68Ga-labeled gold glyconanoparticles for exploring blood-brain barrier permeability: Preparation, biodistribution studies, and improved brain uptake via neuropeptide conjugation

Article abstract of DOI:10.1021/ja411096m

New tools and techniques to improve brain visualization and assess drug permeability across the blood-brain barrier (BBB) are critically needed. Positron emission tomography (PET) is a highly sensitive, noninvasive technique that allows the evaluation of the BBB permeability under normal and disease-state conditions. In this work, we have developed the synthesis of novel water-soluble and biocompatible glucose-coated gold nanoparticles (GNPs) carrying BBB-permeable neuropeptides and a chelator of the positron emitter ⁶⁸Ga as a PET reporter for in vivo tracking biodistribution. The small GNPs (2 nm) are stabilized and solubilized by a glucose conjugate. A NOTA ligand is the chelating agent for the ⁶⁸Ga, and two related opioid peptides are used as targeting ligands for improving BBB crossing. The radioactive labeling of the GNPs is completed in 30 min at 70 C followed by purification via centrifugal filtration. As a proof of principle, a biodistribution study in rats is performed for the different ⁶⁸Ga-GNPs. The accumulation of radioactivity in different organs after intravenous administration is measured by whole body PET imaging and gamma counter measurements of selected organs. The biodistribution of the ⁶⁸Ga-GNPs varies depending on the ligands, as GNPs with the same gold core size show different distribution profiles. One of the targeted ⁶⁸Ga-GNPs improves BBB crossing near 3-fold (0.020 ± 0.0050% ID/g) compared to nontargeted GNPs (0.0073 ± 0.0024% ID/g) as measured by dissection and tissue counting.

Full text of DOI:10.1021/ja411096m

Article
pubs.acs.org/JACS
68Ga-Labeled Gold Glyconanoparticles for Exploring Blood−Brain
Barrier Permeability: Preparation, Biodistribution Studies, and
Improved Brain Uptake via Neuropeptide Conjugation
Jens Frigell,† Isabel García,†,‡ Vanessa Gomez-Vallejo,§ Jordi Llop,§ and Soledad Penades*,†,‡
́
́
‡
§
†Laboratory of GlycoNanotechnology, Biofunctional Nanomaterials Unit, CIC biomaGUNE, CIBER-BBN, and Radiochemistry
Department, Molecular Imaging Unit, CIC biomaGUNE, Parque Tecnologico, Paseo Miramon 182, 20009 San Sebastian, Spain
́
́
S
* Supporting Information
ABSTRACT: New tools and techniques to improve brain
visualization and assess drug permeability across the blood−
brain barrier (BBB) are critically needed. Positron emission
tomography (PET) is a highly sensitive, noninvasive technique
that allows the evaluation of the BBB permeability under
normal and disease-state conditions. In this work, we have
developed the synthesis of novel water-soluble and biocom-
patible glucose-coated gold nanoparticles (GNPs) carrying
BBB-permeable neuropeptides and a chelator of the positron
emitter 68Ga as a PET reporter for in vivo tracking
biodistribution. The small GNPs (2 nm) are stabilized and
solubilized by a glucose conjugate. A NOTA ligand is the
chelating agent for the 68Ga, and two related opioid peptides are used as targeting ligands for improving BBB crossing. The
radioactive labeling of the GNPs is completed in 30 min at 70 °C followed by purification via centrifugal filtration. As a proof of
principle, a biodistribution study in rats is performed for the different 68Ga-GNPs. The accumulation of radioactivity in different
organs after intravenous administration is measured by whole body PET imaging and gamma counter measurements of selected
organs. The biodistribution of the 68Ga-GNPs varies depending on the ligands, as GNPs with the same gold core size show
different distribution profiles. One of the targeted 68Ga-GNPs improves BBB crossing near 3-fold (0.020 0.0050% ID/g)
compared to nontargeted GNPs (0.0073 0.0024% ID/g) as measured by dissection and tissue counting.
loaded with therapeutic agents and deliver cargo selectively to
the site of action, increasing the therapeutic efficacy and
reducing undesired side effects. Additionally, compared to
molecular scale agents, NPs have prolonged circulation times
and enable multivalent interactions. Numerous reports exist on
molecules and macromolecules that have been anchored to
liposomes, dendrimers, and polymeric nanoparticles in order to
interact with transporters or receptors of endothelial cells and
thus improve BBB penetration. Examples include proteins
(lactoferrin),10 antibodies (apolipoprotein E11 or antitransfer-
rin12), peptides (TAT,13 bacteriophage Clone 12-2 peptide,14
RVG-9R peptide,15 angiopeptides16), and bioadhesive surfac-
tants such as polysorbate 80.17 However, there is still a need for
tools with efficient BBB permeability and enhanced targeting
properties.
