KR20220138985A - A nanostructure with enhanced photo-fenton-like reaction and uses thereof - Google Patents
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
Description
광-펜톤 유사반응이 향상된 나노 구조체 및 이의 용도에 관한 것이다.It relates to a nanostructure with improved Fenton-like reaction and uses thereof.
나노자임(nanozyme)이란 유기 효소의 활성을 보이는 나노 구조체를 의미한다. 기존 단백질 효소는 특정 생물체에서 발현하여 정제과정을 거치기 때문에 대량생산이 불가능하여 그 생산가격이 일반적으로 비싸며, 주변 환경 및 시간에 의해 그 활성이 필연적으로 감소한다. 그에 반해 나노자임은 화학적으로 대량생산이 가능하기 때문에 생산가격이 크게 저렴하고, 외부환경 등의 변화에 대해 안정한 활성을 보여, 이와 같은 나노자임의 특성을 이용하여 질병 진단, 치료에 응용하려는 연구가 활발히 진행되고 있다.Nanozyme refers to a nanostructure showing the activity of an organic enzyme. Existing protein enzymes cannot be mass-produced because they are expressed in a specific organism and undergo a purification process, so their production price is generally high, and their activity is inevitably reduced by the surrounding environment and time. On the other hand, nanozymes are chemically mass-produced, so their production prices are very low and they show stable activity against changes in the external environment, etc. is being actively pursued.
최근에는 이와 같은 나노자임 개발에 초분자(supramolecule)를 이용하고자 하는 시도가 이루어지고 있다. 초분자란, 수소결합, 정전기적 상호작용 또는 반데르발스 인력과 같은 비공유 결합을 통해 분자나 이온이 모여 형성된 분자복합체를 의미한다. 초분자 촉매는 손님(guest) 분자를 받아들일 수 있는 특성을 가지며, 구성 분자의 입체적인 배열에 따라 분자 단독으로는 가질 수 없는 독특한 성질을 가질 수 있다. 그러나, 초분자 나노자임(supramolecular nanozyme)이 높은 촉매 효율을 갖기 위해서는 빌딩 블록(building block)의 선택, 효소 활성을 갖는 최종 나노구조(nanostructure)로의 합성이 한계로 작용하며, 나노자임을 실제로 적용하기 위해서는 이와 같은 효율성 문제를 해결해야 한다.Recently, attempts have been made to use supramolecules to develop such nanozymes. A supramolecular refers to a molecular complex formed by gathering molecules or ions through non-covalent bonds such as hydrogen bonding, electrostatic interaction, or van der Waals attraction. The supramolecular catalyst has the property to accept guest molecules, and depending on the three-dimensional arrangement of the constituent molecules, it may have unique properties that cannot be possessed by the molecule alone. However, in order for a supramolecular nanozyme to have high catalytic efficiency, selection of building blocks and synthesis into a final nanostructure with enzymatic activity are limitations. Efficiency issues like this need to be addressed.
한편, 페로토시스(ferroptosis)는 세포 내 철에 의존하는 세포사멸의 한 형태를 의미하며, 철이 활성산소(Reactive Oxygen Species: ROS)와 만나 펜톤 반응(Fenton reaction)이 유도되고, 이를 통해 지질과산화물이 세포 내에 축적되어 세포가 사멸하는 방식이다.On the other hand, ferrotosis (ferroptosis) refers to a form of apoptosis dependent on intracellular iron, iron meets reactive oxygen species (ROS) to induce a Fenton reaction, through which lipid peroxide It accumulates in these cells and the cells die.
본 발명자들은 배위결합이 원동력인(coordination-driven) 시아닌(cyanine) 및 철 이온(ferrous ion)의 조립을 기반으로 한 초분자 광-페노자임(photo-fenozyme)을 제조하고, 상기 나노자임이 광산화효소(photooxidase) 유사 활성을 나타내며, 특히 광-펜톤 유사반응이 향상되어 페로토시스를 통한 암세포 사멸 효과를 나타낼 수 있음을 확인함으로써 본 발명을 완성하였다.The present inventors have prepared a supramolecular photo-phenozyme based on the assembly of cyanine and ferrous ions in which the coordination-driven binding is driven, and the nanozyme is photooxidized. The present invention was completed by confirming that the enzyme (photooxidase)-like activity was exhibited, and in particular, the photo-Fenton-like reaction was improved, thereby exhibiting the effect of killing cancer cells through ferrotosis.
일 양상은 하기 화학식 1의 시아닌 및 철 이온을 포함하는 나노 구조체로서,One aspect is a nanostructure comprising cyanine and iron ions of Formula 1 below,
상기 나노 구조체는 철 이온이 시아닌과 배위 결합하여 자기조립된 것인 나노 구조체를 제공하는 것이다.The nanostructure is to provide a nanostructure that is self-assembled by coordinating iron ions with cyanine.
[화학식 1][Formula 1]
다른 양상은 상기 나노 구조체를 포함하는 광역학 치료용 조성물을 제공하는 것이다.Another aspect is to provide a composition for photodynamic therapy comprising the nanostructure.
또 다른 양상은 나노 구조체를 포함하는 암의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another aspect is to provide a pharmaceutical composition for preventing or treating cancer comprising a nanostructure.
또 다른 양상은 1) 시아닌계 화합물 용액을 준비하는 단계; 및Another aspect is 1) preparing a cyanine-based compound solution; and
2) 상기 단계 1)에서 제조한 용액에 철 용액을 첨가하여 혼합하는 단계를 포함하는 시아닌 및 철 이온을 포함하는 나노 구조체를 제조하는 방법을 제공하는 것이다.2) to provide a method for manufacturing a nanostructure containing cyanine and iron ions, including the step of adding and mixing an iron solution to the solution prepared in step 1).
일 양상은 하기 화학식 1의 시아닌 및 철 이온을 포함하는 나노 구조체로서,One aspect is a nanostructure comprising cyanine and iron ions of Formula 1 below,
상기 나노 구조체는 철 이온이 시아닌과 배위 결합하여 자기조립된 것인 나노 구조체를 제공한다.The nanostructure provides a nanostructure that is self-assembled by coordinating iron ions with cyanine.
[화학식 1][Formula 1]
본 명세서에서 "포함"이라는 용어는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 추가 또는/및 개재할 수 있음을 나타내도록 사용된다.In the present specification, the term “including” is used to indicate that other components may be added and/or interposed, rather than excluding other components, unless specifically stated otherwise.
본 명세서에서 "이들의 조합"이라는 용어는 기재된 구성요소들 하나 이상과의 혼합 또는 조합되는 것을 의미한다.As used herein, the term “combination thereof” means mixing or combining with one or more of the described components.
본 명세서에서 용어 "상호작용"은 직접 또는 간접적일 수 있고, 직접 결합을 포함하거나 또는 간접적으로 결합할 수 있으며, 결합은 다른 분자에 의해 매개될 수도 있다.As used herein, the term “interaction” may be direct or indirect, and may include a direct bond or may bind indirectly, and the binding may be mediated by another molecule.
본 명세서에서 용어 "Fe(Ⅱ) 및 Fe(Ⅲ)"는 Fe가 산화된 형태를 나타내는 것으로서, 상기 Fe(Ⅱ) 및 Fe(Ⅲ)는 2가 Fe(Fe2+) 및 3가 Fe(Fe3+)를 의미한다.As used herein, the terms "Fe(II) and Fe(III)" refer to an oxidized form of Fe, wherein Fe(II) and Fe(III) are divalent Fe(Fe 2+ ) and trivalent Fe(Fe). 3+ ).
본 명세서에서 용어 "나노 구조체"는 나노미터(nanometer) 단위의 구조체를 의미하며, 특히 본원에서는 광감작성 빌딩 블록으로서 시아닌(cyanine)을 갖는 나노 구조체를 총칭하는 것일 수 있다. 일 구체예에서는, CyH 용액 및 금속 이온 용액을 혼합하여 CyFe(Ⅱ), CyZn(Ⅱ) 및 CyEu(Ⅱ)의 나노 구조체를 제조하였다.As used herein, the term “nanostructure” refers to a nanometer-scale structure, and in particular, herein, may be a generic term for a nanostructure having cyanine as a photosensitive building block. In one embodiment, a nanostructure of CyFe(II), CyZn(II), and CyEu(II) was prepared by mixing a CyH solution and a metal ion solution.
상기 시아닌은 안정성이 낮고 단량체(monomer)로 불용성인 특징을 가지나, 일 구체예에 따른 나노 구조체는 시아닌과 금속 이온이 배위결합 기반으로 자기조립하여 합성됨으로써 높은 안정성 및 향상된 촉매 활성을 가질 수 있다. 특히, 시아닌과 철 이온(Fe(Ⅱ))이 배위결합 기반으로 자기조립하여 합성된 CyFe(Ⅱ) 나노구조체는 자연에 존재하는 광산화제를 구조 및 활성 면에서 모방할 뿐만 아니라, 향상된 광-펜톤 반응을 가져 페로토시스 활성을 나타내는 광-페노자임으로 사용될 수 있다.The cyanine has low stability and is insoluble as a monomer, but the nanostructure according to an embodiment is synthesized by self-assembly of cyanine and metal ions based on a coordination bond. It can have high stability and improved catalytic activity. In particular, the CyFe(II) nanostructure synthesized by self-assembly of cyanine and iron ions (Fe(II)) based on coordination bonds not only mimics natural photooxidizers in structure and activity, but also improves photo-Fenton It can be used as a photo-phenozyme that has a reaction and exhibits ferrototic activity.
본 명세서에서 용어 "광-페노자임"은 상기 나노 구조체 중 CyFe(Ⅱ) 나노 구조체를 의미할 수 있고, 또는 단백질과 결합한 전효소(Holoenzyme)로서의 CyFe(Ⅱ) 나노 구조체를 의미할 수도 있다. 상기 광-페노자임은 photo-fenozyme, photo-ferritin nanozyme, 또는 광-페리틴 나노자임과 혼용하여 사용될 수 있다.As used herein, the term “photo-phenozyme” may mean a CyFe(II) nanostructure among the nanostructures, or a CyFe(II) nanostructure as a Holoenzyme bound to a protein. The photo-phenozyme may be used in combination with photo-fenozyme, photo-ferritin nanozyme, or photo-ferritin nanozyme.
본 명세서에서 용어 "초분자(supramolecule)"는 다른 분자와 결합하지 않고 스스로 수소결합, 정전기적 상호작용(electrostatic interaction), 반데르발스 인력(van der Waals force) 등과 같은 분자간 비공유성 상호작용(non-covalent interaction)을 통해 둘 또는 그 이상의 작은 분자들이 모여 생성된 분자들의 집합인 것을 의미한다. As used herein, the term "supramolecule" refers to intermolecular non-covalent interactions such as hydrogen bonding, electrostatic interaction, van der Waals force, etc. without binding to other molecules. Covalent interaction) refers to a set of molecules formed by the gathering of two or more small molecules.
일 구체예에 따른 나노 구조체는 수용액 상에서 초분자 상호작용에 의한 자기조립을 이용하여 제조하기 때문에, 친환경적이면서도, 제조과정 중 독성물질을 수반하지 않아 매우 안정하며, 용액을 혼합하는 단순하면서 쉬운 제조방식을 이용하므로 비용을 현저히 절감할 수 있다.Since the nanostructure according to one embodiment is manufactured using self-assembly by supramolecular interaction in an aqueous solution, it is eco-friendly and very stable because it does not involve toxic substances during the manufacturing process, and a simple and easy manufacturing method of mixing a solution By using it, the cost can be significantly reduced.
본 명세서에서 용어 "펜톤 반응(fenton reaction)"은 2가 철염과 과산화수소 혼합 용액의 환원과정을 거쳐 히드록실 라디칼(OH radical, ·OH)을 생성하는 과정을 의미한다.As used herein, the term “fenton reaction” refers to a process of generating hydroxyl radicals (OH radicals, OH) through a reduction process of a divalent iron salt and a mixed solution of hydrogen peroxide.
일 양상에 따른 나노 구조체는 자연에 존재하는 광산화제(photooxidase)를 모방하도록 제조한 것으로, 구조 면에서 자연에 존재하는 광산화제의 활성 구조와 유사한 분자 구조를 가지도록 하였다.The nanostructure according to an aspect is manufactured to imitate a photooxidase in nature, and has a molecular structure similar to the active structure of a photooxidase existing in nature in terms of structure.
예를 들어, Fe-함유 헴(heme)은 이미다졸(imidazole) 측쇄에 의해 비스-히스티딘(bis-histidine) 분자와 배위결합을 형성하며, 안정한 구조를 위해 여러 잔기로 분리된다. 이미다졸 모티프의 금속 결합 부위는 손님(guest) 분자를 받아들일 수 있는 강력한 배위 자리 및 공-조립(co-assembly)을 통해 광산화제의 촉매 중심을 위한 물리화학적 공간을 제공할 수 있다.For example, Fe-containing heme forms a coordination bond with a bis-histidine molecule by an imidazole side chain, and is separated into several residues for a stable structure. The metal binding site of the imidazole motif can provide a physicochemical space for the catalytic center of the photooxidizer through co-assembly and a strong coordination site that can accept guest molecules.
