KR102270784B1 - Novel blue fluorescent substance based on dextran polymer - Google Patents
Novel blue fluorescent substance based on dextran polymer Download PDFInfo
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Abstract
Description
본 발명은 새로운 덱스트란 고분자 기반의 청색 형광체에 관한 것으로, 보다 구체적으로 7-히드록시-쿠마린이 결합된 다이벤조사이클로옥틴으로 기능화되어 생체 내 혈관 영상화에 활용될 수 있는 덱스트란 고분자에 관한 것이다.The present invention relates to a novel dextran polymer-based blue phosphor, and more particularly, to a dextran polymer functionalized with 7-hydroxy-coumarin-coupled dibenzocyclooctine and utilized for in vivo vascular imaging.
혈관 영상화 기술은 혈관 전체의 영상 또는 혈관 내의 특정 물질 및 세포체를 표지하는 것으로 혈관 관련 질병의 이해와 더불어 기초 연구에 있어 다양하게 활용될 수 있다. 생체 내에서 일어나는 신부전증, 폐혈관 그리고 심혈관계 관련 질병들은 비정상적인 혈류의 흐름을 관찰함으로써 진단, 치료, 임상 등의 연구가 진행될 할 수 있다. 특히, 형광을 기반으로 한 혈관 영상기술은 접근이 용이하며 관련 정보를 직관적으로 제공받을 수 있다는 점에서 다양한 연구 분야에 기본적으로 활용되고 있다. 따라서, 생체 내 혈관을 선택적으로 영상화할 수 있는 소재의 개발은 중요하다. Vascular imaging technology can be used in various ways in basic research along with the understanding of blood vessel-related diseases as an image of the entire blood vessel or labeling of specific substances and cell bodies in the blood vessel. Diagnosis, treatment, and clinical research can be conducted by observing abnormal blood flow for renal failure, pulmonary vascular and cardiovascular diseases occurring in vivo. In particular, fluorescence-based vascular imaging technology is basically used in various research fields in that it is easy to access and can intuitively receive related information. Therefore, it is important to develop a material that can selectively image blood vessels in vivo.
최근에는 기초과학 분야에서 생체 내 영상화 연구에 이광자 현미경(two-photon microscopy, TPM)이 활용되고 있다. 이광자 현미경은 다른 영상화 기술과 비교하여 영상 제공 시간이 빠르며, 높은 해상도의 3차원 해상도를 제공하고, 약 650-1000 nm의 긴 여기 파장대(excitation wavelength)를 사용하기 때문에 광 손상(photo-damage)과 광 퇴색(photo-bleaching)이 낮고 또한 조직에 깊이 침투할 수 있다는 장점이 있다(Denk. W. et al., Science 1990, 248, 73; Kim H.M. et al., Chem. Rev. 2015, 115, 5014; Kim. D. et al., J. Am. Chem. Soc. 2015, 137, 6781; Kim. D. et al., Adv. Mater. 2017, 29, 1703309; Kim D. et al., Org. Biomol. Chem. 2014, 12, 4550). Recently, two-photon microscopy (TPM) has been used for in vivo imaging research in the field of basic science. Compared to other imaging technologies, two-photon microscopy has a fast image delivery time, provides high-resolution three-dimensional resolution, and uses a long excitation wavelength of about 650-1000 nm to prevent photo-damage and damage. It has the advantage of low photo-bleaching and deep penetration into the tissue (Denk. W. et al., Science 1990, 248, 73; Kim HM et al., Chem. Rev. 2015, 115, 5014; Kim. D. et al., J. Am. Chem. Soc. 2015, 137, 6781; Kim. D. et al., Adv. Mater. 2017, 29, 1703309; Kim D. et al., Org. (. Biomol. Chem. 2014, 12, 4550).
이에 이광자 현미경에 활용될 수 있는 혈관 영상화 용도의 소재도 다양하게 개발되어왔다. 하지만, 현재까지 개발된 혈관 영상화 용도의 형광 소재들은 주로 녹색과 적색을 나타내는 것들이 대부분이다. 이는 생체 형광 영상화 시 세포 또는 영상화 소재가 청색의 짧은 파장대의 레이저에 낮은 안정성을 가짐에 따라 쉽게 광 손상 및 광 퇴색이 일어나게 되기 때문이다.Accordingly, various materials for vascular imaging that can be used in two-photon microscopy have been developed. However, most of the fluorescent materials for vascular imaging that have been developed so far are mainly green and red. This is because, during biofluorescence imaging, light damage and photobleaching easily occur as cells or imaging materials have low stability to a blue, short wavelength laser.
일부 상업적으로 판매되고 있는 청색의 형광 혈관 조영제가 있지만, 이러한 조영제의 경우 맥아 균 응집소(wheat germ agglutinin, WGA)에 형광체를 부착되어 있어, 많을 양을 사용해야만 혈관 전체를 염색하는 것이 가능하고, 항체를 이용하기 때문에 혈관 벽만 영상을 제공하거나, 다른 세포체와 결합하여 일정 부분에서만 형광을 제공한다는 단점을 가진다.There are some commercially available blue fluorescent angiography agents, but in the case of these contrast agents, a phosphor is attached to wheat germ agglutinin (WGA), so it is possible to stain the entire blood vessel only by using a large amount of antibody, It has the disadvantage of providing an image of only the vessel wall or providing fluorescence only in a certain portion by combining with other cell bodies.
이로 인해, 세포체 또는 특정 기질은 녹색 또는 적색의 형광체로 표지하는 경우가 일반적이며, 혈관을 같이 염색하여 형광 분석을 해야 하는 경우 기존 혈관 영상용 형광 조영제와 신호 파장대가 겹쳐 결과분석이 불가능하다는 문제가 있다. For this reason, it is common for cell bodies or specific substrates to be labeled with green or red fluorescent substances, and when fluorescence analysis is performed by staining blood vessels together, there is a problem that the signal wavelength band overlaps with the existing vascular imaging fluorescence contrast agent, making it impossible to analyze the results have.
