HA-AuNPs/FDF用于透明质酸酶的高灵敏检测、肿瘤靶向细胞荧光成像和光疗

doi:10.6023/A23060283

摘要/Abstract

本工作构建了一种新型复合纳米诊疗剂HA-AuNPs/FDF, 用于透明质酸酶(HAase)的高灵敏荧光检测、肿瘤靶向荧光成像和光动力/光热协同治疗. 吡咯并吡咯二酮基共轭小分子(FDF)与肿瘤靶向生物分子透明质酸(HA)功能化的金纳米粒子(HA-AuNPs)通过静电作用自组装形成HA-AuNPs/FDF. FDF在近红外光激发下产生较强的荧光, HA-AuNPs会通过荧光共振能量转移效应(FRET)猝灭FDF的荧光. 然而, 肿瘤细胞中过表达的透明质酸酶(HAase)能使HA逐渐降解, FDF被释放, 从而荧光逐渐恢复. HA-AuNPs/FDF的荧光恢复程度与HAase的浓度有很好的线性关系, 可用于快速定量检测HAase. 而且, HA-AuNPs/FDF作为透明质酸酶激活的荧光探针成功地用于人宫颈癌肿瘤HeLa细胞的靶向荧光成像, 细胞实验结果证实它能通过光动力/光热协同治疗有效抑制HeLa细胞的增殖. 该体系为实现精准高效的肿瘤诊疗拓展了思路.

关键词: 吡咯并吡咯二酮, 纳米诊疗剂, 透明质酸酶, 荧光成像, 光疗

In this paper, a novel composite nano diagnostic and therapeutic agent HA-AuNPs/FDF was constructed for highly sensitive fluorescence detection of hyaluronidase (HAase), tumor-targeting fluorescence cell imaging and photodynamic/photothermal synergistic therapy. HA-AuNPs/FDF was formed by the electrostatic self-assembly of FDF, a diketopyrrolopyrrole-based conjugated small molecule, and HA-AuNPs, the gold nanoparticles functionalized with the tumor-targeting biomolecule hyaluronan (HA). FDF generated strong fluorescence under the excitation of near-infrared (NIR) light, and HA-AuNPs will quench the fluorescence of FDF through fluorescence resonance energy transfer (FRET). However, the overexpression of hyaluronidase (HAase) in tumor cells can gradually degrade HA, so FDF was released and the fluorescence of FDF was gradually recovered. Therefore, the fluorescence recovery of HA-AuNPs/FDF can be used for the rapid quantification of HAase. Experiments have shown that this method can achieve good linear response within the range of 0.25~2.25 U/mL with a detection limit of 0.04 U/mL. On this basis, after respective incubation with HA-AuNPs/FDF for 4 h, NIR fluorescence imaging was performed on human cervical cancer tumor cells (HeLa cells), HeLa cells pretreated with HA, and mouse embryonic fibroblasts (NIH-3T3 cells) to study the tumor-targeting capability of HA-AuNPs/FDF. In addition, after incubation with HA-AuNPs/FDF for 20 min, NIR fluorescence imaging was also performed on HeLa cells to detect the changes in fluorescence intensity over time and study the capability of HAase to activate fluorescence. All the results showed that HA-AuNPs/FDF was readily endocytosed by HeLa cells via the receptor CD44 and degraded by intracellular overexpressed HAase, and that it can be successfully used as a fluorescence probe activated by HAase for fluorescence cell imaging of HeLa cells. Furthermore, as FDF is a material with photothermal and photodynamic therapeutic potential, HA-AuNPs/FDF exhibited the single linear oxygen yield of 23.7%, with a photothermal conversion efficiency of 21.3% and good photothermal stability. The cytotoxicity of HA-AuNPs/FDF was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay and live/dead cell staining experiments using calcein-acetoxymethyl ester (AM) and propidium iodide (PI) double staining kit. The results showed that the number of dead HeLa cells was significantly increased under laser irradiation as compared to that under dark conditions, confirming that HA-AuNPs/FDF can effectively inhibit the proliferation of HeLa cells by photodynamic/photothermal synergistic therapy. This system expands the ideas for achieving precise and efficient tumor diagnosis and therapy.

