meso-Substituted BODIPY fluorescent probes for cellular bio-imaging and anticancer activity

Abstract

A series of meso-substituted thienyl BODIPY analogues (1, 1a–g) have been designed and synthesized. Among those, compounds 1c, 1d and 1e show biocompatibility towards endothelial cells whereas 1c and 1d show significant cytotoxicity towards cancerous cells. The formation of intracellular reactive oxygen species (ROS) and the activation of the apoptotic protein may be a plausible mechanism for the anti-proliferative nature of 1c and 1d. Additionally, compounds 1c, 1d, and 1e fluoresce inside the cancer cells. The combined results suggest the future theranostic (therapeutics + diagnostics) applications of 1c and 1d in cancer diseases.

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Fluorescence imaging has been playing a vital role in numerous fields of modern science. BODIPY (4,4-difluoro-4-bora-3a,4a-diaza indacene) dyes are known as excellent fluorophores for their applications in fluorescent materials.¹ Besides this, BODIPY dyes have been employed in the fields of light harvesting arrays,² ion sensors,³ supramolecular fluorescent gels,⁴ biological probes,⁵ fluorescent stains,⁶ chemo sensors,⁷ energy transfer cassettes,^8,9 logic gates,¹⁰ dye-sensitized solar cells,¹¹ photodynamic therapy^12a etc.

Photodynamic therapy (PDT) has been one of the effective treatments of cancers and tumours; in which photosensitizers are decisive. BODIPY derivatives possess significant ability to generate singlet oxygen and thus they have been under study as photosensitizers for PDT.^12b Many BODIPY analogues are well known as in vitro fluorescent probes.¹³ However, synthesis, fluorescence properties and anticancer activity (theranostic properties) of these meso-BODIPY derivatives have not been reported elsewhere, to the best of our knowledge.

Numerous contributions have emerged to conquer the challenging functionalization of the BODIPY core system.¹⁴ Regardless, an efficient synthesis of the meso-aryl substituted BODIPY dyes has been difficult. meso (C-8)-substituted BODIPYs were reported, with Pd catalyzed and Cu(I) mediated cross-coupling reactions of 8-thiomethyl BODIPY with organoboron derivatives under neutral conditions.¹⁵ Nevertheless, the extension of thioether substitutions remains much less extensive in contrast to the super abundance of reactions accessible for aromatic halides.¹⁶

The analogues of bromothienyl BODIPY find much scope in the array of material science.^17,19 We functionalized 1 at meso position with different substituents bearing various functional groups via Suzuki cross-coupling reactions with fair to excellent yields (Scheme 1). Here we assembled a series of meso-substituted BODIPY derivatives implying a Suzuki cross-coupling reaction catalyzed by Pd₂(dba)₃, using P(t-Bu)₃HBF₄ as ligand with K₃PO₄ base in THF–H₂O (9 : 1) solvent (synthetic procedures and NMR spectra of all new molecules are included in ESI†). The classical way of Suzuki coupling using Pd(PPh₃)₄ as catalyst with Na₂CO₃ base in 4 : 1 DME–H₂O solvent has been screened out in this case. However, 1 was synthesized following the classic Lindsey method with an overall yield of 41%.^2a

Scheme 1 Synthesis of meso-substituted BODIPY dyes.

Owing to the various functional groups of the synthesized BODIPY derivatives, we studied optical properties in different solvents. Absorption, emission spectra and quantum yields were recorded for all the molecules in three solvents ranging from non-polar to polar i.e., cyclohexane (CH), THF and DMF (ESI†). The low quantum yields of the above analogues can be ascribed to the presence of free rotating meso-substituted group which causes the energy loss from the excited state through non-radiative molecular motions.¹⁸ The absorption spectra of all the mentioned dyes followed a similar pattern with a sharp absorption peak around 510 nm regardless the nature of substituent. No considerable shifts in absorption maxima were observed in meso-substituted analogues with reference to 1. The geometrical isomers 1c and 1d possessed similar properties with considerable change in their extinction coefficients. The absorption and emission maxima were not changed significantly with the change of solvents. UV and fluorescence spectra of all the compounds were recorded in THF (Fig. 1), CH, and DMF (Fig. 15 and 16 respectively, ESI†). The optical properties of all compounds in various solvents were reported in Table 1.

