醌氧化还原酶响应型光声探针的合成及其对乳腺癌的成像

doi:10.6023/A20100459

摘要/Abstract

醌氧化还原酶(NQO1)是生物体内的一种双电子还原反应的专性催化还原酶. 在一些疾病如乳腺癌中NQO1过度表达, 因此NQO1可作为生物标志物以观察乳腺癌肿瘤的产生和发展. 目前已有一些文献报导用于NQO1检测和荧光成像的荧光探针, 但大部分这些探针具有吸收波长较短、背景干扰较强、组织穿透深度较低等不足. 为克服荧光探针成像的上述缺陷, 我们构建出一种具有推拉电子结构的可对NQO1响应的新型近红外光声探针TPA-X-Q. 实验结果表明, 该探针可在生物体内和体外对NQO1进行专一性的增强型光声检测和成像, 并可提供高分辨率的实时光声信号图像. 此外, 这种新型的光声探针能够成功应用于乳腺癌小鼠模型, 对乳腺癌肿瘤中过量表达的NQO1进行灵敏准确的光声检测与成像, 借助多光谱光声断层扫描技术(MSOT)的三维光声图像重建功能, 可实现肿瘤病灶区域的定位.

关键词: 醌氧化还原酶, 探针, 近红外, 乳腺癌, 多光谱光声断层扫描

NAD(P)H:quinone oxidoreductase-1 (NQO1) is a cytosolic two-electron-specific reductase whose abnormal expression is associated with breast cancer, and NQO1 has been regarded as an important biomarker for monitoring the occurrence and development of breast tumors. Although there are some research reports involving fluorescent probes for imaging NQO1 in vivo, they generally suffer from the strong light scattering by tissues which would cause the spatial resolution of fluorescent signals degrade rapidly with imaging depth. On the other hand, as an emerging detection and imaging modality, optoacoustic tomography (OAT) is capable of providing more detailed information in deep biological tissues than fluorescent imaging, since in OAT ultrasound signals are the reporting signals. To overcome the inherent defects of fluorescent imaging, in this work, we developed a novel near-infrared (NIR) NQO1-activatable optoacoustic (OA) probe TPA-X-Q based on the push-pull internal charge transfer (ICT) scaffold. TPA-X-Q consists of a NQO1-responsive moiety, quinone propionate group and a NIR chromophore TPA-X-OH. In the absence of the enzyme NQO1, the probe displays nearly no noticeable OA signal upon NIR excitation. Whereas in the presence of NQO1, the quinone propionate group of TPA-X-Q is reduced and cleaved by the enzymatic reaction triggered by NQO1, and thus TPA-X-Q is transformed into TPA-X-OH, consequently evident OA signal is generated, thereby realizing the detection and imaging of NQO1. As for the probe, the limit of detection (LOD) is determined to be 0.193 μg·mL ^–1. Furthermore, the probe TPA-X-Q displays several advantages, such as quite good selectivity towards NQO1, low cytotoxicity and excellent photostability. Moreover, the probe has been successfully utilized to detect and image the overexpressed NQO1 in the breast cancer-bearing mouse model. Orthogonal-view three-dimensional (3D) images can also be obtained by using multispectral optoacoustic tomography (MSOT), and thus precise localization of the breast cancer tumors can be achieved. This probe holds great potential for being employed as an efficacious tool for diagnosing breast cancer via responding to NQO1.

Key words: NQO1, optoacoustic probe, near-infrared, breast cancer, MSOT

引用此文

黄靖, 王超, 林敏刚, 曾钫, 吴水珠. 醌氧化还原酶响应型光声探针的合成及其对乳腺癌的成像[J]. 化学学报, 2021, 79(3): 331-337.

Jing Huang, Chao Wang, Mingang Lin, Fang Zeng, Shuizhu Wu. Synthesis of NQO1-activatable Optoacoustic Probe and Its Imaging of Breast Cancer[J]. Acta Chimica Sinica, 2021, 79(3): 331-337.

