the brain of SD rats using stereotaxic surgery. Anesthesia was performed using ketamine and rompun before transplantation, and the skull was partially removed after shaving of the scalp. After that, the implants were stereotaxic at coordinates AP = +0.7, R = +2, and V = -5.5.

C. MRI  shooting

MRI was used for in vivo observation of transplanted NSCs and ferrocenecarboxylic-pep1. MRI was performed 3 years after transplantation and was taken using Philips Best 3T MRI equipment. Anesthesia also used ketamine and rompun, and the multiplanar gradient-recalled (MPGR) pulse sequence (TR = 596 ms, TE = 16 ms, section thickness = 0.7 mm, phosphorus) In-plane resolution: photographed using 292 × 290 μm (pixel size 0.0593 mm ³ ), and number of acquisitions = 1.

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

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

MRI was used for in vivo observation of transplanted NSCs and ferrocenecarboxylic-pep1. MRI was performed three years after transplantation and was taken using Philips Best 3T MRI equipment. Anesthesia was also used with ketamin and rompun, 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). FIG. 13 shows the contents of different cell amounts of rats for MRI detection of ferrocenecarboxylic-pep1. 14 shows MRI photographs taken up to 5 weeks. For verification, the detection point was sectioned to confirm that the correct Fe was detected by Prussian blue staining. FIG. 15 shows a picture of reconfirming fe detection because the portion corresponding to the control portion and the Fe detected portion were stained with Prussian blue stain in the MRI tissue, and the portion corresponding to Fe was blue. As confirmed by in-vivo, in-vitro was observed with Prussian blue stain.

In FIG. 16, ferrocene carboxy-pep1 was observed on the cell membrane as shown in the photograph, and it was confirmed by electron microscopy which organelle was located in the cell in more detail. As shown in the NSCs electron micrographs, the cell membranes were located on the cell membrane as well as the Prussian blue stains.

Example 2 Permeation of Ferrocenecarboxylic-pep1 Conjugate in Mesenchymal Stem Cells

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

Ferrocenecarboxylic-pep1 conjugates were prepared and prepared according to the method described in Example 2 above.

(2) Toxicity Test

The results of measuring the pro-apoptotic signal in mesenchymal stem cells (MSC) by Western blot and FACS are shown in FIG. 17. As shown in FIG. 17, it was confirmed that no special change occurred in pro-apoptotic signals, and it was confirmed that Fe-PEP1 did not exhibit toxicity.

(3) Ferrocene carboxylic - pep1 of MSC Check for impact on

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

The proliferation of stem cells induced by ferrocene carboxyl-pep1 was measured by the cell survival signals pAkt and BrdU. 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. Results for mobility confirmation are shown in FIG. 20.

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

Oxidative stress can damage all cells. If the ROS increases, there will be a problem to use as a stem cell contrast medium. DCF-DA staining samples were used to observe the change in the generation of free radicals after ferrocene-carboxyl-pep1 treatment. As can be seen from FIG. 21, no change in free radicals was observed for MSC.

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

MSCs were prepared by the method as described in Example 1, and confirmed using MRI for in vivo observation of transplanted MSCs and ferrocenecarboxylic-pep1. MSC + Fe was observed with Prussian blue stain in in-vitro as shown in In-vivo and shown in FIG. 22. In FIG. 23, as shown in the photograph, ferrocene-carboxyl-pep1 was observed on the cell membrane, and it was confirmed by electron microscopy which organelle was located in the cell in more detail. As shown in the electron micrographs of MSC, the cell membrane was located in the cell membrane as well as the Prussian blue stain, and autophagosome was observed.

<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 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 Ser 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 Valle 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 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 cell penetrating peptides and contrast materials,
The cell penetrating peptide,
A peptide having a sequence of SEQ ID NO: 1, a peptide that is a fragment of the sequence of SEQ ID NO: 1, or a peptide having at least 80% sequence homology with the peptide sequence,
Conjugate for Detection of Stem Cells.
The method according to claim 1,
The peptide is a conjugate for the detection of stem cells consisting of up to 30 amino acids.
The method of claim 1,
Wherein the contrast material is selected from the group consisting of radiopaque contrast agents, paramagnetic contrast materials, superparamagnetic contrast materials, and CT contrast materials.
The conjugate of claim 1, wherein the contrast material is an iron-based material.
The conjugate of claim 4, wherein the contrast agent is ferrocene carboxylate.
The conjugate of 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 agent are for detecting the stem cells conjugated by non-covalent bonds.
The conjugate of claim 1, wherein the stem cells for detection are neural stem cells or mesenchymal stem cells.
Contrast agent for the detection of stem cells comprising the conjugate according to any one of claims 1 to 8.

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