KR102643240B1 - Nanostructure comprising metal organic frameworks, glucose oxidases, and peroxidase mimetics, and use thereof - Google Patents
Nanostructure comprising metal organic frameworks, glucose oxidases, and peroxidase mimetics, and use thereof Download PDFInfo
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- KR102643240B1 KR102643240B1 KR1020210143333A KR20210143333A KR102643240B1 KR 102643240 B1 KR102643240 B1 KR 102643240B1 KR 1020210143333 A KR1020210143333 A KR 1020210143333A KR 20210143333 A KR20210143333 A KR 20210143333A KR 102643240 B1 KR102643240 B1 KR 102643240B1
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- nanostructure
- cancer
- zif
- gox
- glucose oxidase
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
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- A61K9/51—Nanocapsules; Nanoparticles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/28—Compounds containing heavy metals
- A61K31/295—Iron group metal compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/44—Oxidoreductases (1)
- A61K38/443—Oxidoreductases (1) acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
본 발명은 포도당 산화효소 (glucose oxidase) 및 과산화효소 모방 효소 (peroxidase mimetic)를 포함하는 금속-유기골격체 (metal organic framework)를 포함하는 나노구조체와 이를 포함하는 암 예방 또는 치료용 약학적 조성물 등에 관한 것이다. 상기 나노구조체는 포도당의 공급을 차단하면서 활성산소종을 생성함으로써 정상 세포에서는 매우 안정하면서도 암세포를 특이적으로 사멸할 수 있으며, 종양 미세 환경 (TME)이 가지는 특이적인 조건 (과량의 포도당, 약산성 환경) 하에 촉매 반응이 개시된다는 특징을 가지므로, 레이저, 초음파, 전자기, 방사선 등의 외부 자극 없이도 연속 반응을 통한 암세포의 사멸 유도가 가능하다. 또한, 상기 나노구조체는 유기 합성 과정이나 장시간의 추출 공정을 필요로 하지 않고 물을 용매로 하여 제조할 수 있으므로, 이를 포함하는 암 예방 또는 치료용 약학적 조성물을 효율적, 경제적으로 제공할 수 있다.The present invention relates to a nanostructure containing a metal organic framework containing glucose oxidase and peroxidase mimetic, and a pharmaceutical composition containing the same for preventing or treating cancer. It's about. The nanostructure is very stable in normal cells and can specifically kill cancer cells by blocking the supply of glucose and generating reactive oxygen species, and is capable of specifically killing cancer cells under the specific conditions of the tumor microenvironment (TME) (excessive glucose, weakly acidic environment). ), it is possible to induce death of cancer cells through a continuous reaction without external stimulation such as laser, ultrasound, electromagnetic, or radiation. In addition, since the nanostructure can be manufactured using water as a solvent without requiring an organic synthesis process or a long-time extraction process, a pharmaceutical composition for preventing or treating cancer containing the nanostructure can be provided efficiently and economically.
Description
본 발명은 금속-유기골격체, 포도당 산화효소, 및 과산화효소 모방 효소를 포함하는 나노구조체, 및 이를 포함하는 암 예방 또는 치료용 조성물 등에 관한 것이다.The present invention relates to a nanostructure containing a metal-organic framework, glucose oxidase, and peroxidase mimicking enzyme, and a composition for preventing or treating cancer containing the same.
암은 국내 사망원인 1위, 전 세계 사망원인 2위에 달하는 질병으로, 암을 치료하기 위한 일반적인 방법으로 크게 외과적 수술, 방사선 치료, 항암 화학요법 등이 행해지고 있다. 하지만 기존의 단일 항암 치료 요법은 심각한 약물 부작용 우려에도 불구하고 항암 효능이 상대적으로 낮다는 단점이 있어 왔다. 때문에 두 가지 혹은 그 이상의 치료법을 결합시켜 다수의 단점을 극복하고 안전성 및 효능 면에서 우월한 복합 요법 (multimodal therapy)을 개발하여 여러 치료적 접근법 간에 시너지 효과를 내도록 하는 연구가 계속되고 있다. Cancer is the leading cause of death in Korea and the second leading cause of death worldwide. Common methods for treating cancer include surgery, radiation therapy, and chemotherapy. However, existing single anti-cancer treatment regimens have the disadvantage of relatively low anti-cancer efficacy, despite concerns about serious drug side effects. Therefore, research is continuing to combine two or more treatments to develop a multimodal therapy that overcomes many shortcomings and is superior in terms of safety and efficacy, thereby creating a synergy effect among various therapeutic approaches.
그 중 하나로, 특정 물질의 처리를 통해 암세포 내부에서 해로운 활성산소종 (ROS)를 유발하고 세포 내부의 불균형을 초래하여 결국에는 사멸에 이르게 하는 치료법인 화학역학 치료법 (chemodynamic therapy; CDT) 와 포도당 산화효소를 이용한 단식 치료법 (starvation therapy)를 함께 결합시켜 연속 효소/촉매 반응을 통한 시너지 효과를 낼 수 있다.One of them is chemodynamic therapy (CDT), which is a treatment that induces harmful reactive oxygen species (ROS) inside cancer cells through treatment of certain substances, causes imbalance inside the cells, and eventually leads to death, and glucose oxidation. By combining starvation therapy using enzymes, a synergistic effect can be achieved through continuous enzyme/catalyst reactions.
구체적으로, 암세포 내외부에 암세포의 에너지원인 포도당이 많이 분포해 있다는 점과 암세포는 항상 약산성 환경을 유지한다는 특성을 이용하여, 포도당 산화효소를 통해 포도당과 산소를 소비함으로써 암세포의 에너지 공급을 차단함으로써 사멸시킬 수 있다.Specifically, taking advantage of the fact that a large amount of glucose, the energy source of cancer cells, is distributed inside and outside cancer cells and the characteristic that cancer cells always maintain a slightly acidic environment, they consume glucose and oxygen through glucose oxidase, thereby blocking the energy supply of cancer cells and killing them. You can do it.
이처럼 펜톤 반응 (fenton reaction), 저산소증 활성 치료법, pH-반응성 약물 방출 치료법 등의 화학역학 치료법을 단식 치료법과 결합시킨 치료법들이 많은 관심을 끌어오고 있지만, 나노 물질의 독성, 부작용, 유효성에 관한 우려 등 고려해야 할 사항은 여전히 많기 때문에, 암 치료를 목적으로 하는 나노 물질의 도입에는 아직까지 한계가 있는 실정이다.In this way, treatments that combine fasting treatment with chemokinetic treatments such as the Fenton reaction, hypoxia activation therapy, and pH-responsive drug release therapy are attracting a lot of attention, but there are concerns about the toxicity, side effects, and effectiveness of nanomaterials. Because there are still many things to consider, there are still limits to the introduction of nanomaterials for cancer treatment.
이에, 본 발명자들은 과산화효소 모방 효소 (peroxidase mimetic)와 포도당 산화효소 (glucose oxidase; GOx)를 각각 화학역학 치료법과 단식 치료법의 제제로 활용하고, 이를 아연 (Zn) 기반의 금속-유기골격체 (metal organic framework; MOF)로 포집하여 나노구조체를 제조하였으며, 상기 나노구조체를 항암 치료에 응용함으로써 본 발명을 완성하였다.Accordingly, the present inventors used peroxidase mimetic and glucose oxidase (GOx) as agents for chemodynamic therapy and fasting therapy, respectively, and used them as agents for zinc (Zn)-based metal-organic framework ( A nanostructure was manufactured by encapsulating it in a metal organic framework (MOF), and the present invention was completed by applying the nanostructure to anticancer treatment.
본 발명이 이루고자 하는 기술적 과제는 포도당 산화효소 (glucose oxidase) 및 과산화효소 모방 효소 (peroxidase mimetic)를 포함하는 금속-유기골격체 (metal organic framework)를 포함하는 나노구조체를 제공하는 것이다.The technical problem to be achieved by the present invention is to provide a nanostructure containing a metal organic framework containing glucose oxidase and peroxidase mimetic.
본 발명의 다른 기술적 과제는 상기 나노구조체를 포함하는 암 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another technical object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer containing the nanostructure.
본 발명의 또 다른 기술적 과제는 상기 나노구조체의 제조방법을 제공하는 것이다.Another technical problem of the present invention is to provide a method for manufacturing the nanostructure.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당해 기술분야의 통상의 기술자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.
상기 과제를 해결하기 위하여, 본 발명은 포도당 산화효소 (glucose oxidase; GOx) 및 과산화효소 모방 효소 (peroxidase mimetic)를 포함하는 금속-유기골격체 (metal organic framework; MOF)를 포함하는 나노구조체를 제공한다.In order to solve the above problems, the present invention provides a nanostructure containing a metal organic framework (MOF) containing glucose oxidase (GOx) and peroxidase mimetic. do.
본 발명의 일 구현예로서, 상기 과산화효소 모방 효소는 프루시안 블루 나노효소 (Prussian blue nanoparticle; PB)일 수 있다.In one embodiment of the present invention, the peroxidase mimicking enzyme may be Prussian blue nanoparticle (PB).
본 발명의 다른 구현예로서, 상기 금속-유기골격체는 제올라이트형 이미다졸레이트 골격체 (zeolitic imidazole framework; ZIF)일 수 있다.As another embodiment of the present invention, the metal-organic framework may be a zeolitic imidazolate framework (ZIF).
본 발명의 또 다른 구현예로서, 상기 나노구조체는 포도당의 공급을 차단하면서 활성산소종 (reactive oxygen species)을 생성하여 암세포를 특이적으로 사멸시키는 것일 수 있다.As another embodiment of the present invention, the nanostructure may specifically kill cancer cells by blocking the supply of glucose and generating reactive oxygen species.
본 발명의 또 다른 구현예로서, 상기 나노구조체는 pH 3 내지 7에서 분해되는 것일 수 있으며, 바람직하게는 pH 5 내지 6에서 분해되는 것일 수 있다.As another embodiment of the present invention, the nanostructure may be decomposed at pH 3 to 7, and preferably may be decomposed at pH 5 to 6.
본 발명의 또 다른 구현예로서, 상기 나노구조체의 크기는 200 내지 400 nm일 수 있다.As another embodiment of the present invention, the size of the nanostructure may be 200 to 400 nm.
본 발명의 또 다른 구현예로서, 상기 나노구조체의 제타 전위는 -10 내지 -30 mV일 수 있다.As another embodiment of the present invention, the zeta potential of the nanostructure may be -10 to -30 mV.
또한, 본 발명은 상기 나노구조체를 유효성분으로 포함하는 암 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer containing the nanostructure as an active ingredient.
또한 본 발명은 암 예방 또는 치료용 약제 제조를 위한 상기 나노구조체의 용도를 제공한다.The present invention also provides the use of the nanostructure for manufacturing drugs for preventing or treating cancer.
또한, 본 발명은 상기 나노구조체를 개체에 투여하는 단계를 포함하는 암 예방 또는 치료방법을 제공한다.Additionally, the present invention provides a method for preventing or treating cancer comprising administering the nanostructure to an individual.
