KR102194155B1 - Thermo-responsive biomaterial comprising thermo-responsive protein conjugated-mussel adhesive protein - Google Patents

Abstract

The present invention relates to a temperature-sensitive biological material comprising a mussel adhesive protein to which a temperature-sensitive polymer is conjugated, a composition for tissue regeneration, a cell support, a carrier for drug delivery, and a method for producing a temperature-sensitive biological material. The mussel adhesive protein to which the temperature-sensitive polymer is bonded according to the present invention can be hydrogelized immediately in the body temperature range, and the biggest drawback of the existing temperature-sensitive polymer, which is the dramatic volume reduction that occurs during phase transition, moisture loss, and the resulting lack of porosity The problem is solved and the mussel adhesive protein is excellent in biocompatibility and tissue adhesion, so it can be usefully used as a biomaterial useful for regeneration of a soft tissue defect.

Description

Temperature-sensitive biomaterial comprising thermo-responsive protein conjugated-mussel adhesive protein {Thermo-responsive biomaterial comprising thermo-responsive protein conjugated-mussel adhesive protein}

The present invention relates to a temperature-sensitive biological material comprising a mussel adhesive protein to which a temperature-sensitive polymer is conjugated, a composition for tissue regeneration, a cell support, a carrier for drug delivery, and a method for producing a temperature-sensitive biological material.

Tissue engineering is an applied study aimed at maintaining, improving or restoring the function of the human body by making artificial tissues that can be implanted in the body to replace or regenerate damaged tissues or organs with normal tissues. For example, mastectomy , It refers to the study to reconstruct a large number of soft tissue defects caused by burns, accidents and tumor removal. According to a report by the American Society of Plastic Surgeons, tumor removal was the number one in the annual tissue reconstruction surgery performed in 2015, with a number of 45 million cases. Currently, methods for regenerating such a soft tissue defect area include autologous fat transplantation, permanent structure insertion, and non-permanent structure injection, respectively, which have side effects such as rapid fat resorption and necrosis and the need for reconstruction procedures. I have. Therefore, it is necessary to develop a biorenewable material that can be ultimately integrated with existing original tissues by inducing soft tissue regeneration through not only simply filling defective tissues, but also stem cell mounting and differentiation.

Hydrogel, which is widely used in the development of biorenewable materials, is a material that absorbs a large amount of water or body fluid into a crosslinked grid in water or body fluid and swells, does not scatter in water, and maintains a three-dimensional structure. do. Even after swelling, it is thermodynamically stable and has mechanical and physicochemical properties corresponding to an intermediate form between a liquid and a solid. These hydrogels generally exhibit biocompatibility, high porosity, and oxygen permeability, and may exhibit physical properties similar to those of living soft tissue. The initial applications of hydrogels based on natural and synthetic polymers were in lenses and wound dressings, but nowadays, tissue engineering fields such as hemostatic agents, tissue adhesives, drug delivery carriers, tissue fillers, and tissue regeneration agents including cells and growth factors. The width is widening.

Various crosslinking methods are used to prepare such a hydrogel, and most of the crosslinking methods use a chemical crosslinking agent. For example, a protein crosslinking method using glutaraldehyde is known. However, most chemical crosslinking methods, including glutaraldehyde, can not be used with cells because they have a large change in protein structure and have cellular and tissue toxicity. Therefore, in order to avoid cytotoxicity, the chemical crosslinking method has a disadvantage in that it is impossible to immediately obtain a support having uniformly distributed cells because the cells are coated or injected onto a support having already formed crosslinking.

As a method to solve this problem, a method of inducing a phase transition according to a temperature change is being studied. This is a temperature-sensitive polymer, and the temperature-sensitive polymer mainly has an amphiphilic nature and a solution-solid phase transition occurs as the temperature increases. However, the method of using a temperature-sensitive polymer also has limited application to a tissue engineering material for mounting cells, which is a problem such as dramatic volume reduction, moisture loss, and lack of porosity that occur during phase transition due to hydrophobic interactions of the temperature-sensitive polymer. Although it is easy to mount raw cells, it is due to an environment where cell survival is difficult. In addition, there are limitations such as lack of bioactivity, biocompatibility and bioadhesiveness.

In addition, even if gelation occurs quickly when a substance is injected into the living body, temperature-sensitive polymers do not exhibit bio-adhesiveness, although they must be adhered and maintained within the targeted soft tissue defects in order to function as a tissue filling and tissue regeneration agent for a long period of time. As a result, there is a problem in that the characteristics are insufficient for use in tissue regeneration.

Therefore, there is a need for a new biomaterial having both mechanical properties and bioadhesion ability that can be used as a biomaterial for purposes such as tissue regeneration.

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Korean Patent Publication No. 10-2016-0026441 (published on March 9, 2016) Korean Patent Publication No. 10-1103423 (announced on January 6, 2012) Korean Patent Publication No. 10-1250923 (announced on April 4, 2013)

Li L et al., “Novel mussel-inspired injectable self-healing hydrogel with anti-biofouling property”, Adv Mater (2015), Vol. 27(7), pp.1294-1299

The present inventors believe that the mussel adhesive protein conjugated with a temperature-sensitive polymer not only has excellent temperature-sensitivity at which phase transition occurs in vivo, but also improves bio-adhesion and biocompatibility, which are problems of existing temperature-sensitive polymers, It was confirmed that the problem of volume reduction, moisture loss, and insufficient porosity can be solved, and the present invention was completed.

Accordingly, it is an object of the present invention to provide a temperature-sensitive biological material comprising a mussel adhesive protein to which a temperature-sensitive polymer is conjugated, a composition for tissue regeneration, a cell support, a carrier for drug delivery, and a method for producing the same.

In order to achieve the above object, the present invention provides a temperature-sensitive biomaterial comprising a mussel adhesive protein to which a temperature-sensitive polymer is bonded.

In addition, the present invention provides a composition for tissue regeneration comprising a mussel adhesive protein to which a temperature-sensitive polymer is conjugated.

In addition, the present invention provides a cell support comprising a mussel adhesive protein to which a temperature sensitive polymer is conjugated.

In addition, the present invention provides a carrier for drug delivery comprising a mussel adhesive protein to which a temperature sensitive polymer is conjugated.

In addition, the present invention comprises the steps of forming an amide bond by reacting a temperature-sensitive polymer with a mussel adhesive protein; It provides a method of manufacturing a temperature-sensitive biological material comprising a.

The mussel adhesive protein to which the temperature-sensitive polymer is bonded according to the present invention can be hydrogelized immediately in the body temperature range, and the biggest drawback of the existing temperature-sensitive polymer, which is the dramatic volume reduction that occurs during phase transition, moisture loss, and the resulting lack of porosity The problem is solved and the mussel adhesive protein is excellent in biocompatibility and tissue adhesion, so it can be usefully used as a biomaterial useful for regeneration of a soft tissue defect.

