Establishment of a Trimodality Analytical Platform for Tracing, Imaging and Quantification of Gold Nanoparticles in Animals by Radiotracer Techniques

Abstract

This study aims to establish a (198)Au-radiotracer technique for in vivo tracing, rapid quantification, and ex vivo visualization of PEGylated gold nanoparticles (GNPs) in animals, organs and tissue dissections. The advantages of GNPs lie in its superior optical property, biocompatibility and versatile conjugation chemistry, which are promising to develop diagnostic probes and drug delivery systems. (198)Au is used as a radiotracer because it simultaneously emits beta and gamma radiations with proper energy and half-life; therefore, (198)Au can be used for bioanalytical purposes. The (198)Au-tagged radioactive gold nanoparticles ((198)Au-GNPs) were prepared simply by irradiating the GNPs in a nuclear reactor through the (197)Au(n,γ)(198)Au reaction and subsequently the (198)Au-GNPs were subjected to surface modification with polyethylene glycol to form PEGylated (198)Au-GNPs. The (198)Au-GNPs retained physicochemical properties that were the same as those of GNP before neutron irradiation. Pharmacokinetic and biodisposition studies were performed by intravenously injecting three types of (198)Au-GNPs with or without PEGylation into mice; the γ radiation in blood specimens and dissected organs was then measured. The (198)Au-radiotracer technique enables rapid quantification freed from tedious sample preparation and shows more than 95% recovery of injected GNPs. Clinical gamma scintigraphy was proved feasible to explore spatial- and temporal-resolved biodisposition of (198)Au-GNPs in living animals. Moreover, autoradiography, which recorded beta particles from (198)Au, enabled visualizing the heterogeneous biodisposition of (198)Au-GNPs in different microenvironments and tissues. In this study, the (198)Au-radiotracer technique facilitated creating a trimodality analytical platform for tracing, quantifying and imaging GNPs in animals.

Full text

Establishment of a Trimodality Analytical Platform for Tracing,
Imaging and Quantification of Gold Nanoparticles in Animals by
Radiotracer Techniques
Chien-Hung Chen,
†
Fong-Sian Lin,
†
Wei-Neng Liao,
†
Sanching L. Liang,
†
Min-Hua Chen,
†
Yo-Wen Chen,
‡
Wan-Yu Lin,
‡
Ming-Hua Hsu,
§
Mei-Ya Wang,
§
Jinn-Jer Peir,
§
Fong-In Chou,
§
Ching-Ya Chen,
†
Sih-Yu Chen,
†
Su-Chin Huang,
†
Mo-Hsiung Yang,
∥
Dueng-Yuan Hueng,
⊥
Yeukuang Hwu,
#
Chung-Shi Yang,*
,†
and Jen-Kun Chen*
,†
†
Institute of Biomedical Engineering & Nanomedicine, National Health Research Institutes, Miaoli 35053, Taiwan
‡
Department of Nuclear Medicine, Taichung Veterans General Hospital, Taichung 40705, Taiwan
§
Nuclear Science & Technology Development Center and
∥
Department of Biomedical Engineering and Environmental Sciences,
National Tsing Hua University, Hsinchu 30013, Taiwan
⊥
Department of Neurological Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan
#
Institute of Physics, Academia Sinica, Taipei 11529, Taiwan
ABSTRACT: This study aims to establish a 198Au-radiotracer
technique for in vivo tracing, rapid quantification, and ex vivo
visualization of PEGylated gold nanoparticles (GNPs) in
animals, organs and tissue dissections. The advantages of
GNPs lie in its superior optical property, biocompatibility and
versatile conjugation chemistry, which are promising to develop
diagnostic probes and drug delivery systems. 198Au is used as a
radiotracer because it simultaneously emits beta and gamma
radiations with proper energy and half-life; therefore, 198Au can
be used for bioanalytical purposes. The 198Au-tagged radioactive
gold nanoparticles (198Au-GNPs) were prepared simply by
irradiating the GNPs in a nuclear reactor through the 197Au-
(n,γ)198Au reaction and subsequently the 198Au-GNPs were
subjected to surface modification with polyethylene glycol to form PEGylated 198Au-GNPs. The 198Au-GNPs retained
physicochemical properties that were the same as those of GNP before neutron irradiation. Pharmacokinetic and biodisposition
studies were performed by intravenously injecting three types of 198Au-GNPs with or without PEGylation into mice; the γ
radiation in blood specimens and dissected organs was then measured. The 198Au-radiotracer technique enables rapid
quantification freed from tedious sample preparation and shows more than 95% recovery of injected GNPs. Clinical gamma
scintigraphy was proved feasible to explore spatial- and temporal-resolved biodisposition of 198Au-GNPs in living animals.
Moreover, autoradiography, which recorded beta particles from 198Au, enabled visualizing the heterogeneous biodisposition of
198Au-GNPs in different microenvironments and tissues. In this study, the 198Au-radiotracer technique facilitated creating a
trimodality analytical platform for tracing, quantifying and imaging GNPs in animals.
Gold nanoparticles (GNPs) have been developed for uses
of sensing devices because of outstanding optical
1−4
and
electrical properties.
5,6
Colloidal gold based pregnancy test has
been developed and went on the market in 1980s for the
detection of human chorionic gonadotropin (hCG) in
urine.
7−10
In this decade, GNPs have been reported to detect
and quantify nucleic acid with high-sensitivity.
