KR20100005589A - Hyaluronic acid immobilized gold nanoparticles for detection of reactive oxygen species and preparing method thereof - Google Patents

Hyaluronic acid immobilized gold nanoparticles for detection of reactive oxygen species and preparing method thereof Download PDF

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KR20100005589A
KR20100005589A KR1020080065683A KR20080065683A KR20100005589A KR 20100005589 A KR20100005589 A KR 20100005589A KR 1020080065683 A KR1020080065683 A KR 1020080065683A KR 20080065683 A KR20080065683 A KR 20080065683A KR 20100005589 A KR20100005589 A KR 20100005589A
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박태관
이혁진
이규리
김인경
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Abstract

PURPOSE: A method for preparing hyaluronic acid gold nanoparticel sensing oxygen free radical is provided to sensitively react to intracellular free radical and accurately measure the amount of free radical. CONSTITUTION: A method for preparing hyaluronic acid gold nanoparticle sensing oxygen free radical comprises: a step of adding hyaluronidase to hyaluronic acid to obtain low molecular hyaluronic acid; a step of introducing flurescence dye to carboxylic group of the low molecular hyaluronic acid; a step of preparing low molecular hyaluronic acid synthesized material; and a step of fixing the low molecular hyaluronic acid synthesized material on the surface of golf nanoparticle.

Description

활성산소를 감지하는 히알루론산 금 나노입자 및 이의 제조방법{Hyaluronic acid immobilized gold nanoparticles for detection of reactive oxygen species and preparing method thereof}Hyaluronic acid immobilized gold nanoparticles for detection of reactive oxygen species and preparing method

본 발명은 활성산소를 감지하는 히알루론산 금 나노입자(Hyaluronic acid immobilized gold nanoparticles) 및 이의 제조방법에 관한 것으로서, 보다 상세하게는 생분해성과 생체적합성을 가진 히알루론산에 히알루로니데이즈를 첨가하여 저분량 히알루론산을 제조하는 단계;와 상기 저분자량 히알루론산의 카르복실기에 형광염료를 도입하며, 환원 말단에는 금 나노입자 표면에 고정화 시킬 수 있는 기능기를 도입하여 저분자량 히알루론산 합성물을 제조하는 단계; 및 상기 저분자량 히알루론산 합성물을 금 나노입자 표면에 고정하는 단계;를 포함하는 것을 특징으로 하는 활성산소를 감지하는 히알루론산 금 나노입자 및 이의 제조방법에 관한 것이다.The present invention relates to hyaluronic acid gold nanoparticles (hyaluronic acid immobilized gold nanoparticles) for detecting active oxygen and a method for manufacturing the same, more specifically, hyaluronic acid having a biodegradable and biocompatible with a low amount of hyaluronic acid Preparing a low molecular weight hyaluronic acid compound by introducing a fluorescent dye into the carboxyl group of the low molecular weight hyaluronic acid and introducing a functional group which can be immobilized on the surface of the gold nanoparticles; And fixing the low molecular weight hyaluronic acid composite to the surface of the gold nanoparticles. The present invention relates to gold hyaluronic acid nanoparticles for detecting active oxygen, and a method of manufacturing the same.

활성산소는 호흡과정에서 몸속으로 들어간 산소가 산화과정에 이용되면서 여러 대사과정에서 생성되어 생체조직을 공격하고 세포를 손상시키는 산화력이 강한 산소를 말한다. 이를 유해산소라고도 하며, 우리가 호흡하는 산소와는 완전히 다르게 불안정한 상태에 있는 산소이다. 환경오염과 화학물질, 자외선, 혈액순환장애, 스트레스 등으로 산소가 과잉생산된 것이다. 이렇게 과잉생산된 활성산소는 사람 몸속에서 산화작용을 일으킨다. 이렇게 되면 세포막, DNA, 그 외의 모든 세포 구조가 손상당하고 손상의 범위에 따라 세포가 기능을 잃거나 변질된다.Active oxygen refers to oxygen that has strong oxidative power, which is produced by various metabolic processes as oxygen entered into the body during the breathing process is used for oxidation. This is also called harmful oxygen, and oxygen is in an unstable state completely different from the oxygen we breathe. Oxygen is overproduced due to environmental pollution, chemicals, ultraviolet rays, blood circulation disorders, and stress. This overproduced free radicals cause oxidation in the human body. This damages the cell membrane, DNA, and all other cellular structures, and the cells lose their function or change depending on the extent of the damage.

이와 함께 몸속의 여러 아미노산을 산화시켜 단백질의 기능 저하도 가져온다. 그리고 핵산을 손상시켜 핵산 염기의 변형과 유리, 결합의 절단, 당의 산화분해 등을 일으켜 돌연변이나 암의 원인이 되기도 한다. 또한 생리적 기능이 저하되어 각종 질병과 노화의 원인이 되기도 한다. At the same time, it oxidizes several amino acids in the body, leading to a decrease in protein function. In addition, the nucleic acid may be damaged, resulting in modification and release of nucleic acid bases, cleavage of bonds, and oxidative degradation of sugars, resulting in mutations and cancer. In addition, the physiological function is lowered, causing various diseases and aging.

그러나 활성산소가 나쁜 영향을 주는 것만은 아니다. 병원체나 이물질을 제거하기 위한 생체방어과정에서 산소·과산화수소와 같은 활성산소가 많이 발생하는데, 이들의 강한 살균작용으로 병원체로부터 인체를 보호하기도 한다. But free radicals aren't just bad. In the process of biological defense to remove pathogens and foreign substances, free radicals such as oxygen and hydrogen peroxide are generated, and their strong sterilization action also protects the human body from pathogens.

