KR20230020362A - Novel Suckerin-based Hydrogel and Uses Thereof - Google Patents
Novel Suckerin-based Hydrogel and Uses Thereof Download PDFInfo
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- KR20230020362A KR20230020362A KR1020220094949A KR20220094949A KR20230020362A KR 20230020362 A KR20230020362 A KR 20230020362A KR 1020220094949 A KR1020220094949 A KR 1020220094949A KR 20220094949 A KR20220094949 A KR 20220094949A KR 20230020362 A KR20230020362 A KR 20230020362A
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- hydrogel
- protein
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- squid sucker
- squid
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Abstract
Description
본 발명은 신규한 오징어 빨판 이빨 단백질-기반 하이드로겔 및 이의 용도에 관한 것이다.The present invention relates to novel squid sucker teeth protein-based hydrogels and uses thereof.
일상 생활 및 작업 현장에서 상해가 발생할 경우 신속하고 안전한 응급 지혈이 과다 출혈과 이에 수반되는 생명의 위협을 막는 데에 중요한 역할을 한다. 외과 수술에서 환부의 효율적인 지혈은 환자의 출혈량을 줄이고 수혈량을 절약할 수 있으며 수술 의사의 시야를 확보함으로써 수술에 소요되는 시간과 비용을 절감할 수 있다. 지혈을 위해서는 붕대, 거즈, 수술용 봉합사, 스테이플러 등 물리적 압박 또는 전기, 초음파, 레이저 등 기기를 이용하는 방법이 사용되기도 하나 출혈 부위와 손상과 감염 등을 유발할 수 있다. 지혈의약품은 출혈 부위에 도입시 제품에 따라 물리적, 화학적, 생리활성 등의 성질을 이용해 출혈을 차단하며 상처 조직의 손상과 감염을 방지할 수 있어 안전성 및 효율성 측면에서 비교우위에 있다.When an injury occurs in daily life and at work, prompt and safe emergency hemostasis plays an important role in preventing excessive bleeding and the subsequent threat to life. Efficient hemostasis in the affected area in surgery can reduce the amount of blood loss in the patient, save the amount of blood transfusion, and save the time and cost required for surgery by securing the surgeon's field of view. For hemostasis, physical pressure such as bandages, gauze, surgical sutures, and staplers, or methods using devices such as electricity, ultrasound, and lasers are sometimes used, but they can cause bleeding and damage and infection. Hemostatic drugs have a comparative advantage in terms of safety and efficiency as they block bleeding using properties such as physical, chemical, and physiological activity depending on the product when introduced to the bleeding site and can prevent wound tissue damage and infection.
그러나, 현재 의료용 생체 접착소재는 외과 수술 시에 발생하는 상처를 봉합하기 위한 보조제 역할을 할 뿐, 실제 의료용 생체 접착소재로 활용하기에 그 기능성과 물성이 부족한 수준이다. 따라서, 가장 기본적으로 의료용 접착제는 조직에 직접 접촉하므로 생체 적합성이 요구되며, 체내 환경 속에서 순간적으로 접착이 종결될 수 있는 접착력과 용이성 뿐만 아니라 오랜 기간 그 기능을 유지해야 한다.However, the current medical bioadhesive material only serves as an auxiliary for suturing wounds generated during surgery, and its functionality and physical properties are insufficient to be used as actual medical bioadhesive materials. Therefore, most basically, medical adhesives require biocompatibility because they come into direct contact with tissues, and they must maintain their functions for a long period of time as well as adhesive strength and ease in which adhesion can be terminated instantaneously in the body environment.
현재 상용화 및 실용화되고 있는 대표적인 생체 접착제로는 시아노아크릴레이트 계열의 순간 접착제, 피브린(fibrin) 글루 및 폴리우레탄계 접착제 등이 있다. 시아노아크릴레이트는 빠른 시간에 개시제 없이 경화되고 높은 접착 강도를 갖지만, 충격에 약하며 내열성 및 내수성이 떨어지고 독성으로 인한 면역 반응을 야기시킨다. 또한, 피브린 기반의 생체 접착제는 실제의 실제 혈액응고 과정을 이용한 방법이기 때문에 비교적 우수한 생체 적합성과 생분해성을 가지나, 합성 고분자 기반 접착제에 비해 현저히 낮은 수준의 접착력을 가지므로 수중 접착을 요하는 부위에는 사용이 매우 제한적이다.Representative bioadhesives currently commercialized and put into practical use include cyanoacrylate-based instant adhesives, fibrin glues, polyurethane-based adhesives, and the like. Cyanoacrylate cures quickly without an initiator and has high adhesive strength, but is weak against impact, has poor heat resistance and water resistance, and causes an immune response due to toxicity. In addition, since the fibrin-based bioadhesive is a method using the actual blood coagulation process, it has relatively excellent biocompatibility and biodegradability. Use is very limited.
또한, 폴리우레탄계 생체 접착제는 조직과의 높은 밀착성 및 유연성을 가지나, 합성 원료의 생체 독성을 줄여야 하는 문제점이 남아있다. 이처럼 현재 접착 소재들은 대부분이 화학합성 기반의 소재로 수분에 약하고 독성을 갖고 그 대안으로 제기되고 있는 생합성 기반의 바이오 접착 소재는 접착력 측면에서 크게 부족하다. In addition, polyurethane-based bioadhesives have high adhesion and flexibility to tissues, but there remains a problem of reducing biotoxicity of synthetic raw materials. As such, most of the current adhesive materials are chemical synthesis-based materials, which are weak and toxic to moisture, and the biosynthesis-based bio-adhesive materials proposed as alternatives are largely insufficient in terms of adhesive strength.
따라서, 기존의 시아노아크릴레이트 계열의 순간 접착제, 피브린 글루 및 폴리우레탄계 접착제와 같은 화학합성 기반의 생체 접착제의 문제점을 개선하고, 화학적 가교 시 문제점을 해소한, 천연 물질 기반의 생체 접착제 및 지혈 소재에 대한 요구가 절실하다.Therefore, natural material-based bioadhesives and hemostatic materials that improve the problems of chemical synthesis-based bioadhesives such as existing cyanoacrylate-based instant adhesives, fibrin glues, and polyurethane-based adhesives, and solve problems during chemical crosslinking. There is an urgent need for
상술한 상황하에서, 본 발명자들은 종래 생체 접착제의 문제점들을 극복하고, 생체 적합성과 물성이 우수한 해양 생체 유래 소재를 이용하여 상처 부위의 지혈을 도와줄 수 있는 신규한 복합 기능성 접착제 및 지혈제를 개발하고자 예의 연구 노력하였다. 그 결과, 본 발명자들은 해양 생물인 오징어의 빨판 이빨을 이루는 단백질인 오징어 빨판 이빨(Sucker ring tooth, SRT) 단백질, 즉, 서커린(suckerin)과 특정 광개시제를 최적 조합하여 하이드로겔을 제작했을 때, 이와 같이 제조된 오징어 빨판 이빨 단백질 기반의 하이드로겔이 생체 내 면역반응을 유발하지 않아 생체적합성이 우수할 뿐만 아니라, 생체접착제 및 지혈제로서 우수한 지혈 효능을 발휘할 수 있음을 규명함으로써, 본 발명을 완성하였다.Under the circumstances described above, the present inventors are eager to overcome the problems of conventional bioadhesives and to develop novel multi-functional adhesives and hemostatic agents that can help hemostasis in wounds using materials derived from marine organisms with excellent biocompatibility and physical properties. research effort. As a result, when the present inventors optimally combined the squid sucker ring tooth (SRT) protein, that is, suckerin and a specific photoinitiator, which is a protein constituting the sucker teeth of the marine creature squid, the hydrogel was produced, The present invention was completed by identifying that the squid sucker tooth protein-based hydrogel prepared as described above has excellent biocompatibility as it does not induce an immune response in vivo and can exhibit excellent hemostatic efficacy as a bioadhesive and hemostatic agent. .
따라서, 본 발명의 일 목적은 오징어 빨판 이빨 단백질(Suckerin); 및 광개시제를 포함하는 하이드로겔 제조용 조성물을 제공하는 데 있다.Therefore, one object of the present invention is squid sucker tooth protein (Suckerin); And to provide a composition for preparing a hydrogel containing a photoinitiator.
또한, 본 발명의 다른 목적은 하이드로겔 제조용 조성물에 자외선-조사로 다이-타이로신(di-tyrosine) 잔기를 형성하여 가교화하는 단계를 포함하는 하이드로겔의 제조 방법을 제공하는 데 있다.In addition, another object of the present invention is to provide a method for preparing a hydrogel comprising the step of crosslinking a composition for preparing a hydrogel by forming a di-tyrosine residue by irradiation with ultraviolet rays.
또한, 본 발명의 또 다른 목적은 상기 제조 방법에 의해 제조된 하이드로겔을 제공하는 데 있다.In addition, another object of the present invention is to provide a hydrogel prepared by the above production method.
또한, 본 발명의 또 다른 목적은 상기 하이드로겔을 포함하는 상처 치유 또는 지혈용 조성물을 제공하는 데 있다.In addition, another object of the present invention is to provide a composition for wound healing or hemostasis comprising the hydrogel.
또한, 본 발명의 또 다른 목적은 상기 하이드로겔을 포함하는 생체접착제 조성물을 제공하는 데 있다.In addition, another object of the present invention is to provide a bioadhesive composition comprising the hydrogel.
또한, 본 발명의 또 다른 목적은 상기 하이드로겔을 포함하는 상처 치유 또는 지혈용 습윤 드레싱제를 제공하는 데 있다.In addition, another object of the present invention is to provide a wet dressing for wound healing or hemostasis comprising the hydrogel.
또한, 본 발명의 또 다른 목적은 상기 하이드로겔을 포함하는 조직공학용 지지체를 제공하는 데 있다.In addition, another object of the present invention is to provide a scaffold for tissue engineering comprising the hydrogel.
본 발명의 다른 목적 및 이점은 하기에 기재된 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
본 명세서에서 사용한 용어는 단지 설명을 목적으로 사용된 것으로, 한정하려는 의도로 해석되어서는 안된다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 명세서에서, "포함하다" 또는 "가지다" 등의 용어는 명세서 상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.The terms used herein are used for descriptive purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 실시예가 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥 상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't
이하, 본 발명에 대하여 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 양태에 따르면, 본 발명은 오징어 빨판 이빨 단백질(Suckerin); 및 광개시제를 포함하는 하이드로겔(gel) 제조용 조성물을 제공한다.According to one aspect of the present invention, the present invention squid sucker tooth protein (Suckerin); And it provides a composition for preparing a hydrogel (gel) containing a photoinitiator.
본 발명에서는 오징어 빨판 이빨 단백질(Suckerin)의 타이로신 잔기를 고려하여, 광개시제를 이용하여 자외선 영역 대의 빛에 의한 타이로신 잔기 간의 결합을 유도하여 광가교물을 제조하고, 이를 포함하는 하이드로겔을 형성하였다. 이와 같이 제조된 오징어 빨판 이빨 단백질 기반 하이드로겔이 생체 접착제로서의 우수한 효능을 나타낸다는 것을 확인하여 본 발명을 완성하였다.In the present invention, considering the tyrosine residues of squid sucker protein (Suckerin), a photoinitiator was used to induce bonding between tyrosine residues by light in the ultraviolet range to prepare a photocrosslinked product, and a hydrogel containing it was formed. The present invention was completed by confirming that the squid sucker tooth protein-based hydrogel prepared as described above exhibits excellent efficacy as a bioadhesive.
본 발명자들은 의료용 생체접착소재에 적합한 제형 개발을 위해 오징어 빨판 이빨 단백질 기반의 광가교성 하이드로겔을 제조하였다. 상기 하이드로겔은 생체 안정성 및 우수한 접착 능력을 갖추면서도, 신속하고 효율적으로 제조가 가능하며, 하이드로겔 자체가 실제 조직과 비슷한 기계적 유연성을 가지고 있기 때문에 외과용 봉합사를 대신하는 등 실제 의료용 소재로의 개발 및 다양한 응용이 가능하다.The present inventors prepared a photocrosslinkable hydrogel based on squid sucker tooth protein to develop a formulation suitable for medical bioadhesive materials. The hydrogel has biostability and excellent adhesive ability, can be manufactured quickly and efficiently, and since the hydrogel itself has mechanical flexibility similar to that of actual tissue, it can be developed as an actual medical material, such as replacing surgical sutures. and various applications are possible.
본 발명에서 용어, "하이드로겔(hydrogel)"이란, 물 또는 체액 내에서 가교된 격자 안으로 많은 양의 물 또는 체액을 흡수하여 팽윤되며 삼차원적인 구조를 유지하는 재료를 의미한다. 팽윤된 이후에도, 열역학적으로 안정하게 존재하여 액체와 고체의 중간 형태에 해당하는 기계적 및 물리화학적 특성을 갖는다. 이러한 하이드로겔은 대개 생체 적합성, 높은 다공성 및 산소 투과도를 보이며, 생체 연조직과의 비슷한 물리적 특성을 나타낼 수 있다.In the present invention, the term "hydrogel" refers to a material that absorbs a large amount of water or body fluid into a crosslinked lattice in water or body fluid, swells, and maintains a three-dimensional structure. Even after being swollen, it remains thermodynamically stable and has mechanical and physicochemical properties corresponding to an intermediate form between a liquid and a solid. These hydrogels usually exhibit biocompatibility, high porosity and oxygen permeability, and may exhibit similar physical properties to living soft tissues.
