Design, Synthesis and Photodynamic Therapy of a H2O2-Activatable Near Infrared Borondipyrromethene （BODIPY） Photosensitizer

doi:10.6023/A22120487

Abstract

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

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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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Fig. & Tab. 16

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

Scheme 2. The synthetic routes of photosensitizer FP-IBDP

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

Design, Synthesis and Photodynamic Therapy of a H₂O₂-Activatable Near Infrared Borondipyrromethene （BODIPY） Photosensitizer

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