현대인의 질병 중 약 90%가 활성산소와 관련이 있다고 알려져 있으며, 구체적으로 그러한 질병에는 암·동맥경화증·당뇨병·뇌졸중·심근경색증·간염·신장염·아토피·파킨슨병, 자외선과 방사선에 의한 질병 등이 있다. 따라서 이러한 질병에 걸리지 않으려면 몸속의 활성산소를 없애주면 된다. 활성산소를 없애주는 물질인 항산화물에는 비타민E·비타민C·요산·빌리루빈·글루타티온·카로틴 등이 포함된다. 이러한 항산화물을 자연적인 방법으로 섭취하면 큰 효과가 있다. About 90% of modern diseases are known to be related to free radicals.In particular, such diseases include cancer, arteriosclerosis, diabetes, stroke, myocardial infarction, hepatitis, nephritis, atopy, Parkinson's disease, ultraviolet and radiation-related diseases. There is this. Therefore, if you do not want to get sick, you can get rid of free radicals in the body. Antioxidants, which are free radicals, include vitamin E, vitamin C, uric acid, bilirubin, glutathione, and carotene. Ingesting these antioxidants in a natural way has a great effect.

기존에 제조된 활성산소를 측정하는 진단제로는 2’,7’- dichlorodihydrofluorescein diacetate (H2DCFDA) 와 Aminophenyl fluorescein (APF) 와 hydroxyphenyl fluorescein (HPF) 등이 있다. 이러한 활성 산소 진단제는 안정성 및 세포 독성에 많은 문제점을 일으킨다. 예를 들어, 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA)의 경우 진단제의 안정성과 활성산소 특이성이 현저히 낮고, Aminophenyl fluorescein (APF) 와 hydroxyphenyl fluorescein (HPF)은 활성 산소의 특이성이 향상되었지만 세포 독성이 높은 것으로 알려져 있다. Existing diagnostic agents for measuring active oxygen include 2 ', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA), Aminophenyl fluorescein (APF) and hydroxyphenyl fluorescein (HPF). Such reactive oxygen diagnostic agents cause many problems in stability and cytotoxicity. For example, 2 ', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA) has significantly lower diagnostic stability and free radical specificity, while Aminophenyl fluorescein (APF) and hydroxyphenyl fluorescein (HPF) have improved cytotoxicity but This is known to be high.

천연 고분자인 히알루론산 (HA)은 넌설페이티드 (nonsulfated) 글라이코사미노글리칸 (glycosaminoglycan;GAG)로서 N-acetyl-D-glucosamine 과 D-glucuronic acid로 구성되어 있으며, 세포 밖 구조체의 주요 성분으로서 높은 생체 적합성을 가지고 있다. 또한 피하조직과 연골조직 같은 연결조직의 세포 외측 기질뿐 아니라 안구의 유리체 및 관절 동동의 활액에 존재하며, 히알루론산과 히알루론산 수용체인 CD44/RHAMM의 상호작용은 세포 이동성 및 세포증식과 특정 생리학적 과정들을 제어하는 것으로 알려져 있다. 이러한 특징으로 히알루론산은 치료적 용도를 가지며, 미국식품의약품(FDA)에서 정맥주사, 구강투여, 피부투여 등으로 인간에게 사용하는 것을 승인받은 천연 고분자이다 (P. Bulpitt, et al., J. Biomed. Mater. Res. 47, 152(1999)). 현재로는 조직공학용 지지체와 약물전달용 하이드로젤로서 그 사용 범위가 증대되고 있다. 또한 활성산소에 의해 히알루론산이 분해되는 것이 최근 밝혀짐으로서 히알루론산 분해를 통한 활성산소 측정이 가능하게 되었다. (L. Soltes, et.al., Biomacromolecules 7, 659(2006)).The natural polymer, hyaluronic acid (HA), is a nonsulfated glycosaminoglycan (GAG) and consists of N-acetyl-D-glucosamine and D-glucuronic acid. As it has high biocompatibility. It is also present in the synovial fluid of the eye's vitreous and joint sinus as well as the extracellular matrix of connective tissues such as subcutaneous and cartilage tissues. It is known to control the processes. Because of these features, hyaluronic acid is a natural polymer that has therapeutic uses and is approved for use in humans for intravenous injection, oral administration, and skin administration in the US Food and Drug Administration (PDA) (P. Bulpitt, et al., J. Biomed. Mater. Res. 47, 152 (1999)). At present, the scope of use is increasing as a support for tissue engineering and hydrogel for drug delivery. In addition, it has been recently discovered that hyaluronic acid is decomposed by active oxygen, thereby making it possible to measure active oxygen through decomposition of hyaluronic acid. (L. Soltes, et. Al., Biomacromolecules 7, 659 (2006)).

활성산소를 측정하는 구체적 메카니즘에 있어서, 나노입자표면 에너지전이(NSET: nanoparticle surface energy transfer)의 방법이 이용되고 있다. 이러한 나노입자표면 에너지전이를 통한 형광 소멸 및 형광 복원은 그 동안 많은 연구 결과와 효용성이 발표되었다 (Guarise, C., Proc . Natl . Acad . Sci . U. S. A. 103, 3978-3982 (2006)).In a specific mechanism for measuring active oxygen, a method of nanoparticle surface energy transfer (NSET) is used. Fluorescence annihilation and fluorescence recovery through the nanoparticle surface energy transfer have been published (Guarise, C., Proc . Natl . Acad . Sci . USA 103, 3978-3982 (2006)).