상기 오징어 빨판 이빨 단백질의 농도가 10wt% 미만인 경우, 절대적인 타이로신 잔기의 양이 적어 전자 수용체의 농도를 높인다해도 견고한 하이드로겔을 만들 수 없고, 단백질 농도가 30wt% 초과인 경우 점도가 급격히 높아져 다루기 불편하고, 전체적인 광가교가 아니라 빛을 쬐어주기 전부터 강렬하고 국소적인 결합이 일어나므로 균일한 결합으로 이루어진 하이드로겔을 만들 수 없다. 따라서, 다루기 쉽고 균일한 광가교를 유도할 수 있는 바람직한 오징어 빨판 이빨 단백질의 농도는 전체 조성물을 기준으로 10 내지 30wt%이고, 바람직하게는 15wt%이다.When the concentration of the squid sucker tooth protein is less than 10 wt%, the absolute amount of tyrosine residues is low, and even if the concentration of the electron acceptor is increased, a solid hydrogel cannot be made. , It is not possible to make a hydrogel with uniform bonding because intense and local bonding occurs even before exposure to light, rather than overall photocrosslinking. Therefore, the preferred concentration of squid sucker tooth protein, which is easy to handle and can induce uniform photocrosslinking, is 10 to 30 wt%, preferably 15 wt%, based on the total composition.
본 발명에서 "오징어 빨판 이빨 단백질(Suckerin)"은 오징어의 빨판 및 이빨에서 유래한 접착 단백질로, 본 발명의 목적을 달성할 수 있는 한, 당업계에 공지된 임의의 종류의 오징어 빨판 이빨 단백질을 이용할 수 있으며, 본 발명의 일 실시예에서는 서열번호 2로 기재되는 아미노산 서열의 펩티드를 이용하였으나, 이에 제한되지 않는다.In the present invention, "squid sucker tooth protein (Suckerin)" is an adhesive protein derived from suckers and teeth of squid, and any kind of squid sucker tooth protein known in the art can be used as long as the object of the present invention can be achieved. It can be used, and in one embodiment of the present invention, a peptide having an amino acid sequence represented by SEQ ID NO: 2 was used, but is not limited thereto.
상기 서열번호 2로 기재되는 아미노산 서열은 서열번호 1에 기재된 염기서열로 코딩된다.The amino acid sequence of SEQ ID NO: 2 is encoded by the nucleotide sequence of SEQ ID NO: 1.
상기 본 발명의 오징어 빨판 이빨 단백질은 수중 조건 하에서 생체적합성에 최적인 나노입자 상태로 존재 및/또는 형성하는 것을 특징으로 한다.The squid sucker tooth protein of the present invention is characterized in that it exists and/or forms nanoparticles optimal for biocompatibility under aquatic conditions.
또한, 본 발명에서 오징어 빨판 이빨 단백질은 접착력을 유지하는 전제 하에 상기 오징어 빨판 이빨 단백질의 카르복실 말단이나 아미노 말단에 추가적인 서열을 포함하거나 일부 아미노산이 다른 아미노산으로 치환된 것일 수 있다. In addition, the squid sucker tooth protein in the present invention may contain an additional sequence at the carboxyl or amino end of the squid sucker tooth protein, or some amino acids may be substituted with other amino acids, on the premise of maintaining adhesiveness.
또한, 오징어 빨판 이빨 단백질을 용매에 용해시킬 때 산성 수용액에 20wt% 이상의 우수한 용해도로 용해될 수 있는 오징어 빨판 이빨 단백질을 이용하는 것이 바람직하다. 상기 용매는 산성 수성용매 또는 중성 수성용매일 수 있고, 상기 산성 수성용매는 인산, 아세트산, 포름산, 염산, 황산, 질산 및 시트르산으로 이루어진 군으로부터 선택된 1 종 이상일 수 있으며, 바람직하게는 아세트산이나, 이에 한정되지 않는다.In addition, when the squid sucker tooth protein is dissolved in a solvent, it is preferable to use a squid sucker tooth protein that can be dissolved with an excellent solubility of 20 wt% or more in an acidic aqueous solution. The solvent may be an acidic aqueous solvent or a neutral aqueous solvent, and the acidic aqueous solvent may be at least one selected from the group consisting of phosphoric acid, acetic acid, formic acid, hydrochloric acid, sulfuric acid, nitric acid and citric acid, preferably acetic acid. Not limited.
상기 광개시제는, 본 발명의 목적을 달성할 수 있는 한, 임의의 광개시제를 이용할 수 있으며, 예컨대, 리보플라빈 (Riboflavin, vitamin B2), 리튬 페닐-2,4,6-트리메틸벤조일포스피네이트(lithium phenyl-(2,4,6-trimethyl benzoyl) phosphinate, LAP), 벤질디메틸케탈(benzyl dimethyl ketal), 아세토페논(acetophenone), 벤조인메틸에테르(benzoin methylether), 디에톡시아세토페논(diethoxyacetophenone), 벤조일 포스핀 옥사이드(benzoyl phosphine oxide), 1-하이드록시사이클로헥실 페닐 케톤(1-hydroxycyclohexyl phenyl ketone), tris(bipyridine)ruthenium(II) chloride (Ru(bpy3)2+), APS (ammonium persulfate), HRP (horse radish peroxide) 및 H2O2 (hydrogen peroxide)으로 이루어진 군으로부터 선택된 1 종 이상일 수 있으며, 바람직하게는 리보플라빈이나, 이에 제한되지 않는다.Any photoinitiator may be used as long as the object of the present invention can be achieved, for example, riboflavin (vitamin B2), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (lithium phenyl -(2,4,6-trimethyl benzoyl) phosphinate, LAP), benzyl dimethyl ketal, acetophenone, benzoin methylether, diethoxyacetophenone, benzoyl phos benzoyl phosphine oxide, 1-hydroxycyclohexyl phenyl ketone, tris(bipyridine)ruthenium(II) chloride (Ru(bpy3) 2+ ), APS (ammonium persulfate), HRP ( horse radish peroxide) and H 2 O 2 (hydrogen peroxide), and preferably riboflavin, but is not limited thereto.
상기 광개시제의 농도가 광가교의 속도나 견고함에 미치는 영향은 미미하나 광가교가 견고하게 이루어지는 최적농도는 0.5 mM이고, 이 이상으로 사용해도 광가교에는 영향을 주지 않으나 세포를 활용하여 함께 사용될 경우 세포의 생존율을 높이기 위해 낮은 농도로 사용하는 것이 바람직하다. 따라서, 광개시제의 농도는 0.1 내지 10 mM이고, 바람직하게는 0.5 mM이다.Although the concentration of the photoinitiator has little effect on the speed or robustness of photocrosslinking, the optimal concentration for photocrosslinking to be robust is 0.5 mM, and even if used above this concentration, it does not affect photocrosslinking. It is preferable to use it in a low concentration to increase the survival rate. Therefore, the concentration of the photoinitiator is 0.1 to 10 mM, preferably 0.5 mM.
또한, 상기 오징어 빨판 이빨 단백질을 포함하는 하이드로겔 제조용 조성물은 생리활성물질을 추가로 포함할 수 있으며, 상기 생리활성 물질은 세포, 단백질, 효소, 및 당으로 이루어진 군에서 선택된 1종 이상일 수 있으나 이에 한정되지 않는다.In addition, the composition for preparing a hydrogel containing squid sucker teeth protein may further include a physiologically active material, and the physiologically active material may be at least one selected from the group consisting of cells, proteins, enzymes, and sugars, but Not limited.
본 발명의 일 구현예는 광가교성 및 생체접착성을 갖는 오징어 빨판 이빨 단백질 기반의 하이드로겔을 제공하는 것이다.One embodiment of the present invention is to provide a squid sucker tooth protein-based hydrogel having photocrosslinkability and bioadhesiveness.
바람직하게, 본 발명에서 오징어 빨판 이빨 단백질 기반의 하이드로겔은 오징어 빨판 이빨 단백질에 포함된 타이로신 잔기들 사이의 가교결합으로 형성된 하이드로겔일 수 있다.Preferably, in the present invention, the squid sucker tooth protein-based hydrogel may be a hydrogel formed by cross-linking between tyrosine residues included in the squid sucker tooth protein.
본 발명의 원료가 되는 해양 생명체인 오징어의 빨판의 수중접착력의 효능성분으로 예상되는 오징어 빨판 이빨 (Sucker Ring Teeth, SRT) 단백질, 즉, 서커린(suckerin)을 생산 및 분비함으로써 바다 속 고체 표면에 단단히 부착할 수 있어, 파도와 같은 격변하는 환경 속에서 영향을 받지 않는다. 또한, 오징어 빨판 이빨 단백질 및 이를 기반으로 제작한 하이드로겔은 플라스틱, 유리, 금속 및 생체 물질 등의 다양한 표면에 접착뿐만 아니라 젖은 표면에서의 접착도 가능하다.By producing and secreting Sucker Ring Teeth (SRT) protein, that is, suckerin, which is expected to be an effective component of the underwater adhesive force of the sucker of the squid, which is the raw material of the present invention, on the solid surface in the sea It can be firmly attached, so it is not affected in a rapidly changing environment such as waves. In addition, squid sucker teeth proteins and hydrogels made based on them can adhere to various surfaces such as plastic, glass, metal, and biomaterials, as well as adhere to wet surfaces.
또한, 본 발명의 다른 양태에 따르면, 본 발명은 하이드로겔 제조용 조성물에 자외선-조사로 다이-타이로신(di-tyrosine) 잔기를 형성하여 가교화하는 단계를 포함하는 하이드로겔의 제조 방법을 제공한다.In addition, according to another aspect of the present invention, the present invention provides a method for preparing a hydrogel comprising the step of forming a di-tyrosine residue in a composition for preparing a hydrogel by UV-irradiation to crosslink it.
상기 자외선은, 바람직하게는 파장범위 300 내지 400nm의 자외선일 수 있다.The ultraviolet light may be ultraviolet light having a wavelength range of 300 to 400 nm.
상기 방법은 오징어 빨판 이빨 단백질에 포함된 타이로신 잔기들 사이의 결합을 유도하여 하이드로겔을 제조하는 것이다.The method is to prepare a hydrogel by inducing a bond between tyrosine residues included in squid sucker tooth protein.
보다 바람직하게는, 상기 오징어 빨판 이빨 단백질이 15wt%로 용해되어 있는 용액에 리보플라빈 0.5mM을 첨가시키고, 368 nm 파장 대의 빛을 쬐어주면 수 초, 수 분 또는 수 시간내에 하이드로겔을 형성시킬 수 있다.More preferably, when 0.5 mM of riboflavin is added to a solution in which the squid sucker tooth protein is dissolved at 15 wt% and irradiated with light in the 368 nm wavelength band, a hydrogel can be formed within seconds, minutes, or hours. .
본 발명의 구체적인 실시예에서는 광반응을 통해 형성된 하이드로겔의 사진을 도 5에 나타내었다.In a specific embodiment of the present invention, a photograph of a hydrogel formed through photoreaction is shown in FIG. 5 .
또한, 상기 방법에 의해서 제조된 하이드로겔의 유변학적 성질로서, 저장탄성율(storage modulus) 및 손실 탄성율(loss modulus) 값을 알 수 있으며, 고체-유사(solid like)한 특성을 나타냄을 알 수 있다. In addition, as the rheological properties of the hydrogel prepared by the method, storage modulus and loss modulus values can be known, and it can be seen that it exhibits solid-like properties .
하이드로겔은 생체 내에서도 결합이 빠른 시간 내에 끊어지지 않고 그 형태를 유지하며 실제 조직과 비슷한 기계적 유연함을 지니므로 내수성 및 우수한 접착력을 필요로 하는 의료용 접착제 등으로의 응용이 활발하게 이루어지고 있다. Since the hydrogel maintains its shape without breaking the bond in a short time in vivo and has mechanical flexibility similar to that of actual tissue, it is actively applied as a medical adhesive that requires water resistance and excellent adhesive strength.
따라서, 본 발명에 따른 광반응성 및 조직 접착성을 갖는 하이드로겔은 원하는 부위에 즉각적으로 겔화가 가능하여 조직 접착제뿐만 아니라 지혈제, 조직 충진재 및 상처치료 등의 다양한 생의학적 응용이 가능하다.Therefore, the hydrogel having photoreactivity and tissue adhesiveness according to the present invention can be instantly gelated at a desired site, and thus various biomedical applications such as hemostatic agents, tissue filling materials, and wound healing as well as tissue adhesives are possible.
또한, 본 발명의 또 다른 양태에 따르면, 본 발명은 오징어 빨판 이빨 단백질(Suckerin); 및 광개시제를 포함하는 하이드로겔을 제공하고, 상기 하이드로겔은 상기 생산 방법에 의해 제조된 오징어 빨판 이빨 단백질(Suckerin)-기반 하이드로겔을 제공한다.In addition, according to another aspect of the present invention, the present invention squid sucker tooth protein (Suckerin); and a photoinitiator, wherein the hydrogel provides a squid suckerin tooth protein (Suckerin)-based hydrogel prepared by the production method.