이에, 본 발명은 상기 종래기술의 문제점을 해결하기 위하여, 3-하이드록시-쿠마린(7-hydroxy-coumarin)이 결합된 다이벤조사이클로옥틴(DBCO; Dibenzocyclooctyne)을 덱스트란(dextran) 고분자에 부착한 혈관 영상화용 청색 형광체를 제공하는 것을 목적으로 한다.Accordingly, the present invention is a method of attaching dibenzocyclooctyne (DBCO) to a dextran polymer to which 3-hydroxy-coumarin (7-hydroxy-coumarin) is bound to solve the problems of the prior art. An object of the present invention is to provide a blue phosphor for imaging blood vessels.
또한, 본 발명은 상기 청색 형광체을 포함하는 혈관 영상화용 조성물을 제공하는 것을 목적으로 한다.Another object of the present invention is to provide a composition for imaging blood vessels comprising the blue phosphor.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.
상기와 같은 목적을 달성하기 위하여, 본 발명은 하기 화학식 1로 표시되는 화합물과 이의 레지오아이소머의 혼합물인 청색 형광체를 제공한다. In order to achieve the above object, the present invention provides a blue phosphor, which is a mixture of a compound represented by the following Chemical Formula 1 and a regioisomer thereof.
[화학식 1][Formula 1]
상기에서 m 은 23 내지 24의 정수이고, n은 365 내지 366의 정수이다. In the above, m is an integer from 23 to 24, and n is an integer from 365 to 366.
또한, 본 발명은 상기 청색 형광체을 포함하는, 혈관 영상화용 조성물을 제공한다.In addition, the present invention provides a composition for imaging blood vessels comprising the blue phosphor.
또한, 본 발명은 상기 청색 형광체를 세포 또는 조직에 처리하고 관측하는 단계를 포함하는, 혈관 영상화 방법을 제공한다.In addition, the present invention provides a blood vessel imaging method comprising the step of processing and observing the blue phosphor in cells or tissues.
또한, 본 발명은 하기 화학식 2로 표시되는 다이벤조-사이클로옥틴 그룹이 부착된 덱스트란 (DBCO-functionalized Dextran)에 메탄올(methanol)을 첨가한 후, 하기 화학식 3으로 표시되는 3-아지도-7-하이드록시쿠마린(3-azido-7-hydroxycoumarin)을 혼합하고 교반기를 사용하여 교반하는 단계를 포함하는 상기 청색 형광체의 제조방법을 제공한다. In addition, in the present invention, after adding methanol to dextran to which a dibenzo-cyclooctin group is attached (DBCO-functionalized Dextran) represented by the following formula (2), 3-azido-7 represented by the following formula (3) -Provides a method for producing the blue phosphor, comprising mixing 3-azido-7-hydroxycoumarin and stirring the mixture using a stirrer.
[화학식 2][Formula 2]
상기에서 m 은 23 내지 24의 정수이고, n은 365 내지 366의 정수이다. In the above, m is an integer from 23 to 24, and n is an integer from 365 to 366.
[화학식 3] [Formula 3]
본 발명에 따른 혈관 영상화용 신규 청색 형광체는 이광자 여기 특성을 가지기 때문에 깊은 세포 투과성, 낮은 세포 파괴성, 생체 내 헤모글로빈 등에 의한 소광 등에 영향을 적게 받을 뿐 아니라, 초점 부위만 여기 시키므로 매우 높은 해상도를 구현할 수 있다. Since the new blue phosphor for imaging blood vessels according to the present invention has two-photon excitation properties, it is not only less affected by deep cell permeability, low cell destruction, and quenching by hemoglobin in vivo, but also excites only the focal region, so that very high resolution can be realized. have.
또한, 70 kDa의 분자량을 가지는 고분자 덱스트란을 뼈대로 사용함으로써, 혈관 밖으로 누출되지 않으며, 혈관 내부를 전부 염색할 수 있어 혈관 내부 또는 혈관 외부의 세포체들 또는 물질들 간의 상호작용을 효과적으로 관찰할 수 있다. In addition, by using the polymer dextran having a molecular weight of 70 kDa as a skeleton, it does not leak out of the blood vessel and the inside of the blood vessel can be completely stained, so that the interaction between cell bodies or substances inside or outside the blood vessel can be effectively observed. have.
또한, 녹색 및 적색으로 표지되어 있는 세포 또는 특정 생체 내 기질과 상호 보완적으로 혈관 형광 영상화에 활용 가능하므로 혈관 관련 질병 연구 및 치료제 개발, 임상학적 적용연구, 생체면역 관련 연구 등과 같은 분야에 폭넓게 활용될 수 있다.In addition, since it can be used for vascular fluorescence imaging complementary to cells labeled with green and red or specific in vivo substrates, it is widely used in fields such as vascular-related disease research and therapeutic development, clinical application research, and bioimmunity-related research. can be
도 1은 본 발명에 따른 청색 형광체의 합성 방법을 나타낸 것이다.
도 2는 본 발명에 따른 청색 형광체(HCD-70K)와 3-아지도-7-하이드록시쿠마린 (AHC: 3-azido-7-hydroxycoumarin, 10 μM) 각각의 흡광 및 형광 그래프를 측정한 결과를 나타낸 것이다.
도 3은 다이벤조사이클로옥틴(DBCO) 그룹만이 부착된 덱스트란(DBCO-Dextran, 70 kDa, 1 mg/mL)의 흡광 및 형광 그래프를 측정한 결과를 나타낸 것이다.
도 4는 본 발명에 따른 청색 형광체(HCD-70K)의 자외선 하에서 형광 특성을 측정한 결과이다.
도 5는 본 발명에 따른 청색 형광체(HCD-70K)의 사람 혈청(human serum) 내에서의 흡광 및 형광 측정 결과를 나타낸 그래프이다.
도 6은 본 발명에 따른 청색 형광체(HCD-70K)의 사람 혈청에서의 안정성과 광 안정성(photo-stability)을 형광 세기로 나타낸 그래프이다.
도 7은 본 발명에 따른 청색 형광체(HCD-70K)의 HeLa 암세포에서 세포 내 섭취(endocytosis) 여부를 일광자 형광 현미경을 통해 관찰한 결과이다.