导出引用 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

分享此文

图/表 5

图1. 探针TPA-X-Q结构及其响应成像机理示意图

Figure 1. The response and imaging mechanism of TPA-X-Q

图2. (A) 探针TPA-X-Q(10 μmol·L –1)、生色团化合物TPA-X-OH(10 μmol·L –1)溶液、探针和NQO1反应液(探针浓度10 μmol·L –1, NQO1浓度15 μg·mL –1, 反应时间40 min)的吸收光谱. (B) 探针与NQO1反应液的HRMS图谱(探针浓度10 μmol·L –1, NQO1浓度15 μg·mL –1, 反应时间10 min). (C) 探针(10 μmol·L –1)在15 μg·mL –1 NQO1作用下, 不同响应时间(0~40 min)的吸收光谱变化. (D) 探针(10 μmol·L –1)在不同浓度(0~15 μg·mL –1)NQO1作用下40 min后的吸收光谱变化. 所有测试均在37 ℃下的磷酸盐缓冲液(pH=7.4, 含DMSO(φ=1%)和100 μmol·L–1辅酶因子NADH)中进行

Figure 2. (A) Absorption spectra of TPA-X-Q (10 μmol·L –1), TPA-X-OH (10 μmol·L –1), TPA-X-Q upon reaction with NQO1 (the concentration of probe was 10 μmol·L –1; that of NQO1 was 15 μg·mL –1; the reaction time was 40 min). (B) The HR mass spectrum of TPA-X-Q upon reaction with NQO1 (the concentration of probe was 10 μmol·L –1; that of NQO1 was 15 μg·mL –1; the reaction time was 10 min). (C) Time-dependent absorption spectra (0~40 min) of the probe in the presence of NQO1 (15 μg·mL –1). (D) Absorption spectra at 40 min of the probe (10 μmol·L –1) in the presence of varied levels of NQO1 (0~15 μg·mL –1). These measurements were performed in PBS (pH=7.4, containing DMSO(φ=1%) and 100 μmol·L–1 NADH cofactor) at 37 ℃

图3. (A) 15 μg·mL –1NQO1作用下探针(10 μmol·L –1)的归一化光声强度与波长关系图. (B) 探针(10 μmol·L –1)对不同浓度(0~15 μg·mL –1) NQO1的相对光声强度响应(样本量n=3); 插图中为探针与不同浓度NQO1在假体中的光声图像(激发波长: 710 nm). (C) 探针(10 μmol· L –1)对NQO1(15 μg·mL –1)及干扰物的光声响应,及(D)抗干扰测试(n=3); 测试浓度: 氯化钠(NaCl)、氯化钾(KCl)、丙氨酸(Ala)、精氨酸(Arg)、酪氨酸(Tyr)、高半胱氨酸(Hcy)、半胱氨酸(Cys)、二硫代苏糖醇(DTT)、谷胱甘肽(GSH)、尿素、抗坏血酸(AA)和丝氨酸为1 mmol· L–1; 牛血清蛋白(BSA)为20 mg·mL–1; 葡萄糖为10 mmol·L–1. 所有测试均在37 ℃下的磷酸盐缓冲液(pH=7.4, 含DMSO(φ=1%)和100 μmol·L–1辅酶因子NADH)中进行

Figure 3. (A) The wavelength dependence of probe’s (10 μmol·L–1) normalized optoacoustic(OA) intensity in the presence of NQO1(15 μg·mL –1). (B) OA response of the probe (10 μmol·L –1) to NQO1 of varied levels (sample size n=3); The inset shows the representative optoacoustic images of the probe in phantom at varied NQO1 level; Excitation wavelength: 710 nm. (C) OA response of the probe (10 μmol·L –1) in the presence of NQO1(15 μg·mL –1) and (D) anti-interference test (n=3); The concentration of NaCl, KCl, alanine (Ala), arginine (Arg), tyrosine (Tyr), homocysteine (Hcy), cysteine (Cys), DL-dithiothreitol (DTT), glutathione (GSH), Urea, ascorbic acid (AA), serine was 1 mmol·L–1; that of BSA was 20 mg·mL–1; that of glucose was 10 mmol·L–1. These measurements were performed in PBS (pH=7.4, containing DMSO(φ=1%) and 100 μmol·L–1 NADH cofactor) at 37 ℃

图4. (A) 注射PBS缓冲液(对照)或含4T1细胞悬浮缓冲液(乳腺癌模型)1周后小鼠的明场图像. (B) 乳腺癌小鼠模型在注射探针溶液之前(0 min)和之后10, 30和60 min时的横截面光声图像; 右侧彩条代表不同的OA信号强度. (上排：激活探针的光声信号图; 下排：激活探针与灰度背景的光声信号叠加图)