Key words: diketopyrrolopyrrole (DPP), nano diagnostic and therapeutic agent, hyaluronidase, fluorescence imaging, phototherapy

引用此文

黄艳琴, 栗丽君, 杨书培, 张瑞, 刘兴奋, 范曲立, 黄维. HA-AuNPs/FDF用于透明质酸酶的高灵敏检测、肿瘤靶向细胞荧光成像和光疗[J]. 化学学报, 2023, 81(12): 1687-1694.

Yanqin Huang, Lijun Li, Shupei Yang, Rui Zhang, Xingfen Liu, Quli Fan, Wei Huang. HA-AuNPs/FDF for Highly Sensitive Detection of Hyaluronidase, Tumor-targeting Fluorescence Cell Imaging and Phototherapy[J]. Acta Chimica Sinica, 2023, 81(12): 1687-1694.

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

分享此文

图/表 10

图1. (a) FDF和HA的结构式; (b) HA-AuNPs的制备; (c)检测HAase并对肿瘤细胞进行靶向荧光成像和光疗的原理

Figure 1. (a) Structural formula of FDF and HA; (b) preparation of HA-AuNPs; (c) schematic diagram of the detection of HAase and tumor-targeting fluorescence imaging and phototherapy for tumor cells

图2. (a) HA-AuNPs/FDF、HA-AuNPs和FDF的紫外-可见-近红外吸收光谱(FDF浓度均为1 mg/mL); (b) HA-AuNPs/FDF、HA-AuNPs和FDF的Zeta电位

Figure 2. (a) UV-Vis-NIR absorption spectra of HA-AuNPs/FDF, HA-AuNPs and FDF (the concentration of FDF is 1 mg/mL); (b) Zeta potential of HA-AuNPs/FDF, HA-AuNPs and FDF

图3. (a) HA-AuNPs的TEM图像; (b) HA-AuNPs/FDF的TEM图像; (c) HA-AuNPs的流体动力学尺寸; (d) HA-AuNPs/FDF的流体动力学尺寸

Figure 3. (a) TEM image of HA-AuNPs; (b) TEM image of HA-AuNPs/FDF; (c) hydrodynamic sizes of HA-AuNPs; (d) hydrodynamic sizes of HA-AuNPs/FDF

图4. (a) FDF的荧光光谱(黑色实线), HA-AuNPs/FDF的荧光光谱(红色实线)以及随着HAase浓度增加的荧光光谱(蓝色虚线); 检测在磷酸盐缓冲溶液(10 mmol/L, pH 5.6)中进行, [FDF]=0.025 mg/mL; (b) ΔI与HAase浓度的函数关系

Figure 4. (a) Fluorescence spectra of FDF (black solid line), fluorescence spectra of HA-AuNPs/FDF (red solid line), and fluorescence spectra of HA-AuNPs/FDF with the increase of HAase concentration (blue dash dot line); measurements were performed in phosphate buffer solution (PBS) (10 mmol/L, pH 5.6), [FDF]=0.025 mg/mL; (b) ΔI as a function of HAase concentration

检测方法	线性范围/(U•mL⁻¹)	检测限/(U•mL⁻¹)	文献
比色法	0~240	24	[7]
酶谱法	0.625~5	0.625	[12]
荧光法	0.01~10	0.004	[19]
荧光法	0.05~2	0.02	[18]
荧光法	0~1.3	0.075	[17]
荧光法	0.5~100	0.14	[9]
荧光法	1.25~50	0.63	[16]
荧光法	0.25~2.25	0.04	本文

表1. 不同透明质酸酶检测方法的比较

Table 1. Comparison of different methods for HAase detection

图5. HAase检测时各种潜在干扰物对ΔI的影响(透明质酸酶: 2.0 U/mL; 限制性核酸内切酶、溶菌酶、胰蛋白酶和凝血酶: 10 U/mL)

Figure 5. Effects of various potential interferents on ΔI during the detection of HAase (HAase: 2.0 U/mL; EcoRI, Lysozyme, Trypsin and Thrombin: 10 U/mL)