11−15
In addition
to in vitro diagnostic devices, conjugating anticancer drugs with
GNPs have demonstrated the enhanced permeability and
retention (EPR) effect, which give rise to accumulate
therapeutic molecules in tumor and lead to amelioration of
therapy. The CytImmune Sciences Inc. developed the first
GNP-based anticancer drug, CYT-6091, and completed phase I
clinical trial in 2010.
16
CYT-6091 comprises 30 nm-diameter
gold nanoparticle and immune-avoiding molecule (thiol-
derivatized polyethylene glycol) to deliver tumor necrosis
factor-alpha (TNF-α),
17−19
which can be used to treat patients
with advanced-stage tumors effectively. More recently, Wang et
al. and You et al. bound doxorubicin with GNPs to treat cancer
because GNPs can carry the doxorubicin into the tumor
region.
20−22
To develop diagnostic agents for in vivo
biomedical applications, Zheng and colleagues produced
Received: August 31, 2014
Accepted: November 26, 2014
Published: November 26, 2014
Article
pubs.acs.org/ac
© 2014 American Chemical Society 601 dx.doi.org/10.1021/ac503260f |Anal. Chem. 2015, 87, 601−608

ultrasmall (2.5 nm-diameter) gold nanoparticles conjugated
with glutathione (GS) and/or polyethylene glycol (PEG).
23−25
The ultrasmall GS-GNPs presented superior features in rapid
diffusion, relatively long circulation time and predominantly
renal clearance. The distribution half-life (T1/2α)and
elimination half-life (T1/2β) of GS-GNPs are 5.0 min and 10.0
h, comparable to small-molecule diagnostic probes, suggesting
potential for clinical application.
23
As for PEG conjugated with
ultrasmall GNPs (PEG-GNPs), the nanoparticle showed
significant increase of area under curve (AUC) in pharmaco-
kinetic analysis, which may change disposition of GNPs in
tumor region.
24
The percentage of injection dose per gram
tissue weight (%ID/g) for the PEG-GNPs (8.3%ID/g) was
three times that of GS-GNPs (2.3%ID/g) in a tumor 12 h
postinjection.
24,25
Targeting delivery of long-circulating anticancer drugs should
be a promising strategy for cancer therapy. With the EPR effect,
drugs encapsulated or conjugated with nanoparticles have been
proved effectively delivered and accumulated in solid
tumor.
17−19,26
More recently, Kataoka and colleagues indicated
delivering nanomedicine through EPR effect should depend on
not only particle size but also tumor permeability.
27
When used
to treat hypervascular and hyperpermeable colon adenocarci-
noma, drug-loaded nanoparticles 30, 50, 70, and 100 nm in
diameter exhibited neither size-dependent penetration nor
differences in tumor suppression. Regarding pancreatic
adenocarcinoma BxPC3, a type of hypovascular tumor that is
difficult to penetrate, nanomedicine 30 nm in diameter had the
highest penetration capability, which facilitated achieving the
optimal tumor suppression efficacy. Although GNPs have been
extensively applied for in vitro biomedical applications, the
particle size and surface modification of GNPs should be
carefully adjusted for in vivo use in compatible patients with
different tumor vasculature and permeability. To design the
optimal compatible drug formulations for different tumors,
evaluating how physicochemical properties of GNPs can alter
the biological responses and efficacy of the formulations is
essential. Therefore, bioanalytical approaches are necessary for
determining the pharmacokinetics and biodispositions of GNPs
in blood, tissues, and organs.
This study aims to establish and evaluate 198Au-radiotracer
technique by using PEGylated 198Au-GNPs to accomplish
trimodality analytical application of GNPs, including in vivo
imaging/tracing, rapid quantification, and ex vivo visualization
of GNPs in animals, organs, and tissue dissections. We tried to
produce 198Au-GNPs without the requirement of radioactive
precursors. Scheme 1 illustrates an approach used in this work,
in which gold nanoparticles were prepared by reducing auric
acid (H197AuCl4) based on the Turkevich method,
28−30
and
were subsequently subjected to neutron irradiation in a nuclear
reactor. A particular portion of the 197Au atoms in GNP could
capture thermal neutrons, and were converted into radioactive
198Au atoms. Two types of polyethylene glycols were
subsequently immobilized onto the surface of 198Au-GNPs for
exploring biological fates of GNP formulations.
The 198Au-GNP simultaneously emits γrays (412 keV) and
beta particles (Emax = 0.96 MeV) that have a half-life of 2.69
days.
31,32
γ-Rays facilitate imaging, tracing and rapidly
quantifying GNP-based drug carriers in biological systems.
Gamma scintigraphy of 198Au-GNPs for living animals was
performed using a clinical equipment of single photon emission
computed tomography (SPECT). γ-Rays can readily penetrate
soft tissues free from interferences; thus, determining 198Au-
GNPs in biological specimens is feasible, and sample
preparation does not require labor-intensive procedures. In
terms of beta radiations emitting from 198Au-GNPs, the kinetic
energy of beta particles can sensitize commercially available
imaging plate, enabling the presence of GNPs to be visualized
through autoradiography. Autoradiography of 198Au-GNPs can
also reveal the heterogeneous biodisposition of GNPs in the
microenvironments of various dissected tissues. Therefore, we
anticipated that, by using an 198Au-radiotracer technique as an
analytical platform, we could comprehensively evaluate of the
optimal GNPs formulation for the specific tumor model.