Figure 4. (A) Representative bright-field images of nude mouse 1 week after injection of PBS (Control) or 4T1 cell suspension (Breast cancer model). (B) Representative cross-sectional OA images of the breast cancer-bearing nude mouse model before injection of the probe (0 min) and 10, 30, 60 min after injection of the probe; the color bar on the right side reflects different OA intensities. (Upper row: the OA signal image of activated probe; lower row: the overlay of the OA signal images of activated probe and grayscale background)

图5. 乳腺癌小鼠模型在注射探针溶液之前(0 min)和之后10, 30 和60 min时的正交视图三维光声图像; 右侧彩条代表不同的OA信号强度. (上排：激活探针的光声信号图; 下排：激活探针与灰度背景的光声信号叠加图)

Figure 5. Representative 3D OA images of the breast cancer-bearing nude mouse model before injection of the probe (0 min) and 10, 30, 60 min after injection of the probe; the color bar on the right side reflects different OA intensities. (Upper row: the OA signal image of activated probe; lower row: the overlay of the OA signal images of activated probe and grayscale background)

参考文献 50

[1]	Asher, G.; Reuven, N.; Shaul, Y. BioEssays 2006, 28,844. doi: 10.1002/bies.20447 pmid: 16927316
[2]	Clavería-Gimeno, R.; Velazquez-Campoy, A.; Pey, A. L. Arch. Biochem. Biophys. 2017, 636,17. doi: 10.1016/j.abb.2017.10.020 pmid: 29100982
[3]	Pey, A. L. Int. J. Biol. Macromol. 2019, 126,1223. pmid: 30615965
[4]	Zhao, Z.; Li, X.; Wu, B.; Zhou, Y. Chin. J. Chem. 2020, 38,714. doi: 10.1002/cjoc.v38.7
[5]	Wang, N.; Zhao, J. Chin. J. Org. Chem. 2006, 26,775. (in Chinese) doi: 10.1002/(ISSN)1614-7065
	( 王乃兴, 赵嘉, 有机化学, 2006, 26,775.) 83d27848-ce6f-4042-bcec-2202ef50c36e
[6]	Mizumoto, A.; Ohashi, S.; Kamada, M.; Saito, T.; Nakai, Y.; Baba, K.; Hirohashi, K.; Mitani, Y.; Kikuchi, O.; Matsubara, J.; Yamada, A.; Takahashi, T.; Lee, H.; Okuno, Y.; Kanai, M.; Muto, M. J. Gastroenterol 2019, 54,687. pmid: 30737573
[7]	Luo, S.; Lei, K.; Xiang, D.; Ye, K. Oxid. Med. Cell. Longevity 2018, 2018,1.
[8]	Sies, H.; Berndt, C.; Jones, D. P. Annu. Rev. Biochem. 2017, 86,715. pmid: 28441057
[9]	Sabharwal, S. S.; Schumacker, P. T. Nat. Rev. Cancer 2014, 14,709. doi: 10.1038/nrc3803 pmid: 25342630
[10]	Hayes, J. D.; Dinkova-Kostova, A. T.; Tew, K. D. Cancer Cell 2020, 38,167. pmid: 32649885
[11]	Sharma, C.; Wang, H.-X.; Li, Q.; Knoblich, K.; Reisenbichler, E. S.; Richardson, A. L.; Hemler, M. E. Cancer Res. 2017, 77,6880. pmid: 29055014
[12]	Danson, S.; Ward, T. H.; Butler, J.; Ranson, M. Cancer Treat. Rev. 2004, 30,437. pmid: 15245776
[13]	Dinkova-Kostova, A. T.; Talalay, P. Arch. Biochem. Biophys. 2010, 501,116. doi: 10.1016/j.abb.2010.03.019 pmid: 20361926
[14]	Li, Z.; Zhang, Y.; Jin, T.; Men, J.; Lin, Z.; Qi, P.; Piao, Y.; Yan, G.. BMC Cancer 2015, 15,207. pmid: 25880877