이와 같은 광산화제에 착안하여, 본 발명자들은 일 구체예에서 광감작성(photosensitive) 빌딩 블록으로서 시아닌(cyanine)이, 히스티딘 유도체이자 금속-결합 부위를 갖는 2-(4-이미다졸일) 에틸아민(히스타민)과 연결된 광감작성(photosensitive) 구조체 CyH를 제조하고(도 2, 도 3), 상기 CyH와 금속 이온을 포함하는 용액을 혼합하여 일 양상에 따른 나노 구조체를 제조하였다.Focusing on such a photooxidizer, the present inventors in one embodiment, as a photosensitive (photosensitive) building block cyanine (cyanine), a histidine derivative and 2-(4-imidazolyl) ethylamine having a metal-binding site ( Histamine) and a photosensitive structure linked to CyH were prepared ( FIGS. 2 and 3 ), and a solution containing CyH and a metal ion was mixed to prepare a nanostructure according to an aspect.
상기 일 양상에 따른 나노 구조체는, 예를 들어 하기와 같은 화학식으로 나타낼 수 있다. 우측의 CyFe(Ⅱ)는 C41N5H51Fe의 분자식으로 나타낼 수 있다.The nanostructure according to the aspect may be represented by the following chemical formula, for example. CyFe(II) on the right may be represented by a molecular formula of C 41 N 5 H 51 Fe.
투과전자현미경(TEM) 및 동적광산란법(DLS) 분석을 통해, 제조된 나노 구조체(CyH, CyZn(Ⅱ), CyFe(Ⅱ) 또는 CyEu(Ⅱ))들은 초분자를 구성하는 빌딩 블록인 금속에 의존적인 크기 분포를 가지고 있음을 확인하였다. 상기 결과를 통해, CyH 친수성(hydrophilic) 그룹과의 매크로사이클(macrocycle) π-컨쥬게이션 시스템(conjugation system) 및 이의 금속 이온과의 배위 결합이 구조체의 용해도를 높이고, 초분자 나노자임의 형성을 가능하게 함을 확인하였다. Through transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis, the prepared nanostructures (CyH, CyZn(II), CyFe(II), or CyEu(II)) are metal-dependent, which is a building block of supramolecules. It was confirmed that it has a phosphorus size distribution. Through the above results, the macrocycle π-conjugation system with CyH hydrophilic group and its coordination bond with metal ions increase the solubility of the structure and enable the formation of supramolecular nanozymes was confirmed.
상기 시아닌은 시아닌(cyanine)계 화합물일 수 있으며, 바람직하게는 인도시아닌계, 티아시아닌계, 프탈로시아닌계 화합물일 수 있고, 예를 들어 IR-140, IR-746, IR-768 퍼클로레이트(perchlorate), IR775 클로라이드(chloride), IR-780 요오드화물(iodide), IR-780 퍼클로레이트, IR-783, IR-786 퍼클로레이트, IR-786, IR-792 퍼클로레이트, IR-797 클로라이드, IR-797 퍼클로레이트, IR-806, IR-813 클로라이드, IR-813 퍼클로레이트 및 IR820으로 이루어진 군으로부터 선택되는 어느 하나일 수 있으나, 이에 제한되는 것은 아니다.The cyanine may be a cyanine-based compound, preferably an indocyanine-based, thiasianin-based, or phthalocyanine-based compound, for example, IR-140, IR-746, IR-768 perchlorate (perchlorate). ), IR775 chloride, IR-780 iodide, IR-780 perchlorate, IR-783, IR-786 perchlorate, IR-786, IR-792 perchlorate, IR-797 chloride, IR-797 perchlorate, It may be any one selected from the group consisting of IR-806, IR-813 chloride, IR-813 perchlorate and IR820, but is not limited thereto.
일 구체예에서, 상기 시아닌은 2-[2-[2-클로로-3-[(1,3-디히드로-3,3-디메틸-1-프로필-2H-인돌-2-일리덴)에틸리덴]-1-시클로헥센-1-일]에테닐]-3,3-디메틸-1-프로필인돌륨 아이오다이드(IR-780 iodide)를 사용할 수 있다.In one embodiment, the cyanine is 2-[2-[2-chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene ]-1-cyclohexen-1-yl]ethenyl]-3,3-dimethyl-1-propylindolium iodide (IR-780 iodide) can be used.
상기 철(iron) 이온은 수용액에서 가용성인, FeX2 형태로서의 철 이온을 제한없이 포함하며, 예를 들어 철 브로마이드(Ⅱ)(FeBr2), 질산 철(Ⅲ)(Fe(NO3)2), 염화철(Ⅱ)(FeCl2) 또는 염화철(Ⅲ)(FeCl3)일 수 있고, 바람직하게는 염화철(Ⅱ)(FeCl2) 또는 염화철(Ⅲ)(FeCl3)일 수 있으나, 이에 제한되는 것은 아니다. 상기 철이온은 CyH와 배위결합을 형성하여, 페로토시스를 야기할 수 있다.The iron (iron) ions include, without limitation, iron ions in the form of FeX 2 , soluble in aqueous solution, for example, iron bromide (II) (FeBr 2 ), iron (III) nitrate (Fe(NO 3 ) 2 ) , iron (II) chloride (FeCl 2 ) or iron (III) chloride (FeCl 3 ), preferably iron (II) chloride (FeCl 2 ) or iron (III) chloride (FeCl 3 ), but is limited thereto not. The iron ion may form a coordination bond with CyH, causing ferrotosis.
상기 나노 구조체는 650 nm 내지 900 nm 파장의 영역에서 광 조사에 의해 활성산소종을 생성할 수 있다. 상기 650 nm 내지 900 nm 파장의 영역은 가시광선 내지 근적외선 영역으로, 일 구체예에서는 상기 나노 구조체가 근적외선 조사 하에서 활성산소종(히드록실 라디칼)을 생성할 수 있음을 확인하였다.The nanostructure may generate reactive oxygen species by light irradiation in a wavelength region of 650 nm to 900 nm. The 650 nm to 900 nm wavelength region is a visible ray to near-infrared region, and in one embodiment, it was confirmed that the nanostructure can generate active oxygen species (hydroxyl radical) under near-infrared irradiation.
상기 나노 구조체는 나노 구조체 그 자체, 이의 염, 또는 이의 용매화물의 형태를 모두 포함할 수 있다.The nanostructure may include all of the nanostructure itself, a salt thereof, or a solvate thereof.
상기 나노 구조체는 암 조직 또는 표적 세포를 표적화하기 위한 하나 이상의 표적화 리간드 또는 모이어티(moiety)를 포함할 수 있다. 이러한 표적화 리간드로는 소분자(예컨대, 폴레이트, 염료, 등), 압타머, 항체, 항체 단편, 세포 표면의 특정 수용체와 결합하는 것으로 알려져 있는 화합물 등을 사용할 수 있으나, 이에 제한되는 것은 아니며 당업계에 알려져 있는 표적화 리간드라면 제한 없이 사용할 수 있다.The nanostructure may include one or more targeting ligands or moieties for targeting cancer tissue or target cells. The targeting ligand may include, but is not limited to, small molecules (eg, folate, dye, etc.), aptamers, antibodies, antibody fragments, compounds known to bind to specific receptors on the cell surface, and the like. Any targeting ligand known in the art can be used without limitation.
다른 양상은 상기 나노 구조체를 포함하는 광역학 치료용 조성물을 제공한다.Another aspect provides a composition for photodynamic therapy comprising the nanostructure.
상기 조성물은 용매, 버퍼 용액 또는 이들의 혼합물에 전술한 나노 구조체를 첨가하고, 여기에 산, 및/또는 염기를 첨가하여 준비될 수 있다. 또한 상기 조성물은 당업계에서 잘 알려진 다른 첨가제를 추가적으로 포함할 수 있다. 상기 조성물이 포함하는 용매, 산, 염기, 및 버퍼 용액의 함량은 요구되는 성능에 따라 적절히 조절될 수 있다. The composition may be prepared by adding the above-described nanostructures to a solvent, a buffer solution or a mixture thereof, and adding an acid, and/or a base thereto. In addition, the composition may further include other additives well known in the art. The content of the solvent, acid, base, and buffer solution included in the composition may be appropriately adjusted according to the required performance.
또한, 상기 조성물은 시료(sample)와 혼합될 수 있다. 상기 시료는 미생물(microorganism), 세포(cell) 및 조직(tissue)으로 이루어진 군으로부터 선택되는 하나 이상을 포함하는 생물학적 시료일 수 있으나, 반드시 이들로 한정되지 않으며 당업계에서 생물학적 시료로 사용할 수 있는 것이라면 모두 가능하다.In addition, the composition may be mixed with a sample. The sample may be a biological sample including at least one selected from the group consisting of microorganisms, cells, and tissues, but is not limited thereto, and any biological sample that can be used in the art All is possible.
상기 조성물은 총 중량에 대하여 상기 나노 구조체를 0.0001 내지 50 중량%로 포함할 수 있으며, 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 더 포함할 수 있다.The composition may include the nanostructure in an amount of 0.0001 to 50% by weight based on the total weight, and may further include one or more active ingredients having the same or similar function.
상기 조성물은 각각의 사용 목적에 맞게 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁제, 에멀젼, 시럽, 에어로졸 등의 경구 제형, 멸균 주사용액의 주사제 등 다양한 형태로 제형화하여 사용할 수 있으며, 경구 투여하거나 정맥 내, 복강 내, 피하, 직장, 국소 투여 등을 포함한 다양한 경로를 통해 투여될 수 있다.The composition can be formulated and used in various forms, such as oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and injections of sterile injection solutions, according to conventional methods for each purpose of use. and may be administered through various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.
또 다른 양상은 상기 나노 구조체를 포함하는 암의 예방 또는 치료용 약학적 조성물을 제공한다.Another aspect provides a pharmaceutical composition for preventing or treating cancer comprising the nanostructure.
상기 나노 구조체는 페로토시스를 통해 암세포의 사멸을 유도할 수 있다. 페로토시스는 철 의존성 세포 사멸로 세포 사멸의 한 형태이며, 철 의존적인 활성산소종의 발생, 지질과산화물의 발생 및 축적에 의해 발생하는 모든 세포 사멸을 통칭한다.The nanostructure may induce apoptosis of cancer cells through ferrotosis. Ferrotosis is iron-dependent cell death, a form of cell death, and collectively refers to all cell death caused by iron-dependent generation of reactive oxygen species, generation and accumulation of lipid peroxides.
상기 암은 폐암, 비소세포성 폐암, 결장암, 골암, 췌장암, 피부암, 두부 또는 경부암, 피부 또는 안구 내 흑색종, 자궁암, 난소암, 직장암, 위암, 항문부근암, 결장암, 유방암, 나팔관암종, 자궁내막암종, 자궁경부암종, 질암종, 음문암종, 호지킨병(Hodgkin's disease), 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계 종양, 1차 중추신경계 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.The cancer is lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, perianal cancer, colon cancer, breast cancer, fallopian tube carcinoma, uterus Endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic Or at least one selected from the group consisting of acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma can
상기 약학적 조성물에는 추가적으로 담체, 부형제 또는 희석제 등이 더 포함될 수 있으며, 포함될 수 있는 적합한 담체, 부형제 또는 희석제의 예로는 락토오스, 덱스트로오스, 수크로오스, 솔비톨, 만니톨, 자일리톨, 에리쓰리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로스, 메틸 셀룰로스, 비정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 들 수 있다.The pharmaceutical composition may further include a carrier, excipient, or diluent, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. and the like.
또한, 상기 약학적 조성물은 충전제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 더 포함할 수도 있다.In addition, the pharmaceutical composition may further include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a fragrance, an emulsifier, a preservative, and the like.
예를 들어, 경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 상기 약학적 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 탄산칼슘, 수크로오스, 락토오스, 젤라틴 등을 혼합하여 제형화할 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 등과 같은 윤활제가 사용될 수도 있다.For example, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the pharmaceutical composition, for example, starch, calcium carbonate, sucrose. , lactose, gelatin, etc. can be mixed to formulate it. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like may be used.
경구용 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 예시될 수 있으며, 흔히 사용되는 단순 희석제인 물, 액체 파라핀 이외에 여러 가지 부형제, 예를 들면, 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Liquid formulations for oral use may include suspensions, solutions, emulsions, syrups, etc., and various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc., in addition to commonly used simple diluents such as water and liquid paraffin. may be included.
비경구 투여를 위한 제제에는 멸균된 수용액제, 비수성용제, 현탁제, 유제, 동결건조제, 좌제 등이 예시될 수 있다. 비수성용제, 현탁제에는 프로필렌글리콜, 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 포함될 수 있다. 주사제에는 용해제, 등장화제, 현탁화제, 유화제, 안정화제, 방부제 등과 같은 종래의 첨가제가 포함될 수 있다. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized agents, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Injections may contain conventional additives such as solubilizing agents, isotonic agents, suspending agents, emulsifying agents, stabilizing agents, and preservatives.
상기 약학적 조성물은 약제학적으로 유효한 양이 대상체에 투여될 수 있다. 상기 "약제학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. The pharmaceutical composition may be administered to a subject in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the patient's type, severity, drug activity, and drug. Sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field.
상기 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용이 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.
상기 약학적 조성물은 다양한 경로를 통하여 대상에 투여될 수 있다. 투여방법에는 제한이 없으며, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관내 주사에 의해 투여될 수 있다.The pharmaceutical composition may be administered to a subject through various routes. The administration method is not limited, and for example, it may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection.