1 is a diagram showing the result of confirming the MAP-PNIPAM conjugate to which fp-151 and the temperature-sensitive polymer PNIPAM are conjugated through an electrophoresis experiment.
2 is a diagram showing the result of confirming the change of the phase transition ability according to the number of reaction moles of the MAP-PNIPAM conjugate through light transmittance.
3 is a diagram showing the result of confirming the phase transition ability according to the number of reaction moles of the MAP-PNIPAM conjugate and the protein concentration of the conjugate.
4 is a diagram showing the results of confirming the change in storage modulus according to the number of reaction moles and temperature of the MAP-PNIPAM conjugate.
5 is a diagram showing the results of an injection experiment for confirming the formation of a hydrogel at a biological temperature of the MAP-PNIPAM conjugate.
6 is a diagram showing the results of evaluating the moisture retention ability according to the number of reaction moles and polymer concentration of the MAP-PNIPAM conjugate.
7 is a diagram showing the results of confirming the porous structure of the PNIPAM hydrogel and the MAP-PNIPAM conjugate through a scanning electron microscope.
8 is a diagram showing the results of confirming the tissue adhesion ability of the MAP-PNIPAM conjugate.
9 is a diagram showing the results of comparing stem cell viability in collagen hydrogel and MAP-PNIPAM conjugate hydrogel.
10 is a diagram showing the results of confirming the ease of injection and immediate hydrogelation under body temperature conditions when injecting the MAP-PNIPAM conjugate solution and the mesenchymal stem cell solution into the rat.
FIG. 11 is a diagram showing the results of comparing volume maintenance and inflammatory responses by H&E staining tissues on days 14 and 21 after in vivo injection of MAP-PNIPAM conjugates or alginate including stem cells.
FIG. 12 is a diagram showing a result of comparing the effect of generating new blood vessels by staining CD31 tissues on days 14 and 21 after in vivo injection of MAP-PNIPAM conjugates or alginate including stem cells.
13 is a diagram showing the results of observing the differentiation of stem cells by performing optical microscopy or fluorescent staining on the tissue at day 21 after in vivo injection of MAP-PNIPAM conjugate or alginate including stem cells.
14 is a diagram showing the results of confirming the MAP-Pluronic conjugate through an electrophoresis experiment (lane 1: maker, lane 2-4: concentrations of the amine group of fp-1 and the carboxy group of Pluronic-127, respectively MAP-Pluronic conjugates reacted with 0.5:1, 1: 1 and 2:1, lane 5: unreacted fp-1).
15 is a result of confirming the state at room temperature (left) and the state at 37°C (right) of the MAP-Pluronic conjugate in which the concentration of the amine group of fp-1 and the carboxy group of Pluronic-127 were reacted at 2:1. Is a diagram showing.

The present invention provides a temperature-sensitive biological material comprising a mussel adhesive protein to which a temperature-sensitive polymer is conjugated.

The temperature-sensitive biomaterial exhibits ease of bioinjection and cell introduction through immediate phase transition near body temperature, which is an advantage of conventional temperature-sensitive polymers, and improved water retention ability, bioadhesion ability, porosity, and It has excellent advantages that show all of the advantages such as cell survival.

In the present invention, the term “living material” means including all materials directly inserted into the human body.

In the present invention, the temperature-sensitive polymer refers to a polymer capable of instantaneous phase transition at a phase transition temperature according to a temperature change, and has amphiphilic nature and a solution-solid phase transition occurs as the temperature increases. In particular, the temperature-sensitive polymer of the present invention may preferably include a polymer whose phase transition temperature is near body temperature.

More specifically, the temperature-sensitive polymer of the present invention may include, without limitation, polyacrylates, polyesters, and pluronic polymers capable of immediate phase transition near body temperature, and preferably poly(N-isopropylacrylamide) (PNIPAM ), polymethacrylic acid, poly(2-hydromethacrylic acid), poly(N,N'diethylaminoethyl)acrylate, poly(lacticco-glycolic acid), polyfumaric acid And it may be one or more selected from the group consisting of Pluronic-127.

The temperature-sensitive biological material of the present invention contains a temperature-sensitive polymer, so that it exists in the form of an aqueous solution at room temperature, and has increased hydrophobicity near a body temperature of 30 to 50°C, preferably 30 to 40°C, more preferably 35 to 38°C. The interaction can form a gel state. Therefore, the biomaterial of the present invention may be a biomaterial characterized in that it gels at 30 to 50°C.

In this case, if the gel is rapidly formed near body temperature, it can be more usefully used in tissue engineering applications. Since the temperature-sensitive biological material of the present invention exists as an aqueous solution at room temperature and gels due to an immediate phase transition near body temperature, the structure can be maintained even in a body environment with high moisture without treatment with a separate crosslinking agent. In addition, since cell and tissue toxicity can be avoided, it is easy to mount active factors such as cells and stem cells, growth factors, etc., and it can be easily injected into the body using a syringe, which is a minimal invasive method, due to immediate phase transition according to temperature. I can.

In the present invention, "mussel adhesion protein" is an adhesion protein derived from mussels, preferably Mytilus edulis, Mytilus galloprovincialis, or Mytilus co. Rusucus (Mytilus coruscus) derived from the mussel adhesion protein, including, but not limited to, a variant thereof.

For example, the mussel adhesion protein of the present invention is Mefp (Mytilus edulis foot protein)-1, Mgfp (Mytilus galloprovincialis foot protein)-1, Mcfp (Mytilus coruscus foot protein)-1, Mefp- respectively derived from the mussel species. 2, Mefp-3, Mgfp-3 and Mgfp-5 or a variant thereof may be included, preferably fp (foot protein)-1 (SEQ ID NO: 1), fp-2 (SEQ ID NO: 5), fp-3 (SEQ ID NO: 6), fp-4 (SEQ ID NO: 7), fp-5 (SEQ ID NO: 8), and fp-6 (SEQ ID NO: 9) A protein selected from the group consisting of, or a fusion in which two or more proteins are linked Protein, or a variant of the protein, but is not limited thereto.

In addition, the mussel adhesive protein of the present invention includes all mussel adhesive proteins described in International Publication No. WO2006/107183 or WO2005/092920. Preferably, the mussel adhesive protein is fp-151 (SEQ ID NO: 10), fp-131 (SEQ ID NO: 12), fp-353 (SEQ ID NO: 13), fp-153 (SEQ ID NO: 14), fp-351 (SEQ ID NO: 15) may include a fusion protein such as, but is not limited thereto, preferably fp-151 (SEQ ID NO: 10).

In addition, the biomaterial of the present invention may be in the form of a hydrogel.

The term “hydrogel” refers to a three-dimensional structure composed of a hydrophilic polymer network, and exhibits properties such as high moisture content, porous structure, soft physical properties, and biocompatibility. The biomaterial of the present invention can be immediately changed into a hydrogel form through a phase transition at 30 to 50°C. Therefore, the mussel adhesive protein to which the temperature-sensitive polymer of the present invention is conjugated can be immediately phase-transferred from the body to the hydrogel by the temperature-sensitive polymer, and at the same time have a high moisture content, a porous structure, and biocompatibility due to the mussel adhesive protein.

The mussel adhesive protein to which the temperature-sensitive polymer is bonded according to the present invention can easily adjust its physical properties according to the purpose, and is not limited thereto in order to manufacture a biomaterial having excellent moisture-retaining ability and volume-retaining ability, but the total temperature-sensitive polymer The concentration of the conjugated mussel adhesive protein may be preferably adjusted to 40 wt% to 55 wt%, and the protein content in the conjugate may be adjusted to 12 wt% to 20 wt%.

In addition, the temperature-sensitive biological material comprising the mussel adhesive protein to which the temperature-sensitive polymer of the present invention is bonded can be used for adhesion to the tissue, and the present invention is a temperature for tissue adhesion including the mussel adhesive protein to which the temperature-sensitive polymer is bonded. It provides a sensitive biomaterial and a composition for tissue regeneration.

The temperature-sensitive biomaterial of the present invention is excellent in biocompatibility because it does not induce inflammation at the injection site, and is applied locally in vivo so that it can be adhered to the desired soft tissue defect site and maintained for a long time, and maintains the volume at the injection site. Since it induces a high level of neovascularization, it can be usefully used for tissue adhesion or regeneration. In the present invention, the tissues are skin, cartilage, bone, blood vessels, brain, liver, heart, ligaments, muscles, spinal cord, blood, bone marrow, lungs, teeth, nerves, cornea, retina, esophagus, spine, kidney, pancreas and urethra It may be selected from the group consisting of, and when the tissue is soft tissue, the soft tissue is muscle, fat, blood vessel, nerve, tendon, ligament, articular membrane, skin, dermis, pericardium, fascia, cartilage, dura mater, endocardial membrane , Mucosal tissue may be selected from the group consisting of placenta membrane, periosteum, bladder, small intestine or large intestine, urethra and placenta, but is not limited thereto.