[15]	Li, L.-S.; Reddy, S.; Lin, Z.-H.; Liu, S.; Park, H.; Chun, S. G.; Bornmann, W. G.; Thibodeaux, J.; Yan, J.; Chakrabarti, G.; Xie, X.; Sumer, B. D.; Boothman, D. A.; Yordy, J. S. Mol. Cancer Ther. 2016, 15,1757. pmid: 27196777
[16]	Pani, G.; Giannoni, E.; Galeotti, T.; Chiarugi, P. Antioxid. Redox Signaling 2009, 11,2791. doi: 10.1089/ars.2009.2739
[17]	Yang, Y.; Zhang, Y.; Wu, Q.; Cui, X.; Lin, Z.; Liu, S.; Chen, L. J. Exp. Clin. Cancer Res. 2014, 33,14. doi: 10.1186/1756-9966-33-14 pmid: 24499631
[18]	Fagerholm, R.; Hofstetter, B.; Tommiska, J.; Aaltonen, K.; Vrtel, R.; Syrjäkoski, K.; Kallioniemi, A.; Kilpivaara, O.; Mannermaa, A.; Kosma, V.-M.; Uusitupa, M.; Eskelinen, M.; Kataja, V.; Aittomäki, K.; von Smitten, K.; Heikkilä, P.; Lukas, J.; Holli, K.; Bartkova, J.; Blomqvist, C.; Bartek, J.; Nevanlinna, H. Nat. Genet. 2008, 40,844. pmid: 18511948
[19]	Jamshidi, M.; Bartkova, J.; Greco, D.; Tommiska, J.; Fagerholm, R.; Aittomäki, K.; Mattson, J.; Villman, K.; Vrtel, R.; Lukas, J.; Heikkilä, P.; Blomqvist, C.; Bartek, J.; Nevanlinna, H. Breast Cancer Res. Treat. 2012, 132,955. doi: 10.1007/s10549-011-1629-5 pmid: 21706157
[20]	Qi, Y.; Huang, Y.; Li, B.; Zeng, F.; Wu, S. Anal. Chem. 2018, 90,1014. doi: 10.1021/acs.analchem.7b04407 pmid: 29182316
[21]	Xiong, L.; Fan, Y.; Zhang, F. Acta Chim. Sinica 2019, 77,1239. (in Chinese) doi: 10.6023/A19080305 pmid: 73282259-D05E-4539-8A88-A678B317D50D
	( 熊麟, 凡勇, 张凡, 化学学报, 2019, 77, 1239.) pmid: 73282259-D05E-4539-8A88-A678B317D50D
[22]	Niu, H.; Tang, J.; Zhu, X.; Li, Z.; Zhang, Y.; Ye, Y.; Zhao, Y. Chem. Commun. 2020, 56,7710. doi: 10.1039/D0CC02668A
[23]	Specht, E. A.; Braselmann, E.; Palmer, A. E. Annu. Rev. Physiol. 2017, 79,93. pmid: 27860833
[24]	Wu, Y.; Wang, J.; Zeng, F.; Huang, S.; Huang, J.; Xie, H.; Yu, C.; Wu, S. ACS Appl. Mater. Interfaces 2016, 8,1511. pmid: 26701212
[25]	Chang, Y.; Li, B.; Guo, M.; Cai, Y.; Xu, K. Chin. J. Org. Chem. 2019, 39,2485. (in Chinese) doi: 10.6023/cjoc201903010
	( 常永新, 李白, 郭淼, 蔡永红, 徐括喜, 有机化学, 2019, 39,2485.) ce3fcf14-0b02-4e50-a864-d4e0aa974467 doi: 10.6023/cjoc201903010
[26]	Christensen, P. R.; Wolf, M. O. Adv. Funct. Mater. 2016, 26,8471. doi: 10.1002/adfm.v26.46
[27]	Wang, J.; Wu, Y.; Sun, L.; Zeng, F.; Wu, S. Acta Chim. Sinica 2016, 74,910. (in Chinese) doi: 10.6023/A16070342 pmid: 038E5B9E-3975-4E43-99F1-BAB63AF9C904
	( 王俊, 武英龙, 孙立和, 曾钫, 吴水珠, 化学学报, 2016, 74,910.) 6d05ea64-37bd-41b8-8490-f1e8f5c3f335 doi: 10.6023/A16070342 pmid: 038E5B9E-3975-4E43-99F1-BAB63AF9C904
[28]	Liu, P.; Xu, J.; Yan, D.; Zhang, P.; Zeng, F.; Li, B.; Wu, S. Chem. Commun. 2015, 51,9567. doi: 10.1039/C5CC02149A
[29]	Yang, Q.; Wen, Y.; Xu, J.; Shao, S. Talanta 2020, 216,120982. doi: 10.1016/j.talanta.2020.120982 pmid: 32456908
