Bisphosphonate-functionalized gold nanoparticles for contrast-enhanced X-ray detection of breast microcalcifications

Abstract

Microcalcifications are one of the most common abnormalities detected by mammography for the diagnosis of breast cancer. However, the detection of microcalcifications and correct diagnosis of breast cancer are limited by the sensitivity and specificity of mammography. Therefore, the objective of this study was to investigate the potential of bisphosphonate-functionalized gold nanoparticles (BP-Au NPs) for contrast-enhanced radiographic detection of breast microcalcifications using two models of breast microcalcifications, which allowed for precise control over levels of hydroxyapatite (HA) mineral within a low attenuating matrix. First, an in vitro imaging phantom was prepared with varying concentrations of HA uniformly dispersed in an agarose hydrogel. The X-ray attenuation of HA-agarose compositions labeled by BP-Au NPs was increased by up to 26 HU compared to unlabeled compositions for HA concentrations ranging from 1 to 10 mg/mL. Second, an ex vivo tissue model was developed to more closely mimic the heterogeneity of breast tissue by injecting varying concentrations of HA in a Matrigel carrier into murine mammary glands. The X-ray attenuation of HA-Matrigel compositions labeled by BP-Au NPs was increased by up to 289 HU compared to unlabeled compositions for HA concentrations ranging from 0.5 to 25 mg/mL, which included an HA concentration (0.5 mg/mL) that was otherwise undetectable by micro-computed tomography. Cumulatively, both models demonstrated the ability of BP-Au NPs to enhance contrast for radiographic detection of microcalcifications, including at a clinically-relevant imaging resolution. Therefore, BP-Au NPs may have potential to improve clinical detection of breast microcalcifications by mammography.

Keywords: Breast cancer; Computed tomography; Contrast agent; Gold nanoparticles; Mammary gland; Microcalcification.

Fig. 1

(a) Gold nanoparticles were surface functionalized with alendronate, a bisphosphonate with a primary amine for binding to gold surfaces opposite phosphonate groups for targeting calcium ions on HA crystal surfaces. (b) Representative TEM micrograph showing spherical and monodispersed BP-Au NPs. (c) The particle diameter distribution measured by TEM and the hydrodynamic diameter distribution measured by DLS. The mean (±standard deviation) physical particle diameter and hydrodynamic diameter were 12.8 (1.6) nm and 45.8 (1.5) nm, respectively. (d) UV-vis spectra of BP-Au NPs showing the surface plasmon resonance peak at ~527 nm.

Fig. 2

The expected HA concentration (mg/mL) within 10 μL HA-agarose pellets of the in vitro imaging phantom was validated against the measured HA concentration (mg/mL) from ICP-OES. Error bars show one standard deviation of the mean (n = 3/group) and error bars not shown lie within the data point. The dashed gray line shows a one-to-one equivalence. Differences between expected and measured HA concentrations were significantly different (p < 0.05, paired t-test), but the mean error was −8.3%, indicating reasonable accuracy. More importantly, linear least squares regression indicated that HA concentrations were highly reproducible (R² = 0.992).

Fig. 3

The X-ray attenuation (HU) measured by micro-CT for the in vitro imaging phantom comprising unlabeled and BP-Au NP labeled HA crystals at varying HA concentrations within an agarose hydrogel. Error bars show one standard deviation of the mean (n = 5/group). X-ray attenuation increased with increasing HA concentration for both labeled and unlabeled HA-agarose pellets (p < 0.001, ANOVA). All unlabeled HA concentrations exhibited significantly greater X-ray attenuation compared to the 0 mg/mL agarose control (*p < 0.005, t-test). BP-Au NPs enhanced the X-ray attenuation of HA concentrations ranging from 1–10 mg/mL compared to unlabeled HA at the same concentration (**p < 0.005, t-test).

Fig. 4

BP-Au NP binding to varying concentrations of HA crystals in HA-agarose pellets was characterized by measuring the concentration of unbound BP-Au NPs using ICP-OES. (a) The percent of BP-Au NPs bound to HA crystals showed complete binding of the BP-Au NP dose at HA concentrations ≥ 2.5 mg/mL and decreased percent binding with decreasing HA concentration (p < 0.0001, ANOVA). (b) The mass of BP-Au NPs bound per HA surface area (mg Au/m² HA) showed that HA crystals surfaces were saturated with BP-Au NPs at HA concentrations ≤ 2.5 mg/mL and the surface density decreased with increasing HA concentration (p < 0.0001, ANOVA). Error bars show one standard deviation of the mean (n = 3) and error bars not shown were negligibly small.

Fig. 5

Representative FE-SEM micrographs showing the surface density of BP-Au NPs (arrows) on HA crystal surfaces. The surface density appeared to increase with decreasing HA concentration and became saturated at HA concentrations ≤ 2.5 mg/mL, in agreement with quantitative measurements of the mass of BP-Au NPs bound per HA surface area (Fig. 4b).

Fig. 6

The X-ray attenuation (HU) measured by micro-CT for the ex vivo tissue model comprising unlabeled and BP-Au NP labeled HA crystals at varying HA concentrations within HA-Matrigel compositions injected into murine MGs and imaged at (a) high (10 μm) and (b) low (100 μm) resolution. Error bars show one standard deviation of the mean (n = 5). X-ray attenuation increased with increasing HA concentration for both labeled and unlabeled HA-Matrigel compositions imaged at both high and low resolution (p < 0.0001, ANOVA). (a) Using high resolution imaging (10 μm), BP-Au NPs enhanced the X-ray attenuation of HA-Matrigel compositions at all HA concentrations (**p < 0.05 labeled vs. unlabeled, t-test), including at levels that were otherwise undetectable by micro-CT (*p < 0.005 vs. control 0 mg/mL, t-test). (b) Using low imaging resolution, BP-Au NPs enhanced the X-ray attenuation of HA-Matrigel compositions at HA concentrations ≥ 5.0 mg/mL compared to unlabeled HA at the same concentration (**p < 0.05 labeled vs. unlabeled, t-test). All differences in X-ray attenuation between the low (10 mg/L) and high (74 mg/L) BP-Au NP dose at the same HA concentration were not statistically significant (p > 0.23, t-test).

Fig. 7

Representative grayscale micro-CT images taken at high (10 μm) and (b) low (100 μm) resolution, showing unlabeled and BP-Au NP labeled (arrows) HA-Matrigel compositions within excised murine MGs. Enhanced contrast was visually apparent for BP-Au NP labeled HA compared to unlabeled HA at HA concentrations of 0.5 mg/mL and 5 mg/mL at high resolution and 5 mg/mL HA at low resolution. The lymph node (LN) was used as an anatomical landmark for injections.

Fig. 8

BP-Au NP binding to varying concentrations of HA crystals in HA-Matrigel compositions was characterized by measuring the concentration of unbound BP-Au NPs using ICP-OES. (a) Complete binding occurred at HA concentrations ≥ 5.0 mg/mL for either BP-Au NP dose, but the percent binding was significantly lower (*p < 0.001, t-test) for the higher BP-Au NP dose when binding was incomplete at a lower HA concentration (0.5 mg/mL). (b) The mass of BP-Au NPs bound per HA surface area (mg Au/m² HA) showed that the surface density of BP-Au NPs was significantly greater for the higher BP-Au NP at each HA concentration (*p < 0.0001, t-test) and decreased with increasing HA concentration for either BP-Au NP dose (p < 0.0001, ANOVA). Error bars show one standard deviation of the mean (n = 3) and error bars not shown were negligibly small.