Water-soluble, deep-red fluorescent squaraine rotaxanes

Abstract

Eight fluorescent squaraine rotaxanes with deep-red absorption/emission wavelengths were prepared and assessed for chemical stability and suitability as water-soluble, fluorescent tracers. The most stable squaraine rotaxanes have four large stopper groups attached to the ends of the encapsulated squaraine, and two members of this structural class have promise as highly fluorescent tracers with rapid renal clearance and very low tissue uptake in living mice.

Fig. 1

Change in squaraine rotaxane absorption (5 μM) at wavelength maxima in pure water (top) and 10% FBS in water (bottom), T=25° C. Samples exposed to laboratory lights throughout the experiment. Note that the fluorescence spectra of 7 and 8 were also unchanged over time indicating no longitudinal change in fluorescence quantum yield.

Fig. 2

Absorption and emission intensities at different concentrations in phosphate buffered saline (sodium phosphate 10 mM, NaCl 150 mM, pH 7.4): (a) 7 em (red open squares); (b) 8 em (red open circles); (c) 7 abs (blue filled squares); (d) 8 abs (blue filled circles). Linear fits of the two absorption plots have R² values >0.99; the curved lines for the fluorescence plots are simply to guide the eye.

Fig. 3

Fluorescence intensity maps (ex: 630±10 nm, em: 700±20 nm) and brightfield images (50 mm field of view) of the exposed abdominopelvic cavities of representative mice treated intravenously with 40 pmol/g of 7 (A,D), 8 (B,E), or ICG (C,F) and sacrificed at two hours post-injection of fluorophore. Abbreviations: bladder (Bl), intestines (In), liver (Li). Note that the physical area of the bladder signal in A is smaller than B but the pixel intensity is higher. The fluorescence intensity scale for each image is in arbitrary units.

Fig. 4

Comparative fluorescence intensity maps (ex: 630±10 nm, em: 700±20 nm) of excised tissue and organs of mice treated intravenously with 40 pmol/g of 7 (A) or 8 (B) and sacrificed at two hours post-injection of fluorophore. Abbreviations: liver (Lvr), kidney (Kdn), intenstines (Int), spleen (Spl), lung (Lng), heart (Hrt), muscle (Msc). Fluorescence intensity scale in arbitrary units.

Scheme 1

Structures of squaraine rotaxanes and the trend for aqueous stability.

Scheme 2

Molecular model of squaraine rotaxane 8 at physiological pH showing electrostatic surface with positive centers colored blue and negative centers red. The longest diagonal length of the extended molecule is ~ 4.6 nm.