KR102052418B1 - Fluorescence probe compound having beta-galactosidase selectiveity and visualizing beta-galactosidase in vivo using the same - Google Patents
Fluorescence probe compound having beta-galactosidase selectiveity and visualizing beta-galactosidase in vivo using the same Download PDFInfo
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- KR102052418B1 KR102052418B1 KR1020180003712A KR20180003712A KR102052418B1 KR 102052418 B1 KR102052418 B1 KR 102052418B1 KR 1020180003712 A KR1020180003712 A KR 1020180003712A KR 20180003712 A KR20180003712 A KR 20180003712A KR 102052418 B1 KR102052418 B1 KR 102052418B1
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- dcdhf
- galactose
- gal
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Abstract
하기 [화학식 1]로 표시되는 베타-갈락토오스 분해효소 선택성을 갖는 형광 프로브 화합물에 관한 것이다:
[화학식 1]
본 발명에 따른 베타-갈락토오스 분해효소 선택성을 갖는 형광 프로브 화합물은 베타-갈락토오스 분해효소에 대해서 높은 선택성을 나타내며, 우수한 발광 특성을 보이고, 생체 내에서, 특히 간세포에서 베타-갈락토오스 분해효소를 직접적이면서도 실시간으로 시각화하는 것을 가능케 한다.It relates to a fluorescent probe compound having beta-galactose degrading enzyme selectivity represented by the following [Formula 1]:
[Formula 1]
The fluorescent probe compound having beta-galactose degrading enzyme selectivity according to the present invention exhibits high selectivity to beta-galactose degrading enzyme, shows excellent luminescent properties, and direct and real-time beta-galactose degrading enzyme in vivo, especially in hepatocytes. Makes it possible to visualize
Description
본 발명은 베타-갈락토오스 분해효소 선택성을 갖는 형광 프로브 화합물 및 이를 이용한 생체 내 베타-갈락토오스 분해효소 시각화 방법에 관한 것이다.The present invention relates to a fluorescent probe compound having beta-galactose degrading enzyme selectivity and a method for visualizing beta-galactose degrading enzyme in vivo using the same.
일부 유전적 장애를 위한 효과적인 치료는 중재가 일찍 시작된다면 유익할 수 있다. 빠르고, 비침습적인 전략에 기초한 사전 개입은 장애가 통제될 수 없게 되기 전에 장애 유형, 빠른 진단, 장애 예방 및 적절한 치료에 대한 결정에 대해 보다 나은 이해를 가능하게 할 수 있다. 비침습적인 형광 이미지화는 생체 시스템 에서 다양한 생체분자들의 기능적 활성의 시각화를 위한 이상적인 도구로서 부각되고 있다(비특허문헌 1-2). 형광 탐지체에 의한 분자 이미지화는 기관 수준에서 생체내 이미지화 및 세포내 수준에서의 상이한 동적인 생물학적 사건을 탐지하기 위한 매우 민감한 접근에 해당한다(비특허문헌 3). 특히, 특정한 기관의 생체내 이미지화는 의도된 부위로의 사전적인 근적외선 (NIR) 형광 탐지체의 표적화된 전달 후 특정한 환경에서 일부 생체분자에 반응한 그들의 활성에 의해 달성될 수 있다(비특허문헌 4). NIR 형광 탐지체는 그들의 감소된 조직 흡수도, 높은 광안정성, 넓은 스토크스 이동, 높은 양자 수율, 및 긴 방출 파장으로 인해 특정한 기관의 생체내 이미지화를 위한 좋은 방법이 될 수 있다(비특허문헌 5-9). 또한, NIR 형광체는 조직 내로 깊게 침투할 수 있고, 생물학적 샘플에 대해 최소의 간섭 및 광손상을 나타내므로 NIR 형광체는 고적적인 방사성 동위원소 탐지 방법(비특허문헌 10-11)에 대한 대안으로서 가치 있고, 비침습적이며 정밀한 진단적 이미지화를 가능하게 한다.Effective treatment for some genetic disorders can be beneficial if intervention is started early. Proactive intervention based on a fast, non-invasive strategy can enable a better understanding of the type of disorder, the early diagnosis, the prevention of the disorder and the appropriate treatment before the disorder becomes uncontrolled. Noninvasive fluorescence imaging has emerged as an ideal tool for the visualization of the functional activity of various biomolecules in biological systems (Non-Patent Documents 1-2). Molecular imaging by fluorescence detectors corresponds to very sensitive approaches for in vivo imaging at the organ level and for detecting different dynamic biological events at the intracellular level (Non-Patent Document 3). In particular, in vivo imaging of specific organs can be achieved by their activity in response to some biomolecules in a particular environment after targeted delivery of a prior near infrared (NIR) fluorescence detector to the intended site (Non-Patent Document 4). ). NIR fluorescence detectors may be a good method for in vivo imaging of specific organs due to their reduced tissue absorption, high photostability, wide Stokes shift, high quantum yield, and long emission wavelengths (Non-Patent Document 5). -9). In addition, NIR phosphors can penetrate deep into tissues and exhibit minimal interference and photodamage to biological samples, which makes NIR phosphors valuable and valuable as an alternative to conventional radioisotope detection methods (Non Patent Literature 10-11). This enables non-invasive and precise diagnostic imaging.
특정 질병의 진단 및 적용가능한 약물의 치료 반응을 향상시키기 위한 종래 방법에 대해 효율적인 대안으로서 NIR 형광 탐지체에 의한 표적화된 이미지화가 발전되어 왔다(비특허문헌 12). 항체, 펩타이드, 및 저분자량의 비펩타이드 리간드를 포함하는 모이어티에 대한 다양한 표적화가 진단시약 및/또는 약물의 전달을 위해 종양 부위를 선택적으로 표적화하기 위한 다양한 제제에 사용되고 있다. 표적-특이 담체 분자 중에, β-갈락토오스 분해효소(β-galactosidase)의 갈락토오스 잔기, 아시알로당단백질 수용체(asialoglycoprotein receptor, ASGPR)의 리간드가 리포터(reporter)로 널리 사용되어 왔고, 효소를 활성화시켜 전사 조절 및 효소 면역 분석을 면밀하게 조사하였으며, 분자 및 세포 수준에서 집중적으로 연구되어 왔다(비특허문헌 13-15). 핵심적인 효소로서, β-갈락토오스 분해효소의 더 높은 발현이 다양한 암, 특히 난소암 및 직장암의 진행과 관련된 것으로 밝혀졌다. 몇 가지 형광 탐지체가 상이한 암세포에서 β-갈락토오스 분해효소의 발현을 모니터링하기 위해 발전되어 왔지만; 이들은 낮은 세포 침투성, 세포 및 조직의 배경 자가형광을 방해하는 좁은 방출 파장, 및 낮은 침투 깊이와 같은 몇 가지 제한이 있다(비특허문헌 16-24). 최근에, Zhu 등은 NIR 형광 탐지체 (DCM-βgal)를 이용한 직장 종양 모델에서 β-갈락토오스 분해효소의 생체내 추적 및 시각화를 보고한바 있고(비특허문헌 25), 또한, Urbano 등은 β-갈락토오스 분해효소의 발현을 시각화함으로써 복막 전이성 종양을 탐지하였다(비특허문헌 26).Targeted imaging by NIR fluorescence detectors has been developed as an efficient alternative to conventional methods for diagnosing specific diseases and improving the therapeutic response of applicable drugs (Non-Patent Document 12). Various targeting of moieties including antibodies, peptides, and low molecular weight non-peptide ligands have been used in various agents for selectively targeting tumor sites for delivery of diagnostic reagents and / or drugs. Among the target-specific carrier molecules, galactose residues of β-galactosidase, ligands of asialoglycoprotein receptor (ASGPR), have been widely used as reporters, and enzymes are activated to transcription. Regulatory and enzymatic immunoassays have been closely investigated and studied intensively at the molecular and cellular level (Non-Patent Document 13-15). As a key enzyme, higher expression of β-galactose degrading enzymes has been found to be associated with the progression of various cancers, particularly ovarian and rectal cancers. Several fluorescence detectors have been developed to monitor the expression of β-galactose degrading enzymes in different cancer cells; These have some limitations such as low cell permeability, narrow emission wavelengths that interfere with background autofluorescence of cells and tissues, and low penetration depth (Non-Patent Documents 16-24). Recently, Zhu et al. In vivo tracking and visualization of β-galactose degrading enzymes in a rectal tumor model using a NIR fluorescence detector (DCM-βgal) has been reported (Non-Patent Document 25), and Urbano et al. Also described the expression of β-galactose degrading enzymes. Peritoneal metastatic tumors were detected by visualization (Non Patent Literature 26).
간세포암종 (HCC)은 전세계적으로 만성 간 질병으로 고통 받는 환자들에서 빈도가 증가된 가장 널리 알려져 있는 간암 유형이다(비특허문헌 27-29). B/C형 간염 및 아플라톡신-B1 (AFB1) 노출이 HCC와 관련된 일반적인 위험 요인이다(비특허문헌 30-33). 수용체인, 간세포 상의 ASGPR이 간기능 부전증의 평가로서 사용되어 왔다(비특허문헌 34). 간은 암전이와 관련된 가장 빈번한 기관에 해당한다. 따라서, 초기 단계에서의 악성 간질환의 정확한 인지가 암 치료 요법에 있어 매우 중요하다. 과발현된 ASGPR 수용체가 간세포에 선택적으로 갈락토오스 작용화된 담체를 세포내 섭취함으로써(endocytosed), 다른 기관에 대한 그의 비선택적 분배를 감소시켰다(비특허문헌 35). 결과적으로, 높은 β-갈락토오스 분해효소 발현과 관련된 간세포암종 세포는 일반적인 간 조직에 비해 시각화된다(비특허문헌 36-37). 지금까지 컴퓨터 단층촬영 (CT), 자기 공명화상법 (MRI), 양전자 방사 단층촬영 (PET), 및 초음파검사 (US)가 주요 종양 병변의 탐지를 위해 사용되어 왔지만(비특허문헌 38), HCC를 이용한 환자의 진단은 여전히 쉽지 않은 상황이다.Hepatocellular carcinoma (HCC) is the most widely known type of liver cancer with increased frequency in patients suffering from chronic liver disease worldwide (Non-Patent Document 27-29). Hepatitis B / C and aflatoxin-B 1 (AFB 1 ) exposure are common risk factors associated with HCC (Non-Patent Documents 30-33). ASGPR on hepatocytes, which is a receptor, has been used as an evaluation of liver failure (Non Patent Literature 34). The liver is the most frequent organ involved in cancer metastasis. Therefore, accurate recognition of malignant liver disease at an early stage is very important for cancer therapy. The overexpressed ASGPR receptor endocytosed the galactose functionalized carrier selectively to hepatocytes, thereby reducing its non-selective distribution to other organs (Non-Patent Document 35). As a result, hepatocellular carcinoma cells associated with high β-galactose degrading enzyme expression are visualized compared to normal liver tissue (Non-Patent Documents 36-37). Until now, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and ultrasonography (US) have been used for the detection of major tumor lesions (Non-Patent Document 38). Diagnosis of the patient using a still difficult situation.
