Azaphosphinate Dyes: A Low Molecular Weight Near-Infrared Scaffold for Development of Photoacoustic or Fluorescence Imaging Probes

Abstract

Near-infrared (NIR) dyes are desirable for biological imaging applications including photoacoustic (PA) and fluorescence imaging. Nonetheless, current NIR dyes are often plagued by relatively large molecular weights, poor water solubility, and limited photostability. Herein, we provide the first examples of azaphosphinate dyes which display desirable properties such as low molecular weight, absorption/emission above 750 nm, and remarkable water solubility. In PA imaging, an azaphosphinate dye exhibited a 4.1-fold enhancement in intensity compared to commonly used standards, the ability to multiplex with existing dyes in whole blood, imaging depths of 2.75 cm in a tissue model, and contrast in mice. An improved derivative for fluorescence imaging displayed a >10-fold reduction in photobleaching in water compared to the FDA-approved indocyanine green dye and could be visualized in mice. This new dye class provides a robust scaffold for the development of photoacoustic or NIR fluorescence imaging agents.

Keywords: Fluorescence; Fluorescent probes; Imaging agents; Near-Infrared dyes; Photoacoustic probes.

Figure 1.

a) A previously reported structure of MB dimers in aqueous solution. b) The core azaphosphinate dye from this work. Phosphinate substitution inhibits dye aggregation.

Figure 2.

a) Normalized absorption and emission spectra of NR₇₅₁ in PBS. b) Photophysical properties of NR₇₅₁ in PBS.

Figure 3.

a) The relationship between concentration and absorbance for MB and NR₇₅₁ in PBS. b) Normalized absorbance spectra for increasing concentrations of NR₇₅₁ and MB in PBS.

Figure 4.

Normalized absorbance (a) and emission (b) spectra of NR₇₅₁ in the indicated solvents. c) The correlation between either NR₇₅₁ absorbance (red) or emission (blue) maxima in wavenumber versus E_T(30) for each solvent. d) Fluorescence brightness of NR₇₅₁ in the indicated solvent. e) Photophysical properties of NR₇₅₁ in the indicated solvent.

Figure 5.

a) PA images of PBS solutions containing 50 μM NR₇₅₁ (left) or no dye (right) in tissue-mimicking phantoms consisting of 5% agarose and 2.5% milk. b) Overlay of normalized PA and absorbance spectra of NR₇₅₁. c) PA spectra from the indicated dyes (50 μM each) in tissue-mimicking phantoms.

Figure 6.

a) Spectral unmixing of 100 μM NR₇₅₁ (green) or MB (magenta) PA signal in defibrinated sheep’s blood using a two-channel tissue mimicking phantom consisting of 5% agarose and 2.5% milk. b) Mean pixel intensity of NR₇₅₁ and MB within the indicated region of interest (red circle) from panel a.

Figure 7.

a) PA images of PBS solutions with or without 50 μM NR₇₅₁ in chicken breast cylinders of increasing thickness. b) Average PA signal intensity from 7 positions, scanned in triplicate within the indicated region of interest (red circle) from panel a. **** indicates p<0.0001 (two sample independent t-test). c) PA signal-to-noise ratios (SNR) at the indicated imaging depths.

Figure 8.

Representative cross-sectional (a) or maximal intensity projections of cross-sectional images from the mouse chest (top) to abdomen (bottom, b) PA images of nude mice following subcutaneous injection of 100 μL saline without (left) or with 1 mg/kg NR₇₅₁ (right). c) Average PA signal intensity for NR₇₅₁ after spectral unmixing within the indicated region of interest (red circle) from panel a (n = 3). *** indicates p<0.001 (two-tailed independent t-test).

Figure 9.

a) Normalized absorbance and emission spectra of NR₇₅₁-Az in PBS. b) Photophysical properties of NR₇₅₁-Az in PBS.

Figure 10.

Confocal microscopy images of HeLa cells incubated with 20 μM NR₇₅₁-Az (a) or NR₇₅₁-Az-CTAB (b). c) Confocal microscopy images of the indicated organelle tracker and 50 μM NR₇₅₁-Az-CTAB. Merged images show predominant localization to the mitochondria. Scale bars = 20 μm.

Figure 11.

a) A representative fluorescence image of a nude mice with intramuscular injection sites for saline (left hindlimb) or 50 μM NR₇₅₁-Az in saline containing 0.5% DMSO (right hindlimb) indicated. b) Average fluorescence intensity within the indicated region of interest (red circle) from panel a (n = 3 mice in each group). Mice were imaged immediately following injection. *** indicates p<0.001 (two-tailed independent t-test).

Scheme 1.

Synthesis of NR₇₅₁.

Scheme 2.

Synthesis of NR₇₅₁-AZ.