过氧化氢激活型近红外氟硼二吡咯光敏剂的设计、合成及光动力治疗研究

doi:10.6023/A22120487

摘要/Abstract

基于传统的氟硼二吡咯(BODIPY)荧光染料, 设计合成了一种过氧化氢(H₂O₂)激活型近红外光敏剂中位-N-(4-硼酸苄基)吡啶鎓盐取代的碘化双苯乙烯基氟硼二吡咯(FP-IBDP). FP-IBDP在乙腈中的吸收和发射波长均达到近红外区, 最大吸收和发射波长分别为681 nm和740 nm, 对应的荧光量子效率和单线态氧效率分别为0.01和0.09. 在被H₂O₂激活后, FP-IBDP转变为IBDP, 其在乙腈中的最大吸收和发射波长分别为661 nm和701 nm. 与FP-IBDP相比, IBDP的荧光量子效率和单线态氧效率大幅提升, 分别达到0.11和0.48. 细胞水平的荧光影像实验表明FP-IBDP对癌细胞内的H₂O₂具有灵敏的响应, 并能通过明显的荧光增强变化实现癌细胞与正常细胞的有效区分. 活性氧检测实验证明FP-IBDP能够被癌细胞内过表达的H₂O₂激活, 并能在660 nm光照射下在癌细胞内产生单线态氧. 噻唑蓝(MTT)比色法测试表明了FP-IBDP具有低的细胞暗毒性和好的生物相容性. 细胞光毒性测试及活/死细胞染色标记实验则表明FP-IBDP对癌细胞具有更高的光毒性, 而对正常细胞具有低的光损伤. 细胞划痕实验进一步表明FP-IBDP在光照下能够有效抑制癌细胞增殖. 此外, 荧光共定位及溶酶体完整性检测实验表明FP-IBDP主要作用于细胞溶酶体, 光照下产生的单线态氧通过破坏溶酶体导致溶酶体相关的细胞死亡. 上述结果为氟硼二吡咯光敏剂FP-IBDP实现近红外光激发下荧光成像指导的光动力学治疗奠定了基础.

关键词: 激活型光敏剂, 近红外光, 氟硼二吡咯光敏剂, 光动力治疗, 荧光成像

In this work, a borondipyrromethene (BODIPY)-based activatable photosensitizer FP-IBDP was designed and synthesized, which has absorption and emission wavelengths in the near infrared (NIR) region. The maximum absorption and emission of FP-IBDP are 681 nm and 740 nm in acetonitrile. The fluorescence quantum yield and singlet oxygen yield of FP-IBDP is 0.01 and 0.09, respectively. After activated by H₂O₂, FP-IBDP was transformed to photosensitizer IBDP, which has maximum absorption and emission peak at 661 nm and 701 nm in acetonitrile. Compared with FP-IBDP, the fluorescence quantum yield and singlet oxygen yield of IBDP are much increased, up to 0.11 and 0.48 respectively. It is demonstrated that FP-IBDP can respond H₂O₂ sensitively in cancer cells and has the ability to distinguish cancer cells from normal cells by a fluorescence enhancement way. Reactive oxygen species (ROS) detection indicated that FP-IBDP could be activated by the endogenous H₂O₂ in cancer cells and produce highly toxic singlet oxygen under 660 nm light excitation with dichlorodihydrofluorescein diacetate (DCFH-DA) as ROS indicator. Tetrazolium (MTT) assay indicated that FP-IBDP has good biocompatibility and low dark toxicity to both cancer cells and normal cells, while phototoxicity test and living/dead cell double staining experiment proved that FP-IBDP has higher phototoxicity to cancer cells than to normal cells. Besides, wounding healing assay confirmed its good ability to inhibit cancer cell proliferation. Fluorescence localization and lysosome disruption assays further indicated that FP-IBDP was specifically located in lysosomes of living cells and the produced singlet oxygen could induce cancer cell death in a lysosome disruption associated pathway. These features are expected to lay good foundation for the application of FP-IBDP in imaging guided photodynamic therapy under NIR excitation.

Key words: activatable photosensitizer, near infrared light, borondipyrromethene (BODIPY) photosensitizer, photodynamic therapy, fluorescence imaging

引用此文

吕鑫, 吴仪, 张勃然, 郭炜. 过氧化氢激活型近红外氟硼二吡咯光敏剂的设计、合成及光动力治疗研究[J]. 化学学报, 2023, 81(4): 359-370.

