Mitochondria-targeting biocompatible fluorescent BODIPY probes

Abstract

An increase in the mitochondrial membrane potential (MMP) is a characteristic feature of cancer and cardiovascular disease. Therefore, it remains of crucial importance to develop new and improved fluorescent probes that are sensitive to the MMP, to report on mitochondrial health and function. Reported here are the design, synthesis, photophysical properties and biological characterisation of a series of BODIPY dyes, BODIPY-Mito-n, for mitochondria-targeted fluorescence imaging applications. Six BODIPY-Mito-n analogues were synthesised under mild conditions, and displayed excellent fluorescence quantum yields of between 0.59 and 0.72 in aqueous environments at physiological pH (pH = 7.4). The incorporation of poly(ethylene glycol) (PEG) chains to the triarylphosphonium cation moiety significantly improved the biocompatibility of the probes (BODIPY-Mito-6, IC₅₀ > 50 μM). All BODIPY-Mito-n compounds demonstrated a high MMP-sensitive localisation in the mitochondria, with Pearson's correlation coefficients (PCC) of between 0.76 and 0.96. Compounds BODIPY-Mito-2 and BODIPY-Mito-6 revealed the highest sensitivity to the MMP, with a decrease in the emission intensity of 62% and 75%, respectively following MMP depolarisation. It is anticipated that the highest MMP sensitivity and enhanced biocompatibility of BODIPY-Mito-6 could lead to the development of new probes for mitochondrial imaging in the future.

Fig. 1. Phosphonium cation-functionalised BODIPY compounds. (A) Previously reported compounds by Archibald and co-workers, and (B) BODIPY-Mito-n compounds investigated in this work.

Scheme 1. Synthesis of BODIPY-Mito-n. Conditions: (i) PR¹R²R³, toluene, 110 °C, 3 d (BODIPY-Mito-1–3) and (ii) PR¹R²R³, CH₃CN, 85 °C, 3 d (BODIPY-Mito-4–6).

Fig. 2. Absorption (blue), emission (orange) and excitation (green) spectra of BODIPY-Mito-1. [BODIPY] = 10 μM, PBS (pH = 7.4), 298 K. λ_ex = 497 nm, λ_em = 511 nm.

Fig. 3. (A) LSCM images of live HeLa cells incubated with BODIPY-Mito-n (500 nM, 1 h) at 37 °C. λ_ex = 488 nm, λ_em = 501–521 nm. Scale bar = 25 μm. (B) Flow cytometric results of HeLa cells incubated with blank medium or BODIPY-Mito-n (500 nM, 1 h) at 37 °C.

Fig. 4. LSCM images of live HeLa cells incubated with BODIPY-Mito-1–4 (500 nM, 1 h; λ_ex = 488 nm, λ_em = 501–521 nm) and then MitoTracker Deep Red (100 nM, 30 min; λ_ex = 635 nm, λ_em = 655–675 nm) at 37 °C. Scale bar = 10 μm.

Fig. 5. (A) LSCM images of live HeLa cells incubated with BODIPY-Mito-5 and BODIPY-Mito-6 at different incubation concentrations for 1 h at 37 °C. λ_ex = 488 nm, λ_em = 501–521 nm. Scale bar = 25 μm. (B) LSCM images of live HeLa cells incubated with BODIPY-Mito-5 (5 μM, 1 h; λ_ex = 488 nm, λ_em = 501–521 nm) and BODIPY-Mito-6 (25 μM, 1 h; λ_ex = 488 nm, λ_em = 501–521 nm) and then MitoTracker Deep Red (100 nM, 30 min; λ_ex = 635 nm, λ_em = 655–675 nm) at 37 °C. Scale bar = 10 μm. (C) LSCM images of live HeLa cells incubated with BODIPY-Et (500 nM, 1 h; λ_ex = 488 nm, λ_em = 501–521 nm) and then MitoTracker Deep Red (100 nM, 30 min; λ_ex = 635 nm, λ_em = 655–675 nm) (top) or Lipi-Blue (500 nM, 30 min; λ_ex = 405 nm, λ_em = 420–470 nm) (bottom) at 37 °C. Scale bar = 10 μm.

Fig. 6. (A) LSCM images of live HeLa cells incubated with BODIPY-Mito-1–4 (500 nM, 1 h), BODIPY-Mito-5 (5 μM, 1 h) or BODIPY-Mito-6 (25 μM, 1 h) without (top) or with (bottom) post-treatment with CCCP (10 μM, 30 min) at 37 °C. λ_ex = 488 nm, λ_em = 501–521 nm. Scale bar = 25 μm. (B) Flow cytometric results of HeLa cells incubated with BODIPY-Mito-n. The cells were treated with blank medium for 1 h (black); BODIPY-Mito-1–4 (500 nM), BODIPY-Mito-5 (5 μM) or BODIPY-Mito-6 (25 μM) for 1 h (red); and BODIPY-Mito-1–4 (500 nM), BODIPY-Mito-5 (5 μM) or BODIPY-Mito-6 (25 μM) for 1 h and then CCCP (10 μM) for 30 min (blue).