Many metallic nanoparticles have been designed to be
biocompatible and furthermore to be used as contrast agents
for in vivo imaging by different techniques. There are also
reports on NPs that have been functionalized with targeting
ligands in order to facilitate BBB crossing by selective or
preferential interaction with receptors or transporters.16,18−23
INTRODUCTION
■
Neurological diseases include a range of disorders that affect a
large percentage of the world’s population. Even though the
biological basis underlying many of such disorders is often
known, the therapeutic efficacy is limited due to the selectivity
of the blood−brain barrier (BBB).1,2 The BBB consists of
endothelial cells separated by tight junctions, and it plays an
effective role in protecting the brain from harmful substances
and microorganisms. At the same time, the BBB hampers the
systemic delivery of therapeutic agents from the blood into the
brain.3,4 Drugs should be small (molecular weight below 400
Da) and lipophilic to traverse the BBB, and they should not be
a substrate of the efflux system such as P-glycoproteins. In
practice, few drugs meet these specifications, and most
therapeutic agents targeting the central nervous system
(CNS) must cross the BBB through interaction with specific
transporters or receptors expressed at the luminal side of the
endothelial cells.5 Additionally, poor stability in vivo of drugs
such as peptides and nucleosides also limits systemic drug
administration targeting the CNS.
Engineered biomaterials and polymeric nanoparticles have
arisen as promising alternatives to promote brain protection
and repair and to improve the delivery of therapeutic agents
into the CNS.6−9 Nanoparticles (NPs) have the capacity to be
Received: October 30, 2013
Published: December 9, 2013
© 2013 American Chemical Society
449
dx.doi.org/10.1021/ja411096m | J. Am. Chem. Soc. 2014, 136, 449−457

Journal of the American Chemical Society
Article
Figure 1. (A) Control and precursor glyconanoparticles (GNPs) employed in the preparation of the different 68Ga- labeled GNPs. (B) Prepared
nontargeted and targeted 68Ga-GNPs for in vivo studies. (C) Ligands used for the synthesis of 68Ga-GNPs.
Among metallic nanoparticles, gold nanoclusters are an
excellent platform to combine therapeutic and diagnostic
agents for noninvasive visualization of drug delivery.24
Our experience in the design and preparation of biocompat-
ible sugar-coated gold nanoparticles (glyconanoparticles,
GNPs) for a variety of biomedical applications25−27 prompted
us to address the preparation of gold GNPs able to reach an
intact brain. GNP methodology allows the incorporation of
multiple ligands in a controlled fashion.26 The hydrophilicity of
sugars confers biocompatibility27 and resistance to protein
adhesion to nanomaterials.28 We reasoned that decorating
GNPs with molecules that can access the brain (i.e., glucose,
neuropeptides) in combination with a gallium-68 (68Ga)
chelator might result in highly sensitive PET probes as a tool
450
dx.doi.org/10.1021/ja411096m | J. Am. Chem. Soc. 2014, 136, 449−457

Journal of the American Chemical Society
Article
a
Table 1. Properties and Average Chemical Composition of GNPs
b
c
GNPs
TEM d(nm)
ligands per GNP
glucoseC5SH per GNP
peptides per GNP
NOTA per GNP
MW (kDa)
GlcC5S−Au
LipGNP
1.8 0.2
2.2 0.2
2.4 0.2
2.1 0.2
2.7 0.3
2.2 0.2
3.2 0.3
87
80
87
67
64
87
d
13
d
C11GNP
130
80
110
20
132
94
e
e
Lip-EnkGNP
C11-EnkGNP
Lip-Glycopep GNP
C11-Glycopep GNP
59
13
10
6
8
f
135
106
19
162
92
e
e
80
60
14
f
221
174
15
32
329
a
b
All GNPs are coated with and stabilized by GlcC5SH ligand. The amount of peptide on the GNPs was assessed by quantitative 1H NMR with the
