An acidic pH independent piperazine-TPE AIEgen as a unique bioprobe for lysosome tracing

Abstract

Lysosomes are involved in a multitude of cellular processes and their dysfunction is associated with various diseases. They are the most acidic organelles (pH 3.8-6.6, size 0.1-1.2 μm) with the highest viscosity (47-190 cP at 25 °C) in the cell. Because of their acidity, pH dependent non-AIE active fluorescent lysosomal probes have been developed that rely on protonation inhibited photoinduced electron transfer (PET). In this work, an acidic pH independent lysosome targetable piperazine-TPE (PIP-TPE) AIEgen has been designed with unique photophysical properties making it a suitable probe for quantifying viscosity. In a non-aggregated state PIP-TPE shows deep-blue emission as opposed to its yellowish-green emission in the bulk. It possesses high specificity for lysosomes with negligible cytotoxicity and good tracing ability due to its better photostability compared to LysoTracker Red. In contrast to most known lysosome probes that rely solely on PET, restriction of intramolecular motion (RIM) due to the larger viscosity inside the lysosomes is the mechanism responsible for PIP-TPE's fluorescence. PIP-TPE's high selectivity is attributed to its unique molecular design that features piperazine fragments providing a perfect balance between lipophilicity and polarity.

Fig. 1. PIP–TPE’s fluorescence turns on blue due to the higher viscosity that restricts intramolecular motion (RIM) in lysosomes but red-shifts in the bulk (λ_ex = 360 nm).

Scheme 1. Synthetic route to PIP–TPE.

Fig. 2. ORTEP drawing of PIP–TPE (CCDC: 1555412). All thermal ellipsoids are shown at 50% thermal probability. All H atoms are omitted for clarity.

Fig. 3. (A) Normalized UV absorption of PIP–TPE in different solvents (0.8 × 10^–5 M) and as a thin film. (B) Normalized PL spectra of PIP–TPE in different solvents (0.8 × 10^–5 M) and as a crystalline powder (λ_ex = 360 nm). Photographs of PIP–TPE in different solvents, as an amorphous powder obtained from an evaporated DMSO solution on filter paper and as a crystalline powder taken under 365 nm UV irradiation using a hand-held UV lamp. Inserted table: the absolute photoluminescence quantum yields (PLQYs) of PIP–TPE in THF solution, as a thin film and as a crystalline powder (λ_ex = 303 nm).

Fig. 4. (A) Photoluminescence (PL) intensity and (B) absolute fluorescence quantum yields of PIP–TPE in pH aqueous buffer solutions (λ_ex = 360 nm), inset: photographs of PIP–TPE in pH buffer solutions taken under illumination of a UV lamp at 365 nm; (C) the absolute fluorescence quantum yields of PIP–TPE in different acidic buffer solutions at various viscosities (λ_ex = 360 nm), inset: photographs of PIP–TPE in pH 5.59 solution at a viscosity of 32.46 cP and in pH 5.35 solution at a viscosity of 7.94 cP taken under illumination of a UV lamp at 365 nm; (D) the normalized PL intensities of PIP–TPE in acidic buffer solutions of different viscosities (λ_ex = 360 nm), and protonated PIP–TPE in a thin film (λ_ex = 360 nm) and in THF (λ_ex = 300 nm). All of the data for solutions in 0.8 × 10^–5 M were collected at 25 °C.

Fig. 5. (A and B) Confocal images of HeLa cells co-stained with 1 μM PIP–TPE and 200 nM LysoTracker Red for 15 min, (C) merged image of (A) and (B) and (D) bright field; excitation wavelength: 405 nm for PIP–TPE; 561 nm for LysoTracker Red. Scale bar = 20 μm.

Fig. 6. (A–E) Confocal images of HeLa cells incubated with 1 μM PIP–TPE for 15 min and then stimulated using 3 μM chloroquine for (A) 0 min, (B) 1 min, (C) 2 min, (D) 3 min, and (E) 4 min. Merged images at two different points in time: (F) 0 and 1 min, (G) 1 and 2 min, (H) 2 and 3 min, (I) 3 and 4 min, and (J) 0 and 4 min, in the yellow squares: enlarged image for a specific lysosome with a red arrow indicating its direction of movement. Excitation wavelength: 405 nm. Scale bar = 20 μm.

Fig. 7. Overlays of (a) ground state (S₀) optimized structures of PIP–TPE and PIP–TPEH⁺_(soln) (yellow = PIP–TPE; green = PIP–TPEH⁺_(soln)) and (b) ground state (S₀) and first excited state (S₁) optimized structures of PIP–TPEH⁺_(soln) (green = S₀; red = S₁); (c) labeling scheme for PIP–TPE and related species.

Fig. 8. (a) HOMO and (b) LUMO for PIP–TPEH⁺_(soln) (iso value of 0.04); hydrogen atoms are omitted for clarity.