본 발명의 일 구현예로서, 상기 암은 피부암, 유방암, 자궁암, 식도암, 위암, 뇌 종양, 결장암, 직장암, 대장암, 폐암, 난소암, 자궁경부암, 자궁내막암, 외음부암, 신장암, 혈액암, 췌장암, 전립선암, 고환암, 후두암, 두경부암, 갑상선암, 간암, 방광암, 골육종, 림프종, 백혈병, 흉선암, 요도암, 및 기관지암으로 구성된 군으로부터 선택된 어느 하나 이상일 수 있다.In one embodiment of the present invention, the cancer includes skin cancer, breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain tumor, colon cancer, rectal cancer, colon cancer, lung cancer, ovarian cancer, cervical cancer, endometrial cancer, vulvar cancer, kidney cancer, and blood. It may be any one or more selected from the group consisting of cancer, pancreatic cancer, prostate cancer, testicular cancer, laryngeal cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, leukemia, thymic cancer, urethral cancer, and bronchial cancer.
또한, 본 발명은 하기 단계를 포함하는 상기 나노구조체의 제조방법을 제공한다.Additionally, the present invention provides a method for manufacturing the nanostructure comprising the following steps.
(1) 질산아연 헥사수화물 (zinc nitrate hexahydrate)에 포도당 산화효소 (glucose oxidase) 및 과산화효소 모방 효소 (peroxidase mimetic)를 첨가하는 단계;(1) adding glucose oxidase and peroxidase mimetic to zinc nitrate hexahydrate;
(2) 상기 (1) 단계에서 생성된 용액에 2-메틸이미다졸 (2-methylimidazole)을 첨가하여 반응시키는 단계; 및(2) adding 2-methylimidazole to the solution produced in step (1) and reacting it; and
(3) 상기 포도당 산화효소 및 과산화효소 모방 효소를 포함하는 금속-유기골격체 (metal organic framework)를 포함하는 나노구조체를 제조하는 단계.(3) manufacturing a nanostructure containing a metal organic framework containing the glucose oxidase and peroxidase mimic enzymes.
본 발명의 일 구현예로서, 상기 과산화효소 모방 효소는 프루시안 블루 나노효소 (Prussian blue nanoparticle)일 수 있다.In one embodiment of the present invention, the peroxidase mimicking enzyme may be Prussian blue nanoparticle.
본 발명의 다른 구현예로서, 상기 프루시안 블루 나노효소는 FeCl3·6H2O와 citric acid가 담긴 수용액 및 K4[Fe(CN)6]와 citric acid가 담긴 수용액을 반응시키고, 아세톤을 추가로 첨가한 후, 원심분리를 진행하여 제조된 것일 수 있다.As another embodiment of the present invention, the Prussian blue nanoenzyme reacts an aqueous solution containing FeCl 3 ·6H 2 O and citric acid and an aqueous solution containing K 4 [Fe(CN) 6 ] and citric acid, and adds acetone. It may be manufactured by centrifugation after addition.
본 발명의 또 다른 구현예로서, 상기 나노구조체는 나노구조체 100 중량부 기준으로, 포도당 산화효소 0.01 내지 0.1 중량부와 과산화효소 모방 효소 0.001 내지 0.01 중량부를 포함하는 것일 수 있다.As another embodiment of the present invention, the nanostructure may include 0.01 to 0.1 parts by weight of glucose oxidase and 0.001 to 0.01 parts by weight of peroxidase mimetic enzyme, based on 100 parts by weight of the nanostructure.
본 발명의 또 다른 구현예로서, 상기 (1) 단계에서 질산아연 헥사수화물의 농도는 10 내지 50 mM, 바람직하게는 40 mM일 수 있고, 상기 (2) 단계에서 2-메틸이미다졸의 농도는 2.5 내지 5 M, 바람직하게는 2.8 M일 수 있다.As another embodiment of the present invention, the concentration of zinc nitrate hexahydrate in step (1) may be 10 to 50 mM, preferably 40 mM, and the concentration of 2-methylimidazole in step (2) may be 2.5 to 5 M, preferably 2.8 M.
본 발명의 또 다른 구현예로서, 상기 (2) 단계는 상온에서 1 내지 10 분 동안 반응시키는 것일 수 있다.As another embodiment of the present invention, step (2) may be performed at room temperature for 1 to 10 minutes.
본 발명은 포도당 산화효소 (glucose oxidase) 및 과산화효소 모방 효소 (peroxidase mimetic)를 포함하는 금속-유기골격체 (metal organic framework)를 포함하는 나노구조체와 이를 포함하는 암 예방 또는 치료용 약학적 조성물 등에 관한 것이다. 상기 나노구조체는 포도당의 공급을 차단하면서 활성산소종을 생성함으로써 정상 세포에서는 매우 안정하면서도 암세포를 특이적으로 사멸할 수 있으며, 종양 미세 환경 (TME)이 가지는 특이적인 조건 (과량의 포도당, 약산성 환경) 하에 촉매 반응이 개시된다는 특징을 가지므로, 레이저, 초음파, 전자기, 방사선 등의 외부 자극 없이도 연속 반응을 통한 암세포의 사멸 유도가 가능하다. 또한, 상기 나노구조체는 유기 합성 과정이나 장시간의 추출 공정을 필요로 하지 않고 물을 용매로 하여 제조할 수 있으므로, 이를 포함하는 암 예방 또는 치료용 약학적 조성물을 효율적, 경제적으로 제공할 수 있다.The present invention relates to a nanostructure containing a metal organic framework containing glucose oxidase and peroxidase mimetic, and a pharmaceutical composition containing the same for preventing or treating cancer. It's about. The nanostructure is very stable in normal cells and can specifically kill cancer cells by blocking the supply of glucose and generating reactive oxygen species, and is capable of specifically killing cancer cells under the specific conditions of the tumor microenvironment (TME) (excessive glucose, weakly acidic environment). ), it is possible to induce death of cancer cells through a continuous reaction without external stimulation such as laser, ultrasound, electromagnetic, or radiation. In addition, since the nanostructure can be manufactured using water as a solvent without requiring an organic synthesis process or a long-time extraction process, a pharmaceutical composition for preventing or treating cancer containing the nanostructure can be provided efficiently and economically.
도 1A는 프루시안 블루 나노효소 (PB)의 합성 과정, 및 상기 합성된 프루시안 블루 나노효소와 포도당 산화효소를 동시에 포집하는 금속-유기골격체를 포함하는 나노구조체 (ZIF@GOx@PB)를 제조하는 과정을 나타낸 모식도이다. 도 1B는 암세포 환경에서 포도당 산화효소와 프루시안 블루 나노효소가 점차적으로 방출되는 과정의 메커니즘을 나타낸 모식도이다.
도 2는 균일한 크기로 합성된 ZIF-8 입자의 (A), (B) 주사 전자 현미경 (SEM) 사진과 (C) 투과 전자 현미경 (TEM) 사진을 나타낸 것이다.
도 3은 최적화 조건이 아닌 경우 합성된 ZIF-8 입자의 주사 전자 현미경 (SEM) 사진으로, 왼쪽은 아세트산 아연과 2-메틸이미다졸을 상온에서 5 분간 반응시켜 제조한 것이고, 오른쪽은 질산 아연과 2-메틸이미다졸을 80℃에서 5 분간 반응시켜 제조한 것이다.
도 4는 다양한 농도의 포도당 산화효소 (GOx)와 프루시안 블루 나노효소 (PB)를 이용하여 합성한 ZIF@GOx@PB 나노구조체의 크기를 나타내는 DLS 측정 결과를 나타낸 것이다.
도 5는 다양한 농도의 포도당 산화효소 (GOx)와 프루시안 블루 나노효소 (PB)를 이용하여 합성한 ZIF@GOx@PB 나노구조체의 촉매 활성 결과를 나타낸 것이다.
도 6은 본 발명의 나노구조체 제조를 위해 필요한 금속 및 유기물의 농도와 부피, 합성 조건과 과정을 나타낸 모식도이다.
도 7은 제조예 1에서 합성한 4종의 나노구조체 (ZIF-8, ZIF@GOx, ZIF@PB, 및 ZIF@GOx@PB)의 주사 전자 현미경 (SEM) 사진을 나타낸 것이다.
도 8은 제조예 1에서 합성한 4종의 나노구조체 (ZIF-8, ZIF@GOx, ZIF@PB, 및 ZIF@GOx@PB)의 입자 크기를 나타내는 동적 광산란(DLS) 측정 결과를 나타낸 것이다.
도 9는 제조예 1에서 합성한 4종의 나노구조체 (ZIF-8, ZIF@GOx, ZIF@PB, 및 ZIF@GOx@PB)의 표면 전하를 나타내는 제타 전위 (zeta potential) 값을 나타낸 것이다.
도 10은 제조예 1에서 합성한 4종의 나노구조체 (ZIF-8, ZIF@GOx, ZIF@PB, 및 ZIF@GOx@PB)의 열중량분석 (thermogravity analysis) 결과를 나타낸 것이다.
도 11은 프루시안 블루 나노효소와 포도당 산화효소를 포함하는 금속-유기골격체를 포함하는 나노구조체 (ZIF@GOx@PB)의 주사 투과 전자 현미경 (STEM) 사진 및 원소 맵핑 (elemental mapping) 사진을 나타낸 것이다.
도 12는 ZIF@GOx@PB 나노구조체의 pH 7.4, 6.0, 5.0 버퍼에서의 투과 전자 현미경 (TEM) 사진을 나타낸 것이다.
도 13은 다양한 농도의 포도당 수용액 내에 ZIF@GOx@PB 나노구조체를 분산시켰을 때에, 시간에 따른 pH 변화를 측정한 결과이다.
도 14는 다양한 농도의 포도당 수용액 내에 분산된 ZIF@GOx@PB 나노구조체가 시간이 지남에 따라 점차적으로 분해되면서 누적되는 Zn 이온의 방출량을 유도 결합 플라즈마 (ICP) 로 측정한 결과이다.
도 15A는 pH에 따라 프루시안 블루 나노효소에 의해 과산화수소가 환원, TMB가 산화되면서 일으키는 색 변화 (O.D.652 nm)를 UV-Vis 흡광도 스펙트럼으로 측정한 결과이고, 도 15B는 상기 반응에 대한 반응식을 나타낸 것이다.
도 16은 과산화수소의 농도에 따라 프루시안 블루 나노효소에 의해 과산화수소가 환원, TMB가 산화되면서 일으키는 색 변화를 (O.D.652 nm)를 UV-Vis 흡광도 값으로 측정한 결과이다.
