Fluorescent Arylphosphonic Acids: Synergic Interactions between Bone and the Fluorescent Core

Abstract

Herein, we report the third generation of fluorescent probes (arylphosphonic acids) to target calcifications, particularly hydroxyapatite (HAP). In this study, we use highly conjugated porphyrin-based arylphosphonic acids and their diesters, namely 5,10,15,20-tetrakis[m-(diethoxyphosphoryl)phenyl]porphyrin (m-H₈ TPPA-OEt₈ ) and 5,10,15,20-tetrakis [m-phenylphosphonic acid]porphyrin (m-H₈ TPPA), in comparison with their positional isomers 5,10,15,20-tetrakis[p-(diisopropoxyphosphoryl)phenyl]porphyrin (p-H₈ TPPA-iPr₈ ) and 5,10,15,20-tetrakis [p-phenylphosphonic acid]porphyrin (p-H₈ TPPA), which have phosphonic acid units bonded to sp² carbon atoms of the fluorescent core. The conjugation of the fluorescent core is thus extended to the (HAP) through sp² -bonded -PO₃ H₂ units, which generates increased fluorescence upon HAP binding. The resulting fluorescent probes are highly sensitive towards the HAP in rat bone sections. The designed probes are readily taken up by cells. Due to the lower reported toxicity of (p-H₈ TPPA), these probes could find applications in monitoring bone resorption or adsorption, or imaging vascular or soft tissue calcifications for breast cancer diagnosis etc.

Keywords: Alzheimer's disease; calcifications; fluorescent imaging; ligand synthesis; vascular calcifications.

Scheme 1

The fluorescent probes used in this study: A) 5,10,15,20‐Tetrakis[p‐(diisopropoxyphosphoryl)phenyl]porphyrin (p ‐H₈TPPA‐iPr₈). B) 5,10,15,20‐Tetrakis [p‐phenylphosphonic acid]porphyrin (p ‐H₈TPPA). C) 5,10,15,20‐Tetrakis[m‐(diethoxyphosphoryl)phenyl]porphyrin (m ‐H₈TPPA‐OEt₈). D) 5,10,15,20‐Tetrakis [m‐phenylphosphonic acid]porphyrin (m ‐H₈TPPA).

Figure 1

Mouse ribs incubated with either p ‐H₈TPPA (λ _ex/em 595/613 nm; exposure time −200 ms) or m ‐H₈TPPA (λ _ex/em 578/603 nm; exposure time −100 ms) diluted to 1 mg mL⁻¹ in HEPES (A–E or F–J, respectively) and counter‐stained with DAPI (λ _ex/em 359/461 nm; exposure time: −200 or 100 ms, respectively). Images were captured on the day of staining (A and F), as well as after 4 days (B and G), 7 days (C and H), and 14 days (D and I). A negative control of mouse ribs incubated with each buffer alone is also included (E and J, respectively). Scale bar=100 μm.

Figure 2

The absorbance (A) and fluorescence (B) spectra of 0.01 mg mL⁻¹ p ‐H₈TPPA in the presence (dashed lines) and absence (solid lines) of HAP. The dye was diluted to 0.01 mg mL⁻¹ in PBS (pH 7.4, green), TBS (pH 7.4, orange), HEPES (pH 7.4, purple) and d H₂O with absorbance and fluorescence spectra obtained using the Flexstation 3 microplate reader.

Figure 3

Fluorescence spectra of THP‐1 monocytes cells incubated with p ‐H₈TPPA.