A 2,7-dichlorofluorescein derivative to monitor microcalcifications

Abstract

Herein, we report the crystal structure of 2,7-dichlorofluorescein methyl ester (DCF-ME) and its fluorescence response to hydroxyapatite binding. The reported fluorophore is very selective for staining the bone matrix and provides turn-on fluorescence upon hydroxyapatite binding. The reported fluorophore can readily pass the cell membrane of the C2C12 cell line, and it is non-toxic for the cell line. The reported fluorophore DCF-ME may find applications in monitoring bone remodeling and microcalcification as an early diagnosis tool for breast cancer and age-related macular degeneration.

Fig. 1

Crystal structure of DCF-ME in a unit cell (A), potential Ca-HAP coordination modes (B) and one-dimensional hydrogen bonding pattern (C).

Fig. 2

Fluorescence characteristics of 12 μM DCF-ME in the absence and presence of various calcium phosphate minerals. A) The absorbance spectrum of DCF-ME in TBS. B) Normalized fluorescence intensity of DCF-ME, excited at λ_ex 460 nm, when diluted in different buffers, the presence and absence of CerHAP. C) Normalized fluorescence intensity of the mean peak emission of DCF-ME in TBS when incubated with various calcium phosphate minerals, excited at λ_ex 460 nm. D) Normalized fluorescence intensity of the mean corrected peak excitation of DCF-ME in TBS when incubated with various calcium phosphate minerals, using λ_em 570 nm. Data in A, C and D represent mean values, whilst C and D are ± S.D. (n = 3); for these experiments emission was measured with excitation at shorter wavelength (460 nm) than peak absorbance, and excitation with emission at longer wavelength (570 nm) than peak emission to minimize the effects of Rayleigh scattering.

Fig. 3

Cancellous mouse ribs incubated with 1.2 mM (A), 120 μM (B) and 12 μM (C) DCF-ME (λ_ex/em 480/527 nm; exposure time – 135 ms), diluted in TBS and counterstained with DAPI (λ_ex/em 360/470 nm; exposure time – 50 ms). Scale bar = 100 μm.

Fig. 4

Cancellous mouse ribs were incubated with 120 μM DCF-ME (λ_ex/em 480/527 nm; exposure time – 135 ms), diluted in TBS, and counterstained with DAPI (λ_ex/em 360/470 nm; exposure time – 50 ms). Incubation times of DCF-ME varied between 120, 60, 30, 20 and 10 min (A–E, respectively). Negative control of mouse ribs incubated with TBS alone is also included (F). Scale bar = 100 μm.

Fig. 5

Cell culture cytotoxicity in differentiated osteoblast cells, which were differentiated with BMP-2. A) ALP activity of C2C12 cells, with and without the addition of BMP-2 for 6 days. B) Cell viability following 24 h DCF-ME treatment was measured by a tetrazolium-based metabolic activity assay. Triton X-100 (0.5%) was used as a positive control, causing around 95.5% cytotoxicity (not shown). C) Fluorescence spectra of DCF-ME-exposed C2C12 cells in comparison to 10 μM DCF-ME dissolved in incubation buffer, using λ_ex 492 nm. D) Dose-dependency of cellular DCF-ME labeling (λ_ex/em 492/530 nm). Data represent mean values ± S.D. from n = 3 independent experiments.