본 발명에서 용어 "투여"는 임의의 적절한 방법으로 환자에게 소정의 물질을 제공하는 것을 의미하며, 본 발명의 약학적 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 일반적인 모든 경로를 통하여 경구 또는 비경구 투여될 수 있다. 또한, 상기 조성물은 유효성분을 표적 세포로 전달할 수 있는 임의의 장치를 이용해 투여될 수도 있다.In the present invention, the term "administration" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention is oral or through all general routes as long as it can reach the target tissue. It may be administered parenterally. In addition, the composition may be administered using any device capable of delivering an active ingredient to a target cell.
본 발명에서 용어 "대상체"는, 특별히 한정되는 것은 아니지만, 예를 들어, 인간, 원숭이, 소, 말, 양, 돼지, 닭, 칠면조, 메추라기, 고양이, 개, 마우스, 쥐, 토끼 또는 기니 피그를 포함할 수 있다.In the present invention, the term "subject" is not particularly limited, but includes, for example, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs. may include
상기 약학적 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물 형태, 투여 경로, 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 바람직하게는, 1일 0.001 내지 100mg/체중kg으로, 보다 바람직하게는 0.01 내지 30mg/체중kg으로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 여러번 나누어 투여할 수도 있다.The preferred dosage of the pharmaceutical composition varies depending on the condition and weight of the patient, the severity of the disease, the drug form, the route of administration, and the duration, but may be appropriately selected by those skilled in the art. Preferably, it can be administered at 0.001 to 100 mg/kg of body weight per day, and more preferably at 0.01 to 30 mg/kg of body weight. Administration may be administered once a day, or may be administered in several divided doses.
또 다른 양상은 1) 시아닌계 화합물 용액을 준비하는 단계; 및Another aspect is 1) preparing a cyanine-based compound solution; and
2) 상기 단계 1)에서 제조한 용액에 철 용액을 첨가하여 혼합하는 단계를 포함하는 시아닌 및 철 이온을 포함하는 나노 구조체를 제조하는 방법을 제공한다.2) It provides a method for preparing a nanostructure containing cyanine and iron ions, which includes the step of adding and mixing an iron solution to the solution prepared in step 1).
또 다른 양상은 상기 약학적 조성물을 약제학적으로 유효한 양으로 개체에 투여하여 암을 예방 또는 치료하는 방법을 제공한다.Another aspect provides a method for preventing or treating cancer by administering the pharmaceutical composition to a subject in a pharmaceutically effective amount.
본 명세서에서 개체(subject)란 질병의 치료를 필요로 하는 대상을 의미하고, 보다 구체적으로는 인간, 또는 비-인간인 영장류, 생쥐(mouse), 쥐(rat), 개, 고양이, 말, 및 소 등의 포유류를 의미한다. 또한, 본 발명에서 약제학적 유효량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율, 및 질환의 중증도 등에 따라 그 범위가 다양하게 조절될 수 있음은 당업자에게 명백하다.As used herein, a subject means a subject in need of treatment for a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, and mammals such as cattle. In addition, it is apparent to those skilled in the art that the pharmaceutically effective amount in the present invention can be variously adjusted according to the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and the severity of the disease. do.
상기 나노 구조체의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물 형태, 투여경로, 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나 바람직하게는, 1일 0.001 내지 550mg/체중kg으로, 보다 바람직하게는 0.01 내지 30mg/체중kg으로 투여한다. 투여는 하루에 한번 투여할 수도 있고, 여러 번 나누어 투여할 수도 있다. 일 양상에 따른 나노 구조체는 전체 조성물 총 중량에 대하여 0.0001 내지 10 중량%, 바람직하게는 0.001 내지 1 중량%의 양으로 존재할 수 있다.The preferred dosage of the nanostructure varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route of administration, and the period, but may be appropriately selected by those skilled in the art. However, preferably, it is administered at 0.001 to 550 mg/kg of body weight per day, and more preferably at 0.01 to 30 mg/kg of body weight. Administration may be administered once a day, or may be administered in several divided doses. The nanostructure according to one aspect may be present in an amount of 0.0001 to 10 wt%, preferably 0.001 to 1 wt%, based on the total weight of the total composition.
상기 약학적 조성물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여방법에는 제한이 없으며, 예를 들면, 경구, 직장, 또는 정맥, 근육, 피하, 자궁내 경막, 또는 뇌혈관(intracerbroventricular) 주사에 의해 투여될 수 있다.The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, and humans by various routes. The administration method is not limited, and for example, it may be administered by oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine, or intracerbroventricular injection.
일 양상에 따른 나노 구조체는 시아닌 및 철 이온의 조립을 기반으로 한 초분자 나노자임으로서, 금속이온 주변의 배위결합 구조에 의해 히드록실 라디칼을 생산하는 광-펜톤 유사반응을 더욱 향상시키고, 과산화수소(H2O2)와 같은 종양 대사산물의 촉매 분해를 촉진시켜 효율적인 페로토시스를 유도할 수 있다. 상기 초분자 나노자임은 암세포만을 특이적으로 사멸시키고, 기존 항암제가 갖는 독성이나 부작용 없이 안전한 치료가 가능하여 다양한 암의 치료에 효과적으로 사용될 수 있다.The nanostructure according to an aspect is a supramolecular nanozyme based on the assembly of cyanine and iron ions, and further improves the photo-Fenton-like reaction that produces hydroxyl radicals by the coordination structure around metal ions, and hydrogen peroxide (H 2 O 2 ), it can induce efficient ferrotosis by promoting the catalytic degradation of tumor metabolites. The supramolecular nanozyme specifically kills only cancer cells, and can be safely treated without the toxicity or side effects of existing anticancer drugs, so it can be effectively used in the treatment of various cancers.
도 1은 자연에 존재하는 광산화제(a), 배위결합 자기조립(coordination self-assembly)에 의해 합성된 광-페노자임(b) 및 (c) 상기 광-페노자임의 페로토시스를 통한 암세포 사멸 기작을 나타낸 개략도이다.
도 2는 합성예 1-1에 의해 얻은 CyH의 분자 구조를 나타낸 도이다.
도 3은 합성예 1-1에 의해 얻은 CyH의 1H NMR 결과를 나타낸 도이다.
도 4 내지 도 6은 합성예 1-2에 의해 얻은 CyFe(Ⅱ), CyZn(Ⅱ) 및 CyEu(Ⅱ) 나노 구조체의 동적광산란법 및 투과전자현미경 분석 결과를 나타낸 도이다.
도 7은 CyH와 FeCl2 용액을 몰 비율 1:0.5로 혼합하여 제조한 CyFe(Ⅱ) 나노 구조체의 biomedium에서의 크기 분포를 분석한 결과를 나타낸 도이다.
도 8은 단백질(protein), 합성예 1-1에 의해 얻은 CyH 및 합성예 1-2에 의해 얻은 CyFe(Ⅱ) 나노 구조체의 푸리에 변환 적외선(Fourier Transform Infrared: FT-IR) 분석 결과를 나타낸 것이다.
도 9는 [CyH] + [Fe(Ⅱ)] = 24 μM인 CyFe(Ⅱ) 나노 구조체의 job's plot(a) 및 CyH를 포함하는 수용액(pH 6.36)을 10배 희석한 용액에 2:1 내지 6:1의 서로 다른 몰 비율로 Fe(Ⅱ) 수용액을 첨가하고 4배 희석하여 제조한 나노 구조체의 job's plot을 나타낸 도이다(b).
도 10은 50 μg·mL-1 CyH를 포함하는 수용액(pH 3.15~10.98)의 형광 스펙트럼(a, b) 및 50 μg·mL-1 CyH 및 Fe(Ⅱ)를 포함하는 수용액(pH 3.15~10.98)의 형광 스펙트럼을 나타낸 도이다(c, d).
도 11은 CyFe(Ⅱ) 나노 구조체의 최적 구조를 나타낸 도이다.
도 12는 Fe(Ⅱ) 농도 0.04~0.32 mM인 CyFe(Ⅱ) 나노 구조체의 자외선 가시광 흡수(UV-vis absorption) 스펙트럼(a) 및 자연의 광산화제의 촉매 중심(catalytic core)에서의 항간교차율 조절 메커니즘(b)을 나타낸 도이다. ES: 여기상태(Excited-state); Et: 에너지 교환(Energy transfer); ISC: 항간교차율(Intersystem Crossing rate); Fluorescence: 형광; Phosphorescence: 인광(燐光).
도 13은 CyFe(Ⅱ) 나노 구조체의 시간-의존적 형광 스펙트럼을 나타낸 도이다.
도 14는 CyH의 탈양성자화(deprotonation) 및 후속하는 CyFe(Ⅱ) 나노 구조체의 형성 과정(a), 수용액(pH=4.0) 내 CyH 및 CyFe(Ⅱ)의 자외선 가시광 흡수 스펙트럼(b), 물에서의 CyH, CyFe(Ⅱ) 및 Cy의 흡수 스펙트럼(c), CyH 및 Cy의 방출 스펙트럼(d, e)을 나타낸 도이다. 한편, (c)의 경우 CyH, 578 nm에서 S0 → S1 전이(transition); Cy, 452 nm에서 S0 → S1 전이; CyFe(Ⅱ), 460 nm에서 S0 → S8 전이를 나타내고, (d)의 경우 722 nm에서, CyH S1 → S0 방출(emission)을 나타내고, (e)의 경우 543 nm에서, Cy S1 → S0 방출을 나타낸다.
도 15는 CyH, Cy 및 CyFe(Ⅱ)의 HOMO, LOMO 에너지 준위를 나타낸 도이다.
도 16은 S0 → S8 전이에 대한 CyFe(Ⅱ)의 최고 점유 자연 전이궤도 함수 HOTO(hole wavefunction) 및 최저 점유 자연 전이궤도 함수 LUTO(electron wavefunction)를 나타낸 도이다. 비교를 위해, S0 → S1 전이에 대한 CyH 및 Cy의 자연 전이궤도 함수를 함께 나타내었다.
도 17은 촉매 중심으로서의 CyFe(Ⅱ) 및 단백질과 결합한 전효소(Holoenzyme)로서의 CyFe(Ⅱ) 광-페노자임의 크기 분포를 분석한 결과를 나타낸 도이다.
도 18은 0.1 내지 100μM의 다양한 농도의 FBS 단백질과 유리(free) CyH의 자외선 가시광 흡수(UV-vis absorption) 스펙트럼(a) 및 CyFe(Ⅱ) 광-페노자임의 자외선 가시광 흡수 스펙트럼(b)을 나타낸 도이다.
도 19는 시간(0~24시간)에 따른 CyFe(Ⅱ) 광-페노자임의 자외선 가시광 흡수 스펙트럼을 나타낸 도이다.
도 20은 50 μg·mL-1 CyH를 포함하는 수용액 pH(3.15~10.98)의 자외선 가시광 흡수 스펙트럼을 나타낸 도이다.
도 21은 50 μg·mL-1 CyH를 포함하는 수용액 pH(3.15~10.98)을 제조한 뒤, 이어서 Fe(Ⅱ) 용액을 첨가한 경우의 자외선 가시광 흡수 스펙트럼을 나타낸 도이다.
도 22는 50 μg·mL-1 CyFe(Ⅱ)를 포함하는 수용액 pH(3.15~10.98)의 자외선 가시광 흡수 스펙트럼을 나타낸 도이다.
도 23은 CyH 및 CyFe(Ⅱ) 광-페노자임의 광열 특성(photothermal properties)을 나타낸 도이다.
도 24는 광-페노자임(0.5 mg·mL-1), HCl(5 mM) 및 H2O2(10 mM)를 포함하는 용액에 655 nm 레이저(0.7 W·cm-2)를 2분 동안 조사한 뒤 EPR 스펙트럼을 분석한 결과를 나타낸 도이다.
도 25는 광-페노자임(0.5 mg·mL-1) 및 H2O2(10 mM)를 포함하는 용액에 레이저(0.7 W·cm-2)를 0분, 2분, 5분 조사한 뒤 EPR 스펙트럼을 분석한 결과(a) 및 촉매 반응 동안 반응 시스템의 온도 변화를 측정한 결과를 나타낸 도이다(b).
도 26은 H2O2(10 mM) 부재 또는 존재 하에 광 조사 시 일중항 산소 발생량을 나타낸 도이다.
도 27은 활성산소종(ROS) 생성 여부를 확인하기 위해, CyH의 λem = 520nm에서의 시간-의존적 형광 강도의 변화를 측정한 결과를 나타낸 도이다.
도 28은 활성산소종(ROS) 생성 여부를 확인하기 위해, CyFe(Ⅱ) 광-페노자임의 λem = 520nm에서의 시간-의존적 형광 강도의 변화를 측정한 결과를 나타낸 도이다.
도 29는 서로 다른 농도의 H2O2(0, 50, 100, 200 μM) 존재 하에서 CyFe(Ⅱ) 광-페노자임(5 μg·mL-1) 처리 시의 공초점 현미경 이미지를 나타낸 도이다.
도 30은 HeLa 세포를 CyFe(Ⅱ) 광-페노자임과 24시간 동안 배양한 후의 세포 생존율(cell viability)(a) 및 암실 또는 광 조사 하에서 4시간 동안 배양한 후의 세포 생존율(b)을 나타낸 도이다.
도 31은 서로 다른 배양 시간(2시간, 암실 또는 광 조사/4시간, 암실 또는 광 조사)으로 CyFe(Ⅱ) 광-페노자임(5 μg·mL-1) 처리 시의 공초점 현미경 이미지를 나타낸 도이다.