본 발명에서는 베타-갈락토오스 분해효소에 대해서 높은 선택성을 나타내며, 우수한 발광 특성을 보이고, 특히, 생체 내에서 직접적으로, 또한 실시간으로 베타-갈락토오스 분해효소를 시각화할 수 있는 베타-갈락토오스 분해효소 선택성을 갖는 형광 프로브 화합물을 제공하고자 한다.In the present invention, it exhibits high selectivity for beta-galactose degrading enzyme, shows excellent luminescence properties, and in particular, has beta-galactose degrading enzyme selectivity capable of visualizing beta-galactose degrading enzyme directly and in real time in vivo. It is intended to provide fluorescent probe compounds.
또한, 본 발명에서는 상기 형광 프로브 화합물을 이용한 생체 내 베타-갈락토오스 분해효소 시각화 방법을 제공하고자 한다.In addition, the present invention is to provide a method for visualizing beta-galactose degrading enzyme in vivo using the fluorescent probe compound.
상기 과제를 해결하기 위해, 본 발명은 하기 [화학식 1]로 표시되는 베타-갈락토오스 분해효소 선택성을 갖는 형광 프로브 화합물을 제공한다:In order to solve the above problems, the present invention provides a fluorescent probe compound having beta-galactose degrading enzyme selectivity represented by the following [Formula 1]:
[화학식 1][Formula 1]
본 발명에 따르면, 상기 베타-갈락토오스 분해효소는 간세포에서 과발현되는 것일 수 있다.According to the present invention, the beta-galactose degrading enzyme may be overexpressed in hepatocytes.
또한, 본 발명은 상기 제1항에 따른 형광 프로브 화합물을 생체 내에 주입한 후, 방출되는 형광을 모니터링하는 단계;를 포함하는 생체 내 베타-갈락토오스 분해효소 시각화 방법을 제공한다.In another aspect, the present invention provides a method for visualizing beta-galactose degrading enzyme in vivo, comprising the step of injecting the fluorescent probe compound according to
본 발명에 따른 베타-갈락토오스 분해효소 선택성을 갖는 형광 프로브 화합물은 베타-갈락토오스 분해효소에 대해서 높은 선택성을 나타내며, 우수한 발광 특성을 보이고, 생체 내에서, 특히 간세포에서 베타-갈락토오스 분해효소를 직접적이면서도 실시간으로 시각화하는 것을 가능케 한다.The fluorescent probe compound having beta-galactose degrading enzyme selectivity according to the present invention exhibits high selectivity for beta-galactose degrading enzyme, shows excellent luminescence properties, and direct and real-time beta-galactose degrading enzyme in vivo, especially in hepatocytes. Makes it possible to visualize
도 1은 β-갈락토오스 분해효소와 만났을 때 본 발명에 따른 형광 프로브 화합물(DCDHF-βgal)의 개념적인 반응식 및 이에 따른 형광 변화를 나타낸 것이다.
도 2는 (a) β-갈락토오스 분해효소(50 U)를 첨가한 DCDHF-βgal (20 μM)의 흡수 스펙트럼을 나타내고, (b)는 다양한 양의 β-갈락토오스 분해효소 (0 U-50 U)에서 DCDHF-βgal (5 μM)의 형광 방출을 나타내며, (c 및 d)는 β-갈락토오스 분해효소 (50 U)가 첨가된 DCDHF-βgal (5 μM)의 시간 의존성 형광 스펙트럼을 나타낸다(0-30분). 모든 스펙트럼은 37 ℃, λex = 580 nm에서 PBS 완충액 (pH 7.4) 중에서 기록되었다.
도 3은 DCDHF-βgal의 세포 특이적 표적화를 나타낸 것으로, HepG2, A549, 및 KB 세포 중 DCDHF-βgal의 세포 형광 이미지이다. 세포들은 10 μM의 DCDHF-βgal로 24시간 동안 처리되었다. 필터 세트: Texas-Red (Yellow, λex:589/λem: 610), mPlum (Red, λex:588/λem:649), Hoechst (Blue, λex:359/λem:457) 스케일바: 20 μm.
도 4는 β-갈락토오스 분해효소의 양에 의해 강화된 DCDHF-βgal의 형광 강도를 나타낸 것이다. (a)는 β-갈락토오스 분해효소로 과발현된 HepG2 세포에서의 DCDHF-βgal의 세포 형광 이미지이다. pCMV-LacZ로 형질주입한 후, 세포들을 4 μM의 DCDHF-βgal로 12시간 동안 처리하였다. 필터 세트: mPlum (Red, λex:588/λem:649), Hoechst (Blue, λex:359/λem:457) 스케일바: 20 μm. DCDHF-βgal-처리된 HepG2 및 pCMV-LacZ로 형질주입된 HepG2 세포의 형광 강도의 비교가 막대그래프 플롯 (b) 및 유동 세포 분석법의 평균 형광값 (c)에 제시되어 있다.
도 5는 DCDHF-βgal의 생체내 형광 이미지화 결과이다. β-갈락토오스 분해효소를 과발현시키는 HepG2 이종이식 종양을 갖는 마우스 내로 DCDHF-βgal (1 mg/kg)를 정맥 내 주입하거나 하지 않았다. 24시간 후에, Maestro 생체내 이미지화 시스템을 이용하여 종양 부분의 형광 이미지(a)를 얻었다. (b)는 동결절편된 종양으로부터 수득한 현미경 이미지이다. 필터 세트: mPlum (Red, λex:588/λem:649), DAPI (Blue, λex:405/λem:450-490) 스케일바: 20 μm.
도 6은 다양한 pH에서 DCDHF-βgal (5 μM)의 형광 스펙트럼을 나타낸다. 모든 스펙트럼은 β-갈락토시다제를 첨가한 후에 수득하였고, λex = 580 nm인 PBS 완충액에서 기록하였다.
도 7은 다양한 티올 (각각 10 mM) 및 활성 산소 (ROS) (각각 200 μM) 에스테라아제 (100U), 리소자임 (100U), 셀룰라아제 (100U)에 대한 DCDHF-βgal(5 μM) 형광 반응을 나타낸 막대 그래프이다; (1) control, (2) glutathiol (GSH), (3) cysteine, (4) homocysteine, (4) Na2S (5) superoxide (O2-), (6) tert-butyl hydroperoxide (TBHP), (7) Hydroxyl radical (OH.), (8) hypochlorite ion (OCl-), (9) Esterase (10) Lysozyme (11) Cellulase and (12) Galactosidase. 모든 스펙트럼은 37 ℃, PBS 완충액 (10 mM, pH = 7.4)에서 분석 물을 첨가한 후 1 시간에 획득하였다. λex/λem = 615nm/665nm
도 8은 HepG2 세포에서 DCDHF-βgal의 세포 생존 능력을 나타낸 것이다. HepG2 세포를 24 시간동안 다양한 농도의 DCDHF-βgal으로 처리하였다.
도 9는 HepG2 세포에서 DCDHF-βgal의 세포 형광 이미지를 나타낸 것이다. 세포를 1, 2, 6 및 12 시간동안 10 μM의 DCDHF-βgal으로 처리하였다. 필터 세트: Texas-Red (황색, λex:589/λem:610), mPlum (적색, λex:588/λem:649), Hoechst (청색, λex:359/λem:457) 스케일바: 20 μm
도 10은 유동 세포 계측법을 이용한 경쟁 분석 결과를 나타낸 것이다. HepG2 세포는 10 mM의 갈락토오스 전처리(적색 선) 또는 전처리 없이(파란색 선), 6 시간 동안 10 μM의 DCDHF-βgal으로 처리하였다.
도 11은 유동 세포 계측법을 이용한 표적 세포 흡수를 측정한 결과이다. KB, A549 및 HepG2 세포에서 DCDHF-βgal의 (a) 도트 플롯 및 (b) 형광 강도 비교 결과이다. 세포를 10 μM의 DCDHF-βgal으로 24 시간 동안 처리하였다.
도 12는 β-갈락토시다제를 과발현하는 형질 감염되지 않은 HepG2 세포의 Western blot 분석 결과이다. β-갈락토시다제 과발현은 monoclonal anti-β-갈락토시다제로 Western blotting하여 평가하였다. α-tubulin을 loading control로 사용하였다.
도 13은 DCDHF-βgal의 생체 내 형광 이미징 결과이다. β-갈락토시다제를 과발현하는 HepG2 이종 이식 종양이있는 마우스에 DCDHF-βgal (1 mg / kg)을 정맥 주사하였다. 4 시간과 24 시간 후 마우스의 전신 (a)과 해부 된 종양 조직 (b)의 형광 이미지를 Maestro 생체 내 이미징 시스템을 사용하여 얻었다. (c)는 β-갈락토시다아제-과발현 및 대조군 종양으로부터의 생체 외 형광에서 신호 대 배경 비(background ratio)의 비교결과이다.Figure 1 shows a conceptual diagram of the fluorescent probe compound (DCDHF- β gal) according to the present invention and the fluorescence change according to the present invention when it encounters β-galactose degrading enzyme.
Figure 2 shows (a) the absorption spectrum of DCDHF- β gal (20 μM) to which β-galactose degrading enzyme (50 U) was added, and (b) shows various amounts of β-galactose degrading enzyme (0 U-50 U). ) Shows fluorescence emission of DCDHF- β gal (5 μM), and (c and d) show time-dependent fluorescence spectra of DCDHF- β gal (5 μM) to which β-galactose degrading enzyme (50 U) was added ( 0-30 minutes). All spectra were recorded in PBS buffer (pH 7.4) at 37 ° C., λ ex = 580 nm.
3 is a fluorescent image of a cell of DCDHF- β gal illustrates the cell-specific targeting of DCDHF- β gal, HepG2, A549, and KB cells. Cells were treated with 10 μM of DCDHF- β gal for 24 hours. Filter set: Texas-Red (Yellow, λ ex : 589 / λ em : 610), mPlum (Red, λ ex : 588 / λ em : 649), Hoechst (Blue, λ ex : 359 / λ em : 457) scale Bar: 20 μm.