In the present invention, the composition for tissue regeneration may preferably be a composition for regeneration of soft tissue, and the bonding ability of the mussel adhesive protein itself to which a temperature-sensitive polymer is bonded is used to induce the bonding of biological tissues to regenerate the tissue or tissue regeneration. After loading a drug, a cell therapy, etc., it is administered to the tissue and immediately gelled at a human body temperature, thereby sustaining the intended effect at the site for a long time so that the tissue can be smoothly regenerated.

The mussel adhesive protein to which the temperature-sensitive polymer is conjugated for tissue regeneration may be included in a concentration of 20 to 50 wt%, and preferably may be included in a concentration of 30 to 40 wt%. The higher the concentration of the mussel adhesive protein to which the total temperature-sensitive polymer is conjugated, and the higher the total protein content, the better the tissue adhesion.

The composition for tissue regeneration of the present invention may be applied in various forms, and in particular, may be injected directly into the human body in the form of an injection composition. Since the composition for tissue regeneration of the present invention can be changed into an aqueous solution state at room temperature and a gel state in the body due to the conjugation of a temperature-sensitive polymer, when applied as an injection, it can be easily injected into a local part of the body through an injection needle without clogging. Once injected, it can gel and allow the desired effect to last for a long time. When the composition of the present invention is injected through an injection needle, for example, it may be injected in vivo using a 15 to 26G, 15 to 21G, preferably 18G injection needle.

In addition, the composition for tissue regeneration of the present invention may additionally include stem cells in addition to the mussel adhesive protein to which the temperature-sensitive polymer is conjugated.

When stem cells are additionally included in the composition for tissue regeneration according to the present invention, the stem cells can be injected into a local area in a living body in a mixed state with the temperature-sensitive polymer of the present invention, and differentiation is induced at the injection site without biotoxicity. As a result, the therapeutic effect as a target cell therapy can be achieved. In the present invention, the stem cells are stem cells derived from adipose tissue, bone marrow, umbilical cord tissue, Wharton's jelly, amniotic fluid, placenta, peripheral blood, fallopian tubes, corneal stroma, lung, muscle, skin, bone, tooth tissue, and fetal liver. May include without limitation.

In addition, the present invention provides a cell support comprising a mussel adhesive protein to which a temperature sensitive polymer is conjugated.

The mussel adhesive protein to which the temperature-sensitive polymer of the present invention is conjugated can be immediately hydrogelated without treatment with a chemical crosslinking agent, so it is not cytotoxic and can be used as a cell support.

The "cell support" is a physical support made to enable in vitro culture and implantation of cells. The physicochemical properties of the scaffold can attach and transfer cells, induce differentiation and promote cell growth, and are preferably biocompatible and biodegradable.

"Cell" applicable to the cell support of the present invention is a functional and structural basic unit of all organisms, including thymic epithelial cells, epithelial cells, skin cells, endothelial cells, vascular cells, bone cells, osteoblasts, chondrocytes, fibroblasts, In addition to various adult cells including muscle cells, adipocytes, nerve cells, immune cells, blood cells, and connective tissue cells, it is used in a broad sense to include cells that can differentiate into various cells such as stem cells and hematopoietic stem cells. Not limited.

According to the present invention, it is possible to easily prepare a cell support on which cells are supported by simply mixing the mussel adhesive protein conjugated with a temperature-sensitive polymer with cells, and for immediate hydrogelization at a temperature near body temperature and maintaining a solid gel shape, The concentration of the mussel adhesive protein to which the total temperature-sensitive polymer is conjugated may be finally adjusted to 30 wt% to 50 wt%, preferably 40 wt% to 45 wt%. In addition, the final cell concentration for securing a sufficient amount of cells and differentiation into soft tissue may be 10 ⁵ cells/ml to 10 ⁷ cells/ml, preferably 10 ⁶ ells/ml to 10 ⁷ cells/ml.

In addition, the present invention provides a carrier for drug delivery comprising a mussel adhesive protein to which a temperature sensitive polymer is conjugated.

The mussel adhesive protein to which the temperature-sensitive polymer of the present invention is conjugated may be present in an aqueous solution state at room temperature by the temperature-sensitive polymer, and may be changed to a gel state by a strong hydrophobic interaction near body temperature. Therefore, when the drug is loaded at room temperature and then enters the body and becomes a gel, it contracts and simultaneously releases the supported drug, so that it can be used as a drug delivery carrier that can control the drug release behavior.

In addition, the present invention comprises the steps of forming an amide bond by reacting a temperature-sensitive polymer with a mussel adhesive protein; It provides a method of manufacturing a temperature-sensitive biological material comprising a.

More specifically, the amide bond can be induced by a temperature-sensitive polymer having a functional group such as a carboxy group and a mussel adhesive protein having an amine group through an EDC/NHS-based amide bond formation method. In addition, in order to prevent self-binding in the protein, the reactivity of unreacted EDC can be suppressed by adding a deactivator such as 2-mercaptoethanol after the activation of the polymer functional group. In addition, after the amide bond is formed, a step of separating the unreacted polymer and EDC/NHS through dialysis may be additionally performed in order to separate the formed protein-polymer conjugate.

The step of forming an amide bond by reacting the temperature-sensitive polymer with the mussel adhesive protein may specifically include adjusting the pH to 5 to 6 to activate the carboxy group of the temperature-sensitive polymer; Adding a deactivating agent to remove the reactivity of unreacted EDC; inducing the formation of an amide bond between the carboxy group of the polymer and the mussel adhesive protein at pH7 to pH8; It may include performing sequentially. The reaction time for forming the amide bond may be about 10 to 20 hours, preferably about 15 to about 20 hours, more preferably about 16 hours, but the reaction amount of the temperature-sensitive polymer and mussel adhesive protein to be conjugated It can be appropriately adjusted according to.

The numerical values described in the present specification should be construed as including an equivalent range unless otherwise specified.

Hereinafter, the present invention will be described in detail by way of manufacturing examples and examples. However, the following Preparation Examples and Examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following Preparation Examples and Examples.

Example 1. Temperature Sensitivity Polymer Spliced Production of mussel adhesive protein

1.1 Recombinant mussel adhesive protein fp -151 production

Mussel adhesion protein fp-151 is an fp-1 variant consisting of six decapeptides so that decapeptide, composed of 10 amino acids repeated 80 times, can be expressed in E. coli among the natural mussel adhesion protein fp-1. Was synthesized, and the gene of Mgfp-5 (Genbank No. AAS00463 or AY521220) was inserted between the two fp-1 variants, and then successfully expressed in E. coli and produced through a simple purification and separation process using acetic acid (DS Hwang et al. al., Biomaterials 28, 3560-3568, 2007). Specifically, in the amino acid sequence of fp-1 (Genbank No. Q27409 or S23760), a fp-1 variant (hereinafter referred to as 6xAKPSYPPTYK) in which the peptide consisting of AKPSYPPTYK is repeatedly linked 6 times was prepared, and the N-terminus of Mgfp-5 Fp-151 was prepared by combining 6xAKPSYPPTYK and also combining 6xAKPSYPPTYK at the C-terminus of Mgfp-5. The specific preparation of the mussel adhesive protein is the same as that shown in International Patent Publication No. WO2006/107183 or WO2005/092920, and the prepared fp-151 is shown in SEQ ID NO: 10.

