噻吩基氟硼二吡咯近红外光敏染料的合成、双光子荧光成像及光动力治疗研究

doi:10.6023/A22040141

摘要/Abstract

设计并合成了两种新型噻吩基氟硼二吡咯(Thienyl-BODIPY)近红外光敏染料ITBDP-1和ITBDP-2. 两种光敏染料的吸收和发射波长均达到近红外区, ITBDP-1的吸收与发射峰分别是617 nm和650 nm; ITBDP-2的吸收与发射峰分别是687 nm和731 nm. 两种光敏剂均具有较高的单线态氧产率, ITBDP-1与ITBDP-2的单线态氧产率(Φ_Δ)分别为51%和24%. 通过密度泛函理论(DFT)计算研究了光敏染料激发态下的能量变化, 理论计算表明, ITBDP-1和ITBDP-2在激发至单重态后可通过系间窜越(ISC)到达三重态, 从而提高单线态氧产率. ITBDP-1和ITBDP-2在A549细胞内具有良好的的荧光成像效果, 并且在900 nm激光激发下, ITBDP-1能够在斑马鱼体内显示出清晰的双光子荧光成像. 单线态氧成像实验证明了光敏染料在光激发下可以在肿瘤细胞和斑马鱼中产生单线态氧. 通过噻唑蓝(MTT)比色法测定了两种光敏染料的光毒性和暗毒性, ITBDP-1和ITBDP-2的最大半抑制浓度(IC₅₀)分别为2.22 μmol•L^-1和2.86 μmol•L^-1, 并且无光照条件下细胞的存活率在80%以上, 证明了两种光敏染料均具有较高的光毒性和良好的生物相容性. ITBDP-1和ITBDP-2可以在近红外光激发下实现荧光成像指导的光动力学治疗, 并且可以实现生物体内的双光子荧光成像, 这一结果也为噻吩基氟硼二吡咯光敏染料在长波激发下的双光子光动力学治疗应用打下了基础.

关键词: 近红外光敏剂, 噻吩基氟硼二吡咯, 光动力学治疗, 双光子荧光成像

In this paper, two novel thienyl boron dipyrromethene (thienyl-BODIPY) near infrared (NIR) photosensitive dyes, ITBDP-1 and ITBDP-2 were designed and synthesized. The absorption and emission wavelengths of two photosensitizers reach near infrared region. The absorption and emission peaks of ITBDP-1 are 617 and 650 nm while the absorption and emission peaks of ITBDP-2 are 687 and 731 nm respectively. The singlet oxygen yields (Φ_Δ) of photosensitizers were determined using 1,3-diphenyliso- benzofuran (DPBF) as singlet oxygen scavenger. The results turned out that two photosensitizers both could generate singlet oxygen under 660 nm light irradiation, and the absorption of DPBF decreased over 50% within 1 min. Φ_Δof ITBDP-1 and ITBDP-2 are 51% and 24% respectively. Excited state energy level of photosensitizers were studied by density functional theory (DFT) calculation. Theoretical calculation indicates that ITBDP-1 and ITBDP-2 can reach triplet state through intersystem crossing (ISC) after being excited to singlet state, thus improving singlet oxygen yield. ITBDP-1 and ITBDP-2 showed satisfying imaging effect in A549 cells, and ITBDP-1 could show distinct two-photon fluorescence in zebrafish under 900 nm excitation. Apart from good cell imaging effect, these two photosensitizers showed abilities to generate reactive oxygen species (ROS) in tumor cells as well. It can be proved that photosensitizers could produce singlet oxygen in tumor cells and zebrafish under light excitation with dichlorodihydrofluorescein diacetate (DCFH-DA) as ROS indicator. Besides, ITBDP-1 and ITBDP-2 also presented high cytotoxicity under 660 nm light irradiation. In the tetrazolium (MTT) experiment, the maximum half inhibitory concentration (IC₅₀) of ITBDP-1 and TBDP-2 were 2.22 and 2.86 μmol•L^-1 respectively, and the cell viability was over 80% without irradiation, which proved that both photosensitizers possess high phototoxicity and good biocompatibility. ITBDP-1 and ITBDP-2 could realize imaging-guided photodynamic therapy under excitation of NIR light in tumor cells, and displayed two-photon fluorescence imaging in vivo. Photosensitizers ITBDP-1 and ITBDP-2 are excepted to lay good foundation for the application of thienyl-BODIPY photosensitizers in two-photon photodynamic therapy under NIR excitation.