본 발명의 목적은 상기에서 설명한 것과 같이 기존에 제조된 활성산소를 측정하는 진단제가 많은 문제점을 일으킨 바, 활성산소를 보다 안전하고 효율적으로 측정하기 위한 진단 조형제를 제공하고자 함이며, 그의 제조를 위한 제조방법을 제공하고자 함이다. It is an object of the present invention to provide a diagnostic molding agent for measuring the active oxygen more safely and efficiently, as the diagnostic agent for measuring the active oxygen produced as described above has caused a lot of problems, It is to provide a manufacturing method.

상기의 과제를 해결하기 위하여In order to solve the above problems

본 발명은 히알루론산에 히알루로니데이즈를 첨가하여 저분자량 히알루론산을 제조하는 단계;The present invention comprises the steps of preparing a low molecular weight hyaluronic acid by adding hyaluronic acid to hyaluronic acid;

상기 저분자량 히알루론산의 카복실기에 형광염료를 도입하며, 환원 말단에 는 금 나노입자 표면에 고정화 시킬 수 있는 기능기를 도입하여 저분자량 히알루론산 합성물을 제조하는 단계; 및 Preparing a low molecular weight hyaluronic acid compound by introducing a fluorescent dye into the carboxyl group of the low molecular weight hyaluronic acid and introducing a functional group which can be immobilized on the surface of the gold nanoparticles at the reducing end; And

상기 저분자량 히알루론산 합성물을 금 나노입자 표면에 고정하는 단계;를 포함하는 것을 특징으로 하는 활성산소를 감지하는 히알루론산 금 나노입자의 제조방법을 제공한다.Fixing the low molecular weight hyaluronic acid composite on the surface of the gold nanoparticles provides a method for producing hyaluronic acid gold nanoparticles for detecting the active oxygen comprising a.

또한 본 발명은 상기의 방법으로 제조된 활성산소를 감지하는 히알루론산 금 나노입자를 제공한다. In another aspect, the present invention provides hyaluronic acid gold nanoparticles for detecting the active oxygen produced by the above method.

상기의 과제 해결 수단을 통하여Through the above problem solving means

본 발명인 히알루론산이 고정된 금 나노입자는 세포내의 활성산소와 민감하게 반응하여 올리고 히알루론산이 분해됨으로써, 세포내에서 특이적인 상황에 의해 발생하는 활성산소의 양을 민감하게 측정할 수 있다. 또한 본 발명은 FDA의 승인을 받은 생체적합성 천연 고분자인 히알루론산 (HA)을 재료로 하여 금 나노입자와 나노입자표면 에너지전이(nanoparticle surface energy transfer)를 이용한 활성산소 진단 조형제를 제조하여 제공함으로써, 기존의 활성산소 진단 조형제에 비하여 그 안정성 및 세포독성에서 뛰어나며 활성산소 민감성 및 특이성에서도 더욱 큰 효과가 기대되어 질 수 있다. The gold nanoparticles to which the hyaluronic acid is immobilized according to the present invention are sensitively reacted with the active oxygen in the cell to decompose the oligo hyaluronic acid, thereby sensitively measuring the amount of the active oxygen generated by the specific situation in the cell. In another aspect, the present invention by using the hyaluronic acid (HA), a biocompatible natural polymer approved by the FDA as a material to prepare and provide an active oxygen diagnostic molding agent using gold nanoparticles and nanoparticle surface energy transfer, Compared with the existing reactive oxygen diagnostic agents, it is superior in the stability and cytotoxicity and can be expected to be more effective in reactive oxygen sensitivity and specificity.

본 발명은 a) 히알루론산에 히알루로니데이즈(hyaluronidase)를 첨가하여 저분자량 히알루론산을 제조하는 단계;The present invention comprises the steps of: a) adding hyaluronic acid to hyaluronic acid (hyaluronidase) to prepare a low molecular weight hyaluronic acid;

b) 상기 저분자량 히알루론산의 카복실기에 형광염료를 도입하며, 환원 말단에 금 나노입자 표면에 고정화 시킬 수 있는 기능기를 도입하여 저분자량 히알루론산 합성물을 제조하는 단계; 및 b) preparing a low molecular weight hyaluronic acid compound by introducing a fluorescent dye into the carboxyl group of the low molecular weight hyaluronic acid and introducing a functional group which can be immobilized on the surface of the gold nanoparticles at the reducing end; And

c) 상기 저분자량 히알루론산 합성물을 금 나노입자 표면에 고정하는 단계;를 포함하는 활성산소를 감지하는 히알루론산 금 나노입자의 제조방법을 제공한다. c) fixing the low molecular weight hyaluronic acid composite to the surface of the gold nanoparticles, and provides a method for preparing hyaluronic acid gold nanoparticles that detects active oxygen.

본 발명에서 상기 a) 단계의 저분자량 히알루론산의 분자량은 1000~100000 Da 일 수 있다. In the present invention, the molecular weight of the low molecular weight hyaluronic acid of step a) may be 1000 to 100,000 Da.

본 발명에서 상기 b) 단계의 기능기는 유리 황화기, 도파민기 및 아민기로 이루어진 군에서 선택된 어느 하나이며, 히알루론산의 환원 말단에 히알루론산의 몰비의 10~30배로 첨가되어 질 수 있다. In the present invention, the functional group of step b) is any one selected from the group consisting of a free sulfide group, a dopamine group, and an amine group, and may be added at 10 to 30 times the molar ratio of hyaluronic acid to the reduced terminal of the hyaluronic acid.