도 8은 본 발명에 따른 청색 형광체(HCD-70K)의 세포독성을 확인한 결과이다.
도 9는 본 발명에 따른 청색 형광체(HCD-70K)를 정맥 주사한 쥐의 등 진피(dorsal dermis)의 생체 내 혈관을 이광자 현미경을 이용하여 촬영한 결과이다.
도 10은 본 발명에 따른 청색 형광체 (HCD-70K)를 정맥 주사한 쥐의 대퇴골 골수(calvarial bone marrow), 등 진피(dorsal dermis), 및 고환거근(cremaster muscle)의 생체 내 혈관을 촬영한 결과와 50분 내의 형광 세기를 이광자 현미경을 이용하여 측정한 그래프이다.
도 11은 본 발명에 따른 청색 형광체(HCD-70K)의 혈관 누출 정도를 측정한 결과이다.
도 12는 본 발명에 따른 청색 형광체(HCD-70K)를 정맥 주사한 쥐의 뇌 혈관을 2가지 색의 추가적인 세포체들과 함께 이광자 현미경을 이용하여 생체 내 혈관을 촬영한 결과이다. 1 shows a method for synthesizing a blue phosphor according to the present invention.
Figure 2 shows the results of measuring the absorption and fluorescence graphs of the blue phosphor (HCD-70K) and 3-azido-7-hydroxycoumarin (AHC: 3-azido-7-hydroxycoumarin, 10 μM) according to the present invention. it has been shown
3 shows the results of measuring the absorption and fluorescence graphs of dextran (DBCO-Dextran, 70 kDa, 1 mg/mL) to which only the dibenzocyclooctyne (DBCO) group is attached.
4 is a result of measuring the fluorescence characteristics of the blue phosphor (HCD-70K) according to the present invention under ultraviolet light.
5 is a graph showing the results of measurement of absorption and fluorescence in human serum of the blue phosphor (HCD-70K) according to the present invention.
6 is a graph showing the stability and photo-stability in human serum of the blue phosphor (HCD-70K) according to the present invention as fluorescence intensity.
7 is a result of observing the endocytosis of the blue phosphor (HCD-70K) in HeLa cancer cells according to the present invention through a one-photon fluorescence microscope.
8 is a result confirming the cytotoxicity of the blue phosphor (HCD-70K) according to the present invention.
9 shows the results of photographing using a two-photon microscope in vivo blood vessels in the dorsal dermis of a rat intravenously injected with a blue phosphor (HCD-70K) according to the present invention.
10 is a result of photographing blood vessels in vivo of the calvarial bone marrow, dorsal dermis, and cremaster muscle of a rat intravenously injected with a blue phosphor (HCD-70K) according to the present invention. and the fluorescence intensity within 50 min was measured using a two-photon microscope.
11 is a result of measuring the degree of vascular leakage of the blue phosphor (HCD-70K) according to the present invention.
12 shows the results of photographing blood vessels in vivo using a two-photon microscope together with additional cell bodies of two colors in the brain blood vessels of mice intravenously injected with the blue phosphor (HCD-70K) according to the present invention.
상기한 기술적 과제를 해결하기 위하여, 본 발명은 하기 화학식 1로 표시되는 화합물과 이의 레지오아이소머의 혼합물인 청색 형광체를 제공한다. In order to solve the above technical problem, the present invention provides a blue phosphor, which is a mixture of a compound represented by the following Chemical Formula 1 and a regioisomer thereof.
[화학식 1][Formula 1]
상기에서 m 은 23 내지 24의 정수이고, n은 365 내지 366의 정수이다. In the above, m is an integer from 23 to 24, and n is an integer from 365 to 366.
본 발명의 일 실시예로, 상기 화합물은 7-히드록시-쿠마린(7-hydroxy-coumarin)이 결합된 다이벤조사이클로옥틴(DBCO; Dibenzocyclooctyne)이 부착된 덱스트란(dextran) 고분자로, 상기 덱스트란 고분자는 몰 질량이 70kDa인 것이 바람직하다.In an embodiment of the present invention, the compound is a dextran polymer to which 7-hydroxy-coumarin is bound to dibenzocyclooctyne (DBCO; Dibenzocyclooctyne), and the dextran The polymer preferably has a molar mass of 70 kDa.
또한, 상기 청색 형광체는 이광자 흡수 형광체일 수 있다.In addition, the blue phosphor may be a two-photon absorbing phosphor.
본 발명에 따른 상기 청색 형광체는 혈관 내에서 장시간 누출되지 않으며, 항체를 사용하지 않으므로 여타의 세포체들과 결합하거나 세포 내로 섭취되지 않고, 혈관 내에서 순환하므로 혈관 전체를 염색할 수 있다.The blue phosphor according to the present invention does not leak in the blood vessel for a long time, does not use an antibody, does not bind to other cell bodies or ingested into the cell, and circulates in the blood vessel, so that the entire blood vessel can be stained.
또한, 상기 청색 형광체를 포함하는, 혈관 영상화(imaging)용 조성물을 제공한다.In addition, there is provided a composition for imaging blood vessels comprising the blue phosphor.
상기 조성물은 생체 내에서 혈관 특이적으로 청색 형광을 나타낸다.The composition exhibits blood vessel-specific blue fluorescence in vivo.
상기 영상화용 조성물 내의 유효성분으로서 상기 청색 형광체의 함량은 사용 형태 및 목적 등에 따라 적절하게 조정될 수 있다.The content of the blue phosphor as an active ingredient in the composition for imaging may be appropriately adjusted according to the type and purpose of use.
상기 영상화용 조성물 내의 유효성분으로서 상기 청색 형광체의 함량은 적용 동물에 따라 적절하게 조정될 수 있으며, 이에 한정되지 않으나, 바람직하게는 5-100 mg/kg, 보다 바람직하게는 10-50 mg/kg, 가장 바람직하게는 25 mg/kg일 수 있다. The content of the blue phosphor as an active ingredient in the imaging composition may be appropriately adjusted according to the applied animal, but is not limited thereto, but preferably 5-100 mg/kg, more preferably 10-50 mg/kg, Most preferably, it may be 25 mg/kg.