Key words: near infrared photosensitizer, thienyl-BODIPY, photodynamic therapy, two-photon imaging

引用此文

刘巴蒂, 王承俊, 钱鹰. 噻吩基氟硼二吡咯近红外光敏染料的合成、双光子荧光成像及光动力治疗研究[J]. 化学学报, 2022, 80(8): 1071-1083.

Badi Liu, Chengjun Wang, Ying Qian. Synthesis, Two-photon Fluorescence Imaging and Photodynamic Therapy of Near Infrared Thienyl-BODIPY Photosensitizer[J]. Acta Chimica Sinica, 2022, 80(8): 1071-1083.

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图/表 16

图式1. (a)已报道的不同类型光敏剂; (b)噻吩基氟硼二吡咯近红外光敏剂ITBDP-1和ITBDP-2及其成像指导的光动力学治疗

Scheme 1. (a) Different types of reported photosensitizers; (b) NIR photosensitizer ITBDP-1, ITBDP-2 and their imaging-guided photodynamic therapy

图1. 噻吩基BODIPY系列化合物在二氯甲烷中的吸收及发射光谱(浓度均为10-5 mol•L-1). (a) ITBDP-1的紫外-可见吸收及荧光发射光谱; (b) ITBDP-2的紫外-可见吸收及荧光发射光谱; (c) TBDP的紫外-可见吸收及荧光发射光谱; (d) ITBDP的紫外-可见吸收及荧光发射光谱

Figure 1. The absorption and emission spectra of thienyl-BODIPY compounds in dichloromethane (all concentrations are 10-5 mol•L-1). (a) UV-Vis and fluorescence emission spectra of ITBDP-1 (10-5 mol•L-1); (b) UV-Vis and fluorescence emission spectra of ITBDP-2; (c) UV-Vis and fluorescence emission spectra of TBDP; (d) UV-Vis and fluorescence emission spectra of ITBDP

图式2. 近红外噻吩基氟硼二吡咯光敏剂ITBDP-1和ITBDP-2的合成路线

Scheme 2. Synthesis routine of NIR thienyl-BODIPY photosensitizers ITBDP-1 and ITBDP-2

图2. (a) ITBDP-1溶液中DPBF的紫外-可见吸收强度随时间下降示意图及DPBF在ITBDP-1溶液和亚甲基蓝溶液中414 nm处吸光度的(A/A0)值随时间变化示意图; (b) ITBDP-2溶液中DPBF的紫外-可见吸收强度随时间下降示意图及DPBF在ITBDP-2溶液和亚甲基蓝溶液中414 nm处吸光度的(A/A0)值随时间变化示意图

Figure 2. (a) Time-dependent plot of UV-Vis intensity of DPBF in ITBDP-1 solution; time-dependent plot of (A/A0) value of DPBF at 414 nm in ITBDP-1 solution and methylene blue solution; (b) time-dependent plot of UV-Vis intensity of DPBF in ITBDP-2 solution; time-dependent plot of (A/A0) value of DPBF at 414 nm in ITBDP-2 solution and methylene blue solution

Compound	$\lambda _{\max }^{\text{abs}}$/ nm	$\varepsilon _{\max }^{\text{abs}}$/(10⁴L•mol^-1•cm^-1)	$\lambda _{\max }^{em}$/nm	Φ_Δ	IC₅₀/ (μmol•L^-1)
ITBDP-1	617	0.62	650	0.51	2.22
ITBDP-2	687	7.60	731	0.24	2.86
TBDP	513	3.50	523	—	—
ITBDP	546	3.90	562	0.63	—