즉, 본 발명의 오징어 빨판 이빨 단백질 기반 하이드로겔은 하이드로겔 제조용 조성물에 자외선-조사로 다이-타이로신(di-tyrosine) 잔기를 형성하여 가교화된 하이드로겔이고, 오징어 빨판 이빨 단백질(Suckerin)을 광개시제로 광가교하여 겔화시킨 소재로서, 생체에 적합한 높은 기계적 물성 및 접착 능력을 지니게 된다.That is, the squid sucker tooth protein-based hydrogel of the present invention is a hydrogel cross-linked by forming a di-tyrosine residue by UV-irradiation in a composition for preparing a hydrogel, and the squid sucker tooth protein (Suckerin) as a photoinitiator As a material that is gelled by optical crosslinking, it has high mechanical properties and adhesive ability suitable for living organisms.
또한, 본 발명의 또 다른 양태에 따르면, 본 발명은 오징어 빨판 이빨 단백질(Suckerin); 및 광개시제를 포함하는 상처 치유 또는 지혈용 조성물을 제공한다.In addition, according to another aspect of the present invention, the present invention squid sucker tooth protein (Suckerin); And it provides a composition for wound healing or hemostasis comprising a photoinitiator.
본 발명의 조성물은 염화칼슘(CaCl2), 아데노신이인산(ADP), 비타민 K, 트롬보플라스틴, 포도당, 탈크, 탄산나트륨, 스테아린산마그네슘 및 폴리비닐알코올로 이루어진 군에서 1종 이상 선택되는 첨가제를 더 포함할 수 있으나, 이에 한정되지는 않으며, 본 발명의 조성물의 형태, 용도, 투여 부위 및 경로, 사용량에 따라 필요한 약학적 성분들을 추가로 포함할 수 있다. The composition of the present invention further contains at least one additive selected from the group consisting of calcium chloride (CaCl 2 ), adenosine diphosphate (ADP), vitamin K, thromboplastin, glucose, talc, sodium carbonate, magnesium stearate, and polyvinyl alcohol. It may include, but is not limited thereto, and may further include necessary pharmaceutical ingredients depending on the form, use, administration site and route, and amount of the composition of the present invention.
예컨대, 염화칼슘을 염 또는 수용액의 형태로 조성물 리터당 약 5~25 mM을 첨가하여 지혈 경로 활성에 필요한 칼슘 이온을 제공할 수 있다. 아데노신이인산을 염 또는 수용액의 형태로 상처 치유 또는 지혈용 조성물 리터당 약 0.5~5 μM을 첨가하여 지혈 경로를 활성화할 수 있다. For example, calcium chloride may be added in the form of a salt or aqueous solution at about 5 to 25 mM per liter of the composition to provide calcium ions necessary for activity of the hemostatic pathway. The hemostatic pathway can be activated by adding about 0.5 to 5 μM per liter of adenosine diphosphate in the form of a salt or aqueous solution for wound healing or hemostasis.
본 발명에 따른 상처 치유 또는 지혈용 조성물은 하이드로겔, 콜로이드 용액, 나노섬유, 용액, 분말 또는 스프레이 형태로 제공될 수 있다.The composition for wound healing or hemostasis according to the present invention may be provided in the form of a hydrogel, colloidal solution, nanofiber, solution, powder or spray.
본 발명의 조성물이 하이드로겔 형태로 제공되는 경우, 상기 하이드로겔은 오징어 빨판 이빨 단백질(Suckerin) 및 광개시제 혼합물이 광가교되어 형성된 것일 수 있다. 오징어 빨판 이빨 단백질(Suckerin) 및 광개시제를 광가교하여 겔화시킨 소재는 높은 기계적 물성과 다공성 구조 및 접착 능력을 지니게 된다. 이 하이드로겔은 세척 후 습윤 상태의 지혈 패치 또는 동결 건조한 스펀지 형태로 이용될 수 있다.When the composition of the present invention is provided in the form of a hydrogel, the hydrogel may be formed by photocrosslinking a mixture of squid sucker tooth protein (Suckerin) and a photoinitiator. A gelled material obtained by photocrosslinking squid sucker tooth protein (Suckerin) and a photoinitiator has high mechanical properties, porous structure, and adhesive ability. This hydrogel can be used in the form of a hemostatic patch in a wet state after washing or a freeze-dried sponge.
본 발명의 조성물이 콜로이드 용액의 형태로 제공되는 경우, 상기 콜로이드 용액은 오징어 빨판 이빨 단백질(Suckerin) 및 광개시제로 형성된 코아세르베이트를 포함할 수 있다. 이 경우 음이온성 고분자가 더 첨가된 것일 수 있다. 음이온성 고분자는 페레독신(ferredoxin), 폴리스티렌술폰산(poly styrene sulfonic acid), 아라비아 검(gum arabic), 카보폴(carbopol), 고 또는 저 메톡실 펙틴(high or low methoxyl pectin), 카르복시메틸 구아검 나트륨(sodium carboxymethyl guar gum), 잔탄 검(xanthan gum), 유청 단백질(whey protein), 레구민(faba bean legumin), 카르복시메틸 셀룰로오스(carboxymethyl cellulose), 알긴산(alginate), 캐러지넌(carrageenan), 헥사메타인산나트륨(sodium hexametaphosphate), 카제인 나트륨(sodium casinate), 헤모글로빈(hemoglobin), 헤파린(heparin) 및 세포외 다당체 B40(exopolysaccharide B40)으로 구성된 군에서 선택되는 것일 수 있다. When the composition of the present invention is provided in the form of a colloidal solution, the colloidal solution may include a coacervate formed of squid suckerin and a photoinitiator. In this case, an anionic polymer may be further added. Anionic polymers include ferredoxin, poly styrene sulfonic acid, gum arabic, carbopol, high or low methoxyl pectin, carboxymethyl guar gum Sodium carboxymethyl guar gum, xanthan gum, whey protein, faba bean legumin, carboxymethyl cellulose, alginate, carrageenan, hexa It may be one selected from the group consisting of sodium hexametaphosphate, sodium casinate, hemoglobin, heparin, and exopolysaccharide B40.
본 발명의 조성물이 나노섬유 형태로 제공되는 경우, 나노섬유는 오징어 빨판 이빨 단백질(Suckerin) 및 광개시제의 혼합물을 전기방사하여 제조한 것이거나, 전기방사하여 제조한 나노섬유에 오징어 빨판 이빨 단백질(Suckerin) 및 광개시제를 흡착시키거나 코팅한 것일 수 있다.When the composition of the present invention is provided in the form of nanofibers, the nanofibers are prepared by electrospinning a mixture of squid sucker tooth protein (Suckerin) and a photoinitiator, or in the nanofibers prepared by electrospinning, squid sucker tooth protein (Suckerin ) and a photoinitiator may be adsorbed or coated.
본 발명의 조성물이 용액 형태로 제공되는 경우, 용액은 오징어 빨판 이빨 단백질(Suckerin) 및 광개시제를 멸균수, 식염수, 완충액 및 초산나트륨 수용액으로 이루어진 군에서 선택되는 용매와 혼합한 것일 수 있다. 상기 용액은 약학적으로 허용되는 첨가제를 더 포함할 수 있고, 투여 부위 및 경로에 따라 주사액, 드레싱, 실란트, 농축액, 젤 등의 형태로 활용될 수 있다.When the composition of the present invention is provided in the form of a solution, the solution may be a mixture of squid sucker tooth protein (Suckerin) and a photoinitiator with a solvent selected from the group consisting of sterilized water, saline, buffer, and sodium acetate aqueous solution. The solution may further contain pharmaceutically acceptable additives, and may be used in the form of an injection solution, dressing, sealant, concentrate, gel or the like depending on the site and route of administration.
본 발명의 조성물이 분말 형태로 제공되는 경우, 분말은 오징어 빨판 이빨 단백질(Suckerin) 및 광개시제의 혼합물을 1 mm 이상의 입체, 1 μm 내지 1 mm의 분체, 0.1 내지 1 μm의 미분체 또는 1 μm 미만의 초미분체로 파쇄한 것일 수 있다. 상기 혼합물은 약학적으로 허용되는 첨가제를 더 포함할 수 있다.When the composition of the present invention is provided in powder form, the powder is a mixture of squid sucker tooth protein (Suckerin) and a photoinitiator with a size of 1 mm or more, a powder of 1 μm to 1 mm, a fine powder of 0.1 to 1 μm, or less than 1 μm. It may be crushed into an ultrafine powder of. The mixture may further contain pharmaceutically acceptable additives.
본 발명의 조성물이 스프레이 형태로 제공되는 경우, 스프레이는 오징어 빨판 이빨 단백질(Suckerin) 및 광개시제의 혼합물을 외상 및 외과 수술 부위에 스프레이로 도포하는 것일 수 있다.When the composition of the present invention is provided in the form of a spray, the spray may be applied by spraying a mixture of squid sucker tooth protein (Suckerin) and a photoinitiator to a traumatic or surgical site.
또한, 본 발명의 또 다른 양태에 따르면, 본 발명은 오징어 빨판 이빨 단백질(Suckerin)-기반 하이드로겔을 포함하는 생체접착제용 조성물을 제공한다.In addition, according to another aspect of the present invention, the present invention provides a bioadhesive composition comprising a squid suckerin protein (Suckerin)-based hydrogel.
본 발명의 생체 접착제 조성물은 생체 내에 국소적으로 적용되어 외과 수술용 봉합사를 대체해 손쉽고, 즉각적으로 상처를 봉합할 수 있고, 피부, 혈관, 신경 등의 연조직과 뼈, 치아 등의 경조직을 포함하는 생체 조직의 접합에 이용될 수 있다. 본 명세서에서 용어 "생체 조직"은 특별하게 제한되지 않으며, 예를 들어 피부, 신경, 뇌, 폐, 간, 신장, 위, 소장, 직장 및 뼈 등을 포함한다.The bioadhesive composition of the present invention can be applied locally in a living body to replace surgical sutures to easily and immediately suture wounds, and can be applied to soft tissues such as skin, blood vessels, and nerves and hard tissues such as bones and teeth. It can be used for joining biological tissues. In this specification, the term "biological tissue" is not particularly limited, and includes, for example, skin, nerve, brain, lung, liver, kidney, stomach, small intestine, rectum, and bone.
본 발명의 생체 적합성 생체접착제의 다양한 응용 분야를 정리하면 다음과 같다. 상기 생체접착제는 봉합사를 대체하여 즉각적으로 상처를 봉합할 수 있고, 조직의 손상된 부분을 접착시키거나, 조직에서 공기 및 혈액의 누출을 즉각적으로 봉하거나, 의료기구를 생체 조직에 접착시키거나, 조직의 결함부분을 채우는 구조물로서 이용될 수 있다.Summarizing various fields of application of the biocompatible bioadhesive of the present invention are as follows. The bioadhesive can substitute for a suture to immediately suture a wound, adhere a damaged part of a tissue, immediately seal leakage of air and blood from a tissue, adhere a medical device to a living tissue, or It can be used as a structure that fills the defective part of
다른 구현 예로, 본 발명의 생체 접착제는 상처 치유에 이용될 수 있다. 예를 들어, 본 발명의 생체 접착제는 상처에 적용되는 드레싱 및 혈관의 지혈 및 봉합 등에 사용될 수 있다.In another embodiment, the bioadhesive of the present invention can be used for wound healing. For example, the bioadhesive of the present invention can be used for hemostasis and suturing of dressings applied to wounds and blood vessels.
또한, 본 발명의 또 다른 양태에 따르면, 본 발명은 오징어 빨판 이빨 단백질(Suckerin)-기반 하이드로겔을 포함하는 상처 치유 또는 지혈용 습윤 드레싱제를 제공한다.In addition, according to another aspect of the present invention, the present invention provides a wet dressing for wound healing or hemostasis comprising a squid sucker tooth protein (Suckerin)-based hydrogel.
또한, 본 발명의 또 다른 양태에 따르면, 본 발명은 오징어 빨판 이빨 단백질(Suckerin)-기반 하이드로겔을 포함하는 조직공학용 지지체를 제공한다.In addition, according to another aspect of the present invention, the present invention provides a scaffold for tissue engineering comprising a squid suckerin-based hydrogel.
조직 공학 기술이란 환자의 조직으로부터 분리된 세포를 지지체 위에 배양하여 또는 지지체가 되는 구성물과 함께 지지체를 형성하여 세포를 포함한 지지 복합체를 제조한 후 다시 이를 인체 내에 이식하는 것을 말한다. 조직 공학 기술은 인공피부, 인공연골, 인공뼈, 인공혈관, 인공근육 등 인체의 거의 모든 장기의 재생에 적용될 수 있으며, 세포에 무해하고 실제 생체조직과 비슷한 물성을 갖는 지지체를 형성하는 것이 중요하다. 본 발명의 광반응성 및 생체 접착성 하이드로겔은 조직공학 기술에서 조직의 결함을 채우는 역할뿐만 아니라 생체 조직 및 장기의 재생을 촉진시키는 생체조직과 유사한 지지체를 제공할 수 있다.Tissue engineering technology refers to culturing cells isolated from a patient's tissue on a scaffold or forming a scaffold together with components to be a scaffold to prepare a scaffold complex including cells and then transplanting it into the human body. Tissue engineering technology can be applied to the regeneration of almost all organs in the human body, such as artificial skin, artificial cartilage, artificial bone, artificial blood vessels, and artificial muscles, and it is important to form scaffolds that are harmless to cells and have properties similar to those of actual living tissue. . The photoreactive and bioadhesive hydrogel of the present invention can provide a scaffold similar to a living tissue that promotes regeneration of living tissue and organs as well as filling in tissue defects in tissue engineering technology.