■EXPERIMENTAL SECTION
Preparation of GNPs and 198Au-GNPs. Auric acid
(H197AuCl4, Sigma-Aldrich) aqueous solution (1.0 mM, 500
mL) was heated to boil with vigorous stir and sodium citrate
solution (38.8 mM, 62.5 mL) was subsequently added for
reducing Au(III) to Au0. This solution was boiled with stirring
for another 10 min until its color turned into burgundy (λmax:
520 ±1 nm). Average size of gold nanoparticles was confirmed
by both of dynamic light scattering (DLS) spectroscopy and
transmission electron microscope (TEM). These GNPs were
encapsulated in polyethylene containers and brought to Tsing
Hua Open-pool Nuclear Reactor (THOR, National Tsing Hua
University, Hsinchu, Taiwan) for 15 min neutron irradiation.
The thermal neutron flux and fast neutron flux were 1.23 ×1012
and 2.93 ×1011 neutrons·cm−2·s−1, respectively. The non-
radioactive gold nanoparticles were transformed to 198Au-GNPs
through 197Au(n,γ)198Au reaction.
32−34
Characterization of 198Au-GNPs. The solutions of gold
nanoparticles before and after neutron activation were
dispensed into cuvettes (SARSTEDT, Germany) with a 2
mm light path. The UV−vis spectra were recorded using a
NanoDrop 2000c spectrophotometer (Thermo Fisher Scien-
tific, Wilmington, DE 19810, USA) within 300 to 840 nm.
Aliquot of gold nanoparticles (0.2 mL) after neutron activation
was filtered using an ultrafiltration device (Vivaspin 500, GE
Healthcare, Uppsala, Sweden) with 20 kDa molecular weight
cutoffmembrane at 5000 gfor 10 min. The concentrated
particulate fraction on ultrafiltration membrane was obtained
and subjected to a high-purity germanium (HPGe) detector
(GC1020, CANBERRA, CT, USA) to acquire gamma energy
spectrum of 198Au-GNPs. Characteristic peaks in energy
spectrum were compared with the decay scheme of 198Au.
31
Furthermore, the concentrated 198Au-GNP was resuspended
into double deionized water for determination of hydrodynamic
size. The 198Au-GNP suspension was dispensed into a cuvette
at 25 °C for 2 min of thermal equilibrium. Dynamic light
scattering measurements (Zetasizer Nano ZS, Malvern Instru-
ments, Worcestershire, UK) were performed using a He−Ne
laser (633 nm) at an angle of 173°to inspect the consistency of
hydrodynamic sizes before and after neutron activation. The
Scheme 1. Approach for Preparation of Radioactive Gold
Nanoparticles
Analytical Chemistry Article
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above-mentioned 198Au-GNPs were further dripped and dried
on copper grids for TEM measurement (H-7650, Hitachi,
Tokyo, Japan). All of the copper grids were preserved in a dry
cabinet before analysis. Primary particle size of GNPs and
198Au-GNPs were counted using SigmaScan Pro 5 software
(Systat Software Inc., San Jose, CA) and averaged for at least
300 nanoparticles.
Surface Modifications of 198Au-GNPs. Two types of
polyethylene glycols were conjugated onto the surface of 198Au-
GNPs through ligand exchange procedure. The carboxymethyl-
poly(ethylene glycol)-thiol (cPEG, Laysan Bio Inc., Alabama,
USA) and methoxyl-poly(ethylene glycol)-thiol (mPEG, NOF
Co., Tokyo, Japan) molecules with 5 kDa of average molecular
weight were, respectively, employed to exchange citrate ions on
198Au-GNPs. The cPEG or mPEG solution (2.0 mM, 0.6 mL)
was added into 30 mL of 198Au-GNPs solution for 10 min of
vigorous mixing and another 10 min of incubation. After
PEGylation, double deionized water was employed to wash
twice. The PEGylated 198Au-GNPs were consequently
suspended in double deionized water to obtain a desired
radioactivity (10 μCi in 0.1 mL) prior to intravenous injection.
Pharmacokinetics and Biodisposition. The male ICR
mice (6 weeks old) were purchased from BioLASCO (Taiwan)
and acclimated in laboratory animal center for 2 weeks at the
National Health Research Institutes (NHRI). All mice were
under 12-h light/dark cycle, 23 ±1°C, 39−43% relative
humidity; water and food were available ad libitum. Treatments
and experimental protocols for this study were reviewed and
approved by the Institutional Animal Care and Use Committee
(IACUC) of NHRI (approval number: NHRI-IACUC-097045-
A). Mice were randomly divided into three groups with five
mice in each group. The mice were anesthetized using
isoflurane and subsequently intravenous injected with 10 μCi
(0.37 MBq) of 198Au-GNPs, cPEG-198Au-GNPs, and mPE-
G-198Au-GNPs through tail vein. Mice were sacrificed at 5 min,
30 min, 1 h, 1 day, 2 days, 3 days, 4 days, and 7 days after
dosing gold nanoparticles. Blood and tissue samples including
brain, heart, lung, liver, spleen, gastrointestinal (GI) track,
kidney, pancreas, and carcass were collected for counting γrays
of 198Au.
Determination of 198Au-GNPs in Organs. Liver, GI track
and carcass, were collected into 20 mL plastic vials; blood,
heart, lung, spleen, kidney, and pancreas were collected into 3
mL plastic tubes for measuring tissue wet weights and counting
rate (counts per minute) of 198Au. Counting rates of 198Au,
proportional to amounts of 198Au-GNPs in organs, were
determined by automatic gamma counter (2480 WIZARD2,
PerkinElmer, Turku, Finland). Data were collected for 60 s with
82 keV energy window centered at the 412 keV. Percentage of
injection dose (%ID) was used to present biodisposition of
198Au-GNPs, cPEG-198Au-GNPs, and mPEG-198Au-GNPs. The
%ID was calculated based on the radioactivity measured for
each organ divided by the initially injected radioactivity to each
mouse. For pharmacokinetic study, concentration of radioactive
gold nanoparticles in blood was presented by percentage of
injection dose per gram sample wet weight (%ID/g).