도 32는 대조군(control), CyFe(Ⅱ) 광-페노자임과 24시간 배양한 후 및 광 조사 하에서 4시간 동안 배양한 후의 투과전자현미경 분석 결과를 나타낸 도이다.1 is a photo-oxidizer (a) present in nature, photo-phenozyme synthesized by coordination self-assembly (b) and (c) photo-phenozyme through ferrotosis It is a schematic diagram showing the mechanism of cancer cell death.
2 is a diagram showing the molecular structure of CyH obtained by Synthesis Example 1-1.
3 is a diagram showing 1 H NMR results of CyH obtained by Synthesis Example 1-1.
4 to 6 are diagrams showing the results of dynamic light scattering and transmission electron microscopy analysis of the CyFe(II), CyZn(II) and CyEu(II) nanostructures obtained in Synthesis Example 1-2.
7 is a diagram showing the results of analyzing the size distribution in the biomedium of CyFe(II) nanostructures prepared by mixing CyH and FeCl 2 solutions at a molar ratio of 1:0.5.
8 shows the Fourier Transform Infrared (FT-IR) analysis results of the protein, CyH obtained by Synthesis Example 1-1, and CyFe(II) nanostructure obtained by Synthesis Example 1-2 .
9 is a job's plot (a) of a CyFe(II) nanostructure with [CyH] + [Fe(II)] = 24 μM and a solution containing CyH (pH 6.36) diluted 10 times from 2:1 to It is a diagram showing a job's plot of a nanostructure prepared by adding an aqueous solution of Fe(II) at a different molar ratio of 6:1 and diluting it 4 times (b).
10 is a fluorescence spectrum (a, b) of an aqueous solution (pH 3.15 to 10.98) containing 50 μg·mL -1 CyH and an aqueous solution containing 50 μg·mL -1 CyH and Fe(II) (pH 3.15 to 10.98) ) is a diagram showing the fluorescence spectrum of (c, d).
11 is a diagram illustrating an optimal structure of a CyFe(II) nanostructure.
12 is a UV-vis absorption spectrum (a) of a CyFe(II) nanostructure having an Fe(II) concentration of 0.04 to 0.32 mM (a) and control of the inter-crossing rate in the catalytic core of a natural photooxidizer; It is a figure which shows the mechanism (b). ES: Excited-state; Et: Energy transfer; ISC: Intersystem Crossing rate; Fluorescence: fluorescence; Phosphorescence: Phosphorescence.
13 is a diagram illustrating a time-dependent fluorescence spectrum of a CyFe(II) nanostructure.
FIG. 14 is an ultraviolet visible light absorption spectrum of CyH and CyFe(II) in an aqueous solution (pH=4.0) (a) of the deprotonation of CyH and the subsequent formation of a CyFe(II) nanostructure (b), water Absorption spectra of CyH, CyFe(II) and Cy in (c), and emission spectra of CyH and Cy (d, e) are shown. On the other hand, in the case of (c), CyH, S 0 → S 1 transition (transition) at 578 nm; Cy, S 0 → S 1 transition at 452 nm; CyFe(II) shows S 0 → S 8 transition at 460 nm, (d) shows CyHS 1 → S 0 emission at 722 nm, and (e) at 543 nm, Cy S 1 → S 0 represents emission.
15 is a diagram showing HOMO and LOMO energy levels of CyH, Cy, and CyFe(II).
16 is a diagram showing the highest occupied natural transition orbital function HOTO (hole wavefunction) and the lowest occupied natural transition orbital function LUTO (electron wavefunction) of CyFe(II) for the S 0 → S 8 transition. For comparison, the natural transition orbital functions of CyH and Cy for the S 0 → S 1 transition are also shown.
17 is a diagram showing the results of analyzing the size distribution of CyFe(II) as a catalyst center and CyFe(II) photo-phenozyme as a protein-bound proenzyme (Holoenzyme).
18 is a UV-vis absorption spectrum (a) and CyFe(II) photo-Fenozyme of various concentrations of FBS protein and free (free) CyH (a) and UV-visible absorption spectrum (b) of FBS protein at various concentrations of 0.1 to 100 μM is the diagram shown.
19 is a diagram showing the ultraviolet visible light absorption spectrum of CyFe(II) photo-phenozyme according to time (0 to 24 hours).
20 is a view showing the ultraviolet visible light absorption spectrum of an aqueous solution containing 50 μg·mL -1 CyH (3.15 to 10.98).
FIG. 21 is a view showing an ultraviolet visible light absorption spectrum when an aqueous solution pH (3.15 to 10.98) containing 50 μg·mL -1 CyH is prepared and then Fe(II) solution is added thereto.
22 is a view showing an ultraviolet visible light absorption spectrum of an aqueous solution containing 50 μg·mL -1 CyFe(II) at pH (3.15 to 10.98).
23 is a diagram showing the photothermal properties of CyH and CyFe(II) photo-phenozyme.
24 is a photo-phenozyme (0.5 mg·mL −1 ), HCl (5 mM), and a 655 nm laser (0.7 W·cm −2 ) in a solution containing H 2 O 2 (10 mM) for 2 min. It is a diagram showing the results of analyzing the EPR spectrum after irradiation.
25 is a photo-phenozyme (0.5 mg·mL -1 ) and H 2 O 2 (10 mM) after irradiating a laser (0.7 W·cm -2 ) to a solution containing 0 min, 2 min, 5 min. It is a diagram showing the result of analyzing the EPR spectrum (a) and the result of measuring the temperature change of the reaction system during the catalytic reaction (b).
26 is a diagram showing the amount of singlet oxygen generated when irradiated with light in the absence or presence of H 2 O 2 (10 mM).
27 is a diagram showing the result of measuring the time-dependent change in fluorescence intensity at λ em = 520 nm of CyH in order to check whether reactive oxygen species (ROS) is generated.
28 is a diagram showing the results of measuring the time-dependent change in fluorescence intensity at λ em = 520 nm of CyFe(II) photo-phenozyme in order to check whether reactive oxygen species (ROS) is generated.
29 is a view showing a confocal microscope image during treatment with CyFe(II) photo-phenozyme (5 μg·mL -1 ) in the presence of different concentrations of H 2 O 2 (0, 50, 100, 200 μM) to be.
Figure 30 shows HeLa cells after culturing with CyFe(II) photo-phenozyme for 24 hours (a) and cell viability (b) after culturing for 4 hours in the dark or under light irradiation. it is do
Figure 31 is a confocal microscope image of CyFe(II) photo-phenozyme (5 μg mL -1 ) treatment with different incubation times (2 hours, dark room or light irradiation / 4 hours, dark room or light irradiation) is the diagram shown.
32 is a view showing the results of transmission electron microscopy analysis after incubation with a control, CyFe(II) photo-phenozyme for 24 hours and after culturing for 4 hours under light irradiation.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these examples.
합성예 1. 광-페노자임Synthesis Example 1. Photo-phenozyme
배위 결합이 원동력인 시아닌 및 철 이온(Fe(Ⅱ))의 조립에 기반한 초분자 광-페노자임(photo-fenozyme, photo-ferritin nanozyme, 또는 광-페리틴 나노자임)을 합성하였다.A supramolecular photo-phenozyme (photo-fenozyme, photo-ferritin nanozyme, or photo-ferritin nanozyme) based on the assembly of cyanine and iron ions (Fe(II)), driven by coordination bonds, was synthesized.
1-1. CyH의 합성1-1. Synthesis of CyH
2-[2-[2-클로로-3-[(1,3-디히드로-3,3-디메틸-1-프로필-2H-인돌-2-일리덴)에틸리덴]-1-시클로헥센-1-일]에테닐]-3,3-디메틸-1-프로필인돌륨 아이오다이드(IR-780 iodide)(1 mmol, 667.11 mg) 및 2-(4-이미다졸일) 에틸아민(1 mmol, 111.45 mg)을 무수 DMF(30 mL)에 용해시켰다. 상기 혼합물을 아르곤(Ar) 하에서 90℃, 4시간 동안 교반하였다. 감압 하에서 용매를 제거한 후, 용출액으로 DCM/메탄올(10:1, v/v)을 사용한 실리카겔 크로마토그래피로 광택이 있는 푸른색 고체를 정제하였다. 정제 후, 이를 수득 및 농축시킨 뒤 진공건조하였고, 붉은색이 도는 청색(reddish-blue)의 고체로서 CyH를 수득하였다(0.316 mmol, 31.6 %)(도 2). 하기 1H NMR 스펙트럼을 통하여 CyH가 합성되었음을 확인하였다(도 3).2-[2-[2-chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indol-2-ylidene)ethylidene]-1-cyclohexene-1 -yl]ethenyl]-3,3-dimethyl-1-propylindolium iodide (IR-780 iodide) (1 mmol, 667.11 mg) and 2- (4-imidazolyl) ethylamine (1 mmol, 111.45 mg) was dissolved in anhydrous DMF (30 mL). The mixture was stirred under argon (Ar) at 90° C. for 4 hours. After removing the solvent under reduced pressure, a glossy blue solid was purified by silica gel chromatography using DCM/methanol (10:1, v/v) as an eluent. After purification, it was obtained and concentrated and dried in vacuo to obtain CyH as a reddish-blue solid (0.316 mmol, 31.6 %) (FIG. 2). It was confirmed that CyH was synthesized through the following 1 H NMR spectrum (FIG. 3).
1H NMR(CDCl3, 300 MHz)(Bruker AM 300) δ 1.02-1.07 (t, 6H, J=15.0 Hz), 1.27 (s, 4H), 1.61 (m, 2H), 1.73-1.87 (m, 12H), 2.49 (t, 4H, J=6 Hz), 3.08 (s, 2H), 3.64 (s, 1H), 3.77-3.82 (t, 4H, 6.0 Hz), 4.04 (s, 2H), 5.62 (d, 2H, J=12.0 Hz), 6.86 (d, 2H, J=9.0 Hz), 7.07 (t, 3H, 9.0 Hz), 7.26-7.34 (m, 3H), 7.73 (d, 2H, J=12.0 Hz), 7.91 (s, 1H). 1 H NMR (CDCl 3 , 300 MHz) (Bruker AM 300) δ 1.02-1.07 (t, 6H, J=15.0 Hz), 1.27 (s, 4H), 1.61 (m, 2H), 1.73-1.87 (m, 12H), 2.49 (t, 4H, J=6 Hz), 3.08 (s, 2H), 3.64 (s, 1H), 3.77-3.82 (t, 4H, 6.0 Hz), 4.04 (s, 2H), 5.62 ( d, 2H, J=12.0 Hz), 6.86 (d, 2H, J=9.0 Hz), 7.07 (t, 3H, 9.0 Hz), 7.26-7.34 (m, 3H), 7.73 (d, 2H, J=12.0) Hz), 7.91 (s, 1H).
1-2. 광-페노자임 및 다른 금속 기반 나노 구조체의 합성1-2. Synthesis of photo-phenozyme and other metal-based nanostructures
상기 실시예 1-1에서 제조한 CyH는 에탄올(16mM)에 용해시키고, FeCl2(10 mM)는 증류된 탈이온수(dd H2O)에 용해시켰다. CyH 용액 50μL를 40μL FeCl2이 포함된 수용액 910μL과 혼합하여, CyH 및 금속 이온의 최종 농도는 각각 0.8mM 및 0.4mM이며 몰 비율은 1:0.5인 시료를 제조하였다. 동일한 방법으로, FeCl2의 농도를 달리한 서로 다른 몰 비율(1:1, 1:2, 1:4)의 시료 및 다른 금속 이온(ZnCl2 and EuCl2)을 사용한 시료를 제조하였다.CyH prepared in Example 1-1 was dissolved in ethanol (16 mM), FeCl 2 (10 mM) was dissolved in distilled deionized water (dd H 2 O). 50 μL of CyH solution was mixed with 910 μL of an aqueous solution containing 40 μL FeCl 2 , to prepare a sample having a final concentration of CyH and metal ions of 0.8 mM and 0.4 mM, respectively, and a molar ratio of 1:0.5. In the same way, samples of different molar ratios (1:1, 1:2, 1:4) with different concentrations of FeCl 2 and samples using different metal ions (ZnCl 2 and EuCl 2 ) were prepared.
분석예 1. 동적광산란법 및 투과전자현미경 분석Analysis Example 1. Dynamic light scattering and transmission electron microscopy analysis
합성예 1의 용액 내 광-페노자임의 입자 크기 분포를 Nanotrac Wave를 사용하여 동적광산란법(Dynamic Light Scattering： DLS)으로 확인하였다. 또한, 합성예 1에 의해 얻은 광-페노자임에 대하여 투과전자현미경(Transmission Electron Microscopy: TEM) 분석을 수행하였다. 100kV에서 JEM-2100F(JEOL) 투과전자현미경을 사용하여 TEM 사진을 얻었다.Photo-Penozyme particle size distribution in the solution of Synthesis Example 1 was confirmed by Dynamic Light Scattering (DLS) using Nanorac Wave. In addition, the photo-phenozyme obtained in Synthesis Example 1 was subjected to transmission electron microscopy (TEM) analysis. TEM images were obtained using a JEM-2100F (JEOL) transmission electron microscope at 100 kV.