또한, 상기 하이드로겔에 세포의 성장 및 분화를 촉진시키고, 주변 조직과의 상호작용을 통해 조직의 재생을 도와주는 역할을 하는 각종 생리활성물질들을 쉽게 부착시킬 수 있다. 상기 생리활성 물질은 세포, 단백질, 효소, 당 등을 포함하며, 바람직하게는 세포를 들 수 있다. 상기 세포는 원핵세포 및 진핵 세포를 포함하는 모든 세포일 수 있고, 일 예로 섬유세포(fibroblast), 조골세포(osteoblast), 신경세포(neurons), B cell 등을 포함한 면역세포 및 배아세포 등일 수도 있다. 본 발명의 지지체가세포를 포함하여 배양이 가능하므로 다양한 장기로의 이식 및 재생에 이용될 수 있다.In addition, various physiologically active substances that promote cell growth and differentiation and help tissue regeneration through interaction with surrounding tissues can be easily attached to the hydrogel. The physiologically active substance includes cells, proteins, enzymes, sugars, and the like, preferably cells. The cells may be any cell including prokaryotic and eukaryotic cells, and may be, for example, fibroblasts, osteoblasts, neurons, immune cells including B cells, and embryonic cells. . Since the scaffold of the present invention can be cultured including cells, it can be used for transplantation and regeneration into various organs.
본 발명은 상술한 오징어 빨판 이빨 단백질(Suckerin)-기반 하이드로겔을 이용하므로, 중복된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재 를 생략한다. Since the present invention uses the above-described squid suckerin protein (Suckerin)-based hydrogel, duplicate descriptions are omitted to avoid excessive complexity in the present specification.
결론적으로, 본 발명의 오징어 빨판 이빨 단백질-하이드로겔 제조용 조성물은 강한 수중 접착력을 가질 뿐만 아니라, 즉각적인 겔화 반응으로 형성될 수 있으며, 적용 부위의 특성에 따라 다양한 형태로의 제형이 가능하고, 우수한 지혈 효능을 가지므로 원하는 생체 내에 생체 접착제, 조직 충진제, 조직공학용 지지체 등의 조성물로 이용하여 뛰어난 지혈 작용으로 빠르게 출혈을 막고 상처를 치유할 수 있다. In conclusion, the composition for preparing squid sucker teeth protein-hydrogel of the present invention not only has strong adhesion in water, but can be formed through an immediate gelation reaction, can be formulated in various forms depending on the characteristics of the application site, and has excellent hemostasis. Since it has efficacy, it can be used as a composition such as a bioadhesive, a tissue filler, and a support for tissue engineering in a desired living body to quickly stop bleeding and heal wounds with excellent hemostasis.
본 발명에 따른 오징어 빨판 이빨 단백질(Suckerin)에 광개시제를 처리하여 광중합(광가교)된 오징어 빨판 이빨 단백질-기반 하이드로겔(SRT hydrogel)의 탁월한 생체접착능, 우수한 응고 및 창상 회복 효과를 확인하였으므로, 의료용 생체 재료, 화장료 소재 분야 등에 다양하게 적용가능하며, 특히, 생체 접착제 및 창상 부위에 대한 지혈제로서 유용하게 사용할 수 있을 것으로 기대된다.The excellent bioadhesive ability, excellent coagulation and wound healing effect of the photopolymerized (photocrosslinked) squid sucker tooth protein-based hydrogel (SRT hydrogel) by treating the squid sucker tooth protein (Suckerin) according to the present invention with a photoinitiator was confirmed. It can be applied in various fields such as medical biomaterials and cosmetic materials, and is expected to be useful especially as a bioadhesive and a hemostatic agent for wounds.
도 1은 본 발명의 오징어 빨판 이빨 단백질의 수중 나노 파티클 형성을 보여준다.
도 2는 본 발명의 오징어 빨판 이빨 단백질의 크기를 보여준다.
도 3은 L929 세포주에서의 본 발명의 오징어 빨판 이빨 단백질의 세포독성 평가를 보여준다.( NT: 공시험액 실험군, PE film(Polyethylene Film), PUR file(Polyurethane Film)
도 4a는 Raw 264.7 세포주에서의 본 발명의 오징어 빨판 이빨 단백질의 세포독성 평가를 보여준다.
도 4b는 Raw 264.7 세포주에서의 본 발명의 오징어 빨판 이빨 단백질의 염증성 사이토카인 분비 활성 평가를 보여준다.
도 5는 개시제에 따른 본 발명의 오징어 빨판 이빨 단백질-기반 하이드로겔 형성을 비교한 결과를 보여준다.
도 6은 본 발명의 오징어 빨판 이빨 단백질-기반 하이드로겔(SRT hydrogel)의 가교와 관련된 리보플라빈에 의해 파란 형광을 방출하여 다이-타이로신(di-tyrosine) 잔기가 형성된 것을 확인한 결과를 보여준다.
도 7은 본 발명의 오징어 빨판 이빨 단백질-기반 하이드로겔의 점탄성을 확인한 결과를 보여준다.
도 8은 본 발명의 오징어 빨판 이빨 단백질-기반 하이드로겔의 돼지 껍데기를 이용한 수중습윤환경에서의 조직 접착력을 확인한 결과를 보여준다.
도 9는 본 발명의 빨판 이빨 단백질-기반 하이드로겔의 지혈 효과를 보여준다.
도 10은 본 발명의 오징어 빨판 이빨 단백질-기반 하이드로겔의 비용혈 효과를 보여준다.
도 11a는 랫드 피부 창상 실험 모델에서의 본 발명의 오징어 빨판 이빨 단백질-기반 하이드로겔의 창상 회복 효과를 보여준다. 도 11b는 랫드 피부 창상 실험 모델에서의 본 발명의 오징어 빨판 이빨 단백질-기반 하이드로겔의 염증 반응 수준을 보여준다. 도 11c는 랫드 피부 창상 실험 모델에서의 본 발명의 오징어 빨판 이빨 단백질-기반 하이드로겔의 콜라겐 재생 효과를 보여준다.Figure 1 shows the formation of nanoparticles in water of the squid sucker tooth protein of the present invention.
Figure 2 shows the size of squid sucker tooth protein of the present invention.
Figure 3 shows the cytotoxicity evaluation of the squid sucker tooth protein of the present invention in the L929 cell line. (NT: blank test solution experimental group, PE film (Polyethylene Film), PUR file (Polyurethane Film)
Figure 4a shows the evaluation of the cytotoxicity of the squid sucker tooth protein of the present invention in the Raw 264.7 cell line.
Figure 4b shows the evaluation of the inflammatory cytokine secretion activity of the squid sucker tooth protein of the present invention in the Raw 264.7 cell line.
Figure 5 shows the results of comparison of squid sucker tooth protein-based hydrogel formation according to the present invention according to the initiator.
Figure 6 shows the results confirming that di-tyrosine residues are formed by emitting blue fluorescence by riboflavin associated with crosslinking of the squid sucker tooth protein-based hydrogel (SRT hydrogel) of the present invention.
Figure 7 shows the result of confirming the viscoelasticity of the squid sucker tooth protein-based hydrogel of the present invention.
8 shows the result of confirming the tissue adhesiveness of the squid sucker teeth protein-based hydrogel of the present invention in a wet environment using pig skin.
9 shows the hemostatic effect of the sucker tooth protein-based hydrogel of the present invention.
Figure 10 shows the non-bleeding effect of the squid sucker teeth protein-based hydrogel of the present invention.
Figure 11a shows the wound healing effect of the squid sucker tooth protein-based hydrogel of the present invention in a rat skin wound experimental model. Figure 11b shows the inflammatory response level of the squid sucker tooth protein-based hydrogel of the present invention in a rat skin wound experimental model. Figure 11c shows the collagen regeneration effect of the squid sucker tooth protein-based hydrogel of the present invention in a rat skin wound experimental model.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.
실시예Example
실시예 1. 실험 준비 및 실험 방법Example 1. Experiment preparation and experiment method
1-1. 오징어 빨판 이빨 단백질의 수중 파티클 형성 확인1-1. Confirmation of underwater particle formation of squid sucker teeth proteins
오징어 빨판 이빨 단백질(Suckerin-12, 서열번호 2)을 1 mg/mL 농도로 5% 아세트산에 녹인 후 negative stain을 위해 플라즈마 처리 탄소 코팅 TEM 그리드(Ted Pella, Inc)에 1 mg/ml superin 용액 4 ul을 도포하고 60초 동안 배양한 후 여과지로 제거하였다. 2% 초산 우라늄 용액 4ul을 그리드에 도포하여 블랏 처리하였다. TEM 및 Bright-field STEM 모드에서 200 kV의 전계방출 전자총을 장착한 JEM 2100F 전자현미경(Jeol, 일본)에서 이미징을 수행하였다. Squid sucker tooth protein (Suckerin-12, SEQ ID NO: 2) was dissolved in 5% acetic acid at a concentration of 1 mg/mL, and then 1 mg/ml superin solution 4 was placed on a plasma-treated carbon-coated TEM grid (Ted Pella, Inc) for negative staining. ul was applied and incubated for 60 seconds, and then removed with filter paper. 4ul of a 2% uranium acetate solution was applied to the grid and blotted. Imaging was performed on a JEM 2100F electron microscope (Jeol, Japan) equipped with a 200 kV field emission electron gun in TEM and Bright-field STEM modes.
1-2. 수중 나노파티클 입자 크기 분석1-2. Particle size analysis of nanoparticles in water
오징어 빨판 이빨 단백질을 5% 아세트산에 10wt%와 15wt% 농도로 녹인 후 dynamic light scattering (DLS)을 통해 hydrodynamic diameter를 측정하였다. 658nm 단색 레이저가 장착된 90 Plus particle size analyser (Brookhaven Instruments)를 이용하였으며 반사 효과를 최소화하기 위해 모든 측정은 90°의 산란 각도에서 진행하였다.After dissolving squid sucker tooth protein in 5% acetic acid at concentrations of 10 wt% and 15 wt%, the hydrodynamic diameter was measured by dynamic light scattering (DLS). A 90 Plus particle size analyzer (Brookhaven Instruments) equipped with a 658 nm monochromatic laser was used and all measurements were performed at a scattering angle of 90° to minimize reflection effects.
1-3. 세포독성 L929 세포 MTT 어세이 1-3. Cytotoxic L929 cell MTT assay
MTT 세포독성 테스트는 ISO 10993-5, 용출 표준은 ISO 10993-12를 따라 진행했다. L929 세포는 태아 소 혈청(FBS), 4 mML-글루타민, 100 IU mL-1 페니실린, 100 μg mL-1 스트렙토마이신(Gibco, 미국)을 함유한 MEM 배지에서 CO2 인큐베이터(Vision Scientific, 한국) 37℃에서 배양하였다. 오징어 빨판 이빨 단백질 필름은 5% 아세트산에 용해된 단백질 용액을 건조하여 제조하였다. 오징어 빨판 이빨 단백질 필름은 37℃에서 24시간 동안 Shaking incubator에서 표면적/세포배양용액 6 cm2/mL로 용출하였다. 희석하지 않은 용리액을 가장 높은 농도인 100%으로 하여 50, 25, 12.5%로 용리액을 희석한 후 사용하였다. 1 X 104 세포를 96well에 넣고 용출액을 첨가한 후 24시간 동안 배양하였다. 배지를 well에서 제거한 후 MTT 용액(50 μg/well)을 첨가하여 2시간 배양하였다. MTT 용액을 제거하고 이소프로판올(100 μL/well)을 첨가한 후 용출액(elute formazan)을 제조한다. 흡광도는 570nm에서 마이크로 플레이트 리더(Tecan, Swiss)로 측정하였다. 음성 대조군으로는 고밀도 폴리에틸렌 필름(하타노 연구소, 식품의약품안전센터), 양성 대조군으로는 0.1%의 ZDEC 폴리우레탄 필름(하타노 연구소, 식품의약품안전센터)을 사용하였다.The MTT cytotoxicity test was carried out according to ISO 10993-5 and the dissolution standard according to ISO 10993-12. L929 cells were cultured in MEM medium containing fetal bovine serum (FBS), 4 mM L-glutamine, 100 IU mL-1 penicillin, and 100 μg mL-1 streptomycin (Gibco, USA) in a CO 2 incubator (Vision Scientific, Korea) 37 Incubated at °C. Squid sucker tooth protein film was prepared by drying a protein solution dissolved in 5% acetic acid. Squid sucker tooth protein film was eluted with a surface area/cell culture solution of 6 cm 2 /mL in a shaking incubator at 37°C for 24 hours. The undiluted eluent was used after diluting the eluent to 50, 25, and 12.5% with the highest concentration of 100%. 1
1-4. Raw 264.7 대식 세포 MTT 어세이 1-4. Raw 264.7 macrophage MTT assay
Raw 264.7 세포를 10% FBS를 포함한 DMEM에 현탁시킨 후 48 well plate에 1Х105 cells/ml 세포수가 되도록 분주하여 CO2 incubator에서 12시간 37℃배양 후 5% 아세트산에 녹인 오징어 빨판 단백질과 5% 아세트산을 0.1, 1, 10 μg/ml 농도로 처리하고 24시간 동안 배양하였다. 이후 각 well에 MTT를 넣고 잘 섞어 준 후 90분 동안 37℃에서 배양한 후 상층액을 제거하고 DMSO를 분주하여 흔들어 준 후 plate reader(spectrostar nano, BMG Labtech, Germany)로 570nm에서 흡광도를 측정하였다. After suspending Raw 264.7 cells in DMEM containing 10% FBS, dispensing to 1Х10 5 cells/ml cell count in a 48 well plate and culturing in CO 2 incubator for 12 hours at 37℃, squid sucker protein dissolved in 5% acetic acid and 5% acetic acid were treated at concentrations of 0.1, 1, and 10 μg/ml and incubated for 24 hours. Then, MTT was added to each well, mixed well, incubated at 37 ° C for 90 minutes, the supernatant was removed, DMSO was dispensed and shaken, and the absorbance was measured at 570 nm with a plate reader (spectrostar nano, BMG Labtech, Germany). .
