KR20180099205A - Hydrogel composition for carbon monoxide release and preparation method thereof - Google Patents

Abstract

The present invention relates to a hydrogel composition for releasing carbon monoxide comprising a gellant and a co-magnetic assembly of a carbon monoxide releasing molecule bonded to a peptide, and a method for producing the same.
The synthesized carbon monoxide releasing hydrogel (CORH) according to the present invention has excellent mechanical properties and exhibits an excellent effect against damage caused by oxidative stress and has a characteristic of being slowly released in the body, It is possible to obtain the effect of reducing the cell protection and cell death, and thus it is expected to be widely used as a therapeutic agent.

Description

[0001] The present invention relates to a hydrogel composition for carbon monoxide release,

The present invention relates to a hydrogel composition for releasing carbon monoxide comprising a gellant and a co-magnetic assembly of a carbon monoxide releasing molecule bonded to a peptide, and a method for producing the same.

Carbon monoxide is well known as a silent killer because of its strong affinity for hemoglobin, which is responsible for oxygen transfer from the blood. However, carbon monoxide is also recognized as an essential biological signaling molecule. Carbon monoxide was produced as one of the byproducts of hemeification through the heme oxygenase-1 (HO-1) enzyme. Carbon monoxide plays a protective role in tissues, for example, anti-inflammatory, anti-proliferative and anti-apoptotic activities, Based on the effectiveness of the treatment can be attractive for therapeutic purposes.

However, carbon monoxide has a problem that handling and delivery are not easy due to toxicity at a high concentration. Thus, the importance of developing a CO-releasing molecule (CORM) has been raised. Among the carbon monoxide releasing molecules, CORM3 has excellent solubility in water and carbon monoxide emission due to ligand substitution. Nonetheless, small-sized drugs such as carbon monoxide-releasing molecules rapidly spread after being injected into the body, resulting in a poor therapeutic effect and a risk of side effects to the tissue.

In order to solve the above problems, researchers have suggested that a carbon monoxide emitter containing polymers, peptides, and proteins provides carbon monoxide transport control capability, so that the carbon monoxide emitter enables local carbon monoxide transport, In the form of an injection.

U.S. Published Patent Application No. 2016/0008291 A1 (Jan. 14, 2014).

 Silvia Cavalli et al. Chem. Soc. Rev. 2010, 39: 241-263.  John B. Matson et al., Soft Matter. 2012, 8 (25): 2689-2692.

In order to solve the above problems, the present invention intends to effectively transfer carbon monoxide through a self-assembling peptide-based hydrogel. Peptide-based hydrogels have potential in biomedical fields such as cell culture, drug delivery and regenerative medicine. Synthetic peptides with 20 amino acids have advantages such as simplicity of synthesis, bioactivity, biocompatibility and biodegradability, but peptide-based hydrogels usually have mechanical properties due to self-assembly behavior due to weak intermolecular noncovalent interactions There are disadvantages that are not good.

In order to solve this problem, the present invention aims to form an ordered nanostructure having improved mechanical properties through a π-π stack or a non-covalent interaction of short-length aromatic peptides. In order to prepare a self-assembled hydrogel, a method of using a nanofiber incorporating a sequence for forming a β-sheet in a peptide was approached.

The present invention relates to a hydrogel composition for releasing carbon monoxide comprising a hydrogelator and a co-magnetic assembly of a carbon monoxide releasing molecule bonded to a peptide represented by the following formula (I).

(I)

Phe-Phe-X-Asp

(In the above peptide,

X is a water-soluble peptide composed of 2 to 6 water-soluble amino acids.)

Also,

Synthesizing a peptide represented by the following formula (I);

(I)

Phe-Phe-X-Asp

(In the above peptide,

X is a water-soluble peptide composed of 2 to 6 water-soluble amino acids.)

Reacting the synthesized peptide with a carbon monoxide releasing molecule (CORM); And

Mixing the hydrogeling agent;

To a method for producing a hydrogel composition for carbon monoxide release.

The present invention also relates to an injection comprising the hydrogel composition for carbon monoxide release.

The synthesized carbon monoxide releasing hydrogel (CORH) according to the present invention has excellent mechanical properties and exhibits an excellent effect against damage caused by oxidative stress and has a characteristic of being slowly released in the body, It is possible to obtain the effect of protecting the cells and decreasing the cell death, and thus it is expected to be widely used as a therapeutic agent.

