KR20190134204A - Substance p peptide-fixed fibrin gel for tissue regeneration and preparation method thereof - Google Patents

Abstract

The present invention relates to fibrin gel, a method for preparing the fibrin gel, and a composition for tissue regeneration comprising the fibrin gel. The fibrin gel of the present invention is biocompatible and at the same time, has a short manufacturing process, and forms a more stable conjugation due to the enzymatic conjugation, and thus can be advantageously used for tissue regeneration.

Description

SUBSTANCE P PEPTIDE-FIXED FIBRIN GEL FOR TISSUE REGENERATION AND PREPARATION METHOD THEREOF}

The present invention relates to a fibrin gel, a preparation method thereof, and a composition for tissue regeneration including the fibrin gel.

As research for tissue regeneration of body organs is actively conducted, it is necessary to develop factors that have better regeneration efficiency and biomaterials that can stably transfer them to tissues in need of regeneration.

Existing acrylic group attached hyaluronic acid-based hydrogel (hydrogel) has the advantage that can be delivered by conjugating (growth) growth factors and cells, but the manufacturing process is long, has a hydration power and can not be applied to all organs There is this.

In addition, heparin-based fibrin gel, which was developed to compensate for the disadvantages of the hydrogel, has the advantage of having biocompatibility, but it is difficult to obtain a stable fibrin gel due to solidification by protein binding.

Accordingly, there is an urgent need to develop biomaterials that can maintain biocompatibility and maintain growth factor and cell activity, have a short manufacturing process, are applicable to all organs, and can maintain stable binding.

Korean Patent Publication No. 10-2007-0073008

The present inventors have made intensive research efforts to develop biomaterials that maintain growth factors and cell activity, and have biocompatibility, and which have a short manufacturing process and are applicable to all organs. As a result, when manufacturing fibrin gel using a synthetic polypeptide in which a substance P peptide (substance P) peptide is bound to the peptide of the plasmin inhibitor, it is biocompatible and has a short manufacturing process and more stable binding due to enzyme binding. By identifying the formation, the present invention has been completed.

Accordingly, an object of the present invention is to provide a fibrin gel comprising a polypeptide comprising a amino acid sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 2 and a fibrinogen.

Another object of the present invention is to provide a composition for tissue regeneration.

Still another object of the present invention is to provide a method for preparing fibrin gel.

It is another object of the present invention to provide a method for tissue regeneration comprising administering a composition for tissue regeneration to a subject.

Another object of the present invention is to provide a tissue regeneration use of the composition for tissue regeneration.

The present inventors have made intensive research efforts to develop biomaterials that maintain growth factors and cell activity, and have biocompatibility, and which have a short manufacturing process and are applicable to all organs. As a result, when manufacturing fibrin gel using a synthetic polypeptide in which a substance P peptide (substance P) peptide is bound to the peptide of the plasmin inhibitor, it is biocompatible and has a short manufacturing process and more stable binding due to enzyme binding. Formation was found.

The present invention relates to a fibrin gel, a preparation method thereof, and a composition for tissue regeneration including the fibrin gel.

Hereinafter, the present invention will be described in more detail.

One aspect of the invention relates to a fibrin gel comprising a polypeptide and a fibrinogen comprising an amino acid sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 2.

As used herein, "Fibrin gel" refers to a gel-type material in which fibrin (Fibrin, a biocompatible natural coagulation material) is mixed. Since the fibrin gel has excellent biocompatibility as well as excellent biocompatibility, the fibrin gel has been widely used for the production of a cell-mounted structure. In addition, since the fibrin gel does not require a separate synthesis, specific factors for tissue regeneration can be easily fixed, and the number of specific factors that can be immobilized is not limited. There is an advantage in minimizing cellular damage in the manufacture of the structure.

The cells may be, for example, stem cells, cardiac cells, vascular endothelial cells, or fibroblasts, but are not limited thereto.

The polypeptide may be one in which the peptide comprising the amino acid sequence of SEQ ID NO: 1 and the peptide comprising the amino acid sequence of SEQ ID NO: 2 are linked by a linker.

The linker may include any linker used to link peptides in the art.

The polypeptide may be composed of the amino acid sequence of SEQ ID NO: 3.

