ESIPT-Based Photoactivatable Fluorescent Probe for Ratiometric Spatiotemporal Bioimaging

2.1. Reagents and Apparatus

All chemicals were obtained from commercial suppliers and without any further purification. Water used in all experiments was doubly distilled. Liquid chromatography-mass spectrometry (LC-MS) analyses were performed using the Agilent 6120 Quadrupole LC/MS from Agilent Technologies. The pH was measured using a pH meter (pH_S-3C, Shanghai Leici). ¹H-NMR and ¹³C-NMR spectra were recorded on a Bruker DRX-500 spectrometer operating at 500 MHz. All chemical shifts are reported in the standard notation of parts per million. All fluorescence measurements were carried out on a Hitachi-F4600 fluorescence spectrometer with both excitation and emission bandwidths set at 5.0 nm. Fluorescence images of MDA-MB-231 cells were obtained using a Nikon Te-2500 microscope (Japan).

2.2. Spectroscopic Materials and Methods

Stock solution of probe PHBT (1 mM) was prepared by dissolving probe PHBT in dimethyl sulfoxide (DMSO). The test solution of probe PHBT was prepared by placing 10 µL probe PHBT stock solution in 990 µL phosphate-buffered saline (PBS) solution (10 mM, pH = 7.4). Experiments to measure the fluorescence of PHBT were conducted in (10 mmol PBS, pH = 7.4) water/DMSO (99/1, v/v) solution. Unless otherwise stated, the excitation wavelength was 365 nm; the excitation slit width was 5 nm; and the emission slit width was 5 nm for all measurements.

2.3. Probe Synthesis

The Synthetic route to HBT and PHBT is depicted in Scheme 1. A detailed description of the synthesis can be found in the Supporting Information (Figure S1). The product was characterized by ¹H-NMR, ¹³C-NMR and ESI mass spectra (Figures S2–S4). Compound PHBT: ¹H-NMR (500 MHz, DMSO) δ = 8.471–8.447 (dd, 1H), 8.227–8.206 (d, 1H), 8.063 (m, 2H), 7.868–7.826 (m, 2H), 7.681 (t, 1H), 7.535 (t, 2H), 7.420–7.418 (m, 2H), 7.369–7.205 (t, 1H), 5.797 (s, 2H); ¹³C-NMR (500 MHz, DMSO) δ = 162.474, 156.037, 152.041, 148.042, 135.756, 134.754, 132.888, 132.189, 130.391, 130.072, 129.603, 126.789, 125.657, 125.484, 122.970, 122.224, 122.186, 121.914, 114.218, 68.108; MS (ESI-MS): m/z 100% Calcd. for C₂₀H₁₅N₂O₂S⁺ [(M + H)¹⁺] 363.1; found 363.1.

2.4. Cytotoxicity Test

Cell viability was evaluated by the reduction of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) to formazan crystals by mitochondrial dehydrogenases (Mosmanm, 1983) (Figure S5). A sample of 1 × 10⁵ MDA-MB-231 cells in 50 μL of washing buffer was seeded into each test well on a 96-well plate. After an overnight culture, PHBT (0–10 μmol/L) in 50 μL of washing buffer was added to the respective test wells. After 2 h of treatment, 10 μL of MTT solution (5 mg/mL in phosphate-buffered solution) were added to each well. After 4 h of incubation at 37 °C, 100 μL of a solution containing 10% sodium dodecyl sulfate (SDS) and 0.01 mol HCl were added to dissolve the purple crystals. After 12 h of incubation, the optical density readings at 595 nm were recorded using a plate reader. Each of the experiments was performed at least three times.

2.5. Bovine Serum Test

The test solution of probe PHBT (10 μM) in bovine serum was prepared by placing 10 μL probe PHBT stock solution in 790 μL PBS buffer (10 mM, pH = 7.4) and 200 μL bovine serum. The resulting solution was incubated for 30 min at 37 °C and irradiated under UV light for 30 min before the recording of the spectrum.