Xin Lv, Yi Wu, Boran Zhang, Wei Guo. Design, Synthesis and Photodynamic Therapy of a H₂O₂-Activatable Near Infrared Borondipyrromethene （BODIPY） Photosensitizer[J]. Acta Chimica Sinica, 2023, 81(4): 359-370.

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图/表 16

图式1. 氟硼二吡咯光敏剂FP-IBDP的结构及其激活机理

Scheme 1. The chemical structure of photosensitizer FP-IBDP and the proposed activation mechanism

图式2. 氟硼二吡咯光敏剂FP-IBDP的合成路线

Scheme 2. The synthetic routes of photosensitizer FP-IBDP

图1. (a) IBDP (2 μmol/L)在乙腈溶液中的归一化紫外-可见吸收光谱和荧光发射光谱. (b) FP-IBDP (2 μmol/L)在乙腈溶液中的归一化紫外-可见吸收光谱和荧光发射光谱. 激发波长630 nm

Figure 1. (a) Normalized absorption and fluorescence emission spectra of IBDP (2 μmol/L) in CH3CN. (b) Normalized absorption and fluorescence emission spectra of FP-IBDP (2 μmol/L) in CH3CN. λex: 630 nm

Photosensitizer	λ_abs/nm	λ_em/nm	ε_max/ (L•mol⁻¹•cm⁻¹)	Ф_f^a	Ф_△^b
IBDP	661	701	1.01×10⁵	0.11	0.48
FP-IBDP	681	740	0.91×10⁵	0.01	0.09

表1. 光敏剂IBDP和FP-IBDP在乙腈溶液中的光物理性质数据

Table 1. Photophysical properties of IBDP and FP-IBDP in CH3CN

图2. (a) IBDP (2 μmol/L)溶液中DPBF的紫外-可见吸收光谱随光照时间变化示意图; (b) FP-IBDP (2 μmol/L)溶液中DPBF的紫外-可见吸收光谱随光照时间变化示意图; (c) MB (2 μmol/L)溶液中DPBF的紫外-可见吸收光谱随光照时间变化示意图; (d) DPBF在IBDP, FP-IBDP 和 MB溶液中415 nm处的吸光度随光照时间变化示意图

Figure 2. (a) Time-dependent absorption spectral changes of DPBF in the presence of IBDP (2 μmol/L); (b) time-dependent absorption spectral changes of DPBF in the presence of FP-IBDP (2 μmol/L); (c) time-dependent absorption spectral changes of DPBF in the presence of MB (2 μmol/L); (d) time-dependent plot of absorbance of DPBF at 415 nm in IBDP solution, FP-IBDP solution and MB solution respectively

图3. (a) FP-IBDP (2 μmol/L)与H2O2 (100 μmol/L)在PBS溶液(10 mmol/L, pH=7.4, 含50% 体积分数的CH3CN)中的荧光光谱随反应时间变化图; (b) FP-IBDP (2 μmol/L)与H2O2 (100 μmol/L)作用前后的紫外-可见吸收光谱与IBDP的吸收光谱对比

Figure 3. (a) Fluorescence spectra changes of FP-IBDP (2 μmol/L) in PBS (10 mmol/L, pH=7.4, containing 50% CH3CN) upon addition of H2O2 (100 μmol/L) for varying time intervals (0~60 min); (b) absorption spectra of FP-IBDP (2 μmol/L) before and after reaction with H2O2 (100 μmol/L), and the absorption spectra of IBDP

图4. (a) PBS溶液中(10 mmol/L, pH=7.4, 含50% 体积分数的CH3CN), FP-IBDP (2 μmol/L)与不同浓度H2O2 (0~100 μmol/L)反应后的荧光光谱, 反应时间为60 min; (b) FP-IBDP在701 nm处的荧光强度随H2O2浓度变化的线性关系

Figure 4. (a) Fluorescence spectra changes of FP-IBDP (2 μmol/L) in PBS (10 mmol/L, pH=7.4, containing 50% CH3CN) upon addition of H2O2 (0~100 μmol/L) with a reaction time of 60 min; (b) the linear relationship between fluorescence intensity of FP-IBDP at 701 nm and H2O2 concentration

图5. (a) 在H2O2处理后的FP-IBDP (2 μmol/L)溶液中, 荧光探针SOSG (10 μmol/L)的荧光发射光谱随光照时间(0~150 s)变化示意图; (b)在FP-IBDP (2 μmol/L)溶液中, 荧光探针SOSG (10 μmol/L)的荧光发射光谱随光照时间(0~150 s)变化示意图