图6. (a) 在含有HA-AuNPs/FDF的水溶液中, DPBF在不同辐照时间(635 nm, 30 mW/cm2)的紫外-可见吸收光谱; (b) DPBF在含有HA-AuNPs/FDF或MB的水溶液中的光降解速率; (c)在与HA-AuNPs/FDF孵育后的HeLa细胞内检测1O2, 采用DCFH-DA作为荧光探针

Figure 6. (a) UV-Vis absorption spectra of DPBF in the aqueous solutions containing HA-AuNPs/FDF at different irradiation time (635 nm, 30 mW/cm2); (b) photodegradation rate of DPBF in the aqueous solutions containing HA-AuNPs/FDF or MB; (c) 1O2 detection in HeLa cells after incubation with HA-AuNPs/FDF, using DCFH-DA as a fluorescent probe

图7. (a) 同一浓度HA-AuNPs/FDF (1 mg/mL)在不同激光功率照射下的升温曲线; (b) HA-AuNPs/FDF (1 mg/mL)在激光照射600 s下(635 nm, 1 W/cm2)的光热响应, 然后关闭激光器, 重复五遍; (c) (b)图中的单次光热循环; (d) 从图(c)的冷却周期得到的时间与-ln θ的线性关系

Figure 7. (a) Temperature elevation curve of HA-AuNPs/FDF at the same concentration (1 mg/mL) under laser irradiation with different powers; (b) photothermal response of HA-AuNPs/FDF (1 mg/mL) under laser irradiation (635 nm, 1 W/cm2) for 600 s and then the laser was shut off, and this operation was cycled five times; (c) single photothermal cycle of the process in (b); (d) linear time versus -ln θ obtained from the cooling period of (c)

图8. (a) 用HA-AuNPs/FDF孵育4 h后的近红外荧光成像(1) HeLa细胞, (2)用HA 预处理的HeLa细胞, (3) NIH-3T3 细胞; (b)在用HA-AuNPs/FDF孵育20 min后的HeLa细胞内荧光强度随时间的变化

Figure 8. (a) NIR fluorescence imaging of (1) HeLa cells, (2) HeLa cells pretreated with HA, and (3) NIH-3T3 cells after incubation with HA-AuNPs/FDF for 4 h; (b) changes in fluorescence intensity over time in HeLa cells incubated with HA-AuNPs/FDF for 20 min

图9. (a) 黑暗条件下与HA-AuNPs/FDF共孵育的HeLa细胞存活率; (b)与HA-AuNPs/FDF共孵育后光照8 min条件下(635 nm, 0.8 W/cm2)的HeLa细胞存活率; (c) 与HA-AuNPs/FDF共孵育的HeLa细胞在黑暗和光照(635 nm, 0.8 W/cm2) 10 min 条件下的活死细胞染色图

Figure 9. (a) Cell viability of HeLa cells treated with HA-AuNPs/FDF under dark conditions; (b) cell viability of HeLa cells treated with HA-AuNPs/FDF under light irradiation (635 nm, 0.8 W/cm2) for 8 min; (c) live/dead staining of HeLa cells treated with HA-AuNPs/FDF without or with light irradiation (635 nm, 0.8 W/cm2) for 10 min

