Water-soluble chromenylium dyes for shortwave infrared imaging in mice

Abstract

In vivo imaging using shortwave infrared light (SWIR, 1000-2000 nm) benefits from deeper penetration and higher resolution compared to using visible and near-infrared wavelengths. However, the development of biocompatible SWIR contrast agents remains challenging. Despite recent advancements, small molecule SWIR fluorophores are often hindered by their significant hydrophobicity. We report a platform for generating a panel of soluble and functional dyes for SWIR imaging by late-stage functionalization of a versatile fluorophore intermediate, affording water-soluble dyes with bright SWIR fluorescence in serum. Specifically, a tetra-sulfonate derivative enables clear video-rate imaging of vasculature with only 0.05 nmol dye, and a tetra-ammonium dye shows strong cellular retention for tracking of tumor growth. Additionally, incorporation of phosphonate functionality enables imaging of bone in awake mice. This modular design provides insights for facile derivatization of existing SWIR fluorophores to introduce both solubility and bioactivity towards in vivo bioimaging.

Figure 1

Design of hydrophilic and versatile Chrom7 derivatives. (a) Water-soluble polymethine dyes used for SWIR imaging and their emission wavelengths. (b) Structure of ICG. (c) Structures of Flav7 and Chrom7. (d) Derivatization of PropChrom7 into a series of water-soluble, functional SWIR imaging agents.

Figure 2

Photophysical comparisons of water-soluble SWIR fluorophores. (a) Table of photophysical properties. See Figure S8a for error values. ^a Peak at the second maximum absorption (monomer absorption). ^b Not determined due to strong aggregation. (b) Brightness comparison by SWIR imaging of each water-soluble dye (2 μM) in FBS in capillaries under 975 nm illumination (100 mW/cm²), with 2 μM of ICG in FBS under 785 nm illumination (50 mW/cm²) as a benchmark (1100 nm long-pass filter, 4 ms/frame). (c) Quantification of (b). (d,e) Overlay of absorption (solid line) and emission (dotted line) spectra of 2 μM Sulfo-, Ammon- or ZwitChrom7 in methanol (d) or FBS (e). Zoomed-in emission spectra in FBS between 1400–1500 nm are shown as inset in (e).

Figure 3

Video-rate imaging of mouse vasculature with i.v. injected SulfoChrom7. (a) Schematics of i.v. injection of SulfoChrom7 for vasculature imaging under different filters. (b-d) Fluorescence images of mice recorded at 30 ms/frame (b) under 1100 nm LP filter with 0.05 nmol dye injected as a 50 μL solution (20 frames averaged, max brightness 646 excluding tail region), or (c) under 1400 nm LP filter with 5.0 nmol dye injected as a 100 μL solution (20 frames averaged, max brightness 289 excluding tail region); their raw brightness profiles along the highlighted line is shown in (d). See Video S1 and S2 for injection video. (e) Schematics of co-injection of SulfoChrom7 and ICG for their direct comparison under two-channel imaging. (f-h) Fluorescent images of a mouse at 10 ms/frame under 1300 nm LP filter (f) 3 min [84 frames averaged, max brightness 4302 (green) and 3960 (blue)] or (g) 32 min [87 frames averaged, max brightness 3692 (green) and 4027 (blue)] after i.v. injection of SulfoChrom7 (10 nmol, shown in green) and ICG (10 nmol, shown in blue); their raw brightness profiles along the highlighted line is shown in (h). Illumination was provided at 100 mW/cm² for 975 nm and 50 mW/cm² for 785 mm. Scale-bar: 2 cm.

Figure 4

Tracking of tumor growth using AmmonChrom7. (a) Schematics of s.c. injection of AmmonChrom7 stained cells for tracking of their in vivo growth. (b) Uptake and retention of AmmonChrom7 in different cell lines. (c) Comparison of the growth curves of stained A375 as determined by fluorescence imaging and by caliper measurement in three mice. (d) In vivo images of A375 tumors 34 days after xenograft on Mouse 1 (1 ms/frame, 31 frames averaged, max brightness 7957). (e, f) Ex vivo fluorescent images of A375 tumors and organs (e) and carcass (f) of Mouse 1 34 days after xenograft [0.8 ms/frame, 51 frames averaged, max brightness 10873 (e) and 2920 (f)]. See Figure S16e,f for corresponding bright field images. Lu: lung, Sp: spleen, St: stomach, In: intestine, He: heart, Ste: sternum, Li: liver, Ki: kidney. TuL: left, stained tumor. TuR: right, unstained tumor. Sc: subcutaneous tissue around TuL. (g) Maximum brightness of ex vivo fluorescent tumor, liver and subcutaneous tissue surrounding the tumor (mean ± s.d., Mouse 1: 34 days, Mouse 2: 15 days, Mouse 3: 23 days after xenograft). (h) Ex vivo fluorescent images of SK-OV-3 tumors and organs 39 days after xenograft and 4 h after i.v. injection of OTL-38 [1 ms/frame, 112 frames averaged, max brightness 9928 (green) and 2095 (blue)]. See Figure S17g for corresponding bright field image. Tu1: abdomen s.c. tumor. Tu2: flank s.c. tumor. (i) Maximum fluorescence intensity of ex vivo tumors and their surrounding hypodermis after tumor removal from the sacrificed mice (n = 3, mean ± s.d., 39 days after xenograft). Illumination was provided at 100 mW/cm² for 975 nm and 50 mW/cm² for 785 mm. Scale bar: 2 cm.

Figure 5

Imaging of bone using PhosphoChrom7. (a) Schematics of bone imaging by s.c. injection of PhosphoChrom7. (b) Absorption and emission spectra of 2 μM PhosphoChrom7 in FBS. (c) Fluorescent image of calcium hydroxyapatite suspension in bovine serum treated with PhosphoChrom7, and its subsequent washes with bovine serum (0.5 ms/frame, 101 frames averaged, max brightness 11338); quantification is shown below (mean ± s.d., n = 3). (d-f) Fluorescence images of a mouse 24 h after i.v. injection of 20 nmol PhosphoChrom7 on (d) ventral, (e) dorsal or (f) lateral view. M: mandible, Mx: maxilla, R: Rib, Ste: sternum, T: tibia, P: phalange, V: vertebrae, C: carpus, E: elbow, Kn: knee. (g) Representative single frame from Video S3 showing fluorescence image of awake mice 48 h after injection. (h) Fluorescence images of a mouse after removal of abdominal organs and most of the skin on dorsal view. Images were captured under 100 mW/cm² 975 nm illumination at 3 ms/frame (d-h); 81 frames (d-e), single frame (f-g) or 201 frames (h) averaged. Max brightness: 7096 (d), 4985 (e), 6379 (f), 6962 (g) and 12313 (h). Scale-bar: 2 cm.

Scheme 1

Synthesis of PropChrom7 and post-synthetic CuAACs to afford SulfoChrom7, AmmonChrom7, ZwitChrom7, as well as the control dye (8’) without o-methyl substitution.