Intramolecular charge transfer of molecular rotor reduces phototoxicity of BODIPY compound

Iida Haapalehto 1 , Nikita Durandin 1 , Elina Vuorimaa-Laukkanen 1 , Timo Laaksonen 1,2 , Ekaterina Lisitsyna 1

  • 1 Tampere University, Tampere
  • 2 University of Helsinki, Helsinki

Abstract

Boron-dipyrromethene (BODIPY) derivatives are commonly used to label different cellular structures for live-cell fluorescence imaging due to their high extinction coefficients in VIS-region, high fluorescence quantum yields and photostability. [1] More recently BODIPY-derived molecular rotos have been introduced as organelle viscosity sensors. Their sensing ability is based on the molecular structure which contains electron donor and acceptor parts linked together and capable to rotate around the linker. The structure allows twisted intramolecular charge transfer (TICT) state formation upon photoexcitation. [2] As the probability of TICT formation strongly depends on its immediate microenvironment (solvent viscosity) the molecules can be used e.g. as intracellular viscosity sensors. [3] However, the BODIPY derivatives may possess significant phototoxicity that is especially detrimental in long-term live cell imaging. The dyes can undergo intersystem-crossing (ISC) leading to the formation of their triplet state, which in turn can react with a molecular oxygen (O₂) to form a singlet oxygen (¹O₂). [4] ¹O₂ is known to cause an oxidative stress and consequently cell death. [5]

In this study we hypothesized that the compounds with TICT may show lower phototoxicity due to competing ISC and TICT resulting in reduced triplet state formation. Three different BODIPY derivatives were examined: BP, BPC12 and BPC3+. Two of the studied compounds (BPC12 and BPC3+) are molecular rotors possessing the TICT, while third one, i.e. BP, does not.

Firstly, singlet oxygen quantum yields of the dyes were indirectly determined in three different solvents by using 9,10-dimethylanthracene as a singlet oxygen trap and eosin Y as a reference molecule with high 0.611 singlet oxygen quantum yield [6]. The singlet oxygen quantum yield was the highest for BP, while BPC3+ and BPC12 showed lower values regardless the used solvent. In cellular phototoxicity experiments with a human prostate cancer cell line (PC-3), the cells treated with the BP demonstrated the lowest viability compared to moderate one for BPC3+ and the highest cell viability for BPC12. The phototoxicity effect was shown to be concentration- and light dose-dependent meaning that the higher concentration and light dose the more severe photodamage. Moreover, cell viability was substantially improved in poor oxygen conditions (reduction of phototoxic effect) thus confirming the oxygen-related origin of the phototoxicity for the studied molecules. The results of the cellular experiments agree with the indirect singlet oxygen measurements, i.e. BP leads to the highest singlet oxygen generation and causes the strongest toxicity.

Thus, we concluded that the BODIPY derivatives with TICT turned out to be less phototoxic for the cells making the dyes suitable for long-term imaging experiments without sacrificing fluorescence quantum yields and sensing properties.

[1] J. Karolin, L.B.A. Johansson, L. Strandberg, T. Ny, Journal of the American Chemical Society, 1994, 116, 7801-7806.
[2] M.A. Haidekker, E.A. Theodorakis, Journal of Biological Engineering, 2010, 4, 1-14.
[3] M.K. Kuimova, Physical Chemistry Chemical Physics, 2012, 14, 12671-12686.
[4] X. Zhang, J. Zhu, Journal of Luminescence, 2019, 212, 286-292.
[5] K. Briviba, L.O. Klotz, H. Sies, Biological Chemistry, 1997, 378, 1259-1265.
[6] L.V. Lutkus, S.S. Rickenbach, T.M. McCormick, Journal of Photochemistry and Photobiology A: Chemistry, 2019, 378, 131-135.

Acknowledgements: The research is funded by Academy of Finland (project nos. 323669 and 311362). BPC3+ rotor was kindly provided by Dr. Marina Kuimova (Imperial College London).