또한 본 발명에서 상기 b) 단계의 형광염료는 로다민(rhodamine), 시아닌(cyanine)3, 시아닌(cyanine)5, 시아닌(cyanine)7, 알렉사 염료(Alexa dyes), 하일라이트 플루오르 염료(Hylite fluor dyes), 스틸벤 유도체, 이미다졸 유도체 및 쿠마린 유도체로 이루어진 군에서 선택된 어느 하나일 수 있다. In the present invention, the fluorescent dye in step b) is rhodamine, cyanine 3, cyanine 5, cyanine 5, cyanine 7, Alexa dyes, highlight fluorine dyes (Hylite fluor) dyes), stilbene derivatives, imidazole derivatives and coumarin derivatives.

또한 본 발명에서 상기 c) 단계의 금 나노입자와 히알루론산의 몰 비는 1:100 ~ 1:10000 으로 할 수 있으며, 상기 c) 단계의 금 나노입자는 입자의 크기가 4 ~ 200 ㎚ 의 지름을 가지며 히알루론산이 황화금속 결합으로 고정화되어 질 수 있다.In addition, in the present invention, the molar ratio of the gold nanoparticles and hyaluronic acid of step c) may be 1: 100 to 1: 10000, and the gold nanoparticles of step c) have a diameter of 4 to 200 nm. And hyaluronic acid can be immobilized with a metal sulfide bond.

본 발명은 상기의 방법으로 제조된 히알루론산 금 나노입자를 제공한다.The present invention provides hyaluronic acid gold nanoparticles prepared by the above method.

이하 본 발명인 활성산소를 감지하는 히알루론산 금 나노입자 및 이의 제조방법을 구체적으로 설명한다. Hereinafter, gold hyaluronic acid nanoparticles for detecting active oxygen of the present invention and a preparation method thereof will be described in detail.

본 발명은 생체적합성 천연 고분자인 히알루론산을 재료로 하여 히알루론산이 접목된 금 나노 입자 및 이를 제조하는 방법에 관한 것이다.  The present invention relates to gold nanoparticles grafted with hyaluronic acid using a hyaluronic acid, which is a biocompatible natural polymer, and a method of manufacturing the same.

히알루론산이 접목된 금 나노 입자의 제조단계는 히알루론산에 히알루로니데이즈(hyaluronidase)를 첨가하여 저분자량 히알루론산을 제조하는 단계와 상기 저 분자량 히알루론산의 카복실기에 형광염료를 도입하며, 환원 말단에 금 나노입자 표면에 고정화 시킬 수 있는 기능기를 도입하여 저분자량 히알루론산 합성물을 제조하는 단계 및 상기 저분자량 히알루론산 합성물을 금 나노입자 표면에 고정하는 단계를 포함한다. Hyaluronic acid-grafted gold nanoparticles are prepared by adding hyaluronic acid to hyaluronic acid (hyaluronidase) to prepare a low molecular weight hyaluronic acid and a fluorescent dye in the carboxyl group of the low molecular weight hyaluronic acid, and at the reduction end Preparing a low molecular weight hyaluronic acid compound by introducing a functional group that can be immobilized on the surface of the gold nanoparticles, and fixing the low molecular weight hyaluronic acid compound to the surface of the gold nanoparticles.

상기 저분자량 히알루론산은 다양한 분포의 분자량이 사용될 수 있으며, 바람직하게는 분자량이 1000~100000 Da 인 것이 사용될 수 있다. The low molecular weight hyaluronic acid may be used in a variety of molecular weight distribution, preferably a molecular weight of 1000 ~ 100,000 Da may be used.

상기 기능기는 유리 황화기, 도파민기 및 아민기로 이루어진 군에서 선택된 어느 하나이나 이에 한정되는 것은 아니며, 히알루론산의 환원 말단에 히알루론산의 몰비의 10~30배로 첨가되어 질 수 있다. 바람직하게는 상기 기능기는 유리 황화기인 경우에는 시스타민(cystamine), 도파기인 경우에는 도파민(dopamine)이 이용되고, 히알루론산의 환원 말단에 히알루론산의 몰비의 20배로 첨가되어 질 수 있다. The functional group is not limited to any one selected from the group consisting of a free sulfide group, a dopamine group and an amine group, and may be added at 10 to 30 times the molar ratio of hyaluronic acid to the reduction terminal of hyaluronic acid. Preferably, the functional group is cystamine (cystamine) in the case of free sulfide group, dopamine (dopamine) in the case of the dopant, and may be added at 20 times the molar ratio of hyaluronic acid to the reducing end of hyaluronic acid.

또한 상기 형광염료는 로다민, 시아닌3, 시아닌5, 시아닌7, 알렉사 염료, 하일라이트 플루오르 염료, 스틸벤 유도체, 이미다졸 유도체 및 쿠마린 유도체로 이루어진 군에서 선택된 어느 하나이나 이에 한정되는 것은 아니다.In addition, the fluorescent dye is not limited to any one selected from the group consisting of rhodamine, cyanine 3, cyanine 5, cyanine 7, Alexa dye, highlight fluorine dye, stilbene derivative, imidazole derivative and coumarin derivative.

