Rhodamine-Based Cyclic Hydroxamate as Fluorescent pH Probe for Imaging of Lysosomes

Abstract

Monitoring the microenvironment within specific cellular regions is crucial for a comprehensive understanding of life events. Fluorescent probes working in different ranges of pH regions have been developed for the local imaging of different pH environments. Especially, rhodamine-based fluorescent pH probes have been of great interest due to their ON/OFF fluorescence depending on the spirolactam ring's opening/closure. By introducing the N-alkyl-hydroxamic acid instead of the alkyl amines in the spirolactam of rhodamine, we were able to tune the pH range where the ring opening and closing of the spirolactam occurs. This six-membered cyclic hydroxamate spirolactam ring of rhodamine B proved to be highly fluorescent in acidic pH environments. In addition, we could monitor pH changes of lysosomes in live cells and zebrafish.

Keywords: acidic pH; fluorescent imaging; fluorescent probe; hydroxamate; rhodamine B.

Scheme 1

The proposed H⁺-sensing mechanism of rhodamine cyclic hydroxamate probe.

Scheme 2

Synthesis of probe 1.

Figure 1

(a) Fluorescence intensity changes of probe 1 (2 μM) in aqueous solution (DMSO 1% v/v) of varying pH values (25 °C, Ex. 520 nm; Em. 585 nm). (b) Plot of fluorescence intensities at 585 nm depending on the different pH values. The aqueous solution (DMSO 1% v/v) of varying pH values were prepared using sodium chloride solution and sodium hydroxide solution. A solution of 1 (2.0 mL) was placed in a quartz cell (10.0 mm width) and the fluorescence spectrum was recorded. Fluorescence intensity changes (at 585 nm) were recorded after 30 min at 25 °C every time.

Figure 2

Color changes of probe 1 (20 μM) in aqueous solution (DMSO 1% v/v) of varying pH values: 1, pH = 1.0; 2, pH = 1.2; 3, pH = 1.4; 4, pH = 1.6; 5, pH = 1.8; 6, pH = 2.0; 7, pH = 2.2; 8, pH = 2.4; 9, pH = 2.6; 10, pH = 2.8; 11, pH = 3.0; 12, pH = 3.2; 13, pH = 3.4; 14, pH = 3.6; 15, pH = 3.8; 16, pH = 4.0; 17, pH = 4.2; 18, pH = 4.4; 19, pH = 4.6; 20, pH = 4.8; 21, pH = 5.0; 22, pH = 5.2; 23, pH = 5.4; 24, pH = 5.6; 25, pH = 5.8; 26, pH = 6.0; 27, pH = 7.0; 28, pH = 8.0; 29, pH = 9.0; 30, pH = 10.0; 31, pH = 11.0; and 32, pH = 12.0.

Figure 3

(a) Fluorescence spectra of 1 (2 μM) were recorded (aqueous DMSO 1% v/v) in the presence of metal ions (blank, Fe³⁺, Fe²⁺, Zn²⁺, Ca²⁺, Mn², Mg²⁺, Cu²⁺, Na⁺, K⁺, Ba²⁺). (b) Plot of fluorescence intensities at 585 nm at pH = 4.4 and pH = 7.0 (aqueous DMSO 1% v/v) in the presence of metal ions (0, blank; 1, Fe³⁺; 2, Fe²⁺; 3, Zn²⁺; 4, Ca²⁺; 5, Mn²⁺; 6, Mg²⁺; 7, Cu²⁺; 8, Na⁺; 9, K⁺; 10, Ba²⁺). The aqueous solutions (DMSO 1% v/v) of different pH values were prepared using 0.1 M citric acid, 0.2 M sodium phosphate dibasic anhydrous, and 0.2 M sodium phosphate monobasic monohydrate. A solution of 1 (2.0 mL) was placed in a quartz cell (10.0 mm width) and the fluorescence spectrum was recorded. Fluorescence intensity changes (at 585 nm) were recorded after 30 min at 25 °C every time.

Figure 4

Cytotoxicity of probe 1 for living PC3 and A549 Cells. Cells were treated with the indicated concentration of probe 1. Cell viability was measured using the EZ-Cytox colorimetric kit in PC3 (a) and A549 (b) cells (mean ± SD, n = 3).

Figure 5

Real-time monitoring of changes in intracellular fluorescence. (a) PC-3 cells and (b) A549 cells were treated with various concentrations (0.5 (◆), 1 (▲), 2 (●) μM) of probe 1. The fluorescence intensity was measured using the IncuCyte™ live content imaging system (Essen BioScience, Hertfordshire, UK). (b) PC3 and A549 cells were treated with various concentrations of probe 1 for a duration of 1 h. Subsequent to the 1 h incubation period, the intracellular fluorescence levels were measured using a flow cytometer. (c) Cells were treated with 1 μM of probe 1 for different time periods. After incubation for the indicated time, the intracellular fluorescence levels were measured using a flow cytometer.

Figure 6

Fluorescence microscope images of living PC3 and A549 cells. (a) PC3 cells were co-strained with 1 μM of probe 1 and Lysotracker green or Mitotracker green for 1 h at 37 °C (Scale bar = 10 μm). (Pearson coefficient between probe 1 and lysotracker = 0.884; Pearson coefficient between probe 1 and mitotracker = 0.0647). (b) A549 Cells were co-strained with 1 μM of probe 1 and Lysotracker green or Mitotracker green for 1 h at 37 °C (Scale bar = 10 μm). (Pearson coefficient between probe 1 and lysotracker = 0.874; Pearson coefficient between probe 1 and mitotracker = −0.218). Graph shows correlation between probe 1 and FITC fluorescence in left images. Hoechst 33342, λ_ex = 405 nm, λ_em = 450 nm; Lysotracker green, λ_ex = 443 nm, λ_em = 505 nm; Mitotracker, λ_ex = 490 nm, λ_em = 516 nm; Probe 1, λ_ex = 520 nm, λ_em = 585 nm.

Figure 7

Microscopic and fluorescent images of zebrafish. Images of 5 dpf zebrafish were treated with 1 μM probe 1 for 1 h. The images were obtained using fluorescence microscope. (Top, phase contrast image; bottom, fluorescence image). Red fluorescence: λ_ex = 520 nm, λ_em = 585 nm. Scale bars = 500 μm.