Figure 5. (a) Time-dependent fluorescence spectra changes of SOSG (10 μmol/L) in the presence of H2O2 treated FP-IBDP (2 μmol/L) under light irradiation for 0~150 s; (b) time-dependent fluorescence spectra changes of SOSG (10 μmol/L) in the presence of FP-IBDP (2 μmol/L) under light irradiation for 0~150 s

图6. HeLa细胞在不同处理条件下的共聚焦荧光图像: (a) HeLa细胞; (b) FP-IBDP (2 μmol/L)与HeLa细胞共孵育2 h; (c) H2O2 (100 μmol/L)处理HeLa细胞1 h后再与FP-IBDP (2 μmol/L)共孵育2 h; (d) PMA (2 μg /mL)处理HeLa细胞12 h后再与FP-IBDP (2 μmol/L)共孵育2 h; (e) PMA (2 μg/mL)处理HeLa细胞12 h后用NAC (2 mmol/L)处理1 h, 再与FP-IBDP (2 μmol/L)共孵育2 h; (a1~e1) 荧光图像(a)~(e)对应的明场图像. 激发波长为633 nm, 收集范围为650~750 nm. 标尺: 20 μm

Figure 6. Confocal fluorescence images of HeLa cells under different treatments. (a) Blank HeLa cells; (b) HeLa cells incubated with FP-IBDP (2 μmol/L) for 2 h; (c) HeLa cells treated with H2O2 (100 μmol/L) for 1 h and then incubated with FP-IBDP (2 μmol/L) for 2 h; (d) HeLa cells treated with PMA (2 μg/mL) for 12 h and then incubated with FP-IBDP (2 μmol/L) for 2 h; (e) HeLa cells treated with PMA (2 μg/mL) for 12 h and then treated with NAC (2 mmol/L) for 1 h, further incubated with FP-IBDP (2 μmol/L) for 2 h; (a1~e1) the corresponding bright field of images (a)~(e). The emission was collected at 650~750 nm with excitation at 633 nm. Scale bar: 20 μm

图7. (a~e)光敏剂FP-IBDP (2 μmol/L)分别与HeLa, HCT116, HepG2, HL-7702以及COS-7 五种细胞孵育2 h、3 h、4 h、5 h后的荧光图像; (f)荧光图像(a)~(e)内的相对荧光强度. 激发波长为633 nm, 收集范围为650~750 nm. 标尺: 20 μm

Figure 7. (a~e) Confocal fluorescence images of HeLa cells, HCT116 cells, HepG2 cells, HL-7702 cells and COS-7 cells after incubation with FP-IBDP (2 μmol/L) for 2 h, 3 h, 4 h and 5 h, respectively; (f) relative pixel intensity from images (a)~(e). The emission was collected at 650~750 nm with excitation at 633 nm. Scale bar: 20 μm

图8. 孵育有FP-IBDP (2 μmol/L)和DCFH-DA (10 μmol/L)的HeLa细胞在660 nm光照射前后的荧光图像; FP-IBDP对应的激发波长为633 nm, 收集范围为650~750 nm; DCFH-DA对应的激发波长为488 nm, 收集范围为500~550 nm. 标尺: 20 μm

Figure 8. Fluorescence images of FP-IBDP (2 μmol/L) and DCFH-DA (10 μmol/L) co-incubated HeLa cells before and after 660 nm light irradiation. The emission was collected at 650~750 nm for FP-IBDP under excitation at 633 nm and 500~550 nm for DCFH-DA under excitation at 488 nm. Scale bar: 20 μm

图9. 孵育有FP-IBDP (2 μmol/L)的HeLa细胞在660 nm光 (10 mW/cm2)照射前后的Calcein-AM/PI (10 μmol/L)染色图像; Calcein- AM对应的收集范围为500~550 nm, PI对应的收集范围为600~650 nm, 激发波长为488 nm. 标尺: 100 μm

Figure 9. Calcein-AM/PI (10 μmol/L) staining images of FP-IBDP (2 μmol/L) treated HeLa cells before and after light irradiation (660 nm, 10 mW/cm2). The emission was collected at 500~550 nm for Calcein-AM and 600~650 nm for PI under excitation at 488 nm. Scale bar: 100 μm

图10. (a)不同浓度FP-IBDP孵育的HepG2细胞在660 nm光(10 mW/cm2)照射5 min后的细胞存活率; (b)不同浓度FP-IBDP孵育的HL-7702细胞在660 nm光(10 mW/cm2)照射5 min后的细胞存活率