상기 금 나노입자와 히알루론산의 몰 비는 1: 100 ~ 1: 10000 으로 할 수 있으며, 상기 금 나노입자는 입자의 크기가 4 ~ 200 ㎚ 의 지름을 가지며 히알루론산이 황화금속 결합으로 고정화되어 질 수 있다. 고정화시는 소금을 첨가함으로써 히알루론산과 금 나노입자간의 정전기적 반발작용(electrostatic repulsion)을 줄일 수 있다. The molar ratio of the gold nanoparticles and hyaluronic acid can be 1: 100 ~ 1: 10000, the gold nanoparticles have a diameter of 4 ~ 200 ㎚ size and the hyaluronic acid to be immobilized by a metal sulfide bond Can be. During immobilization, the addition of salt can reduce the electrostatic repulsion between hyaluronic acid and gold nanoparticles.

본 발명은 상기의 방법으로 제조된 활성산소를 감지하는 히알루론산 금 나노입자에 관한 것이다.The present invention relates to hyaluronic acid gold nanoparticles for detecting the active oxygen produced by the above method.

이하 실시예를 통하여 활성산소를 감지하는 히알루론산 금 나노입자의 제조방법을 설명한다. 다만, 이하의 실시예가 본 발명의 기술적 범위를 제한하는 것은 아니다. Hereinafter, a method of preparing hyaluronic acid gold nanoparticles for detecting active oxygen will be described. However, the following examples do not limit the technical scope of the present invention.

<실시예 1> 저분자량 히알루론산의 제조 Example 1 Preparation of Low Molecular Weight Hyaluronic Acid

히알루론산 (HA, MW:1.79x107) 을 pH 7.0 의 phosphate buffer saline (PBS) 에 녹인 후, 0.44 um 의 나일론 필터 (Minisart)를 이용하여 필터링을 했다. 필터링이 된 히알루론산 용액에 히알루로니데이즈 (hyaluronidase)를 1밀리그램 히알루론산 당, 히알루로니데니즈 100유닛을 첨가하여 반응을 시켰다. 시간이 지남에 따라 히알루론산의 분자량이 감소함으로 대략 12 시간 후에 히알루론산 용액을 가열하여 히알루로니데이즈의 활성을 억제시켰다. 침강된 히알루로니데이즈는 0.2 um 나일론 필터 (Minisart) 를 이용해 제거한 후, 분해된 저분자량 히알루론산의 분자량을 GPC(Gel Permeation Chromatography)를 통해 확인했다. Hyaluronic acid (HA, MW: 1.79x10 7 ) was dissolved in phosphate buffer saline (PBS) at pH 7.0 and filtered using a 0.44 um nylon filter (Minisart). Hyaluronic acid (hyaluronidase) was added to the filtered hyaluronic acid solution by adding 1 unit of hyaluronic acid sugar and 100 units of hyaluronidenide to react. As the molecular weight of hyaluronic acid decreased over time, the hyaluronic acid solution was heated after approximately 12 hours to inhibit the activity of hyaluronides. After the precipitated hyaluronides were removed using a 0.2 um nylon filter (Minisart), the molecular weight of the degraded low molecular weight hyaluronic acid was confirmed by GPC (Gel Permeation Chromatography).

<실시예 2> 히알루론산에 기능기 및 형광염료를 도입하여 히알루론산 합성물을 제조하는 단계 Example 2 Preparation of Hyaluronic Acid Composite by Incorporating Functional Group and Fluorescent Dye into Hyaluronic Acid

실시예 1에서 준비한 히알루론산에 존재하는 환원 말단 (reducing ends)에 히알루론산의 몰비(molar ratio)의 20배에 해당하는 시스타민 (cystamine hydrochloride) 또는 도파민 (dopamine hydrochloride)을 첨가한 후 2시간 동안 잘 섞어주었다. 그 후 sodium cyanoborohydrate (NaBH3CN) 첨가한 후 약 5일간 반응을 시켰다. 반응 물질에 남아있는 NaBH3CN, 시스타민 및 도파민을 제거하기 위해 투석막(MWCO(Molecular Weight Cut Off): 1000)을 이용하여 증류수에서 24시간 동안 투석시켰다. 시스타민을 도입한 히알루론산인 경우, 투석된 용액에 100 밀리몰 (mM)에 해당하는 DTT(1,4-dithiothretitol)를 첨가하여 12시간 동안 반응을 시켰다. 이때 첨가된 DTT는 시스타민과 반응하여 유리 황화기 (free thiol group)를 만드는데 이용되었다. 황화기가 말단에 도입된 히알루론산 용액은 다시 한번 투석막 (MWCO: 1000)을 이용하여 증류수에서 이틀간 투석시킨 후 동결건조 했다. 2 hours after cystamine or dopamine hydrochloride corresponding to 20 times the molar ratio of hyaluronic acid was added to reducing ends present in the hyaluronic acid prepared in Example 1 Mix well. Thereafter, sodium cyanoborohydrate (NaBH 3 CN) was added and reacted for about 5 days. The dialysis membrane (Molecular Weight Cut Off (MWCO): 1000) was dialyzed in distilled water for 24 hours to remove NaBH 3 CN, cystamine and dopamine remaining in the reaction material. In the case of hyaluronic acid introduced with cystamine, DTT (1,4-dithiothretitol) corresponding to 100 mmol (mM) was added to the dialyzed solution and reacted for 12 hours. The added DTT was used to react with cystamine to form a free thiol group. The hyaluronic acid solution in which the sulfidizer was introduced at the end was once again dialyzed in distilled water for two days using a dialysis membrane (MWCO: 1000) and then lyophilized.