상기 영상화용 조성물은 포유동물을 비롯한 기타 동물에 투여될 수 있다. 투여 방식은 통상적으로 정맥 주사에 의해 투여될 수 있으며, 통상의 방법에 따라 멸균 주사 용액의 형태의 비경구 제형으로 사용될 수도 있다.The composition for imaging may be administered to other animals including mammals. The mode of administration may be administered by intravenous injection, and may be used as a parenteral formulation in the form of a sterile injection solution according to a conventional method.
또한, 본 발명은 상기 청색 형광체를 세포 또는 조직에 처리하고 관측하는 단계를 포함하는, 혈관의 영상화 방법을 제공한다.In addition, the present invention provides a method for imaging blood vessels, comprising the step of processing and observing the blue phosphor to a cell or tissue.
또한, 본 발명은 하기 화학식 2로 표시되는 다이벤조-사이클로옥틴 그룹이 부착된 덱스트란 (DBCO-functionalized Dextran)에 메탄올(methanol)을 첨가한 후, 하기 화학식 3으로 표시되는 3-아지도-7-하이드록시쿠마린(3-azido-7-hydroxycoumarin)을 혼합하고 교반기를 사용하여 교반하는 단계를 포함하는 하기 화학식 1로 표시되는 화합물과 이의 레지오아이소머의 혼합물인 청색 형광체의 제조방법을 제공한다. In addition, in the present invention, after adding methanol to dextran to which a dibenzo-cyclooctin group is attached (DBCO-functionalized Dextran) represented by the following formula (2), 3-azido-7 represented by the following formula (3) -Provides a method for preparing a blue phosphor, which is a mixture of a compound represented by the following formula (1) and a regioisomer thereof, including mixing with hydroxycoumarin (3-azido-7-hydroxycoumarin) and stirring using a stirrer.
[화학식 1][Formula 1]
상기에서 m 은 23 내지 24의 정수이고, n은 365 내지 366의 정수이다. In the above, m is an integer from 23 to 24, and n is an integer from 365 to 366.
[화학식 2] [Formula 2]
상기에서 m 은 23 내지 24의 정수이고, n은 365 내지 366의 정수이다. In the above, m is an integer from 23 to 24, and n is an integer from 365 to 366.
[화학식 3] [Formula 3]
이하, 본 발명의 이해를 돕기 위하여 실시예 등을 들어 상세하게 설명하기로 한다. 그러나 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 하기 실시예들에 한정되는 것으로 해석되어서는 안 된다. 본 발명의 실시예들은 당 업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples and the like will be described in detail to help the understanding of the present invention. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.
실시예 1: 청색 형광체(HCD-70K)의 합성Example 1: Synthesis of blue phosphor (HCD-70K)
본 발명의 일실시예에 따른 3-하이드록시-쿠마린이 결합된 다이벤조사이클로옥틴 기반의 덱스트란 구조의 청색 형광체(hydroxy-coumarine-dextran; HCD-70K)를 하기 방법에 따라 합성하였다. 합성과정은 도 1에 나타낸 바와 같다.A blue phosphor (hydroxy-coumarine-dextran; HCD-70K) having a dextran structure based on dibenzocyclooctin to which 3-hydroxy-coumarin is bound according to an embodiment of the present invention was synthesized according to the following method. The synthesis process is as shown in FIG. 1 .
합성 시작 물질인 다이벤조-사이클로옥틴 그룹이 부착된 덱스트란 (DBCO-functionalized Dextran, 70 kDa)(2.5mg)이 들어있는 4 mL 바이알 (vial)에 메탄올 (methanol) 200 μL를 첨가하였다. 그리고 3-아지도-7-하이드록시쿠마린(AHC; 3-azido-7-hydroxycoumarin, 40 μg, 10 mM) 25 μL를 추가하고, 이를 교반기를 사용하여 37
실시예 2: 청색 형광체(HCD-70K)의 흡광 및 형광 특성 확인Example 2: Confirmation of absorption and fluorescence properties of blue phosphor (HCD-70K)
상기 실시예 1에 따라 제조된 청색 형광체(HCD-70K)(1 mg/mL)의 흡광 및 형광 변화를 증류수 조건하에서 측정하였다. 흡수 스펙트럼(UV/Vis absorption spectra) 분석을 위해서 애질런트(Agilent)사의 UV/Vis 분광광도계(UV/Vis spectrophotometer)를 사용하였고, 형광 스펙트럼(fluorescence spectra) 분석을 위해서 시마즈(SHIMADZU0)사의 분광형광 광도계(spectro-fluorophotometer)를 사용하였다. 이때, 각 기기에 HCD-70K를 넣어주는 셀(cell)로는 1 cm 두께의 석영표준셀(standard quartz cell)을 이용하였다. Changes in absorption and fluorescence of the blue phosphor (HCD-70K) (1 mg/mL) prepared according to Example 1 were measured under distilled water conditions. For UV/Vis absorption spectra analysis, Agilent's UV/Vis spectrophotometer was used, and for fluorescence spectra analysis, Shimadzu's spectrofluorophotometer (SHIMADZU0) spectro-fluorophotometer) was used. In this case, a standard quartz cell having a thickness of 1 cm was used as a cell for putting HCD-70K in each device.
도 2는 본 발명에 따른 청색 형광체(HCD-70K)와 3-아지도-7-하이드록시쿠마린(AHC: 3-azido-7-hydroxycoumarin, 10 μM) 각각의 흡광 및 형광 그래프를 측정한 결과를 나타낸 것이다. 도 3은 반응 전 시작 물질인 다이벤조사이클로옥틴(DBCO) 그룹만이 부착된 덱스트란(DBCO-Dextran, 70 kDa, 1 mg/mL)의 흡광 및 형광 그래프를 측정한 결과를 나타낸 것이다. Figure 2 shows the results of measuring the absorption and fluorescence graphs of the blue phosphor (HCD-70K) and 3-azido-7-hydroxycoumarin (AHC: 3-azido-7-hydroxycoumarin, 10 μM) according to the present invention. it has been shown 3 shows the measurement results of absorption and fluorescence graphs of dextran (DBCO-Dextran, 70 kDa, 1 mg/mL) to which only the dibenzocyclooctin (DBCO) group, which is the starting material, is attached before the reaction.
