Phosphonated near-infrared fluorophores for biomedical imaging of bone

Abstract

The conventional method for creating targeted contrast agents is to conjugate separate targeting and fluorophore domains. A new strategy is based on the incorporation of targeting moieties into the non-delocalized structure of pentamethine and heptamethine indocyanines. Using the known affinity of phosphonates for bone minerals in a model system, two families of bifunctional molecules that target bone without requiring a traditional bisphosphonate are synthesized. With peak fluorescence emissions at approximately 700 or 800 nm, these molecules can be used for fluorescence-assisted resection and exploration (FLARE) dual-channel imaging. Longitudinal FLARE studies in mice demonstrate that phosphonated near-infrared fluorophores remain stable in bone for over five weeks, and histological analysis confirms their incorporation into the bone matrix. Taken together, a new strategy for creating ultra-compact, targeted near-infrared fluorophores for various bioimaging applications is described.

Keywords: fluorophores; imaging agents; medicinal chemistry; near-infrared fluorescence.

Figure 1

Synthetic scheme (a) and optical and physicochemical properties (b) of P700 and P800 NIR fluorophores.

Figure 2

Calcium salt binding properties of (a) P700 and (b) P800 NIR fluorophores. SBR was calculated by the fluorescence intensity of each fluorophore sample versus the signal intensity of each control sample. All NIR fluorescence images have identical exposure and normalizations. Abbreviations used are: HA, hydroxyapatite; CC, calcium carbonate; CP, calcium phosphate; CO, calcium oxalate, and CPP, calcium pyrophosphate. Images are representative of n = 3 independent experiments.

Figure 3

In vivo biodistribution and bone tissue imaging using P700 (a) and P800 (b) NIR phosphonates in mice. Each NIR fluorophore was intravenously injected into 20 g CD-1 or NCRNU nude mice (10 nmol; 0.4 mg/kg) 4 h and 24 h prior to imaging. Abbreviations used are: BD, bile duct; Bl, bladder; Du, duodenum; He, Heart; In, intestine; JB, jaw bone; Ki, kidneys; Li, liver; Lu, lungs; Mu, muscle; Pa, pancreas; SJ, shoulder joint; Sp, spleen; St, stomach, and Ur, ureter. Scale bars = 1 cm. Images are representative of n = 3 independent experiments.

Figure 4

In vivo biodistribution and bone tissue imaging using P700SO3 and P800SO3 NIR phosphonates in pigs. (a) 30 pmol/g of P700SO3 and P800SO3 were intravenously injected into 35 kg Yorkshire pigs 4 h prior to imaging. (b) Blood clearance (%ID/g) and blood half-life (mean ± 95% confidence intervals) of P700SO3 and P800SO3 in pigs. All NIR fluorescence images have identical exposure and normalizations. Scale bars = 1 cm. Images are representative of n = 3 independent experiments.

Figure 5

Stable incorporation of phosphonated NIR fluorophores into bone matrix. (a) P700SO3 and P800SO3 were injected intravenously into 20 g NCRNU nude mice (10 nmol; 0.4 mg/kg) 5 wk prior to imaging. Scale bars = 1 cm. (b) H&E and NIR imaging of resected bone tissues at day 1 and 5 wk post-injection of P700SO3 and P800SO3. Scale bars = 100 μm. All NIR fluorescence images for each condition have identical exposure times and normalizations.