형광염료의 도입방법으로 히알루론산에 존재하는 카복실기의 몰비(molar ratio)의 3배에 해당하는 EDC(1-ethyl-3-[3-(dimethlamino)propylcarbodiimide) 와 Hobt(1-hydroxybenzotriazole)를 첨가한 후 2시간 동안 잘 섞어주었다. 그 후 형광염료를 첨가한 후 12시간 동안 반응을 시켰다. 반응 물질에 남아있는 EDC/Hobt 와 형광염료를 제거하기 위해 투석막(MWCO: 2500)을 이용하여 증류수에서 24시간 동안 투석했다. As an introduction method of fluorescent dye, EDC (1-ethyl-3- [3- (dimethlamino) propylcarbodiimide) and Hobt (1-hydroxybenzotriazole) corresponding to three times the molar ratio of the carboxyl group present in hyaluronic acid are added. After mixing well for 2 hours. After the addition of the fluorescent dye it was reacted for 12 hours. Dialysis membrane (MWCO: 2500) was used for dialysis in distilled water for 24 hours to remove EDC / Hobt and fluorescent dye remaining in the reaction material.

유리 황화기(free thiol group)가 히알루론산 말단에 도입된 양을 확인하기 위하여, 엘만 측정법(Elman's assay)을 사용하였다. 엘만 측정법에 따라, 히알루론 산 말단에 유리 황화기의 도입 정도는 반응 가능한 환원 말단의 70~99% 로 측정되었다.In order to confirm the amount of free thiol group introduced into the hyaluronic acid terminal, Elman's assay was used. According to the Elman measurement, the degree of introduction of the free sulfide group at the hyaluronic acid terminal was determined to be 70 to 99% of the reactable reducing terminal.

<실시예 3> 히알루론산 합성물을 금 나노 입자 표면에 고정하는 단계Example 3 Fixing Hyaluronic Acid Composite to Gold Nanoparticle Surface

실시예 2에서 제조한 히알루론산 합성물을 이용하여 금 나노입자 표면에 히알루론산의 말단을 고정하여 보았다. 유리 황화기(-SH)와 금 나노입자의 강한 접촉 및 고정성(high binding affinity)을 이용한 금속 황화 결합(sulfur metal bond)을 통해 히알루론산 합성물을 금 나노입자 표면에 고정하였다. <도 1 참조>The hyaluronic acid composite prepared in Example 2 was used to fix the terminal of hyaluronic acid on the surface of gold nanoparticles. The hyaluronic acid composite was immobilized on the surface of the gold nanoparticles through a sulfur metal bond using a strong contact and high binding affinity of the free sulfide (-SH) and the gold nanoparticles. <See FIG. 1>

금 나노 입자와 히알루론산의 몰 비는 1:100 ~ 1:10000 으로 금 나노입자의 크기에 따라 몰 비의 변화를 주었다. 접목 과정 중에 소금을 첨가함으로써 히알루론산과 금 나노입자 간의 정전기적 반발작용 (electrostatic repulsion)을 줄일 수 있었다. 최종 소금 농도는 약 0.3 ~ 2 M 농도를 유지하도록 하였다. 고정된 히알루론산의 양을 정량하였을 때, 황화기인 경우 대략 40 개의 히알루론산이 16 nm 금 나노입자에 고정된 것으로 확인이 되었으며, 도파기인 경우 대략 130개의 히알루론산이 16 nm 금 나노 입자에 고정된 것으로 나타났다 . The molar ratio of gold nanoparticles to hyaluronic acid ranges from 1: 100 to 1: 10000, depending on the size of the gold nanoparticles. Adding salt during the grafting process reduced the electrostatic repulsion between hyaluronic acid and gold nanoparticles. The final salt concentration was maintained at about 0.3-2 M. When the amount of immobilized hyaluronic acid was quantified, it was confirmed that about 40 hyaluronic acids were fixed to 16 nm gold nanoparticles in the sulfide group, and about 130 hyaluronic acids were fixed to 16 nm gold nanoparticles in the waveguide group. It appeared that.

<실시예 4> 히알루론산이 고정된 금 나노입자의 안정성 확인Example 4 Confirmation of Stability of Gold Nanoparticles Fixing Hyaluronic Acid

실시예 3에서 제조한 히알루론산이 고정된 금 나노입자의 안정성을 확인하는 실험을 해보았다. 다양한 산도와 소금 농도 그리고 혈청 포함 용액에서 그 안정성이 우수하다는 것을 보여주었다. <도 2 참조>An experiment was conducted to confirm the stability of the gold nanoparticles to which hyaluronic acid prepared in Example 3 was fixed. It has been shown to have excellent stability in various acid and salt concentrations and serum-containing solutions. <See FIG. 2>

도 2에서, 처음 4개의 시약병들은 HHAuNPs(형광염료로 하일라이트 염료가 사용된 히알루론산 금 나노입자)를 이용하여 pH 2.0, pH 12.0, 염화나트륨 2M 및 세럼 10% 에서의 안정성 실험결과이다. 실험결과에서 보듯이 아무런 반응이 없는 것으로 확인되어 안정성이 증명되었다. 반면에 나머지 시약병들은 단순히 AuNPs(금 나노입자)을 이용하여 control 과 염화나트륨 0.1M 하에서의 안정성 실험결과를 나타낸 것이다. 실험결과에서 보듯이 특히 염화나트륨 0.1M 하에서는 반응이 일어난 바,(색깔의 뚜렷한 변화 관찰됨) 안정성에 문제가 있다는 결론을 얻을 수 있었다. In Figure 2, the first four reagent bottles are the results of stability experiments at pH 2.0, pH 12.0, sodium chloride 2M and serum 10% using HHAuNPs (hyaluronic acid gold nanoparticles using a highlight dye as a fluorescent dye). As shown in the experimental results, it was confirmed that there was no reaction and the stability was proved. On the other hand, the remaining reagent bottles simply showed AuNPs (gold nanoparticles) for control and stability under 0.1M sodium chloride. As can be seen from the experimental results, it was concluded that there was a problem with stability, especially under 0.1 M sodium chloride (a distinct change in color was observed).