도 2와 도 3에 나타난 바와 같이 시작 물질인 3-아지도-7-하이드록시쿠마린(AHC)과 다이벤조사이클로옥틴(DBCO) 그룹만이 부착된 덱스트란(DBCO-functionalized Dextran)은 형광 특성을 나타내지 않았다. 본 발명의 실시예 1에 따라 제조된 물질인 HCD-70K는 증류수에서 410 nm 파장에서 최고 흡광도를, 464 nm 파장에서 최고 형광 값을 나타내는 것으로 확인되었다. 그리고 도 4에 나타낸 바와 같이, HCD-70K는 자외선 하에서 형광 특성을 나타냄을 확인할 수 있다. As shown in FIGS. 2 and 3, the starting materials, 3-azido-7-hydroxycoumarin (AHC) and dextran (DBCO-functionalized Dextran) to which only dibenzocyclooctyne (DBCO) groups are attached, exhibited fluorescence properties. did not show It was confirmed that HCD-70K, a material prepared according to Example 1 of the present invention, exhibited the highest absorbance at a wavelength of 410 nm and the highest fluorescence value at a wavelength of 464 nm in distilled water. And as shown in FIG. 4 , it can be confirmed that HCD-70K exhibits fluorescence properties under ultraviolet light.
실시예 3: 청색 형광체(HCD-70K)의 혈액 내 안정성 확인Example 3: Confirmation of stability in blood of blue phosphor (HCD-70K)
상기 실시예 1에 따라 제조된 청색 형광체(HCD-70K)의 사람 혈청 내에서의 안정성을 확인하였다. HCD-70K(1 mg/mL)가 혈액 내에서 형광 특성을 가지는지 여부를 확인하기 위하여, 증류수 내에 10%의 사람 혈청(human serum)이 포함된 조건에서 흡수 및 형광 그래프를 측정하였다. 사람 혈청은 시그마(Sigma)사의 제품을 사용하였으며, 흡수 스펙트럼(UV/Vis absorption spectra) 분석을 위해서 애질런트(Agilent)사의 UV/Vis 분광광도계(UV/Vis spectrophotometer)를 사용하였고, 형광 스펙트럼(fluorescence spectra) 분석을 위해서 시마즈(SHIMADZU0)사의 분광형광 광도계(spectro-fluorophotometer)를 사용하였다. 이때, 각 기기에 HCD-70K를 넣어주는 셀(cell)은 1 cm 두께의 석영표준셀(standard quartz cell)을 이용하였다. 그 결과를 도 5에 나타내었다.The stability of the blue phosphor (HCD-70K) prepared according to Example 1 in human serum was confirmed. To determine whether HCD-70K (1 mg/mL) has fluorescence properties in blood, absorption and fluorescence graphs were measured in distilled water containing 10% of human serum. For human serum, Sigma's product was used, and Agilent's UV/Vis spectrophotometer was used for UV/Vis absorption spectra analysis, and fluorescence spectra were used. ) for the analysis, a spectro-fluorophotometer manufactured by SHIMADZU0 was used. At this time, a 1 cm thick standard quartz cell was used as a cell for putting HCD-70K in each device. The results are shown in FIG. 5 .
도 5에 나타난 바와 같이, 상기 실시예 2(증류수)에서처럼 사람 혈청에서도 안정적으로 410 nm 파장에서 최고 흡광도를, 464 nm 파장에서 최고 형광 값을 나타내는 것을 확인하였다.As shown in FIG. 5 , it was confirmed that human serum stably exhibited the highest absorbance at a wavelength of 410 nm and the highest fluorescence value at a wavelength of 464 nm, as in Example 2 (distilled water).
실시예 4: 청색 형광체(HCD-70K)의 사람 혈청 내 및 자외선 조사 하에서의 안정성 확인Example 4: Confirmation of stability of blue phosphor (HCD-70K) in human serum and under UV irradiation
상기 실시예 1에 따라 제조된 청색 형광체(HCD-70K)의 사람 혈청 내 및 자외선 조사 하에서의 안정성을 확인하였다. 상기 실시예 3에서와 같은 조건의 용매에서 60분간 37℃에서 교반하면서 형광의 변화를 측정하였다. 그리고 3 W 세기의 강한 자외선을 60분간 조사하여 광 안정성(photo-stability)실험을 수행하였다. 그 결과는 도 6에 나타내었다.The stability of the blue phosphor (HCD-70K) prepared according to Example 1 in human serum and under UV irradiation was confirmed. The change in fluorescence was measured while stirring at 37° C. for 60 minutes in a solvent under the same conditions as in Example 3. Then, a photo-stability test was performed by irradiating a strong ultraviolet light of 3 W intensity for 60 minutes. The results are shown in FIG. 6 .
도 6에 나타난 바와 같이, 본 발명에 따른 청색 형광체(HCD-70K)는 사람 혈청 내의 조건에서 일정 시간이 지나더라도 형광 세기에 변화를 거의 일으키지 않는다는 것을 확인하였다. 그리고 3 W 세기의 강한 자외선을 60분간 조사한 이후에도 형광 강도가 약 70%로 유지됨을 확인하였다.As shown in FIG. 6 , it was confirmed that the blue phosphor (HCD-70K) according to the present invention hardly causes a change in fluorescence intensity even after a certain period of time has passed under the conditions in human serum. And it was confirmed that the fluorescence intensity was maintained at about 70% even after being irradiated with strong ultraviolet rays of 3 W intensity for 60 minutes.