Figure 4 shows the fluorescence intensity of DCDHF- β gal enhanced by the amount of β-galactose degrading enzyme. (a) is a cell fluorescence image of DCDHF- β gal in HepG2 cells overexpressed with β-galactose degrading enzyme. After transfection with pCMV-LacZ, cells were treated with 4 μM of DCDHF- β gal for 12 hours. Filter set: mPlum (Red, λ ex : 588 / λ em : 649), Hoechst (Blue, λ ex : 359 / λ em : 457) Scale bar: 20 μm. A comparison of the fluorescence intensity of HepG2 cells transfected with DCDHF- β gal-treated HepG2 and pCMV-LacZ is shown in the histogram plots (b) and mean fluorescence values (c) of flow cytometry.
5 shows in vivo fluorescence imaging results of DCDHF- β gal. DCDHF- β gal (1 mg / kg) was not injected intravenously into mice with HepG2 xenograft tumors overexpressing β-galactose degrading enzymes. After 24 hours, a fluorescent image (a) of the tumor part was obtained using a Maestro in vivo imaging system. (b) is a microscopic image obtained from cryosectioned tumor. Filter set: mPlum (Red, λ ex : 588 / λ em : 649), DAPI (Blue, λ ex : 405 / λ em : 450-490) Scale bar: 20 μm.
6 shows fluorescence spectra of DCDHF-βgal (5 μM) at various pHs. All spectra were obtained after addition of β-galactosidase and recorded in PBS buffer with λ ex = 580 nm.
FIG. 7 is a bar graph showing DCDHF-βgal (5 μM) fluorescence response to various thiols (10 mM each) and active oxygen (ROS) (200 μM each) esterase (100U), lysozyme (100U), cellulase (100U) to be; (1) control, (2) glutathiol (GSH), (3) cysteine, (4) homocysteine, (4) Na 2 S (5) superoxide (O 2- ), (6) tert-butyl hydroperoxide (TBHP), (7) Hydroxyl radical (OH. ), (8) hypochlorite ion (OCl -), (9) Esterase (10) Lysozyme (11) Cellulase and (12) Galactosidase. All spectra were acquired at 37 ° C., 1 hour after addition of the analytes in PBS buffer (10 mM, pH = 7.4). λ ex / λ em = 615 nm / 665 nm
8 shows the cell viability of DCDHF-βgal in HepG2 cells. HepG2 cells were treated with various concentrations of DCDHF-βgal for 24 hours.
9 shows cell fluorescence images of DCDHF-βgal in HepG2 cells. Cells were treated with 10 μM of DCDHF-βgal for 1, 2, 6 and 12 hours. Filter set: Texas-Red (yellow, λ ex : 589 / λ em : 610), mPlum (red, λ ex : 588 / λ em : 649), Hoechst (blue, λ ex : 359 / λ em : 457) scale Bar: 20 μm
10 shows the results of competition analysis using flow cytometry. HepG2 cells were treated with 10 μM of DCDHF-βgal for 6 hours with or without 10 mM galactose pretreatment (red line) or without pretreatment (blue line).
11 shows the results of measuring target cell uptake using flow cytometry. (A) dot plot of DCDHF-βgal and (b) fluorescence intensity comparison results in KB, A549 and HepG2 cells. Cells were treated with 10 μM of DCDHF-βgal for 24 hours.
12 shows Western blot analysis of untransfected HepG2 cells overexpressing β-galactosidase. β-galactosidase overexpression was evaluated by Western blotting with monoclonal anti-β-galactosidase. α-tubulin was used as loading control.
13 shows in vivo fluorescence imaging results of DCDHF-βgal. Mice with HepG2 xenograft tumors overexpressing β-galactosidase were intravenously injected with DCDHF-βgal (1 mg / kg). After 4 and 24 hours, fluorescence images of the whole body (a) and dissected tumor tissue (b) of the mice were obtained using the Maestro in vivo imaging system. (c) is a comparison of the signal to background ratio in β-galactosidase-overexpression and in vitro fluorescence from control tumors.
이하에서는 바람직한 실시예 등을 들어 본 발명을 더욱 상세하게 설명한다. 그러나 이들 실시예 등은 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이에 의하여 제한되지 않는다는 것은 당업계의 통상의 지식을 가진 자에게 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these examples and the like are intended to explain the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited thereto.
실험방법Experiment method
재료 및 기기장치Materials and Equipment
4-하이드록시-3-나이트로벤즈알데하이드 (Aldrich), 산화은 (Aldrich), 아세토브로모-α-D-갈락토오스 (Aldrich), 소듐 보로하이드라이드 (Aldrich), 포스포러스 트리브로마이드 (Aldrich), 요오드화 구리(I) (Aldrich), 4-포르밀페닐보론산 (Aldrich), 소듐 아자이드 (Aldrich), 트리페닐포스핀 (Aldrich), 3-하이드록시-3-메틸-2-부탄온 (Aldrich), 말론나이트릴 (Aldrich), 포타슘 카보네이트 (Aldrich), 소듐 메톡사이드 (Aldrich)가 정제 없이 그대로 사용되었다. 갈락토오스 모이어티 및 형광체가 하기 보고된 절차에 의해 합성되었다. 칼럼 크로마토그래피는 실리카 겔 60을 사용하여 화합물을 정제하는 데 사용되었다. 반응 진행은 박층 크로마토 그래피 (TLC)에 의해 60 실리카 겔을 사용하여 모니터링 하였다. 질량 및 NMR 스펙트럼은 Ion Spec Hi Res ESI 질량 분석기 및 300 및 400 MHz 분광기 (AS400, 300, Varian, US)를 사용하여 수득하였다.4-hydroxy-3-nitrobenzaldehyde (Aldrich), silver oxide (Aldrich), acetobromo-α-D-galactose (Aldrich), sodium borohydride (Aldrich), phosphorus tribromide (Aldrich), iodide Copper (I) (Aldrich), 4-formylphenylboronic acid (Aldrich), sodium azide (Aldrich), triphenylphosphine (Aldrich), 3-hydroxy-3-methyl-2-butanone (Aldrich) , Malonitril (Aldrich), potassium carbonate (Aldrich), sodium methoxide (Aldrich) were used as such without purification. Galactose moieties and phosphors were synthesized by the procedure reported below. Column chromatography was used to purify the compound using
[화학식 Formula 1]로1] 표시되는 화합물( The compound represented ( DCDHFDCDHF -- ββ galgal )의 합성) Synthesis
하기 [합성 경로]에 표시된 방법에 따라, 본 발명의 [화학식 1]로 표시되는 화합물(DCDHF-βgal, 화합물 6)을 합성하였다.According to the method shown in the following [synthetic route], the compound represented by the formula (1) of the present invention (DCDHF- β gal, Compound 6) was synthesized.
[합성 경로][Synthetic path]
a) NaBH4, MC/MeOH, 0 ℃; b) PBr3, MC, 1 h; c) K2CO3, DMF, RT, 2 days; d) Sodium methoxide, MeOH, RT, 24 h.a) NaBH 4 , MC / MeOH, 0 ° C; b) PBr 3 , MC, 1 h; c) K 2 CO 3 , DMF, RT, 2 days; d) Sodium methoxide, MeOH, RT, 24 h.
(1) 화합물 1의 합성(1) Synthesis of
무수 아세토니트릴(173 mL) 중의 아세토브로모-β-D-갈락토오스 (6.9g, 16mmol)가 자기적으로 교반된 용액에 4-하이드록시-3-나이트로벤즈알데하이드 (4.0g, 24mmol) 및 산화은 (5.6 g, 24 mmol)을 첨가하였다. 상기 혼합물을 어두운 곳에서 실온에서 22 시간 동안 자기적으로 교반하였으며, 침전물을 셀 라이트를 사용하여 용액을 여과하여 완전히 제거하였다. 용매를 증발시킨 후, 고체를 에틸 아세테이트에 용해시킨 후 용액을 소듐 바이카보네이트 용액 및 염수로 세척하였다. 유기층을 MgSO4상에서 건조시키고 증발시켰다. 이 조(crude) 생성물을 헥산으로 세척 및 여과시켜 백색 고체의 화합물 1을 수득하였다(7.0g, 88% 수율). 1H NMR (300 MHz, CDCl3): δ 2.03 (s, 3H), 2.09 (s, 3H), 2.14 (s, 3H), 2.20 (s, 3H), 4.14-4.28 (m, 3H), 5.13 (dd, 1H, J = 10.4, 3.4 Hz), 5.21 (d, 1H, J = 7.8 Hz), 5.50 (d, 1H, J = 3.7 Hz), 5.60 (dd, 1H, J = 10.7, 7.9 Hz), 7.49 (d, 1H, J = 8.6 Hz), 8.07 (dd, 1H, J = 8.6, 2.1 Hz), 8.31 (d, 1H, J = 2.0 Hz), 9.99 (s, 1H).Acetobromo-β-D-galactose (6.9 g, 16 mmol) in anhydrous acetonitrile (173 mL) was magnetically stirred in 4-hydroxy-3-nitrobenzaldehyde (4.0 g, 24 mmol) and silver oxide. (5.6 g, 24 mmol) was added. The mixture was magnetically stirred for 22 hours at room temperature in the dark, and the precipitate was completely removed by filtration of the solution using Celite. After evaporating the solvent, the solid was dissolved in ethyl acetate and then the solution was washed with sodium bicarbonate solution and brine. The organic layer was dried over MgSO 4 and evaporated. This crude product was washed with hexane and filtered to give
(2) 화합물 2의 합성(2) Synthesis of