<실시예 5> 히알루론산이 고정된 금 나노 입자를 이용한 활성산소 측정Example 5 Measurement of Active Oxygen Using Gold Nanoparticles Fixed with Hyaluronic Acid

다양한 활성산소를 이용하여 실시예 3에서 제조한 히알루론산이 고정된 금 나노 입자를 이용한 활성산소 측정도를 확인해 보았다. 각각의 활성산소 발생군 100 uM을 금 나노입자와 3시간 동안 반응시킨 결과, 활성산소에 의한 히알루론산의 분해를 형광 복원 정도에 따라 확인할 수 있었다.<도 3 참조>The active oxygen measurement using the gold nanoparticles fixed hyaluronic acid prepared in Example 3 using a variety of active oxygen was confirmed. When 100 uM of each of the active oxygen generation groups were reacted with the gold nanoparticles for 3 hours, the decomposition of hyaluronic acid by the active oxygen was confirmed according to the degree of fluorescence recovery.

도 3에서, 각각의 활성산소는 수페록사이드(superoxide), 하이드로젠 페록사이드(hydrogen peroxide) 및 하이드록시 라디칼(hydroxyl radical)을 나타내며, HAdase는 히알루로니데이즈(hyaluronidase)를, GSH는 글루타치온(glutathione)를 , DTT는 디타이어트레이톨(Dithiothreitol)를 나타낸다. 실험결과 본 발명이 가장 민감하게 반응하여 검출가능한 활성산소는 수페록사이드(superoxide)이며, 히알루론산을 분해하는 히알루로니데이즈(hyaluronidase) 역시 검출이 용이함을 알 수 있었다. In FIG. 3, each active oxygen represents a superoxide, a hydrogen peroxide and a hydroxyl radical, HAdase is hyaluronidase, and GSH is glutathione. ), And DTT stands for Dithiothreitol. As a result of the experiment, the most sensitive reaction of the present invention was detectable active oxygen (superoxide), and hyaluronatease (hyaluronidase) that decomposes hyaluronic acid was also easily detected.

또한 활성산소(superoxide radical)의 농도에 따른 형광 복원 정도도 확인할 수 있었다.<도 4 참조> In addition, the degree of fluorescence recovery according to the concentration of the active oxygen (superoxide radical) was also confirmed.

<실시예 6> 히알루론산이 고정된 금 나노 입자를 이용한 세포내 활성산소 측정 Example 6 Measurement of Intracellular Active Oxygen Using Gold Nanoparticles Fixed with Hyaluronic Acid

실시예 3에서 제조한 히알루론산이 고정된 금 나노 입자를 이용하여 세포내의 활성산소 증가를 측정해 보았다. 대식 세포 내의 (J774.A1 macrophages) 활성산소 증가를 리포폴리사카라이드(lipopolysaccharide)를 이용하여 증가시켰을 경우 그 변화를 확인할 수 있는지를 측정해 보았다. 96 well plate에 약 50000 개의 세포를 분주하였을 경우, 0.5 nM의 히알루론산이 고정된 금 나노 입자를 이용하여 시간의 따른 활성 산소 증가를 측정하였다. < 도 5 참조> Using the hyaluronic acid-immobilized gold nanoparticles prepared in Example 3 was measured the increase of free radicals in the cell. We measured whether the increase in free radicals (J774.A1 macrophages) in macrophages was increased by using lipopolysaccharide. When 50000 cells were dispensed into a 96 well plate, 0.5 nM hyaluronic acid-immobilized gold nanoparticles were used to measure the increase in free radicals over time. <See FIG. 5>

본 발명은 활성산소를 보다 효과적으로 감지하며 인체에 무해한 히알루론산을 이용함으로써, 공학분야뿐 아니라 의학분야의 계통에서도 응용이 가능하다. The present invention is more effective in detecting free radicals and by using hyaluronic acid that is harmless to the human body, it is possible to apply not only in engineering but also in the field of medicine.

또한 본 발명은 발명자들에게는 더욱 향상된 추가 발명의 의욕을 고취시키며, 이를 이용한 기업 등에게는 연구개발의 투자 동기를 부여함으로써, 결국 국가의 산업발전에 이바지할 수 있을 것이다.In addition, the present invention inspires the inventors to further improve the motivation of the invention, and by using the same to motivate the investment in research and development to companies, etc., will eventually contribute to the industrial development of the country.

도 1은 히알루론산이 금 나노입자 표면에 접목되어 있는 것을 나타낸 것으로 나노입자표면 에너지전이(nanoparticle surface energy transer: NSET)를 통한 히알루론산에 도입된 형광염료의 형광소멸 및 접목된 히알루론산의 분해를 통한 형광복원을 설명하고 있다. Figure 1 shows that hyaluronic acid is grafted on the surface of the gold nanoparticles, the fluorescent disappearance of the fluorescent dye introduced into the hyaluronic acid through the nanoparticle surface energy transfer (NSET) and decomposition of the grafted hyaluronic acid Fluorescence restoration is described.

도 2는 본 발명에서 제조된 히알루론산이 접목된 금 나노입자의 안정성을 산성도의 변화 및 염도 그리고 혈청 상태에서 확인한 것을 나타내고 있다.Figure 2 shows that the stability of the gold nanoparticles grafted hyaluronic acid prepared in the present invention confirmed the change in acidity and salinity and serum state.