1 is a) amino acid sequence of peptide P2 according to Example 2 b) HPLC spectrum and c) MALDI-TOF / TOF mass spectrometry results.
FIG. 2 is a reaction formula a) of the peptide-carbon monoxide releasing molecule conjugate P2-CORM according to Example 3 and b) a result of MALDI-TOF / TOF mass spectrometry.
3 is an FT-IR spectrum of the peptide-carbon monoxide releasing molecule conjugate P2-CORM according to Example 3 under acidic and basic conditions.
4 is a carbon monoxide emission dynamic graph of a hydrogel (CORH) which is a co-magnetic assembly of a carbon monoxide releasing molecular conjugate (P2-CORM) bonded with a hydrogelating agent and a peptide according to Example 4. Fig.
5 is an FT-IR spectrum of a hydrogel (CORH) which is a co-magnetic assembly of a carbon monoxide releasing molecule conjugate (X-CORM) bonded with a hydrogelling agent and a peptide according to Example 4. FIG.
Figure 6 shows a) strain sweep of a hydrogel (CORH) which is a co-magnetic assembly of a carbon monoxide releasing molecule conjugate (P2-CORM) bonded with a hydrogelator and peptide according to Test Example 4 b) a frequency sweep ).
7 is a TEM image of negative staining with 2 wt% uranyl acetate of a hydrogel (CORH) which is a co-magnetic assembly of a carbon monoxide releasing molecule conjugate (P2-CORM) bonded with a hydrogelating agent and a peptide according to Test Example 4. Fig.
FIG. 8 is a graph of 2D ¹ H-NMR (NOESY) at 600 MHz D ₂ O of a hydrogel (CORH) which is a co-magnetic assembly of a carbon monoxide releasing molecular conjugate (P2-CORM) bonded with a hydrogelating agent and peptide according to Test Example 4. [ Results.
FIG. 9 shows the effect of hydrogel (CORH) which is a co-magnetic assembly of a carbon monoxide releasing molecule conjugate (P2-CORM) bound to a hydrogelating agent and a peptide according to Test Example 5 and a control group with the hydrogen peroxide treatment.
FIG. 10 is a graph showing the results of a comparison between a hydrogel (CORH) which is a co-magnetic assembly of a carbon monoxide releasing molecule conjugate (P2-CORM) bound to a hydrogelating agent and a peptide according to Test Example 5 and a reactive oxygen species (ROS) by hydrogen peroxide treatment in H9c2 cells of a control group. The effect on the generation is shown.

The present invention relates to a hydrogel composition for releasing carbon monoxide comprising a hydrogelator and a co-magnetic assembly of a carbon monoxide releasing molecule bonded to a peptide represented by the following formula (I).

(I)

Phe-Phe-X-Asp

(In the above peptide,

X is a water-soluble peptide composed of 2 to 6 water-soluble amino acids.)

In the present invention, the water-soluble peptide is not particularly limited, but may be selected from the group consisting of serine, threonine, tyrosine, cystine, asparagine, glutamine, lysine, arginine ), Histidine (His), aspartic acid (Asp) and glutamic acid (Glu), and preferably glutamic acid (Glu), serine , And more preferably, when glutamic acid (Glu) -glutamic acid (Glu) -lysine (Lys) is used, water solubility is effectively increased.

In the present invention, the hydrogelating agent is not particularly limited but may be selected from the group consisting of fluorenylmethoxycarbonyl peptides (Fmoc-peptides), substances having an amphipathic low molecular skeleton, substances having a skeleton , And a material having a semi-conjugated low-molecular structure as a skeleton. Among them, fluorenylmethoxycarbonyl peptides (Fmoc-peptides) may be used. Most preferably, Fmoc-FF or Fmoc-PhePhe having the structure of the following formula (II) is used.

≪ RTI ID = 0.0 &

Examples of the material having an amphipathic low molecular skeleton include a surfactant-type gelling agent having a quaternary ammonium salt moiety as a hydrophilic moiety and an alkyl long chain as a hydrophobic moiety modeled on an artificial lipid membrane, a surfactant-type gelling agent having two surfactant- Type gelling agents. As an example of the hydrogel by such a gelling agent, a molecular-structured hydrogel formed by adding an anion having a molecular weight of 90 or more to an aqueous dispersion solution of a cationic amphipathic compound having a branched alkyl group in a hydrophobic portion can be used.

Examples of the substance having a skeleton with an in-vivo component as an motive include a gelling agent using association between molecular aggregates by a peptide secondary structure skeleton (? -Helix structure,? -Sheet structure, etc.).

In addition, the substance having a backbone small molecule as a backbone is composed of a combination of in vivo components (hydrophilic part) such as DNA base, peptide chain, and sugar chain, and an alkyl chain (hydrophobic part), and the two gelling agents Gelling agent that combines the characteristics of the gelling agent. Here, DNA bases, peptide chains and sugar chains not only increase the hydrophilicity but also play a role in imparting intermolecular interactions such as hydrogen bonding. For example, a hydrogelating agent comprising a glycoside amino acid derivative having a sugar structure moiety having a glycoside structure of an N-acetylated monosaccharide or a disaccharide, a peptide lipid represented by the general formula &quot; RCO (NHCH ₂ CO) _m OH &quot; And fine hollow fibers formed by self-aggregation from metals. In addition, an amphipathic peptide having a structure of a <fractional-cysteine residue (disulfide bond formation at network formation) -glycerin residue (impart flexibility) -phosphorylated serine residue-cell adhesive peptide> A fiber network can be used, and a glycolipid type supramolecular hydrogel can be prepared and used using a chemical library.