도 17A는 ZIF@GOx@PB 나노구조체에 포함된 포도당 산화효소에 의해 포도당이 산화되면서 과산화수소가 발생하고, 프루시안 블루 나노효소에 의해 발생한 과산화수소가 환원, TMB가 산화되면서 일으키는 색 변화 (O.D.652 nm)를 UV-Vis 흡광도 스펙트럼으로 측정한 결과이며, 이 때, ZGP는 ZIF@GOx@PB 나노구조체를 가리킨다. 도 17B는 상기 연속 반응에 대한 반응식을 나타낸다.
도 18은 포도당의 농도에 따라 ZIF@GOx@PB 나노구조체에 포함된 포도당 산화효소에 의해 발생한 과산화수소가 프루시안 블루 나노효소에 의해 환원, TMB가 산화되면서 일으키는 색 변화 (O.D.652 nm)를 시간에 따른 UV-Vis 흡광도 값으로 측정한 결과이다.
도 19는 ZIF@GOx@PB 나노구조체의 포도당의 산화로 인해 개시된 연속 반응의 결과로 생성된 활성산소종 (ROS)을 형광 신호로 측정 가능하도록 정량화한 terephthalic acid (TA) assay 결과를 나타낸 것이다. 이 때, Z는 ZIF-8 나노구조체, ZG는 ZIF@GOx 나노구조체, ZP는 ZIF@PB 나노구조체, ZGP는 ZIF@GOx@PB 나노구조체를 가리킨다.
도 20A는 정상세포인 섬유아세포 (3T3-L1)에, 도 20B는 피부암세포 (SK-MEL28)에 제조예 1에서 합성한 4종의 나노구조체 (ZIF-8, ZIF@GOx, ZIF@PB, 및 ZIF@GOx@PB)를 처리한 경우, 세포 생존율을 나타낸 것이다. 이 때, Z는 ZIF-8 나노구조체, ZG는 ZIF@GOx 나노구조체, ZP는 ZIF@PB 나노구조체, ZGP는 ZIF@GOx@PB 나노구조체를 가리킨다.
도 21은 제조예 1에서 합성한 4종의 나노구조체 (ZIF-8, ZIF@GOx, ZIF@PB, 및 ZIF@GOx@PB)가 피부암세포에 미치는 영향을 생존/사멸세포 염색법 (Live/dead staining)을 통해 정성적으로 나타낸 형광 현미경 사진이다.
도 22는 제조예 1에서 합성한 4종의 나노구조체 (ZIF-8, ZIF@GOx, ZIF@PB, 및 ZIF@GOx@PB)를 피부암세포에 처리한 후, 암세포 내부에 형성된 활성산소종을 DCFH-DA staining을 통해 정성적으로 나타낸 형광 현미경 사진이다.
도 23은 피부암세포에 제조예 1에서 합성한 (i) ZIF@GOx@PB 나노구조체를 처리한 경우 (ii) ZIF@PB 나노구조체와 포도당 산화효소 (GOx)를 병용 투여한 경우, (iii) ZIF@GOx 나노구조체와 프루시안 블루 나노효소 (PB)를 병용 투여한 경우, 세포 생존율을 나타낸 것이다. 이 때, ZG는 ZIF@GOx 나노구조체, ZP는 ZIF@PB 나노구조체, ZGP는 ZIF@GOx@PB 나노구조체를 가리킨다.Figure 1A shows the synthesis process of Prussian blue nanoenzyme (PB), and a nanostructure (ZIF@GOx@PB) containing a metal-organic framework that simultaneously captures the synthesized Prussian blue nanoenzyme and glucose oxidase. This is a schematic diagram showing the manufacturing process. Figure 1B is a schematic diagram showing the mechanism of the process by which glucose oxidase and Prussian blue nanoenzyme are gradually released in the cancer cell environment.
Figure 2 shows (A), (B) scanning electron microscopy (SEM) photographs and (C) transmission electron microscopy (TEM) photographs of ZIF-8 particles synthesized with uniform sizes.
Figure 3 is a scanning electron microscope (SEM) photograph of ZIF-8 particles synthesized under non-optimized conditions. The left side is prepared by reacting zinc acetate and 2-methylimidazole at room temperature for 5 minutes, and the right side is zinc nitrate. It was prepared by reacting 2-methylimidazole at 80°C for 5 minutes.
Figure 4 shows DLS measurement results showing the size of ZIF@GOx@PB nanostructures synthesized using various concentrations of glucose oxidase (GOx) and Prussian blue nanoenzyme (PB).
Figure 5 shows the catalytic activity results of ZIF@GOx@PB nanostructures synthesized using various concentrations of glucose oxidase (GOx) and Prussian blue nanoenzyme (PB).
Figure 6 is a schematic diagram showing the concentration and volume of metals and organic materials, synthesis conditions and processes required for manufacturing the nanostructure of the present invention.
Figure 7 shows scanning electron microscopy (SEM) photographs of four types of nanostructures (ZIF-8, ZIF@GOx, ZIF@PB, and ZIF@GOx@PB) synthesized in Preparation Example 1.
Figure 8 shows dynamic light scattering (DLS) measurement results showing the particle sizes of four types of nanostructures (ZIF-8, ZIF@GOx, ZIF@PB, and ZIF@GOx@PB) synthesized in Preparation Example 1.
Figure 9 shows zeta potential values indicating the surface charge of four types of nanostructures (ZIF-8, ZIF@GOx, ZIF@PB, and ZIF@GOx@PB) synthesized in Preparation Example 1.
Figure 10 shows the thermogravity analysis results of four types of nanostructures (ZIF-8, ZIF@GOx, ZIF@PB, and ZIF@GOx@PB) synthesized in Preparation Example 1.
Figure 11 shows a scanning transmission electron microscope (STEM) photograph and elemental mapping photograph of a nanostructure (ZIF@GOx@PB) containing a metal-organic framework containing Prussian blue nanoenzyme and glucose oxidase. It is shown.
Figure 12 shows transmission electron microscopy (TEM) images of ZIF@GOx@PB nanostructures in buffers of pH 7.4, 6.0, and 5.0.
Figure 13 shows the results of measuring the pH change over time when ZIF@GOx@PB nanostructures were dispersed in aqueous glucose solutions of various concentrations.
Figure 14 shows the results of measuring the release amount of Zn ions accumulated as ZIF@GOx@PB nanostructures dispersed in glucose aqueous solutions of various concentrations gradually decompose over time using inductively coupled plasma (ICP).
Figure 15A shows the results of measuring the color change (OD652 nm) caused by reduction of hydrogen peroxide and oxidation of TMB by Prussian blue nanoenzyme depending on pH using a UV-Vis absorbance spectrum, and Figure 15B shows the reaction formula for the reaction. will be.
Figure 16 shows the results of measuring the color change (OD652 nm) caused by reduction of hydrogen peroxide and oxidation of TMB by Prussian blue nanoenzyme according to the concentration of hydrogen peroxide as a UV-Vis absorbance value.
Figure 17A shows the color change (OD652 nm) that occurs when glucose is oxidized by the glucose oxidase contained in the ZIF@GOx@PB nanostructure, generating hydrogen peroxide, hydrogen peroxide generated by the Prussian blue nanoenzyme is reduced, and TMB is oxidized. This is the result measured by UV-Vis absorbance spectrum, where ZGP refers to the ZIF@GOx@PB nanostructure. Figure 17B shows the reaction scheme for the above continuous reaction.
Figure 18 shows the color change (OD652 nm) over time caused when hydrogen peroxide generated by the glucose oxidase contained in the ZIF@GOx@PB nanostructure is reduced by the Prussian blue nanoenzyme and TMB is oxidized depending on the concentration of glucose. This is the result measured by UV-Vis absorbance value.
Figure 19 shows the results of a terephthalic acid (TA) assay in which reactive oxygen species (ROS) generated as a result of a continuous reaction initiated by the oxidation of glucose in the ZIF@GOx@PB nanostructure can be measured as a fluorescence signal. At this time, Z refers to the ZIF-8 nanostructure, ZG refers to the ZIF@GOx nanostructure, ZP refers to the ZIF@PB nanostructure, and ZGP refers to the ZIF@GOx@PB nanostructure.
Figure 20A shows four types of nanostructures synthesized in Preparation Example 1 (ZIF-8, ZIF@GOx, ZIF@PB, and ZIF@GOx@PB), cell viability is shown. At this time, Z refers to the ZIF-8 nanostructure, ZG refers to the ZIF@GOx nanostructure, ZP refers to the ZIF@PB nanostructure, and ZGP refers to the ZIF@GOx@PB nanostructure.
Figure 21 shows the effect of four types of nanostructures (ZIF-8, ZIF@GOx, ZIF@PB, and ZIF@GOx@PB) synthesized in Preparation Example 1 on skin cancer cells using live/dead cell staining. This is a fluorescence micrograph qualitatively shown through staining.
Figure 22 shows the treatment of four types of nanostructures (ZIF-8, ZIF@GOx, ZIF@PB, and ZIF@GOx@PB) synthesized in Preparation Example 1 to skin cancer cells, and the reactive oxygen species formed inside the cancer cells. This is a fluorescence micrograph qualitatively shown through DCFH-DA staining.
Figure 23 shows skin cancer cells treated with (i) the ZIF@GOx@PB nanostructure synthesized in Preparation Example 1, (ii) when the ZIF@PB nanostructure and glucose oxidase (GOx) were administered in combination, (iii) This shows the cell survival rate when ZIF@GOx nanostructure and Prussian blue nanoenzyme (PB) were administered together. At this time, ZG refers to ZIF@GOx nanostructure, ZP refers to ZIF@PB nanostructure, and ZGP refers to ZIF@GOx@PB nanostructure.
본 발명자들은 과산화효소 모방 효소 중 프루시안 블루 나노효소 (prussian blue nanoparticle; PB)와 포도당 산화 효소 (glucose oxidase; GOx)를 함께 포집하는 금속-유기 골격체 (MOF)의 일종인 제올라이트형 이미다졸레이트 골격체 (ZIF-8)를 포함하는 나노구조체를 개발하였으며, 상기 나노구조체가 상기 나노구조체가 프루시안 블루 나노효소에 의한 화학역학 치료법과 포도당 산화 효소에 의한 단식 치료법의 시너지 효과로 암세포 내에서 pH 감소에 따라 붕괴되고, 일련의 연속적인 효소 반응을 통해 암세포를 사멸한다는 점을 확인하였다.The present inventors developed a zeolite-type imidazolate, a type of metal-organic framework (MOF) that captures Prussian blue nanoparticle (PB) and glucose oxidase (GOx), among peroxidase mimicking enzymes. We developed a nanostructure containing a scaffold (ZIF-8), and the nanostructure is a synergistic effect of chemokinetic therapy using Prussian blue nanoenzyme and fasting therapy using glucose oxidase, thereby reducing pH within cancer cells. It was confirmed that it collapses as it decreases and kills cancer cells through a series of sequential enzymatic reactions.