1-5. 면역원성 실험1-5. Immunogenicity experiment
Raw 264.7 세포를 10% FBS를 포함한 DMEM에 현탁시킨 후 48 well plate에 1Х105 cells/ml 세포수가 되도록 분주하여 4시간 배양 후 LPS (10 μg/ml)와 5% 아세트산에 녹인 오징어 빨판 단백질과 5% 아세트산을 0.1, 1, 10 μg/ml 농도로 처리하고 24시간 동안 배양하였다. 세포 배양액을 취하여 염증성 cytokine인 TNF-α와 IL-6의 함량을 ELISA kit를 이용하여 측정하였다. After suspending Raw 264.7 cells in DMEM containing 10% FBS, distributing them to 1Х10 5 cells/ml in a 48-well plate and incubating for 4 hours, squid sucker protein dissolved in LPS (10 μg/ml) and 5% acetic acid and 5 % acetic acid was treated at concentrations of 0.1, 1, and 10 μg/ml and cultured for 24 hours. The cell culture medium was taken and the contents of TNF-α and IL-6, which are inflammatory cytokines, were measured using an ELISA kit.
1-6. 오징어 빨판 이빨 단백질을 이용한 하이드로겔 제작 1-6. Fabrication of hydrogel using squid sucker tooth protein
오징어 빨판 이빨 재조합 단백질을 분리 정제 후 동결 건조한 상태로 제공받아 사용하였다. 오징어 빨판 이빨 단백질을 10% 아세트산에 10 wt%로 용해시키고, 광개시제로서 1 mM 리보플라빈 (riboflavin); 0.2 mM tris(bipyridine)ruthenium(II) chloride (Ru(bpy3)2+)와 1mM ammonium persulfate (APS); 및 0.8 U/mL의 horse radish peroxide (HRP)와 0.04wt% hydrogen peroxide (H2O2);를 각각 처리한 후 2시간 동안 UV 중합 진행하여 하이드로겔 형성 효과를 비교하였다.Squid sucker tooth recombinant protein was separated and purified and then provided in a freeze-dried state for use. Squid sucker tooth protein was dissolved at 10 wt% in 10% acetic acid, and 1 mM riboflavin as a photoinitiator; 0.2 mM tris(bipyridine)ruthenium(II) chloride (Ru(bpy3) 2+ ) and 1 mM ammonium persulfate (APS); And 0.8 U/mL of horse radish peroxide (HRP) and 0.04wt% hydrogen peroxide (H 2 O 2 ) respectively; UV polymerization was performed for 2 hours to compare the hydrogel formation effect.
1-7. 점탄성 평가1-7. Viscoelastic evaluation
다양한 농도로 용해시킨 오징어 빨판 이빨 단백질에, 다양한 농도로 리보플라빈을 처리하여 광중합한 오징어 빨판 이빨 단백질이 하이드로겔을 형성하는지 확인하기 위해 Rheometer (Discovery HR 10, TA instruments, USA) 장비에 직경 8mm, 간격 1 mm의 parallel plate geometry 를 이용하여 점탄성을 평가하였다. 오징어 빨판 이빨 단백질을 10% 아세트산에 10wt%, 15wt% 농도로 용해시키고 리보플라빈을 0.5, 1.0 mM 농도로 처리하여 혼합한 후 UV 광(light)에서 광중합하여 하이드로겔을 제작하였다. Squid sucker tooth proteins dissolved in various concentrations were treated with riboflavin at various concentrations to check whether the photopolymerized squid sucker tooth proteins formed a hydrogel. Viscoelasticity was evaluated using a 1 mm parallel plate geometry. Squid sucker tooth protein was dissolved in 10% acetic acid at concentrations of 10wt% and 15wt%, and riboflavin was treated and mixed at concentrations of 0.5 and 1.0 mM, and then photopolymerized under UV light to prepare a hydrogel.
각주파수 (angular frequency)를 10 rad/s로 고정하고 0.01-10%의 응력(strain) 범위에서 amplitude sweeps 를 측정하여 샘플의 linear viscoelastic region (LVE region)를 확인하였다. 앞서 측정된 LVE region을 바탕으로 0.1-100 rad/s 범위에서 oscillation frequency sweep를 측정하여, storage modulus(G') 와 loss modulus(G'') 값을 얻었다.The angular frequency was fixed at 10 rad/s, and the linear viscoelastic region (LVE region) of the sample was confirmed by measuring amplitude sweeps in the strain range of 0.01-10%. Based on the previously measured LVE region, an oscillation frequency sweep was measured in the range of 0.1-100 rad/s to obtain storage modulus (G') and loss modulus (G'') values.
1-8. 돼지껍데기 접착 실험 1-8. Pork skin adhesion test
하이드로겔의 조직 접착 강도는 ASTM 표준에 따라 universal testing machine (Instron 5544, Norwood, MA, US)를 이용한 전단응력 측정을 통해 평가하였다. 돼지 껍데기 (stellen Medical, USA)는 10X10 mm 크기로 잘라 실험 1 시간 전 0.1 M PBS에 넣어 37℃에서 수화하였다. 돼지 껍데기는 10X100 mm 크기의 알루미늄 바에 순간접착제(3M)를 이용하여 부착하였으며, 돼지껍데기 표면에 10% 아세트산에 15wt%로 용해한 오징어 빨판 이빨 단백질 및 오징어 빨판 이빨 단백질-기반 하이드로겔 40mg을 올려주었다. 시료를 올리지 않은 돼지껍데기를 붙인 알루미늄 바를 겹쳐준 후 클립을 사용하여 고정하였다. 고정된 알루미늄 바는 습윤 환경에서 RT 조건에서 2시간 접착 후 UTM 장비에서 10kN load cell을 사용하여 10 mm/min의 cross head 속도로 두 알루미늄 바가 완전히 분리될 때까지 측정하였다. 대조군으로 사용한 피브린 글루(녹십자, 한국)도 동일한 방법으로 측정하였다. Tissue adhesive strength of the hydrogel was evaluated by shear stress measurement using a universal testing machine (Instron 5544, Norwood, MA, US) according to ASTM standards. Pork skin (stellen Medical, USA) was cut into 10X10 mm size and hydrated at 37°C in 0.1
1-9. 지혈 실험1-9. hemostasis experiment
지혈 실험은 대구 실험동물센터에서 유지되고 있는 토끼 혈액을 채혈하여 진행되었으며 지혈 실험 전체 과정은 대구경북 첨단의료산업진흥재단의 동물실험 윤리위원회의 승인을 받아 진행하였다. 10% 아세트산에 15wt%로 용해한 오징어 빨판 이빨 단백질과 리보플라빈 0.5mM 넣어 제작한 하이드로겔을 시료로 준비하였다. 준비된 시료는 10 mg씩 50ml 튜브에 담은 후 1500rpm으로 3분동안 원심분리 하였다. 토끼 혈액은 채혈 즉시 항응고제인 anticoagulant-citrate-dextrose 용액과 9:1 비율로 희석하여 준비한 후 실험 직전에 1.25 당량의 1M CaCl2를 처리했다. 튜브에 담긴 시료에 50μl의 토끼 혈액을 넣은 후 RT에서 60초 경과 후 증류수 10ml을 넣어 희석하였다. 상등액 5ml에 증류수 5ml을 넣어 추가 희석시킨 후 RT에서 1시간 경과 후 542nm에서 흡광도를 측정하였다. 전혈에서 총 헤모글로빈 흡광도; Blank (Abs)와 시료에 포획되지 않아 상등액에 존재하는 헤모글로빈의 흡광도; Sample(Abs)를 측정하여 하기 식 1을 이용하여 상대 혈액 응고 지수 (Relative whole blood clotting index, Relative BCI)를 계산하였다. The hemostasis experiment was conducted by collecting blood from rabbits maintained at the Daegu Experimental Animal Center, and the entire process of the hemostasis experiment was approved by the Animal Experiment Ethics Committee of the Daegu-Gyeongbuk Advanced Medical Industry Promotion Foundation. A hydrogel made of squid sucker tooth protein dissolved in 10% acetic acid at 15 wt% and 0.5 mM riboflavin was prepared as a sample. The prepared sample was placed in a 50ml tube of 10 mg each and then centrifuged at 1500 rpm for 3 minutes. Rabbit blood was prepared by diluting it with an anticoagulant-citrate-dextrose solution in a 9:1 ratio immediately after blood collection, and then treated with 1.25 equivalents of 1M CaCl2 immediately before the experiment. After adding 50 μl of rabbit blood to the sample in the tube, it was diluted with 10 ml of distilled water after 60 seconds at RT. After further dilution by adding 5 ml of distilled water to 5 ml of the supernatant, the absorbance was measured at 542 nm after 1 hour at RT. total hemoglobin absorbance in whole blood; Absorbance of blank (Abs) and hemoglobin present in the supernatant not captured by the sample; Sample (Abs) was measured and relative whole blood clotting index (Relative BCI) was calculated using
[식 1][Equation 1]
1-10. 용혈 실험1-10. hemolysis experiment
혈액을 15분간 4000rpm으로 원심분리하고 상등액은 폐기했으며 적혈구는 PBS로 3회 세척하였다. 적혈구는 PBS로 10v/v%로 희석하였으며, 희석된 적혈구는 하이드로겔 100 mg 당 500 μL씩 첨가하였다. PBS는 음성 대조군으로 사용되었고 0.1% Triton-x는 양성 대조군으로 사용되었다. 37℃에서 1시간 배양한 후 3500 rpm에서 10분간 원심분리하여 상등액의 흡광도를 540 nm로 측정하였다. 용혈률(%)은 다음 식 2에 따라 계산하였다.The blood was centrifuged at 4000 rpm for 15 minutes, the supernatant was discarded, and the red blood cells were washed three times with PBS. Red blood cells were diluted to 10 v/v% with PBS, and 500 μL of the diluted red blood cells was added per 100 mg of the hydrogel. PBS was used as a negative control and 0.1% Triton-x was used as a positive control. After incubation at 37 ° C. for 1 hour, centrifugation was performed at 3500 rpm for 10 minutes, and the absorbance of the supernatant was measured at 540 nm. The hemolysis rate (%) was calculated according to
[식 2][Equation 2]
1-11.1-11. In vivo In vivo 창상 회복 실험 wound healing experiment
Sprague Dawley (SD) 랫드는 포항공과대학교 생명공학연구센터의 실험 동물실에서 유지되었으며 창상 회복 실험 과정은 포항공과대학교 동물실험윤리위원회의 승인을 받아 진행하였다. 모든 실험과정에서 성별과 주령이 일치하는 7주령 이내의 랫드를 사용하였다. 실험 전 모든 수술 도구는 멸균하여 준비하였으며 10% 아세트산에 15wt%로 녹인 오징어 빨판 이빨 단백질과 0.5mM 리보플라빈을 처리하여 광중합한 하이드로겔은 자외선 살균하였다. 랫드는 아이프란액을 넣은 호흡마취기를 사용하여 마취 후 실험을 진행하였다. 랫드의 등을 면도기로 제모한 후 직경 8mm의 바이옵시 펀치(Kai medical, Japan)를 이용하여 만든 원형 창상 부위에 시료를 발라준 후 긁음 방지를 위해 멸균방수필름을 부착하였다. 창상 회복 경과는 카메라를 이용하여 21일 동안 같은 시간에 촬영 후 Image J 프로그램을 이용하여 창상 부위를 계산하였다. 실험 시작 7일과 14일에 이산화탄소를 이용한 안락사 시킨 후 창상 부위 주변 10Х10 mm 크기로 피부 조직을 잘라 포르말린 용액에 고정시킨 후 hematoxylin and eosin (H&E) 염색과 Masson's trichrome (MT) 염색을 진행하였으며 이미지 분석은 Leica microscope를 이용하여 진행했다. Sprague Dawley (SD) rats were maintained in the Laboratory Animal Room of the Biotechnology Research Center, Pohang University of Science and Technology, and the wound healing experiment was conducted with the approval of the Animal Research Ethics Committee of Pohang University of Science and Technology. In all experiments, rats within 7 weeks of age matching gender and age were used. Before the experiment, all surgical tools were sterilized and prepared, and the hydrogel photopolymerized by treating squid sucker tooth protein dissolved in 10% acetic acid at 15 wt% and 0.5 mM riboflavin was sterilized by ultraviolet rays. The rats were anesthetized using a respiratory anesthetic machine containing Ipran solution and then the experiment was conducted. After depilating the back of the rat with a razor, the sample was applied to the circular wound site made using a biopsy punch (Kai medical, Japan) with a diameter of 8 mm, and then a sterilized waterproof film was attached to prevent scratching. The wound healing progress was taken using a camera at the same time for 21 days, and the wound area was calculated using the Image J program. After euthanasia using carbon dioxide on the 7th and 14th days of the experiment, the skin tissue was cut to a size of 10Х10 mm around the wound site, fixed in formalin solution, and hematoxylin and eosin (H&E) staining and Masson's trichrome (MT) staining were performed. Image analysis It was performed using a Leica microscope.