In the present invention, the structure of the carbon monoxide releasing molecule (CORM) is not particularly limited, but the use of a compound represented by the following formula (III) can increase the solubility in water and promote the release of carbon monoxide through ligand substitution .

(III)

Ru (CO) ₃ Cl (glycinate)

The glycinate in the above formula may have the structure of the formula (IV).

(IV)

In the present invention, the hydrogel composition for releasing carbon monoxide is not particularly limited, but may have a solubility in a pH of 2 to 10 and a solubility of 0.065 to 0.085 g in 100 g of distilled water, The solubility may be in the form of 0.072 to 0.080 g per 100 g of distilled water. In this range, administration into living body is easy, and the effect of delaying the release of carbon monoxide when administered in vivo can be appropriately maintained.

In another aspect,

Synthesizing a peptide represented by the following formula (I);

(I)

Phe-Phe-X-Asp

(In the above peptide,

X is a water-soluble peptide composed of 2 to 6 water-soluble amino acids.)

Reacting the synthesized peptide with a carbon monoxide releasing molecule (CORM); And

Mixing the hydrogeling agent;

To a method for producing a hydrogel composition for carbon monoxide release.

In the present invention, the water-soluble peptide is not particularly limited, but may be selected from the group consisting of serine, threonine, tyrosine, cystine, asparagine, glutamine, lysine, arginine ), Histidine (His), aspartic acid (Asp) and glutamic acid (Glu), and preferably glutamic acid (Glu), serine , And more preferably, when glutamic acid (Glu) -glutamic acid (Glu) -lysine (Lys) is used, water solubility is effectively increased.

In the present invention, the hydrogelating agent is not particularly limited but may be selected from the group consisting of fluorenylmethoxycarbonyl peptides (Fmoc-peptides), substances having an amphipathic low molecular skeleton, substances having a skeleton , And a material having a semi-conjugated low-molecular structure as a skeleton. Among them, fluorenylmethoxycarbonyl peptides (Fmoc-peptides) may be used. Most preferably, Fmoc-FF or Fmoc-PhePhe having the structure of the following formula (II) is used.

&Lt; RTI ID = 0.0 &

In the present invention, the structure of the carbon monoxide releasing molecule (CORM) is not particularly limited, but the use of a compound represented by the following formula (III) can increase the solubility in water and promote the release of carbon monoxide through ligand substitution .

(III)

Ru (CO) ₃ Cl (glycinate)

The glycinate in the above formula may have the structure of the formula (IV).

(IV)

In the present invention, the step of reacting the synthesized peptide with a carbon monoxide releasing molecule (CORM) is not particularly limited, but may be carried out at a pH of 2 to 10, preferably at a pH of 3 to 9, more preferably at a pH of 4 To 8, and in this case, the solubility in a water-soluble solvent is increased, so that an effect meeting the purpose of the present invention can be obtained.

In still another aspect, the present invention relates to a therapeutic product comprising the carbon monoxide releasing hydrogel composition. The therapeutic product is not particularly limited, but may be in any form as long as it is a generally accepted form, and may have a form such as an injection, a cutaneous permeable agent, or the like.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. It is to be understood that the embodiments are illustrative of the present invention in more detail, and the scope of the present invention is not limited thereto.

[Example 1] Synthesis of Fmoc-FF (hydrogeling agent)

Fmoc-FF represented by the following formula (II) was synthesized by a solid phase peptide synthesis using a 2-chlorotrityl chloride resin with a microwave.

Fmoc-FF (Fmoc-PhePhe)

First, the resin was swelled in dichloromethane (DCM) for 30 minutes or more in a shaking incubator. Fmoc-Phe-OH (Merck) (2 equiv.) And N, N-diisopropylethylamine (20 mM) in dichloromethane (DCM, Daejung Chemical & The first amino acid was attached to the resin using N, N-Diisopropylethylamine (DIPEA, Merck) (4 equiv.).

The mixture was filtered, washed with dichloromethane and further covered with N, N-diisopropylethylamine: methanol: dichloromethane mixed in a volume ratio of 1: 2: 17 for 30 minutes in a shaking incubator. Fmoc removal was carried out using 20% piperidine solution in dimethylformamide (DMF, Fisher) using microwave. After filtering the solution, the resin was washed with dichloromethane and dimethylformamide. To a solution of N, N-diisopropylethylamine (6 equiv.) In N-methyl-pyrrolidine was added with Fmoc-Phe-OH (3 equiv.) To the peptide coupling HBTU (Sigma-Aldrich) Was mixed with 2-pyrrolidone (NMP, Sigma-Aldrich) solution and microwave-treated for 10 minutes. After filtering the mixture, the resin was washed with N-methyl-2-pyrrolidone and dichloromethane.

