Introduction: Microcalcifications are the most common tissue abnormality observed on a mammogram and are early markers for breast cancer [1]. However, the detection of microcalcifications and correct diagnosis of breast cancer are limited by the sensitivity and specificity of mammography, especially in women with radiographically dense breast tissue[2],[3]. Therefore, bisphosphonate-functionalized gold nanoparticles (BP-Au NPs) were developed as a targeted X-ray contrast agent for microcalcifications due to the high binding affinity of BP for hydroxyapatite (HA) mineral in microcalcifications [1] and were shown to enable contrast-enhanced detection of breast microcalcifications in both normal [4] and radiographically dense [5] mammary tissue in vivo. The aims of this study were to (1) insert a polyethylene glycol (PEG) spacer between the Au NP surface and BP ligands (Fig. 1a) to improve in vivo delivery and clearance, and (2) verify microcalcification targeting by BP ligands by comparing BP-PEG-Au NPs with PEG-Au NPs (Fig. 1b).
Methods: Au NPs were synthesized using the citrate reduction method [6] and surface functionalized with thiol-PEG via near-covalent thiol-gold bonding. BP-PEG-Au NPs (Fig. 1a) were prepared using a heterobifunctional PEG (HS-PEG-COOH) and carboiimide chemistry to link the carboxyl to the free amine in BP (alendronate). PEG-Au NPs (Fig. 1a) were prepared as a nontageted control using a monofunctional PEG. All Au NP preparations were characterized by TEM, DLS, UV-vis, and ICP-OES. The binding affinity for HA was measured by incubating known concentrations of functionalized Au NPs with HA crystals. Radiographic detection of breast microcalcifications was investigated with computed tomography (CT) prior to delivery (0 h) and longitudinally at 3, 6, 24, and 48 h after injecting BP-PEG-Au NPs or PEG-Au NPs (100 µL, 50 mM) into the mammary glands of mice bearing microcalcifications (Fig. 1b) [3],[4].
Results: BP-PEG-Au NPs and PEG-Au NPs were both spherical and monodispersed, exhibiting a particle diameter of ~13 nm, a hydrodynamic diameter of ~30 nm, and colloidal stability for up to 7 d in water, PBS, or 10% fetal bovine serum. BP-PEG-Au NPs exhibited similar binding affinity to HA mineral compared with BP-Au NPs,6 while PEG-Au NPs exhibited negligible binding affinity to HA. Delivery of BP-PEG-Au NPs to microcalcifications in vivo resulted in increased X-ray contrast of microcalcifications compared with surrounding tissue (Fig. 2). Importantly, non-specifically bound BP-PEG-Au NPs were more readily cleared from the mammary tissue by 48 h after delivery compared with previous results for BP-Au NPs.
Figure 1: (a) Schematic diagrams showing (a) BP-PEG-Au NPs and PEG-Au NPs preparations, and (b) a preclinical model for creating and targeting microcalcifications within murine mammary glands for imaging.
Figure 2: X-ray attenuation (HU) measured in vivo by CT for microcalcifications compared with controls before and after delivery of BP-PEG-Au NPs. At all time points after delivery of BP-PEG-Au NPs, the X-ray attenuation of microcalcifications was increased compared with prior to delivery and controls.
Conclusions: BP-PEG-Au NPs exhibited similar binding affinity to HA in vitro and similar targeting and contrast-enhancement of microcalcifications in vivo compared with BP-Au NPs, but improved clearance after intramammary delivery. PEG-Au NPs exhibited negligible binding affinity to HA and targeting for microcalcifications confirming specific binding by BP ligands.
References:
[1] M.G. Morgan et al., J Mammary Gland Biol., 10:181-87, 2005
[2] H.D. Cheng et al., Pattern Recogn., 36:2967-91, 2003
[3] N.F. Boyd et al., Breast Cancer Res., 13:223, 2011
[4] L.E. Cole et al., ACS Nano, 8:7486-96, 2014
[5] L.E. Cole et al., ACS Nano, In press
[6] R.D. Ross and R.K. Roeder, J Biomed Mater Res., 99A:58-66, 2011