2.6. Preparation and Staining of Cell Cultures

Immediately prior to the imaging experiments, the MDA-MB-231 cells were washed with PBS, incubated with 10 μM PHBT for 30 min at 37 °C, washed with PBS three times, irradiated by UV 30 min and then imaged. Fluorescence imaging was observed under a Nikon Te-2500 microscope. The laser excitation wavelength was 405 nm, and emissions were centered at 415–460 nm and 470–550 nm.

3.1. Design and Synthesis of PHBT

Inspired by the dual-emission optical properties of HBT, a photocaged PHBT was then designed and synthesized. PHBT contains a 2-nitrobenzyl group as a photocaged moiety, as shown in Scheme 1. Upon irradiation with UV light, the 2-nitrobenzyl group of PHBT was cleaved from the probe molecule to release an ESIPT HBT, affording a ratiometric fluorescence signal for imaging in vivo.

3.2. Photoactivatable Response Property of PHBT

As shown in Figure 1, the dual emission bands of PHBT were observed under UV light irradiation. The enol tautomer gave emission at 465 nm, and the keto tautomer gave emission at 512 nm. After 30 min, a clear green color could be observed by the naked eyes under a UV lamp (Figure 1). Measurement of the intensity showed a 73-fold enhancement at 512 nm, indicating that PHBT had a good photoactivatable response within 30 min. The quantum yield for the caged probe PHBT was calculated to be 0.0019, and the quantum yield for the uncaged PHBT was 0.17, corresponding to an 89-fold enhancement after photoconversion. Both the photolysis of PHBT and the liberation of HBT were confirmed by the HPLC and UV absorption spectra (Figures S6 and S7).

3.3. Bovine Serum Test

To test the feasibility of the practical application of the probe PHBT, we further conducted the UV light detection in 20% bovine serum (Figure 2). With irradiation under UV light, the solution of PHBT in bovine serum showed dual emission, as well. However, the fluorescence emission was stronger at 452 nm than that at 508 nm, which is because the forms of the enol tautomer and the keto tautomer are sensitive to the solvent [38,39,40,41,42]. Moreover the interaction between HBT and serum protein can cause the enol form to be much more pronounced [43]. Measurement of the intensity showed a 69-fold enhancement at 452 nm. As shown in Figure 2b, an excellent linear relationship was obtained both at 452 nm and 508 nm.

The fluorescence intensity increased linearly with the time of UV irradiation from 0 min to 30 min, which confirmed that the probe PHBT was applicable for practical phototriggered spatiotemporal imaging in real samples with satisfactory results.

3.4. Cell Viability and Imaging in Living MDA-MB-231 Cells

Previous results demonstrated that PHBT had good chemical and spectral performances. The cytotoxicity of PHBT, HBT and UV irradiation at 365 nm (0–30 min) was then evaluated using an MTT assay (Figure S5). Experimental results showed that negligible cytotoxicity was observed when up to 10 μM PHBT or HBT were added to the culture medium. Moreover, the cytotoxicity of UV irradiation of less than 10 min is also negligible; when the UV irradiation time was more than 10 min, an increased cytotoxicity was observed. The effect of some relevant ions and neutral molecules on the response of the PHBT probe was also investigated. The results are shown in Figure S8; 100 μM of common biologically-relevant species, such as K⁺, Na⁺, Ca²⁺, ClO⁻, H₂O₂, vitamin C and cysteine, exhibited a negligible effect on the response of PHBT. In addition, the effect of pH on the fluorescence response of PHBT was also studied, with results added in Figure S9. It is obvious that the probe could achieve the best performance at neutral pH, which is favorable for its applications. MDA-MB-231 cells were incubated with PHBT (10 μM), irradiated by UV 30 min and then imaged with a 405 nm laser line. As shown in Figure 3, the irradiated cell grew bright after irradiation.