表1. 噻吩基氟硼二吡咯系列化合物的光物理性质数据

Table 1. Photophysical properties of thienyl-BODIPY derivatives

图3. 基于DFT//B3LYP/6-31G(d)和DFT//B3LYP/6-31G(d)/LANL2DZ的基态结构优化结果以及基于TD-DFT//cam-B3LYP/6-31G(d)和TD-DFT//cam-B3LYP/6-31G(d)/LANL2DZ计算得到的(a) ITBDP-1和(b) ITBDP-2的激发态能量及前线轨道图

Figure 3. Ground state structure optimization based on DFT//B3LYP/6-31G(d) and DFT//B3LYP/6-31G(d)/LANL2DZ. (a) calculated excited state energy and frontier orbital diagram of ITBDP-1; (b) calculated excited state energy and frontier orbital diagram of ITBDP-2 based on TD-DFT//cam-B3LYP/6-31G(d) and TD-DFT// cam-B3LYP/6-31G(d)/LANL2DZ

图4. ITBDP-1 (2 μmol•L-1, λex=900 nm)与斑马鱼共同孵育2 h后的双光子荧光成像图: (a)斑马鱼头部成像; (b)斑马鱼身体中段成像; (c)斑马鱼尾部成像

Figure 4. Two-photon fluorescence imaging of ITBDP-1 (2 μmol•L-1, λex=900 nm) incubated with zebrafish for 2 h: (a) head imaging of zebrafish; (b) body imaging of zebrafish; (c) tail imaging of zebrafish

图5. (a1~a3) ITBDP-1 (2 μmol•L-1)在A549细胞中的荧光成像; (b1~b3) ITBDP-2 (2 μmol•L-1)在A549细胞中的荧光成像

Figure 5. (a1~a3) Fluorescence imaging of ITBDP-1 (2 μmol•L-1) in A549 cells; (b1~b3) fluorescence imaging of ITBDP-2 (2 μmol•L-1) in A549 cells (a1~a3) Fluorescence imaging of ITBDP-1 (2 μmol•L-1) in A549 cells; (b1~b3) fluorescence imaging of ITBDP-2 (2 μmol•L-1) in A549 cells

图6. (a) 光照15 min (660 nm, 4 mW•cm-2)和黑暗条件下与ITBDP-1共孵育的A549细胞存活率; (b) 光照15 min (660 nm, 4 mW•cm-2)和黑暗条件下与ITBDP-2 共孵育的A549细胞存活率

Figure 6. (a) Cell viability of A549 cells treated with ITBDP-1 under light irradiation for 15 min (660 nm, 4 mW•cm-2) and dark conditions; (b) cell viability of A549 cells treated with ITBDP-2 under light irradiation for 15 min (660 nm, 4 mW•cm-2) and dark conditions

图7. 光照15 min条件下(660 nm, 4 mW•cm-2)与ITBDP-1共孵育的A549细胞生长曲线; (b) 光照15 min (660 nm, 4 mW•cm-2)条件下与ITBDP-2 共孵育的A549细胞生长曲线

Figure 7. (a) Cell growth curve of A549 cells treated with ITBDP-1 under light irradiation for 15 min (660 nm, 4 mW•cm-2); (b) cell growth curve of A549 cells treated with ITBDP-2 under light irradiation for 15 min (660 nm, 4 mW•cm-2)

图8. ITBDP-1在A549细胞内的ROS成像. (a1~a4) A549细胞在DCFH-DA和ITBDP-1存在下于黑暗中孵化3 h成像; (b1~b4) A549细胞在DCFH-DA和ITBDP-1存在下于黑暗中孵化3 h 后, 光照(660 nm, 4 mW•cm-2) 10 min成像

Figure 8. ROS imaging of ITBDP-1 in A549 cells. (a1~a4) A549 cells incubated in dark for 3 h in the presence of DCFH-DA and ITBDP-1; (b1~b4) A549 cells incubated in dark for 3 h in the presence of DCFH-DA and ITBDP-1, then irradiated under light (660 nm, 4 mW•cm-2) for 10 min