Fig. 1 Normalized absorption and fluorescence (FL) emission spectra of BODIPY dyes 1, 1a–g in tetrahydrofuran (THF).

Table 1 Optical properties of meso-substituted BODIPY dyes

Product	λabs^a (nm)			λem^b (nm)			QF^c			ε^d (in THF) mol^−1 cm^−1 × 10^4
	CH	THF	DMF	CH	THF	DMF	CH	THF	DMF
a Absorption maximum.b Fluorescence emission maximum (obtained by exciting at the absorption maximum of the dye).c Fluorescence quantum yield.d Extinction coefficient at absorption maximum. The fluorescence quantum yields were obtained by using Rhodamine B (0.31 in water) as a standard.^20
1	515	513	513	628	628	627	0.00283	0.00186	0.00252	1.86
1a	516	513	513	644	644	633	0.00202	0.00183	0.00010	4.3
1b	514	512	513	645	642	633	0.00211	0.00203	0.00141	3.6
1c	514	514	514	650	622	644	0.00133	0.00291	0.00033	5.8
1d	514	516	514	650	625	645	0.00122	0.00273	0.00034	6.7
1e	511	510	510	625	622	620	0.01261	0.00170	0.00050	7.5
1f	514	514	515	618	618	640	0.00192	0.00161	0.00061	6.9
1g	514	514	512	640	640	635	0.00333	0.00264	0.00210	6.5

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A screening has been carried out to find out the prominent compounds for bio-imaging purpose with all the synthesized BODIPY analogues (1, 1a–g) in cancerous B16 F10 cells. Initially the cells have been treated with 1 and 1a–g molecules for 24 h to check their fluorescence properties inside the cells. It has been found that untreated control B16 F10 cells do not show any fluorescence whereas the cells treated with compounds 1c, 1d and 1e show intense red fluorescence even at low concentrations compared to 1, 1f and 1g (ESI-Fig. 1–8†). However, 1a and 1b do not show any fluorescence properties inside the cells. It has been observed that the cells are getting ruptured for 24 h treatment which may be due to the cytotoxicity of those molecules towards B16 F10 cell line. In order to obtain better fluorescence images, B16 F10 cells have been incubated with compounds 1c, 1d and 1e as well as compounds 1, 1f and 1g for only 6 h. Fig. 2 demonstrates the fluorescence imaging of compounds 1c, 1d and 1e in cancerous B16 F10 cells. The control untreated cells do not exhibit any fluorescence while cells treated with 1c, 1d and 1e (20 μM) show nice red fluorescence with high intensity even after 6 h of treatment. Also, the cells treated with those three compounds exhibit red fluorescence even at lower concentration (5–10 μM) (see ESI-Fig. 9: 1c; ESI-Fig. 10: 1d and ESI-Fig. 11: 1e†). ESI-Fig. 12–14 represents the weak red fluorescence of B16 F10 cells treated with compounds 1, 1f and 1g respectively for 6 h of treatment. Cell viability assay of compounds 1, 1a–g shows their anti-proliferative activity towards various cancerous cells (B16 F10, SKOV3 and MCF-7) except 1e (Fig. 3). IC₅₀ values of all the materials in different cancer cells have been shown in Table 2. However, all compounds are found to be biocompatible in non-cancerous ECV-304 cell lines (Fig. 4a). The biocompatibility of the lead molecules 1c, 1d and 1e in non-cancerous cells has further been confirmed towards endothelial HUVECs (Fig. 4b) indicating the feasibility of applications of 1c and 1d molecules in both bio-imaging and therapeutic (anti-cancer) purposes. However, considering the biocompatibility of 1e in both normal and cancerous cell lines, it seems that it would be better fluorescent probe for live cell imaging than 1c or 1d. Additionally, the cell cycle analysis (Fig. 5) in B16 F10 cells treated with lead molecules 1c and 1d reveals the significant increase in cell population of sub-G1 phase and subsequently decrease in cell population of G0/G1 phase compared to untreated control cells. This result suggests the sub-G1 phase arrest in treated B16 F10 cells leading to the induction of apoptosis as reported in earlier literatures.²¹

Fig. 2 Fluorescence microscopic images of B16 F10 cells. Row 1 (a1–a4): untreated control cells; row 2 (b1–b4): cells treated with 20 μM 1c; row 3 (c1–c4): cells treated with 20 μM 1d and row 4 (d1–d4): cells treated with 20 μM 1e. Column 1: bright field images; column 2: red fluorescence images of BODIPY analogues; column 3: blue fluorescence images of Hoechst stained nucleus; column 4: merging of red and blue fluorescence images. The incubation time period of all the compounds (1c, 1d and 1e) in B16 F10 cells is 6 h.