그 결과, CyFe(Ⅱ), CyZn(Ⅱ) 및 CyEu(Ⅱ) 광-페노자임의 크기는 각각 104.8 ± 39.18 nm, 61.35 ± 20.26 nm 및 136 ± 46.93 nm로 나타나, 금속 의존적인 크기 분포를 나타냄을 확인하였다(도 4a, 도 5a, 도 6a). As a result, the sizes of CyFe(II), CyZn(II) and CyEu(II) photo-phenozymes were 104.8 ± 39.18 nm, 61.35 ± 20.26 nm, and 136 ± 46.93 nm, respectively, indicating a metal-dependent size distribution. was confirmed (Fig. 4a, Fig. 5a, Fig. 6a).
또한, CyH와 FeCl2 용액을 몰 비율 1:0.5로 혼합하여 제조한 CyFe(Ⅱ) 광-페노자임의 경우, TEM 분석으로 확인한 입자 평균 크기가 104.8 ± 39.18 nm(PDI: 0.102)로 확인되어 DLS 방법으로 측정한 결과와 일치함을 확인하였다(도 4b). 동일한 방법으로 제조한 CyZn(Ⅱ) 및 CyEu(Ⅱ)의 경우에도 입자 평균 크기는 각각 61.35 ± 20.26 nm 및 136 ± 46.93 nm로 나타나, DLS 결과와 일치함을 확인하였다(도 5b, 도 6b).In addition, in the case of CyFe(II) photo-phenozyme prepared by mixing CyH and FeCl 2 solution in a molar ratio of 1:0.5, the average particle size confirmed by TEM analysis was 104.8 ± 39.18 nm (PDI: 0.102), and DLS It was confirmed that it is consistent with the results measured by the method (FIG. 4b). In the case of CyZn(II) and CyEu(II) prepared by the same method, the average particle size was 61.35 ± 20.26 nm and 136 ± 46.93 nm, respectively, confirming that it was consistent with the DLS results (Figs. 5b and 6b).
추가적으로, 합성예 1에 의해 얻은 광-페노자임의 안정성(stability) 분석을 수행하였다. 상기 광-페노자임을 생물학적 배지(5% FBS)에서 24시간 내지 120시간 동안 방치한 뒤 크기 분포를 측정한 결과, 구조체는 유의한 수준의 크기 변화를 나타내지 않음을 확인하였다. 상기 결과를 통해, 광-페노자임은 콜로이드 안정성(colloidal stability)이 현저히 우수하여, 생체 내에서 안정적인 촉매 활성을 발휘할 수 있음을 확인하였다(도 7).Additionally, photo-obtained by Synthesis Example 1 - Stability analysis of phenozyme was performed. The photo-phenozyme was left for 24 hours to 120 hours in a biological medium (5% FBS) and the size distribution was measured. As a result, it was confirmed that the structure did not show a significant level of size change. Through the above results, it was confirmed that photo-phenozyme has remarkably excellent colloidal stability, and thus can exhibit stable catalytic activity in vivo (FIG. 7).
분석예 2. FT-IR 분석Analysis Example 2. FT-IR analysis
합성예 1에 의해 얻은 광-페노자임에 대하여 VERTEX 80/80v FTIR 분광계(BRUKER)를 사용하여 푸리에 변환 적외선(Fourier Transform Infrared: FT-IR) 분석을 수행하였다.Photo-Penozyme obtained by Synthesis Example 1 was subjected to Fourier Transform Infrared (FT-IR) analysis using a
그 결과, 처음에는 이미다졸 그룹의 N-H 신축(stretching) 및 변형(deformation) 진동에 의해 각각 3,348 cm-1 및 1,525 cm-1에서 나타나는 피크(peak)가 관찰되었고, 이는 각각 3,276 cm-1 및 1,538 cm-1에서 나타나는 피크로 이동하여 피크의 폭이 넓어짐이 관찰되었다. 이는 수소 결합의 형성 또는 Fe ←: N-Cy 형태로서 양성자 수용체 Fe(Ⅱ)와의 배위 결합 형성에 기인한 것으로 판단되었다(도 8).As a result, at first, peaks appearing at 3,348 cm -1 and 1,525 cm -1 by NH stretching and deformation vibrations of the imidazole group were observed, which were 3,276 cm -1 and 1,538, respectively, respectively. It was observed that the width of the peak moved to the peak appearing at cm -1 . This was determined to be due to the formation of a hydrogen bond or the formation of a coordination bond with the proton acceptor Fe(II) in the form of Fe ←: N-Cy (FIG. 8).
상기 결과를 통해, 광-페노자임 구조체의 촉매 중심(catalytic core)은 자연의 광산화제가 갖는 전형적인 배위결합 패턴과 동일한 구조 패턴을 가짐을 알 수 있었다.From the above results, it was found that the catalytic core of the photo-phenozyme structure had the same structural pattern as the typical coordination pattern of a natural photooxidizer.
분석예 3. 형광발광 스펙트럼 분석Analysis Example 3. Fluorescence emission spectrum analysis
광-페노자임에서의 시아닌(Cy)과 금속 이온의 결합 비율을 검증하기 위하여 합성예 1에 의해 얻은 광-페노자임에 대하여 형광발광 스펙트럼(Fluorescence spectra) 분석을 수행하였다. 상기 형광발광 스펙트럼은 Edinburgh FL900/FS900 형광광도계로 기록하였다.In order to verify the binding ratio of cyanine (Cy) and metal ions in photo-phenozyme, photo-phenozyme obtained in Synthesis Example 1 was subjected to fluorescence spectra analysis. The fluorescence spectra were recorded with an Edinburgh FL900/FS900 fluorometer.
구체적으로, Fe(Ⅱ) 및 CyH를 포함하는 수용액(pH 6.36)을 1:23 내지 23:1의 서로 다른 몰 비율로 하여 실험을 위해 80배 희석하고 형광 스펙트럼을 측정하였다. 또한, 초분자 화학에서 배위된 나노입자의 응집 조건은 화학 양론(stoichiometry)에서 하나 이상의 job's plot을 포함함을 고려하여, 다른 조성비에서도 실험을 수행하였다. CyH를 포함하는 수용액(pH 6.36)을 10배 희석한 용액을 신속하게 준비한 다음, 이어서 2:1 내지 6:1의 서로 다른 몰 비율로 Fe(Ⅱ) 수용액을 첨가한 뒤, 실험을 위해 4배 희석하여 형광 스펙트럼을 측정하였다.Specifically, an aqueous solution (pH 6.36) containing Fe(II) and CyH was diluted 80-fold for the experiment in different molar ratios of 1:23 to 23:1, and the fluorescence spectrum was measured. In addition, considering that the aggregation conditions of nanoparticles coordinated in supramolecular chemistry include one or more job's plots in stoichiometry, experiments were performed at different composition ratios. A solution of a 10-fold dilution of an aqueous solution (pH 6.36) containing CyH was quickly prepared, and then an aqueous solution of Fe(II) was added in different molar ratios of 2:1 to 6:1, and then 4 times for the experiment. After dilution, the fluorescence spectrum was measured.
그 결과, [CyH] + [Fe(Ⅱ)] = 24 μM인 CyFe(Ⅱ) 광-페노자임은 Fe(Ⅱ)-착물(complex)이 1:0.5로 형성되며(도 9a), CyFe(Ⅱ) 광-페노자임에서 CyH:Fe(Ⅱ)의 몰 비율은 2:1이 보다 일반적임을 확인하였다(도 9b).As a result, in CyFe(II) photo-phenozyme with [CyH] + [Fe(II)] = 24 μM, Fe(II)-complex is formed at 1:0.5 (FIG. 9a), and CyFe( II) It was confirmed that the molar ratio of CyH:Fe(II) in photo-phenozyme was 2:1 (Fig. 9b).
나아가, 상기 결과를 CyH와 비교하였다. 구체적으로, 50 μg·mL-1 CyH를 포함하는 3.15 내지 10.98의 다양한 pH의 수용액을 제조하고 이의 형광 스펙트럼을 측정하였다. 그 결과, CyH는 760 nm에서 강한 흡수를 보였다(도 10a). FL 강도(intensity)는 처음에 pH=4에서 최대값까지 증가한 다음 pH=7에서 최소값으로 감소하고, pH=9에서 두번째 최대값으로 증가하고 pH가 11로 증가함에 따라 다시 감소하는 양상을 보였다(도 10b). Furthermore, the above results were compared with CyH. Specifically, aqueous solutions of various pHs of 3.15 to 10.98 containing 50 μg·mL -1 CyH were prepared and their fluorescence spectra were measured. As a result, CyH showed strong absorption at 760 nm (FIG. 10a). FL intensity initially increased from pH = 4 to the maximum value, then decreased to the minimum value at pH = 7, increased to the second maximum value at pH = 9, and decreased again as the pH increased to 11 ( Fig. 10b).
이번에는, 50 μg·mL-1 CyH 및 Fe(Ⅱ)를 포함하는 3.15 내지 10.98의 다양한 pH의 수용액을 제조하고 이의 형광 스펙트럼을 측정하였다. 그 결과, CyH는 760 nm에서 강한 흡수를 보이고(도 10c), FL 강도는 처음에 pH=5에서 최대값까지 증가한 다음 pH가 11로 증가함에 따라 천천히 감소하는 양상을 보였다(도 10d).This time, 50 μg·mL −1 aqueous solutions of various pHs of 3.15 to 10.98 containing CyH and Fe(II) were prepared and their fluorescence spectra were measured. As a result, CyH showed strong absorption at 760 nm (FIG. 10c), and the FL intensity initially increased from pH=5 to a maximum value and then slowly decreased as the pH increased to 11 (FIG. 10d).
상기 결과를 통해, pH 7에서 CyFe(Ⅱ)의 FL 강도는 CyH의 FL 강도보다 14배 더 큼을 확인하였다. 이는 비방사 채널(non-radiative channels)을 통한 광-페노자임의 강력한 π-π 적층(stacking)에 따른 바닥 상태(ground state)로의 복귀가 광-페노자임의 형광 발생을 촉발시킴을 의미하며, 이를 통해 CyFe(Ⅱ) 구조체 내부 공간은 분할되어 있음이 예상되었다. 이로써 CyH의 이미다졸 그룹이 Fe(Ⅱ)와 배위되고, CyFe(Ⅱ)는 촉매 중심인 금속 부위를 보호하기 위하여 소수성 공간을 형성하였음을 알 수 있다.From the above results, it was confirmed that the FL intensity of CyFe(II) at pH 7 was 14 times greater than that of CyH. This means that the return to the ground state following strong π-π stacking of photo-phenozyme through non-radiative channels triggers fluorescence of photo-phenozyme, Through this, it was expected that the inner space of the CyFe(II) structure was divided. As a result, it can be seen that the imidazole group of CyH is coordinated with Fe(II), and CyFe(II) forms a hydrophobic space to protect the metal site, which is the center of the catalyst.
분석예 4. 자외선 가시광 흡수 스펙트럼 분석Analysis Example 4. Ultraviolet visible light absorption spectrum analysis
합성예 1에 의해 얻은 광-페노자임에 대하여 자외선 가시광 흡수 스펙트럼(UV-visible absorption spectra) 분석을 수행하였다. 구체적으로, 시료를 1㎝ 경로거리 석영 큐벳에 주입한 후 Evolution 201 UV/vis 분광계(Thermo Fisher Scientific)를 이용하여 시료의 빛 투과도를 측정하였다.Photo-obtained by Synthesis Example 1 - UV-visible absorption spectra analysis was performed for the phenozyme. Specifically, after injecting the sample into a 1 cm path distance quartz cuvette, the light transmittance of the sample was measured using an Evolution 201 UV/vis spectrometer (Thermo Fisher Scientific).
그 결과, CyFe(Ⅱ) 광-페노자임의 최적 구조에서 Fe(Ⅱ)와 N 원자간 사이의 거리가 각각 Fe-Na(0.194 nm), Fe-Nb(0.408 nm) 및 Fe-Nc(0.188 nm)로 확인되어 Fe←Nc의 거리가 짧아졌음을 확인하였다(도 11).As a result, in the optimal structure of CyFe(II) photo-phenozyme, the distances between Fe(II) and N atoms were Fe-Na (0.194 nm), Fe-Nb (0.408 nm) and Fe-Nc (0.188 nm), respectively. ) to confirm that the distance of Fe←Nc was shortened (FIG. 11).
또한, 0.04 내지 0.32 mM의 다양한 Fe(Ⅱ) 농도의 CyFe(Ⅱ) 나노 구조체의 자외선 가시광 흡수(UV-vis absorption) 스펙트럼을 분석한 결과, CyFe(Ⅱ) 광-페노자임은 Fe(Ⅱ) 이온의 첨가에 따라 CyH(635nm)와 비교하여 670 nm에서의 흡수 피크를 감소시키고, 478 nm, 512 nm 및 781 nm에서의 흡수 피크를 증가시킴을 확인하였다. 상기 결과를 통해, CyFe(Ⅱ)의 광수확능은 가시광선 및 근적외선(near-infrared) 파장 영역으로 확장되었음을 확인하였다(도 12a). In addition, as a result of analyzing UV-vis absorption spectra of CyFe(II) nanostructures with various Fe(II) concentrations of 0.04 to 0.32 mM, CyFe(II) photo-phenozyme is Fe(II) It was confirmed that the absorption peak at 670 nm was decreased and the absorption peaks at 478 nm, 512 nm and 781 nm were increased compared to CyH (635 nm) according to the addition of the ion. Through the above results, it was confirmed that the light harvesting power of CyFe(II) was extended to the visible and near-infrared wavelength regions (FIG. 12a).