2. General

Chart 1. Molecular structures of A) the BODIPY derivatives; BP, BPC12 and BPC3+. B) A reaction of the DMA with a singlet oxygen (1O2) to form the endoperoxide compound.

In this study, we investigate three different BODIPY derivatives of which two are molecular rotors (BPC12 and BPC3+, Chart 1A) and one is not (BP, Chart 1A). The phototoxic nature of all the dyes was examined with following studies:

  • Absorption and fluorescence spectra in ethanol (EtOH), dimethyl sulfoxide (DMSO), toluene and polyethylene glycol (200 g/mol, PEG-200).
  • Fluorescence quantum yields in EtOH, DMSO, toluene and PEG-200 by using BP in EtOH as a reference.
  • Singlet oxygen quantum yields in DMSO, toluene and PEG-200 by using 2-(2,4,5,7-tetrabromo-6-oxido-3-oxo-3H-xanthen-9-yl)benzoate (Eosin Y) in DMSO as a reference and dimethyl anthracene (DMA, Chart 1B) as a singlet oxygen trap.
  • The morphology changes and viabilities of human prostate cancer cell line (PC-3) in four different concentrations (0.6, 1.5, 3.0 and 6.0 μM), three different light dosages (13.4, 26.8 and 53.5 J/cm2) and two different oxygen levels (normoxia: ≈20 %; hypoxia: ≈0.08 %).

4. Visualization of phototoxic effect

Figure 2. Merged phase contrast and fluorescence images of PC-3 cells treated with 6.0 μM of BP, BPC12 and BPC3+ in darkness and after illumination with 53.5 J/cm2 in both normoxic and hypoxic conditions.

One day after the illumination procedure, fluorescence images were obtained from the cells treated with the highest dye concentration of 6.0 μM. The fluorescence images in three different conditions: darkness and illumination in normoxic and hypoxic conditions with the highest light dosage (53.5 J/cm2) are presented in Figure 2. With all the dyes, cells are having typical morphology in darkness (upper row). Illumination in normoxic conditions (middle row) clearly shows rounding and swelling of the cells, which is a sign of cell necrosis. Phototoxicity is certainly caused by oxygen-presence since illumination in hypoxic conditions (bottom row) causes the cells to resemble the morphology of the dark-control.

3. Quantum yields of fluorescence and singlet oxygen

Fluorescence quantum yields

The fluorescence quantum yields for all three dyes in EtOH, DMSO, toluene and PEG-200 are presented in Table 1. The BP owns the highest fluorescence quantum yields in every solvent. The high viscosity of PEG-200 increases the fluorescence quantum yields of the BPC12 and BPC3+, since the rotation of the molecules is slower and possibility of TICT is reduced.

Table 1. The fluorescence quantum yields for BP, BPC12 and BPC3+ in EtOH, DMSO, toluene and PEG-200. 1G. Durán-Sampedro, A. R. Agarrabeitia, L. Cerdán, M. E. Pérez-Ojeda, A. Costela, I. García-Moreno, I. Esnal, J. Bañuelos, I. L. Arbeloa and M. J. Ortiz, Carboxylates versus Fluorines: Boosting the Emission Properties of Commercial BODIPYs in Liquid and Solid Media, Adv. Funct. Mater., 2013, 23, 4195–4205.

Singlet oxygen quantum yields

The singlet oxygen quantum yields for BP, BPC12 and BPC3+ in DMSO, toluene and PEG-200 are presented in Table 2. In PEG-200 both the BP and the BPC12 own the highest values of singlet oxygen quantum yields and BPC3+ owns the same value as BPC12. Singlet oxygen quantum yields are high in toluene, the most viscose solvent since, TICT formation is reduced. For BPC3+ the highest value is in toluene, which is the most polarizable solvent of all.

Table 2. The singlet oxygen quantum yields in percentages for BP, BPC12 and BPC3+ in DMSO, toluene and PEG-200. Number is presented by mean ± standard deviation calculated from triplicates.