1-12. 통계분석1-12. statistical analysis
통계 분석은 Origin 소프트웨어를 이용하였다. 실험의 모든 분석 결과는 unpaired Student's t-test를 이용하였다. P-value > 0.05 일 때 통계적으로 유의미하다고 판단하였다.Statistical analysis was performed using Origin software. All analysis results of the experiment were performed using unpaired Student's t-test. When P-value > 0.05, it was judged to be statistically significant.
실시예 2. 오징어 빨판 이빨 단백질의 특성 평가 Example 2. Characteristic evaluation of squid sucker tooth protein
2-1. 수중 나노파티클 형성 평가2-1. Evaluation of nanoparticle formation in water
본 발명자들은 본 발명의 오징어 빨판 이빨 단백질(SRT 단백질)의 수중에서의 상태를 확인하기 위해, 오징어 빨판 이빨 단백질을 2mg/mL로 5% 아세트산에 희석하여 수성 분산액 상태로 만든 후 cryo-TEM을 측정하였다.In order to confirm the state of the squid sucker tooth protein (SRT protein) of the present invention in water, the present inventors diluted the squid sucker tooth protein to 2 mg / mL in 5% acetic acid to form an aqueous dispersion, and then measured by cryo-TEM did
그 결과, 도 1에 나타낸 바와 같이, 본 발명의 오징어 빨판 이빨 단백질은 수중에서 나노입자로 존재하는 것을 확인하였다(scale bar; 100 nm).As a result, as shown in FIG. 1, it was confirmed that the squid sucker tooth protein of the present invention exists as nanoparticles in water (scale bar; 100 nm).
또한, 본 발명자들은 DLS를 이용하여 나노입자 크기를 측정하였다.In addition, the present inventors measured the nanoparticle size using DLS.
그 결과, 도 2에 나타낸 바와 같이, 평균 크기는 10wt% 농도에서 144 ± 3 nm (10 wt%), 15wt% 농도에서 168 ± 4 nm로 측정되었다. As a result, as shown in Figure 2, the average size was measured as 144 ± 3 nm (10 wt%) at 10wt% concentration and 168 ± 4 nm at 15wt% concentration.
2-2. L929 세포주를 이용한 오징어 빨판 이빨 단백질의 세포독성 평가2-2. Cytotoxicity evaluation of squid sucker tooth protein using L929 cell line
본 발명자들은 분리 정제하여 얻은 오징어 빨판 이빨 단백질의 세포 독성 여부를 L929 세포주에서 평가하였다. L929 세포주를 이용한 세포독성 실험은 경북테크노파크 메디컬융합소재실용화센터를 통해 ISO 10993-5을 토대로 진행하였다. ISO 10993-12에 따라 SRT 단백질을 필름 형태로 제작하고 양쪽 표면적 비(6cm2/ml)로 용출하였다. 용출물을 2배씩 희석하여 100%, 50%, 25%, 12.5% 농도로 L929 세포에 처리한 후 MTT 테스트를 진행하였다. The present inventors evaluated the cytotoxicity of squid sucker tooth protein obtained by isolation and purification in the L929 cell line. The cytotoxicity test using the L929 cell line was conducted based on ISO 10993-5 through the Gyeongbuk Techno Park Medical Convergence Material Commercialization Center. According to ISO 10993-12, the SRT protein was produced in the form of a film and eluted at a surface area ratio (6 cm 2 /ml) on both sides. After diluting the lysate twice and treating L929 cells at concentrations of 100%, 50%, 25%, and 12.5%, the MTT test was performed.
본 실시예와 관련하여 대조군 종류 및 용출액 농도에 따라, 하기와 같이 총 10개의 실험군으로 나누었다:In relation to this Example, a total of 10 experimental groups were divided according to the type of control group and the concentration of the eluate as follows:
(1) 공시험액 실험군(NT);(1) blank test solution experimental group (NT);
(2) 음성 대조군 High Density Polyethylene Film (RM-C) 용출물 100% 실험군;(2) Negative control High Density Polyethylene Film (RM-C) 100% extract test group;
(3) 양성 대조군 0.1% ZDEC Polyurethane Film (RM-A) 용출물 100% 실험군; (3) positive control 0.1% ZDEC Polyurethane Film (RM-A) 100% elution test group;
(4) 양성 대조군 0.1% ZDEC Polyurethane Film (RM-A) 용출물 50% 실험군;(4) positive control 0.1% ZDEC Polyurethane Film (RM-A) 50% elution test group;
(5) 양성 대조군 0.1% ZDEC Polyurethane Film (RM-A) 용출물 25% 실험군;(5) positive control 0.1% ZDEC Polyurethane Film (RM-A) elution 25% experimental group;
(6) 양성 대조군 0.1% ZDEC Polyurethane Film (RM-A) 12.5% 실험군;(6) positive control 0.1% ZDEC Polyurethane Film (RM-A) 12.5% experimental group;
(7) 오징어 빨판 이빨 단백질(Suckerin) 필름 용출물 100% 실험군; (7) 100% test group of squid sucker tooth protein (Suckerin) film extract;
(8) 오징어 빨판 이빨 단백질(Suckerin) 필름 용출물 50% 실험군; (8) 50% test group of squid sucker tooth protein (Suckerin) film extract;
(9) 오징어 빨판 이빨 단백질(Suckerin) 필름 용출물 25% 실험군; 및(9) 25% test group of squid sucker tooth protein (Suckerin) film extract; and
(10) 오징어 빨판 이빨 단백질(Suckerin) 필름 용출물 12.5% 실험군.(10) Experimental group with 12.5% of squid sucker tooth protein (Suckerin) film extract.
ISO 10993-5를 기준으로 세포 생존율이 70% 이상일 때 음성으로 간주하였다. Based on ISO 10993-5, when the cell viability was 70% or more, it was considered negative.
그 결과, 도 3에 나타낸 바와 같이, (7) 내지 (10)번 실험군에서 용출액 농도가 감소함에 따라 세포 생존율이 증가하였으며, 이는 오징어 빨판 이빨 단백질의 세포 독성이 약하거나 거의 없음을 의미한다((1)번 실험군과 t-test ****p<0.0001).As a result, as shown in FIG. 3, the cell viability increased as the concentration of the eluate decreased in the experimental groups (7) to (10), which means that the cytotoxicity of the squid sucker tooth protein was weak or almost absent ((( 1) experimental group and t-test ****p<0.0001).
2-3. Raw 264.7 세포주를 이용한 오징어 빨판 이빨 단백질의 세포 독성 평가2-3. Cytotoxicity evaluation of squid sucker tooth protein using Raw 264.7 cell line
본 발명자들은 Raw 264.7 세포주에서의 면역세포 생존율(viability)을 확인하고자 MTT 어세이를 실시하였다. 5% 아세트산에 용해시킨 오징어 빨판 이빨 단백질 용액과 단백질 용매인 5% 아세트산을 0.1, 1, 10 μg/ml 농도로 희석한 후 흡광도를 측정하였다.The present inventors performed MTT assay to confirm the viability of immune cells in the Raw 264.7 cell line. Absorbance was measured after diluting squid sucker teeth protein solution dissolved in 5% acetic acid and 5% acetic acid as a protein solvent at concentrations of 0.1, 1, and 10 μg/ml.
흡광도가 높을수록 많은 수의 세포가 살아있고 대사가 활발한 것을 의미한다. The higher the absorbance, the more cells are alive and the metabolism is active.
그 결과, 도 4a에서 나타낸 바와 같이, 본 발명의 오징어 빨판 이빨 단백질(Suckerin) 처리군들의 경우, 정상 대조군인 배지 단독처리군과 비교하여 유사하거나 더 높은 수준으로 흡광도 값이 측정되었고, 이는 오징어 빨판 이빨 단백질이 세포 독성을 나타내지 않음을 의미한다(**p<0.01, ***p<0.001).As a result, as shown in FIG. 4a, in the case of the squid sucker tooth protein (Suckerin) treatment group of the present invention, the absorbance value was measured at a similar or higher level compared to the medium alone treatment group, which is a normal control group. It means that the tooth protein did not show cytotoxicity (**p<0.01, ***p<0.001).
2-4. 오징어 빨판 이빨 단백질의 염증성 사이토카인 분비 활성 확인 2-4. Confirmation of inflammatory cytokine secretion activity of squid sucker tooth protein
본 발명자들은, 본 발명의 오징어 빨판 이빨 단백질을 생체 재료로 응용하는 데 중요한 요소인 면역원성을 확인하기 위하여, 12시간 배양한 Raw 264.7 세포주에, 5% 아세트산에 용해시킨 오징어 빨판 이빨 단백질 용액을 다시 5% 아세트산으로 희석하여 0.1, 1, 10 μg/ml 농도로 제작한 오징어 빨판 이빨 단백질 용액을 처리한 후, 대표적인 염증성 사이토카인인 TNF-α와 IL-6의 발현 수준을 측정하였다.In order to confirm immunogenicity, which is an important factor in applying the squid sucker tooth protein of the present invention as a biomaterial, the present inventors added a solution of squid sucker tooth protein dissolved in 5% acetic acid to Raw 264.7 cell line cultured for 12 hours. After treating squid sucker tooth protein solutions prepared at concentrations of 0.1, 1, and 10 μg/ml diluted with 5% acetic acid, the expression levels of TNF-α and IL-6, which are representative inflammatory cytokines, were measured.
그 결과, 도 4b에 나타낸 바와 같이, TNF-α의 경우 오징어 빨판 이빨 단백질 10 μg/ml 처리군에서만 약간 발현되었을 뿐, TNF-α 및 IL-6 대부분은 양성 대조군인 LPS 10 μg/ml 처리군에 비해 거의 발현되지 않았다(****p<0.0001). As a result, as shown in FIG. 4b, TNF-α was only slightly expressed in the squid
따라서, 이는 본 발명의 오징어 빨판 이빨 단백질이 사이토카인에 의한 염증 반응을 유발하지 않아 생체 소재로 적용하기에 적합함을 보여준다Therefore, this shows that the squid sucker tooth protein of the present invention does not induce an inflammatory response by cytokines and is suitable for application as a biomaterial.
실시예 3. 오징어 빨판 이빨 단백질을 이용한 하이드로겔 형성 최적 조건 정립Example 3. Establishment of optimal conditions for hydrogel formation using squid sucker tooth protein
3-1. 개시제 종류에 따른 하이드로겔 형성 시간 비교3-1. Comparison of hydrogel formation time according to the type of initiator
본 발명자들은 오징어 빨판 이빨 단백질에 높은 분포로 존재하는 타이로신(tyr)을 di-tyr으로 가교결합(crosslinking)시켜 하이드로겔을 제작하였다. 이때, 오징어 빨판 이빨 단백질 하이드로겔 제작에 가장 적합한 물질을 찾기 위해, 가교결합제(crosslinking agent) 종류에 따라 리보플라빈(riboflavin) 처리군, Ru(bpy3)2+/APS 처리군 및 HRP/H2O2 처리군으로 나누고, 각 그룹에 동일하게 2 시간 동안 UV 중합한 후 겔화(gelation) 정도를 비교하였다. 다이-타이로신(di-tyrosine) 잔기가 형성되는 경우, UVA 조사시 샘플에서 410nm 파장의 파란색 형광이 방출된다. The present inventors prepared a hydrogel by crosslinking tyrosine (tyr), which is present in a high distribution in squid sucker tooth protein, with di-tyr. At this time, in order to find the most suitable material for producing squid sucker tooth protein hydrogel, riboflavin treatment group, Ru(bpy3) 2+ /APS treatment group and HRP/H 2 O 2 according to the type of crosslinking agent Divided into treatment groups, UV polymerization was performed for 2 hours in the same way for each group, and then the degree of gelation was compared. When a di-tyrosine residue is formed, blue fluorescence with a wavelength of 410 nm is emitted from the sample upon UVA irradiation.
그 결과, 도 5에 나타낸 바와 같이, 리보플라빈(riboflavin) 처리군에서 파란색 형광이 방출되어 다이-타이로신(di-tyrosine) 잔기가 형성되었음을 확인하였고, 실험 튜브를 뒤집었을 때 그 상태로 고정된 것을 확인하였다. 이는, 리보플라빈(riboflavin) 처리군에서 중합이 신속히 진행되어 겔화된 것을 의미한다. As a result, as shown in FIG. 5, it was confirmed that blue fluorescence was emitted from the riboflavin-treated group to form a di-tyrosine residue, and it was confirmed that it was fixed in that state when the experimental tube was inverted. did This means that polymerization proceeds rapidly in the riboflavin-treated group and gelation occurs.