c
internal standard TSP and integration of the aromatic proton region of the spectrum. Calculated from the number of Au atoms as derived from the
TEM diameter plus the number of ligands and their molecular weight.63 Determined by elemental analysis. Derived from known ratio of GlcC5SH
d
e
f
and NOTA ligand in the precursor GNP and assuming that the total amount of ligands does not change. The total ligands per GNPs were estimated
based on the average gold diameter and according to the literature.63
to visualize biodistribution and evaluate brain uptake in small
animals. To date, different in vitro and in vivo methods have
been applied to assess BBB permeation of drugs. However,
noninvasive molecular imaging techniques such as positron
emission tomography (PET), combined PET/computed
tomography (PET/CT), and magnetic resonance imaging
(MRI) provide the most accurate information regarding in
vivo biodistribution and brain uptake in animals with
uncompromised BBB.29,30
one example of 68Ga-labeled magnetic NPs has been reported
to date.19
Two types of 68Ga-GNPs (nontargeted and targeted GNPs)
were prepared (Figure 1). The nontargeted GNPs, GlcC5S-Au-
C11NOTA (C11GNP) and GlcC5S-Au-LipNOTA (LipGNP)
incorporate glucose and the cyclic chelator 1,4,7-triazacyclono-
nane-1,4,7-triacetic acid (NOTA), which forms a stable
complex with Ga(III) (log K = 30.98).40 The targeted GNPs,
GlcC5S-Au-C11NOTAEnkephalin (C11-EnkGNP), GlcC5S-Au-
LipNOTAEnkephalin (Lip-EnkGNP), GlcC5S-Au-
C11NOTAGlycopep (C11GlycopepGNP), and GlcC5S-Au-
LipNOTAGlycopep (Lip-GlycopepGNP) incorporate two
peptides, the pentapeptide Tyr-Gly-Gly-Phe-Leu (Enk) or the
glycosylated peptide Gly-Gly-Tyr-Thr-Gly-Phe-Leu-Ser-O-β-
glucoside (Glycopep) (Figure 1B). Control and precursor
GNPs (Figure 1A) bearing either glucose (GlcC5S-Au), glucose
and the Enk peptide (GlcC5S-Au-Enk) or glucose and the
glycosylated peptide (GlcC5S-Au-Glycopep) were also pre-
pared following a one-pot protocol. All prepared GNPs and the
thiol-ending ligands used in their preparation are shown in
Figure 1.
In this work, we report the preparation of nontargeted and
targeted glucose/68Ga-coated gold nanoparticles (68Ga-GNPs)
as novel nanosystems for the assessment of in vivo
biodistribution and BBB permeation. Nontargeted GNPs
incorporate a glucose conjugate (GlcC5S) and a thiol-ending
derivative of a cyclic 68Ga chelator. To convert nontargeted
GNPs into targeted GNPs in order to achieve BBB crossing,
two neuropeptide derivatives were selected, either the
pentapeptide Tyr-Gly-Gly-Phe-Leu (Enk) or the glycosylated
peptide Gly-Gly-Tyr-Thr-Gly-Phe-Leu-Ser-O-β-glucoside (Gly-
copep). Small neuropeptide derivatives from the enkephalin
family ([H2N-Tyr-Gly-Gly-Phe-Leu(Met)-COOH]) and re-
lated glycopeptides have frequently been used as suitable
vectors for BBB passage.31−34 However, their low stability and
poor bioavailability in vivo due to enzymatic degradation have
limited their efficacy. Different strategies to achieve improved
stability and bioavailability of the peptides, including glyco-
sylation, lipidation, and pegylation among others, have been
reported.33 Here, we propose that the incorporation of small,
modified neuropeptides onto the GNP surface improves the
peptide stability and enhances the capability to reach and bind
to their natural targets. After the GNPs were labeled, in vivo
biodistribution PET studies in rodents were performed in
combination with dissection and tissue counting experiments. A
significant improvement of BBB penetration for one of the
targeted GNPs was observed.