Pharmacokinetic parameters for three types of 198Au-GNPs
were analyzed by noncompartment model of WinNonlin
software (Pharsight Corp., Mountain View, CA) and results
were presented in mean ±standard deviations.
In Vivo Imaging of 198Au-GNPs by Gamma Scintig-
raphy. Tumor-bearing mice (male NU/NU, 6 weeks old,
BioLASCO, Taiwan) were established by subcutaneous trans-
plantation 2 ×106cells of C6 glioma cell line (BCRC, Taiwan)
in 200 μL of phosphate buffer saline at the dorsal region of the
right thigh. Mice were subjected to gamma scintigraphy while
tumor size grew to near 2000 mm3. Mice were anesthetized
with Zoletil (12.5 mg/kg) and Xylazine (5.0 mg/kg) through
intraperitoneal injection. Then the mPEG-198Au-GNPs (60 μCi
in 50 μL) was subsequently intravenously injected for acquiring
gamma scintigraphy at one and 120 h post injection by a
clinical SPECT system (E. CAM plus HD3 Detector,
SIEMENS) equipped single camera head and pinhole
collimator (5 mm I.D.). Mice were positioned on their backs
with legs extended and 11.5 cm below the pinhole collimator
for whole body imaging. Events of 412 keV gamma photons
were accumulated for reaching 3 ×104counts to explore the
biodisposition of mPEG-198Au-GNPs.
Ex Vivo Imaging of 198Au-GNPs by Autoradiography.
Two types of tumor-bearing mice (male NU/NU, 6 weeks old,
BioLASCO, Taiwan) were established by subcutaneous trans-
plantation of C6 glioma and CT26 colon carcinoma cells (2 ×
106cells in 200 μL of phosphate buffer saline) at the dorsal
region of the right thigh. Mice were injected with mPEG-198Au-
GNPs (60 μCi in 50 μL) through tail vein while tumor size
grew to near 2000 mm3. Furthermore, mice were sacrificed at
144-h postinjection of mPEG-198Au-GNPs followed by
immersed into 2.5% of carboxymethyl cellulose sodium salt
(Sigma-Aldrich) solution and then frozen at −20 °C for 3 h.
Animal bodies were subjected to a cryostat (OTF-5000, Bright
Instrument Co. Ltd., England) and frozen sliced into 30 μm-
thick specimens. The sliced specimens covered with PE film
were placed onto imaging plate (BAS-IP SR 2040, Fuji Photo
Film Co. Ltd., Japan) for sensitization in a light-shielding
cassette at −20 °C for 120 h. Imaging plates after sensitization
were subsequently scanned using an Image Analyzing System
(FLA-5000, Fuji Photo Film Co. Ltd., Japan) to obtain 16 bits
planar images with 50 μm of pixel size.
■RESULTS AND DISCUSSION
Critical Role of 198Au-GNPs in Bioanalytical Studies.
Radiotracing is among the optimal methods for investigating
the absorption, distribution, metabolism, and excretion of
molecules of interests.
35−38
Radioactive nuclides, such as 3H
and 14C, have been incorporated into numerous compounds
that are commercially available for tracing molecules and their
metabolites in biological systems. Recently, Georgin and
colleagues fabricated 14C-labeled multiwall carbon nanotubes
(14C-MWNT) to extend the use of radiolabeling technique for
tracing nanomaterials.
39
However, 14C emits pure beta radiation
and biodispositions of 14C-MWNT in tissue dissections can
only be presented by autoradiography. In this study, we
introduced the use of 198Au as a tracer incorporated into GNPs
to conduct a series of bioanalytical studies. By measuring the
characteristic γradiation of 198Au, we traced, imaged, and
quantified 198Au-GNPs in biological systems without encoun-
tering interference caused by matrix substances.
198Au-GNPs are effective radiotracers because of the
characteristic properties of 198Au, which simultaneously emits
γrays (412 keV) and beta particles (Emax: 0.96 MeV) with a
reasonable half-life of 2.69 days. The half-life of 198Au is
comparable to 67Ga (t1/2 = 3.26 days), 90Y(t1/2 = 2.67 days),
111In (t1/2 = 2.80 days), and 201Tl (t1/2 = 3.04 days), which have
been frequently employed for clinical diagnosis. The emitted
gamma photons can readily penetrate tissue free from
Analytical Chemistry Article
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interferences, enabling rapid quantification of 198Au-GNPs in
biological specimens. Gamma scintigraphy is also feasible for
tracing 198Au-GNPs in vivo because the gamma energy of 198Au
is comparable to that of 131I (364 keV, t1/2 = 8.02 days) which
has been popularly used for clinical gamma scintigraphy.
Furthermore, autoradiography is feasible for imaging 198Au
nuclide because the emission of beta particles can sensitize
imaging plate to generate two-dimensional images, and can thus
be used for elucidating the biodisposition of 198Au-GNPs in
microenvironments. Therefore, 198Au-GNPs can facilitate rapid
quantification and visualization of the dynamics of GNP-based
drug carriers in living animals, and be used as a trimodality
analytical platform for imaging, tracing and quantifying GNPs.