반면, HRP/H2O2 처리군은 중합이 되지 않아 용액 상태 그대로였고 Ru(bpy3)2+/APS 처리군은 겔화가 덜 진행되어 실험 튜브를 뒤집었을 때 내용물이 흘러 고정되지 않았다.On the other hand, the HRP/H 2 O 2 treatment group did not polymerize and remained in the solution state, and the Ru(bpy3) 2+ /APS treatment group showed less gelation and the contents did not flow and were not fixed when the test tube was turned over.
따라서, 겔화에 진행되는 시간을 고려했을 때 HRP/H2O2는 중합 시간을 조절하기 어렵고, Ru(bpy3)2+/APS는 리보플라빈을 처리했을 때보다 오랜 시간이 소요되었으므로, 리보플라빈이 가장 효과적인 개시제임을 알 수 있다.Therefore, considering the gelation time, it is difficult to control the polymerization time for HRP/H 2 O 2 and Ru(bpy3) 2+ /APS takes longer than when riboflavin was treated, so riboflavin is the most effective. It can be seen that it is an initiator.
3-2. 리보플라빈을 이용한 오징어 빨판 이빨 단백질 기반 하이드로겔 형성 최적 조건 정립3-2. Establishment of optimal conditions for squid sucker tooth protein-based hydrogel formation using riboflavin
본 발명자들은 오징어 빨판 이빨 단백질을 10wt% 와 15wt% 농도로 10% 아세트산에 용해시킨 후, 상기 실시예 3-1에서 선별된 개시제인 리보플라빈 0.5mM, 1mM을 추가하여 UV 광중합을 진행하여 di-tyr이 충분히 형성되도록 하였다. The present inventors dissolved squid sucker tooth protein in 10% acetic acid at concentrations of 10wt% and 15wt%, and then proceeded with UV photopolymerization by adding 0.5mM and 1mM of riboflavin, the initiator selected in Example 3-1, to obtain di-tyr was sufficiently formed.
그 결과, 도 6에 나타낸 바와 같이, 비교 대조군인 1% 아가로스겔의 경우 UVA에서 파란 형광을 발산하지 못하는 반면, 본 발명의 오징어 빨판 이빨 단백질 기반 하이드로겔(SRT hydrogel)의 경우 파란 형광을 방출하여 di-tyr이 형성된 것을 확인하였다.As a result, as shown in FIG. 6, the 1% agarose gel as a comparative control did not emit blue fluorescence under UVA, whereas the squid sucker tooth protein-based hydrogel (SRT hydrogel) of the present invention emitted blue fluorescence. Thus, it was confirmed that di-tyr was formed.
3-3. 오징어 빨판 이빨 단백질 기반 하이드로겔의 점탄성 확인 3-3. Confirmation of viscoelastic properties of squid sucker teeth protein-based hydrogels
본 발명자들은, 본 발명의 광중합한 오징어 빨판 이빨 단백질 기반 하이드로겔의 점탄성(viscoelasticity)을 확인하기 위해, 단백질 농도와 리보플라빈 농도에 따라 하기와 같이 총 6개의 실험군으로 나누었다:In order to confirm the viscoelasticity of the photopolymerized squid sucker teeth protein-based hydrogel of the present invention, the present inventors divided into six experimental groups according to the protein concentration and riboflavin concentration as follows:
오징어 빨판 이빨 단백질 10wt% 단독처리군(10wt%_G', 10wt%_G'');Squid sucker tooth protein 10wt% alone treatment group (10wt%_G', 10wt%_G'');
오징어 빨판 이빨 단백질 10wt% + 리보플라빈 0.5mM 처리군(10wt%_0.5mM_G', 10wt%_0.5mM_G'');Squid sucker tooth protein 10wt% + riboflavin 0.5mM treatment group (10wt%_0.5mM_G', 10wt%_0.5mM_G'');
오징어 빨판 이빨 단백질 10wt% + 리보플라빈 1mM 처리군(10wt%_1mM_G', 10wt%_1mM_G'');Squid sucker tooth protein 10wt% + riboflavin 1mM treatment group (10wt%_1mM_G', 10wt%_1mM_G'');
오징어 빨판 이빨 단백질 15wt% 단독처리군(15wt%_G', 15wt%_G'');Squid sucker tooth protein 15wt% alone treatment group (15wt%_G', 15wt%_G'');
오징어 빨판 이빨 단백질 15wt% + 리보플라빈 0.5mM 처리군(15wt%_0.5mM_G', 15wt%_0.5mM_G''); 및Squid sucker tooth protein 15wt% + riboflavin 0.5mM treatment group (15wt%_0.5mM_G', 15wt%_0.5mM_G''); and
오징어 빨판 이빨 단백질 15wt% + 리보플라빈 1mM 처리군(15wt%_1mM_G', 15wt%_1mM_G'').Squid sucker tooth protein 15wt% + riboflavin 1mM treatment group (15wt%_1mM_G', 15wt%_1mM_G'').
점탄성 물질의 탄성을 나타내는 G'(저장 탄성률; storage modulus)과 점성을 나타내는 G''(손실 탄성률; loss modulus)을 통해 고체에 가까운 성질을 나타내는지, 액체에 가까운 성질을 나타내는지 알 수 있다.It can be seen whether a viscoelastic material exhibits a property close to a solid or a liquid through G' (storage modulus) representing elasticity and G'' (loss modulus) representing viscosity.
그 결과, 도 7에 나타낸 바와 같이, 모든 실험군에서 storage modulus가 loss modulus보다 높게 나와 Loss tangent(G''/G')가 모두 유체-유사(fluid like)에서 고체-유사(solid like) 양상으로 변하는 1보다 작은 값을 가지는 것을 확인하였다. As a result, as shown in FIG. 7, the storage modulus was higher than the loss modulus in all experimental groups, and the loss tangent (G''/G') changed from fluid-like to solid-like. It was confirmed that the variable had a value less than 1.
상기 결과들은 실험한 frequency 범위의 모든 시료들이 탄성 성질이 우세하게 나타나는 하이드로겔로 만들어졌음을 보여준다.The above results show that all samples in the frequency range tested were made of hydrogels with predominantly elastic properties.
특히, 오징어 빨판 이빨 단백질 15wt%에 리보플라빈을 0.5mM 처리한 경우, 리보플라빈을 처리하지 않았거나 1mM 처리한 경우보다 높은 storage modulus (G') 값을 가지는 것을 확인할 수 있다.In particular, when 0.5mM of riboflavin was treated with 15wt% of squid sucker tooth protein, it was confirmed that it had a higher storage modulus (G') value than when riboflavin was not treated or 1mM was treated.
따라서, 본 발명의 오징어 빨판 이빨 단백질 기반 하이드로겔은 오징어 빨판 이빨 단백질의 농도가 15wt%이고, 리보플라빈의 농도가 0.5mM일 때 가장 고체-유사(solid like)한 특성을 가지는 것을 확인하였다. Therefore, it was confirmed that the squid sucker tooth protein-based hydrogel of the present invention has the most solid-like characteristics when the concentration of squid sucker tooth protein is 15 wt% and the concentration of riboflavin is 0.5 mM.
실시예 4. 돼지 껍데기를 이용한 습윤환경에서 조직 접착능 확인Example 4. Confirmation of tissue adhesiveness in a wet environment using pig skin
본 발명자들은, 상기 실시예 3에서의 점탄성 실험을 통해 가장 고체-유사(solid like)한 특성을 가진 것으로 나타난 15wt% 농도의 오징어 빨판 이빨 단백질을 10% 아세트산 수용액에 용해시킨 후, 리보플라빈을 0, 0.5, 1mM 처리하여 제조한 하이드로겔을 돼지 껍데기에 적용하여 습윤 환경에서의 조직 접착능을 확인하였다. The present inventors, after dissolving squid sucker tooth protein at a concentration of 15 wt%, which was shown to have the most solid-like properties through the viscoelasticity test in Example 3, in 10% aqueous acetic acid solution, riboflavin to 0, The hydrogel prepared by treatment with 0.5 or 1 mM was applied to pig skin to confirm tissue adhesion in a wet environment.
그 결과, 도 8에서 나타낸 바와 같이, 리보플라빈 0.5mM 처리군이, 리보플라빈 비첨가군(0 mM) 및 리보플라빈 1mM 첨가군보다 높은 접착력을 보였으나 차이가 유의하지는 않았다. As a result, as shown in FIG. 8, the riboflavin 0.5 mM treated group showed higher adhesive strength than the riboflavin non-added group (0 mM) and the
반면, 대조군으로 사용한 시판 피브린 글루의 접착력보다 본 발명의 오징어 빨판 이빨 단백질 기반 하이드로겔의 접착력이 우수한 것을 확인하였다. (*p<0.05, ***p<0.001, ****p<0.0001)On the other hand, it was confirmed that the adhesive strength of the squid sucker tooth protein-based hydrogel of the present invention was superior to that of the commercially available fibrin glue used as a control. (*p<0.05, ***p<0.001, ****p<0.0001)
실시예 5. 토끼 혈액을 이용한 오징어 빨판 이빨 단백질의 특성 평가Example 5. Characteristic evaluation of squid sucker tooth protein using rabbit blood
5-1. 오징어 빨판 이빨 단백질 기반 하이드로겔의 지혈 효과 5-1. Hemostatic effect of squid sucker tooth protein-based hydrogel
본 발명자들은 오징어 빨판 이빨 단백질 기반 하이드로겔의 지혈 효과를 확인하기 위하여, 토끼 혈액을 이용하는 총 4개의 실험군을 나눠 실험을 진행하였다:In order to confirm the hemostatic effect of the squid sucker teeth protein-based hydrogel, the present inventors conducted experiments by dividing a total of four experimental groups using rabbit blood:
아무 처리하지 않은 비처리 실험군 (NT);No treatment, untreated experimental group (NT);
시중에 판매중인 피브린 글루 처리군 (FG); a commercially available fibrin glue treated group (FG);
15wt%로 용해한 오징어 빨판 이빨 단백질 처리군(Suckerin); 및Squid sucker tooth protein treatment group dissolved at 15 wt% (Suckerin); and
오징어 빨판 이빨 단백질을 15wt%로 용해한 후 리보플라빈을 0.5mM 첨가하여 제작한 하이드로겔 처리군(Hydrogel).Hydrogel treatment group (Hydrogel) prepared by dissolving squid sucker tooth protein at 15 wt% and adding 0.5 mM riboflavin.
4개 실험군에 동일한 양의 토끼 혈액을 넣고 60초 후 희석하여 시료에 응집되지 않고 상등액에 존재하는 적혈구의 흡광도를 측정하였다. 이를 바탕으로 얼마나 많은 적혈구가 응집되지 않았는지 상대적 BCI 값을 계산하여 오징어 빨판 이빨 단백질과 이의 하이드로겔이 지혈 효과를 측정하였다. The same amount of rabbit blood was added to the four experimental groups, diluted after 60 seconds, and the absorbance of red blood cells present in the supernatant without aggregation was measured. Based on this, the hemostatic effect of the squid sucker tooth protein and its hydrogel was measured by calculating the relative BCI value of how many red blood cells were not aggregated.
상대적 BCI 값이 낮을수록 시료에 응집된 혈구가 많이 존재하여 시료의 지혈 효과가 높은 것으로 알려져 있다. It is known that the lower the relative BCI value, the higher the hemostatic effect of the sample because more aggregated blood cells are present in the sample.
그 결과, 도 9에서 나타난 바와 같이, 혈액만 넣은 대조군(control)인 비처리 실험군 및 종래 지혈제로 이용되는 시판용 피브린 글루 처리군보다, 본 발명의 오징어 빨판 이빨 단백질 15wt% 처리군(Suckerin)과 오징어 빨판 이빨 단백질 15wt%에 리보플라빈을 0.5mM 첨가하여 제작한 하이드로겔 처리군(Hydrogel)이 현저하게 향상된 지혈 효과를 발휘하는 것을 확인하였다.As a result, as shown in FIG. 9, the squid sucker tooth protein 15wt% treatment group (Suckerin) and squid of the present invention were more It was confirmed that the hydrogel treated group (Hydrogel) prepared by adding 0.5 mM of riboflavin to 15 wt% of sucker tooth protein exhibited a remarkably improved hemostatic effect.
5-2. 오징어 빨판 이빨 단백질 기반 하이드로겔의 용혈 효과5-2. Hemolytic effect of squid sucker tooth protein-based hydrogel
본 발명자들은 본 발명의 오징어 빨판 이빨 단백질 기반 하이드로겔의 용혈 효과를 확인하기 위하여, 오징어 빨판 이빨 단백질을 15wt%로 용해한 후 리보플라빈을 0.5mM 첨가하여 제작한 하이드로겔(Hydrogel)과 15wt%로 용해한 오징어 빨판 이빨 단백질(Suckerin)에, 동일한 양으로 희석한 적혈구를 첨가하여 인큐베이션한 후, 용혈되어 상등액에 존재하는 적혈구 용해물의 흡광도를 측정하였다.In order to confirm the hemolytic effect of the squid sucker tooth protein-based hydrogel of the present invention, the present inventors dissolved 15 wt% of squid sucker tooth protein and then added 0.5 mM riboflavin to a hydrogel and squid dissolved at 15 wt%. Red blood cells diluted in the same amount were added to sucker tooth protein (Suckerin) and incubated, and then hemolyzed and the absorbance of the red blood cell lysate present in the supernatant was measured.