For the preparation of the nontargeted GNPs, the
glycoconjugate 5-mercaptopentyl β-D-glucopyranoside
(GlcC5S)41,42 and two different NOTA conjugates, LipNOTA
and C11NOTA (Figure 1C), were synthesized. The NOTA was
functionalized with linkers containing a thiol-ending group for
attaching the chelator to the gold surface. Two linkers, an 11-
carbon atom aliphatic chain (C11) and a lipoic acid (Lip), were
selected for the preparation of the C11NOTA and LipNOTA
conjugates (Figure 1C and Scheme S1, Supporting Informa-
tion), in order to investigate their effect on the in vivo 68Ga-
GNPs biodistribution pattern. The C11GNP and LipGNP
(Figure 1B) were prepared by in situ reduction (one-pot
reaction) of HAuCl4 with NaBH4 in the presence of an excess
of thiol-ending compounds following a previously described
protocol.42 The GNPs were purified by centrifugal filtration and
RESULTS AND DISCUSSION
■
1
dialysis and characterized by H NMR, IR, UV−vis, TEM, and
Synthesis of Glyconanoparticles and Incorporation of
the Chelating Agent. Our 68Ga-labeled glyconanoparticles
(68Ga-GNPs) are based on 2 nm gold nanoparticles coated with
a glucose conjugate (GlcC5S). Among all radionuclides,
gallium-68 (68Ga) was selected because, contrary to other
positron emitters that have to be produced in cyclotrons, 68Ga
can be obtained from a 68Ge−68Ga generator and can be eluted
on a daily basis.35 In addition, it has 89% positron branching
and low photon emission. As far as we know, only a few
examples of 68Ga-labeled polymeric NPs36−39 exist, and only
elemental analysis (see the Supporting Information). The ratio
between the glucoside and the NOTA conjugates on the GNPs
was determined by elemental analysis of the GNP, since the
NOTA conjugate contains nitrogen atoms whereas the
glucoside does not. An average percentage of 84/15 of glucose
to NOTA per GNP was determined on the basis of the number
of ligands (Table 1). The ratio of glucose/NOTA conjugates
1
was also confirmed by comparing the H NMR spectra of the
conjugates mixture before and after formation of the GNPs.
451
dx.doi.org/10.1021/ja411096m | J. Am. Chem. Soc. 2014, 136, 449−457

Journal of the American Chemical Society
Article
Preparation of GNPs Incorporating the Peptides. In
order to prepare targeted GNPs and to test the capacity of the
small-sized (2 nm) GNPs to penetrate the BBB in rodents, the
Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu, Enk) peptide, modified
with a thiolated PEG linker, and the glycosylated peptide, Gly-
Gly-Tyr-Thr-Gly-Phe-Leu-Ser-O-β-glucoside (Glycopep),
modified with a thiopentyl chain, were prepared (Figure 1C
and Figure S1, Supporting Information). Similar peptides have
been shown to be able to cross the BBB.43,44 Although Leu-
enkephalin is known to rapidly undergo enzymatic cleavage in
vivo,45,46 improved stability toward enzymatic hydrolysis can be
expected because of the attachment onto the GNPs surface.
Enhanced resistance toward E. coli β-galactosidase hydrolysis
was observed for lactose on GNPs compared to the free
disaccharide.47
reaction, nonchelated 68Ga was removed by centrifugal filtration
and repeated centrifugal washings with PBS buffer (pH 7.4)
until no radioactivity was detected in the washings. The overall
preparation, including washing, could be performed in less than
60 min. The high number of NOTA molecules (8−32 per
GNP, Table 1) ensured 68Ga incorporation to obtain specific
activity values higher than 0.11 MBq/μg GNP. Such high
specific activities are essential in order to decrease the
administered dose in the in vivo investigations, which in our
case was 150−200 μg GNP/180−250 g body weight (3.7−14.8
MBq), thus minimizing potential toxicological effects. In
addition, in vitro stability test (2 h, at 37 °C in saline buffer)
showed a very slow release of 68Ga from GNPs (after 2 h of
incubation, radiochemical purity >85%), again pronouncing the
suitability of 68Ga-labeled GNPs for in vivo PET studies.
Finally, control labeling experiments (70 °C, pH 3.5, 30 min)
were carried out with GNPs containing only glucose (GlcC5S−
Au) or glucose and the peptides (GlcC5S-Au-Enk or GlcC5S-
Au-Glycopep, Figure 1A) in order to test that 68Ga is only
attached to NOTA and that no radiolabeling of the GNPs
occurred due to unspecific complexation of 68Ga ion to glucose
and/or peptides. After labeling, the free 68Ga was removed from
the GNPs by centrifugal filtration and washing. Gamma counter
analysis of the washings and the GNPs confirmed the lack of
radioactive labeling of glucose bearing GNPs and a very low
68Ga incorporation in the peptide-bearing GNPs. This result
clearly indicates that effective radiolabeling is only achieved via
the formation of 68Ga-NOTA complex (Figures S6 and S7,
Supporting Information).