The polypeptide may be immobilized in a fibrin gel through enzyme-linked. By immobilizing the polypeptide to the alpha chain of fibrinogen in the fibrin gel through enzyme-substrate binding, more stable binding can be induced than protein binding, which is a binding between peptide sequences.

The fibrin gel may comprise a polypeptide comprising a amino acid sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 2, fibrinogen, Ca ^2+, and thrombin.

The concentration of the polypeptide in the fibrin gel can be, for example, 1 to 50 μM, 1 to 45 μM, 1 to 40 μM, 1 to 35 μM, 1 to 30 μM, 1 to 25 μM, 1 to 20 μM, 1 to 15 μM, 1 to 10 μM, 10 to 50 μM, 15 to 50 μM, 20 to 50 μM, 25 to 50 μM, 30 to 50 μM, 35 to 50 μM, 40 to 50 μM, 45 to 50 μM, It may be 10 to 40 μM, 20 to 30 μM or 10 μM, but is not limited thereto.

The fibrinogen concentration in the fibrin gel is, for example, 1 to 20 mg / mL, 1 to 15 mg / mL, 1 to 10 mg / mL, 1 to 5 mg / mL, 5 to 20 mg / mL, 10 To 20 mg / mL, 15 to 20 mg / mL, 5 to 15 mg / mL, or 5 mg / mL, but is not limited thereto.

The concentration of Ca ²⁺ in the fibrin gel may be, for example, 2.5 mM, but is not limited thereto.

The thrombin concentration in the fibrin gel may be, for example, 1 to 5 U / mL, 1 to 4 U / mL, 1 to 3 U / mL, 1 to 2 U / mL, or 2 U / mL. It is not limited to this.

The fibrin gel comprises 30 to 80% by weight of the polypeptide, 0.01 to 1% by weight of fibrinogen, 10% by weight of Ca ²⁺ in 0.1 and 0.1 to 10% by weight of thrombin based on 100% by weight of the fibrin gel. Can be.

Yet another aspect of the present invention relates to a fibrin gel manufacturing method comprising the following steps.

A first mixing step of mixing the polypeptide and the fibrinogen comprising the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2;

A second mixing step of mixing Ca ²⁺ ; And

Third mixing step of mixing thrombin.

The preparation method may further include a preparation step of preparing a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.

The first mixing step may be performed at room temperature, but is not limited thereto.

The first mixing step may be performed for one hour, but is not limited thereto.

The second mixing step may be performed at room temperature, but is not limited thereto.

The second mixing step may be performed within 5 minutes, but is not limited thereto.

The third mixing step may be performed at 37 ° C., but is not limited thereto.

The third mixing step may be performed within 5 minutes, but is not limited thereto. Since it can be solidified within a few seconds, it is possible to shorten the manufacturing time than the conventional hydrogel, there is an advantage that can be solidified in a short time to minimize cell damage.

The overlapping contents of the polypeptide and fibrin gel including the same are omitted in consideration of the complexity of the present specification.

Another aspect of the invention relates to a composition for tissue regeneration comprising a polypeptide and fibrinogen comprising an amino acid sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 2.

The tissue may be tissue of any organ that requires regeneration, for example, epithelial tissue, muscle tissue, neural tissue or connective tissue. ), But is not limited thereto.

The muscle may be a skeletal muscle, a cadiac mucscle or a smooth muscle.

The composition for tissue regeneration may be formulated in a suitable form with a pharmaceutical carrier generally used for the treatment of tissue regeneration. 'Pharmaceutical' refers to a physiologically acceptable and, when administered to a human, usually does not cause an allergic reaction or the like, such as gastrointestinal disorders, dizziness and the like. Pharmaceutical carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid, sucrose, albumin and the like. Suitable preservatives include DMSO, glycerol, ethylene glycol, sucrose, trehalose, dextrose, polyvinylpyrrolidone, and the like.

Another aspect of the invention relates to a method for tissue regeneration comprising administering to a subject a composition comprising a polypeptide comprising a amino acid sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 2 and fibrinogen.

The subject refers to a mammal that is the subject of treatment, observation or experiment, preferably human.

The composition for tissue regeneration can be administered by any device that can move the composition to target cells.