1.2 temperature Sensitivity Polymer Spliced Preparation of recombinant mussel adhesive protein

In order to prepare a recombinant mussel adhesive protein conjugated with a temperature-sensitive polymer, PNIPAM (poly(N-isopropylacrylamide; poly(N-isopropylacrylamide)) was selected among the temperature-sensitive polymers and used in subsequent experiments.

The reaction between the amine group (-NH ₂ ) of fp-151 of Example 1-1 and the carboxy group (-COOH) of PNIPAM was induced to form an amide bond, which is a stable bond.

More specifically, EDC and NHS were added to form an amide bond, and the pH was adjusted to 5.5 to activate the carboxy group of the polymer, and a deactivator such as 2-mercaptoethanol was added to eliminate the reactivity of unreacted EDC. And inducing the formation of an amide bond between the carboxy group of the polymer activated at pH 7.4 and the amine group of the protein. After about 16 hours of reaction, a protein-polymer conjugate was obtained through dialysis. The recombinant mussel protein conjugated with the temperature-sensitive polymer prepared as described above was named MAP-PNIPAM conjugate. During the reaction, according to the concentration of the amine group of the protein and the carboxy reaction concentration of the polymer, it was named as 3:1, 2:1 and 1:1. Specifically, the carboxy concentration of the polymer was fixed to 16 mM, and the amine group concentration of the protein was changed to 48, 32, and 16 mM. After dialysis, the degree of reaction of the protein and the MAP-PNIPAM conjugate were confirmed through an electrophoresis experiment, which is shown in FIG. 1.

As shown in FIG. 1, in the case of the unreacted protein, a clear protein band was found near the expected molecular weight of 22.6 kDa, whereas in the case of the protein reacted with a polymer having a carboxy group, the amine group of the protein and the carboxy group of the polymer were It was confirmed that the molecular weight of the protein increased and the molecular weight range was also widely distributed according to the degree of binding of the polymer and protein according to the control of the number of moles of the reaction.

Example 2. MAP- PNIPAM Temperature of conjugate Sensitivity And phase transition check

2.1. Low protein concentration Polymer Temperature of conjugate Sensitivity Phase change confirmation experiment

PNIPAM, a temperature-sensitive polymer, exhibits a phase transition characteristic of reducing light transmittance at the phase transition temperature. In order to confirm whether the MAP-PNIPAM conjugate produced through Example 1.2 also maintains the temperature-sensitive property as it is, the temperature-sensitive phase transition was confirmed. The MAP-PNIPAM conjugate was dissolved in distilled water at a low concentration, and a temperature-sensitive phase transition was confirmed using light transmittance. Specifically, in order to prepare a low-concentration temperature-sensitive protein-based material, freeze-dried 3:1, 2:1, and 1:1 MAP-PNIPAM conjugates are dissolved in distilled water, respectively, and final concentrations based on protein 0.8, 1.2 and 2.0 mg/ml Sample solutions containing a were prepared. Thereafter, the temperature was increased from 25°C to 45°C at a rate of 1.8°C in the 600 nm wavelength band, and it was confirmed whether or not the light transmittance of the solution was decreased, and the results are shown in FIG.

As shown in FIG. 2, it was confirmed that the 1:1 MAP-PNIPAM conjugate in which the polymer is most bound in the conjugate solution containing the same protein concentration (2 mg/ml) has the lowest phase transition temperature, through which the amine group of the protein and By controlling the number of moles of reaction of the carboxy group of the polymer, it was confirmed that the higher the degree of binding of the polymer to the protein, the lower the hydrophilic effect by the protein, and thus the lower the phase transition temperature.

In addition, even with the same MAP-PNIPAM conjugate, it was confirmed whether the phase transition temperature was lowered by increasing the phase transition concentration of PNIPAM as the concentration of the conjugate protein increased, and the results are shown in FIG. 3.

As shown in FIG. 3, as the protein concentration of the conjugate increases from 0.8 to 2 mg/ml, that is, as the total conjugate concentration increases, the phase transition temperature of PNIPAM easily occurs.

2.2. High protein concentration Polymer Temperature of conjugate Sensitivity Phase change confirmation experiment

The temperature-sensitive phase transition of the temperature-sensitive protein-polymer conjugate MAP-PNIPAM produced in Example 1.2 was confirmed by using the increase in mechanical properties at the phase transition temperature, which is characterized by the phase transition of the temperature-sensitive polymer PNIPAM. MAP-PNIPAM was dissolved in distilled water at a high concentration, and a temperature-sensitive phase transition was confirmed using a rheometer.

Specifically, in order to prepare a high-concentration temperature-sensitive protein-based material, freeze-dried MAP-PNIPAM was dissolved in distilled water to prepare sample solutions containing a final polymer concentration of 36.8 mg/ml. Apply this to a 20 mm flat plate and increase the temperature at a rate of 5°C for a constant strain of 15% and a temperature range of 15°C to 45°C at a constant frequency of 1 rad/s. The experiment was conducted, and the results are shown in Table 1 and FIG. 4.

[Table 1]

As shown in Table 1 and Figure 4, the higher the degree to which the polymer is bound to the protein in the conjugate solutions containing the same polymer concentration (36.8 mg/ml), the faster the phase transition occurs. . In addition, the phase transition temperature can be obtained as a median value between the maximum value of the storage modulus and the minimum value. It was confirmed that the higher the degree of binding of the polymer to the protein, the lower the phase transition temperature.

Example 3. Protein- Polymer Conjugate Phase change And check the ease of injection

In order to further confirm the temperature-sensitive phase transition of the mussel adhesive protein conjugate MAP-PNIPAM to which the temperature-sensitive polymer produced in Example 1.2 was conjugated, an experiment was performed in which a solution was injected with physiological saline at 37°C. Specifically, in order to prepare a high-concentration temperature-sensitive protein-based material, a freeze-dried MAP-PNIPAM conjugate was dissolved in distilled water at 25° C. to prepare a conjugate solution having a final concentration of 50 wt%. This was directly injected into physiological saline at 37° C. through a 26G syringe, and the results are shown in FIG. 5.

As shown in FIG. 5, the prepared high concentration MAP-PNIPAM conjugate solution was injected with physiological saline at 37° C. without clogging the syringe needle, and a hydrogel was formed immediately and rapidly by increasing the temperature in the physiological saline solution. Through these results, it was confirmed that the MAP-PNIPAM conjugate of the present invention has a temperature-sensitive phase transition ability and can be used as a material based on a temperature-sensitive protein.

Example 4. Temperature Sensitivity Polymer -Based on protein conjugates Hydrogel Water retention capacity within

In the case of PNIPAM, which is a representative temperature-sensitive polymer, it has a technical limitation that is difficult to easily apply to applications such as tissue engineering as a cell support or carrier because of the dramatic volume reduction and water loss resulting from the phase transition. Therefore, compared with the conventional polymer, the water retention ability in the hydrogel based on the mussel adhesive protein conjugated with the temperature-sensitive polymer of the present invention was evaluated, and water retention ability was evaluated to confirm the effect of the mussel adhesive protein.

Specifically, in order to prepare a high-concentration temperature-sensitive protein-based material to be used in cell and animal experiments, the lyophilized MAP-PNIPAM conjugate of Example 2.1 was dissolved in distilled water, and the sample containing the final polymer concentrations 22.1 and 36.8 mg/ml The solutions were prepared. After storing this at 37° C. for 48 hours, the amount of water that came out was measured, and the difference from the initial volume was calculated to evaluate the water retention ability, and the results are shown in FIG. Without chemical bonding between the protein and the polymer, the solution simply mixed was named simple mixing and used as a control.