Figure 10. (a) Viability of HepG2 cells treated with different concentrations of FP-IBDP under 660 nm light irradiation (10 mW/cm2) for 5 min; (b) viability of HL-7702 cells treated with different concentrations of FP-IBDP under 660 nm light irradiation (10 mW/cm2) for 5 min

图11. (a) FP-IBDP (2 μmol/L)存在时, 无光照情况下24 h内的划痕状态; (b) FP-IBDP (2 μmol/L )存在时, 用660 nm光(10 mW/cm2)照射5 min后24 h内的划痕状态. 标尺: 200 μm

Figure 11. (a) Scratch state within 24 h without illumination in the presence of FP-IBDP (2 μmol/L); (b) scratch state within 24 h after illumination with 660 nm light (10 mW/cm2) for 5 min in the presence of FP-IBDP (2 μmol/L). Scale bar: 200 μm

图12. (a) FP-IBDP (2.0 μmol/L)与溶酶体染料Lyso Tracker Green DND-26 (1.0 μmol/L)共孵育HeLa细胞的荧光图像; (b) FP-IBDP (2.0 μmol/L)与线粒体染料Mito Tracker Green FM (1.0 μmol/L)共孵育HeLa细胞的荧光图像; FP-IBDP对应的激发波长为633 nm, 收集范围为650~750 nm; 线粒体染料和溶酶体染料对应的激发波长为488 nm, 收集范围为500~550 nm. 标尺: 20 μm

Figure 12. (a) Fluorescence images of HeLa cells co-stained with FP-IBDP (2.0 μmol/L) and Lyso Tracker Green DND-26 (1.0 μmol/L); (b) fluorescence images of HeLa cells co-stained with FP-IBDP (2.0 μmol/L) and Mito Tracker Green FM (1.0 μmol/L). The emission was collected at 650~750 nm for FP-IBDP under excitation at 633 nm and 500~550 nm for Mito Tracker and Lyso Tracker under excitation at 488 nm. Scale bar: 20 μm

图13. 孵育有吖啶橙(AO)荧光染料(10 μmol/L)的HeLa细胞在不同处理条件下的荧光图像. 红色荧光通道的收集范围为600~650 nm, 绿色荧光通道收集范围为500~550 nm, 激发波长为488 nm. 标尺: 20 μm

Figure 13. Confocal fluorescence images of AO (10 μmol/L) treated HeLa cells under different treatment conditions. The emission was collected at 600~650 nm for red channel and 500~550 nm for green channel under excitation at 488 nm. Scale bar: 20 μm