실시예 5: 청색 형광체(HCD-70K)의 HCD-70K의 세포 내 섭취 확인Example 5: Confirmation of intracellular uptake of HCD-70K by blue phosphor (HCD-70K)
상기 실시예 1에 따라 제조된 청색 형광체(HCD-70K)가 세포 내로 투과하는지를 확인하였다. 구체적으로, HeLa 세포(사람 자궁암 세포)를 96-웰 플레이트(96-well plate)에 100,000 cells/well 밀도로, 5% 이산화탄소(CO2) 조건의 37 
도 7의 첫 번째 줄은 HCD-70K 처리 후 1시간 그리고 두 번째 줄은 6시간 동안 배양 후 촬영한 일광자 형광 현미경 결과를 나타낸 것이다. 또한, 가장 좌측 열은 밝은 세포 사진 (bright field image)이며, 두 번째 열은 청색 파장, 세 번째 열은 적색 파장, 그리고 마지막 가장 우측 열은 각 열의 영상을 가로로 합친 결과이다. 이때, 영상의 눈금 막대 바는 50 μm의 길이를 뜻한다.The first line of FIG. 7 shows the results of one-photon fluorescence microscopy taken after incubation for 1 hour after HCD-70K treatment, and the second line for 6 hours. In addition, the leftmost column is a bright field image, the second column is a blue wavelength, the third column is a red wavelength, and the last rightmost column is the result of horizontally merging the images of each column. In this case, the scale bar of the image means the length of 50 μm.
도 7에 나타난 바와 같이, HCD-70K는 낮은 세포 흡수율을 가지며, 이는 HCD-70K가 세포 섭취를 통해 혈액 외부로 유출되지 않는다는 것을 알 수 있다.As shown in FIG. 7 , HCD-70K has a low cell uptake rate, indicating that HCD-70K does not leak out of the blood through cell uptake.
실시예 6: 청색 형광체(HCD-70K)의 세포 독성 확인Example 6: Confirmation of Cytotoxicity of Blue Phosphor (HCD-70K)
상기 실시예 1에 따라 제조된 청색 형광체(HCD-70K)의 세포 독성을 MTT 세포 증식 분석 킷(MTT cell proliferation assay kit, Thermo Fisher, US)을 사용하여 HeLa 세포에서 확인하였다. 구체적으로, HeLa 세포(사람 자궁암 세포)를 96-웰 플레이트(96-well plate)에 100,000 cells/well 밀도로, 5% 이산화탄소(CO2) 조건의 37℃ 공기 중에서 24시간 배양하였다. HCD-70K를 버퍼용액(산도 7.4, phosphate-buffered saline; PBS)에 녹인 뒤 0.5~2.5 mg/mL 농도로 세포에 처리하고 다시 24시간 동안 배양하였다. 이후, 10 μL의 MTT 용액을 처리하고 4시간 동안 추가로 세포를 배양하였다. 이후, 마이크로플레이트 리더(microplate reader, Multiskan EX, Thermo Eletron)에서 550 nm 파장을 이용하여 흡수를 관찰함으로써 세포 독성 여부를 확인하였다. MTT 세포 증식 분석 킷 기반 세포 독성 검사는 제조사에서 제공하는 프로토콜을 따라 진행되었다. 그 결과를 도 8에 나타내었다.The cytotoxicity of the blue phosphor (HCD-70K) prepared according to Example 1 was confirmed in HeLa cells using an MTT cell proliferation assay kit (Thermo Fisher, US). Specifically, HeLa cells (human uterine cancer cells) were cultured in a 96-well plate at a density of 100,000 cells/well in 5% carbon dioxide (CO 2 ) air at 37° C. for 24 hours. After dissolving HCD-70K in a buffer solution (pH 7.4, phosphate-buffered saline; PBS), the cells were treated at a concentration of 0.5-2.5 mg/mL and incubated again for 24 hours. Then, 10 μL of MTT solution was treated and the cells were further cultured for 4 hours. Thereafter, cytotoxicity was confirmed by observing absorption using a 550 nm wavelength in a microplate reader (microplate reader, Multiskan EX, Thermo Eletron). The MTT cell proliferation assay kit-based cytotoxicity test was performed according to the protocol provided by the manufacturer. The results are shown in FIG. 8 .
도 8에 나타난 바와 같이, HCD-70K는 0~2.5 mg/mL 농도에 대하여 90% 이상의 세포 생존율을 보였으며, 이를 통해 HCD-70K가 낮은 세포독성을 나타냄을 확인하였다.As shown in FIG. 8 , HCD-70K showed a cell viability of 90% or more at a concentration of 0 to 2.5 mg/mL, confirming that HCD-70K exhibited low cytotoxicity.
실시예 7: 청색 형광체(HCD-70K) 유무에 따른 세포 내 혈관의 이광자 현미경 촬영 결과 확인Example 7: Confirmation of two-photon microscopy results of intracellular blood vessels with or without blue phosphor (HCD-70K)
상기 실시예 1에 따라 제조된 청색 형광체(HCD-70K)를 쥐의 꼬리 정맥에 주입한 후 생체 내 혈관을 이광자 현미경 영상을 통해 관측하였다. HCD-70K를 주입하지 않은 쥐를 대조군으로 비교하였다. 구체적으로, 실험에는 생후 2개월 수컷 쥐(C57BL/6 mice, Orient Bio, Rep. of Korea)를 사용하였다. 실험 전 50 mg/kg 제올라이트(zoletile, Virbac, France)와 30 mg/kg의 럼푼(rompun, Bayer, Germany)을 주사해 마취한 뒤 실험을 수행하였다. 마취 30분 후 대조군 쥐의 꼬리 정맥에 버퍼용액(산도 7.4, phosphate-buffered saline; PBS)를 200 μL 주입하고, 실험군 쥐의 꼬리 정맥에 버퍼용액(산도 7.4, phosphate-buffered saline; PBS) 25 mg/kg로 용해된 HCD-70K를 100 μL 주입하였다. 이후, 쥐의 등쪽 진피층(dorsar dermis)의 혈관에 780, 800, 820, 및 840 nm의 여기 파장대를 조사하고 이광자 형광 현미경(LSM 7 MP, Carl Zeiss, Germany)을 통해 촬영하였다. 렌즈는 W Plan-Apochromat 20´/1.0 water immersion을 사용하였고, 1 μm 슬라이스(slice)로 45-50 μm의 깊이에서 60초 간격으로 총 30분에서 1시간 동안 영상을 획득하였다. 그 결과를 도 9에 나타내었다. After the blue phosphor (HCD-70K) prepared according to Example 1 was injected into the tail vein of a rat, blood vessels in vivo were observed through a two-photon microscope image. Mice not injected with HCD-70K were compared as controls. Specifically, two-month-old male mice (C57BL/6 mice, Orient Bio, Rep. of Korea) were used for the experiment. Before the experiment, 50 mg/kg of zeolite (zoletile, Virbac, France) and 30 mg/kg of rumpun (rompun, Bayer, Germany) were injected and anesthetized, and the experiment was performed. After 30 minutes of anesthesia, 200 μL of buffer solution (pH 7.4, phosphate-buffered saline; PBS) was injected into the tail vein of control mice, and 25 mg of buffer solution (pH 7.4, phosphate-buffered saline; PBS) was injected into the tail vein of