According to recent literatures, Katti et al. has first prepared
radioactive gold nanoparticles in which they put gold foil in
nuclear reactor for neutron capture reaction to transform 197Au
to 198Au and followed by dissolving radioactive gold foil in aqua
regia to generate radioactive auric acid (H198AuCl4).
40
The
resulted 198Au(III) solution was further reacted with reducing
agent in a heated aqueous system to reduce 198Au(III) to form
radioactive gold nanoparticles. With this method, it is inevitable
to involve a series of chemical treatments, and would thus raise
risk of radiation exposure. In this study, we demonstrated a
direct approach to prepare 198Au-GNPs simply by irradiating
gold nanoparticles in a nuclear reactor. PEGylation of 198Au-
GNPs is conducted after the irradiation of gold nanoparticles to
avoid damage to PEG molecules from intense radiation field in
the nuclear reactor.
Characterization of Physicochemical Properties for
198Au-GNPs. The 197Au is the only stable isotope of natural
gold element, which can be transformed to radioactive 198Au by
capturing thermal neutrons through 197Au(n,γ)198Au reaction in
a nuclear reactor. Therefore, ensuring that the physical and
chemical identities of 198Au-GNPs remain the same as those of
the original GNPs is critical. The following is a discussion of the
physicochemical properties of GNPs before and after neutron
irradiation. As shown in Figure 1a, the surface plasmon
resonance (SPR) band of GNPs at 519−520 nm did not
present any perceivable wavelength shift before and after
neutron irradiation. Subsequently, ultrafiltration was employed
to separate the nanoparticle fraction from the neutron-activated
aqueous fraction. The radionuclidic purity of nanoparticle
fraction was measured by a high-purity germanium detector.
37
Figure 1b shows the energy spectrum of the gamma photons
emitted from the nanoparticle fraction, displaying a major peak
at 412 keV, accompanied by minor peaks at 674 and 1082 keV.
All of these peaks are in good agreement with the decay scheme
of 198Au, demonstrating perfect radionuclidic purity of 198Au-
GNPs prepared in this study.
31,32
Furthermore, transmission
electron microscopy (TEM) and dynamic light scattering
(DLS) spectroscopy were also used to confirm the size and size
distribution of gold nanoparticles before and after the nuclear
reaction. The TEM micrograph in Figure 1c shows that the
particle size and size distribution of the 198Au-GNPs (13.2 ±
1.0 nm) were comparable to that of the GNPs (13.4 ±1.0 nm)
before neutron irradiation; this similarity can be cross-validated
according to the SPR bands shown in Figure 1a. Additionally,
Figure 1d displays the hydrodynamic sizes of the GNPs and
198Au-GNPs; z-average values are 14.0 nm (PDI = 0.031) and
14.4 nm (PDI = 0.054), respectively. These results clearly
indicate that the 198Au-GNPs remained equivalent to the
original nonradioactive GNPs, and therefore can be employed
as a radiotracer for evaluating the biological fates, including
absorption, distribution, metabolism, and excretion of various
types of GNPs in biological systems.
Pharmacokinetics and Biodisposition of PEGylated
198Au-GNPs. Fluorescent tags conjugated with nanoparticles
have been frequently used for reporting the presence of
nanoparticles in cells and tissues, which facilitate in vivo tracing
of nanoparticles.
41−43
In addition, chemical analysis can be
performed to evaluate concentrations of the component
elements of nanoparticles in of biological specimens. As for
gold nanoparticles, the excitation or emission wavelengths of
fluorescent tags might be attenuated by GNPs or masked by the
autofluorescence of living animals. For chemical analysis, acidic
digestion of tissue samples is necessary before gold can be
quantified using atomic emission spectroscopy or mass
spectrometry, which cannot be used to determine spatial- or
temporal-resolved information on GNPs in tissues.
44,45
In this study, we used an 198Au-radiotracer to rapidly quantify
of GNPs in biological specimens without encountering the
aforementioned drawbacks. The PEGylated-GNPs were used as
model drug carriers by conjugating the 198Au-GNPs with
carboxymethyl-poly(ethylene glycol)-thiol (cPEG) and me-
thoxyl-poly(ethylene glycol)-thiol (mPEG) molecules, respec-
tively. As is well-known, PEG-conjugated pharmaceuticals can
provide a shielding layer to prevent nonspecific adhesion of
numerous molecules in blood and avoid capturing by immune
system, which consequently lead to prolonging circulation of
PEGylated molecules.
46,47
Since PEGylation is necessary to
render GNP a practical anticancer carrier,
17−19
both cPEG and
mPEG molecules (averagely 5 kDa of molecular weight) were
immobilized on 198Au-GNPs in this study through Au−S
binding. The material characterization indicated that the
hydrodynamic sizes of cPEG-198Au-GNPs and mPEG-198Au-
GNPs were 34.8 ±3.4 and 37.3 ±1.3 nm, respectively. The ζ
potential values were −39.1 ±8.9 and −26.5 ±8.5 mV for the
cPEG-198Au-GNPs and mPEG-198Au-GNPs, respectively.
The effectiveness of using 198Au-radiotracing techniques for
pharmacokinetics and biodisposition analysis was evaluated.
Three types of 198Au-GNPs (with different surface moieties)
Figure 1. Physicochemical characterization using (a) UV−vis spectra
of bare GNPs before and after neutron irradiation, (b) gamma photon
energy spectrum of bare GNPs after neutron irradiation, (c)TEM
micrograph of bare GNPs after neutron irradiation, and (d) particle
size determined by dynamic light scattering before and after neutron
activation.