보다 구체적으로, 상기 나노구조체는 금속-유기 골격체를 포함하여 정상 세포에서 프루시안 블루 나노효소와 포도당 산화 효소를 안정적으로 저장하고 전달할 수 있으며, 생체 적합성을 향상시킬 수 있다. 한편, 상기 나노구조체는 암세포에서 포도당 산화 효소가 암세포의 에너지원인 포도당의 공급을 차단하면서, 글루콘산 (gluconic acid)과 과산화수소 (H2O2)를 생성하고, 글루콘산은 pH를 감소시키고, 과산화수소는 프루시안 블루 나노효소에 의해 암세포 내부에 해로운 활성산소를 생성함으로써 암세포에 대해서 특이적으로 독성을 나타낸다.More specifically, the nanostructure includes a metal-organic framework and can stably store and deliver Prussian blue nanoenzyme and glucose oxidase in normal cells and improve biocompatibility. Meanwhile, the nanostructure generates gluconic acid and hydrogen peroxide (H 2 O 2 ) while blocking the supply of glucose, the energy source of cancer cells, to glucose oxidase enzyme in cancer cells, and gluconic acid reduces pH and hydrogen peroxide is specifically toxic to cancer cells by generating harmful oxygen radicals inside cancer cells through Prussian blue nanoenzyme.
이에, 본 발명은 포도당 산화효소 및 과산화효소 모방 효소를 포함하는 금속-유기골격체를 포함하는 나노구조체를 제공한다.Accordingly, the present invention provides a nanostructure containing a metal-organic framework containing glucose oxidase and peroxidase mimicking enzymes.
본 발명에서, 용어 “포도당 산화효소 (glucose oxidase; GOx)”는 포도당을 글루콘산으로 산화시키는 효소를 의미하며, "프루시안 블루 나노효소 (Prussian blue nanoparticle; PB)”는 철 기반의 나노물질로 산성 조건에서 활성산소종 (ROS)를 효과적으로 유발하는 과산화효소 (peroxidase) 활성을 가진 효소를 의미한다.In the present invention, the term “glucose oxidase (GOx)” refers to an enzyme that oxidizes glucose to gluconic acid, and “Prussian blue nanoparticle (PB)” is an iron-based nanomaterial. It refers to an enzyme with peroxidase activity that effectively induces reactive oxygen species (ROS) under acidic conditions.
본 발명에서, 용어 “나노구조체”는 적어도 하나의 영역 또는 특성 치수(예컨대, 높이, 길이, 두께, 직경 등)가 1 μm 미만인 구조체를 의미하고, 일부 실시형태들에 있어서, 나노구조체는 약 500 nm 미만, 약 200 nm 미만, 약 100 nm 미만, 약 50 nm 미만, 약 20 nm 미만, 또는 약 10 nm 미만의 치수를 갖는다. 통상적으로, 영역 또는 특성 치수는 구조체의 가장 작은 축을 따라 있을 것이다. 그러한 구조체들의 예들은, 나노와이어들, 나노로드들, 나노튜브들, 분기된 나노구조체들, 나노테트라포드들, 트리포드들, 바이포드들, 나노결정들, 나노도트들, QD들, 나노입자들 등을 포함한다. 나노구조체들은, 예를 들면, 실질적으로 결정질, 실질적으로 단결정질, 다결정질, 비정질, 또는 이들의 조합일 수 있다.As used herein, the term “nanostructure” refers to a structure in which at least one area or characteristic dimension (e.g., height, length, thickness, diameter, etc.) is less than 1 μm, and in some embodiments, a nanostructure is about 500 μm. and has dimensions of less than about 200 nm, less than about 100 nm, less than about 50 nm, less than about 20 nm, or less than about 10 nm. Typically, the area or characteristic dimension will be along the smallest axis of the structure. Examples of such structures include nanowires, nanorods, nanotubes, branched nanostructures, nanotetrapods, tripods, bipods, nanocrystals, nanodots, QDs, nanoparticles. Includes etc. Nanostructures can be, for example, substantially crystalline, substantially monocrystalline, polycrystalline, amorphous, or combinations thereof.
또한, 본 발명은 상기 나노구조체를 유효성분으로 포함하는 암 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer containing the nanostructure as an active ingredient.
본 발명에서, 용어 "예방"은 본 발명의 조성물의 투여로 암의 발생, 확산 또는 재발을 억제시키거나 지연시키는 모든 행위를 의미하고, "치료"는 본 발명의 조성물의 투여로 암의 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.In the present invention, the term "prevention" refers to any action that inhibits or delays the occurrence, spread, or recurrence of cancer by administration of the composition of the present invention, and "treatment" refers to the prevention of symptoms of cancer by administration of the composition of the present invention. It refers to any action that improves or changes to advantage.
본 발명에서, 용어 "유효성분으로 포함"은 원하는 생물학적 효과를 실현하는데 필요하거나 또는 충분한 양으로 해당 성분이 포함되는 것을 의미한다. 실제 적용에 있어서 유효 성분으로 포함되는 양의 결정은 대상 질병을 치료하기 위한 양으로서, 다른 독성을 야기하지 않는 사항을 고려해서 결정될 수 있으며, 예를 들어 치료되는 질병 또는 병태, 투여되는 조성물의 형태, 피험체의 크기, 또는 질병 또는 병태의 심각도 등과 같은 다양한 인자에 따라서 변화될 수 있다. 본 발명이 속하는 분야에서 통상의 기술을 지닌 기술자라면 과도한 실험을 동반하지 않고 개별적 조성물의 유효량을 경험적으로 결정할 수 있다.In the present invention, the term “included as an active ingredient” means that the ingredient is included in an amount necessary or sufficient to achieve the desired biological effect. In actual application, the amount to be included as an active ingredient can be determined by considering the amount to treat the target disease and not causing other toxicity, such as the disease or condition being treated, the form of the composition to be administered, It may vary depending on various factors such as the size of the subject, or the severity of the disease or condition. A person skilled in the art can empirically determine the effective amount of an individual composition without undue experimentation.
본 발명에서, 용어 "약학적 조성물 (pharmaceutical composition)"은 질병의 예방 또는 치료를 목적으로 제조된 것을 의미하며, 약학적 조성물의 제조에 통상적으로 사용되는 적절한 담체, 부형제, 및 희석제를 더 포함할 수 있다. In the present invention, the term "pharmaceutical composition" means prepared for the purpose of preventing or treating diseases, and may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. You can.
본 발명에서, 용어 “개체”는 유방암의 예방 또는 치료가 필요한 가축, 인간 등의 포유류라면 제한되지 아니하나, 바람직하게는 인간일 수 있다. 본 발명의 약학적 조성물은 개체에 투여하기 위해 통상의 방법에 따라 다양한 형태로 제형화하여 사용될 수 있으며 예컨대, 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽 등의 경구형 제형으로 제형화할 수 있고, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.In the present invention, the term “individual” is not limited to any mammal such as livestock or human that requires prevention or treatment of breast cancer, but is preferably a human. The pharmaceutical composition of the present invention can be formulated and used in various forms according to conventional methods for administration to an individual. For example, it can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc. It can be formulated and used in the form of external preparations, suppositories, and sterile injectable solutions.
본 발명에서, "담체 (carrier)"란 비이클 (vehicle)이라고도 불리며, 세포 또는 조직 내로의 단백질 또는 펩타이드의 부가를 용이하게 하는 화합물을 의미하는 것으로서, 예를 들어 디메틸술폭사이드 (DMSO)는 생물체의 세포 또는 조직 내로의 많은 유기물의 투입을 용이하게 하는 통상 사용되는 담체이다. 상기 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로오스 용액, 말토덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 하나 이상의 혼합물일 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 더 포함할 수도 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수도 있다. 더 나아가, 당분야의 적정한 방법으로, 또는 Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수도 있다.In the present invention, “carrier” is also called a vehicle and refers to a compound that facilitates the addition of a protein or peptide into a cell or tissue. For example, dimethyl sulfoxide (DMSO) is used to It is a commonly used carrier that facilitates the introduction of many organic substances into cells or tissues. The carrier may be saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these ingredients, and if necessary, other common additives such as antioxidants, buffers, and bacteriostatic agents. It may also include more. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Furthermore, it may be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA).
본 발명에서, "희석제 (diluent)"란 대상 단백질 또는 펩타이드의 생물학적 활성 형태를 안정화시킬 뿐만 아니라, 단백질 또는 펩타이드를 용해시키게 되는 물에서 희석되는 화합물로 정의된다. 버퍼 용액에 용해되어 있는 염은 당해 분야에서 희석제로 사용된다. 통상 사용되는 버퍼 용액은 포스페이트 버퍼 식염수이며, 이는 인간 용액의 염 상태를 모방하고 있기 때문이다. 버퍼 염은 낮은 농도에서 용액의 pH를 제어할 수 있기 때문에, 버퍼 희석제가 화합물의 생물학적 활성을 변형하는 일은 드물다. 여기에 사용된 아젤라산을 함유하는 화합물들은 인간 환자에게 그 자체로서, 또는 결합 요법에서와 같이 다른 성분들과 함께 또는 적당한 담체나 부형제와 함께 혼합된 약학적 조성물로서, 투여될 수 있다.In the present invention, “diluent” is defined as a compound diluted in water that not only stabilizes the biologically active form of the protein or peptide of interest, but also dissolves the protein or peptide. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline because it mimics the salt conditions of human solutions. Because buffer salts can control the pH of a solution at low concentrations, it is rare for buffer diluents to modify the biological activity of the compound. The compounds containing azelaic acid as used herein can be administered to human patients as such, or as pharmaceutical compositions mixed with other ingredients, such as in combination therapy, or with suitable carriers or excipients.
본 발명의 약학적 조성물은 경구 또는 비경구, 바람직하게는 비경구로 투여할 수 있고, 비경구 투여인 경우에는 근육 주입, 정맥내 주입, 피하 주입, 복강 주입, 국소 투여, 경피 투여 등으로 투여할 수 있다.The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenterally, and in case of parenteral administration, it can be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc. You can.
본 발명의 조성물은 목적하는 방법에 따라 약학적으로 유효한 양으로 경구 투여하거나 비경구 투여할 수 있으며, 본 발명의 용어 “약학적으로 유효한 양”은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미하며, 유효용량 수준은 환자의 건강상태, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.The composition of the present invention can be administered orally or parenterally in a pharmaceutically effective amount according to the desired method, and the term “pharmaceutically effective amount” in the present invention refers to a disease with a reasonable benefit/risk ratio applicable to medical treatment. It refers to an amount that is sufficient to treat and does not cause side effects. The effective dose level refers to the patient's health status, severity, drug activity, sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment. It may be determined based on factors including duration, combination or drugs used simultaneously, and other factors well known in the medical field.
또한, 본 발명에 따른 약학적 조성물은 본 발명의 효과를 보이는 한 그 제형, 투여 경로 및 투여 방법에 특별히 제한되지는 않으며, 본 발명의 약학적 조성물은 본 발명의 나노구조체 이외에 공지된 항암제를 추가로 포함할 수 있고, 이들 질환의 치료를 위해 공지된 다른 치료와 병용될 수 있다.In addition, the pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention, and the pharmaceutical composition of the present invention adds known anticancer agents in addition to the nanostructure of the present invention. and may be used in combination with other known treatments for the treatment of these diseases.