CH2Cl2/MeOH (95:5, 200 mL) 중 상기 화합물 1(2.0 g, 4.02 mmol)의 용액에 소듐 보로하이드라이드 (0.3 g, 8.04 mmol)를 첨가하였다. 용액을 실온에서 1 시간 동안 자기적으로 교반하였으며, 반응을 냉수 (200 mL)로 켄칭시키고, 용매를 증발시켰다. 생성물을 에틸 아세테이트로 추출하였다. 유기층을 염수로 세척하고 MgSO4상에서 건조시켰다. 용매를 제거하고, 잔류물을 디에틸에테르로 세척하여 백색 고체의 화합물 2를 수득하였다(1.42 g, 71% 수율). 1H NMR (300 MHz, CDCl3):δ 2.02 (s, 3H), 2.07 (s, 3H), 2.13 (s, 3H), 2.19 (s, 3H), 4.03-4.28 (m, 3H), 4.73 (d, 2H, J = 5.56 Hz), 5.05 (d, 1H, J = 7.96), 5.10 (dd, 1H, J = 10.66, 3.32 Hz), 5.47 (d, 1H, J = 3.32 Hz), 5.55 (dd, 1H, J = 10.58, 7.88 Hz), 7.35 (d, 1H, J = 8.72 Hz), 7.52 (dd, 1H, J = 8.42, 2.39 Hz),7.81 (d, 1H, J = 1.85 Hz).To a solution of compound 1 (2.0 g, 4.02 mmol) in CH 2 Cl 2 / MeOH (95: 5, 200 mL) was added sodium borohydride (0.3 g, 8.04 mmol). The solution was magnetically stirred at rt for 1 h, the reaction was quenched with cold water (200 mL) and the solvent was evaporated. The product was extracted with ethyl acetate. The organic layer was washed with brine and dried over MgSO 4 . The solvent was removed and the residue was washed with diethyl ether to give
(3) 화합물 3의 합성(3) Synthesis of Compound 3
화합물 2 (1.0 g, 2.00 mmol)을 질소 가스 하 디클로로메탄 (10 mL)에 용해시켰다. 포스포러스 트리브로마이드 (0.2 mL)를 용액에 첨가하고, 혼합물을 실온에서 1시간 동안 자기적으로 교반 하였다. 반응 용매를 진공에서 증발시켰다. 생성물을 용리액으로서 에틸 아세테이트 및 헥산 (1:1)을 사용하는 실리카 겔 크로마토 그래피로 정제하여 화합물 3을 수득하였다(1.0g, 88% 수율). ESI-MS m/z (M+) calcd 561.05 found 584.25 (M+Na+). 1H NMR (300 MHz, CDCl3): δ 2.00 (s, 3H), 2.06 (s, 3H), 2.11 (s, 3H), 2.17 (s, 3H), 4.08-4.27 (m, 3H), 4.46 (s, 2H), 5.07-5.12 (m, 2H), 5.46 (d, 1H, J = 2.69 Hz), 5.53 (dd, 1H, J = 10.53, 7.99 Hz), 7.33 (d, 1H, J = 8.72 Hz), 7.54 (dd, 1H, J = 8.57, 2.18 Hz), 7.82 (d, 1H, J = 2.25 Hz). 13C NMR (100 MHz, CDCl3): 20.78, 20.83, 20.88, 31.05, 61.58, 66.92, 67.94, 70.67, 71.67, 100.69, 120.00, 125.84, 133.96, 134.48, 141.14, 147.30, 169.54, 170.30, 170.37, 170.49 ppm.Compound 2 (1.0 g, 2.00 mmol) was dissolved in dichloromethane (10 mL) under nitrogen gas. Phosphorus tribromide (0.2 mL) was added to the solution and the mixture was magnetically stirred for 1 hour at room temperature. The reaction solvent was evaporated in vacuo. The product was purified by silica gel chromatography using ethyl acetate and hexanes (1: 1) as eluent to afford compound 3 (1.0 g, 88% yield). ESI-MS m / z (M + ) calcd 561.05 found 584.25 (M + Na + ). 1 H NMR (300 MHz, CDCl 3 ): δ 2.00 (s, 3H), 2.06 (s, 3H), 2.11 (s, 3H), 2.17 (s, 3H), 4.08-4.27 (m, 3H), 4.46 (s, 2H), 5.07-5.12 (m, 2H), 5.46 (d, 1H, J = 2.69 Hz), 5.53 (dd, 1H, J = 10.53, 7.99 Hz), 7.33 (d, 1H, J = 8.72 Hz), 7.54 (dd, 1H, J = 8.57, 2.18 Hz), 7.82 (d, 1H , J = 2.25 Hz). 13 C NMR (100 MHz, CDCl 3 ): 20.78, 20.83, 20.88, 31.05, 61.58, 66.92, 67.94, 70.67, 71.67, 100.69, 120.00, 125.84, 133.96, 134.48, 141.14, 147.30, 169.54, 170.30, 170.37, 170.49 ppm.
(4) 화합물 4의 합성(4) Synthesis of
종래 문헌에 보고된 방법(Lord, S. J.; Conley, N. R.; Lee, H. L.; Samuel, R.; Liu, N.; Twieg, R. J.; Moerner, W. E. J. Am. Chem. Soc. 2008, 130, 9204-9205.)에 따라 보라색 고체의 화합물 4를 합성하였다.Methods reported in the prior art (Lord, SJ; Conley, NR; Lee, HL; Samuel, R .; Liu, N .; Twieg, RJ; Moerner, WEJ Am. Chem. Soc. 2008, 130, 9204-9205.
(5) 화합물 5의 합성(5) Synthesis of Compound 5
화합물 1 (0.32 g, 1.0 mmol), 포타슘 카보네이트 (0.24 g, 1.7 mmol) 및 디메틸포름아미드 (13 mL)를 실온에서 1시간 동안 교반하였다. 다음으로, 화합물 4 (0.65 g, 1.2 mmol)를 플라스크 내의 디메틸 포름아미드 (6 mL)에 용해시키고, 반응 용액에 첨가하였다. 반응 혼합물을 실온에서 2일 동안 교반하였다. 생성물을 에틸 아세테이트로 추출하였다. 유기층을 염수로 세척하고 MgSO4상에서 건조시켜 조(crude) 화합물 5를 수득하였다(1.1 g, 122% 수율). ESI-MS m/z (M+) calcd 783.24 found 806.25 (M+Na+), 782.10 (M - H+). 1H NMR (500 MHz, CDCl3): δ 1.81 (s, 6H), 2.01 (s, 3H), 2.09 (s, 3H), 2.15 (s, 3H), 2.21 (s, 3H), 4.10 (m, 1H), 4.16-4.19 (m, 1H), 4.63 (s, 2H), 4.29 (m, 1H), 5.11-5.15 (m, 2H), 5.17 (s, 2H), 5.49-5.50 (m, 1H), 5.56-5.59 (m, 1H), 6.92-6.95 (m, 1H), 7.04 (m, 2H), 7.42 (m, 1H), 7.60-7.67 (m, 4H), 7.92 (s, 1H), 7.92 (s, 1H).13C NMR (125 MHz, CDCl3): 20.58, 20.65, 20.67, 20.69, 30.95, 61.28, 66.69, 67.81, 68.49, 70.49, 71.45. 97.68, 100.66, 110.50, 111.14, 111.84, 113.00, 115.86, 116.01, 119.93, 124.17, 127.43, 128.49, 128.59, 130.39, 131.43, 131.97, 132.12, 132.56, 141.30, 147.00, 149.15, 161.99, 169.40, 170.13, 170.33, 174.21, 175.58 ppm.Compound 1 (0.32 g, 1.0 mmol), potassium carbonate (0.24 g, 1.7 mmol) and dimethylformamide (13 mL) were stirred at rt for 1 h. Next, compound 4 (0.65 g, 1.2 mmol) was dissolved in dimethyl formamide (6 mL) in the flask and added to the reaction solution. The reaction mixture was stirred at rt for 2 days. The product was extracted with ethyl acetate. The organic layer was washed with brine and dried over MgSO 4 to give crude compound 5 (1.1 g, 122% yield). ESI-MS m / z (M + ) calcd 783.24 found 806.25 (M + Na + ), 782.10 (M-H + ). 1 H NMR (500 MHz, CDCl 3 ): δ 1.81 (s, 6H), 2.01 (s, 3H), 2.09 (s, 3H), 2.15 (s, 3H), 2.21 (s, 3H), 4.10 (m , 1H), 4.16-4.19 (m, 1H), 4.63 (s, 2H), 4.29 (m, 1H), 5.11-5.15 (m, 2H), 5.17 (s, 2H), 5.49-5.50 (m, 1H ), 5.56-5.59 (m, 1H), 6.92-6.95 (m, 1H), 7.04 (m, 2H), 7.42 (m, 1H), 7.60-7.67 (m, 4H), 7.92 (s, 1H), 7.92 (s, 1 H). 13 C NMR (125 MHz, CDCl 3 ): 20.58, 20.65, 20.67, 20.69, 30.95, 61.28, 66.69, 67.81, 68.49, 70.49, 71.45. 97.68, 100.66, 110.50, 111.14, 111.84, 113.00, 115.86, 116.01, 119.93, 124.17, 127.43, 128.49, 128.59, 130.39, 131.43, 131.97, 132.12, 132.56, 141.30, 147.00, 149.15, 161.15, 171 174.21, 175.58 ppm.
(6) 화합물 6((6) compound 6 ( DCDHFDCDHF -- ββ galgal )의 합성) Synthesis
Crude 화합물 5 (0.36 g, 0.46 mmol)를 질소 가스하에 메탄올 (6 mL)에 용해시켰다. 메탄올 중 0.5N 나트륨 메톡사이드 (0.4mL)를 용액에 첨가하였다. 혼합물을 어두운 곳에서 실온에서 24시간 동안 자기적으로 교반하였다. 양이온 교환 수지 (H +)를 첨가하고 여과하고 MeOH로 세척하였다. 용매를 진공하에 증발시켰다. 생성물을 HPLC (C18, 5 μm, 고정상 250 x 10 mm; 완충액 A, H2O, 완충액 B, 이동상 CH3CN)를 이용하여 정제하여 화합물 6(DCDHF-βgal)을 수득하였다(63 mg, 8.9% 수율). ESI-MS m/z (M+) calcd 615.20 found 638.30 (M+Na+), 614.15 (M - H+). 1H NMR (500 MHz, DMSO-d6): δ 1.79 (s, 6H), 3.36-3.39 (m, 1H), 3.40-3.42 (m, 2H), 3.48-3.70 (m, 5H), 4.12-4.13 (m, 1H), 4.66-4.70 (m, 2H), 5.05 (d, 1H, J = 5 Hz), 5.21 (s, 2H), 7.11 (d, 1H, J = 16.45 Hz), 7.18 (d, 2H, J = 8.9 Hz), 7.46 (d, 1H, J = 8.7 Hz), 7.73 (m, 1H), 7.91-7.94 (M, 3H), 7.99 (M, 1H). 13C NMR (125 MHz, DMSO-d6): 25.68, 49.07, 54.16, 60.72, 68.45, 68.54, 70.45, 73.79, 98.16, 99.69, 101.47, 111.56, 112.48, 113.32, 113.56, 116.22, 117.65, 117.68, 124.65, 127.98, 129.53, 130.52, 132.34, 132.35, 132.37, 134.08, 140.36, 147.98, 147.99, 149.85, 162.16, 176.09, 177.72, 177.74 ppm.Crude compound 5 (0.36 g, 0.46 mmol) was dissolved in methanol (6 mL) under nitrogen gas. 0.5N sodium methoxide (0.4 mL) in methanol was added to the solution. The mixture was magnetically stirred for 24 hours at room temperature in the dark. Cation exchange resin (H +) was added, filtered and washed with MeOH. The solvent was evaporated in vacuo. The product was purified using HPLC (C18, 5 μιη, fixed phase 250 x 10 mm; buffer A, H 2 O, buffer B, mobile phase CH 3 CN) to give compound 6 (DCDHF- β gal) (63 mg, 8.9% yield). ESI-MS m / z (M + ) calcd 615.20 found 638.30 (M + Na + ), 614.15 (M-H + ). 1 H NMR (500 MHz, DMSO-d 6 ): δ 1.79 (s, 6H), 3.36-3.39 (m, 1H), 3.40-3.42 (m, 2H), 3.48-3.70 (m, 5H), 4.12- 4.13 (m, 1H), 4.66-4.70 (m, 2H), 5.05 (d, 1H, J = 5 Hz), 5.21 (s, 2H), 7.11 (d, 1H, J = 16.45 Hz), 7.18 (d , 2H, J = 8.9 Hz), 7.46 (d, 1H, J = 8.7 Hz), 7.73 (m, 1H), 7.91-7.94 (M, 3H), 7.99 (M, 1H). 13 C NMR (125 MHz, DMSO-d 6 ): 25.68, 49.07, 54.16, 60.72, 68.45, 68.54, 70.45, 73.79, 98.16, 99.69, 101.47, 111.56, 112.48, 113.32, 113.56, 116.22, 117.65, 117.68, 124.65 , 127.98, 129.53, 130.52, 132.34, 132.35, 132.37, 134.08, 140.36, 147.98, 147.99, 149.85, 162.16, 176.09, 177.72, 177.74 ppm.