Fmoc-FF (Fmoc-PhePhe) was obtained by manually cleaving from resin using 20% hexafluoro-2-propanol (HFIP, Alfa-aesar) in dimethylformamide. After filtering the resin, the crude oily product was precipitated in cold diethyl ether (Et ₂ O) and then centrifuged at 4000 rpm for 5 minutes.

Fmoc-FF (Fmoc-PhePhe) was purified by reverse phase high performance liquid chromatography (SUPPLCO, Discovery Bio Wide Pore C-18, 5 um, 10 x 250 mm). At this time, α-cyano-4-hydroxycinnaic acid was dissolved in water (0.1% trifluoroacetic acid) and acetonitrile (0.1% trifluoroacetic acid) Respectively.

The molecular weight of Fmoc-FF (Fmoc-PhePhe) was confirmed using a MALDI-TOF / TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany).

[Example 2] Water-soluble peptide synthesis

Peptides (amino acid sequence: FFEEKD or PhePheGluGluLysAsp) for binding carbon monoxide releasing molecules were synthesized in Rink Amide MBHA (4-methylbenzhydrylamine) resin (Merck) through conventional solid phase peptide synthesis with microwaves (CEM Focused Microwave System, Discover; CEM Corporation, NC, USA). The resin was washed with dichloromethane and swelled in a 1: 1 mixed solution of dimethylformamide and dichloromethane for 30 minutes or more in a shaking incubator.

Fmoc was removed by irradiating microwave for 3 minutes or more using a 20% piperidine solution under dimethylformamide (DMF), and the resin was thoroughly washed with dichloromethane and dimethylformamide. A solution of N, N-diisopropylethylamine (5 equiv.) In N, N-dimethyl-2-pyrrolidone was added with Fmoc-Lys (mtt) -OH (5 equiv.) With HBTU (Merck) N-methyl-2-pyrrolidone (NMP, Daejung Chemical & Metal Co., Ltd.) solution and microwave treatment for 10 minutes. Fmoc-Glu (OtBu) -OH and Fmoc-Phe-OH (Merck) were then coupled to the N-terminus of the peptide with the FFEEK-residue sequence. The methyltrityl group from the amine of lysine (Lys) was dissolved in 4% trifluoroacetic acid (Sigma-Aldrich) and 4% triisopropylsilane (TIPS, TCI Chemicals Co.) in dichloromethane in a shaking incubator. , Ltd.).

After the addition of Boc-Asp-OtBu (Beadtech) to lysine, the resin was treated with a cleavage solution of trifluoroacetic acid: triisopropylsilane: water mixed in a volume ratio of 95: 2.5: 2.5 in a shaking incubator for at least 3 hours Respectively. The excess of trifluoroacetic acid was removed by rotary evaporation and the crude oily product was precipitated in cold diethyl ether (Et ₂ O) and then centrifuged at 4000 rpm for 5 minutes.

The synthesized peptides were purified by reverse phase high performance liquid chromatography (SUPERCO, Discovery Bio Wide Pore C-18, 5 um, 10 x 250 mm). Water (0.1% trifluoroacetic acid) and acetonitrile (0.1% trifluoroacetic acid) were used as mobile phase solvent. Then, the molecular weight of the synthesized peptide was confirmed using a MALDI-TOF / TOF mass spectrometer (Bruker Daltonik GmbH, Bremen Germany).

The results are shown in Fig.

[Comparative Example 1]

The other procedures were the same except that the amino acid sequence of the peptide in Example 1 was FFKD or PhePheLysAsp.

[Comparative Example 2]

The other procedures were the same except that the amino acid sequence of the peptide in Example 1 was GGIILLK or GlyGlyIleIleLeuLeuLys.

[Comparative Example 3]

The other procedures were the same except that the amino acid sequence of the peptide in Example 1 was FFPETSWD or PhePheProGluThrSerTrpAsp.

[Example 3] Synthesis of peptide-carbon monoxide releasing molecule conjugate (X-CORM)

Tricarbonyl dichloro ruthenium (II) dimer, [Ru (CO) ₃ Cl ₂ ] ₂ , Sigma-Aldrich) was used as the carbon monoxide releasing molecules (CORM) Sodium methoxide (CH ₃ ONa; Sigma-Aldrich) was used together to bind the purified peptides synthesized in Example 2 above.

4.5 mg of the purified peptide was dissolved in 450 uL of hexafluoro-2-propanol and then hexafluoro-2-propanol was removed by vacuum evaporation. Then, 1036 uL of dry methanol and 63.2 uL of 0.5 M sodium methoxide solution were mixed to prepare a suspension. To this suspension was added 2.0 mg of trichlorodichloro ruthenium (II) dimer in the amount of 176.7 uL of dry methanol. To prevent emission of carbon monoxide through the ligand substitution in the CORM, Shaking for 36 hours.