실시예에서 사용한 용어는 단지 설명을 목적으로 사용된 것으로, 한정하려는 의도로 해석되어서는 안 된다. 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 in the examples are 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 “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.
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 otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the embodiments belong. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless explicitly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.
이하에서, 첨부된 도면을 참조하여 실시예들을 상세하게 설명한다. 그러나, 실시예들에는 다양한 변경이 가해질 수 있어서 특허출원의 권리 범위가 이러한 실시예들에 의해 제한되거나 한정되는 것은 아니다. 실시예들에 대한 모든 변경, 균등물 내지 대체물이 권리 범위에 포함되는 것으로 이해되어야 한다.Hereinafter, embodiments will be described in detail with reference to the attached drawings. However, various changes can be made to the embodiments, so the scope of the patent application is not limited or limited by these embodiments. It should be understood that all changes, equivalents, or substitutes for the embodiments are included in the scope of rights.
또한, 첨부 도면을 참조하여 설명함에 있어, 도면 부호에 관계없이 동일한 구성 요소는 동일한 참조부호를 부여하고 이에 대한 중복되는 설명은 생략하기로 한다. 실시예를 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 실시예의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.In addition, when describing with reference to the accompanying drawings, identical components will be assigned the same reference numerals regardless of the reference numerals, and overlapping descriptions thereof will be omitted. In describing the embodiments, if it is determined that detailed descriptions of related known technologies may unnecessarily obscure the gist of the embodiments, the detailed descriptions are omitted.
제조예 1. 프루시안 블루 나노효소와 포도당 산화효소를 포집하는 금속-유기골격체를 포함하는 나노구조체 (ZIF@GOx@PB)의 제조Preparation Example 1. Preparation of a nanostructure (ZIF@GOx@PB) containing a metal-organic framework that captures Prussian blue nanoenzyme and glucose oxidase.
1.1.1.1. 프루시안 블루 나노효소 (PB) 합성Prussian blue nanoenzyme (PB) synthesis
먼저, 10 mM의 FeCl3·6H2O와 5 mmol의 citric acid가 담긴 (1) 수용액 20 mL와 10 mM의 K4[Fe(CN)6]와 5 mmol의 citric acid가 담긴 (2) 수용액 20 mL를 60℃로 충분히 가열하였다. (1) 수용액이 담긴 플라스크에 (2) 수용액을 한꺼번에 가하여 1 분을 반응시킨 후, 5 분간 상온에서 냉각하였다. 동량의 아세톤을 가하여 침전시킨 후, 13000 rpm에 25 분간 넉넉히 원심분리를 진행하였다. 상층액은 버리고 에탄올, 물을 차례로 가하여 2번 더 세척을 진행하였고, 마지막 단계에서 일정량의 3차 증류수를 가한 후 충분한 초음파 세척기 처리를 통하여 합성된 프루시안 블루 나노효소 (PB)를 고르게 분포시켰다(도 1A). 합성된 PB의 농도는 유도 결합 플라즈마 (ICP) 분석을 통해 측정하였다.First, 20 mL of (1) aqueous solution containing 10 mM FeCl 3 ·6H 2 O and 5 mmol citric acid and (2) aqueous solution containing 10 mM K 4 [Fe(CN) 6 ] and 5 mmol citric acid. 20 mL was sufficiently heated to 60°C. (1) The aqueous solution (2) was added all at once to the flask containing the aqueous solution, reacted for 1 minute, and then cooled at room temperature for 5 minutes. After adding the same amount of acetone to precipitate, centrifugation was carried out at 13000 rpm for 25 minutes. The supernatant was discarded and washed twice more by adding ethanol and water sequentially. In the final step, a certain amount of tertiary distilled water was added and the synthesized Prussian blue nanoenzyme (PB) was evenly distributed through sufficient ultrasonic washing ( Figure 1A). The concentration of synthesized PB was measured through inductively coupled plasma (ICP) analysis.
1.2.1.2. 금속-유기골격체로서 제올라이트형 이미다졸레이트 골격체 (ZIF-8) 합성Synthesis of zeolite-type imidazolate framework (ZIF-8) as a metal-organic framework
2-메틸이미다졸 500 (2-methylimidazole 500)과 질산아연 헥사수화물 500 (zinc nitrate hexahydrate 500)을 이용하여 금속-유기골격체 중 하나인 제올라이트형 이미다졸레이트 골격체 (ZIF-8)을 제조하였다.Zeolite-type imidazolate framework (ZIF-8), one of the metal-organic frameworks, was prepared using 2-methylimidazole 500 and zinc nitrate hexahydrate 500. did.
하기 표 1과 같이 금속의 종류와 농도, 유기체의 종류와 농도, 합성 온도, 합성 시간에 대한 최적화를 진행하였다.As shown in Table 1 below, optimization was performed on the type and concentration of metal, type and concentration of organism, synthesis temperature, and synthesis time.
(2-Mim.)2-Methylimidazole
(2-Mim.)
그 결과, 주사 전자 현미경 (SEM) 및 투과 전자 현미경 (TEM) 이미징을 통해, 1:70의 몰 비율로 맞춰진 질산아연 40 mM과 2-메틸이미다졸 2.8 mM을 상온 조건에서 5 분간 반응시킬 때에, 가장 균일하고 작은 사이즈의 안정적인 ZIF-8 입자가 합성된다는 점을 확인하였다 (도 2).As a result, through scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging, when 40 mM zinc nitrate and 2.8 mM 2-methylimidazole at a molar ratio of 1:70 were reacted for 5 minutes at room temperature, , it was confirmed that stable ZIF-8 particles of the most uniform and small size were synthesized (Figure 2).
반면, 아세트산아연 (zinc acetate)와 2-메틸이미다졸을 이용하여 상온에서 5 분간 반응시킨 경우, ZIF-8 입자의 크기가 균일하지 않았으며, 질산아연과 2-메틸이미다졸을 이용하여 80℃에서 20 분간 반응시킨 경우, ZIF-8 입자가 안정적으로 형성되지 않았다.On the other hand, when zinc acetate and 2-methylimidazole were used to react at room temperature for 5 minutes, the size of ZIF-8 particles was not uniform, and zinc nitrate and 2-methylimidazole were used to react at room temperature for 5 minutes. When reacted at 80°C for 20 minutes, ZIF-8 particles were not formed stably.
1.3.1.3. ZIF@GOx@PB 나노구조체 합성을 위한 GOx 및 PB 함량 최적화Optimization of GOx and PB contents for synthesis of ZIF@GOx@PB nanostructures
작고 균일한 크기를 가지는 ZIF@GOx@PB 나노구조체의 효율적인 합성을 위하여 포도당 산화효소 (GOx) 및 프루시안 블루 나노효소 (PB)의 함량을 세분화하여 농도 조건을 최적화하였다.For the efficient synthesis of ZIF@GOx@PB nanostructures with small and uniform sizes, the concentration conditions were optimized by subdividing the contents of glucose oxidase (GOx) and Prussian blue nanoenzyme (PB).
각 농도 조건에서 동적 광산란 (DLS)을 이용하여 크기를 측정한 결과, 포도당 산화효소가 0,5 mg, 프루시안 블루 나노효소가 30 μg인 경우, 제조된 ZIF@GOx@PB 나노구조체의 크기는 298 nm (PDI 0.123)이었으며, 포도당 산화효소가 0,75 mg, 프루시안 블루 나노효소가 30 μg인 경우, 제조된 ZIF@GOx@PB 나노구조체의 크기는 296 nm (PDI 0.129)로 가장 작은 크기가 측정되었다 (도 4).As a result of measuring the size using dynamic light scattering (DLS) at each concentration condition, when glucose oxidase is 0.5 mg and Prussian blue nanoenzyme is 30 μg, the size of the prepared ZIF@GOx@PB nanostructure is It was 298 nm (PDI 0.123), and when glucose oxidase was 0.75 mg and Prussian blue nanoenzyme was 30 μg, the size of the manufactured ZIF@GOx@PB nanostructure was the smallest at 296 nm (PDI 0.129). was measured (Figure 4).
한편, 각 농도 조건에서 포도당 산화효소의 촉매 활성을 평가한 결과, 포도당 산화효소가 0,5 mg, 프루시안 블루 나노효소가 30 μg인 경우, 제조된 ZIF@GOx@PB 나노구조체의 흡광도는 O.D. 652 nm에서 2.212였으며, 포도당 산화효소가 0,75 mg, 프루시안 블루 나노효소가 30 μg인 경우, 제조된 ZIF@GOx@PB 나노구조체의 흡광도는 O.D. 652 nm에서 2.140으로 가장 높은 활성을 나타냈다 (도 5).Meanwhile, as a result of evaluating the catalytic activity of glucose oxidase at each concentration condition, when glucose oxidase was 0,5 mg and Prussian blue nanoenzyme was 30 μg, the absorbance of the prepared ZIF@GOx@PB nanostructure was O.D. It was 2.212 at 652 nm, and when glucose oxidase was 0,75 mg and Prussian blue nanoenzyme was 30 μg, the absorbance of the prepared ZIF@GOx@PB nanostructure was O.D. It showed the highest activity at 2.140 at 652 nm (Figure 5).
즉, 크기가 가장 작고 균일하여 세포 내의 함입 (cell internalization)이 용이하며 촉매 활성 효율이 가장 높게 평가된 0.5 mg의 포도당 산화효소와 30 μg의 프루시안 블루 나노효소를 이용하여 ZIF@GOx@PB 나노구조체 합성하는 것으로 최적화하였다.In other words, the ZIF@GOx@PB nano-enzyme is made by using 0.5 mg of glucose oxidase and 30 μg of Prussian blue nanoenzyme, which are the smallest and most uniform in size and easy for cell internalization and have the highest catalytic activity efficiency. It was optimized by synthesizing the structure.
1.4.1.4. 본 발명의 ZIF@GOx@PB 나노구조체 합성Synthesis of ZIF@GOx@PB nanostructure of the present invention
유효물질로서 제조예 1.1에서 합성한 프루시안 블루 나노효소 (PB)와 포도당 산화효소 (GOx)를 각각 0.002% (30 μg), 0.05% (0.5 mg) 내외 함량으로 40 mM의 질산아연 헥사수화물 500 (zinc nitrate hexahydrate 500) (B 수용액) 500 μL에 첨가하고 초음파 세척기 처리를 한다. 상기 B 수용액을 2.8 M의 2-메틸이미다졸 500 (2-methylimidazole 500) (A 수용액) 500 μL에 첨가하고 균일하게 혼합한 후, magnetic bar를 이용한 stirring 과정을 통해 상온에서 5분간 반응시켰다. 3회의 원심분리와 세척 과정을 거쳐 3차 증류수에 고르게 분포시켰고(도 6), 최종적으로 프루시안 블루 나노효소와 포도당 산화효소를 포함하는 ZIF@GOx@PB 나노구조체를 합성하였다. As active substances, Prussian blue nanoenzyme (PB) and glucose oxidase (GOx) synthesized in Preparation Example 1.1 were added to 40 mM zinc nitrate hexahydrate 500 at a content of approximately 0.002% (30 μg) and 0.05% (0.5 mg), respectively. (Zinc nitrate hexahydrate 500) (B aqueous solution) is added to 500 μL and treated with an ultrasonic cleaner. The B aqueous solution was added to 500 μL of 2.8 M 2-methylimidazole 500 (A aqueous solution), mixed uniformly, and reacted at room temperature for 5 minutes through a stirring process using a magnetic bar. After centrifugation and washing three times, it was evenly distributed in distilled water (Figure 6), and finally, ZIF@GOx@PB nanostructure containing Prussian blue nanoenzyme and glucose oxidase was synthesized.