UV/UV / VisVis 및 형광 분광법 And fluorescence spectroscopy
PBS 완충 용액 (pH=7.4) 중에 DCDHF-βgal의 저장 용액을 제조하였다. 580 nm에서 여기를 수행하였고, 모든 여기 및 방출 슬릿 폭은 5 nm이었다. 각각의 β-갈락토오스 분해효소의 농도, pH는 달리 하였으나, 총 부피는 3.0 mL로 고정하였다.A stock solution of DCDHF- β gal was prepared in PBS buffer solution (pH = 7.4). Excitation was performed at 580 nm and all excitation and emission slit widths were 5 nm. The concentration and pH of each β-galactose degrading enzyme were varied, but the total volume was fixed at 3.0 mL.
형광 이미지화 및 유동세포 분석법에 의한 By fluorescence imaging and flow cytometry 세포내Intracellular 흡입 연구 Suction study
10% 소태아혈청 (Hyclone, Thermo Scientific, Logan, UT, USA) 및 1 × 항생제/항진균제 (Gibco, Life Technologies, Grand Island, NY, USA)로 강화된 둘베코수정이글배지 (DMEM, Gibco, Life Technologies, NY, USA)를 간의 간세포암종 (HepG2), 폐 선암종 (A549), 및 경구암 (KB) 세포를 배양하는 데 사용되었다. 2 × 104세포/웰의 농도를 갖는 8 웰 μ-슬라이드 (ibidi, Martinsried, Germany) 내로 세포를 뿌렸고, 5% CO2하에서, 37 ℃에서 24시간 동안 배양하였다. 그 다음에, 1, 2, 6, 12 및 24시간 동안 10 μM의DCDHF-βgal로 세포를 처리하였다. 인산완충식염수 (PBS)로 3회 세척 및 4% 파라포름알데하이드 수용액으로 고정 후에, 세포핵의 시각화를 위해 NucBlue® Fixed Cell Stain Ready ProbeTM 시약 (Molecular probe, Life Technologies, OR, USA)을 사용하여 세포를 대비염색하였다 (counterstrain). 형광 이미지화를 수행하기 위해 공초점 주사레이저 현미경 (LSM 710, Carl Zeiss, Germany)을 사용하였다. 세포 이미지화에 더하여, HepG2 세포에 대해 유동 세포 분석법 (flow cytometry )에 기초한 경쟁 분석을 수행하였다. 웰 당 1 × 105 세포 수로 세포를 12-웰 배양 플레이트에 24시간 동안 뿌린 후에, 세포에 10 mM 갈락토오스를 12시간 동안 사전처리하였다. 그 다음에, 10 μM의 DCDHF-βgal를 세포에 6시간 동안 첨가하였다. PBS로 세척 후에, 유동 세포 분석법 (Attune acoustic focusing cytometer, Applied Biosystems, USA)에 의해 고정된 세포를 분석하였다. Dulbecco's Fertilized Eagle Medium (DMEM, Gibco, Life) enhanced with 10% fetal bovine serum (Hyclone, Thermo Scientific, Logan, UT, USA) and 1 × antibiotic / antifungal (Gibco, Life Technologies, Grand Island, NY, USA) Technologies, NY, USA) were used to culture hepatocellular carcinoma (HepG2), lung adenocarcinoma (A549), and oral cancer (KB) cells. Cells were seeded into 8 well μ-slides (ibidi, Martinsried, Germany) with a concentration of 2 × 10 4 cells / well and incubated for 24 hours at 37 ° C. under 5% CO 2 . Cells were then treated with 10 μM of DCDHF- β gal for 1, 2, 6, 12 and 24 hours. After three washes with phosphate-buffered saline (PBS) and immobilization with 4% aqueous paraformaldehyde solution, the cells are treated with NucBlue ® Fixed Cell Stain Ready Probe ™ reagent (Molecular probe, Life Technologies, OR, USA) for visualization of the nuclei Counterstained. Confocal scanning laser microscope (LSM 710, Carl Zeiss, Germany) was used to perform fluorescence imaging. In addition to cell imaging, competitive assays based on flow cytometry were performed on HepG2 cells. Cells were sprinkled on 12-well culture plates for 24 hours at 1 × 10 5 cell count per well, and then cells were pretreated with 10 mM galactose for 12 hours. Next, 10 μM of DCDHF- β gal was added to the cells for 6 hours. After washing with PBS, fixed cells were analyzed by flow cytometry (Attune acoustic focusing cytometer, Applied Biosystems, USA).
β-갈락토오스 분해효소 과발현된 β-galactose degrading enzyme overexpressed HepG2HepG2 세포에서 In the cell 비율척도적Scale 형광 분석 Fluorescence analysis
HepG2 세포 중에 β-갈락토오스 분해효소를 과발현시키기 위해, β-갈락토오스 분해효소의 과발현을 위해 설계된 pCMV-LacZ 벡터 (Clontech Laboratories, Inc., Mountain View, CA, USA)를 형질 주입 (transfection)을 위해 사용하였다. 세포 이미지화를 위한 8-웰 챔버 또는 유동 세포 분석법을 위한 6-웰 플레이트 내로 각각, HepG2 세포를 뿌리고 70% 융합성 (confluent)이 될 때까지 배양하였다. 그 다음에, 제조자의 지시에 따라, Lipofectamine® 3000 키트 (Invitrogen, Life Technologies, CA, USA)를 사용하여, HepG2 세포 내로 1 μg의 pCMV-LacZ 벡터를 형질주입시켰다. 형질전환 된 HepG2 세포의 β-갈락토오스 분해효소 활성을 확인하기 위한 Western blotting을 시행하기 위해 PBS로 세척한 후 단백질 분해 효소 억제제 칵테일 (Sigma, MO, USA)이 들어있는 RIPA lysis buffer (iNtRON Biotechnology, Korea)로 얼음 위에서 녹였다. 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)로 분리 한 Bradford 분석법 (Bio-Rad Laboratories, Hercules, CA, USA)을 사용하여 세포 용해액의 총 단백질 농도를 측정하고 폴리 비닐디플루오라이드 (PVDF) 멤브레인 (Amersham Pharmacia Biotech, NJ, USA)으로 옮겼다. 차단 후, 막을 마우스 단일 클론 anti-β-갈라토시다제 및 anti-α-튜불린 항체 (Santa Cruz Biotechnology, CA, USA)로 처리한 후, HRP- 접합 된 2 차 항체 (Santa Cruz Biotechnology)와 함께 배양 하였다. 증폭된 화학 발광 (ECL) 검출 시약 (Amersham Pharmacia Biotech)을 사용하여 단백질 밴드를 관찰 하였다. pCMV-LacZ-형질주입된 HepG2 세포를 4 μM의 DCDHF-βgal로 12시간 동안 처리한 후에, 공초점 현미경 (LSM 710)을 사용하여 형광 세포 이미지를 관찰하였고, 유동 세포 분석법 (Attune acoustic focusing cytometer)에 의해 형광 강도를 얻었다.To overexpress β-galactose lyase in HepG2 cells, a pCMV-LacZ vector (Clontech Laboratories, Inc., Mountain View, CA, USA) designed for overexpression of β-galactose lyase is used for transfection It was. HepG2 cells were sprinkled and incubated until 70% confluent, respectively, into 8-well chambers for cell imaging or 6-well plates for flow cytometry. Then, according to the manufacturer's instructions, Lipofectamine 3000 ® kit using (Invitrogen, Life Technologies, CA, USA), were transfected with 1 μg injection of pCMV-LacZ vector into HepG2 cells. RIPA lysis buffer (iNtRON Biotechnology, Korea) containing proteinase inhibitor cocktail (Sigma, MO, USA) after washing with PBS for Western blotting to confirm β-galactose degrading enzyme activity of transformed HepG2 cells Melted on ice. Using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the total protein concentration of the cell lysate was measured and polyvinyldifluoride (PVDF ) Membranes (Amersham Pharmacia Biotech, NJ, USA). After blocking, the membranes were treated with mouse monoclonal anti-β-galatosidase and anti-α-tubulin antibody (Santa Cruz Biotechnology, CA, USA), followed by HRP-conjugated secondary antibody (Santa Cruz Biotechnology) Incubated. Protein bands were observed using an amplified chemiluminescent (ECL) detection reagent (Amersham Pharmacia Biotech). After treatment with pCMV-LacZ-transfected HepG2 cells with 4 μM of DCDHF- β gal for 12 hours, fluorescence cell images were observed using confocal microscopy (LSM 710), and an Attune acoustic focusing cytometer ), The fluorescence intensity was obtained.