参考文献 50

[1]	Li, X.; Lee, S.; Yoon, J. Chem. Soc. Rev. 2018, 47, 1174. doi: 10.1039/C7CS00594F
[2]	Luby, B.; Walsh, C.; Zheng, G. Angew. Chem. Int. Ed. 2019, 58, 2558. doi: 10.1002/anie.v58.9
[3]	Pham, T.; Nguyen, V.; Choi, Y.; Lee, S.; Yoon, J. Chem. Rev. 2021, 121, 13454. doi: 10.1021/acs.chemrev.1c00381
[4]	Zhou, L.; Lv, F.; Liu, L.; Wang, S. Acc. Chem. Res. 2019, 52, 3211. doi: 10.1021/acs.accounts.9b00427
[5]	Yan, T.; Liu, Z.-H.; Song, X.-Y.; Zhang, S.-S. Acta Chim. Sinica 2020, 78, 657. (in Chinese) doi: 10.6023/A20040132
	(闫涛, 刘振华, 宋昕玥, 张书圣, 化学学报, 2020, 78, 657.) doi: 10.6023/A20040132
[6]	Bu, Y.; Zhu, X.; Wang, H.; Zhang, J.; Wang, L.; Yu, Z.; Tian, Y.; Zhou, H.; Xie, Y. Anal. Chem. 2021, 93, 12059. doi: 10.1021/acs.analchem.1c02310
[7]	Chen, D.; Wang, Z.; Dai, H.; Lv, X.-Y.; Ma, Q.; Yang, D.; Shao, J.; Xu, Z.; Dong, X. Small Methods 2020, 2000013.
[8]	Chen, L.; Yang, Y.; Zhang, P.; Wang, S.; Xu, J. F.; Zhang, X. ACS Appl. Bio Mater. 2019, 2, 2920. doi: 10.1021/acsabm.9b00284
[9]	Feng, L.; Betzer, O.; Tao, D.; Sadan, T.; Popovtzer, R.; Liu, Z. CCS Chem. 2019, 1, 239.
[10]	Triesscheijn, M.; Baas, P.; Schellens, J. M.; Stewart, F. A. Oncology 2006, 11, 1034.
[11]	Gross, S.; Gilead, A.; Scherz, A.; Neeman, M.; Salomon, Y. Nat. Med. 2003, 9, 1327. doi: 10.1038/nm940
[12]	Master, A.; Livingston, M.; Sen, A. J. Control. Release 2013, 168, 88.
[13]	Cao, H.; Wang, L.; Yang, Y.; Li, J.; Qi, Y.; Li, Y.; Wang, H.; Li, J.-B. Angew. Chem. Int. Ed. 2018, 57, 7759. doi: 10.1002/anie.v57.26
[14]	Wang, S.-B.; Han, K.; Lei, Q.; Zhu, J.-Y.; Zhang, X.-Z. ACS Nano 2015, 9, 10268. doi: 10.1021/acsnano.5b04243
[15]	Zhai, W.-H.; Zhang, Y.-K.; Liu, M.; Zhang, H.; Zhang, J.-P.; Li, C.-H. Angew. Chem. Int. Ed. 2019, 58, 16601. doi: 10.1002/anie.v58.46
[16]	Tian, J.; Ding, L.; Xu, H.; Shen, Z.; Ju, H.; Jia, L.; Bao, L.; Yu, J. J. Am. Chem. Soc. 2013, 135, 18850. doi: 10.1021/ja408286k
[17]	Ling, D.; Park, W.; Park, S. j.; Lu, Y.; Kim, K.; Hackett, M.; Kim, B.; Yim, H.; Jeon, Y.; Na, K.; Hyeon, T. J. Am. Chem. Soc. 2014, 136, 5647. doi: 10.1021/ja4108287
[18]	Tian, J.; Ding, L.; Ju, H.; Yang, Y.; Li, X.; Shen, Z.; Zhu, Z.; Yu, J.; Yang, C. Angew. Chem. Int. Ed. 2014, 53, 9544. doi: 10.1002/anie.201405490
[19]	Zhang, Y.; Li, X.; Huang, L.; Kim, H.; An, J.; Lan, M.; Cao, Q.; Kim, J. Chem. Commun. 2020, 56, 10317. doi: 10.1039/D0CC02045A
[20]	Xu, F.; Li, H.; Yao, Q.; Ge, H.; Fan, J.; Sun, W.; Wang, J.; Peng, X. Chem. Sci. 2019, 10, 10586. doi: 10.1039/C9SC03355F
[21]	Guo, Z.; Park, S.; Yoon, J.; Shin, I. Chem. Soc. Rev. 2014, 43, 16. doi: 10.1039/C3CS60271K
[22]	Chen, X.; Lee, D.; Yu, S.; Kim, G.; Lee, S.; Cho, Y.; Jeong, H.; Nam, K.; Yoon, J. Biomaterials 2017, 122, 130. doi: 10.1016/j.biomaterials.2017.01.020
[23]	Tian, R.; Sun, W.; Li, M.; Long, S.; Li, M.; Fan, J.; Guo, L.; Peng, X. Chem. Sci. 2019, 10, 10106. doi: 10.1039/C9SC04034J
[24]	Awuah, S.; You, Y. RSC Adv. 2012, 2, 11169. doi: 10.1039/c2ra21404k
[25]	Bessette, A.; Hanan, G. Chem. Soc. Rev. 2014, 43, 3342. doi: 10.1039/c3cs60411j pmid: 24577078
[26]	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