비교군으로 제조예 1.2에서 최적화하여 합성한 ZIF-8 나노구조체와, 포도당 산화효소를 포집한 ZIF-8 금속-유기골격체를 포함하는 ZIF@GOx 나노구조체, 프루시안 블루 나노효소를 포집한 ZIF-8 금속-유기골격체를 포함하는 ZIF@PB 나노구조체를 제조하였다.As a comparison group, the ZIF-8 nanostructure optimized and synthesized in Preparation Example 1.2, the ZIF@GOx nanostructure containing the ZIF-8 metal-organic framework that captured glucose oxidase, and the ZIF that captured Prussian blue nanoenzyme -8 A ZIF@PB nanostructure containing a metal-organic framework was prepared.
실험예 1. ZIF@GOx@PB 나노구조체의 물리적 특성 확인Experimental Example 1. Confirmation of physical properties of ZIF@GOx@PB nanostructure
상기 제조예 1에서 합성된 ZIF@GOx@PB 나노구조체가 잘 형성되었는지 확인하고, 물리적 특성을 평가하기 위하여 주사 전자 현미경 (SEM), 동적 광산란 (dynamic light scattering; DLS), 제타 전위 (Zeta potential), 열중량분석 (Thermogravity analysis; TGA) 및 주사 투과 전자 현미경 (STEM) 분석을 수행하였다.In order to confirm whether the ZIF@GOx@PB nanostructure synthesized in Preparation Example 1 was well formed and to evaluate its physical properties, scanning electron microscopy (SEM), dynamic light scattering (DLS), and Zeta potential were used. , Thermogravity analysis (TGA) and scanning transmission electron microscopy (STEM) analysis were performed.
4종의 나노구조체(ZIF-8, ZIF@GOx, ZIF@PB, 및 ZIF@GOx@PB)를 주사 전자 현미경 (SEM)으로 관찰한 결과, 유효물질과 금속-유기골격체를 5분간 반응시킨 경우 약 200 ~ 400 nm 크기의 결정형 구조가 만들어졌고, 입자 크기는 함유된 유효물질의 구성에 따라 조금씩 달라졌다 (도 7). As a result of observing four types of nanostructures (ZIF-8, ZIF@GOx, ZIF@PB, and ZIF@GOx@PB) using a scanning electron microscope (SEM), the active material and the metal-organic framework were reacted for 5 minutes. In this case, a crystalline structure with a size of approximately 200 to 400 nm was created, and the particle size varied slightly depending on the composition of the active substance contained (Figure 7).
상기 4종의 나노구조체를 동적 광산란 (DLS)으로 측정한 결과, ZIF-8 나노구조체의 입자 크기는 284 nm, ZIF@GOx 나노구조체의 입자 크기는 316 nm, ZIF@PB 나노구조체의 입자 크기는 320 nm, ZIF@GOx@PB 나노구조체의 입자 크기는 367 nm로 (도 8), SEM 관찰 결과와 일치하였다.As a result of measuring the above four types of nanostructures using dynamic light scattering (DLS), the particle size of the ZIF-8 nanostructure was 284 nm, the particle size of the ZIF@GOx nanostructure was 316 nm, and the particle size of the ZIF@PB nanostructure was 284 nm. The particle size of the 320 nm and ZIF@GOx@PB nanostructures was 367 nm (Figure 8), which was consistent with the SEM observation results.
상기 4종의 나노구조체의 표면 전하를 알아보기 위해 제타 전위 (zeta potential)를 측정한 결과, ZIF-8 나노구조체의 표면 전하값은 +13 mV, ZIF@GOx 나노구조체의 표면 전하값은 -31 mV, ZIF@PB 나노구조체의 표면 전하값은 +15 mV, ZIF@GOx@PB 나노구조체의 표면 전하값은 -26 mV였으며, ZIF-8 나노구조체와 ZIF@PB 나노구조체는 (+)의 표면 전하값을, ZIF@GOx 나노구조체와 ZIF@GOx@PB 나노구조체는 (-)의 표면 전하값을 가짐을 확인하였다 (도 9). As a result of measuring the zeta potential to determine the surface charge of the four types of nanostructures, the surface charge value of the ZIF-8 nanostructure was +13 mV, and the surface charge value of the ZIF@GOx nanostructure was -31. mV, the surface charge value of the ZIF@PB nanostructure was +15 mV, the surface charge value of the ZIF@GOx@PB nanostructure was -26 mV, and the ZIF-8 nanostructure and ZIF@PB nanostructure had a (+) surface. In terms of charge value, it was confirmed that the ZIF@GOx nanostructure and ZIF@GOx@PB nanostructure had a surface charge value of (-) (Figure 9).
상기 4종의 나노구조체의 열적 안정성 및 금속-유기골격체에 포집된 유효물질의 포집 비율을 확인하기 위해, 열중량분석(TGA)을 수행한 결과, 금속-유기골격체를 포함하는 경우, 열이 가해져도 유효물질을 안정적으로 저장할 수 있다는 점을 확인하였다 (도 10).In order to confirm the thermal stability of the four types of nanostructures and the capture ratio of the effective substances captured in the metal-organic framework, thermogravimetric analysis (TGA) was performed. As a result, when the metal-organic framework was included, the heat It was confirmed that the active substance can be stored stably even if this is applied (FIG. 10).
프루시안 블루 나노효소 (PB)와 포도당 산화효소 (GOx)를 포함하는 금속-유기골격체 (MOF)를 포함하는 나노구조체 (ZIF@GOx@PB)의 주사 투과 전자 현미경 (STEM) 및 원소 맵핑 (elemental mapping)을 관찰한 결과, 목적하는 유효 물질 두 가지(PB, GOx)가 금속-유기골격체인 제올라이트형 이미다졸레이트 골격체 (ZIF)에 잘 포집되었음을 확인하였다 (도 11).Scanning transmission electron microscopy (STEM) and elemental mapping of a nanostructure (ZIF@GOx@PB) containing Prussian blue nanoenzyme (PB) and a metal-organic framework (MOF) containing glucose oxidase (GOx) ( As a result of observing elemental mapping, it was confirmed that the two target active substances (PB, GOx) were well captured in the zeolite-type imidazolate framework (ZIF), which is a metal-organic framework (FIG. 11).
실험예 2. ZIF@GOx@PB 나노구조체의 pH-반응성 분해 및 약물 방출Experimental Example 2. pH-responsive decomposition and drug release of ZIF@GOx@PB nanostructure
상기 제조예 1에서 합성된 ZIF@GOx@PB 나노구조체가 암세포의 산성 환경 및 과량의 당이 존재하는 환경에서 분해되며 유효물질을 방출하는지 확인하기 위하여, 즉 충분한 pH-반응성을 가지는지 평가하기 위해 투과 전자 현미경 (TEM) 이미징, pH 측정 및 유도 결합 플라즈마 (ICP) 분석을 수행하였다.To determine whether the ZIF@GOx@PB nanostructure synthesized in Preparation Example 1 decomposes in the acidic environment of cancer cells and in an environment where excess sugar is present and releases effective substances, that is, to evaluate whether it has sufficient pH-responsiveness. Transmission electron microscopy (TEM) imaging, pH measurements and inductively coupled plasma (ICP) analysis were performed.
pH 7.4, 6.0, 5.0 버퍼에서 ZIF@GOx@PB 나노구조체를 1 시간씩 담지한 후, 샘플을 채취하여 TEM 이미징을 수행한 결과, pH 값이 감소할수록 나노구조체가 붕괴하는 것을 확인하였다(도 12).After supporting the ZIF@GOx@PB nanostructures in pH 7.4, 6.0, and 5.0 buffers for 1 hour, samples were collected and TEM imaging was performed. As a result, it was confirmed that the nanostructures collapsed as the pH value decreased (Figure 12 ).
다양한 농도의 포도당 수용액 내에 ZIF@GOx@PB 나노구조체를 분산시킨 결과, ZIF@GOx@PB 나노구조체에 포함된 포도당 산화효소와 수용액 속 포도당 간의 반응에 의해 글루콘산 (gluconic acid)이 생성되어 pH가 감소하는 결과를 나타냈으며, 특히 포도당 수용액의 농도가 높을수록 pH가 더 큰 폭으로 급격하게 감소한다는 점을 관찰할 수 있었다(도 13).As a result of dispersing ZIF@GOx@PB nanostructures in glucose aqueous solutions of various concentrations, gluconic acid was generated through a reaction between the glucose oxidase contained in the ZIF@GOx@PB nanostructures and the glucose in the aqueous solution, and the pH increased. The results showed a decrease, and in particular, it was observed that the pH decreased more rapidly as the concentration of the glucose aqueous solution increased (FIG. 13).
다양한 농도의 포도당 수용액 내에 분산된 ZIF@GOx@PB 나노구조체가 분해되면서 누적되는 Zn 이온의 방출량을 유도 결합 플라즈마 (ICP) 로 측정하였다. 상술한 바와 같이 시간이 지날수록 포도당 산화효소와 포도당이 반응하여 글루콘산을 생성함으로써 pH가 감소했으며, ZIF@GOx@PB 나노구조체의 금속-유기골격체는 아연 (Zn)과 메틸 이미다졸로 구성되므로, 상기 나노구조체가 분해되면서 Zn 이온을 방출하였다(도 14).The amount of Zn ions released as the ZIF@GOx@PB nanostructure dispersed in various concentrations of glucose aqueous solution decomposed was measured using inductively coupled plasma (ICP). As described above, the pH decreased over time as glucose oxidase reacted with glucose to produce gluconic acid, and the metal-organic framework of the ZIF@GOx@PB nanostructure was composed of zinc (Zn) and methyl imidazole. Therefore, the nanostructure was decomposed and Zn ions were released (FIG. 14).