参考文献 38

[1]	Lapcik, L.; Lapcik, L.; De Smedt, S.; Demeester, J.; Chabrecek, P. Chem. Rev. 1998, 98, 2663. doi: 10.1021/cr941199z
[2]	Toole, B. P. Nat. Rev. Cancer 2004, 4, 528. doi: 10.1038/nrc1391
[3]	Xie, H. F.; Zeng, F.; Wu, S. Z. Biomacromolecules 2014, 15, 3383. doi: 10.1021/bm500890d
[4]	Lokeshwar, V. B.; Estrella, V.; Lopez, L.; Kramer, M.; Gomez, P.; Soloway, M. S.; Lokeshwar, B. L. Cancer Res. 2006, 66, 11219. pmid: 17145867
[5]	Kolliopoulos, C.; Bounias, D.; Bouga, H.; Kyriakopoulou, D.; Stavropoulos, M.; Vynios, D. H. J. Pharm. Biomed. Anal. 2013, 83, 299. doi: 10.1016/j.jpba.2013.05.037 pmid: 23777618
[6]	Eissa, S.; Shehata, H.; Mansour, A.; Esmat, M.; El-Ahmady, O. Med. Oncol. 2012, 29, 3345. doi: 10.1007/s12032-012-0295-8
[7]	Nossier, A. I.; Eissa, S.; Ismail, M. F.; Hamdy, M. A.; Azzazy, H. M. Biosens. Bioelectron. 2014, 54, 7. doi: 10.1016/j.bios.2013.10.024
[8]	Martinez-Quintanilla, J.; He, D.; Wakimoto, H.; Alemany, R.; Shah, K. Mol. Ther. 2015, 23, 108. doi: 10.1038/mt.2014.204 pmid: 25352242
[9]	Ge, M. H.; Sun, J. J.; Chen, M. L.; Tian, J. J.; Yin, H. C.; Yin, J. Anal. Bioanal. Chem. 2020, 412, 1915. doi: 10.1007/s00216-020-02443-9
[10]	Diferrante, N. J. Biol. Chem. 1956, 220, 303. doi: 10.1016/S0021-9258(18)65354-2
[11]	Vercruysse, K. P.; Lauwers, A. R.; Demeester, J. M. Biochem. J. 1995, 306, 153. doi: 10.1042/bj3060153
[12]	Magalhaes, M. R.; da Silva, N. J.; Ulhoa, C. J. Toxicon 2008, 51, 1060. doi: 10.1016/j.toxicon.2008.01.008
[13]	Jayadev, C.; Rout, R.; Price, A.; Hulley, P.; Mahoney, D. J. Immunol. Methods 2012, 386, 22. doi: 10.1016/j.jim.2012.08.012
[14]	Carter, K. P.; Young, A. M.; Palmer, A. E. Chem. Rev. 2014, 114, 4564. doi: 10.1021/cr400546e pmid: 24588137
[15]	Chen, C.; Tian, R.; Zeng, Y.; Chu, C.; Liu, G. Bioconjug. Chem. 2020, 31, 276. doi: 10.1021/acs.bioconjchem.9b00734
[16]	Cheng, D.; Han, W. Y.; Yang, K. C.; Song, Y.; Jiang, M. D.; Song, E. Q. Talanta 2014, 130, 408. doi: 10.1016/j.talanta.2014.07.005 pmid: 25159428
[17]	Huang, Y. Q.; Song, C. X.; Li, H. C.; Zhang, R.; Jiang, R. C.; Liu, X. F.; Zhang, G. W.; Fan, Q. L.; Wang, L. H.; Huang, W. ACS Appl. Mater. Interfaces 2015, 7, 21529. doi: 10.1021/acsami.5b06799
[18]	Li, X. Q.; Zhou, Z.; Tang, Y. P.; Zhang, C. C.; Zheng, Y. H.; Gao, J. W.; Wang, Q. M. Sens. Actuators 2018, 276, 95. doi: 10.1016/j.snb.2018.08.093
[19]	Ge, J.; Cai, R.; Yang, L.; Zhang, L. L.; Jiang, Y.; Yang, Y.; Cui, C.; Wan, S.; Chu, X.; Tan, W. H. ACS Sustainable Chem. Eng. 2018, 6, 16555. doi: 10.1021/acssuschemeng.8b03684
[20]	Zhang, Z.; Xu, W.; Kang, M.; Wen, H.; Guo, H.; Zhang, P.; Xi, L.; Li, K.; Wang, L.; Wang, D.; Tang, B. Z. Adv. Mater. 2020, 32, 2003210. doi: 10.1002/adma.v32.36