As shown in FIG. 6, compared to the conventional polymer gel in which about 90% of water escapes, the protein-introduced protein-polymer hydrogel, MAP-PNIPAM conjugate, showed much superior water retention ability. In addition, by confirming the highest water retention in the 3:1 MAP-PNIPAM conjugate, in conjugate solutions containing the same polymer concentration (36.8 mg/ml), the higher the protein content, that is, the lower the degree of polymer binding to the protein. , It was confirmed that the water retention ability was improved by increasing the hydrophilic effect by the protein. In addition, it was confirmed that the higher the total conjugate concentration, that is, the higher the total protein content, the better the water retention ability.

Example 5. Temperature Sensitivity Polymer -Protein conjugate-based Hydrogel Microstructure analysis

PNIPAM, a temperature-sensitive polymer, has a technical limitation that is difficult to easily apply to applications such as tissue engineering due to the lack of porosity due to dramatic volume reduction as a phase transition occurs. Compared with conventional polymers, scanning electron microscopy analysis was conducted to determine whether the microstructure in the hydrogel based on the temperature-sensitive polymer conjugated mussel-adhesive protein was changed by conjugation of the mussel-adhesive protein.

Specifically, in order to prepare a high-concentration temperature-sensitive protein-based material to be used in cell and animal experiments, the lyophilized MAP-PNIPAM conjugate of Example 2.1 was dissolved in distilled water to prepare a sample solution containing 36.8 mg/ml of a final polymer concentration. Ready. This was stored at 37° C. for 24 hours to induce phase transition, cooled with nitrogen and then lyophilized to perform electron microscopic analysis, and the results are shown in FIG. 7.

As shown in FIG. 7, it was confirmed that the MAP-PNIPAM conjugate had a porous structure having a larger pore size compared to the conventional polymer gel that showed a dense and collapsed pore of 5 μm or less. From these results, it was confirmed that the MAP-PNIPAM conjugate can solve the problem of lack of porosity of the existing temperature-sensitive polymer PNIPAM by binding the mussel adhesive protein.

Example 6. Temperature Sensitivity protein- Polymer Conjugate based Hydrogel Tissue adhesion evaluation

PNIPAM has the advantage of being able to be conveniently injected into the body because it has a phase transition temperature similar to that of the body temperature, but it is highly likely that the functional material injected into the defective tissue cannot be fixed because it has little bio-adhesive ability. In order to check whether the MAP-PNIPAM conjugate prepared in Example 2 has superior bio-adhesion ability compared to the conventional polymer, tissue adhesion evaluation using pig skin was performed.

Specifically, in order to prepare a high-concentration temperature-sensitive protein-based material to be used in cell and animal experiments, a freeze-dried MAP-PNIPAM conjugate was dissolved in distilled water to prepare sample solutions containing a final concentration of 22.1 and 36.8 mg/ml. Pig skin having a diameter of 8 mm was fixed with double-sided tape on the upper and lower plates of the rheometer, and then the MAP-PNIPAM conjugate solution was applied to the pig skin fixed to the lower plate. Thereafter, after the pig skin of the upper plate was brought into contact, the solution between the plates was waited for 5 minutes at 37° C. based on the solution temperature, and then the maximum force was measured while pulling at a rate of 0.3 mm/min. The results are shown in FIG. 8.

As shown in Figure 8, compared to the conventional polymer PNIPAM alone gel, the protein-introduced MAP-PNIPAM conjugate hydrogel confirmed about 8 times higher tissue adhesion ability. In addition, it was confirmed that the higher the total conjugate concentration, that is, the higher the total protein content, the better the tissue adhesion ability. These results indicate that the tissue adhesion ability of the existing polymer can be improved through the binding of mussel adhesive proteins, and through this, the problem that the existing polymer gel is easily washed away can be improved, and thus it can be usefully used for tissue regeneration.

Example 7. Temperature Sensitivity protein- Polymer Conjugate based Hydrogel Cell culture and in vivo injection

7.1 MAP- PNIPAM Conjugate Hydrogel Used cell culture

The mussel adhesive protein-based hydrogel to which the temperature-sensitive polymer of the present invention is bonded has the temperature sensitivity of the temperature-sensitive polymer and at the same time improves moisture retention, porosity, and tissue adhesion due to the adhesion of the mussel adhesive protein. In order to check whether it can be used as a cell, an experiment was conducted to confirm cell introduction and cell survival.

Specifically, a pre-cultured rat-derived adipose derived mesenchymal stem cell (ASC) solution was mixed with the solution of the MAP-PNIPAM conjugate prepared in Example 2.1 to obtain a final cell concentration of 10 ⁶ cells/ml and a conjugate. The solution concentration was set to 40 wt%. This was placed in an incubator at 37° C. for 5 minutes to become a hydrogel, and then cultured by adding a medium. On the 5th day of cell culture, cell viability was confirmed through the nucleus and actin fluorescence staining of the cells in the hydrogel. At this time, collagen gel was used as a positive control control group, and the results are shown in FIG. 9.

As shown in FIG. 9, it was confirmed that the cells can survive at a level similar to that of the collagen hydrogel in the MAP-PNIPAM conjugate hydrogel.

7.2 MAP- PNIPAM Stem cell injection using conjugate

It was confirmed that stem cells can survive in the MAP-PNIPAM conjugate hydrogel through 7.1, and a rat experiment was performed to confirm whether gelation can be achieved at a desired site when directly injected in vivo.

Specifically, a pre-cultured rat-derived mesenchymal stem cell solution was mixed with the MAP-PNIPAM conjugate solution of Example 2.1 to prepare a final cell concentration of 10 ⁶ cells/ml and a conjugate solution concentration of 40 wt%. This was put into an 18G syringe, and about 200g was injected into the rat dorsal subcutaneous fat, and the results are shown in FIG. 10.

As shown in FIG. 10, when the MAP-PNIPAM conjugate solution and the mesenchymal stem cell solution were mixed and injected through an 18G needle, it was smoothly injected without clogging and escaping the syringe needle, and by body temperature at the site where the syringe was inserted. It was confirmed that hydrogelation was performed immediately.

7.3. After in vivo injection, MAP- PNIPAM Confirmation of conjugate biocompatibility, volume retention and new blood vessel formation

Through the above 7.1 and 7.2, it was confirmed that stem cells can survive in the MAP-PNIPAM conjugate hydrogel, and that immediate hydrogelization is achieved by easily injecting into the body through a syringe needle, MAP-PNIPAM hydrogel including stem cells after in vivo injection The biocompatibility, volume retention ability, and neovascularization effect of the gel were confirmed.

Specifically, according to Example 7.1, MAP-PNIPAM conjugate solution and mesenchymal stem cells were in vivo injected, tissues on days 14 and 21 were recovered, fixed in formalin solution, and H&E staining and CD31 staining were performed using tissue sections. And the results are shown in Figs. 11 and 12, respectively. As a control group, alginate and fat stem cells were mixed and administered.

As shown in Figure 11, in the case of the MAP-PNIPAM hydrogel, it was confirmed that a slight level of inflammatory response and a high volume retention ability were shown, but in the case of the alginate hydrogel used as a control group, a relatively higher level of inflammatory response was observed. I confirmed that it appeared.

In addition, as shown in FIG. 12, in the case of the MAP-PNIPAM hydrogel, a high level of new blood vessel generation was observed, unlike in the case of the MAP-PNIPAM hydrogel, whereas the formation of new blood vessels was not observed and only calcification was observed in the alginate hydrogel.

These results are results showing that the MAP-PNIPAM hydrogel of the present invention not only has high biocompatibility and high volume retention capacity during in vivo injection, but also can induce new blood vessel generation.

7.4. After in vivo injection, MAP- PNIPAM Confirmation of differentiation of stem cells into adipocytes in conjugate

When injecting a mixture of MAP-PNIPAM conjugate hydrogel and adipose stem cells, confirm the differentiation of the injected adipose stem cells into adipocytes (adipogenesis) in order to confirm whether the actual adipose tissue can be regenerated and used for the treatment of soft tissue defects. I did.