Analytical Chemistry Article
dx.doi.org/10.1021/ac503260f |Anal. Chem. 2015, 87, 601−608604

were injected intravenously into mice, followed by withdrawing
blood for counting 412 keV gamma photons. Figure 2 displays
the concentration profiles of bare 198Au-GNPs, cPEG-198Au-
GNPs, and mPEG-198Au-GNPs, in which the concentrations of
GNPs in blood are shown as the percentage of the injected
dose per gram of blood (%ID/g). The concentration of
bare198Au-GNPs in the blood diminished rapidly, and was
approximately 2.0% ID/g at 5 min postinjection. By contrast,
concentrations of cPEG-198Au-GNPs and mPEG-198Au-GNPs
in blood were maintained at the level of 30% ID/g at 5 min
postinjection. Therefore, we could estimate ∼60% of injected
PEGylated-GNPs persisted in blood because of nearly 2 g of
total blood in each mouse. Through radioactivity measurement,
it also showed bare radioactive GNPs were rapidly washed out
of blood within less than 5 min and very quickly accumulated in
liver (Table 2). Table 1 lists comparative results of
pharmacokinetic parameters of bare198Au-GNPs, cPEG-198Au-
GNPs and mPEG-198Au-GNPs formulations. As a drug carrier,
significant improvements occurred for both of cPEG-198Au-
GNPs and mPEG-198Au-GNPs in comparison to bare 198Au-
GNPs. Obviously, two PEGylated-198Au-GNPs show higher
maximum concentration (Cmax) and larger area under curve
(AUC) than the bare 198Au-GNPs. However, evaluating
whether the cPEG-198Au-GNPs or mPEG-198Au-GNPs are
more effective drug carriers only according to pharmacokinetic
information is insufficient.
In addition to pharmacokinetic data, biodisposition informa-
tion is required. Table 2 lists biodisposition data for the bare
198Au-GNPs, cPEG-198Au-GNPs, and mPEG-198Au-GNPs,
showing the percentage of injected dose (% ID) in each
organ at 1 h, 1 day, and 1 week after administration. There was
more than 85% ID of bare 198Au-GNPs accumulated in liver,
persisting for 7 days, which was in good agreement with rapid
decrease of bare 198Au-GNPs in blood (Figure 2). By contrast,
less than 15% ID of PEGylated-198Au-GNPs presented in liver
at 24-h postinjection, accompanied by more than 10%ID/g of
cPEG-198Au-GNPs and mPEG-198Au-GNPs in blood indicated
the practice of prolong circulation. Retention of cPEG-198Au-
GNPs and mPEG-198Au-GNPs in liver, spleen, and carcass
gradually increased for the period from 1 h to 7 days after
administration. More than 85% ID of cPEG-198Au-GNPs and
mPEG-198Au-GNPs were distributed in liver, spleen, and
carcass at 7-day postinjection. For spleen, there was no
significant difference between cPEG-198Au-GNPs and mPE-
G-198Au-GNPs (6.4 ±1.3% ID versus 6.2 ±1.4% ID).
However, biodisposition of cPEG-198Au-GNPs (32.9 ±3.6%
ID) in liver was significantly higher than that of mPEG-198Au-
GNPs (26.0 ±5.4% ID). Conversely, the mPEG-198Au-GNPs
exhibited a marked tendency toward higher accumulation in the
carcass than the cPEG-198Au-GNPs did (65.4 ±5.5% ID versus
49.8 ±4.5% ID). To prevent from overestimating the
biodisposition of GNPs in carcass, we count tail tissue
separated from carcass, showing very few (<3%) GNPs
remained in tail tissue. Also, we do not include radioactivity
of tail tissue into total injection dose to avoid artifact from
performing an i.v. tail vein injection. The amounts of the
cPEG-198Au-GNPs and mPEG-198Au-GNPs in the heart, lung,
kidney, and brain decreased during the period from 1 h to 7
days, which can be attributed to continuous wash-out through
blood circulation. In addition, a small portion of 198Au-GNPs
were excreted from the body within 7 days because the
recovery rates for the three radioactive GNPs in the blood,
organs, and carcass were more than 95% of the injection dose.
In comparison with published works, zinc oxide nanoparticles
(10 and 71 nm),
37,48
CdSe-based quantum dots (13 nm),
49,50
and mesoporous silica nanoparticles (50−100 nm)
51−53
could
be predominantly retained in liver, spleen, lung and kidney.
The above-mentioned nanoparticles might be excreted through
gastrointestinal (GI) track/feces and/or kidney/urine, which
were far different from the observation in this study. Not only
particle size but also physicochemical nature, particle shape,
surface modification and administration route all play a role in
excretion pathways of nanoparticles from the body.
48
Imaging 198Au-GNPs in Vivo and ex Vivo. In addition to
rapid quantification of 198Au-GNPs for pharmacokinetic and
biodisposition investigation, the 198Au-radiotracer technique is
applicable for imaging and tracing GNPs in living animals and
dissected tissues. Simultaneous emission of γrays and beta
particles facilitates imaging 198Au-GNPs by two modalities,
gamma scintigraphy by clinical SPECT and autoradiography by
βparticle responsive imaging plate. Gamma scintigraphy and
autoradiography are not only useful to verify the biodisposition
of PEGylated-198Au-GNPs but also allow screening optimal
portfolio of GNP-based drug carriers for the specific tumor-
bearing animal model. Based on above-mentioned pharmaco-
kinetics and biodisposition evaluations, mPEG-198Au-GNPs was
superior to cPEG-198Au-GNPs for treating animals with
subcutaneous tumors because of longer circulation time and
less retention in liver. Figure 3 shows the gamma scintigraphy
Figure 2. Concentration profiles of bare198Au-GNPs, cPEG-198Au-
GNPs, and mPEG-198Au-GNPs in blood after intravenous injection.