The targeted 68Ga-GNPs, C11-GlycopepGNP, Lip-Glyco-
pepGNP, C11-EnkGNP, and Lip-EnkGNP (Figure 1B),
capped with the peptides were prepared by ligand place
exchange reaction (LPE).48 This methodology allows mod-
ification of the organic shell of previously prepared GNPs by
exchange with other thiolated ligands. Lip-Glycopep GNP and
Lip-EnkGNP were prepared by mixing an aqueous solution of
GlcC5S-Au-LipNOTA GNPs with an aqueous solution of the
respective peptide derivative (0.3−1.2 equiv with respect to the
glucose on the GNP) at 25 °C for 48 h and 180 rpm. In the
case of the C11-GlycopepGNP and the C11-EnkGNP, the LPE
was performed starting from GlcC5S-Au-Glycopep GNP or
GlcC5S-Au-Enk GNP (Figure 1A) and subsequent addition of
an excess of the C11−NOTA conjugate (2−10 equiv with
respect to glucose on the GNP). The functionalized GNPs
were purified as before and characterized by TEM, UV−vis, IR,
In Vivo Studies with 68Ga-Labeled Gold Nano-
particles. It is well-known that systemically administered
nanoparticles accumulate in different organs depending mainly
on particle size. As a general rule, large particles are trapped in
the smallest capillaries of the lungs or engulfed by phagocytic
cells in the organs of the reticulo-endothelial system (RES)
such as the liver, the spleen, and the lungs, while smaller
particles reach various organs by crossing the tight endothelial
junctions and are rapidly excreted through the kidneys.50−52
However, the clearance from the bloodstream and the
accumulation in different organs depends also on other
intrinsic properties such as surface decoration and surface
charge, with opsonization also playing an important role in
macrophage recognition.53 Biodistribution and accumulation of
gold nanoparticles in different organs depend on the size
core,54 but also on the coating and administration route.55 In
the case of 68Ga-GNPs reported here, all particles have similar
core sizes (2−3 nm) as determined by TEM (micrographs in
the Supporting Information). Any differences in biodistribution
should be attributed to the difference in the coating ligands and
not to the variability of the gold core sizes.
1
and H NMR (see the Supporting Information). The amount
of peptide on the GNPs was assessed by quantitative 1H NMR
by integration of the aromatic proton region of the spectrum,
which only contained signals from the peptide, and using
trimethylsilyl propanoic acid (TSP) as internal standard (Figure
S2, Supporting Information). Alternatively, when insertion of
the peptides was performed following the one-pot protocol,
peptide load was determined by comparing the ligand mixture
1
before and after GNPs production using H NMR. Table 1
summarizes the average core size diameter, number of ligands,
organic shell composition, and calculated molecular weights for
the prepared GNPs.
Incorporation of 68Ga to Radiolabel the Nano-
particles. Incorporation of the radionuclide 68Ga was
performed in the very last step. The labeling efficiency was
investigated using different reaction conditions (temperature,
stoichiometry, and reaction time). As previously reported,
Ga(III) chelation worked best at pH ca. 3.5.49 Typically, 200 μg
of GNP were reacted with 37−74 MBq of 68Ga contained in 0.4
mL of 0.6 N aqueous HCl solution as obtained from the
68Ge−68Ga generator (pH of the reaction was adjusted with
HEPES and NaOH). 68Ga incorporation efficiencies ranging
from 85% to 94% were obtained within 30 min at 70 °C for all
GNPs (Figure S3, Supporting Information). Shorter reaction
times at 70 °C did not complete the chelation of Ga-68 as seen
by radio-TLC (Figure S4, Supporting Information). When the
labeling was carried out at 30 °C, lower incorporation
efficiencies were achieved: only 23% of the activity was
chelated to the GNP and 77% was found in the washings
(Figure S5, Supporting Information). Increasing the amount of
radioactivity at the expense of lowering the concentration of
GNP did not enhance the overall radiochemical yield. After the
The biodistribution of 68Ga-GNPs with and without targeting
peptides was investigated in 7−8 week old (180−250 g body
weight) Sprague−Dawley rats. Typically, labeled GNPs (0.75−
1.0 μg/μL) were injected (200 μL, 3.7−14.8 MBq, 100−400
μCi) via one of the lateral tail veins. The dynamic evolution of
the GNP accumulation in different organs was assessed using
PET/CT during 4 h after intravenous (iv) administration.