Specifically, the tissue on the 21st day in which the MAP-PNIPAM conjugate and adipose stem cells were injected according to Example 7.1 was recovered, fixed in a formalin solution, and then the formation of adipose in the tissue was confirmed with an optical microscope using a tissue section. Collagen IV and peroxisome-proliferator activated receptor (PPARγ), which are important markers for differentiation into adipocytes, were stained with a fluorescent antibody method (immunofluorescence staining) and confirmed through a fluorescence microscope, and the results are shown in FIG.

As shown in FIG. 13, in the case of adipocytes in the alginate hydrogel used as a control group, unlike adipocytes, differentiation into adipocytes was hardly observed, in the case of adipocytes in the MAP-PNIPAM hydrogel, a higher amount of fat It was observed that differentiation into fat occurs effectively through production and detection of higher levels of collagen IV and PPARγ fluorescence.

Example 8. Pluronic ( Pluronic )-127 this Spliced Mussel adhesion protein conjugate

8.1 MAP- Pluronic Preparation of conjugate

Since it was confirmed that the mussel adhesive protein conjugate to which the temperature-sensitive polymer was conjugated has an excellent adhesion effect by causing a phase transition at a temperature near body temperature, whether a temperature-sensitive polymer and a conjugate having different types of mussel adhesive protein were additionally prepared to show the same effect. Was confirmed.

Pluronic-127 was used as a temperature-sensitive polymer, and fp-1 of SEQ ID NO: 1 was used as a mussel adhesion protein, and a stable reaction was induced between the amine group of fp-1 and the carboxy group of Pluronic-127. A bond, an amide bond, was formed. More specifically, EDC and NHS were added to form an amide bond, and the pH was adjusted to 5.5 to activate the carboxy group of the polymer, and a deactivator such as 2-mercaptoethanol was added to eliminate the reactivity of unreacted EDC. And inducing the formation of an amide bond between the carboxy group of the polymer activated at pH 7.4 and the amine group of the protein. After about 16 hours of reaction, a protein-polymer conjugate was obtained through dialysis. The recombinant mussel protein to which the temperature-sensitive polymer prepared as described above was conjugated was designated as MAP-pluronic conjugate. During the reaction, each conjugate was named 0.5:1, 1:1, and 2:1 according to the concentration of the amine group of the protein and the carboxy reaction concentration of the polymer. Specifically, the carboxy concentration of the polymer was fixed to 16 mM, and the amine group concentration of the protein was changed to 8, 16 and 32 mM.

After dialysis, the degree of reaction of the protein and the MAP-Pluronic-127 conjugate were confirmed through an electrophoresis experiment, which is shown in FIG. 14.

As shown in FIG. 14, it was confirmed that the molecular weight of the protein increased and the molecular weight range was widely distributed according to the degree of binding of the polymer to the protein according to the control of the number of moles of reaction of the amine group of the protein and the carboxy group of the polymer.

8.2 MAP- Pluronic Confirmation of the phase transition of the conjugate

In order to confirm the temperature-sensitive phase transition of the MAP-Pluronic conjugate, the mussel adhesive protein conjugate to which the temperature-sensitive polymer prepared in Example 8.1 is conjugated, the prepared MAP-Pluronic conjugates 0.5:1, 1:1, and The change of state was measured while adjusting the concentration of 2:1 to 35wt(%) and increasing the temperature per minute by 0.5°C. The results of phase transition measured at 4, 25, and 37°C are shown in Table 2 and FIG. 15.

[Table 2]

As confirmed in Table 2, since the MAP-Pluronic conjugate contains a temperature-sensitive polymer sufficient to cause a phase transition when the total conjugate concentration is 35 wt (%), it is confirmed that the solution-gel phase transition occurs according to the temperature. I did. In addition, even with the same MAP-pluronic conjugate, it was confirmed that the higher the degree of binding of the polymer to the protein, the lower the hydrophilic effect by the protein, so that the phase transition temperature was lower.

FIG. 15 is a result of vertically moving the tube containing the aqueous solution while adding 35wt(%) of MAP-Pluronic conjugate 2:1 in the form of an aqueous solution in a tube and increasing 0.5°C per minute. At 25°C, room temperature (left) It was confirmed that the conjugate was in the form of an aqueous solution and the gel adhered to the bottom of the tube at 37°C (right), which is a body temperature range.