도 9에서 각 그림의 눈금 막대 바는 30 μm를 의미한다. 위쪽 사진은 HCD-70K를 주입하지 않은 사진이고, 아래쪽 사진은 HCD-70K를 주입한 후의 사진이다. 도 9에 나타난 바와 같이, HCD-70K가 주입된 혈관 사진에서 주변보다 밝은 형광 영상이 관측되었으며, 특히, 780 nm의 여기 파장을 조사하였을 때 혈관이 가장 선명하게 관측됨을 확인할 수 있다. 본 실시예를 통해, HCD-70K는 생체 내 이광자 영상화에서 혈관만을 특이적으로 청색 형광으로 영상화함을 확인할 수 있다. 9, the scale bar in each figure means 30 μm. The upper picture is a picture without HCD-70K injection, and the bottom picture is a picture after HCD-70K injection. As shown in FIG. 9 , a fluorescence image brighter than the surrounding was observed in the photograph of blood vessels injected with HCD-70K, and in particular, it can be confirmed that blood vessels are observed most clearly when irradiated with an excitation wavelength of 780 nm. Through this example, it can be confirmed that HCD-70K specifically images only blood vessels with blue fluorescence in in vivo two-photon imaging.
실시예 8: 다양한 장기에서 청색 형광체(HCD-70K) 처리 후, 생체 내 이광자 현미경 촬영 결과 확인Example 8: After treatment with blue phosphor (HCD-70K) in various organs, in vivo two-photon microscopy results confirmed
상기 실시예 1에 따라 제조된 청색 형광체(HCD-70K0를 쥐의 꼬리 정맥에 주입한 후, 세 가지 각기 다른 장기에서 생체 내 혈관을 이광자 현미경 영상을 관측하였다. 구체적으로, 사용된 용매 및 실험 조건은 상기 실시예 7에서 수행된 연구와 동일한 방법을 이용하여, 청색으로 혈관을 표지하는 HCD-70K와 녹색으로 호중구(neutrophil)를 표지하는 염색시약(PE-Anti mouse Ly6G Ab, 2 μg)을 혼합하여 쥐의 꼬리 정맥에 주사하고, 이광자 형광 현미경을 통해 대퇴골 골수(calvarial bone marrow), 등 진피(dorsal dermis), 그리고 고환거근(cremaster muscle)을 관측하였다. 등 진피와 고환거근에는 800 nm, 대퇴골 골수에는 780 nm의 여기 파장을 사용하였다. 그 결과를 도 10에 나타내었다.After injecting the blue phosphor (HCD-70K0) prepared according to Example 1 into the tail vein of a rat, two-photon microscopy images of in vivo blood vessels in three different organs were observed. Specifically, the solvent used and the experimental conditions using the same method as in the study conducted in Example 7 above, mixing HCD-70K, which labels blood vessels in blue, and a staining reagent (PE-Anti mouse Ly6G Ab, 2 μg) that labels neutrophils in green was injected into the tail vein of rats, and the calvarial bone marrow, dorsal dermis, and cremaster muscle were observed through a two-photon fluorescence microscope. An excitation wavelength of 780 nm was used for bone marrow, and the results are shown in FIG.
도 10에 나타난 바와 같이, HCD-70K에 의해서 혈관이 청색으로 영상화되었고, 혈관을 따라 이동하거나 혈관 주변의 존재하는 호중구가 녹색으로 영상화되었다. 그리고 관심 영역(region of interest, ROI)에서의 형광 강도를 50분간 측정하였을 때, 큰 변화가 없는 것으로 나타나 생체 내 이광자 영상 촬영에 HCD-70K가 안정하다는 것을 확인할 수 있다.As shown in FIG. 10 , blood vessels were imaged in blue by HCD-70K, and neutrophils moving along the vessel or existing around the vessel were imaged in green. And when the fluorescence intensity in the region of interest (ROI) was measured for 50 minutes, there was no significant change, confirming that HCD-70K was stable for in vivo two-photon imaging.
실시예 9: 청색 형광체(HCD-70K)의 혈관 외 누출 실험Example 9: Extravascular Leakage Experiment of Blue Phosphor (HCD-70K)
상기 실시예 1에 따라 제조된 청색 형광체(HCD-70K)의 혈관 외 누출 여부를 형광 세기 분석을 통해 확인하였다. 구체적으로, 상기 실시예 7에서 수행된 연구와 동일한 방법을 이용하여, 대조군과 실험군에 각각 버퍼용액(산도 7.4, phosphate-buffered saline; PBS)과 HCD-70K를 처리한 후, 30분 경과 후, 혈액을 순환시킨 다음 피하 복부를 약 1cm 절개하고 버퍼용액(산도 7.4, phosphate-buffered saline; PBS)을 복강 내로 1 mL 주입하였다. PBS가 복강 내에 잘 분포되도록 마사지를 해준 뒤 복강 내 액체를 바늘이 장착되지 않은 주사기로 추출하였다. 추출된 액체의 흡수 및 형광 강도를 측정하기 위해, 원심분리기를 사용해 세포를 제거하고 상층액을 얻었다. 분석 결과를 도 11에 나타내었다.Excessive leakage of the blue phosphor (HCD-70K) prepared according to Example 1 was confirmed through fluorescence intensity analysis. Specifically, using the same method as in the study conducted in Example 7, the control group and the experimental group were each treated with a buffer solution (acidity 7.4, phosphate-buffered saline; PBS) and HCD-70K, after 30 minutes, After circulating the blood, an incision of about 1 cm was made in the subcutaneous abdomen, and 1 mL of a buffer solution (pH 7.4, phosphate-buffered saline; PBS) was injected intraperitoneally. After massaging so that PBS is well distributed in the abdominal cavity, the intraperitoneal fluid was extracted with a syringe without a needle. To measure the absorption and fluorescence intensity of the extracted liquid, the cells were removed using a centrifuge and the supernatant was obtained. The analysis results are shown in FIG. 11 .