Error bars demonstrate standard deviation of GNP concentration in
blood.
Table 1. Pharmacokinetic Parameters of Radioactive Gold
Nanoparticles with and without Conjugation of Polyethylene
Glycols (Mean ±SD, n=5)
a
bare198Au-
GNPs cPEG-198Au-GNPs mPEG-198Au-GNPs
T1/2 (h) 35.64 ±13.15 14.39 ±3.82 14.54 ±1.60i
C0(% ID/g) 2.22 ±1.11 37.26 ±3.19 34.99 ±3.36
AUC
(% ID h/g) 10.19 ±7.10 800.34 ±248.82 1008.58 ±102.35
CL (g/h) 13.13 ±8.66 0.13 ±0.04 0.10 ±0.01
MRT (h) 53.77 ±16.34 13.72 ±6.74 17.27 ±2.36
a
Abbreviations: T1/2 = biological half-life of 198Au-GNPs in blood; C0
= the extrapolated concentration of 198Au-GNPs in blood at t=0;
AUC = the area under the curve of blood concentration−time curve;
CL = total body clearance; MRT = mean residence time.
Analytical Chemistry Article
dx.doi.org/10.1021/ac503260f |Anal. Chem. 2015, 87, 601−608605

acquired by clinical SPECT platform after intravenous injection
of mPEG-198Au-GNPs, which demonstrate the spatial- and
temporal-resolved biodispositions of mPEG-198Au-GNPs in
glioblastoma-bearing mice. The mPEG-198Au-GNPs were
predominantly observed in the circulatory system 1-h
postadministration (left panel, Figure 3); this observation
strongly corresponds to the pharmacokinetic information
shown in Figure 2; more than 70% of the injected
mPEG-198Au-GNPs were estimated to have remained in the
blood. At 120-h postadministration (right panel, Figure 3),
mPEG-198Au-GNPs were evenly distributed in abdomen and
tumor region. The gamma scintigraphy shown in Figure 3
could be performed using a clinical SPECT imaging system.
The centimeter-level spatial resolution is unideal for tracing
GNPs in small animals. Nevertheless, using a clinical SPECT
imaging platform for tracing 198Au-GNPs in humans would be
less problematic, and more GNP-based pharmaceuticals for
clinical treatments are expected to be developed.
In addition to the γrays, the beta particles emitted from the
mPEG-198Au-GNPs were applied in autoradiography, a practical
imaging technique for exploring the biodisposition of GNPs in
microenvironments in tumors and organs. The mPEG-198Au-
GNPs were intravenously injected into mice, and then allowed
to distribute for 144 h before sacrifice for collecting tissue
dissection. By placing a tissue dissection containing mPE-
G-198Au-GNPs onto a beta particles responsive imaging plate,
the kinetic energy of beta particles could be converted to
detectable signals on a planar display. Biodisposition of
mPEG-198Au-GNPs were therefore blotted from the dissected
tissue to a two-dimensional image. Therefore, we can prove the
concept of visualizing mPEG-198Au-GNPs in dissected tissues
from C6 glioblastoma (Figure 4a) and CT26 colorectal
carcinoma (Figure 4b) tumor-bearing animals to distinguish
efficiency of drug delivery in different tumor models. As shown
in the figures, it presents half of mPEG-198Au-GNPs
successfully delivered into C6 tumor, which might imply that
C6 tumor is more hyper-vascular as compared to that of the
CT26 tumor. Regarding the biodisposition of mPEG-198Au-
GNPs in different microenvironments, Figure 4a reveals
absence of mPEG-198Au-GNPs in the center of tumor. Previous
studies have demonstrated the presence of necrotic center of
glioblastoma in human and animal models.
54−56
We determined that the region was necrotic center because
the GNPs could not be delivered. Moreover, the concentrations
of PEGylated-198Au-GNPs in spleen were apparently higher
than that in liver for both of C6 and CT26 tumor-bearing mice
models (Figure 4). Table 2 displays the amounts of gold
nanoparticles in liver much more than that in spleen; however,
the concentration of PEGylated-198Au-GNPs in spleen should
be higher than that in liver for more than 1 day after injection
because liver weights were nearly 20 times more than spleen
weights. Results from autoradiography revealed the heteroge-
neous biodisposition of the mPEG-198Au-GNPs in livers of C6
and CT26 tumor models and afforded abundant information
on GNPs in various microenvironments. The 198Au-radiotracer
technique enables trimodality analytical applications, including
quantification, imaging, and tracing, that are unachievable
through conventional chemical analysis or fluorescent imaging.
■CONCLUSIONS
In this study, an approach for establishing a trimodality
analytical platform for imaging, tracing, and quantifying GNPs
in mice was developed using radiotracer techniques. 198Au-
GNPs were prepared by irradiating nonradioactive GNPs in a
nuclear reactor through the nuclear transformation of 197Au
into 198Au. The obtained 198Au-GNPs retained the same
intrinsic properties as did the original GNPs and were further
synthesized through surface modification with PEG molecules.