[27]	Yogo, T.; Urano, Y.; Ishitsuka, Y.; Maniwa F.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 12162. doi: 10.1021/ja0528533
[28]	Atilgan, S.; Ekmekci, Z.; Dogan, A. L.; Guc, D.; Akkaya, E. Chem. Commun. 2006, 4398.
[29]	Awuah, S. G.; Polreis, J.; Biradar, V.; You, Y. Org. Lett. 2011, 13, 3884. doi: 10.1021/ol2014076
[30]	Batat, P.; Cantuel, M.; Jonusauskas, G.; Scarpantonio, L.; Palma, A.; O’Shea, D.; McClenaghan, N. J. Phys. Chem. A 2011, 115, 14034. doi: 10.1021/jp2077775
[31]	Nguyen, V.; Yim, Y.; Kim, S.; Ryu, B.; Swamy, K.; Kim, G.; Kwon, N.; Kim, C.; Park, S.; Yoon, J. Angew. Chem. Int. Ed. 2020, 59, 8957. doi: 10.1002/anie.v59.23
[32]	Dong, Y.; Dick, B.; Zhao, J. Z. Org. Lett. 2020, 22, 5535. doi: 10.1021/acs.orglett.0c01903 pmid: 32643941
[33]	Raza, M.; Gautam, S.; Howlader, P.; Bhattacharyya, A.; Kondaiah, P.; Chakravarty, A. Inorg. Chem. 2018, 57, 14374. doi: 10.1021/acs.inorgchem.8b02546
[34]	Li, M.; Tian, R.; Fan, J.; Du, J.; Long, S.; Peng, X. Dyes and Pigments 2017, 147, 99. doi: 10.1016/j.dyepig.2017.07.048
[35]	Liu, Y.; Xu, C.; Teng, L.; Liu, H.; Ren, T.; Xu, S.; Lou, X.; Guo, H.; Yuan, L.; Zhang, X. Chem. Commun. 2020, 56, 1956. doi: 10.1039/C9CC09790B
[36]	Teng, K.; Niu, L.; Kang, Y.; Yang, Q. Chem. Sci. 2020, 11, 9703. doi: 10.1039/D0SC01122C
[37]	Lv, X.; Han, T.; Wu, Y.; Zhang, B.; Guo, W. Chem. Commun. 2021, 57, 9744. doi: 10.1039/D1CC03360C
[38]	Yuan, B.; Wang, H.; Xu, J.; Zhang, X. ACS Appl. Mater. Interfaces 2020, 12, 26982. doi: 10.1021/acsami.0c07471
[39]	Zeng, Q.; Zhang, R.; Zhang, T.; Xing, D. Biomaterials 2019, 207, 39. doi: S0142-9612(19)30203-0 pmid: 30953845
[40]	Liu, H.; Hu, X.; Li, K.; Liu, Y.; Rong, Q.; Zhu, L.; Yuan, L.; Qu, F.; Zhang, X.; Tan, W. Chem. Sci. 2017, 8, 7689. doi: 10.1039/C7SC03454G
[41]	Chen, H.; Tian, J.; He, W.; Guo, Z. J. Am. Chem. Soc. 2015, 137, 1539. doi: 10.1021/ja511420n
[42]	Abouelmagd, S. A.; Hyun, H.; Yeo, Y. Expert Opin. Drug Del. 2014, 11, 1601. doi: 10.1517/17425247.2014.930434
[43]	Hamblin, M. R.; Miller, J. L.; Rizvi, I.; Ortel, B.; Maytin, E. V.; Hasan, T. Cancer Res. 2001, 61, 7155. pmid: 11585749
[44]	Sahoo, S. K.; Sawa, T.; Fang, J.; Tanaka, S.; Miyamoto, Y.; Akaike, T.; Maeda, H. Bioconjugate Chem. 2002, 13, 1031. pmid: 12236785
[45]	Rapozzi, V.; Zacchigna, M.; Biffi, S.; Garrovo, C.; Cateni, F.; Stebel, M.; Zorzet, S.; Bonora, G. M.; Drioli, S.; Xodo, L. Cancer Biol. Ther. 2010, 10, 471. doi: 10.4161/cbt.10.5.12536
[46]	Narayanaswamy, N.; Narra, S.; Nair, R.; Saini, D.; Kondaiah, P.; Govindaraju, T. Chem. Sci. 2016, 7, 2832. doi: 10.1039/c5sc03488d pmid: 30090277
[47]	Reja, S.; Gupta, M.; Gupta, N.; Bhalla, V.; Ohri, P.; Kaur, G.; Kumar, M. Chem. Commun. 2017, 53, 3701. doi: 10.1039/C6CC09127J
[48]	Guicciardi, M.; Leist, M.; Gores, G. Oncogene 2004, 23, 2881. pmid: 15077151
[49]	Kroemer, G.; Jaattela, M. Nat. Rev. Cancer 2005, 5, 886. doi: 10.1038/nrc1738 pmid: 16239905
[50]	Chen, H.; Xiao, L.; Anraku, Y.; Mi, P.; Liu, X.; Cabral, H.; Inoue, A.; Nomoto, T.; Kishimura, A.; Nishiyama, N.; Kataoka, K. J. Am. Chem. Soc. 2014, 136, 157. doi: 10.1021/ja406992w