용혈 지수는 다음과 같은 기준에 따라 평가되었다: 지수가 10% 미만일 경우 비용혈성, 25% 미만일 경우 용혈성. The hemolytic index was evaluated according to the following criteria: non-hemolytic if the index was less than 10% and hemolytic if the index was less than 25%.
그 결과, 도 10에 나타낸 바와 같이, 0.1% triton-x군에서는 적혈구가 용혈되어 방출된 헤모글로빈으로 인해 선명한 붉은색을 보였다. Suckerin군은 약간의 용혈현상을 유도하는 것을 확인할 수 있었다. 반면, 하이드로겔군은 비용혈성을 가지는 것을 확인하였다(****p<0.0001).As a result, as shown in FIG. 10, the 0.1% triton-x group showed a vivid red color due to hemoglobin released from hemolysis of red blood cells. It was confirmed that the Suckerin group induced some hemolysis. On the other hand, it was confirmed that the hydrogel group had non-hemorrhagic properties (****p<0.0001).
실시예 6. 랫드 피부 창상 실험 모델을 이용한 창상 치료 효과 확인 Example 6. Confirmation of wound healing effect using rat skin wound experimental model
6-1. 오징어 이빨 단백질의 피부 창상 회복 효과 6-1. Skin wound healing effect of squid tooth protein
본 발명자들은 본 발명의 오징어 빨판 이빨 단백질 기반 하이드로겔의 창상 치료 효과를 확인하기 위하여, 랫드 피부 창상 실험 모델을 이용하였다.The present inventors used a rat skin wound experimental model to confirm the wound healing effect of the squid sucker tooth protein-based hydrogel of the present invention.
등에 원형 창상을 낸 SD 랫드에 4개의 실험군을 나누었다:SD rats with circular wounds on the back were divided into 4 experimental groups:
아무처리도 하지 않은 실험군(NT);Experimental group without any treatment (NT);
시판 피브린 글루를 처리한 실험군(Fibrin);Experimental group treated with commercially available fibrin glue (Fibrin);
15wt%로 용해한 오징어 빨판 이빨 단백질을 처리한 실험군(SRT); 및Experimental group treated with 15wt% dissolved squid sucker tooth protein (SRT); and
15wt%로 용해한 오징어 빨판 이빨 단백질에 0.5mM 리보플라빈을 넣어 준비한 하이드로겔 샘플을 처리한 실험군(Hydrogel).Experimental group (Hydrogel) treated with hydrogel samples prepared by adding 0.5 mM riboflavin to squid sucker tooth protein dissolved at 15 wt%.
창상 실험을 진행한 날을 Day 0으로 하여 21일 동안 창상 회복 정도를 촬영하였다. The degree of wound healing was photographed for 21 days, with the day of the wound experiment as
그 결과, 도 11a에 나타낸 바와 같이, 본 발명의 SRT 처리군과 하이드로겔 처리군의 초기 상처 회복 효과가 비처리군(NT)이나 시판용 피브린 글루 처리군(Fibrin)에 비해, 신속하게 일어난 것을 확인할 수 있다. (scale bar; 3.5mm)As a result, as shown in FIG. 11a, it was confirmed that the initial wound healing effect of the SRT-treated group and the hydrogel-treated group of the present invention occurred more rapidly than that of the non-treated group (NT) or the commercially available fibrin glue-treated group (Fibrin). can (scale bar; 3.5mm)
6-2. 조직 검사를 통한 오징어 빨판 이빨 단백질의 염증 반응 및 피부 세포 재생 효과 확인 6-2. Confirmation of inflammatory response and skin cell regeneration effect of squid sucker tooth protein through biopsy
본 발명자들은 본 발명의 오징어 빨판 이빨 단백질 기반 하이드로겔의 염증 반응 및 피부 세포 재생 효과를 확인하기 위하여, 랫드 피부 창상 실험 모델을 이용하였다.The present inventors used a rat skin wound experimental model to confirm the inflammatory response and skin cell regeneration effect of the squid sucker tooth protein-based hydrogel of the present invention.
실험 시작 후 7, 14일째 되는 날 랫드를 안락사 시켜 H&E, MT 조직 염색을 진행하였다.On the 7th and 14th days after the start of the experiment, the rats were euthanized and H&E and MT tissue staining was performed.
피부가 회복되는 과정에서 상처 부위의 딱지, 표피 생성, 진피에서의 염증세포 침윤, 염증반응, 혈관신생, 등이 관찰되며 상처가 회복될수록 딱지는 없어지고 표피가 피부표면을 완전히 덮으며 염증 세포도 감소하는 것으로 알려져 있다. In the process of skin recovery, scab, epidermis formation, inflammatory cell infiltration in the dermis, inflammatory response, angiogenesis, etc. are observed. As the wound heals, the scab disappears, the epidermis completely covers the skin surface, and inflammatory cells known to decrease.
그 결과, 도 11b에 나타낸 바와 같이, 7일째에 비처리군(NT)이나 시판용 피브린 글루 처리군(Fibrin)에 비해, 본 발명의 SRT 처리군과 하이드로겔 처리군에서 더 적은 염증세포(노란색 화살표)들이 관찰되었다. 모든 실험군 상처에 딱지가 있고 약한 염증 세포 침윤을 확인하였으나 조직 섬유화나 과도한 염증반응은 관찰되지 않았다. As a result, as shown in FIG. 11B, on the 7th day, compared to the untreated group (NT) or the commercially available fibrin glue treated group (Fibrin), fewer inflammatory cells (yellow arrows) were observed in the SRT-treated group and the hydrogel-treated group of the present invention. ) were observed. All experimental groups had scabs and weak inflammatory cell infiltration, but no tissue fibrosis or excessive inflammatory reaction was observed.
14일째에는 모든 실험군에서 표피가 완전히 재생된 것을 확인할 수 있다. 진피의 경우 비처리군(NT)이나 시판용 피브린 글루 처리군(Fibrin)에서 창상 회복이 완전히 이루어지지 않아 피부조직이 느슨하게 배열된 것을 확인할 수 있는 반면, 본 발명의 SRT 처리군과 하이드로겔 처리군의 경우 염증이나 조직 섬유화 없이 회복되고 있으며, 특히, SRT 처리군은 정상 진피와 가장 유사한 수준으로 회복된 것을 확인하였다. (scale bar; 100 μm)On the 14th day, it was confirmed that the epidermis was completely regenerated in all experimental groups. In the case of the dermis, it can be confirmed that the skin tissue is loosely arranged because the wound is not completely healed in the non-treated group (NT) or the commercially available fibrin glue-treated group (Fibrin), whereas the SRT-treated group and the hydrogel-treated group of the present invention In this case, it was recovered without inflammation or tissue fibrosis, and in particular, it was confirmed that the SRT-treated group was restored to the level most similar to that of the normal dermis. (scale bar; 100 µm)
또한, 파란색으로 콜라겐을 염색하는 MT 염색을 통해 상처부위에서 새로 재생된 콜라겐을 확인하였다. In addition, newly regenerated collagen was confirmed at the wound site through MT staining, which stains collagen in blue.
그 결과, 도 11c에 나타낸 바와 같이, 7일째에는 모든 그룹에서 조직이 완전히 재건되지 않은 것을 확인하였다. 반면, 14일째에는 7일 째보다 조직 구조가 향상된 것을 확인하였다. 비처리군(NT)이나 시판용 피브린 글루 처리군(Fibrin)에서 진피층은 여전히 완벽히 회복했다고 보기 어려웠으나, 본 발명의 SRT 처리군과 하이드로겔 처리군에서는 진피층이 상당부분 회복한 것으로 보이며, 특히, SRT 처리군의 경우 콜라겐 밀도가 주변 정상 조직과 유사한 수준으로 회복된 것을 확인하였다. As a result, as shown in FIG. 11c , it was confirmed that tissue was not completely reconstructed in all groups on the 7th day. On the other hand, on the 14th day, it was confirmed that the tissue structure was improved compared to the 7th day. It was difficult to say that the dermis layer was still completely recovered in the non-treated group (NT) or the commercially available fibrin glue-treated group (Fibrin). In the case of the treated group, it was confirmed that the collagen density was restored to a level similar to that of the surrounding normal tissue.
진술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해되어야 한다.The description of the present invention described above is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.
Claims (16)
squid sucker tooth protein (Suckerin); And a composition for preparing a hydrogel (gel) comprising a photoinitiator.
오징어 빨판 이빨 단백질은 수중에서 나노입자 상태인 것을 특징으로 하는,
하이드로겔(gel) 제조용 조성물.
According to claim 1,
Characterized in that the squid sucker tooth protein is in the form of nanoparticles in water,
A composition for producing a hydrogel.
상기 오징어 빨판 이빨 단백질(Suckerin)은 서열번호 2의 아미노산 서열로 이루어진 것을 특징으로 하는,
하이드로겔(gel) 제조용 조성물.
According to claim 1,
Characterized in that the squid sucker tooth protein (Suckerin) consists of the amino acid sequence of SEQ ID NO: 2,
A composition for producing a hydrogel.
상기 오징어 빨판 이빨 단백질(Suckerin)은 인산, 아세트산, 포름산, 염산, 황산, 질산 및 시트르산으로 이루어진 군으로부터 선택된 1 종 이상의 용매로 용해된 것을 특징으로 하는,
하이드로겔(gel) 제조용 조성물.
According to claim 1,
Characterized in that the squid sucker tooth protein (Suckerin) is dissolved in one or more solvents selected from the group consisting of phosphoric acid, acetic acid, formic acid, hydrochloric acid, sulfuric acid, nitric acid and citric acid,
A composition for producing a hydrogel.
상기 오징어 빨판 이빨 단백질(Suckerin)은 전체 조성물 기준으로 10 내지 30wt% 포함된 것을 특징으로 하는,
하이드로겔(gel) 제조용 조성물.
According to claim 1,
Characterized in that the squid sucker tooth protein (Suckerin) is contained in 10 to 30 wt% based on the total composition,
A composition for producing a hydrogel.
상기 광개시제는, 리보플라빈 (Riboflavin, vitamin B2), 리튬 페닐-2,4,6-트리메틸벤조일포스피네이트(lithium phenyl-(2,4,6-trimethyl benzoyl) phosphinate, LAP), 벤질디메틸케탈(benzyl dimethyl ketal), 아세토페논(acetophenone), 벤조인메틸에테르(benzoin methylether), 디에톡시아세토페논(diethoxyacetophenone), 벤조일 포스핀 옥사이드(benzoyl phosphine oxide), 1-하이드록시사이클로헥실 페닐 케톤(1-hydroxycyclohexyl phenyl ketone), tris(bipyridine)ruthenium(II) chloride (Ru(bpy3)2+), APS (ammonium persulfate), HRP (horse radish peroxide) 및 H2O2 (hydrogen peroxide)로 이루어진 군으로부터 선택된 1 종 이상인 것을 특징으로 하는,
하이드로겔(gel) 제조용 조성물.
According to claim 1,
The photoinitiator is riboflavin (vitamin B2), lithium phenyl- (2,4,6-trimethyl benzoyl) phosphinate (LAP), benzyldimethylketal (benzyl dimethyl ketal), acetophenone, benzoin methylether, diethoxyacetophenone, benzoyl phosphine oxide, 1-hydroxycyclohexyl phenyl ketone ketone), tris(bipyridine)ruthenium(II) chloride (Ru(bpy3) 2+ ), APS (ammonium persulfate), HRP (horse radish peroxide) and H 2 O 2 (hydrogen peroxide). characterized in that,
A composition for producing a hydrogel.
상기 광개시제는 0.1 내지 10mM 포함된 것을 특징으로 하는,
하이드로겔(gel) 제조용 조성물.
According to claim 1,
The photoinitiator is characterized in that it contains 0.1 to 10mM,
A composition for producing a hydrogel.
상기 조성물은 세포, 단백질, 효소 및 당으로 이루어진 군에서 선택된 1종 이상의 생리 활성 물질을 추가로 포함하는 것을 특징으로 하는,
하이드로겔(gel) 제조용 조성물.
According to claim 1,
Characterized in that the composition further comprises at least one physiologically active substance selected from the group consisting of cells, proteins, enzymes, and sugars.
A composition for producing a hydrogel.
A method for preparing a hydrogel comprising crosslinking the composition for preparing a hydrogel of claim 1 by forming a di-tyrosine residue by irradiation with ultraviolet rays.
상기 자외선은 파장범위 300 내지 400nm의 자외선인 것을 특징으로 하는, 방법.
According to claim 9,
The ultraviolet light is characterized in that the ultraviolet light in the wavelength range of 300 to 400nm, method.
A hydrogel prepared by the method of claim 9.
squid sucker tooth protein (Suckerin); and a photoinitiator.
A composition for wound healing or hemostasis comprising the hydrogel of claim 11 or 12.
A bioadhesive composition comprising the hydrogel of claim 11 or claim 12.
A wet dressing for wound healing or hemostasis comprising the hydrogel of claim 11 or claim 12.
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