세포독성 분석법Cytotoxicity Assay
3-(4,5-다이메틸티아졸-2-일)-2,5-다이페닐 테트라졸륨 브로마이드 (MTT, Roche Diagnostics GmbH, Mannheim, Germany) 검정을 사용하여 DCDHF-βgal 의 세포독성을 측정하였다. 96-웰 플레이트 중 1 × 104세포/웰의 마이크로플레이트에서 HepG2 세포를 12시간 동안 배양하였다. 그 다음에, 증식 배지를 제거하고 새로운 배양 배지로 교체하였다. 상이한 농도의 DCDHF-βgal의 첨가 후에, 세포를 24시간 동안 배양하였다. 그 다음에, 10 μl의 MTT 시약을 각각의 웰에 첨가하였고, 플레이트를 4시간 동안 배양하였다. 가용화 완충액의 첨가로 바이올렛 포르마잔 결정체 (violet formazan crystals)를 용해시켰고, 마이크로플레이트 리더 (xMark™, Bio-Rad Laboratories, Inc., Hercules, CA, USA)를 사용하여 570 nm에서 흡수율을 측정하였다. Using a 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT, Roche Diagnostics GmbH, Mannheim, Germany) assay The cytotoxicity of DCDHF- β gal was measured. HepG2 cells were incubated for 12 hours in microplates of 1 × 10 4 cells / well in 96-well plates. The growth medium was then removed and replaced with fresh culture medium. After addition of different concentrations of DCDHF- β gal, cells were incubated for 24 hours. Next, 10 μl of MTT reagent was added to each well and the plate was incubated for 4 hours. Violet formazan crystals were dissolved by the addition of solubilization buffer and the absorbance was measured at 570 nm using a microplate reader (xMark ™, Bio-Rad Laboratories, Inc., Hercules, Calif., USA).
마우스 이종이식 종양 모델에서 In a Mouse Xenograft Tumor Model DCDHFDCDHF -- ββ gal의gal's 생체내In vivo 시각화 Visualize
생체내 이미지화를 위해, β-갈락토오스 분해효소-과발현된 HepG2 세포를 종양 이종이식 마우스 모델을 수립하는 데 사용하였다. 모든 동물 실험들은 National Institutes (U.S.) guidelines for Health 및 우리 기관의 동물 연구 위원회의 제안에 따라 수행되었다. 지역 기관의 검토 위원회는 동물 보호에 대한 프로토콜 (KBSI-AEC1009)을 승인하였다. 리포펙타민 3000 시약 (Invitrogen)을 사용하여 LacZ 발현 벡타로 HepG2 세포를 형질주입시켰고, 37 ℃에서 2일 동안 배양하였다. 그 다음에, HepG2 세포 및 컨트롤 (control) HepG2 세포를 과발현시키는 1.2 × 107 β-갈락토오스 분해효소를 6주령 수컷 Balb/c nu/nu 마우스 (Orient Bio, Seungnam, Gyeonggi-do, South Korea)의 양쪽 옆구리 내로 피하 투여시켰다. 접종 7일 후에, 생체내 형광 이미지화 시스템 (Maestro, CRi Inc., Woburn, MA, USA)을 사용하여 1.5 mg/kg의 DCDHF-βgal를 정맥주사한 후, 종양을 갖는 마우스에서 생체내 형광 이미지화를 4시간 및 24시간에 수행하였다. 70% N2O/30% O2 혼합용액 중 2.5% 아이소플루레인으로 마취된 마우스로부터 전신 이미지를 얻었고, Maestro 2.4 소프트웨어로 분석하였다. 절개된 종양 조직의 생체외 형광 이미지를 24시간 희생시킴에 따라 얻었고, 공초점 현미경 (LSM 710)을 통해 동결-절편된 (cryo-sectioned) 종양 조각의 광학 이미지화를 수행하였다. 현미경 이미지에서 종양 형광의 정량화는 이미지 처리 소프트웨어, ImageJ를 사용하여 분석하였다.For in vivo imaging, β-galactose degrading enzyme-overexpressed HepG2 cells were used to establish a tumor xenograft mouse model. All animal experiments were performed under the suggestions of the National Institutes (US) guidelines for Health and our institution's animal research committee. The review committee of the local agency has approved the Protocol on Animal Protection (KBSI-AEC1009). HepG2 cells were transfected with LacZ expressing vector using Lipofectamine 3000 reagent (Invitrogen) and incubated at 37 ° C. for 2 days. Next, 1.2 × 10 7 β-galactose degrading enzymes that overexpress HepG2 cells and control HepG2 cells were used in 6-week-old male Balb / c nu / nu mice (Orient Bio, Seungnam, Gyeonggi-do, South Korea). Subcutaneously administered into both flanks. Seven days after inoculation, 1.5 mg / kg of DCDHF- β gal was injected intravenously using an in vivo fluorescence imaging system (Maestro, CRi Inc., Woburn, MA, USA), followed by in vivo fluorescence imaging in mice with tumors Was carried out at 4 and 24 hours. Systemic images were obtained from mice anesthetized with 2.5% isoflulane in 70% N 2 O / 30% O 2 mixed solution and analyzed by Maestro 2.4 software. In vitro fluorescence images of incised tumor tissue were obtained at 24 hours sacrifice and optical imaging of cryo-sectioned tumor pieces was performed via confocal microscopy (LSM 710). Quantification of tumor fluorescence in microscopic images was analyzed using image processing software, ImageJ.
결과 및 고찰Results and Discussion
DCDHFDCDHF -- ββ gal의gal's 합성 synthesis
전술한 합성 경로 및 방법에 따라 DCDHF-βgal를 제조하였다. 먼저, ASGPR-표적화된 갈락토오스 및 링커를 합성하였다 (Supporting Information, SI) [N.H. Ho, R. Weissleder, C.H. Tung, A Self-immolative Reporter for Beta-galactosidase Sensing. ChemBioChem. 8 (2007) 560-566.). 다음으로, 종래 문헌에 보고된 방법을 적용하여 형광체를 합성하엿다(P. Gopalan, H.E. Katz, D.J. McGee, C. Erben, T. Zielinski, D. Bousquet, D. Muller, J. Grazul, Y. Olsson, Star-shaped Azo-based Dipolar Chromophores: Design, Synthesis, Matrix Compatibility, and Electro-optic Activity. J. Am. Chem. Soc. 126 (2004) 1741-1747., S.J. Lord, N.R. Conley, H.L. Lee, R. Samuel, N. Liu, R.J. Twieg, W.E. Moerner, A Photoactivatable Push-pull Fluorophore for Single-Molecule Imaging in Live Cells. J. Am. Chem. Soc. 130 (2008) 9204-9205.). NIR 형광체 (화합물 4)의 갈락토오스 삽입된 링커 (화합물 3)과의 치환반응을 통해 아세테이트 보호된 화합물 (화합물 5)을 생성하고, NaOMe/MeOH를 사용하여 아세테이트 작용화기의 탈보호를 수행한 후 양이온 교환 수지 처리로 최종 생성물인 화합물 6(DCDHF-βgal)를 수득하였다. 합성된 화합물들을 1H NMR, 13C NMR, 및 ESI-MS에 의해 확인하였다.DCDHF- β gal was prepared according to the synthetic routes and methods described above. First, ASGPR-targeted galactose and linker were synthesized (Supporting Information, SI) [NH Ho, R. Weissleder, CH Tung, A Self-immolative Reporter for Beta-galactosidase Sensing. Chem BioChem. 8 (2007) 560-566.). Next, phosphors were synthesized by applying the method reported in the prior art (P. Gopalan, HE Katz, DJ McGee, C. Erben, T. Zielinski, D. Bousquet, D. Muller, J. Grazul, Y. Olsson , Star-shaped Azo-based Dipolar Chromophores: Design, Synthesis, Matrix Compatibility, and Electro-optic Activity.J. Am. Chem. Soc.126 (2004) 1741-1747., SJ Lord, NR Conley, HL Lee, R Samuel, N. Liu, RJ Twieg, WE Moerner, A Photoactivatable Push-pull Fluorophore for Single-Molecule Imaging in Live Cells.J. Am. Chem. Soc. 130 (2008) 9204-9205. Substitution reaction of the NIR phosphor (Compound 4) with galactose-embedded linker (Compound 3) yielded an acetate protected compound (Compound 5), followed by deprotection of the acetate functional group using NaOMe / MeOH, followed by cation Exchange resin treatment gave the final product Compound 6 (DCDHF- β gal). Synthesized compounds were confirmed by 1 H NMR, 13 C NMR, and ESI-MS.
DCDHF-βgal를 합성한 후에, β-갈락토오스 분해효소에 대한 선택성을 흡수율 및 형광 분광법을 사용하여 시험하였다. 도 2에 나타낸 바와 같이, β-갈락토오스 분해효소에 의한 DCDHF-βgal의 갈락토오스 유닛이 절단되어 세포 내부의 β-갈락토오스 분해효소를 선택적으로 검출할 수 있을 것으로 기대된다. 따라서, 갈락토오스 유닛과 형광단 유닛 사이의 글리코 시드 결합은 내인성으로 생성된 β-갈락토오스 분해효소에 의해 절단될 것으로 예상되었다. DCDHF-βgal은 PBS 완충액 (pH 7.4, 37 ℃,도 2a) 중 540nm에서 강한 흡수 밴드를 보였다. DCDHF-βgal(20 μM)의 시험 용액에 첨가되는 β-갈락토오스 분해효소의 양이 증가함에 따라(0-50 U), 540 nm에서 흡수가 감소되고 595 nm에서 새롭게 편이된 밴드가 형성되었다. 새로운 흡수 밴드는 β-갈락토오스 분해효소에 의한 갈락토오스 유닛의 쪼개짐 뒤에 음이온 종 모이어티의 형성(도 2a)에 기인한 것이다. 또한, DCDHF-βgal의 형광 스펙트럼을 β-갈락토오스 분해효소의 존재하에 분석하였다. DCDHF-βgal (5 μM, 도 2b)의 흡수 스펙트럼 피크에 따라 580 nm의 여기 파장에서 형광 적정을 수행하였고, 615 nm에서 강한 형광 밴드를 나타내었다. 탐지체 DCDHF-βgal (5 μM)의 용액에 첨가되는 β-갈락토오스 분해효소의 양이 증가함에 따라(0-50U) 615 nm에서 방출 밴드는 현저하게 감소되었다 (도 2b). 동시에, β-갈락토오스 분해효소의 비율척도적 탐지를 입증하는 665 nm에서의 새로운 방출 밴드 형성이 관찰되었다. 탐지 범위를 향상시키고 환경적 간섭을 최소화하는 비율척도적 형광 탐지체의 합리적인 신호 출력으로 인해 비율척도적 형광 탐지체(Ratiometric fluorescent probes )가 보통 선호된다. 자유 리간드의 665 nm와 615 nm에서의 형광 방출의 상대적 강도비율 (I665/I615)은 0.12이었고, 이는 50 U의 β-갈락토오스 분해효소의 첨가에 따라 1.26으로 10.7배 증가하였다. 또한, 형광의 증가는 시간 함수(도 2c 및 2d)로서 측정되었고, 이는 665 nm에서 최대로 강화된 형광이 βgal (50 U)의 첨가 20 분 후에 얻어졌음을 보여준다. 이는 DCDHF-βgal에 의해 의한 β-갈락토오스 분해효소의 고감도 검출을 나타낸다.After synthesizing DCDHF- β gal, selectivity to β -galactose degrading enzyme was tested using absorption and fluorescence spectroscopy. As shown in Fig. 2, the galactose unit of DCDHF- β gal by β -galactose degrading enzyme is cleaved to be able to selectively detect β -galactose degrading enzyme inside the cell. Thus, the glycoside bonds between the galactose units and the fluorophore units were expected to be cleaved by endogenously produced β -galactose degrading enzymes. DCDHF- β gal showed a strong absorption band at 540 nm in PBS buffer (pH 7.4, 37 ° C, Figure 2a). As the amount of β -galactose degrading enzyme added to the test solution of DCDHF- β gal (20 μM) increased (0-50 U), absorption was reduced at 540 nm and a newly shifted band was formed at 595 nm. The new absorption band is due to the formation of anionic species moieties following cleavage of the galactose units by β -galactose degrading enzymes (FIG. 2A). In addition, fluorescence spectra of DCDHF- β gal were analyzed in the presence of β -galactose degrading enzyme. DCDHF- β gal Fluorescence titration was performed at an excitation wavelength of 580 nm according to the absorption spectral peak of (5 μM, FIG. 2B), showing a strong fluorescence band at 615 nm. Detector DCDHF- β gal As the amount of β -galactose degrading enzyme added to the (5 μM) solution increased (0-50U), the emission band decreased significantly at 615 nm (FIG. 2B). At the same time, new emission band formation at 665 nm was observed, demonstrating the rational detection of β -galactose degrading enzyme. Ratiometric fluorescence probes are usually preferred because of the rational signal output of the spectral fluorescence detector which improves detection range and minimizes environmental interference. The relative intensity ratio (I 665 / I 615 ) of fluorescence emission at 665 nm and 615 nm of the free ligand was 0.12, which increased by 10.7 fold to 1.26 with the addition of 50 U of β -galactose degrading enzyme. In addition, the increase in fluorescence was measured as a function of time (FIGS. 2C and 2D), showing that maximally enhanced fluorescence at 665 nm was obtained 20 minutes after addition of β gal (50 U). This indicates high sensitivity detection of β -galactose degrading enzyme by DCDHF- β gal.
