P800SO3-PEG: a renal clearable bone-targeted fluorophore for theranostic imaging

Abstract

Background: Due to the deep tissue penetration and reduced scattering, NIR-II fluorescence imaging is advantageous over conventional visible and NIR-I fluorescence imaging for the detection of bone growth, metabolism, metastasis, and other bone-related diseases.

Methods: Bone-targeted heptamethine cyanine fluorophores were synthesized by substituting the meso-carbon with a sulfur atom, resulting in a bathochromic shift and increased fluorescence intensity. The physicochemical, optical, and thermal stability of newly synthesized bone-targeted NIR fluorophores was performed in aqueous solvents. Calcium binding, bone-specific targeting, biodistribution, pharmacokinetics, and 2D and 3D NIR imaging were performed in animal models.

Results: The newly synthesized S-substituted heptamethine fluorophores demonstrated a high affinity for hydroxyapatite and calcium phosphate, which improved bone-specific targeting with signal-background ratios > 3.5. Particularly, P800SO3-PEG showed minimum nonspecific uptake, and most unbound molecules were excreted into the urinary bladder. Histological analyses demonstrated that P800SO3-PEG remained stable in the bone for over two weeks and was incorporated into bone matrices. Interestingly, the flexible thiol ethylene glycol linker on P800SO3-PEG induced a promising photothermal effect upon NIR laser irradiation, demonstrating potential theranostic imaging.

Conclusions: P800SO3-PEG shows a high affinity for bone tissues, deeper tissue imaging capabilities, minimum nonspecific uptake in the major organs, and photothermal effect upon laser irradiation, making it optimal for bone-targeted theranostic imaging.

Keywords: Bone targeting; NIR imaging; Renal clearance; Structure-inherent targeting; Targeted fluorophore.

Fig. 1

Chemical design and optophysical properties of bone-targeted theranostic imaging. a P800SO3-PEG is a bone-targeted heptamethine cyanine fluorophore for NIR-II tail imaging. b Design of P800SO3-PEG using the structure-inherent targeting strategy. c Synthetic routes for bone-targeted agents. d 3D energy minimized chemical structures of the designed fluorophores. e, f Absorbance (Abs) and fluorescence (Fl) spectra of bone-targeted agents in the NIR-1 (e) and NIR-II fluorescence tail imaging (f) recorded for P800SO3-Cl, P800SO3, P800SO3-SH, and P800SO3-PEG (λ_ex = 760 nm) in 10% fetal bovine serum (FBS) solution. g Plasma protein binding assay of bone-targeted NIR fluorophores compared with ICG incubated in 5% bovine serum albumin (BSA)-containing saline for 4 h. h Photothermal effect of bone-targeted NIR fluorophores in PBS (30 µM) under continuous 808 nm exposure for 2 min at a power density of 1.0 W/cm²

Fig. 2

Calcium salt binding assay of the designed bone-targeted agents under NIR-I and NIR-II filters. 760 nm excitation and 785 nm LP filters were used for NIR-I imaging, and 808 nm excitation and 1,070 nm LP filters for NIR-II imaging. a 5 µM of each fluorophore in 10% FBS was imaged. b Calcium-salt binding assay of the bone-targeted agents. SBRs were calculated by dividing the fluorescence intensity of each fluorophore by the signal intensity of each blank (which include only calcium salt and PBS). Data is expressed as mean ± S.D. (n = 3): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. HA, hydroxyapatite; CC, calcium carbonate; CP, calcium phosphate; CO, calcium oxalate; CPP, calcium pyrophosphate

Fig. 3

Biodistribution and bone-targeting of P800SO3-Cl, P800SO3-SH, and P800SO3-PEG in mice. 50 nmol (2.5 mg kg⁻¹) of each NIR fluorophore was injected intravenously to 20 g CD-1 mice 4 h prior to imaging. Whole body bone imaging was conducted (a) as well as knee joint imaging (b) under color (top), NIR-I (middle), and NIR-II (bottom) channels. Scale bars = 5 mm (a) and 2 mm (b). c The SBR of each knee bone over the neighboring muscle tissue was calculated (n = 3, mean ± S.D): **p < 0.01, ***p < 0.001, ****p < 0.0001. d Caudal vertebrae were imaged under the NIR-I and NIR-II window 4 h post-injection of P800SO3-PEG. Scale bars = 3 mm. e FWHM analysis of the tail highlighted by the white dashed box in (d). Based on the cross-sectional intensity profiles in NIR-II, the length of the coccyx condyle spacings was 0.99–1.01 mm

Fig. 4

Stable incorporation of P800SO3-PEG into the bone matrix. a Blood curve and pharmacokinetic parameters obtained from CD-1 mice retro-orbitally injected with 25 nmol of P800SO3-PEG. b Longitudinal biodistribution of P800SO3-PEG fluorophores in nude mice. 50 nmol (2.5 mg kg⁻¹) of P800SO3-PEG was injected intravenously to 20 g athymic NCr nu/nu mice and imaged for 14 d post-injection. Scale bars = 1 cm. SBR was calculated as the fluorescence intensity of each spine divided by the signal intensity of the neighboring skin as a background (n = 3, mean ± S.D). c Long-term signal integrity of P800SO3-PEG. 50 nmol of P800SO3-PEG was injected intravenously to 20 g CD-1 mice 1 d or 14 d prior to imaging (n = 3, mean ± S.D). Scale bars = 4 mm. d H&E and NIR imaging of resected bone tissues taken from mice 1 d and 14 d post-injection (c). Scale bars = 50 µm

Fig. 5

3D fluorescence tomography imaging of P800SO3-PEG in nude mice under the InSyTe FLECT/CT imaging system. 100 nmol (2.5 mg kg⁻¹) of P800SO3-PEG was injected into athymic NCr nu/nu mice 4 d prior to imaging. a Whole-body, b coronal and sagittal, and c transverse sections of fluorescence and CT images. FLECT/CT images were taken with a 780 nm laser excitation and an 853 nm NP filter