Images were acquired in four bed positions in order to cover
the whole body of the animal. Averaged time−activity curves
(TACs) for all GNPs (n = 3 animals per nanoparticle) were
obtained. A steady-state-like behavior was reached for the
majority of the organs and GNPs after 1 h. At the end of the
scans, the animals were sacrificed without recovering from
452
dx.doi.org/10.1021/ja411096m | J. Am. Chem. Soc. 2014, 136, 449−457

Journal of the American Chemical Society
Article
Figure 2. (Left) Biodistribution data of selected organs in Sprague−Dawley rats at time of harvesting. The uptake of different GNPs is represented as
percentage of radioactivity per injected dose, per unit mass of organs, and per animal weight. (Right) Comparison of accumulation of C11GNP and
LipGNP in different organs. Each bar represents the mean SEM of 3 rats. ****P < 0.0001Lip vs Lip-Enk in kidney uptake, **P < 0.01C11 and Lip
vs C11-Glycopep and Lip-Glycopep; ****P < 0.0001C11 vs Lip in liver uptake.
Figure 3. Co-registered coronal PET/CT images of the 68Ga-GNPs: LipGNP (a), Lip-EnkGNP (b), C11GNP (c), and C11-EnkGNP (d) at early
stage (0−10 min) and Lip-EnkGNP (e) and C11-EnkGNP (f) at late stage (60−80 min) after iv injection.
anesthesia, the organs were harvested, and the amount of
radioactivity was determined using a gamma counter
(dissection and tissue counting). The vast majority of the
GNPs were excreted in urine, which was not collected during
the PET scan. After the scan, urine still in the bladder was
collected and measured for radioactivity in the gamma counter.
Figure 2 (left) shows the accumulation of different labeled
GNPs in selected organs at the time of sacrifice (4 h post
injection) as determined by organ removal and gamma
counting. Results showed a major accumulation in liver, spleen,
and kidneys; however, a significant influence of the ligands on
the biodistribution pattern was observed. The presence of the
peptide influenced the biodistribution of the 68Ga-GNPs (Lip-
GNP vs LipEnk-GNP in kidney, Figure 2 left). The
glycopeptide seems to contribute to an increased accumulation
in the liver, especially for those GNPs bearing the C11NOTA
ligand (C11-GlycopepGNP). A comparison of liver accumu-
lation of the nontargeted C11GNP and LipGNP confirms that
the nature of the linker in the NOTA conjugates has a
significant impact on the biodistribution pattern (Figure 2
right). GNPs with a longer aliphatic chain (C11) accumulate to
a greater extent in the liver in comparison to GNPs bearing the
NOTA conjugate with shorter linker (Lip). A similar effect was
previously reported by Sa et al. for non-nanoparticulated
́
Ga(III) chelates,56 suggesting that the organic molecules bound
to very small nanoclusters may present similar physicochemical
and biological properties to those shown when they are free.
The in vivo images showed similar trends with respect to the
gamma counter evaluation. Reconstructed PET/CT images of
rats injected with C11GNP, LipGNP, and their respective
enkephalin targeted GNPs (C11-EnkGNP and Lip-EnkGNP)
at early and late stages after administration also showed a
different behavior depending on the ligands (Figure 3). At the
early stage, Lip-GNP accumulated quickly in the bladder
(∼70−90% ID/cm3 after 10 min) (Figure 3a), while Lip-
EnkGNP showed significant uptake in the kidneys (Figure 3b).