[30]	Yuan, Z.; Xu, M.; Wu, T.; Zhang, X.; Shen, Y.; Ernest, U.; Gui, L.; Wang, F.; He, Q.; Chen, H. Talanta 2019, 198,323. doi: 10.1016/j.talanta.2019.02.009 pmid: 30876568
[31]	Shen, Z.; Prasai, B.; Nakamura, Y.; Kobayashi, H.; Jackson, M. S.; McCarley, R. L. ACS Chem. Biol. 2017, 12,1121. pmid: 28240865
[32]	Hettiarachchi, S. U.; Prasai, B.; McCarley, R. L. J. Am. Chem. Soc. 2014, 136,7575. e701d009-6c49-4c13-aed7-52d3b59ac8c3 doi: 10.1021/ja5030707
[33]	Pan, D.; Luo, F.; Liu, X.; Liu, W.; Chen, W.; Liu, F.; Kuang, Y.-Q.; Jiang, J.-H. Analyst 2017, 142,2624. pmid: 28608874
[34]	Cheng, Z.; Valença, W. O.; Dias, G. G.; Scott, J.; Barth, N. D.; de Moliner, F.; Souza, G. B. P.; Mellanby, R. J.; Vendrell, M.; da Silva Júnior, E.N. Bioorg. Med. Chem. 2019, 27,3938. doi: 10.1016/j.bmc.2019.07.017 pmid: 31327676
[35]	Ovsepian, S. V.; Olefir, I.; Westmeyer, G.; Razansky, D.; Ntziachristos, V. Neuron 2017, 96,966. doi: S0896-6273(17)30990-X pmid: 29216459
[36]	Huang, J.; Wu, Y.; Zeng, F.; Wu, S. Theranostics 2019, 9,7313. pmid: 31695770
[37]	Ouyang, J.; Sun, L.; Zeng, Z.; Zeng, C.; Zeng, F.; Wu, S. Angew. Chem. Int. Ed.Eng. 2020, 59,10111.
[38]	Reber, J.; Willershäuser, M.; Karlas, A.; Paul-Yuan, K.; Diot, G.; Franz, D.; Fromme, T.; Ovsepian, S. V.; Bézière, N.; Dubikovskaya, E.; Karampinos, D. C.; Holzapfel, C.; Hauner, H.; Klingenspor, M.; Ntziachristos, V. Cell Metab. 2018, 27,689. doi: 10.1016/j.cmet.2018.02.002 pmid: 29514074
[39]	Zhan, C.; Huang, Y.; Lin, G.; Huang, S.; Zeng, F.; Wu, S. Small 2019, 15,1900309. doi: 10.1002/smll.v15.33
[40]	Wu, Y.; Chen, J.; Sun, L.; Zeng, F.; Wu, S. Adv. Funct. Mater. 2019, 29,1807960.
[41]	Diot, G.; Metz, S.; Noske, A.; Liapis, E.; Schroeder, B.; Ovsepian, S. V.; Meier, R.; Rummeny, E.; Ntziachristos, V. Clin. Cancer Res. 2017, 23,6912. pmid: 28899968
[42]	Ntziachristos, V.; Razansky, D. Chem. Rev. 2010, 110,2783. doi: 10.1021/cr9002566 pmid: 20387910
[43]	Luís Dean-Ben, X.; Razansky, D. Light: Sci. Appl. 2018, 7,18004.
[44]	Gujrati, V.; Mishra, A.; Ntziachristos, V. Chem. Commun. 2017, 53,4653. doi: 10.1039/C6CC09421J
[45]	Schellenberg, M. W.; Hunt, H. K. Photoacoustics 2018, 11,14. doi: 10.1016/j.pacs.2018.07.001 pmid: 30073147
[46]	Song, X.; Bian, H.; Wang, C.; Hu, M.; Li, N.; Xiao, Y. Org. Biomol. Chem. 2017, 15,8091. pmid: 28905964
[47]	Kim, H.; Lee, M.; Kim, H.; Kim, S.; Yoon, J. Chem. Soc. Rev. 2008, 37,1465. doi: 10.1039/b802497a pmid: 18648672
[48]	Han, J.; Burgess, K.; Chem. Rev. 2010, 110,2709. pmid: 19831417
[49]	Mendoza, M. F.; Hollabaugh, N. M.; Hettiarachchi, S. U.; McCarley, R. L. Biochemistry 2012, 51,8014. pmid: 22989153
[50]	Wu, Y.; Huang, S.; Wang, J.; Sun, L.; Zeng, F.; Wu, S. Nat. Commun. 2018, 9,3983. pmid: 30266905