Through the results as described above, the mussel adhesive protein conjugate to which the temperature-sensitive polymer of the present invention is conjugated can be hydrogelized immediately in the body temperature range, and during phase transition, moisture retention and porosity are maintained, there is no cytotoxicity, and tissue adhesion is excellent. , It was confirmed that it can be used as a useful biomaterial for regeneration of soft tissue defects.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Thermo-responsive biomaterial comprising thermo-responsive protein conjugated-mussel adhesive protein <130> P1-50p-1 <150> KR 2017-0109641 <151> 2017-08-29 <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 800 <212> PRT <213> Artificial Sequence <220> <223> fp-1 <400> 1 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 50 55 60 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 65 70 75 80 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 85 90 95 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 100 105 110 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 115 120 125 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 130 135 140 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 145 150 155 160 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 165 170 175 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 180 185 190 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 195 200 205 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 210 215 220 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 225 230 235 240 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 245 250 255 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 260 265 270 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 275 280 285 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 290 295 300 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 305 310 315 320 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 325 330 335 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 340 345 350 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 355 360 365 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 370 375 380 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 385 390 395 400 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 405 410 415 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 420 425 430 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 435 440 445 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 450 455 460 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 465 470 475 480 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 485 490 495 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 500 505 510 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 515 520 525 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 530 535 540 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 545 550 555 560 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 565 570 575 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 580 585 590 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 595 600 605 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 610 615 620 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 625 630 635 640 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 645 650 655 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 660 665 670 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 675 680 685 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 690 695 700 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 705 710 715 720 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 725 730 735 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 740 745 750 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 755 760 765 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 770 775 780 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 785 790 795 800 <210> 2 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> fragment sequence derived from fp-1 <400> 2 Ala Leu Ala Leu Tyr Ser Pro Arg Ser Glu Arg Thr Tyr Arg Pro Arg 1 5 10 15 Pro Arg Thr His Arg Thr Tyr Arg Leu Tyr Ser 20 25 <210> 3 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> fp-1 variant <400> 3 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 50 55 60 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 65 70 75 80 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 85 90 95 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 100 105 110 Pro Ser Tyr Pro Pro Thr Tyr Lys 115 120 <210> 4 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> fp-1 variant-RGD <400> 4 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 50 55 60 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 65 70 75 80 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 85 90 95 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 100 105 110 Pro Ser Tyr Pro Pro Thr Tyr Lys Gly Arg Gly Asp Ser Pro 115 120 125 <210> 5 <211> 472 <212> PRT <213> Artificial Sequence <220> <223> fp-2 <400> 5 Leu Phe Ser Phe Phe Leu Leu Leu Thr Cys Thr Gln Leu Cys Leu Gly 1 5 10 15 Thr Asn Arg Pro Asp Tyr Asn Asp Asp Glu Glu Asp Asp Tyr Lys Pro 20 25 30 Pro Val Tyr Lys Pro Ser Pro Ser Lys Tyr Arg Pro Val Asn Pro Cys 35 40 45 Leu Lys Lys Pro Cys Lys Tyr Asn Gly Val Cys Lys Pro Arg Gly Gly 50 55 60 Ser Tyr Lys Cys Phe Cys Lys Gly Gly Tyr Tyr Gly Tyr Asn Cys Asn 65 70 75 80 Leu Lys Asn Ala Cys Lys Pro Asn Gln Cys Lys Asn Lys Ser Arg Cys 85 90 95 Val Pro Val Gly Lys Thr Phe Lys Cys Val Cys Arg Asn Gly Asn Phe 100 105 110 Gly Arg Leu Cys Glu Lys Asn Val Cys Ser Pro Asn Pro Cys Lys Asn 115 120 125 Asn Gly Lys Cys Ser Pro Leu Gly Lys Thr Gly Tyr Lys Cys Thr Cys 130 135 140 Ser Gly Gly Tyr Thr Gly Pro Arg Cys Glu Val His Ala Cys Lys Pro 145 150 155 160 Asn Pro Cys Lys Asn Lys Gly Arg Cys Phe Pro Asp Gly Lys Thr Gly 165 170 175 Tyr Lys Cys Arg Cys Val Asp Gly Tyr Ser Gly Pro Thr Cys Gln Glu 180 185 190 Asn Ala Cys Lys Pro Asn Pro Cys Ser Asn Gly Gly Thr Cys Ser Ala 195 200 205 Asp Lys Phe Gly Asp Tyr Ser Cys Glu Cys Arg Pro Gly Tyr Phe Gly 210 215 220 Pro Glu Cys Glu Arg Tyr Val Cys Ala Pro Asn Pro Cys Lys Asn Gly 225 230 235 240 Gly Ile Cys Ser Ser Asp Gly Ser Gly Gly Tyr Arg Cys Arg Cys Lys 245 250 255 Gly Gly Tyr Ser Gly Pro Thr Cys Lys Val Asn Val Cys Lys Pro Thr 260 265 270 Pro Cys Lys Asn Ser Gly Arg Cys Val Asn Lys Gly Ser Ser Tyr Asn 275 280 285 Cys Ile Cys Lys Gly Gly Tyr Ser Gly Pro Thr Cys Gly Glu Asn Val 290 295 300 Cys Lys Pro Asn Pro Cys Gln Asn Arg Gly Arg Cys Tyr Pro Asp Asn 305 310 315 320 Ser Asp Asp Gly Phe Lys Cys Arg Cys Val Gly Gly Tyr Lys Gly Pro 325 330 335 Thr Cys Glu Asp Lys Pro Asn Pro Cys Asn Thr Lys Pro Cys Lys Asn 340 345 350 Gly Gly Lys Cys Asn Tyr Asn Gly Lys Ile Tyr Thr Cys Lys Cys Ala 355 360 365 Tyr Gly Trp Arg Gly Arg His Cys Thr Asp Lys Ala Tyr Lys Pro Asn 370 375 380 Pro Cys Val Val Ser Lys Pro Cys Lys Asn Arg Gly Lys Cys Ile Trp 385 390 395 400 Asn Gly Lys Ala Tyr Arg Cys Lys Cys Ala Tyr Gly Tyr Gly Gly Arg 405 410 415 His Cys Thr Lys Lys Ser Tyr Lys Lys Asn Pro Cys Ala Ser Arg Pro 420 425 430 Cys Lys Asn Arg Gly Lys Cys Thr Asp Lys Gly Asn Gly Tyr Val Cys 435 440 445 Lys Cys Ala Arg Gly Tyr Ser Gly Arg Tyr Cys Ser Leu Lys Ser Pro 450 455 460 Pro Ser Tyr Asp Asp Asp Glu Tyr 465 470 <210> 6 <211> 50 <212> PRT <213> Artificial Sequence <220> <223> fp-3 <400> 6 Pro Trp Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr 1 5 10 15 Gly Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys 20 25 30 Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 Gly Ser 50 <210> 7 <211> 750 <212> PRT <213> Artificial Sequence <220> <223> fp-4 <400> 7 Tyr Gly Arg Arg Tyr Gly Glu Pro Ser Gly Tyr Ala Asn Ile Gly His 1 5 10 15 Arg Arg Tyr Tyr Glu Arg Ala Ile Ser Phe His Arg His Ser His Val 20 25 30 His Gly His His Leu Leu His Arg His Val His Arg His Ser Val Leu 35 40 45 His Gly His Val His Met His Arg Val Ser His Arg Ile Met His Arg 50 55 60 His Arg Val Leu His Gly His Val His Arg His Arg Val Leu His Asn 65 70 75 80 His Val His Arg His Ser Val Leu His Gly His Val His Arg His Arg 85 90 95 Val Leu His Arg His Val His Arg His Asn Val Leu His Gly His Val 100 105 110 His Arg His Arg Val Leu His Lys His Val His Asn His Arg Val Leu 115 120 125 His Lys His Leu His Lys His Gln Val Leu His Gly His Val His Arg 130 135 140 His Gln Val Leu His Lys His Val His Asn His Arg Val Leu His Lys 145 150 155 160 His Leu His Lys His Gln Val Leu His Gly His Val His Thr His Arg 165 170 175 Val Leu His Lys His Val His Lys His Arg Val Leu His Lys His Leu 180 185 190 His Lys His Gln Val Leu His Gly His Ile His Thr His Arg Val Leu 195 200 205 His Lys His Leu His Lys His Gln Val Leu His Gly His Val His Thr 210 215 220 His Arg Val Leu His Lys His Val His Lys His Arg Val Leu His Lys 225 230 235 240 His Leu His Lys His Gln Val Leu His Gly His Val His Met His Arg 245 250 255 Val Leu His Lys His Val His Lys His Arg Val Leu His Lys His Val 260 265 270 His Lys His His Val Val His Lys His Val His Ser His Arg Val Leu 275 280 285 His Lys His Val His Lys His Arg Val Glu His Gln His Val His Lys 290 295 300 His His Val Leu His Arg His Val His Ser His His Val Val His Ser 305 310 315 320 His Val His Lys His Arg Val Val His Ser His Val His Lys His Asn 325 330 335 Val Val His Ser His Val His Arg His Gln Ile Leu His Arg His Val 340 345 350 His Arg His Gln Val Val His Arg His Val His Arg His Leu Ile Ala 355 360 365 His Arg His Ile His Ser His Gln Ala Ala Val His Arg His Val His 370 375 380 Thr His Phe Glu Gly Asn Phe Asn Asp Asp Gly Thr Asp Val Asn Leu 385 390 395 400 Arg Ile Arg His Gly Ile Ile Tyr Phe Gly Gly Asn Thr Tyr Arg Leu 405 410 415 Ser Gly Gly Arg Arg Arg Phe Met Thr Leu Trp Gln Glu Cys Leu Glu 420 425 430 Ser Tyr Gly Asp Ser Asp Glu Cys Phe Val Gln Leu Leu Glu Gly Asn 435 440 445 Gln His Leu Phe Thr Val Val Gln Gly His His Ser Thr Ser Phe Arg 450 455 460 Ser Asp Leu Ser Asn Asp Leu His Pro Asp Asn Asn Ile Glu Gln Ile 465 470 475 480 Ala Asn Asp His Val Asn Asp Ile Ala Gln Ser Thr Asp Gly Asp Ile 485 490 495 Asn Asp Phe Ala Asp Thr His Tyr Asn Asp Val Ala Pro Ile Ala Asp 500 505 510 Val His Val Asp Asn Ile Ala Gln Thr Ala Asp Asn His Val Lys Asn 515 520 525 Ile Ala Gln Thr Ala His His His Val Asn Asp Val Ala Gln Ile Ala 530 535 540 Asp Asp His Val Asn Asp Ile Gly Gln Thr Ala Tyr Asp His Val Asn 545 550 555 560 Asn Ile Gly Gln Thr Ala Asp Asp His Val Asn Asp Ile Ala Gln Thr 565 570 575 Ala Asp Asp His Val Asn Ala Ile Ala Gln Thr Ala Asp Asp His Val 580 585 590 Asn Ala Ile Ala Gln Thr Ala Asp Asp His Val Asn Asp Ile Gly Asp 595 600 605 Thr Ala Asn Ser His Ile Val Arg Val Gln Gly Val Ala Lys Asn His 610 615 620 Leu Tyr Gly Ile Asn Lys Ala Ile Gly Lys His Ile Gln His Leu Lys 625 630 635 640 Asp Val Ser Asn Arg His Ile Glu Lys Leu Asn Asn His Ala Thr Lys 645 650 655 Asn Leu Leu Gln Ser Ala Leu Gln His Lys Gln Gln Thr Ile Glu Arg 660 665 670 Glu Ile Gln His Lys Arg His Leu Ser Glu Lys Glu Asp Ile Asn Leu 675 680 685 Gln His Glu Asn Ala Met Lys Ser Lys Val Ser Tyr Asp Gly Pro Val 690 695 700 Phe Asn Glu Lys Val Ser Val Val Ser Asn Gln Gly Ser Tyr Asn Glu 705 710 715 720 Lys Val Pro Val Leu Ser Asn Gly Gly Gly Tyr Asn Gly Lys Val Ser 725 730 735 Ala Leu Ser Asp Gln Gly Ser Tyr Asn Glu Gly Tyr Ala Tyr 740 745 750 <210> 8 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> fp-5 <400> 8 Lys His His His His His His Ser Ser Ser Glu Glu Tyr Lys Gly Gly Tyr 1 5 10 15 Tyr Pro Gly Asn Thr Tyr His Tyr His Ser Gly Gly Ser Tyr His Gly 20 25 30 Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys 35 40 45 Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys 50 55 60 Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly 65 70 75 80 Ser Ser <210> 9 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> fp-6 <400> 9 Ile Ala Ala Leu Cys Gly Ile Val Lys Ser Ile Asp Ser Asp Asp Ser 1 5 10 15 Asp Tyr Asp Tyr Lys Gly Arg Gly Tyr Cys Thr Asn Lys Gly Cys Arg 20 25 30 Ser Gly Tyr Asn Tyr Phe Gly Asn Lys Gly Tyr Cys Lys Tyr Gly Glu 35 40 45 Lys Ser Tyr Thr Tyr Asn Cys Asn Ser Tyr Ala Gly Cys Cys Leu Pro 50 55 60 Arg Asn Pro Tyr Gly Lys Leu Lys Tyr Tyr Cys Thr Asn Lys Tyr Gly 65 70 75 80 Cys Pro Asn Asn Tyr Tyr Phe Tyr Asn Asn Lys Gly Tyr Tyr Tyr Leu 85 90 95 Glu His His His His His His 100 <210> 10 <211> 200 <212> PRT <213> Artificial Sequence <220> <223> fp-151 <400> 10 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ser Ser 50 55 60 Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His 65 70 75 80 Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly 85 90 95 Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser 100 105 110 Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly 115 120 125 Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Gly Ser Ala Lys Pro Ser Tyr 130 135 140 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala 145 150 155 160 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro 165 170 175 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro 180 185 190 Ser Tyr Pro Pro Thr Tyr Lys Leu 195 200 <210> 11 <211> 202 <212> PRT <213> Artificial Sequence <220> <223> fp-151-RGD <400> 11 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr 1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala 20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro 35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu 50 55 60 Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser 65 70 75 80 Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys 85 90 95 Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly 100 105 110 Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr 115 120 125 Lys Lys Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr 130 135 140 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 145 150 155 160 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 165 170 175 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 180 185 190 Pro Thr Tyr Lys Gly Arg Gly Asp Ser Pro 195 200 <210> 12 <211> 171 <212> PRT <213> Artificial Sequence <220> <223> fp-131 <400> 12 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ala Asp 50 55 60 Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly Asn 65 70 75 80 Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn Asn 85 90 95 Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser Ala Lys 100 105 110 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 115 120 125 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 130 135 140 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 145 150 155 160 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Leu 165 170 <210> 13 <211> 175 <212> PRT <213> Artificial Sequence <220> <223> fp-353 <400> 13 Pro Trp Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr 1 5 10 15 Gly Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys 20 25 30 Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 Pro Trp Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr 50 55 60 Tyr His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly 65 70 75 80 Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys 85 90 95 Tyr Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr 100 105 110 His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Gly Ser Ala 115 120 125 Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly 130 135 140 Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn 145 150 155 160 Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser 165 170 175 <210> 14 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> fp-153 <400> 14 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ser Ser 50 55 60 Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His 65 70 75 80 Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly 85 90 95 Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser 100 105 110 Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly 115 120 125 Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Gly Ser Ala Asp Tyr Tyr Gly 130 135 140 Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly Asn Tyr Asn Arg 145 150 155 160 Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn Asn Gly Trp Lys 165 170 175 Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser 180 185 <210> 15 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> fp-351 <400> 15 Pro Trp Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr 1 5 10 15 Gly Gly Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys 20 25 30 Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 Pro Trp Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr 50 55 60 Tyr His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly 65 70 75 80 Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys 85 90 95 Tyr Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr 100 105 110 His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Gly Ser Ala 115 120 125 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro 130 135 140 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro 145 150 155 160 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr 165 170 175 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 180 185