5. Quantification of phototoxicity

Effect of dye concentration

Viabilities of the cells treated with four different concentrations of all three dyes in darkness and after illumination with the highest light dose (53.5 J/cm2) are presented in Figure 3. With all the dyes, the cell viability decreases with increasing dye concentration. The BPC12 shows much larger viability after illumination than either BP or BPC3+ with the lowest dye concentration of 0.6 μM. With this dye concentration, the BP and the BPC3+ have less than 40 % of cell viability after illumination. However, with all the dyes the highest dye concentration (6.0 μM) gives the very low survival of the cells. None of the dyes give much toxic effect in darkness. 

Figure 3. Cell viabilities of PC-3 cells treated with 0.6 μM, 1.5 μM, 3.0 μM and 6.0 μM BP, BPC12 and BPC3+ in darkness and after illumination with 53.5 J/cm2. Error bars are 95% confidence intervals.

Effect of light dosage

Light dose effects on the phototoxicity were studied for the BP and the BPC12 since those gave the most different results when comparing different dye concentrations. Cell viabilities of 1 % DMSO, and 0.6 μM BP and BPC12 in darkness and after illuminating with three different light dosages are presented in Figure 4. DMSO was used as a solvent in the stock solutions of the dyes and it did not cause any damage to the cells. The BP shows a very light dose dependent behavior when increasing the light dose from 13.4 J/cm2 to 53.5 J/cm2, while the BPC12 is not phototoxic even at the highest light dosage.

Figure 4. Cell viabilities of PC-3 cells treated with 1 % DMSO, 0.6 μM BP and 0.6 μM BPC12 in darkness and after illumination with 3 different light doses: 13.4 J/cm2, 26.8 J/cm2 and 53.5 J/cm2. Error bars are 95% confidence intervals.

Effect of oxygen level

Lastly, the viabilities of the cells treated with the highest dye concentration (6.0 μM) in darkness and illuminated with the highest light dosage (53.5 J/cm2) in normoxic (≈20 % of oxygen) and in hypoxic (≈0.08 % of oxygen) conditions are presented in Figure 5. In hypoxic conditions the viability of the cells increases when comparing with normoxic conditions. Therefore, the phototoxicity is clearly affected by oxygen-presence.

Figure 5. Cell viabilities of PC-3 cells treated with 6.0 μM BP, BPC12 and BPC3+ in darkness and after illumination with 53.5 J/cm2 in both normoxic and hypoxic conditions. Error bars are 95% confidence intervals.

1. Introduction

For long-term live-cell fluorescence imaging it is fundamental to use a label that has good photochemical properties. Boron-dipyrromethene (BODIPY) derivatives are an excellent option to label the lipophilic structures of cells, due to their high fluorescence quantum yield and photostability. However, fluorescence labels might form singlet oxygen upon illumination via their triplet state (Figure 1) resulting in a cell death. In this study, we illustrate that BODIPY based molecular rotors are advantageous options instead of the rigid derivatives of BODIPYs. For molecular rotors twisted intramolecular charge transfer (TICT) is a more probable process from the singlet excited state (S1) than an intersystem crossing (ISC), thus resulting in lower singlet oxygen quantum yields and therefore in lower phototoxicity.

Figure 1. Photophysical processes of the molecular rotors including twisted intramolecular charge transfer (TICT) process, which leads to formation of charge transfer (CT) state. Singlet oxygen (1O2) formation and decay processes are illustrated. Reaction of dimethyl anthracene (DMA) with singlet oxygen to form endoperoxide compound is shown.

6. Conclusions

  • The BP reacts more efficiently with molecular oxygen to form singlet oxygen which is one of the reactive oxygen species.
  • BODIPY-based molecular rotors (e.g. BPC12 and BPC3+) can undergo TICT which reduces the formation of the singlet oxygen.
  • The phototoxicity of all three BODIPY-derivatives is increased in increasing dye concentrations, light dosages and in the presence of oxygen.
  • The BPC12 is the best choice for long-term live-cell studies that require high dye concentrations or light dosages.