도 11서 보는 바와 같이, HCD-70K 용액의 흡수 및 형광 그래프의 세기는 강하게 나타나는 반면, 상기 실험의 복강 내 용액은 형광 방출이 없음을 확인할 수 있다. 이를 통해, HCD-70K가 혈관 외로 누출되지 않는다는 것을 알 수 있다.As shown in FIG. 11 , it can be seen that the intensity of the absorption and fluorescence graph of the HCD-70K solution is strong, whereas the intraperitoneal solution of the experiment does not emit fluorescence. From this, it can be seen that HCD-70K does not leak out of blood vessels.
실시예 10: 청색 형광체(HCD-70K) 처리 후 다중 색상의 생체 내 이광자 형광 현미경 촬영 결과 확인Example 10: Confirmation of results of multi-color in vivo two-photon fluorescence microscopy after treatment with blue phosphor (HCD-70K)
상기 실시예 1에 따라 제조된 청색 형광체(HCD-70K)를 쥐의 꼬리 정맥에 주입하고, 뇌(brain)에서의 다중 색상 생체 내 이광자 현미경 영상을 촬영하였다. 구체적으로, 생후 2개월 CX3CR1-GFP 쥐를 사용했고, 이 쥐는 미세아교세포(microglia)가 녹색 형광 단백질(GFP: green fluorescence protein)로 표지하였다. 상기 실시예 7과 같이 실험이 수행되었으며, 버퍼용액(산도 7.4, phosphate-buffered saline; PBS)에 용해된 HCD-70K(2.5 mg/mL)와 적색 형광을 가지는 호중구 표지 기능의 PE-Anti mouse Ly6G Ab 2 μg을 혼합하여 100 μL를 쥐의 꼬리 정맥에 주입하였다. 30분 경과 후, 상기 실시예 7과 같이 이광자 형광 현미경을 이용하여 뇌의 혈관을 촬영하였다. 여기 파장은 800 nm로 하였다. 그 결과를 도 12에 나타내었다. 도 12에서 각 그림의 눈금 막대 바는 30 μm를 나타낸다.The blue phosphor (HCD-70K) prepared according to Example 1 was injected into the tail vein of a rat, and a multicolor in vivo two-photon microscope image was taken in the brain. Specifically, two-month-old CX3CR1-GFP mice were used, and in these mice, microglia were labeled with green fluorescence protein (GFP). The experiment was performed as in Example 7, and HCD-70K (2.5 mg/mL) dissolved in a buffer solution (pH 7.4, phosphate-buffered saline; PBS) and neutrophil-labeled PE-Anti mouse Ly6G with
도 12에서 보는 바와 같이, HCD-70K가 주사된 혈관은 청색으로, 미세아교세포는 녹색, 그리고 호중구는 적색으로 염색되는 것을 확인하였으며, 세 가지 사진을 합친 영상(우측 아래)을 통해 다중 영상 촬영 시 세 가지 색의 구분이 확연하게 획득될 수 있음을 확인하였다.12, it was confirmed that the blood vessels injected with HCD-70K were stained in blue, microglia were green, and neutrophils were stained red, and multiple images were taken through the combined image (lower right) of the three pictures. It was confirmed that the distinction of three colors can be clearly obtained.
상기 진술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. The description of the present invention stated above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.
Claims (7)
[화학식 1]
(상기에서 m 은 23 내지 24의 정수이고, n은 365 내지 366의 정수이다.)A blue phosphor, which is a mixture of a compound represented by the following formula (1) and a regioisomer thereof:
[Formula 1]
(In the above, m is an integer from 23 to 24, and n is an integer from 365 to 366.)
상기 청색 형광체는 이광자 흡수 형광체인 것을 특징으로 하는, 청색 형광체.The method of claim 1,
The blue phosphor is a two-photon absorbing phosphor, characterized in that the blue phosphor.
상기 화학식 1의 덱스트란(Dextran) 고분자는 몰 질량이 70 kDa인 것을 특징으로 하는, 청색 형광체. The method of claim 1,
The dextran polymer of Formula 1 has a molar mass of 70 kDa, characterized in that the blue phosphor.
상기 관측하는 단계는 일광자 또는 이광자 형광 현미경을 이용하는 것을 특징으로 하는, 혈관의 영상화 방법.6. The method of claim 5,
The observing step is characterized in that using a one-photon or two-photon fluorescence microscope, the imaging method of blood vessels.
하기 화학식 1로 표시되는 화합물과 이의 레지오아이소머의 혼합물인 청색 형광체의 제조방법.
[화학식 1]
(상기에서 m 은 23 내지 24의 정수이고, n은 365 내지 366의 정수이다.)
[화학식 2]
(상기에서 m 은 23 내지 24의 정수이고, n은 365 내지 366의 정수이다.)
[화학식 3]
After the addition of methanol to dextran to which a dibenzo-cyclooctin group is attached (DBCO-functionalized Dextran) represented by the following formula (2), 3-azido-7-hydroxycoumarin represented by the following formula (3) ( 3-azido-7-hydroxycoumarin) and stirring using a stirrer,
A method for producing a blue phosphor, which is a mixture of a compound represented by the following formula (1) and a regioisomer thereof.
[Formula 1]
(In the above, m is an integer from 23 to 24, and n is an integer from 365 to 366.)
[Formula 2]
(In the above, m is an integer from 23 to 24, and n is an integer from 365 to 366.)
[Formula 3]
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| KR20150051009A (en) * | 2013-11-01 | 2015-05-11 | 아주대학교산학협력단 | Two-photon fluorescent probes, preparation method for thereof and methods for imaging of pH using the same |
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