상기 결과를 통해, 초분자 형성에 따라 Fe←Nc의 거리가 짧아지고, 촉매 중심에서 광수확능(lightharvesting capacity) 및 항간 교차율(intersystem crossing rate)이 현저히 증가되었음을 확인하였다. 이는 Fe(Ⅱ) 농도의 증가에 따라 광-페노자임이 성숙하면서 다수의 약한 분자 내 상호작용(intramolecular interaction)이 가속화되었음을 시사한다. 상기 양상은 자연의 광산화제의 항간교차율 조절 메커니즘과 유사함을 알 수 있었다(도 12b).Through the above results, it was confirmed that the distance of Fe←Nc was shortened according to the formation of supramolecules, and lightharvesting capacity and intersystem crossing rate were significantly increased at the catalyst center. This suggests that a number of weak intramolecular interactions (intramolecular interactions) were accelerated as the photo-phenozyme matured with an increase in Fe(II) concentration. It was found that the above aspect was similar to the anti-crossover rate control mechanism of natural photooxidizers (Fig. 12b).
또한, 광-페노자임의 성숙에 따라 초기 CyFe(Ⅱ)의 전형적인 형광강도 증가 현상이 빠르게 감소하며, 광-페노자임의 새로운 배위 패턴(coordination pattern)이 형성됨을 확인하였다(도 13). 이는 탈양성자화된 시아닌(deprotonated Cy)이 새롭게 CyFe(Ⅱ)를 형성하면서 풀(pull)-푸쉬(push) π-컨쥬게이션 시스템이 붕괴되고, 광-페노자임의 촉매 중심의 전자 분포가 재배열된 것에 기인한다(도 14). 마찬가지로, 이와 같은 전자 재배열 양상은 자연의 광산화제인 엽록소와 매우 유사함을 알 수 있었다.In addition, it was confirmed that the typical increase in fluorescence intensity of early CyFe(II) rapidly decreased with the maturation of photo-phenozyme, and a new coordination pattern of photo-phenozyme was formed (FIG. 13). This is because the deprotonated cyanine (deprotonated Cy) newly forms CyFe(II), the pull-push π-conjugation system is disrupted, and the electron distribution in the catalytic center of the photo-phenozyme is rearranged. It is due to the fact that (Fig. 14). Likewise, this electron rearrangement pattern was found to be very similar to chlorophyll, a natural photooxidizer.
분석예 5. 양자 화학 계산(Quantum chemical calculation)Analysis Example 5. Quantum chemical calculation
CyH, Cy 및 CyFe(II)의 최적 분자 구조 및 자외선 가시광 흡수 스펙트럼을 예측하기 위하여 밀도 기능 이론(Density Functional Theory: DFT) 및 시간-의존(time-dependent) DFT 계산(B3LYP/6-31g(d)/IEFPCM(water))을 수행하였다. 항간교차율(Intersystem Crossing(ISC) rate) 상수, k ISC는 하기 식 1에 의해 계산하였다. 스핀-궤도 상호작용(spin-orbit coupling: SOC) 상수는 오픈소스 프로그램인 PySOC를 사용하여 계산하였다.Density Functional Theory (DFT) and time-dependent DFT calculation (B3LYP/6-31g(d) ) / IEFPCM (water)) was performed. Intersystem Crossing (ISC) rate constant, k ISC was calculated by
[식 1][Equation 1]
(여기서, ρFC는 상태 밀도이고, λM은 용매 재구성 에너지(~8000cm-1)이고, EST는 일중항 및 삼중항 상태 간의 에너지 갭(gap)을 의미한다.)(Where ρ FC is the density of states, λ M is the solvent reconstitution energy (~8000 cm −1 ), and E ST is the energy gap between singlet and triplet states.)
CyH, Cy 및 CyFe(Ⅱ)의 분자 오비탈 특성을 확인하였다. Cy는 CyH의 탈양성자화에 따른 π-컨쥬게이션의 조절과 함께, CyH(HOMO = -4.92 eV; LUMO = -2.48 eV)와 비교하여 HOMO(-4.56 eV) 및 LUMO(-1.37 eV) 간에 상대적으로 큰 에너지 갭을 가짐을 확인하였다. Cy가 Fe(Ⅱ)와 배위결합을 형성함에 따라, HOMO-LUMO 에너지 갭이 현저히 감소함을 확인할 수 있었다(도 15a, 15b, 15c).The molecular orbital properties of CyH, Cy and CyFe(II) were confirmed. Cy is relative between HOMO (-4.56 eV) and LUMO (-1.37 eV) compared to CyH (HOMO = -4.92 eV; LUMO = -2.48 eV), with modulation of π-conjugation following deprotonation of CyH. was confirmed to have a large energy gap. As Cy forms a coordination bond with Fe(II), it can be seen that the HOMO-LUMO energy gap is significantly reduced ( FIGS. 15a , 15b and 15c ).
CyH, Cy 및 CyFe(Ⅱ)의 일중항-삼중항 간의 에너지 갭(
(eV)S 1
(eV)
(eV)T 1
(eV)
(eV)T 2
(eV)
(eV)
(eV)
(eV)
(eV)
(eV)S 8
(eV)
(eV)T 9
(eV)
(eV)T 10
(eV)
(eV)
(eV)
(eV)
(eV)
(cm-1)
(cm -1 )
(cm-1)
(cm -1 )
(cm-1)
(cm -1 )
(cm-1)
(cm -1 )
S1 ↔ T1 k ISC (s -1 )
S 1 ↔ T 1
S1 ↔ T2 k ISC (s -1 )
S 1 ↔ T 2
S8 ↔ T9 k ISC (s -1 )
S 8 ↔ T 9
S8 ↔ T10 k ISC (s -1 )
S 8 ↔ T 10
표 1 및 2에 나타낸 바와 같이, CyFe(Ⅱ)의 일중항-삼중항 간의 에너지 갭은 상대적으로 작은 반면, SOC 상수는 34.8 cm-1로, Cy(0.191 cm-1) 및 CyH(0.226 cm-1)에 비해 현저히 큼을 확인하였다. 또한, 표 3에 나타낸 바와 같이, CyH, Cy 및 CyFe(Ⅱ)의 일중항-삼중항 간의 ISC 상수는 각각 2.39 x 106, 1.17 x 106 및 4.41 x 1011 s-1임을 확인하였다. As shown in Tables 1 and 2, the energy gap between singlet-triplet of CyFe(II) is relatively small, while the SOC constant is 34.8 cm -1 , Cy (0.191 cm -1 ) and CyH (0.226 cm -1 ) . It was confirmed that it was significantly larger than that of 1 ). In addition, as shown in Table 3, it was confirmed that the ISC constants between singlets and triplets of CyH, Cy and CyFe(II) were 2.39 x 10 6 , 1.17 x 10 6 and 4.41 x 10 11 s -1 , respectively.
정리하면, CyFe(Ⅱ)는 낮은 
추가적으로, CyH, Cy 및 CyFe(Ⅱ)의 자연 전이궤도 함수(Natural Transition Orbitals: NTO)를 획득하여 전자 전환 특성을 확인한 결과, CyFe(Ⅱ)는 CyH 및 Cy와 비교할 때 흡수에서 상당한 전하 이동 특성을 나타냄을 확인하였다(도 16).In addition, as a result of obtaining the natural transition orbitals (NTO) of CyH, Cy and CyFe(II) and confirming the electron conversion properties, CyFe(II) showed significant charge transfer properties in absorption compared to CyH and Cy. It was confirmed that it shows (FIG. 16).
상기 결과를 통해, 자연의 광산화제의 항간교차율 조절 메커니즘과 유사하게, 광-페노자임은 중심 금속(Fe(Ⅱ))에 대한 축 방향 배위결합(axial coordination)에 의해 항간교차율이 가속화되어 광-펜톤 유사반응을 나타낼 수 있음을 확인하였다.Through the above results, similar to the mechanism of regulating the anti-crossover rate of natural photooxidizers, the photo-phenozyme accelerates the anticrossover rate by axial coordination with respect to the central metal (Fe(II)). - It was confirmed that it can exhibit a Fenton-like reaction.
분석예 6. 구조 안정성 분석Analysis Example 6. Structural stability analysis
6-1. 활성형 광-페노자임의 구조 안정성6-1. Structural stability of active photo-phenozyme
CyFe(Ⅱ)에 FBS(Fetal Bovine Serum)를 추가하여, 단백질과 결합한 전효소(Holoenzyme)로서의 활성형 CyFe(Ⅱ) 광-페노자임의 구조 안정성을 분석하였다. 구체적인 실험은 분석예 1, 4와 동일한 방법으로 수행하였다.By adding Fetal Bovine Serum (FBS) to CyFe(II), the structural stability of the active CyFe(II) photo-phenozyme as a protein-bound proenzyme (Holoenzyme) was analyzed. Specific experiments were performed in the same manner as in Analysis Examples 1 and 4.
그 결과, CyFe(Ⅱ)에 FBS를 추가하면 분자 크기 분포가 104.8 ± 39.18 nm 내지 134.8 ±39.18 nm (PDI:0.102)로 증가함을 확인하였다(도 17).As a result, it was confirmed that when FBS was added to CyFe(II), the molecular size distribution increased from 104.8±39.18 nm to 134.8±39.18 nm (PDI:0.102) ( FIG. 17 ).
다음으로, 0.1 내지 100μM의 다양한 농도의 FBS 단백질과 함께 유리(free) CyH의 자외선 가시광 흡수(UV-vis absorption) 스펙트럼을 측정하였다.Next, UV-vis absorption spectra of free CyH with various concentrations of 0.1 to 100 μM of FBS protein were measured.
그 결과, CyH의 자외선 가시광 흡수 스펙트럼(도 18a)과 비교하여 CyFe(Ⅱ)는 스펙트럼 상의 분열(splitting)이 없음을 확인하였다(도 18b). 이를 통해, CyFe(Ⅱ) 광-페노자임은 단독으로 존재하는 분자(free molecule)가 아니고 촉매 중심이 혈청 단백질로 코팅(coating)되어 있으며, 상기 단백질 코팅을 통해 촉매 구조를 유지함을 알 수 있었다. 나아가, CyFe(Ⅱ) 광-페노자임은 24시간 경과 후에도 약간의 스펙트럼 변화만을 보여, 촉매 중심 구조가 안정하게 유지됨을 확인하였다(도 19). 반면, 단백질 스캐폴드가 없을 때는, 상기에서 확인한 바와 같이 광-페노자임의 촉매 구조가 크게 변경되었다(도 12).As a result, compared with the ultraviolet visible light absorption spectrum of CyH (FIG. 18a), it was confirmed that CyFe(II) had no splitting in the spectrum (FIG. 18b). Through this, it was found that CyFe(II) photo-phenozyme is not a free molecule, but a catalytic center is coated with serum protein, and the catalytic structure is maintained through the protein coating. . Furthermore, CyFe(II) photo-phenozyme showed only a slight spectrum change even after 24 hours, confirming that the catalyst core structure was stably maintained (FIG. 19). On the other hand, in the absence of the protein scaffold, as confirmed above, the photo-phenozyme catalytic structure was significantly changed ( FIG. 12 ).
이를 통해, 활성형 광-페노자임에서 단백질 부분은 촉매 중심 구조를 안정화시키며, 광감작성 빌딩 블록인 시아닌 분자를 활성 구조로 배열하고, 이의 과도한 자가-응집(self-aggregation)을 방지하는 역할을 수행함을 알 수 있었다.Through this, the protein moiety in the active photo-phenozyme stabilizes the catalytic core structure, arranges the photosensitive building block cyanine molecule into the active structure, and prevents excessive self-aggregation thereof. was known to perform.
6-2. 광-페노자임의 pH 안정성6-2. pH stability of photo-phenozyme
이와 같은 광-페노자임의 구조 안정성이 pH 변화에도 유지되는지 확인하였다. 구체적으로, 50 μg·mL-1 CyH를 포함하는 3.15 내지 10.98의 다양한 pH의 수용액을 제조하고 이의 자외선 가시광 흡수 스펙트럼을 측정하였을 때, CyH는 654nm 및 515nm(pH <5)에서 강한 흡수를 나타내며, 654nm의 피크는 증가하는 반면 515nm의 피크는 pH가 3에서 4로 증가함에 따라 감소하였다. pH 범위 5~7에서, CyH는 632 nm에서 강한 흡수를 나타내며, 이 피크는 pH가 증가함에 따라 감소하였다. pH가 7 이상인 경우(pH 7-11), 632nm의 피크가 감소하였고, pH가 증가함에 따라 643 nm (10-11)로 약간 적색편이 되었다. 이 때 413nm 및 305nm의 피크는 증가하고 약간 청색편이 되었다(도 20).As such, it was confirmed whether the structural stability of the photo-phenozyme was maintained even with a change in pH. Specifically, when aqueous solutions of various pHs of 3.15 to 10.98 containing 50 μg mL -1 CyH were prepared and the ultraviolet visible light absorption spectrum thereof was measured, CyH exhibits strong absorption at 654 nm and 515 nm (pH <5), The peak at 654 nm increased while the peak at 515 nm decreased as the pH increased from 3 to 4. In the pH range 5-7, CyH showed strong absorption at 632 nm, and this peak decreased with increasing pH. When the pH was 7 or higher (pH 7-11), the peak at 632 nm decreased, and as the pH increased, it became slightly redshifted to 643 nm (10-11). At this time, the peaks at 413 nm and 305 nm increased and became slightly blue-shifted (FIG. 20).