Claims (14)

A temperature-sensitive biomaterial containing mussel adhesive protein to which a temperature-sensitive polymer is bonded as a single polymer.
The method of claim 1, wherein the temperature-sensitive polymer is poly(N-isopropylacrylamide) (PNIPAM), polymethacrylic acid, poly(2-hydromethacrylic acid), poly(N,N'diethylaminoethyl) Acrylate, polylactic-co-glycolate (poly(lacticco-glycolic acid), polyfumaric acid, and at least one selected from the group consisting of Pluronic-127, a temperature-sensitive biological material.
According to claim 1, The biological material is characterized in that the gelation at 30 to 50 ℃, temperature-sensitive biological material.
The method of claim 1, wherein the mussel adhesion protein is fp-1 (SEQ ID NO: 1), fp-2 (SEQ ID NO: 5), fp-3 (SEQ ID NO: 6), fp-4 (SEQ ID NO: 7), fp-5 (SEQ ID NO: 8), fp-6 (SEQ ID NO: 9), fp-151 (SEQ ID NO: 10), fp-131 (SEQ ID NO: 12), fp-353 (SEQ ID NO: 13), fp-153 (SEQ ID NO: 14) And fp-351 (SEQ ID NO: 15) at least one selected from the group consisting of, temperature-sensitive biological material.

The temperature sensitive biomaterial according to claim 1, wherein the biomaterial is in the form of a hydrogel.
The temperature sensitive biological material according to claim 1, wherein the biological material is for tissue adhesion.
A composition for tissue regeneration, comprising the temperature-sensitive biological material of claim 1.
The method of claim 7, wherein the tissue is skin, cartilage, bone, blood vessel, brain, liver, heart, ligament, muscle, spinal cord, blood, bone marrow, lung, tooth, nerve, cornea, retina, esophagus, spine, kidney, pancreas Or, characterized in that selected from the urethra, tissue regeneration composition.
The composition for tissue regeneration according to claim 7, wherein the mussel adhesive protein to which the temperature-sensitive polymer is conjugated is contained in a concentration of 20 to 50 wt%.
[8] The composition of claim 7, wherein the composition is an injection main substance.
The composition for tissue regeneration according to claim 7, further comprising stem cells.
A cell support comprising the temperature-sensitive biological material of claim 1.
A carrier for drug delivery comprising the temperature-sensitive biological material of claim 1.
Forming an amide bond by reacting a temperature-sensitive polymer with a mussel adhesive protein as a single polymer; Method for producing a temperature-sensitive biological material comprising a.

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