The composition for tissue regeneration can be administered in a conventional manner via rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes.

The composition for tissue regeneration can be administered to a subject to produce a fibrin gel, thus giving minimal damage to bone, nerve, and other tissues, and can be used to regenerate these tissues.

Another aspect of the invention relates to the use of tissue regeneration of a composition comprising a polypeptide comprising a amino acid sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 2 and fibrinogen.

The present invention relates to a fibrin gel, a method for preparing the fibrin gel, and a composition for tissue regeneration including the fibrin gel, wherein the fibrin gel of the present invention has a biocompatibility and a short manufacturing process and is more stable due to enzyme binding. Since it forms a bond, it can be usefully used for tissue regeneration.

1 is a schematic of a fibrin gel immobilized with a synthetic polypeptide.
Figure 2a is a result of confirming the solidification rate of the fibrin gel in order to confirm the optimal concentration of thrombin according to an embodiment of the present invention.
Figure 2b is a result of confirming the degree of solidification of the fibrin gel in order to confirm the optimal concentration of thrombin according to an embodiment of the present invention.
Figure 2c is a result of confirming the Live / Dead analysis of cells in the fibrin gel in order to determine the optimal concentration of fibrinogen according to an embodiment of the present invention.
Figure 3 is a result of AMC fluorescence analysis of the fibrin gel immobilized synthetic polypeptide according to an embodiment of the present invention.
Figure 4 is a result of checking the sprouting (sprouting) of HCAEC spheroids (cultivated) in a synthetic polypeptide fixed fibrin gel according to an embodiment of the present invention.
5 is a result of confirming the sprouting degree of the HCAEC spheroid after F-actin (F-actin) staining for the HCAEC spheroid cultured in the fibrin gel immobilized with the synthetic polypeptide according to an embodiment of the present invention.
Figure 6 is a graph measuring the viscosity of the fibrin gel in accordance with the concentration of the synthetic polypeptide with respect to the fibrin gel fixed to the synthetic polypeptide according to an embodiment of the present invention.
7 is a photograph of a fibrin gel to which a synthetic polypeptide is immobilized according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Preparation Example 1 Preparation of Substance P-Factor XIII Reactant Binding Peptide (Synthetic Polypeptide)

A synthetic polypeptide (SEQ ID NO: 3) was prepared by combining a P substance (Substance P, Anygen, Korea) peptide (SEQ ID NO: 1) and a thirteenth factor (Factor XIII, Anygen) reactive peptide (SEQ ID NO: 2).

Experimental Example 1. Confirmation of Optimal Concentration of Fibrinogen and Thrombin

Fibrin gels at 2.5, 5, 10 or 15 mg / ml fibrinogen (Sigma-Aldrich, Germany) and 1 U / mL or 2 U / mL concentrations of thrombin (Sigma-Aldrich) were mixed at room temperature and left at 37 ° C. Was prepared.

In addition, in order to confirm the optimal concentration of thrombin, fibrin gel strength was measured according to the thrombin concentration (1 U / mL or 2 U / mL). At this time, the concentration of fibrinogen was fixed at 5 mg / ml, and the intensity from the start of the solidification of the gel to the end was measured.

As can be seen in Figures 2a and 2b, it was confirmed that the solidification time of the fibrin gel using a 2 U / mL thrombin concentration than the fibrin gel using a 1 U / mL concentration of thrombin, it was confirmed that using a 1 U / mL concentration of thrombin It was confirmed that the fibrin gel using thrombin at a concentration of 2 U / mL rather than the fibrin gel maintained the degree of solidification firmly. Therefore, in terms of solidification rate, the thrombin concentration was determined to be 2 U / mL, and further experiments were conducted as follows to determine the fibrinogen concentration.

Fibrin gels were prepared by using fibrinogen at a concentration of 5 or 10 mg / mL (thrombin concentration: 2 U / mL), thereby producing human mesenchymal stem cells (hMSCs) (Lonza, # PT-2501). Was inoculated and cultured at 37 ° C. for 14 days, followed by Live / Dead analysis.

As can be seen in Figure 2c, it was confirmed that the adhesion and activity of the cells increased in the fibrin gel using the fibrinogen concentration of 5 mg / mL than the fibrin gel using the fibrinogen concentration of 10 mg / mL. Therefore, the concentration of fibrinogen was determined to be 5 mg / mL in terms of cell adhesion.