실험예 3. 프루시안 블루 나노효소 및 ZIF@GOx@PB 나노구조체의 촉매 활성 평가Experimental Example 3. Evaluation of catalytic activity of Prussian blue nanoenzyme and ZIF@GOx@PB nanostructure
프루시안 블루 나노효소 (PB)와 이를 포함하는 상기 제조예 1에서 합성된 ZIF@GOx@PB 나노구조체의 촉매 활성을 평가하기 위하여 UV-Vis 흡광도 측정 및 TA 분석법을 수행하였다.UV-Vis absorbance measurement and TA analysis were performed to evaluate the catalytic activity of Prussian blue nanoenzyme (PB) and the ZIF@GOx@PB nanostructure synthesized in Preparation Example 1 containing it.
먼저, 프루시안 블루 나노효소를 이용한 과산화수소의 비색 측정을 위해, 0.5 mM TMB, 100 ng/ml PB를 포함하는 1 mM의 과산화수소 수용액을 pH 5.0, 6.0, 7.0 버퍼 바탕으로 준비하였다. 최종 부피 1mL가 되도록 상온에서 5분간 반응시킨 후, 파란색 (O.D. 652 nm)으로 변한 산화 TMB의 흡광도를 측정하였다. 그 결과, pH 5.0에서 1.9627, pH 6.0에서 1.1961, pH 7.0에서 0.4167으로 나타났으며, 과산화수소가 없는 경우에는 아무런 흡광도 변화를 보이지 않았다 (도 15). 이는 프루시안 블루 나노효소가 pH 5.0에서 과산화수소를 가장 잘 환원시킨다는 점을 시사한다.First, for colorimetric measurement of hydrogen peroxide using Prussian blue nanoenzyme, a 1mM aqueous solution of hydrogen peroxide containing 0.5mM TMB and 100ng/ml PB was prepared as a pH 5.0, 6.0, 7.0 buffer background. After reacting at room temperature for 5 minutes to reach a final volume of 1 mL, the absorbance of oxidized TMB, which turned blue (O.D. 652 nm), was measured. As a result, it was 1.9627 at pH 5.0, 1.1961 at pH 6.0, and 0.4167 at pH 7.0, and in the absence of hydrogen peroxide, no change in absorbance was observed (FIG. 15). This suggests that Prussian blue nanoenzyme reduces hydrogen peroxide best at pH 5.0.
한편, pH 5.0에서 다양한 농도의 과산화수소 조건에서 프루시안 블루 나노효소의 비색 측정 결과, 과산화수소의 농도에 비례해 산화된 TMB가 띠는 O.D.652 nm에서 흡광도 세기가 증가함을 알 수 있다 (도 16).Meanwhile, as a result of colorimetric measurement of Prussian blue nanoenzyme under various concentrations of hydrogen peroxide at pH 5.0, it can be seen that the absorbance intensity at OD.652 nm, where oxidized TMB is banded, increases in proportion to the concentration of hydrogen peroxide (Figure 16) .
다음으로, 상기 프루시안 블루 나노효소를 포함하는 ZIF@GOx@PB 나노구조체의 비색 측정을 위해, 1.0 mM TMB, 100 ng/ml ZIF@GOx@PB 나노구조체를 포함하는 20 mM의 포도당 수용액을 pH 5.0 버퍼 바탕으로 준비하였다. 최종 부피 1mL가 되도록 상온에서 5분간 반응시킨 후, 파란색 (O.D. 652 nm)으로 변한 산화 TMB의 흡광도를 측정하였다. 그 결과, pH 5.0에서 0.7774으로 나타났으며, 포도당이 없는 경우에는 아무런 흡광도 변화를 보이지 않았다 (도 17). Next, for colorimetric measurement of the ZIF@GOx@PB nanostructure containing the Prussian blue nanoenzyme, a 20 mM glucose aqueous solution containing 1.0 mM TMB and 100 ng/ml ZIF@GOx@PB nanostructure was adjusted to pH. Prepared as 5.0 buffer blank. After reacting at room temperature for 5 minutes to reach a final volume of 1 mL, the absorbance of oxidized TMB, which turned blue (O.D. 652 nm), was measured. As a result, it was found to be 0.7774 at pH 5.0, and no change in absorbance was observed in the absence of glucose (FIG. 17).
한편, pH 5.0에서 다양한 농도의 포도당 조건에서 ZIF@GOx@PB 나노구조체의 비색 측정 결과, 포도당의 농도에 비례해 산화된 TMB가 띠는 O.D.652 nm에서 흡광도 세기가 증가함을 알 수 있다 (도 18). 이는 포도당이 존재하는 환경에서 ZIF@GOx@PB 나노구조체에 포함된 포도당 산화효소 (GOx)가 포도당을 산화하며 과산화수소를 생성하는 반응, 상기 나노구조체에 포함된 프루시안 블루 나노효소 (PB)가 생성된 과산화수소를 환원하면서 TMB를 산화시키는 반응이 순차적으로 일어난다는 점을 시사한다. Meanwhile, as a result of colorimetric measurement of the ZIF@GOx@PB nanostructure under various concentrations of glucose at pH 5.0, it can be seen that the absorbance intensity at OD.652 nm, where the oxidized TMB is banded, increases in proportion to the concentration of glucose (Figure 18). This is a reaction in which glucose oxidase (GOx) contained in the ZIF@GOx@PB nanostructure oxidizes glucose and generates hydrogen peroxide in an environment where glucose is present, and Prussian blue nanoenzyme (PB) contained in the nanostructure is produced. This suggests that the reaction of reducing hydrogen peroxide and oxidizing TMB occurs sequentially.
ZIF@GOx@PB 나노구조체에 포함된 포도당 산화효소가 촉매한 포도당의 산화로 인한 연속 반응의 결과, 생성된 활성산소종 (ROS)을 형광신호로 측정 가능하도록 terephthalic acid (TA) assay을 수행하였다. 그 결과, ZIF@GOx@PB 나노구조체를 함유한 경우 압도적으로 가장 커다란 세기의 형광이 방출되었고, ZIF@GOx@PB 나노구조체에서 가장 많은 양의 활성산소종을 방출함을 확인하였다 (도 19).As a result of a continuous reaction due to the oxidation of glucose catalyzed by the glucose oxidase contained in the ZIF@GOx@PB nanostructure, a terephthalic acid (TA) assay was performed to measure the reactive oxygen species (ROS) generated as a fluorescence signal. . As a result, it was confirmed that when the ZIF@GOx@PB nanostructure was contained, fluorescence of the highest intensity was emitted, and the ZIF@GOx@PB nanostructure emitted the largest amount of reactive oxygen species (Figure 19). .
실험예 4. ZIF@GOx@PB 나노구조체의 Experimental Example 4. ZIF@GOx@PB nanostructure in vitroin vitro 세포 독성 평가 Cytotoxicity evaluation
ZIF@GOx@PB 나노구조체의 독성 및 항암 효과를 확인하기 위하여 섬유아세포 3T3-L1과 피부암 세포 SK-MEL28을 사용하여 MTT로 세포 독성을 평가하였다. To confirm the toxicity and anticancer effect of ZIF@GOx@PB nanostructure, cytotoxicity was evaluated by MTT using fibroblast 3T3-L1 and skin cancer cells SK-MEL28.
상기 제조예 1에서 합성한 4종의 나노구조체 (ZIF-8, ZIF@GOx, ZIF@PB, 및 ZIF@GOx@PB)를 정상세포인 섬유아세포 (3T3-L1)에 처리한 결과 0 ~ 12.5 μg/mL의 농도에서 아무런 세포 독성을 나타내지 않았다 (도 20A). The results of treating normal fibroblasts (3T3-L1) with the four types of nanostructures (ZIF-8, ZIF@GOx, ZIF@PB, and ZIF@GOx@PB) synthesized in Preparation Example 1 were 0 to 12.5. It showed no cytotoxicity at a concentration of μg/mL (Figure 20A).
한편, 피부암세포 (SK-MEL28)에 처리한 결과, ZIF-8 나노구조체는 모든 농도에서 암세포 사멸을 유발하지 않았으며, ZIF@PB 나노구조체는 12.5 μg/mL의 농도에서 세포 생존율이 20% 정도 감소하였다. ZIF@GOx 나노구조체는 10.0 μg/mL 이상의 농도에서부터 세포 생존율이 감소하였으며, ZIF@GOx@PB 나노구조체는 5.0 μg/mL의 농도에서부터 세포 생존율이 30% 정도 감소해서 6.25 μg/mL에서 약 50%의 세포 생존율을 나타내었다 (도 20B).Meanwhile, when treated with skin cancer cells (SK-MEL28), the ZIF-8 nanostructure did not cause cancer cell death at any concentration, and the cell survival rate of the ZIF@PB nanostructure was about 20% at a concentration of 12.5 μg/mL. decreased. The cell viability of the ZIF@GOx nanostructure decreased at a concentration of 10.0 μg/mL or higher, and the cell viability of the ZIF@GOx@PB nanostructure decreased by about 30% at a concentration of 5.0 μg/mL, to about 50% at 6.25 μg/mL. The cell survival rate was shown (Figure 20B).
상기 결과는 금속-유기골격체를 포함하는 4종의 나노구조체 모두 정상세포에 영향을 주지 않는 높은 생체 적합성을 가지나, 프루시안 블루 나노효소 (PB)와 포도당 산화효소(GOx)를 모두 포함하는 경우, 시너지효과에 의해 향상된 촉매 활성으로 훨씬 큰 암세포 사멸 효과를 나타낸다는 점을 시사한다.The above results show that all four types of nanostructures containing metal-organic frameworks have high biocompatibility without affecting normal cells, but when they contain both Prussian blue nanoenzyme (PB) and glucose oxidase (GOx) , suggesting that the improved catalytic activity resulting from the synergistic effect results in a much greater cancer cell killing effect.
상기 4종의 나노구조체가 피부암세포에 미치는 영향을 생존/사멸세포 염색법 (Live/dead staining)을 통해 정성적으로 분석한 결과, 피부암세포에 낮은 독성을 나타내는 ZIF-8 나노구조체와 ZIF@PB 나노구조체와 달리 ZIF@GOx 나노구조체와 ZIF@GOx@PB 나노구조체는 사멸세포의 형광 염색을 나타냈으며, 특히 ZIF@GOx@PB 나노구조체에서 가장 큰 암세포 사멸 효과를 보였다 (도 21).As a result of qualitative analysis of the effect of the above four types of nanostructures on skin cancer cells through live/dead staining, it was found that ZIF-8 nanostructures and ZIF@PB nanostructures showed low toxicity to skin cancer cells. Unlike the structures, the ZIF@GOx nanostructure and the ZIF@GOx@PB nanostructure showed fluorescent staining of dead cells, and in particular, the ZIF@GOx@PB nanostructure showed the greatest cancer cell killing effect (Figure 21).