도 3은 다양한 활성산소를 이용하여 각각의 활성산소 발생군 100 uM을 금 나노입자와 3시간 동안 반응시킨 결과, 활성산소에 의한 히알루론산의 분해를 형광 복원 정도에 따라 확인한 것을 나타내고 있다.FIG. 3 shows that 100 uM of each active oxygen generation group was reacted with gold nanoparticles for 3 hours using various active oxygen, and the decomposition of hyaluronic acid by active oxygen was confirmed according to the degree of fluorescence recovery.

도 4는 활성산소의 농도에 따른 형광 복원 정도를 나타내고 있다. 4 shows the degree of fluorescence recovery according to the concentration of active oxygen.

도 5는 히알루론산이 접목된 금 나노입자를 이용하여 대식 세포내 (macrophages)의 활성산소의 증가를 측정한 것을 나타내고 있다. FIG. 5 shows the increase of free radicals in macrophages using gold nanoparticles grafted with hyaluronic acid.

Claims (8)

a) 히알루론산에 히알루론니데이즈를 첨가하여 저분자량 히알루론산을 제조하는 단계;a) adding hyaluronic acid to hyaluronic acid to produce low molecular weight hyaluronic acid; b) 상기 저분자량 히알루론산의 카복실기에 형광염료를 도입하며, 상기 저분자량 히알루론산의 환원 말단에 금 나노입자 표면에 고정화 시킬 수 있는 기능기를 도입하여 저분자량 히알루론산 합성물을 제조하는 단계; 및 b) preparing a low molecular weight hyaluronic acid compound by introducing a fluorescent dye into the carboxyl group of the low molecular weight hyaluronic acid and introducing a functional group which can be immobilized on the surface of the gold nanoparticles at the reducing end of the low molecular weight hyaluronic acid; And c) 상기 저분자량 히알루론산 합성물을 금 나노입자 표면에 고정하는 단계;를 포함하는 것을 특징으로 하는 활성산소를 감지하는 히알루론산 금 나노입자의 제조방법.c) fixing the low molecular weight hyaluronic acid composite to the surface of the gold nanoparticles. 제1항에 있어서,The method of claim 1, 상기 a) 단계에서 저분자량 히알루론산의 분자량은 1000~100000 Da 임을 특징으로 하는 히알루론산 금 나노입자의 제조방법.The molecular weight of the low molecular weight hyaluronic acid in the step a) is a method for producing hyaluronic acid gold nanoparticles, characterized in that 1000 ~ 100,000 Da. 제1항에 있어서,The method of claim 1, 상기 b) 단계에서 기능기는 유리 황화기, 도파민기 및 아민기로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하는 히알루론산 금 나노입자의 제조방법.The functional group in the step b) is a method of producing hyaluronic acid gold nanoparticles, characterized in that any one selected from the group consisting of free sulfide groups, dopamine groups and amine groups. 제1항에 있어서,The method of claim 1, 상기 b) 단계에서 기능기는 히알루론산의 환원 말단에 히알루론산의 몰비의 10~30배로 첨가하는 것을 특징으로 하는 히알루론산 금 나노입자의 제조방법.In the step b), the functional group is a method for producing gold hyaluronic acid nanoparticles, characterized in that the addition of 10 to 30 times the molar ratio of hyaluronic acid to the reduction terminal of hyaluronic acid. 제1항에 있어서,The method of claim 1, 상기 b) 단계에서 형광염료는 로다민(rhodamine), 시아닌(cyanine)3, 시아닌(cyanine)5, 시아닌(cyanine)7, 알렉사 염료(Alexa dyes), 하일라이트 플루오르 염료(Hylite fluor dyes), 스틸벤 유도체, 이미다졸 유도체 및 쿠마린 유도체로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하는 히알루론산 금 나노입자의 제조방법.In step b), the fluorescent dye is rhodamine, cyanine 3, cyanine 5, cyanine 5, cyanine 7, Alexa dyes, Hylite fluor dyes, steel Method of producing a hyaluronic acid gold nanoparticles, characterized in that any one selected from the group consisting of ben derivatives, imidazole derivatives and coumarin derivatives. 제1항에 있어서,The method of claim 1, 상기 c) 단계에서 금 나노입자와 히알루론산의 몰 비는 1: 100 ~ 1: 10000 으로 하는 것을 특징으로 하는 히알루론산 금 나노입자의 제조방법.The molar ratio of the gold nanoparticles and hyaluronic acid in the step c) is 1: 100 ~ 1: 10000 The method of producing hyaluronic acid gold nanoparticles, characterized in that. 제1항에 있어서,The method of claim 1, 상기 c) 단계에서 금 나노입자는 입자의 크기가 4 ~ 200 ㎚ 의 지름을 가지며, 히알루론산이 황화금속 결합으로 고정화되는 것을 특징으로 하는 히알루론산 금 나노입자의 제조방법.In the step c), the gold nanoparticles have a particle size of 4 to 200 nm, and hyaluronic acid is a method for producing gold nanoparticles, wherein the hyaluronic acid is fixed by a metal sulfide bond. 상기 제1항 내지 제7항 중 어느 한 항의 방법으로 제조된 히알루론산 금 나 노입자.Gold nanoparticles of hyaluronic acid prepared by the method of any one of claims 1 to 7.
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KR101104775B1 (en) * 2009-10-28 2012-01-12 한국과학기술원 A Method for Modifying a Solid Substrate Surface Using One-Pot Process
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