The product was then obtained via vacuum evaporation. The obtained yellow compound was dissolved in 3 mL of 0.05% ammonia water and then lyophilized to obtain pale yellow powder.

The molecular weight of the synthesized peptide-carbon monoxide releasing molecule conjugate (X-CORM) was confirmed using a MALDI-TOF / TOF mass spectrometer as in the above example.

The results are shown in Fig.

Test Example 1 Dissolution Characteristics of Peptide-Carbon Monoxide Release Molecule Conjugate According to Peptide Structure

For the peptides prepared in Comparative Examples 1 to 3, peptide-carbon monoxide releasing molecule conjugates were prepared in the same manner as in Example 3, instead of the peptides prepared in Example 2, and the solubilities of these peptides in 100 g of water at pH 7 Respectively. The solubility was measured by adding a small amount of the peptide-carbon monoxide releasing molecule conjugate prepared in the above-mentioned manner to water, and at the point of time when the precipitate did not disappear after the production.

The results are shown in Table 1 below.

Determination of the water-solubility of peptide-carbon monoxide releasing molecule conjugate by peptide composition Peptide experimental group (X) Amino acid sequence Solubility (g / g Water) Example 2 (P2) FFEEKD 0.0763 Comparative Example 1 (P1) FFKD 0.0012 Comparative Example 2 (P3) GGIILLK 0.0016 Comparative Example 3 (P4) FFPETSWD 0.0190

From the above results, it was confirmed that when the water-soluble peptide was selectively introduced as in the present invention, the solubility in water was remarkably excellent at a minimum of 4 to 64 times as compared with the control.

[Example 4] Preparation of a co-magnetic assembly of a carbon monoxide releasing molecule conjugate (X-CORM) bonded with a hydrogelating agent and a peptide

The hydrogelating agent Fmoc-FF and the peptide-carbon monoxide releasing molecule conjugate prepared in the above examples were respectively dissolved in hexafluoro-2-propanol (HFIP) and vacuum-vaporized to obtain hexafluoro-2- The propanol was removed.

Fmoc-FF and X-CORM were dissolved in acetone to a concentration of 100 mg / mL at a volume ratio of 1: 1 and then diluted to 50 mg / mL with purified water. The mixed solution was allowed to stand at room temperature for 12 hours, and distilled and purified water was added to give a final concentration of 5.0 mg / mL.

[Test Example 2] Release of carbon monoxide from a carbon monoxide releasing molecule conjugate (X-CORM) bound to a peptide

In order to confirm the stable carbon monoxide binding of the carbon monoxide releasing molecule conjugate (X-CORM) bound to the peptide according to the surrounding pH condition, the pH of the aqueous solution was changed to 2, 6 and 13 using 0.1 mM sodium hydroxide (NaOH) IR spectroscopy was used to analyze the results under acidic and basic conditions.

FT-IR spectroscopy was performed using ZnSe pellets (FTS-175C; Bio-Rad Laboratories, Cambridge, USA). A D ₂ O solution of 0.1 mM sodium chloride (NaCl) was used to measure the background spectrum of the carbon monoxide release molecular conjugate bound to the peptide under the above conditions.

As a result, as shown in FIG. 3, three peaks were observed under acidic conditions and two peaks were observed under basic conditions, and it was confirmed that CO ligands were converted to CO ₂ ligands in basicity, It was confirmed that the carbon monoxide releasing molecular conjugate could release carbon monoxide at the above pH conditions.

[Test Example 3] Myoglobin dynamic property test

For myoglobin, a hemoglobin-like molecule, kinetic assays related to carbon monoxide emission test of carbon monoxide releasing molecules were performed.

All solutions were prepared in 0.1 M phosphate buffer (pH 7.4).

66 uL of a myoglobin solution (Equine Heart) at a concentration of 2.0 mg / mL was prepared in the phosphate buffer solution, and then nitrogen was added for more than 15 minutes to remove the gas. To the degassed solution, 24.0 mg / mL sodium dithionite (Junsei chemicals Co., Ltd.) was added to convert myoglobin to deoxy-myoglobin (deoxy-Mb). The volume ratio of sodium dithionite to the produced dioxy-myoglobin was measured at 1:10 (v: v). The deoxy-myoglobin solution was mixed with the prepared peptide-carbon monoxide releasing molecule conjugate (X-CORM) to obtain a solution of about 40 uM and 80 uM. The solution was quickly transferred to a cuvette and visible spectra were measured at room temperature with a UV-Vis spectrophotometer (NEOSYS-2000 spectrometer, Scinco, Korea) at 500-600 nm.

From this, the emission amount of carbon monoxide was calculated using the following equation (1).