상기 4종의 나노구조체를 처리한 후 피부암세포 내부에 형성된 활성산소종 (ROS)을 DCFH-DA staining을 통해 정성적으로 분석한 결과, ZIF-8 나노구조체, SIF@PB 나노구조체, ZIF@GOx 나노구조체와 비교하여 ZIF@GOx@PB 나노구조체에서 가장 많은 활성산소종이 형성되었음을 형광 염색을 통해 확인하였다 (도 22).As a result of qualitative analysis of reactive oxygen species (ROS) formed inside skin cancer cells after processing the above four types of nanostructures through DCFH-DA staining, ZIF-8 nanostructure, SIF@PB nanostructure, and ZIF@GOx It was confirmed through fluorescence staining that the most reactive oxygen species were formed in the ZIF@GOx@PB nanostructure compared to the nanostructure (Figure 22).
실험예 5. ZIF@GOx@PB 나노구조체와 병용투여의 Experimental Example 5. Combined administration with ZIF@GOx@PB nanostructure in vitroin vitro 세포 독성 평가 비교 Comparison of cytotoxicity assessments
ZIF@GOx@PB 나노구조체의 항암 활성을 확인하기 위해, ZIF@GOx 나노구조체에 프루시안 블루 나노효소를 함께 투여한 비교군 1, ZIF@PB 나노구조체에 포도당 산화효소를 함께 투여한 비교군 2를 피부암 세포 SK-MEL28에 처리한 후, MTT로 세포 독성을 평가하였다. 이 때, 상기 실험군에서 모두 동일한 농도의 금속-유기골격체, 포도당 산화효소, 및 프루시안 블루 나노효소가 투여되도록 하였다.To confirm the anti-cancer activity of ZIF@GOx@PB nanostructure, comparison group 1 was administered ZIF@GOx nanostructure together with Prussian blue nanoenzyme, and comparison group 2 was administered ZIF@PB nanostructure together with glucose oxidase. After treating skin cancer cells SK-MEL28, cytotoxicity was evaluated by MTT. At this time, the same concentrations of metal-organic framework, glucose oxidase, and Prussian blue nanoenzyme were administered to all of the experimental groups.
그 결과, 포도당 산화효소와 프루시안 블루 나노효소를 모두 포집하는 금속-유기골격체를 포함하는 본 발명의 나노구조체가 가장 낮은 세포 생존율을 나타내었으며 (도 23), 이는 병용투여보다 포도당 산화효소와 프루시안 블루 나노효소를 하나의 나노구조체 내에 포함하는 경우, 화학역학 치료법과 단식 치료법의 시너지 효과가 가장 크게 나타난다는 점을 시사한다.As a result, the nanostructure of the present invention containing a metal-organic framework that captures both glucose oxidase and Prussian blue nanoenzyme showed the lowest cell survival rate (FIG. 23), which means that glucose oxidase and This suggests that when Prussian blue nanoenzyme is included in one nanostructure, the synergistic effect of chemodynamic therapy and fasting therapy appears the greatest.
보다 구체적으로, 본 발명의 ZIF@GOx@PB 나노구조체는 포도당 산화효소와 프루시안 블루 나노효소가 금속-유기골격체에 포집되어 있어 기질 간 물리적 거리가 단축되기 때문에 연속적인 촉매 반응 (cascade enzymatic reaction)에 유리하고, 포도당의 산화 결과 생성된 과산화수소가 인접한 프루시안 블루 나노효소와 바로 2차 효소 반응을 진행하기 때문에, 과산화수소가 확산하거나 자기분해하는 것을 최소화할 수 있기 때문에 가장 우수한 세포 독성 효과를 나타낸다.More specifically, in the ZIF@GOx@PB nanostructure of the present invention, glucose oxidase and Prussian blue nanoenzyme are entrapped in a metal-organic framework, thereby shortening the physical distance between substrates, resulting in a cascade enzymatic reaction. ), and since the hydrogen peroxide generated as a result of the oxidation of glucose immediately undergoes a secondary enzyme reaction with the adjacent Prussian blue nanoenzyme, diffusion or self-decomposition of hydrogen peroxide can be minimized, showing the best cytotoxic effect. .
이상과 같이 실시예들이 비록 한정된 도면에 의해 설명되었으나, 해당 기술분야에서 통상의 지식을 가진 자라면 상기를 기초로 다양한 기술적 수정 및 변형을 적용할 수 있다. 예를 들어, 설명된 기술들이 설명된 방법과 다른 순서로 수행되거나, 및/또는 설명된 시스템, 구조, 장치, 회로 등의 구성요소들이 설명된 방법과 다른 형태로 결합 또는 조합되거나, 다른 구성요소 또는 균등물에 의하여 대치되거나 치환되더라도 적절한 결과가 달성될 수 있다. Although the embodiments have been described with limited drawings as described above, those skilled in the art can apply various technical modifications and variations based on the above. For example, the described techniques are performed in a different order than the described method, and/or components of the described system, structure, device, circuit, etc. are combined or combined in a different form than the described method, or other components are used. Alternatively, appropriate results may be achieved even if substituted or substituted by an equivalent.
그러므로, 다른 구현들, 다른 실시예들 및 특허청구범위와 균등한 것들도 후술하는 청구범위의 범위에 속한다.Therefore, other implementations, other embodiments, and equivalents of the claims also fall within the scope of the following claims.
Claims (15)
상기 나노구조체의 상기 포도당 산화효소가 암세포로의 포도당 공급을 차단하면서 글루콘산(gluconic acid)과 과산화수소를 생성하고, 그리고 상기 과산화수소는 상기 프루시안 블루에 의해 활성 산소종((reactive oxygen species)을 생성함으로써 암세포를 특이적으로 사멸시키는 것인, 나노구조체.
A nanostructure containing a zeolitic imidazolate framework containing glucose oxidase and Prussian blue,
The glucose oxidase of the nanostructure blocks the supply of glucose to cancer cells, producing gluconic acid and hydrogen peroxide, and the hydrogen peroxide generates reactive oxygen species by the Prussian blue. A nanostructure that specifically kills cancer cells by doing so.
상기 나노구조체는 pH 3 내지 7에서 분해되는 것을 특징으로 하는, 나노구조체.
According to paragraph 1,
The nanostructure is characterized in that the nanostructure is decomposed at pH 3 to 7.
상기 나노구조체의 크기는 200 내지 400 nm인 것을 특징으로 하는, 나노구조체.
According to paragraph 1,
A nanostructure, characterized in that the size of the nanostructure is 200 to 400 nm.
상기 나노구조체의 제타 전위는 -10 내지 -30 mV인 것을 특징으로 하는, 나노구조체.
According to paragraph 1,
A nanostructure, characterized in that the zeta potential of the nanostructure is -10 to -30 mV.
A pharmaceutical composition for preventing or treating cancer comprising the nanostructure of claim 1.
상기 암은 피부암, 유방암, 자궁암, 식도암, 위암, 뇌 종양, 결장암, 직장암, 대장암, 폐암, 난소암, 자궁경부암, 자궁내막암, 외음부암, 신장암, 혈액암, 췌장암, 전립선암, 고환암, 후두암, 두경부암, 갑상선암, 간암, 방광암, 골육종, 림프종, 백혈병, 흉선암, 요도암, 및 기관지암으로 구성된 군으로부터 선택된 어느 하나 이상인 것인, 약학적 조성물.
According to clause 8,
The above cancers include skin cancer, breast cancer, uterine cancer, esophageal cancer, stomach cancer, brain tumor, colon cancer, rectal cancer, colorectal cancer, lung cancer, ovarian cancer, cervical cancer, endometrial cancer, vulvar cancer, kidney cancer, blood cancer, pancreatic cancer, prostate cancer, and testicular cancer. , a pharmaceutical composition that is at least one selected from the group consisting of laryngeal cancer, head and neck cancer, thyroid cancer, liver cancer, bladder cancer, osteosarcoma, lymphoma, leukemia, thymic cancer, urethral cancer, and bronchial cancer.
(2) 상기 (1) 단계에서 생성된 용액에 2-메틸이미다졸 (2-methylimidazole)을 첨가하여 반응시키는 단계; 및
(3) 상기 포도당 산화효소 및 프루시안 블루를 포함하는 제올라이트형 이미다졸레이트 골격체 (zeolitic imidazole framework)를 포함하는 나노구조체를 제조하는 단계;
를 포함하는 나노구조체의 제조방법으로,
상기 나노구조체의 상기 포도당 산화효소가 암세포로의 포도당 공급을 차단하면서 글루콘산(gluconic acid)과 과산화수소를 생성하고, 그리고 상기 과산화수소는 상기 프루시안 블루에 의해 활성 산소종((reactive oxygen species)을 생성함으로써 암세포를 특이적으로 사멸시키는 것인, 나노구조체의 제조방법.
(1) adding glucose oxidase and Prussian blue to zinc nitrate hexahydrate;
(2) adding 2-methylimidazole to the solution produced in step (1) and reacting it; and
(3) preparing a nanostructure containing a zeolitic imidazole framework containing the glucose oxidase and Prussian blue;
A method of manufacturing a nanostructure containing,
The glucose oxidase of the nanostructure blocks the supply of glucose to cancer cells, producing gluconic acid and hydrogen peroxide, and the hydrogen peroxide generates reactive oxygen species by the Prussian blue. A method of manufacturing a nanostructure that specifically kills cancer cells by doing so.
상기 프루시안 블루 나노효소는 FeCl3·6H2O와 citric acid가 담긴 수용액 및 K4[Fe(CN)6]와 citric acid가 담긴 수용액을 반응시키고, 아세톤을 추가로 첨가한 후, 원심분리를 진행하여 제조된 것을 특징으로 하는, 나노구조체의 제조방법.
According to clause 10,
The Prussian blue nanoenzyme reacts with an aqueous solution containing FeCl 3 6H 2 O and citric acid and an aqueous solution containing K 4 [Fe(CN) 6 ] with citric acid, adds additional acetone, and centrifuges. A method of manufacturing a nanostructure, characterized in that it is manufactured by proceeding.
상기 나노구조체는 나노구조체 100 중량부 기준으로,
포도당 산화효소 0.01 내지 0.1 중량부와 과산화효소 모방 효소 0.001 내지 0.01 중량부를 포함하는 것을 특징으로 하는, 나노구조체의 제조방법.
According to clause 10,
The nanostructure is based on 100 parts by weight of the nanostructure,
A method for producing a nanostructure, comprising 0.01 to 0.1 parts by weight of glucose oxidase and 0.001 to 0.01 parts by weight of peroxidase mimicking enzyme.
상기 (1) 단계에서 질산아연 헥사수화물의 농도는 10 내지 50 mM이고,
상기 (2) 단계에서 2-메틸이미다졸의 농도는 2.5 내지 5 M인 것을 특징으로 하는, 나노구조체의 제조방법.
According to clause 10,
In step (1), the concentration of zinc nitrate hexahydrate is 10 to 50 mM,
A method for producing a nanostructure, characterized in that the concentration of 2-methylimidazole in step (2) is 2.5 to 5 M.
상기 (2) 단계는 상온에서 1 내지 10 분 동안 반응시키는 것을 특징으로 하는, 나노구조체의 제조방법.
According to clause 10,
Step (2) is a method for producing a nanostructure, characterized in that the reaction is performed at room temperature for 1 to 10 minutes.
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