Example 1 Preparation of a Mixture Comprising a Synthetic Polypeptide (P Material) at a Concentration of 10 μM

528 μL of synthetic polypeptide, 5 mg of fibrinogen, and 342 μL of 20 mM Tri-Buffered Saline (TBS) (Bio-RAD, US) dissolved in a flask at 0.5 mg / mL were dissolved at 37 ° C. for 1 hour. Then, 50 mM Ca ²⁺ 50 μL (Sigma-Aldrich) was added and mixed at room temperature to prepare a liquid mixture.

Example 2. Preparation of a Mixture Comprising a Synthetic Polypeptide (P Material) at a Concentration of 50 μM

A liquid mixture was prepared in the same manner as in Example 1, except that the synthetic polypeptide dissolved at a concentration of 2.5 mg / mL was used.

Comparative example. Preparation of mixtures that do not contain synthetic polypeptides (P substances)

5 mg of fibrinogen and 342 μL of 20 mM TBS were added to the flask and dissolved at 37 ° C. for 1 hour. Then, 50 μL of 50 mM Ca ²⁺ was added thereto and mixed at room temperature to prepare a liquid mixture.

Experimental Example 2. Confirmation of Crosslinking of Synthetic Polypeptide in Fibrin Gel

First, for Examples 1 and 2, AMC (7-amino-4-methylcoumarin) peptide (excitation) 360 nm, light emission, which is a fluorescent substance at the N-terminus of the P substance peptide in the synthetic polypeptide; 460 nm).

Fibrin gels were prepared by placing the mixtures of Examples 1 and 2 to which the AMC peptides were attached, and the mixtures of Comparative Examples into 12 wells of 25 μL and solidifying with 80 μL of 100 U thrombin. Then, cross-linking was confirmed using a two-photon laser microscopy (ECLIPSE Ti2, Nikon). When the synthetic polypeptide is simply incorporated into the fibrin gel, it is released by the naturally occurring water in the fibrin gel, and thus color development cannot be confirmed.

As can be seen in FIG. 3, it was developed only in the fibrin gel solidified using a mixture of Example 1 (SP 10 μM) and Example 2 (SP 50 μM) to which the P material was added, thereby synthesizing the two types of fibrin gels. It was found that the polypeptide was bound.

Experimental Example 3. Confirmation of angiogenesis by synthetic polypeptide

Human cardiac aortic endothelial cells in a medium in which endothelial basal medium (EBM) (Lonza) and endothelial growth medium (EGM) (Lonza) are mixed at a ratio of 8: 2; HCAEC) (Lonza, Basel, Swiss) were incubated at 37 ° C. for 24 hours. Cultured HCAEC was Hanging-drop culture (37 ℃, 24 hours) to a concentration of 1,000 cells / 25 uL to form a spheroid (spheroids).

Fibrin gel was prepared by adding the HCAEC spheroid formed above to the mixture of Comparative Example, Example 1 and Example 2, and then solidifying the mixture with 80 μL of 100 U thrombin.

After 6 h and 12 h, respectively, two-photon microscopy was used to confirm the degree of sprouting of the HCAEC spheroid.

As can be seen in FIG. 4, compared to Control, HCAEC in a fibrin gel solidified using a mixture of Example 1 (SP 10 μM) and Example 2 (SP 50 μM) to which P material was added The branches of the spheroids were found to be more and longer.

In addition, after 12 hours, additionally HCAEC spheroid was fixed with 4% paraformaldehyde (Biosesang, # 0694) for 20 minutes and treated with 0.4% Triton X-100 (Life science, # 0694) for 20 minutes, followed by Alexa Fluor F-actin of the HCAEC spheroid was stained at a concentration of 1: 200 488 paloidine (Thermo fisher, US). Then, the degree of sprouting was checked using a confocal microscope (Eclipse TE2000-U, Nikon).

As can be seen in FIG. 5, the branches of HCAEC spheroids were more and longer in the fibrin gel solidified using the mixture of Example 1 (SP 10 μM) and the mixture of Example 2 (SP 50 μM) with the addition of P material. I could see it stretched out.