[Formula 1]

Where A ₅₄₂ , A _iso represents absorption at 542 nm and 550 nm, respectively. Also, ε _d542 , ε _CO542 and ε _iso indicate the extinction coefficients of Mb at 542 nm and MbCO at 542 nm and Mb (and MbCO) at 550 nm, respectively, and [Mb] and [MbCO] MbCO &lt; / RTI &gt;

Deoxy-Mb has a high affinity for carbon monoxide and binds to one molecule of carbon monoxide to form carbonyl myoglobin (MbCO). The maximum absorption difference of dioxy-myoglobin and carbonyl myoglobin can detect carbon monoxide emissions from carbon monoxide-emitting molecules.

In the range of 500 to 600 nm, the maximum absorption peak at 556 nm was observed for dioxy-myoglobin, while the maximum absorption peak at both 542 nm and 578 nm was observed for caribonyl myoglobin.

In addition, for the above-prepared solutions of about 40 uM and 80 uM, the dynamic characteristics for the release time of carbon monoxide were calculated in order to confirm the carbon monoxide emission correlation. According to the results, the half-life of carbon monoxide emission was measured to be 13.7 minutes for 40 uM X-CORM, while it was 1.3 minutes for 80 uM X-CORM.

On the basis of the above measurement results, a CO-releasing hydrogel (CORH) containing a co-magnetic assembly of a hydrogelating agent and a carbon monoxide releasing molecule conjugate (X-CORM) , And analyzed by the same myoglobin dynamic property test method as described above.

As a result, as can be seen from FIG. 4, the carbon monoxide release half-life of the carbon monoxide releasing hydrogel (CORH) was measured to be 20.2 minutes, while the carbon monoxide releasing molecular conjugate (X-CORM) bonded with the peptide before hydrogel formation As a result, it was confirmed that the carbon monoxide releasing hydrogel (CORH) of the present invention has a carbon monoxide retention time of about 20 times that of the X-CORM before hydrogel formation.

[Test Example 4] Co-self assembling property of carbon monoxide releasing hydrogel (CORH)

FT-IR spectroscopy was used to perform a spectroscopic test of carbon monoxide-releasing hydrogel (CORH) to measure intermolecular interactions in CORH. For this, the carbon monoxide releasing molecule conjugate (X-CORM) and the hydrogel (Fmoc-FF) bound to the peptides prepared in the Examples were mixed at a ratio of 5: 5 (v: v) A spectrum contained in a wavenumber of 1500 to 1800 in mg / mL D ₂ O solution was obtained (FIG. 5).

It was confirmed that the CORH in the form of the co-self-assembled Fmoc-FF / X-CORM had two dominant peaks having wavenumbers of 1638 cm ^-1 and 1690 cm ^-1 from which the co-self-assembled carbon monoxide releasing hydrogel (CORH) are all antiparallel beta-sheets. The antiparallel? -Sheet can be observed from Fmoc-FF, which is a hydro gelling agent. From this, it was confirmed that the? -Sheet structure was maintained by co-coupling with Fmoc-FF and X-CORM.

Viscoelastic properties of co-magnetic assemblies

The viscoelasticity of the co-magnetic assembly CORH was measured by rheological measurement at room temperature using a flow meter MARS 40 (HAAKE, Germany) on a 20 mm parallel plate with a time sweep of 1 Hz Respectively. Measurements were made by applying a frequency sweep of 1-100 Hz and a 0.01-1% vibration strain to determine the linear viscoelastic zone.

From this, it was found that the storage modulus (G ') of the hydrogel in the linear region is larger by one digit than the loss modulus (G "), Of the total. In addition, the hydrogel had a storage elastic modulus of 3 kPa or more and thus was confirmed to be very hard. In the case of the co-magnetic assembly CORH, both the storage elastic modulus and the loss elastic modulus were stable regardless of the frequency of 50 Hz or more (FIG. 6).

TEM analysis of co-magnetic assemblies

To confirm the molecular structure of CORH, the prepared co-magnetic assembly, transmission electron microscopy (TEM) was performed using 120 kV JEM-1400 (JEOL, Japan) and analyzed using Gatan Digital Micrograph software Respectively.

From the analysis, it was confirmed that CORH, which is a co-magnetic assembly manufactured through an embodiment of the present invention, has a fiber network structure having a fiber diameter of 10 to 100 nm (FIG. 7).

NMR analysis of co-magnetic assemblies

Dimensional nuclear magnetic resonance (NMR) analysis and 2-dimensional nuclear overhauser spectroscopy (NOESY) were conducted to confirm the molecular structure of CORH, which is a co- (Bruker AVANCE III 600 NMR spectrometer w / BBFO brodband probe; Bruker BioSpin GmbH, Rheinstetten, Germany).