Fig. 3 Cell viability assay in (a) B16 F10, (b) SKOV3 and (c) MCF-7 cell lines using MTT reagent. Results reveal that all of the molecules except 1e show inhibition of cancer cell proliferation (anti-tumor activity) in a dose dependant manner (0.1–20 μM). DMSO and doxorubicin (DOX: 4 μM) has been used as vehicle and positive control experiments, respectively.

Table 2 IC₅₀ values of different BODIPY materials

Materials	IC50 (μM): B16 F10	IC50 (μM): SKOV3	IC50 (μM): MCF-7
1	29.1	177.0	44.8
1a	42.6	14.2	54.7
1b	19.1	33.3	69.0
1c	38.2	13.3	39.2
1d	41.0	28.0	46.6
1e	90.4	76.3	248.5
1f	51.4	24.1	40.6
1g	68.2	24.5	27.5

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Fig. 4 Cell viability assay in (a) ECV-304 and (b) HUVEC cell lines using MTT reagent. All the compounds are biocompatible in nature toward non-cancerous ECV-304 cells. The biocompatibility of the lead molecules 1c, 1d and 1e has also been shown in HUVEC cells.

Fig. 5 Cell cycle assay in B16 F10 cells. Histogram and representative picture show that the cell population of sub-G1 phase increases while that of G0/G1 phase decreases in presence of 1c and 1d compared to untreated control cells. Sub-G1 phase arrest of treated cells indicates the induction of apoptosis.

Recent reports demonstrate that reactive oxygen species (ROS) plays a key role in anticancer activities of different materials.^21c,22 Therefore, we have investigated the generation of ROS, especially H₂O₂ in B16 F10 cells treated with 1c and 1d by using DCFDA reagent. The untreated control cells do not exhibit any green fluorescence suggesting the absence of ROS while cells treated with 1c and 1d show intense green fluorescence indicating the formation of intracellular ROS (Fig. 6a). Furthermore, we have performed western blot analysis (Fig. 6b) which indicates-the upregulation of apoptotic protein caspase 3 in B16 F10 cells treated with 1c and 1d compared to untreated control cells suggesting the activation of apoptotic pathway. This result also corroborates with the cell cycle analysis result where sub-G1 phase arrest suggest the induction of apoptosis. Altogether, it has been found that the generation of intracellular ROS and the activation of the apoptotic protein caspase 3 may be the plausible mechanism for the anti-cancer activity of those materials.

Fig. 6 Plausible mechanism of anti-proliferative activity of 1c and 1d: (a) determination of ROS and (b) western blot analysis in B16 F10 cells. Intracellular formation of ROS (green fluorescence) and upregulation of apoptotic protein caspase 3 have been observed in B16 F10 cells in presence of 1c and 1d.

In summary, a series of meso-substituted BODIPY analogues has been designed and explored for the theranostic applications in cancer cells for the first time. Among all, compounds 1c and 1d are considered as lead molecules showing intense red fluorescence property and anti-tumour activity towards cancerous cells. The intracellular production of ROS and upregulation of apoptotic protein caspase 3 may be the plausible mechanism for the anti-cancer activity of 1c and 1d. Additionally, these lead molecules are biocompatible towards normal endothelial cells. The results may provide the basis for the development of biocompatible highly fluorescent BODIPY analogues that could be useful for future theranostic applications in cancer diseases.

Acknowledgements

We thank director CSIR-IICT for continuous encouragement and support. TG and AKB are thankful to UGC for providing junior research fellowship and senior research fellowship respectively. CRP thanks to DST, India for ‘Ramanujan Fellowship Grant’ (SR/S2/RJN-04/2010; GAP0305). Financial support from CSIR 12^th five year plan (FYP) project CSC 0114 and CSC 0302 are acknowledged. The authors also thank Dr V. J. Rao and Dr P. R. Bangal for the help in fluorescence data.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Synthetic procedures, absorption, emission, NMR spectra and fluorescence microscopic images. See DOI: 10.1039/c4ra07424f

‡ Authors contributed equally.

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