The PEGylated 198Au-GNPs were then injected intravenously
Table 2. Biodispositions of Radioactive Gold Nanoparticles Presenting in % ID for Each Organ (Mean ±SD, n=5)
bare 198Au-GNPs cPEG-198Au-GNPs mPEG-198Au-GNPs
1 h 1 day 7 day 1 h 1 day 7 day 1 h 1 day 7 day
liver 87.6 ±5.5 88.4 ±6.8 87.6 ±3.8 14.5 ±3.3
a
28.9 ±16.7
a
32.9 ±3.6
a
,
b
12.1 ±4.0
a
15.1 ±2.8
a
26.0 ±5.4
a
,
b
spleen 3.8 ±1.0 3.1 ±0.7 3.0 ±0.3 0.2 ±0.1
a
3.3 ±1.8 6.4 ±1.3
a
0.3 ±0.1
a
1.5 ±0.6
a
6.2 ±1.4
a
carcass 4.9 ±2.2 5.8 ±2.6 3.8 ±1.3 39.3 ±4.0
a
47.6 ±7.8
a
49.8 ±4.5
a
,
b
43.3 ±4.4
a
53.8 ±4.6
a
65.4 ±5.5
a
,
b
heart <0.1 <0.1 <0.1 0.7 ±0.2
a
0.4 ±0.1
a
0.3 ±0.1
a
0.8 ±0.3
a
0.6 ±0.3
a
0.3 ±0.1
a
lung 0.9 ±0.6 0.4 ±0.3 0.3 ±0.2 1.6 ±0.4 0.8 ±0.3 0.4 ±0.1 1.7 ±0.4
a
1.1 ±0.3
a
0.6 ±0.2
a
kidney 0.1 ±0.1 0.1 ±0.0 0.1 ±0.0 2.9 ±0.5
a
1.6 ±0.7
a
0.6 ±0.1
a
3.1 ±0.2
a
2.3 ±0.6
a
0.8 ±0.1
a
brain <0.1 <0.1 <0.1 0.3 ±0.1
a
0.1 ±0.1
a
<0.1 0.3 ±0.0
a
0.2 ±0.0
a
<0.1
GI 0.2 ±0.1 0.1 ±0.0 0.2 ±0.1 2.9 ±0.5
a
4.1 ±1.1
a
5.0 ±0.9
a
2.8 ±0.2
a
4.1 ±0.2
a
5.5 ±0.8
a
pancreas <0.1 <0.1 <0.1 0.3 ±0.0
a
0.3 ±0.0
a
0.3 ±0.1
a
0.3 ±0.0
a
0.3 ±0.1
a
0.3 ±0.1
a
recovery 97.7 ±3.6 97.9 ±6.0 95.0 ±3.8 100.0 ±5.9 96.9 ±3.3 95.7 ±3.4 101.9 ±6.1 96.6 ±5.6 105.3 ±8.9
a
Significant difference (pvalue < 0.05) between bare 198Au-GNPs and cPEG-198Au-GNPs or between bare 198Au-GNPs and mPEG-198Au-GNPs for
the same time postinjection.
b
Significant difference (pvalue <0.05) between cPEG-198Au-GNPs and mPEG-198Au-GNPs for the same time
postinjection.
Figure 3. Gamma scintigraphy done by clinical SPECT system for tail
vein injection of mPEG-198Au-GNPs on subcutaneously C6
glioblastoma-bearing nude mouse. The radioactive gold nanoparticles
were circulating in whole body at 1-h postinjection (left panel) and
were almost evenly distributed in abdomen and tumor at 120-h
postinjection (right panel).
Analytical Chemistry Article
dx.doi.org/10.1021/ac503260f |Anal. Chem. 2015, 87, 601−608606

into mice, and gamma photon measurement of blood
specimens and separated tissues was performed to quantify
the GNPs. The simultaneous beta and γradiation emissions
from the 198Au-GNPs can enable exploring the dynamics and
biodisposition of GNPs in animals. Imaging and tracing GNPs
in vivo is possible through gamma scintigraphy, which implies
the potential for clinical use. In addition to being used to
determine the macroscopic distribution of GNPs, auto-
radiography enables visualizing various biodispositions in
microenvironments. 198Au-radiotracer techniques facilitate
comprehensive achievements in rapid quantification, real-time
monitoring in animals, and microscopic visualization of GNPs
in tissues; thus, further development of GNP-based drugs for
clinical applications is feasible.
■AUTHOR INFORMATION
Corresponding Authors
*Fax: +886-37-586447. E-mail: cyang@nhri.org.tw.
*E-mail: jkchen@nhri.org.tw.
Author Contributions
C.-H.C., F.-S.L., amd W.-N.L. contributed equally. The
manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
This work was supported by the grants of National Nano-
science and Nanotechnology Program for the Innovative
Molecular Biomedical Nano-Imaging Open Facility, National
Health Research Institutes (NM-99-PP-11), Nuclear Science
and Technology Development Center of National Tsing Hua
University (NTHU-100N7520E1) and Ministry of Science and
Technology (NSC 101-2113-M-400-001-MY2 and 102-2113-
M-007-010) in Taiwan. This study was supported in part by a
surcharge of tobacco products, funding a grant from the
Ministry of Health and Welfare (MOHW103-TD-B-111-12) of
Taiwan. Authors are grateful of animal housing supports from
Laboratory Animal Center of NHRI (98A1-LAPP01-014) and
Eva Yu-Ching Chen for TEM analysis (NM-101-PP-04,
Electron Microscopes Core Facility, NHRI). We heartedly
thank Jeng-Yan Yang (NTHU), Chien-Tien Liu (NTHU),
Fiona Mei-Hui Shih (NHRI), and Matthew Chih-Hui Liu
(NHRI) for handling radioactive materials.
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