(X-CORM) and hydrogel (Fmoc-FF) bound to the peptides prepared in the above Example were mixed at a ratio of 5: 5 (v: v) mg / mL D ₂ O solution was set at 600 MHz, mixing time tm = 100 ms, and scan number n = 128.

The results show that there is an interaction between the aromatic ring of diphenylalanine and the methylene of the Fmoc linker region. It was also confirmed that an interaction between the aromatic ring of diphenylalanine and the methylene group protons of diphenylalanine was also present (FIG. 8).

From these results, it was newly found that successful co-self assembly of Fmoc-FF and X-CORM in aqueous solution can be induced.

[Test Example 5] Therapeutic effect of carbon monoxide releasing hydrogel (CORH)

Rat cell culture

H9c2 cells derived from embryonic cardiomyoblasts of rats were purchased from Korean Cell Line Bank (Seoul, Korea). Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (Dulbecco's Modified Eagle Medium, Logan, Utah, USA), and all cell lines were maintained at 37 ° C humidified with 5% And cultured in a humidified atmosphere.

Cytotoxicity measurement

Cells were inoculated into a 96-well plate (100 uL per well) and treated with carbon monoxide-releasing hydrogel (CORH) at concentrations of 25 uM, 50 uM and 100 uM. For comparison with the cytotoxicity of the carbon monoxide releasing hydrogel, the same analysis was also performed on the carbon monoxide releasing molecule (CORM3, carbon monoxide releasing molecule conjugate (X-CORM) bound to the peptide used in the hydrogel manufacturing step.

Cell viability and cytotoxicity were measured according to the guidelines recommended by CCK-8 (Cell Counting Kit-8; Dojindo Laboratories, Kumamoto, Japan). 10 uL of CCK-8 solution was added to each well and incubated for 3 hours. Absorbance was measured at a wavelength of 450 nm on a microplate reader (Spectra Max M5, Molecular Device Co.). Cell viability was obtained by comparing the absorbance of the treated cells with the absorbance of the control cells. All experiments were performed independently and repeated three times.

As a result, it was confirmed that the carbon monoxide releasing hydrogel (CORH) showed almost no cytotoxicity even when the concentration was increased.

Measurement of cell viability

Cell viability was measured visually using Live / Dead assay kit. H9c2 cells were treated with a tricarbonyl dichlororuthenium (II) dimer (CORM3), a carbon monoxide releasing molecule conjugate (X-CORM) conjugated with a peptide and a carbon monoxide releasing hydrogel (CORH), hydrogen peroxide was added and cultured for 24 hours Respectively. The surviving cells and dead cells were stained with Live / Dead assay kit and confocal microscope imaging system (LSM710, Carl Zeiss, Germany) installed in Korea Basic Science Institute (Daejeon, Korea) .

As a result, it was confirmed that the cell survival rate was improved by about 30% as compared with the case where only the hydrogen peroxide was added when the carbon monoxide releasing hydrogel (CORH) as the co-magnetic assembly was treated. From this, the co- The inventors of the present invention have found that the protective effect of the present invention is superior to that of the case of oxidative stress due to hydrogen peroxide. And to find out the possibility of using it as a therapeutic agent.

Measurement of apoptosis

For the measurement of intracellular reactive oxygen species (ROS) by hydrogen peroxide treatment, chloromethyl-2,7-dichlorodihydro-fluorescein diacetate (CM) -H2DCFDA) was used. H9c2 cells were plated in 96-well plates at 1 × 10 ⁴ cells / well on the black side of the bottom, and active oxygen species were measured by fluorescence analysis. Cells were divided into three groups, and carbon monoxide releasing particle, tricarbonyl dichlororuthenium (II) dimer (CORM3) and carbon monoxide releasing molecule conjugate (X-CORM) bonded with peptides, together with carbon monoxide releasing hydrogel (CORH) For 2 hours. A mixture of 6-carboxy-2 ', 7'-difluoroscein diacetate (DCF-DA; Thermo Scientific, Rockford, Ill., USA) Min. Fluorescence was then measured using a microplate reader (Spectra max M5, Molecular Device Co .; λ _ex = 405 nm, λ _em = 527 nm).

TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) was performed according to the manufacturer's recommended protocol using the DeadEnd Fluorometric TUNEL System (Promega, Madison, Wis.).

Cell death was assayed using a cell viability kit, NucleoCounter® NC-3000 ™ system, and the amount of free thiol in the reduced glutathione was measured and assayed according to the manufacturer's instructions.

As a result of measuring the protective effect against apoptosis through the above process, the rate of apoptosis in the cell treated with hydrogen peroxide was 44.0%. When carbon monoxide releasing molecules were added, the carbon monoxide releasing particle, tricarbonyldichloro ruthenium (II) 20.0%, and 11.0%, respectively, for carbon monoxide releasing hydrogel (CORH), carbon monoxide releasing molecule conjugate (X-CORM) bound to peptide, and carbon monoxide releasing hydrogel (CORH) , Exhibited the best effect of decreasing cell death (Fig. 9).