[21]	Pan, L. X.; Huang, Y. Q.; Sheng, K.; Zhang, R.; Fan, Q. L.; Huang, W. Acta Chim. Sinica 2021, 79, 1097 (in Chinese). doi: 10.6023/A21050219
	(潘立祥, 黄艳琴, 盛况, 张瑞, 范曲立, 黄维, 化学学报, 2021, 79, 1097.) doi: 10.6023/A21050219
[22]	Gao, D.; Guo, X.; Zhang, X.; Chen, S.; Wang, Y.; Chen, T.; Huang, G.; Gao, Y.; Tian, Z.; Yang, Z. Mater. Today Bio. 2020, 5, 100035.
[23]	Wang, M.; Yan, D.; Wang, M.; Wu, Q.; Song, R.; Huang, Y.; Rao, J.; Wang, D.; Zhou, F.; Tang, B. Z. Adv. Funct. Mater. 2022, 32, 2205371. doi: 10.1002/adfm.v32.36
[24]	Xu, C.; Pu, K. Chem. Soc. Rev. 2021, 50, 1111. doi: 10.1039/D0CS00664E
[25]	Huang, Y. Q.; Sun, L. J.; Zhang, R. ACS Appl. Bio. Mater. 2019, 2, 2421. doi: 10.1021/acsabm.9b00130
[26]	Huang, Y. Q.; Liu, K. L.; Ni, H. L.; Zhang, R.; Liu, X. F.; Fan, Q. L.; Wang, L. H.; Huang, W. ACS Appl. Polym. Mater. 2022, 4, 7739. doi: 10.1021/acsapm.2c01297
[27]	Liu, B. D.; Wang, C. J.; Qian, Y. Acta Chim. Sinica 2022, 80, 1071 (in Chinese). doi: 10.6023/A22040141
	(刘巴蒂, 王承俊, 钱鹰, 化学学报, 2022, 80, 1071.) doi: 10.6023/A22040141
[28]	Li, Y. R.; Wang, Z. G.; Tang, Z. H. Acta Chim. Sinica 2022, 80, 291 (in Chinese). doi: 10.6023/A21120544
	(李嫣然, 王子贵, 汤朝晖, 化学学报, 2022, 80, 291.) doi: 10.6023/A21120544
[29]	Xia, Q.; Chen, Z.; Zhou, Y.; Liu, R. Nanotheranostics 2019, 3, 156. doi: 10.7150/ntno.33536 pmid: 31008024
[30]	Wang, T.; Zhao, L.; Wang, K. W.; Bai, Y. F.; Feng, F. Acta Chim. Sinica 2021, 79, 600 (in Chinese). doi: 10.6023/A20120578
	(王涛, 赵璐, 王科伟, 白云峰, 冯锋, 化学学报, 2021, 79, 600.) doi: 10.6023/A20120578
[31]	Sun, Y.; Wang, Y.; Liu, Y.; Weng, B.; Yang, H.; Xiang, Z.; Ran, J.; Wang, H.; Yang, C. ACS Appl. Mater. Interfaces 2021, 13, 53646. doi: 10.1021/acsami.1c17642
[32]	Song, Y.; Wang, Z.; Li, L.; Shi, W.; Li, X.; Ma, H. Chem. Commun. 2014, 50, 15696. doi: 10.1039/C4CC07565J
[33]	Wu, Z. S.; Zhang, S. B.; Guo, M. M.; Chen, C. R.; Shen, G. L.; Yu, R. Q. Anal. Chim. Acta 2007, 584, 122. doi: 10.1016/j.aca.2006.11.003
[34]	Matsui, J.; Akamatsu, K.; Nishiguchi, S.; Miyoshi, D.; Nawafune, H.; Tamaki, K.; Sugimoto, N. Anal. Chem. 2004, 76, 1310. pmid: 14987086
[35]	Zhu, M. Q.; Wang, L. Q.; Exarhos, G. J.; Li, A. D. J. Am. Chem. Soc. 2004, 126, 2656. doi: 10.1021/ja038544z
[36]	Wu, K.; Zhao, H. H.; Sun, Z. Q.; Wang, B.; Tang, X. Y.; Dai, Y. N.; Li, M. X.; Shen, Q. M.; Zhang, H.; Fan, Q. L.; Huang, W. Theranostics 2019, 9, 7697. doi: 10.7150/thno.38565
[37]	Murphy, C. J.; Gole, A. M.; Stone, J. W.; Sisco, P. N.; Alkilany, A. M.; Goldsmith, E. C.; Baxter, S. C. Acc. Chem. Res. 2008, 41, 1721. doi: 10.1021/ar800035u
[38]	Joris, F.; Manshian, B. B.; Peynshaert, K.; De Smedt, S. C.; Braeckmans, K.; Soenen, S. J. Chem. Soc. Rev. 2013, 42, 8339. doi: 10.1039/c3cs60145e