다음으로, 50 μg·mL-1 CyH를 포함하는 3.15 내지 10.98의 다양한 pH의 수용액을 제조한 뒤, 이어서 Fe(Ⅱ) 용액을 첨가하여 동일한 방법으로 실험하였다. 그 결과, CyH 및 Fe(Ⅱ) 용액은 514nm(pH <5)에서만 강한 흡수를 나타내었고, 514nm의 피크는 감소한 반면, 652nm의 피크는 pH가 5까지 증가함에 따라 증가하였다. pH 범위 6~7에서, 647 nm의 피크가 증가하였고 pH = 5의 피크에 비해 약간 청색편이 되었다. 또한, 517nm에서의 피크는 감소하였다. pH 범위 7~10에서 CyH 및 Fe(Ⅱ) 용액은 642 nm에서 강한 흡수를 나타내었고, 이는 모든 pH 범위(7-10)에서 변하지 않았다. 그러나, 421, 305 및 234 nm에서 피크가 증가하였으며, 이는 염기성 용액에서 Fe(OH)2의 형성이 CyFe(Ⅱ) 광-페노자임 형성과 경쟁할 수 있음을 시사하였다. pH = 11에서는 635nm의 피크가 나타나고 낮은 pH 값의 피크에 비해 약간 청색편이 되었다(도 21).Next, an aqueous solution of 3.15 to 10.98 of various pHs containing 50 μg·mL -1 CyH was prepared, and then Fe(II) solution was added and the same experiment was performed. As a result, the CyH and Fe(II) solutions showed strong absorption only at 514 nm (pH <5), and the peak at 514 nm decreased, while the peak at 652 nm increased as the pH increased to 5. In the pH range 6-7, the peak at 647 nm increased and slightly blueshifted compared to the peak at pH = 5. Also, the peak at 517 nm decreased. CyH and Fe(II) solutions in the pH range 7-10 showed strong absorption at 642 nm, which did not change in all pH ranges (7-10). However, the peaks at 421, 305 and 234 nm increased, suggesting that the formation of Fe(OH) 2 in basic solution may compete with CyFe(II) photo-phenozyme formation. At pH = 11, a peak at 635 nm appeared and slightly blue-shifted compared to the peak at a low pH value (FIG. 21).
반면, CyFe(Ⅱ) 광-페노자임은 동일한 방법으로 실험하였을 때 pH가 3에서 11로 증가하더라도 515nm 및 635nm에서의 피크의 변화가 적었다. 구체적으로, CyFe(Ⅱ)는 653nm 및 514nm(pH <5)에서 강한 흡수를 나타내며, 654nm의 피크는 감소하는 반면 514nm의 피크는 pH가 3에서 4로 증가해도 변하지 않았다. 즉, CyH에 비해 514 nm의 피크는 증가하고 654 nm의 피크는 감소하였다. pH 범위 5~10에서, CyFe(Ⅱ)는 635nm에서 강한 흡수를 나타내며, 이 피크는 pH가 증가함에 따라 감소하였다. pH가 10 이상인 경우, 644nm의 피크가 나타나고 423nm의 피크가 커졌다. 이는 CyFe(Ⅱ)는 Fe(Ⅱ)가 CyH와 배위결합을 형성함에 따라 CyH보다 pH에 대한 민감도가 감소하고, CyH의 이미다졸 고리가 Fe(Ⅱ)에 의해 둘러싸이면서 보호될 수 있음을 시사하였다(도 22).On the other hand, CyFe(II) photo-phenozyme showed little change in peaks at 515 nm and 635 nm even when the pH was increased from 3 to 11 when tested in the same way. Specifically, CyFe(II) shows strong absorption at 653 nm and 514 nm (pH < 5), and the peak at 654 nm decreased while the peak at 514 nm did not change even when the pH was increased from 3 to 4. That is, compared to CyH, the peak at 514 nm increased and the peak at 654 nm decreased. In the pH range 5-10, CyFe(II) shows strong absorption at 635 nm, and this peak decreases with increasing pH. When the pH is 10 or more, a peak at 644 nm appears and the peak at 423 nm becomes larger. This suggests that CyFe(II) has lower sensitivity to pH than CyH as Fe(II) forms a coordination bond with CyH, and that the imidazole ring of CyH can be protected by being surrounded by Fe(II). (Fig. 22).
상기 결과를 통해, 광-페노자임의 구조 안정성은 pH 변화에도 유지됨을 확인하였다. Through the above results, it was confirmed that the structural stability of the photo-phenozyme was maintained even with a change in pH.
분석예 7. 광열 활성 분석Analysis Example 7. Photothermal activity analysis
합성예 1에 의해 얻은 광-페노자임(0.25 mg·mL-1) 수용액 1.0mL을 H2O2, ABDA와 함께 석영 큐벳에 주입한 후 655 nm 레이저를 10분간 조사하였다. 또한, 광-페노자임 또는 CyH 응집체(aggregations)(0.25 mg·mL-1) 수용액 1.0mL에 655 nm 레이저를 3회 조사하였다. 온도와 IR 이미지는 열화상카메라(FLIR E60)로 매 30초마다 기록하였다.Photo-Penozyme (0.25 mg·mL -1 ) obtained by Synthesis Example 1 was injected into a quartz cuvette with 1.0 mL of an aqueous solution of H 2 O 2 and ABDA and then irradiated with a 655 nm laser for 10 minutes. In addition, photo-phenozyme or CyH aggregates (aggregations) (0.25 mg·mL -1 ) 1.0mL of aqueous solution was irradiated with a 655 nm laser three times. Temperature and IR images were recorded every 30 seconds with a thermal imaging camera (FLIR E60).
그 결과, 단백질이 변성 온도에 가깝거나 그 이상으로 가열되었을 때, CyFe(Ⅱ) 광-페노자임은 3 사이클 이후에도 광열 특성이 유지되고, CyH(0.25 mg·mL-1)과 비교하여 광열 특성이 우수함을 확인하였다(도 23). 이는 광-페노자임의 소수성 그룹이 노출되면 이의 구조적 안정성 및 광열 특성이 향상될 수 있음을 시사한다.As a result, when the protein was heated close to or above the denaturation temperature, the photothermal properties of CyFe(II) photo-phenozyme were maintained even after 3 cycles, and compared to that of CyH (0.25 mg mL -1 ). It was confirmed that this was excellent (FIG. 23). This suggests that when the hydrophobic group of photo-phenozyme is exposed, its structural stability and photothermal properties can be improved.
상기 결과를 통해, CyFe(Ⅱ) 광-페노자임은 스캐폴드 단백질의 존재에 따라 항간교차율, 광수확능을 유연하게 조절할 수 있음을 확인하였다.From the above results, it was confirmed that CyFe(II) photo-phenozyme can flexibly control the cross-section rate and light harvesting ability according to the presence of the scaffold protein.
분석예 8. EPR 분석Analysis Example 8. EPR Analysis
합성예 1에 의해 얻은 광-페노자임에 대하여 전자상자성 공명분광기(Electron Paramagnetic Resonance Spectrometer: EPR) 분석을 수행하였다. 구체적으로, 실온에서 광-페노자임(0.5 mg·mL-1), HCl(5 mM) 및 H2O2(10 mM)를 포함하는 용액에 655 nm 레이저(0.7 W·cm-2)를 2분 동안 조사하고, 결과를 EMX-plus(Bruker, KBSI Western Seoul Center)를 사용하여 분석하였다.Photo-Penozyme obtained by Synthesis Example 1 was subjected to Electron Paramagnetic Resonance Spectrometer (EPR) analysis. Specifically, a 655 nm laser (0.7 W·cm −2 ) was applied to a solution containing photo-phenozyme (0.5 mg·mL −1 ), HCl (5 mM) and H 2 O 2 (10 mM) at room temperature. It was irradiated for 2 minutes, and the results were analyzed using EMX-plus (Bruker, KBSI Western Seoul Center).
그 결과, 산성 조건(5 mM H+)에서 광-페노자임과 산소-공급제(H2O2) 존재 시, 일중항 산소 신호가 증가하며 이후 라디칼 생성에도 중요한 역할을 한 반면, 히드록실 라디칼 신호는 작음을 확인하였다(도 24).As a result, in the presence of photo-phenozyme and an oxygen-supplying agent (H 2 O 2 ) under acidic conditions (5 mM H + ), the singlet oxygen signal increased and also played an important role in subsequent radical generation, whereas hydroxyl It was confirmed that the radical signal was small (FIG. 24).
한편, 반응 시스템이 오직 H2O2만을 포함하는 경우, 655 nm 레이저 조사 후 히드록실 라디칼 신호가 검출되었다(도 25a). 또한, 레이저를 5분간 길게 조사하는 경우, 2분간 조사하는 경우에 비해 히드록실 라디칼 신호가 더 강하게 검출되었다. 이로부터 광-페노자임이 근적외선 조사 하에서 히드록실 라디칼을 생성할 수 있음을 알 수 있었다. 또한, 상기 반응 시스템은 반응 중에 온도가 최대 62℃까지 증가함을 확인하였다(도 25b).On the other hand, when the reaction system included only H 2 O 2 , a hydroxyl radical signal was detected after irradiation with a 655 nm laser ( FIG. 25A ). In addition, when the laser was irradiated for 5 minutes for a long time, the hydroxyl radical signal was detected more strongly than when the laser was irradiated for 2 minutes. From this, it was found that photo-phenozyme can generate hydroxyl radicals under near-infrared irradiation. In addition, it was confirmed that the reaction system increased the temperature up to 62° C. during the reaction (FIG. 25b).
상기 결과를 통해, 자연의 광산화효소가 갖는 활성을 효과적으로 모방할 수 있는 것 이외에도, 광-페노자임은 고유한 광역학적, 광열 활성에 따라 향상된 광-펜톤 유사반응을 나타내며, 광-펜톤 유사반응은 H2O2 존재 하에서 라디칼을 생성함을 확인하였다.Through the above results, in addition to being able to effectively mimic the activity of natural photooxidase, photo-phenozyme exhibits enhanced photo-Fenton-like reaction according to its intrinsic photodynamic and photothermal activity, and photo-Fenton-like reaction It was confirmed that a radical is generated in the presence of H 2 O 2 .
분석예 9. 세포 내 일중항 산소(Analysis Example 9. Intracellular singlet oxygen ( 1One OO 22 ) 검출 이미징) detection imaging
9,10-안트라세네디일-비스(메틸렌)디말로닉산(ABDA)을 일중항 산소(singlet oxygen) 인디케이터로 사용하여 세포 내 일중항 산소의 생성 정도를 분석하였다.9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) was used as a singlet oxygen indicator to analyze the degree of intracellular singlet oxygen generation.
실험은 (i) H2O2, 합성예 1에 의해 얻은 광-페노자임(0.8mM) 및 ABDA를 포함하는 경우; 및 (ii) (i)과 동일하되 H2O2가 없는 경우에 대하여 수행하였다. 15 분간 N2 가스를 버블링하여 용액 내 산소를 제거하고, 655nm 레이저(0.7W·cm-2)를 조사한 후 UV-visible 분광광도계로 378nm에서의 흡광도를 측정하였다.Experiments were conducted with (i) H 2 O 2 , photo-phenozyme obtained by Synthesis Example 1 (0.8 mM) and ABDA; and (ii) the same as (i) but without H 2 O 2 . The oxygen in the solution was removed by bubbling N 2 gas for 15 minutes, and the absorbance at 378 nm was measured with a UV-visible spectrophotometer after irradiation with a 655 nm laser (0.7W·cm -2 ).
그 결과, H2O2 존재 하에서 ABDA-O2가 대조군에 비해 2배 이상 생성됨을 확인하였다(도 26). As a result, it was confirmed that in the presence of H 2 O 2 ABDA-O 2 was generated twice or more compared to the control ( FIG. 26 ).
분석예 10. Analysis Example 10. 인 비트로in-vitro 세포 이미징 및 세포 생존율 Cell Imaging and Cell Viability
10-1. 활성산소종 발생 분석10-1. Analysis of reactive oxygen species generation
HeLa(human cervix adenocarcinoma) 세포를 Korean Cell Line Bank(Seoul, Korea)로부터 구입하였다. 상기 세포를 10% FBS, 100 U/ml 페니실린 및 100 U/ml 스트렙토마이신이 보충된 MEM(Minimum Essential Medium Eagle) 배지에서 37℃, 5% CO2 조건으로 배양하였다. 오버나잇 배양 후, HeLa 세포를 서로 다른 농도의 H2O2(0, 50, 100, 200 μM) 및 광-페노자임(5 μg·mL-1)과 함께 2시간 동안 처리한 다음, 디클로로디하이드로플루오레세신 디아세테이트(2,7-Dichlorodihydrofluorescein Diacetate, DCFH2-DA)로 세포 내 활성산소를 측정하였다. 대조군으로는 DCFH2-DA만을 포함하는 수용액을 사용하였다. 공초점 레이저 주사 현미경(Olympus Fluoview FV1200)으로 세포 이미징을 수행하였다.HeLa (human cervix adenocarcinoma) cells were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in MEM (Minimum Essential Medium Eagle) medium supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin at 37° C., 5% CO 2 condition. After overnight incubation, HeLa cells were treated with different concentrations of H 2 O 2 (0, 50, 100, 200 μM) and photo-phenozyme (5 μg mL −1 ) for 2 hours, followed by dichloromethane. Intracellular free radicals were measured with dihydrofluorescein diacetate (2,7-Dichlorodihydrofluorescein Diacetate, DCFH 2 -DA). As a control, an aqueous solution containing only DCFH 2 -DA was used. Cell imaging was performed with a confocal laser scanning microscope (Olympus Fluoview FV1200).