Hydrogen peroxide has the effect of promoting the production of reactive oxygen species (ROS) and promoting apoptosis. After 2 hours from the addition of the synthesized carbon monoxide releasing molecules at 50 uM, the CM-H2DCFDA probe is used to activate intracellular reactive oxygen species The results of measuring the species are shown in Fig.

As a result, it was confirmed that the production of reactive oxygen species by hydrogen peroxide was increased, but the increase of reactive oxygen species was remarkably decreased by the pretreatment of the carbon monoxide Paul molecule. Especially, when the carbon monoxide releasing hydrogel (CORH) was treated, Respectively.

These results indicate that the carbon monoxide-releasing hydrogel (CORH) of the present invention exerts an excellent effect on the damage caused by oxidative stress in the myocardial cells, and that the carbon monoxide releasing hydrogel (CORH) And thus the present invention has been completed.

[Formulation Example 1]

30 mg of the carbon monoxide releasing hydrogel (CORH) prepared in the example of the present invention

25 mL of acetone or ethanol

Distilled water 75 mL

Were mixed and prepared using a conventional method used for preparing injections.

While the invention has been shown and described with respect to the specific embodiments, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. It is obvious that it can be modified and changed.

Claims (15)

A hydrogel composition for releasing carbon monoxide comprising a hydrogelator and a co-magnetic assembly of a carbon monoxide releasing molecule bonded to a peptide represented by formula (I)
(I)
Phe-Phe-X-Asp
(In the above peptide,
X is a water-soluble peptide composed of 2 to 6 water-soluble amino acids.) The method according to claim 1,
The water soluble peptide may be selected from the group consisting of serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine, arginine, histidine, (Asp), glutamic acid (Glu), and the like. The hydrogel composition for releasing carbon monoxide according to claim 1, The method according to claim 1,
Wherein the water-soluble peptide comprises at least one selected from the group consisting of glutamic acid (Glu), serine (Ser), and combinations thereof. 3. The method of claim 2,
Wherein the water-soluble peptide is glutamic acid (Glu) -glutamic acid (Glu) -lysine (Lys). The method according to claim 1,
The hydrogelating agent may be selected from the group consisting of Fluorenylmethoxycarbonyl peptides (Fmoc-peptides), substances having an amphipathic low molecular skeleton, substances having a skeleton based on an in vivo component, Wherein the carbon monoxide releasing agent is at least one selected from the group consisting of carbon monoxide, carbon monoxide and carbon monoxide. 6. The method of claim 5,
Wherein the fluorenylmethoxycarbonyl peptide (Fmoc-peptides) is Fmoc-FF or Fmoc-PhePhe having the structure of the following formula (II).
&Lt; RTI ID = 0.0 &

The method according to claim 1,
Wherein the structure of the carbon monoxide releasing molecule (CORM) is represented by the following formula (III).
(III)
Ru (CO) ₃ Cl (glycinate) The method according to claim 1,
wherein the solubility at pH 5 to 8 is 0.065 to 0.085 g per 100 g of distilled water. Synthesizing a peptide represented by the following formula (I);
(I)
Phe-Phe-X-Asp
(In the above peptide,
X is a water-soluble peptide composed of 2 to 6 water-soluble amino acids.)
Reacting the synthesized peptide with a carbon monoxide releasing molecule (CORM); And
Mixing the hydrogeling agent;
Wherein the carbon monoxide releasing agent is added to the hydrogel composition. 10. The method of claim 9,
The water soluble peptide may be selected from the group consisting of serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine, arginine, histidine, (Asp) and glutamic acid (Glu). The method for producing a hydrogel composition for carbon monoxide releasing according to claim 1, 10. The method of claim 9,
The above-mentioned hydrogelating agent is preferably used in combination with fluorenylmethoxycarbonyl peptides (Fmoc-peptides), a substance having a skeleton of an amphipathic low molecular weight, a substance having a skeleton having an in vivo component as a motif, Wherein the carbon monoxide releasing agent is one or more selected from the group consisting of carbon monoxide, carbon monoxide and carbon monoxide. 12. The method of claim 11,
Wherein the fluorenylmethoxycarbonyl peptide is Fmoc-FF or Fmoc-PhePhe having the structure of the following formula (II).
&Lt; RTI ID = 0.0 &

10. The method of claim 9,
Wherein the structure of the carbon monoxide releasing molecule (CORM) is represented by the following formula (III).
(III)
Ru (CO) ₃ Cl (glycinate)

10. The method of claim 9,
Wherein the step of reacting the synthesized peptide with a carbon monoxide releasing molecule (CORM) is carried out at a pH of 4 to 8. An injection comprising a hydrogel composition for releasing carbon monoxide according to any one of claims 1 to 8.

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