또한, DCDHF-βgal과 β-갈락토오스 분해효소의 활성화를 질량 스펙트럼을 통해 확인하였다. DCDHF-βgal는 614.15의 m/z 값에서 ESI-MS 질량 피크를 나타내었고, 이는 (M-H+)와 대응되는 것이다. β-갈락토오스 분해효소의 첨가 후에, 342.25 (M+K+)에서 새로운 질량 피크가 발견되었다. 이러한 질량 분석은 또한 DCDHF-βgal과 β-갈락토오스 분해효소의 활성화를 가리킨다. β-갈락토오스 분해효소 매개된 쪼개짐 단계의 pH 의존성을 또한 조사하였다(도 6). DCDHF-βgal는 3-10의 pH 범위 내에서 안정하다. 하지만, DCDHF-βgal는 이러한 pH 범위 (3-10) 내에서 β-갈락토오스 분해효소와 즉시 반응한다. 이러한 발견은 또한 pH 효과에 의한 간섭 없는 생물적 조건 하에서의 β-갈락토오스 분해효소의 탐지를 위한 DCDHF-βgal의 유용성을 나타낸다. 추가로, DCDHF-βgal의 반응성에 대한 다른 생물적 피분석물의 간섭 또한 시험하였다. 200 등가물의 환원 효소, 에스테라제, 리소자임, 시스테인, 글루타티온, 다이티오트레이톨 (dithiothreitol), 황화 수소 및 과산화수소의 처리에 의해 현저한 형광성의 변화가 관찰되지 않았다 (도 7).In addition, the activation of DCDHF- β gal and β-galactose degrading enzyme was confirmed by mass spectra. DCDHF- β gal 614.15 ESI-MS mass peaks are shown at m / z values, corresponding to (MH + ). After addition of β-galactose degrading enzyme, a new mass peak was found at 342.25 (M + K + ). This mass spectrometry also indicates the activation of DCDHF- β gal and β-galactose degrading enzymes. The pH dependence of the β-galactose degrading enzyme mediated cleavage step was also investigated (FIG. 6). DCDHF- β gal is stable within the pH range of 3-10. However, DCDHF- β gal reacts immediately with β-galactose degrading enzyme within this pH range (3-10). This finding also indicates the utility of DCDHF- β gal for the detection of β-galactose degrading enzymes under biological conditions without interference by pH effects. In addition, interference of other biological analytes on the reactivity of DCDHF- β gal was also tested. No significant fluorescence change was observed by treatment of 200 equivalents of reductase, esterase, lysozyme, cysteine, glutathione, dithiothreitol, hydrogen sulfide and hydrogen peroxide (FIG. 7).
또한, DCDHF-βgal의 세포독성을 HepG2 세포를 이용한 MTT 분석에 의해 평가하였다 (도 8). 세포가 DCDHF-βgal로 처리되고, 24시간 동안 추가로 배양되는 경우, DCDHF-βgal가 상이한 농도에서 HepG2 세포에 대해 낮은 세포독성을 가짐을 관찰하였다. 종래 연구에서 다른 간세포암 세포계를 포함한 다양한 암세포계 중에 HepG2 세포계가 ASGPR을 매우 과발현시키는 것으로 보고된 바 있다(J.H. Han, Y.K. Oh, D.S. Kim, C.K. Kim, Enhanced Hepatocyte Uptake and Liver Targeting of Methotrexate using Galactosylated Albumin as a Carrier. Int. J. Pharm. 188 (1999) 39-47.). 종합해보면, DCDHF-βgal은 확연한 세포독성 없이 그의 갈락토오스 모이어티를 사용하여 간세포암종 세포를 효율적으로 표적화하는 능력을 가진다.In addition, cytotoxicity of DCDHF- β gal was evaluated by MTT assay using HepG2 cells (FIG. 8). When cells were treated with DCDHF- β gal and further incubated for 24 hours, it was observed that DCDHF- β gal has low cytotoxicity against HepG2 cells at different concentrations. Previous studies have reported that HepG2 cell lines overexpress ASGPR in various cancer cell lines, including other hepatocellular carcinoma cell lines (JH Han, YK Oh, DS Kim, CK Kim, Enhanced Hepatocyte Uptake and Liver Targeting of Methotrexate using Galactosylated Albumin). as a Carrier.Int. J. Pharm. 188 (1999) 39-47.). Taken together, DCDHF- β gal has the ability to efficiently target hepatocellular carcinoma cells using its galactose moiety without pronounced cytotoxicity.
DCDHFDCDHF -- ββ gal의gal's 세포 및 효소 특이성 Cell and Enzyme Specificity
아시알로당단백질 수용체-매개된 세포내 섭취 (endocytosis)를 이용하여 간세포에 대한 DCDHF-βgal의 표적화된 세포 흡입을 조사하기 위해, 몇몇의 암세포계를 DCDHF-βgal로 처리하였다. 세포내 흡입은 공초점 이미지화 및 유동 세포 분석법에 의해 측정하였다. ASGPR을 발현시키는 HepG2 세포를 다양한 시간 (1, 2, 6, 12시간)별로 10 μM의 DCDHF-βgal와 함께 배양시켰고, HepG2 세포에 의해 방출된 형광은 어떠한 간섭 없이 610 nm 및 649 nm에서 측정되었다 (도 9). 측정 결과, 둘 모두의 방출 스펙트럼으로부터의 형광은 2시간에 현저히 나타나기 시작했고, 6시간에 최대로 나타났다. 또한, 배양 24시간이 경과된 이후에도 형광 신호가 A549 및 KB, ASGPR 음성 세포계에서는 탐지되지 않았고, 오직 HepG2 세포에서만 탐지되었다 (도 3). 이 결과는 DCDHF-βgal로 처리 한 세포의 유동 세포 계측법 분석에 의해 뒷받침되었다 (도 10). 또한, 이러한 결과는 10 mM 갈락토오스의 사전처리 후에 DCDHF-βgal (10 μM)로 라벨링된 HepG2 세포를 이용한 유동 세포 분석법에 의해 확인되었다 (도 3). HepG2 세포의 표면 상의 ASGPR에 대한 DCDHF-βgal의 갈락토오스 결합을 차단할 수 있는 HepG2 세포가 과량의 갈락토오스로 사전-첨가된 경우, 형광 막대그래프 플롯에서 감소된 세포 흡입이 관찰되었다. 이러한 세포 흡입 연구는 DCDHF-βgal가 ASGPR에 대한 표적화 능력을 통해 높은 선택성을 가짐을 의미한다.Several cancer cell lines were treated with DCDHF- β gal to investigate targeted cellular uptake of DCDHF- β gal into hepatocytes using asialo glycoprotein receptor-mediated endocytosis. Intracellular aspiration was measured by confocal imaging and flow cytometry. HepG2 cells expressing ASGPR were incubated with 10 μM of DCDHF- β gal at various times (1, 2, 6, 12 hours), and fluorescence emitted by HepG2 cells was measured at 610 nm and 649 nm without any interference. (FIG. 9). As a result of the measurement, fluorescence from both emission spectra began to appear markedly at 2 hours and peaked at 6 hours. In addition, no fluorescence signal was detected in A549 and KB, ASGPR negative cell lines after 24 hours of culture, but only in HepG2 cells (FIG. 3). This result was supported by flow cytometry analysis of cells treated with DCDHF- β gal (FIG. 10). This result was also confirmed by flow cytometry using HepG2 cells labeled with DCDHF- β gal (10 μM) after pretreatment of 10 mM galactose (FIG. 3). Reduced cell uptake was observed in the fluorescent histogram plots when HepG2 cells that could block galactose binding of DCDHF- β gal to ASGPR on the surface of HepG2 cells were pre-added with excess galactose. This cell inhalation study means that DCDHF- β gal has high selectivity through its targeting ability against ASGPR.