图9. ITBDP-2在A549细胞内的ROS成像. (a1~a4) A549细胞在DCFH-DA和ITBDP-1存在下于黑暗中孵化3 h成像; (b1~b4) A549细胞在DCFH-DA和ITBDP-2存在下于黑暗中孵化3 h 后, 光照(660 nm, 4 mW•cm-2) 10 min成像; (c1~c4) A549细胞在DCFH-DA和ITBDP-2存在下于黑暗中孵化3 h 后, 光照(660 nm, 4 mW•cm-2) 30 min成像

Figure 9. ROS imaging of ITBDP-2 in A549 cells. (a1~a4) A549 cells incubated in dark for 3 h in the presence of DCFH-DA and ITBDP-2; (b1~b4) A549 cells incubated in dark for 3 h in the presence of DCFH-DA and ITBDP-2, then irradiated under light (660 nm, 4 mW•cm-2) for 10 min; (c1~c4) A549 cells incubated in dark for 3 h in the presence of DCFH-DA and ITBDP-2, then irradiated under light (660 nm, 4 mW•cm-2) for 30 min

图10. ITBDP-1在斑马鱼内的ROS成像. (a1~a4)斑马鱼在DCFH-DA和ITBDP-1存在下于黑暗中孵化1 h后成像; (b1~b4)斑马鱼在DCFH-DA和ITBDP-1存在下于黑暗中孵化1 h 后, 光照(激发波长660 nm, 4 mW•cm-2) 15 min后成像

Figure 10. ROS imaging of ITBDP-1 in zebrafish. (a1~a4) imaging of zebrafish incubated in the dark for 1 h in the presence of DCFH-DA and ITBDP-1; (b1~b4) imaging of zebrafish incubated in dark for 1 h in the presence of DCFH-DA and ITBDP-1, and then irradiated (660 nm, 4 mW•cm-2) for 15 min

图11. (a1~a4)无光照条件下ITBDP-1与A549细胞共孵育后的AO/EB成像; (b1~b4)光照5 min条件下ITBDP-1与A549细胞共孵育后的AO/EB成像; (c1~c4)光照15 min条件下ITBDP-1与A549细胞共孵育后的AO/EB成像(激发波长为660 nm, 4 mW•cm-2)

Figure 11. (a1~a4) AO/EB imaging of A549 cells incubated with ITBDP-1 in dark; (b1~b4) AO/EB imaging of A549 cells incubated with ITBDP-1 under light irradiation for 5 min; (c1~c4) AO/EB imaging of A549 cells incubated with ITBDP-1 under light irradiation for 15 min (excitation wavelength 660 nm, 4 mW•cm-2)

图12. (a1~a4)无光照条件下ITBDP-2与A549细胞共孵育后成像; (b1~b4)光照15 min条件下ITBDP-2与A549细胞共孵育后的AO/EB成像; (c1~c4)光照30 min条件下ITBDP-2与A549细胞共孵育后的AO/EB成像(激发波长为660 nm, 4 mW•cm-2)

Figure 12. (a1~a4) imaging of A549 cells incubated with ITBDP-2 in dark; (b1~b4) imaging of A549 cells incubated with ITBDP-2 under light irradiation for 15 min; (c1~c4) imaging of A549 cells incubated with ITBDP-2 under light irradiation for 30 min (excitation wavelength 660 nm, 4 mW•cm-2)

图13. (a) ITBDP-1 (2 μmol•L-1)存在下光照15 min (660 nm, 4 mW•cm-2)后24 h内划痕状态; (b) ITBDP-1 (2 μmol•L-1)存在下无光照24 h后划痕状态

Figure 13. (a) Scratch state within 24 h after illumination for 15 min (660 nm, 4 mW•cm-2) in the presence of ITBDP-1 (2 μmol•L-1); (b) scratch state within 24 h in dark in the presence of ITBDP-1 (2 μmol•L-1)

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