Experimental Example 4. Confirmation of Viscosity of Fibrin Gel

80 μL of 100 U thrombin was added to the mixture of Comparative Example, Example 1 and Example 2, and each was 0 (immediately after thrombin) using a rheometer (Brookfield DV-III + programmable rheometer, US), 20, 40, 60 Viscosity was measured at, 80 and 100 seconds (solidification completion point).

As can be seen in Figure 6, the viscosity of the fibrin gel solidified using a mixture of Example 1 (SP 10 μM) and Example 2 (SP 50 μM) to which the P material is added is 2000 ~ 3000 Pas, It has been confirmed that it has a viscosity similar to that of (1500 ~ 2000 Pas).

Preparation Example 2 Preparation of Fibrin Gels Fixed with Synthetic Polypeptide

Based on the results of Experimental Examples 1 to 4, a fibrin gel to which a synthetic polypeptide was bound was prepared as follows. A photograph of the fibrin gel prepared is shown in FIG. 6, and the optimal concentration of each composition used is as follows (final volume: 1 mL): fibrinogen 5 mg / mL, thrombin 2 U / mL, Ca ²⁺ 2.5 mM, And 264 μg / mL (10 μM) of synthetic polypeptide.

Specifically, 5 mg of fibrinogen and 342 μL of 20 mM TBS were added to 528 μL of the synthetic polypeptide dissolved at 0.5 mg / mL, and dissolved at 37 ° C. for 1 hour. Then, 50 μL of 50 mM Ca ²⁺ was added to the mixture to prepare a liquid mixture. 80 μL of 100 U thrombin was added to the prepared mixture to prepare a solidified fibrin gel.

<110> Korea University Research and Business Foundation <120> SUBSTANCE P PEPTIDE-FIXED FIBRIN GEL FOR TISSUE REGENERATION AND          PREPARATION METHOD THEREOF <130> PN180071 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Substance P <400> 1 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met   1 5 10 <210> 2 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> a2 Plasmin inhibitor <400> 2 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met   1 5 10 <210> 3 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> synthesis polypeptide <400> 3 Asn Gln Glu Gln Val Ser Pro Leu Arg Pro Lys Pro Gln Gln Phe Phe   1 5 10 15 Gly leu met            

Claims (18)

Fibrin gel comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2 and fibrinogen. The fibrin gel of claim 1, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO. The fibrin gel of claim 1, wherein the polypeptide is immobilized on an alpha-chain of fibrinogen through enzyme-linked. The fibrin gel of claim 1, wherein the fibrin gel comprises a polypeptide comprising a amino acid sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 2, fibrinogen, Ca ^2+, and thrombin. The fibrin gel of claim 4, wherein the concentration of the polypeptide is 1-50 μM. The fibrin gel of claim 4, wherein the concentration of fibrinogen is between 1 and 20 mg / mL. The fibrin gel of claim 4, wherein the Ca ²⁺ concentration is 2.5 mM. The fibrin gel of claim 4, wherein the concentration of thrombin is 1 to 5 U / mL. The method of claim 4, wherein the fibrin gel is based on 100% by weight of the fibrin gel, 30 to 80% by weight of the polypeptide, 0.01 to 1% by weight of fibrinogen, 10% by weight in Ca ²⁺ 0.1 and 0.1 to 10 Fibrin gel, which comprises by weight. A composition for tissue regeneration comprising a fibrin gel according to any one of claims 1 to 9. Fibrin gel manufacturing method comprising the following steps:
A first mixing step of mixing the polypeptide and the fibrinogen comprising the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2;
A second mixing step of mixing Ca ²⁺ ; And
Third mixing step of mixing thrombin. The method of claim 11, wherein the preparation method further comprises a preparation step of preparing a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2, fibrin gel manufacturing method. The method of claim 11, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 3. 15. The method of claim 11, wherein the concentration of the polypeptide is 1 to 50 μM. The method of claim 11, wherein the concentration of fibrinogen is 1 to 20 mg / mL. The method of claim 11, wherein the Ca ²⁺ concentration is 2.5 mM. The method of claim 11, wherein the concentration of thrombin is 1 to 5 U / mL. The method of claim 11, wherein the third mixing step is performed within 5 minutes.

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