DCDHF-βgal가 선택적으로 β-갈락토오스 분해효소에 대한 비율척도적 형광 탐지체가 될 수 있는지 여부를 추가로 조사하기 위해, 처음에 HepG2 세포에서 pCMV-LacZ 벡터 및 과발현된 β-갈락토오스 분해효소를 이용하여 형질주입 실험을 수행하였다. 음성 대조군 HepG2 세포와 과발현된 β-갈락토오스 분해효소를 지닌 양성 HepG2 세포를 각각 12시간 동안 DCDHF-βgal (4 μM)으로 처리하였고, 약 650 nm에서 상대적 형광 강도를 현미경 및 유동 세포 분석법으로 평가하였다. 도 4a에 나타나는 바와 같이, 강화된 형광은 대조군 세포보다는 오히려 β-갈락토오스 분해효소-과발현된 세포에서 관찰되었다. 이것은 웨스턴 블랏 분석 (도 12)에 의해 더 설명되었다. 또한, 유동 세포 분석법의 막대그래프 플롯은 과발현된 β-갈락토오스 분해효소를 지닌 HepG2 세포에서 증가된 형광을 보여주었고, 이들의 평균 형광 강도는 대조군 세포보다 2배 이상 높았다 (도 4b, 및 c). 이러한 결과들은 DCDHF-βgal이 형질주입된 세포에서 다량의 β-갈락토오스 분해효소에 의해 적절하게 쪼개졌고, 650 nm에서 증가된 형광 신호를 나타나게 했음을 시사하는 것이다. To further investigate whether DCDHF- β gal can optionally be a ratiometric fluorescence detector for β -galactose degrading enzyme, initially using pCMV-LacZ vector and overexpressed β -galactose degrading enzyme in HepG2 cells Transfection experiments were performed. Negative control HepG2 cells and positive HepG2 cells with overexpressed β -galactose degrading enzyme were each treated with DCDHF- β gal (4 μM) for 12 hours, and the relative fluorescence intensity at about 650 nm was evaluated by microscopy and flow cytometry. . As shown in FIG. 4A, enhanced fluorescence was observed in β -galactose degrading enzyme-overexpressed cells rather than control cells. This was further explained by Western blot analysis (FIG. 12). In addition, histogram plots of flow cytometry showed increased fluorescence in HepG2 cells with overexpressed β -galactose degrading enzyme, and their mean fluorescence intensity was more than 2 times higher than control cells (FIGS. 4B, and c). These results suggest that DCDHF- β gal was properly cleaved by a large amount of β -galactose degrading enzyme in the transfected cells and showed an increased fluorescence signal at 650 nm.
DCDHFDCDHF -- ββ gal의gal's 생체내In vivo 표적화Targeting 능력 ability
과발현된 β-갈락토오스 분해효소 HepG2 세포를 갖는 이종이식 모델을 이용하여 DCDHF-βgal의 종양-특이적 표적화 능력을 조사하였다. 플라스미드 형질주입 방법에 의해 과발현된 β-갈락토오스 분해효소를 지닌 HepG2 세포를 제조한 후에, 과발현된 β-갈락토오스 분해효소 및 대조군 HepG2 세포를 마우스의 양쪽 옆구리에 피하 접종시킴으로써 종양을 갖는 마우스 이종이식을 수립하였다. 도 5a에 나타나는 바와 같이, 양쪽 종양에 24시간 동안 정맥내 주입된 DCDHF-βgal (1 mg/kg)의 축적은 600-700 nm에서 방출이 있는 생체내 전신 형광 이미지화에 의해 명백하게 가시화되었다. DCDHF-βgal가 HepG2 종양 상의 ASGPR의 선택적인 전달을 위해 효율적인 표적화 리간드 (갈락토오스)를 이용함에 따라, 상대적으로 이른 시간 지점 (주입 후 4시간, 도 13)에서 두 종양 모두 명백한 형광 신호가 탐지되었다. Maestro 이미지화 시스템에 의해 수행된 생체외 형광 평가는 대조군 종양 보다는 과발현된 β-갈락토오스 분해효소를 지닌 HepG2 세포에서 생성된, 절제된 종양 조직에서 650 nm 내지 700 nm 범위의 형광 방출 스펙트럼의 강도가 현저히 높다는 것을 보여주었다(도 13 (b), (c), 표 1). Tumor-specific targeting capacity of DCDHF- β gal was investigated using a xenograft model with overexpressed β -galactose degrading enzyme HepG2 cells. After preparing HepG2 cells with β -galactose lyase overexpressed by the plasmid transfection method, mouse xenografts with tumors were established by subcutaneously inoculating the overexpressed β -galactose lyase and control HepG2 cells on both sides of the mouse. It was. As shown in FIG. 5A, the accumulation of DCDHF- β gal (1 mg / kg) injected intravenously into both tumors for 24 hours was clearly visualized by in vivo whole body fluorescence imaging with emission at 600-700 nm. As DCDHF- β gal used an efficient targeting ligand (galactose) for the selective delivery of ASGPR on HepG2 tumors, a clear fluorescence signal was detected in both tumors at a relatively early time point (4 hours after injection, Figure 13). . In vitro fluorescence evaluation performed by the Maestro imaging system showed that the intensity of the fluorescence emission spectrum in the range of 650 nm to 700 nm was significantly higher in excised tumor tissue produced in HepG2 cells with overexpressed β -galactose degrading enzyme than in control tumors. It was shown (Fig. 13 (b), (c), Table 1).
700 nm 부근에서 측정된 이러한 비율척도적 형광 탐지는 종양 조직에서 증가된 β-갈락토오스 분해효소의 효소 활성에 의해 유발될 수 있다. 또한, 이러한 결과는 650 nm 부근의 방출 파장에서 동결-절편된 종양 조직의 형광 이미지화에 의해 평가되었고, 과발현된 β-갈락토오스 분해효소를 지닌 종양에서 형광 신호의 현저한 증가를 나타내었으며(도 5b), 이는 종양 형광 이미징의 정량 분석에 의해 뒷받침된다(도 5c). This ratiometric fluorescence detection measured near 700 nm can be caused by increased enzyme activity of β-galactose degrading enzymes in tumor tissues. In addition, these results were evaluated by fluorescence imaging of freeze-sectioned tumor tissue at emission wavelengths near 650 nm and showed a marked increase in fluorescence signal in tumors with overexpressed β-galactose degrading enzymes (FIG. 5B), This is supported by quantitative analysis of tumor fluorescence imaging (FIG. 5C).
세포의 β-갈락토오스 분해효소 또는 그의 효소 활성의 이미지화를 위한 형광 탐지체가 있더라도, 낮은 파장 형광 신호 또는 표적화된 전달과 같은 현실적인 어려움이 내시경 형광 이미지화 또는 β-갈락토오스 분해효소 탐지를 위한 상업적인 종양-표적화 시약을 이용하는 실시간 추적을 통한 그들의 생체내 적용을 방해하였다. 본 발명에서 따른 DCDHF-βgal은 생체내 종양-표적화된 형광 이미지화뿐만 아니라 임의의 수정 없이 β-갈락토오스 분해효소에 대한 비율척도적 형광 반응을 가능하게 하였다. 게다가, 665 nm에서의 표적화된 탐지체의 NIR 형광 방출은 간세포암종으로부터 유래된 종양에서 β-갈락토오스 분해효소의 비침습성 생체내 시각화를 달성할 수 있게 해주었다.Although there are fluorescence detectors for imaging of β-galactose degrading enzymes or their enzymatic activity of cells, practical difficulties such as low wavelength fluorescence signals or targeted delivery can be achieved by commercial tumor-targeting reagents for endoscopic fluorescence imaging or β-galactose degrading enzyme detection. Interfere with their in vivo application via real time tracking using. The DCDHF- β gal according to the present invention enabled not only tumor-targeted fluorescence imaging in vivo but also a rational fluorescence response to β-galactose degrading enzyme without any modification. In addition, the NIR fluorescence emission of the targeted detector at 665 nm enabled to achieve non-invasive in vivo visualization of β-galactose degrading enzymes in tumors derived from hepatocellular carcinoma.
결론conclusion
본 발명에서는 간세포-표적화된 이미지화, 및 β-갈락토오스 분해효소-특이 활성에 대해 이중적인 잠재력이 있는, 신규의 NIR 비율척도적 형광 프로브 화합물인 DCDHF-βgal을 제공한다. 본 발명에 따른 DCDHF-βgal은 비율척도적 형광 반응을 지니고 간세포 상의 ASGPR에 대한 뚜렷한 표적화 능력 및 β-갈락토오스 분해효소에 대한 높은 민감성을 나타내었다 (도 1). DCDHF-βgal 용액에 첨가되는 β-갈락토오스 분해효소의 양을 증가시킬 때, 615 nm에서 방출 밴드의 감소가 일어났으며, 동시에 pH 효과에 의한 간섭 없이 β-갈락토오스 분해효소의 비율척도적 탐지를 입증하는 665 nm에서 새로운 방출 밴드의 형성이 관찰되었다. 이러한 비율척도적 탐지체는 적은 세포 독성과 함께, 간세포암종, HepG2 세포계 내로의 매우 특이적인 ASGPR-매개된 흡입을 나타내었다. DCDHF-βgal의 이러한 종양-표적화 능력 및 NIR 방출은 DCDHF-βgal이 간세포암종 이종이식 마우스 모델에서 생체내 표적화된 이미지를 위해 유용한 탐지체가 될 수 있음을 의미한다. 따라서, DCDHF-βgal는 ASGPR-매개된 세포 흡수에 이어 β-갈락토오스 분해효소에 대해 이작용성 비율척도적 형광 반응을 나타내었고, 생체내 표적화된 시각화 및 치료적 접근을 위한 훌륭한 이미지화 플랫폼이 될 수 있을 것이다.The present invention provides DCDHF- β gal, a novel NIR ratiometric fluorescent probe compound with dual potential for hepatocyte-targeted imaging, and β-galactose degrading enzyme-specific activity. DCDHF- β gal according to the present invention had a ratiometric fluorescence response and showed a clear targeting ability against ASGPR on hepatocytes and high sensitivity to β-galactose degrading enzyme (FIG. 1). Increasing the amount of β-galactose degrading enzyme added to the DCDHF- β gal solution resulted in a decrease in the emission band at 615 nm, while at the same time detecting the proportional detection of β-galactose degrading enzyme without interference by the pH effect. Formation of a new emission band was observed at 665 nm which demonstrates. These ratiometric detectors exhibited very specific ASGPR-mediated inhalation into the HepG2 cell line with hepatocellular carcinoma, with low cytotoxicity. These tumors DCDHF- β gal - targeting ability and NIR emission means that DCDHF- β gal bodies can be useful for detection in vivo targeting images in hepatocellular carcinoma xenograft mouse model. Thus, DCDHF- β gal showed a bifunctional ratiometric fluorescence response to ASGPR-mediated cell uptake followed by β-galactose degrading enzymes and could be an excellent imaging platform for in vivo targeted visualization and therapeutic approaches. There will be.
Claims (3)
[화학식 1]
[Formula 1]
상기 베타-갈락토오스 분해효소는 간세포에서 과발현되는 것을 특징으로 하는 형광 프로브 화합물.The method of claim 1,
The beta-galactose degrading enzyme is characterized in that the overexpression in hepatocytes.
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