Functionalized Gold Nanoparticles for Targeted Labeling of Damaged Bone Tissue in X-Ray Tomography
1
Ross, R D; +1Roeder, R K
+1University of Notre Dame, Notre Dame, IN
rroeder@nd.edu
INTRODUCTION:
Conventional methods used to image and quantify microdamage
accumulation in bone are limited to thin histological sections, which are
inherently invasive, destructive, tedious and two-dimensional [1].
Recent studies have begun to investigate methods for non-destructive,
three-dimensional (3-D) detection and imaging of microdamage in bone
tissue. The presence, spatial location and accumulation of fatigue
microdamage in cortical and trabecular bone specimens was nondestructively detected using micro-CT after staining with barium sulfate
[2-4]; however, specimens were stained in vitro via a precipitation
reaction which was non-specific to damage and not biocompatible.
Therefore, gold nanoparticles (Au NPs) were investigated as a potential
damage-specific X-ray contrast agent due to their relative
biocompatibility, ease of surface functionalization, colloidal stability,
and high X-ray attenuation.
METHODS:
Au NPs were prepared from HAuCl4·3H2O and trisodium citrate
dihydrate to a mean particle size of ~20 nm at [Au] ~0.5 mM using the
citrate reduction method [5]. As-prepared Au NPs were functionalized
by adding 1 mL of 0.01 M solution of either glutamic acid, 2-aminoethylphosphonic acid or alendronate, which exhibit a primary amine for
binding gold opposite carboxylate, phosphonate or bisphosphonate
groups, respectively, for targeting calcium (Fig. 1). Excess
functionalization molecules were removed by dialysis. Samples were
characterized by TEM, DLS, FT-IR, UV-vis, and zeta potential.
Binding affinity to a synthetic mineral analog was investigated by
incubating hydroxyapatite crystals in DI water or FBS solutions
containing functionalized Au NPs. The supernatant solution was
collected after centrifugation and the gold concentration was measured
using ICP-OES. Binding constants were derived using a half-reciprocal
linearization of Langmuir isotherms. Specificity for labeling damaged
bone tissue was demonstrated with bovine cortical bone specimens by
masking machining damage with calcein, scratching the surface with a
scalpel, and soaking in functionalized Au NP solutions. Scratched
specimens were characterized using optical microscopy and
backscattered SEM combined with EDS.
Fatigue microdamage labeled by functionalized Au NPs was imaged
by X-ray tomography at 2.5 µm resolution using a synchrotron light
source at multiple energy levels just above and below the LIII edge of
gold (11.918 keV). Specimens were sectioned from the femoral middiaphyses of a 58 year old female donor, turned down to a 1.5 mm
diameter by 2.5 mm gauge length using a CNC mini-lathe, and
prestained in calcein to mask machining damage. Specimens were
loaded in cyclic uniaxial tension to a 10% reduction in secant modulus.
RESULTS:
Functionalized Au NPs were stable in physiological solutions and
retained a mean particle size of ~20 nm in TEM (Fig. 2a), but the mean
hydrodynamic diameter measured by DLS increased approximately twofold, indicating the presence of functional groups. Binding constants for
functionalized Au NPs in water were 3.82, 0.72 and 0.25 mg/L for
alendronate, glutamic acid and phosphonic acid respectively,
corresponding to a maximum of 7.33, 1.22 and 0.48 mg Au NPs bound
per gram of hydroxyapatite. Thus, alendronate functionalized Au NPs
exhibited the greatest specificity, as expected, and consequently
provided the most visible labeling of damaged tissue (Fig. 2b,c). Fatigue
microcracks labeled with bisphosphonate functionalized Au NPs were
able to be detected by synchrotron X-ray tomography (Fig. 3).
DISCUSSION:
Functionalized Au NPs were prepared as a deliverable, damagespecific X-ray contrast agent. Bisphosphonate functionalized Au NPs
exhibited the strongest binding affinity, followed by glutamic acid and
phosphonic acid, which was in agreement with previous studies that
investigated the hydroxyapatite binding affinity of modified proteins [6].
The small size (~20 nm) and colloidal stability of functionalized Au NPs
enabled the contrast agent to readily diffuse into cortical bone tissue,
which suggests that the contrast agent is deliverable. Diffusion was
verified by epifluorescence after conjugating Au NPs with fluorescein.
However, due to the small size (~20 nm) and concentration required for
deliverability and biocompatibility, damaged tissue labeled with
functionalized Au NPs has not yet been detected using a commercially
available polychromatic micro-CT scanner.
Detection of damage tissue labeled with functionalized Au NPs has
thus far required a high resolution synchrotron light source to tune the
energy of monochromatic X-rays to the LIII edge of gold. Therefore, the
results of this study suggest that the ambitious goal of non-invasive (in
vivo) imaging of microdamage in bone could be feasible with significant
improvements in commercial scientific and clinical CT instruments.
Moreover, a biocompatible, deliverable, damage-specific contrast agent
with greater x-ray attenuation than bone could enable clinical assessment
of bone quality and damage accumulation, and scientific study of
damage processes in situ.
Fig. 1. Schematic diagram showing a Au NP surface functionalized with
alendronate (not to scale). Note that the primary amine binds to the Au
NP surface and is opposite bisphosphonate groups which bind to
exposed bone mineral surfaces in damaged tissue.
Fig. 2. (a) TEM micrograph of bisphosphonate functionalized Au NPs
with a mean particle size of ~20 nm. (b) Optical micrograph and (c)
backscattered SEM micrograph of a scratch on the surface of bovine
cortical bone, where the red color and brightness, respectively, indicates
targeted labeling by bisphosphonate functionalized Au NPs.
Fig. 3. Synchrotron x-ray tomography images of longitudinal crosssections from a human cortical bone specimen loaded in cyclic uniaxial
tension to a 10% reduction in secant modulus showing microcracks
labeled by bisphosphonate functionalized Au NPs (red). The image on
the left was taken at 12.018 keV, just above the LIII edge for gold, and
the image on the right was generated by overlaying in red the subtraction
of images taken above and below the LIII edge.
ACKNOWLEDGEMENTS:
This research was supported by the U.S. Army Medical Research and
Materiel Command (W81XWH-06-1-0196) through the Peer Reviewed
Medical Research Program (PR054672). Use of the Advanced Photon
Source at Argonne National Laboratory was supported by the U.S.
Department of Energy (DE-AC02-06CH11357).
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Biomed. Mater., 1:68-75, 2008. [4] Landrigan MD, et al., Trans. Orthop.
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Poster No. 1368 • 56th Annual Meeting of the Orthopaedic Research Society