λem = 520nm에서의 형광 강도의 변화로 활성산소종(ROS) 생성 여부를 확인한 결과, CyH는 대조군과 달리 활성산소종을 생성함을 확인하였다(도 27). 이는 세포내 활성산소와 DCFH2-DA가 반응하면 형광을 띠는 DCF(2,7-dichlorofluorescein)로 산화되는 원리를 이용한 것이다. CyH는 광-페노자임의 활성산소종 발생가능성을 시사하는 Fe(Ⅱ) 이온의 부재로, 시간에 따라 활성산소종 발생량이 점점 높아짐을 확인할 수 있었다.As a result of confirming whether reactive oxygen species (ROS) was generated by the change in fluorescence intensity at λ em = 520 nm, it was confirmed that CyH produced reactive oxygen species unlike the control group (FIG. 27). This is based on the principle that DCF (2,7-dichlorofluorescein) is oxidized to fluorescent DCF (2,7-dichlorofluorescein) when DCFH 2 -DA reacts with intracellular reactive oxygen species. CyH is the absence of Fe(II) ion, which suggests the possibility of generating reactive oxygen species in photo-phenozyme, and it can be confirmed that the amount of reactive oxygen species generation gradually increases with time.
반면, λem = 543nm에서의 형광 강도를 측정한 결과, 광-페노자임은 시간이 지남에 따라 활성산소종에 의해 매개된 광감작성 산화(photosensitive oxidation) 활성을 나타내었다. 광-페노자임 존재 시, 이의 촉매 활성은 처음 3분 동안 크게 증가 후, 점차 안정적으로 유지되었다. 이는 기질과의 상호작용 후 생성물(ROS)의 분리 및 방출을 수행하는 효소 기반 반응의 전형적인 패턴으로, 광-페노자임이 광산화효소의 기능을 효과적으로 모방할 수 있음을 시사한다(도 28).On the other hand, as a result of measuring the fluorescence intensity at λ em = 543 nm, photo-phenozyme exhibited photosensitive oxidation activity mediated by reactive oxygen species over time. In the presence of photo-phenozyme, its catalytic activity increased significantly during the first 3 minutes and then gradually remained stable. This is a typical pattern of an enzyme-based reaction that carries out the separation and release of the product (ROS) after interaction with the substrate, suggesting that photo-phenozyme can effectively mimic the function of photooxidase (FIG. 28).
동일한 방법으로, HeLa 세포에서 히드록실 라디칼 발생을 확인하였고, 그 결과 광-페노자임 (8 μM)의 형광 강도는 암실에서 H2O2 농도와 양의 상관관계를 보임을 확인하여 히드록실 라디칼은 H2O2 농도 의존적으로 발생이 증가함을 알 수 있었다(도 29). 광 조건 하에서, HeLa 세포의 형광 강도는 H2O2 농도가 증가함에 따라 거의 변하지 않았다. 이는 HeLa 세포에 국재화된 H2O2가 풍부함을 의미하여, 이후 세포 실험에서 H2O2를 별도로 추가하지 않았다.In the same way, generation of hydroxyl radicals was confirmed in HeLa cells, and as a result, it was confirmed that the fluorescence intensity of photo-phenozyme (8 μM) showed a positive correlation with the concentration of H 2 O 2 in the dark. It was found that the occurrence of H 2 O 2 was increased in a concentration-dependent manner (FIG. 29). Under light conditions, the fluorescence intensity of HeLa cells hardly changed with increasing H 2 O 2 concentration. This means that H 2 O 2 localized to HeLa cells is abundant, and H 2 O 2 was not separately added in subsequent cell experiments.
10-2. 세포 생존율 분석 10-2. Cell viability assay
HeLa 세포(1 x 104 cells well-1)를 96-웰 플레이트에 시딩(seeding)한 후, 24시간 동안 두어 플레이트에 부착시켰다. 합성예 1에 의해 얻은 서로 다른 농도의 광-페노자임(0 ~ 8 μg·mL-1)을 각 웰에 첨가하고 4시간 동안 더 배양하였다. 그런 다음, 신선한 배양 배지로 세척하고 655nm 레이저(1.5 W·cm-2)를 2분 동안 조사하였다. 조사 후, 상기 세포를 24시간 동안 암실에서 추가로 배양하였다. 이어서, 각 웰에 10% MTT 용액(5mg·mL-1)을 4시간 동안 첨가하고, 150 μL DMSO를 배지에 첨가한 후 마이크로플레이트 리더(Thermo Fisher Scientific)를 사용하여 490 nm에서 흡광도를 측정하였다. 세포 생존율(cell viability)은 총 대조군 세포에 대한 생존 세포의 백분율로 계산하였다.HeLa cells (1 x 10 4 cells well -1 ) were seeded in a 96-well plate, and then placed for 24 hours to attach to the plate. Photo-phenozyme (0 ~ 8 μg·mL -1 ) of different concentrations obtained by Synthesis Example 1 was added to each well and further cultured for 4 hours. Then, it was washed with fresh culture medium and irradiated with a 655 nm laser (1.5 W·cm -2 ) for 2 minutes. After irradiation, the cells were further cultured in the dark for 24 hours. Then, 10% MTT solution (5mg·mL −1 ) was added to each well for 4 hours, 150 μL DMSO was added to the medium, and absorbance was measured at 490 nm using a microplate reader (Thermo Fisher Scientific). . Cell viability was calculated as the percentage of viable cells relative to total control cells.
이와 별개로, 서로 다른 농도의 광-페노자임(0 ~ 10 μg·mL-1)을 암실에서 배양하였다. 신선한 배양 배지로 세척하여 24시간 동안 추가로 배양하였다. 이어서, 각 웰에 10% MTT 용액(5mg·mL-1)을 4시간 동안 첨가하고, 150 μL DMSO를 배지에 첨가한 후 세포 생존율을 분석하였다.Separately, different concentrations of photo-phenozyme (0 ~ 10 μg·mL -1 ) were cultured in the dark. Washed with fresh culture medium and further incubated for 24 hours. Then, 10% MTT solution (5mg·mL −1 ) was added to each well for 4 hours, and cell viability was analyzed after 150 μL DMSO was added to the medium.
그 결과, 광-페노자임 처리된 HeLa 세포는 Fe(Ⅱ) 농도 의존적으로(0-10 μg·mL-1) 세포사멸이 유도되었다. 세포 생존율은 Fe(Ⅱ) 농도가 가장 높은 10 μg·mL-1에서 13%까지 현저히 감소하였다(도 30a). 또한, 다양한 광-페노자임 농도 조건에서 HeLa 세포를 4시간 동안 배양하고 655 nm 레이저를 2분 동안 조사 시, 광 조사에 따라 세포 사멸능이 최대 6배 증가함을 확인하여 광-페노자임은 레이저 조사 후에 종양 세포 억제능이 보다 향상되었고, 이를 통해 광-펜톤 유사반응을 통해 광-페노자임의 페로토시스를 통한 세포사멸능이 강화되었음을 알 수 있었다(도 30b, 도31).As a result, photo-phenozyme-treated HeLa cells induced apoptosis in a Fe(II) concentration-dependent manner (0-10 μg·mL -1 ). Cell viability was significantly reduced to 13% at the highest Fe(II) concentration of 10 μg·mL -1 (FIG. 30a). In addition, when HeLa cells were cultured for 4 hours under various photo-phenozyme concentration conditions and irradiated with a 655 nm laser for 2 minutes, it was confirmed that apoptosis capacity increased up to 6 times according to light irradiation. After laser irradiation, tumor cell suppression ability was further improved, and through this, it could be seen that the apoptosis ability through photo-phenozyme ferrotosis was enhanced through a light-Fenton-like reaction (FIG. 30b, FIG. 31).
광-페노자임을 처리하지 않은 대조군과의 TEM 이미지를 비교한 결과, 광-페노자임 처리군은 미토콘드리아 수축을 보였다(도 32a, 32b). 흥미롭게도, 광-페노자임은 자기용해소체(autolysosome)의 형성과 막구조의 손상도 유발할 수 있음을 확인하였다. 또한, 전체 세포와 이의 막 구조는 광-페노자임과 4시간 동안 배양 후, 이어지는 레이저 조사에 따른 지질과산화물의 축적으로 심하게 손상됨을 확인하였다(도 32c). 상기 결과를 통해, 일 양상에 따른 나노 구조체, 예를 들어 상기 합성예 1에 의해 얻은 광-페노자임은 페로토시스를 통한 암세포의 선택적 사멸이 가능함을 확인하였다. Photo- As a result of comparing the TEM images with the non-phenozyme-treated control group, the photo-phenozyme-treated group showed mitochondrial contraction ( FIGS. 32a and 32b ). Interestingly, it was confirmed that photo-phenozyme can also induce the formation of autolysosomes and damage to the membrane structure. In addition, it was confirmed that the whole cell and its membrane structure were severely damaged by the accumulation of lipid peroxide following laser irradiation after incubation with photo-phenozyme for 4 hours (FIG. 32c). Through the above results, it was confirmed that the nanostructure according to an aspect, for example, the photo-phenozyme obtained by Synthesis Example 1, can selectively kill cancer cells through ferrotosis.
일 양상에 따른 나노 구조체는 배위 결합이 원동력인 시아닌 및 철 이온(Fe(Ⅱ))의 조립에 기반한 것으로, 생체 내에서 안정적인 촉매 활성을 발휘할 수 있음을 확인하였다. 뿐만 아니라, 중심 금속(Fe(Ⅱ))에 대한 축 방향 배위결합에 의해 항간교차율이 가속화되어 광-펜톤 유사반응을 나타낼 수 있고, 이를 통해 세포 내부의 활성산소종을 증가시킬 수 있음을 확인하였다. 따라서, 정상세포 또는 정상 조직에는 영향을 미치지 않고 암세포 내부의 지질과산화물을 축적시켜 페로토시스를 유도하는 방식으로, 부작용은 최소화하면서도 높은 암 치료 효과를 나타낼 수 있을 것으로 기대된다.It was confirmed that the nanostructure according to an aspect is based on the assembly of cyanine and iron ions (Fe(II)), where coordination bonding is the driving force, and can exhibit stable catalytic activity in vivo. In addition, it was confirmed that the cross-section rate was accelerated by the axial coordination bond to the central metal (Fe(II)), and thus a photo-Fenton-like reaction could be exhibited, thereby increasing the reactive oxygen species inside the cell. . Therefore, it is expected that a high cancer treatment effect can be achieved while minimizing side effects by accumulating lipid peroxides inside cancer cells without affecting normal cells or normal tissues to induce ferrotosis.
전술한 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 이해해야만 한다.The above description is for illustration, and those of ordinary skill in the art to which the present invention pertains will understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.
Claims (10)
상기 나노 구조체는 철 이온이 시아닌과 배위 결합하여 자기조립된 것인 나노 구조체.
[화학식 1]
The nanostructure is a nanostructure that is self-assembled by coordinating iron ions with cyanine.
[Formula 1]
상기 철 이온은 시아닌에 포함된 복수의 질소(N) 원자와 배위결합하는 것인, 나노 구조체.The method according to claim 1,
The iron ion is to coordinate with a plurality of nitrogen (N) atoms contained in cyanine, the nanostructure.
상기 시아닌 및 철 이온의 몰 비는 2:1 내지 6:1인 것인, 나노 구조체.The method according to claim 1,
The molar ratio of the cyanine and iron ions is 2:1 to 6:1, the nanostructure.
상기 나노 구조체는 광-펜톤 유사반응을 나타내는 것인, 나노 구조체.The method according to claim 1,
The nanostructure is a light-Fenton-like reaction, the nanostructure.
상기 나노 구조체는 650 nm 내지 900 nm 파장의 영역에서 광 조사에 의해 활성산소종을 생성하는 것인, 나노 구조체.The method according to claim 1,
The nanostructure will generate reactive oxygen species by light irradiation in a wavelength region of 650 nm to 900 nm.
상기 나노 구조체는 페로토시스를 통해 암세포의 사멸을 유도하는 것인, 약학적 조성물.8. The method of claim 7,
The nanostructure will induce apoptosis of cancer cells through ferrotosis, a pharmaceutical composition.
상기 암은 폐암, 비소세포성 폐암, 결장암, 골암, 췌장암, 피부암, 두부 또는 경부암, 피부 또는 안구 내 흑색종, 자궁암, 난소암, 직장암, 위암, 항문부근암, 결장암, 유방암, 나팔관암종, 자궁내막암종, 자궁경부암종, 질암종, 음문암종, 호지킨병(Hodgkin's disease), 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계 종양, 1차 중추신경계 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것인, 약학적 조성물.8. The method of claim 7,
The cancer is lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, perianal cancer, colon cancer, breast cancer, fallopian tube carcinoma, uterus Endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. that, the pharmaceutical composition.
2) 상기 단계 1)에서 제조한 용액에 철 용액을 첨가하여 혼합하는 단계를 포함하는 시아닌 및 철 이온을 포함하는 나노 구조체를 제조하는 방법.1) preparing a cyanine-based compound solution; and
2) A method for producing a nanostructure containing cyanine and iron ions, comprising the step of adding and mixing an iron solution to the solution prepared in step 1).
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