However, 68Ga-GNPs conjugated to C11NOTA with or without
enkephalin, rapidly accumulated in liver (Figures 3c and 3d). At
a late stage of the scan (60−80 min), the C11-EnkGNP was still
accumulated in the liver (Figure 3f), whereas the Lip-EnkGNP
was almost completely excreted (Figure 3e). The biodistribu-
tion of Lip-EnkGNP followed a similar pattern to that shown
by the nontargeted LipGNP, only with a slightly higher
accumulation in the kidneys (∼20−30% ID/cm3 after 10 min)
453
dx.doi.org/10.1021/ja411096m | J. Am. Chem. Soc. 2014, 136, 449−457

Journal of the American Chemical Society
Article
Figure 4. Brain uptake obtained after administration of the different GNPs using dissection and counting without (left) and with blood perfusion
(right). Results are expressed as percentage of injected dose per gram of tissue. Each bar represents mean SEM of 3 (left) or 5 (right) animals.
Significantly different from no targeted GNP: *p < 0.05, ***p < 0.001.
and a delayed peak in bladder accumulation (20 min after
injection) (Figure 3a and 3b). Rapid renal clearance is expected
for small nanoparticles and is consistent with values reported in
the literature, but the different biodistribution profile between
the 2 nm sized Lip-EnkGNP and C11-EnkGNP should be
attributed to the linker’s nature. The presence of GNPs in other
organs such as spleen, heart, and intestine was also detected.
Even though the vast majority of intravenously injected gold
nanoparticles accumulate in the liver and the spleen,57,58 the
conjugation to sugar molecules can greatly change the
pharmacokinetic behavior by reducing the nonspecific
adsorption of plasma proteins and preventing uptake by the
RES similarly as polyethylene glycol does.59 In this respect,
glucose grafting density on the Lip-GNP might be able to
decrease opsonization leading to low liver accumulation. This is
especially relevant since liver accumulation and subsequent
hepatic toxicity have previously prevented the application of
potentially effective drug delivery systems.
Gamma counter measurements performed on harvested
brains showed almost 2.5-fold higher uptake values in the brain
of animals injected with Lip-EnkGNP compared to those
receiving the nontargeted nanoparticles, Lip-GNP (Figure 4,
left). PET/CT image quantification also validated the higher
brain uptake for animals injected with Lip-EnkGNP. For the
quantification, frames at t > 54 min after injection were
averaged and uptake values expressed as % of injected dose per
body weight (%ID/g) (Figure S8, Supporting Information).
These results might have been influenced to some extent by the
presence of 68Ga-GNPs in blood. Therefore, building on these
encouraging results, the experiments were repeated with Lip-
EnkGNP and LipGNP, but the animals (n = 5) were perfused
before organ harvesting using physiologic saline solution. As
seen in Figure 4 (right), the targeted GNPs have a 2.7-fold
higher accumulation in brain compared to nontargeted particles
(p < 0.001). To further validate these results, the gold content
in the brain was determined by ICP-MS. The result also
confirms a higher accumulation of gold in extracted brains of
rats injected with Lip-EnkGNP (0.07 ppm) when compared to
animals injected with the nontargeted LipGNP where gold was
not detected (<0.05 ppm, detection limit of the technique).
An evaluation of the brain permeability obtained with the
novel gold 68Ga-GNPs in comparison with other targeted
nanoparticles is not straightforward. Most of the reported NPs
designed to target the brain are liposomes or polymeric NPs.
They have so far reached the brain ranging from 0.01% to 0.5%
of the ID when vascular rectification was applied.28,60−62 In the
case of the gold Lip-EnkGNP (∼13 peptides/GNP), a
∼0.025% of the injected dose accumulated in the brain after
iv injection of a maximal dose of 200 μg GNP/rat (Figure 4,
right). This result is similar to that obtained (0.024%) with
high-loading amphipathic peptide gold nanoparticles (400
peptides/NP) after intraperitoneal injection.20 Notably, in our
experiments as little as 28.8 μg of peptide (injected dose = 0.12
mg peptide/kg) was injected. This value is 2 orders of
magnitude lower than the doses previously utilized with
enkephalin peptide analogues (30−122 mg/kg) to achieve an
analgesic effect.31,32 Despite the low peptide dose injected, the
accumulation of the targeted GNPs in the brain was almost 3-
fold to that of nontargeted GNPs. The improved bioavailability
of the peptide is probably a result of an improved stability after
conjugation to the nanoplatform. Preliminary in vitro results
showed an enhanced stability for the peptide conjugated to the
GNP compared to the free peptide when both were subjected
to an endogenous peptidase under the same experimental
conditions (data not shown).
The use of gold glyconanoparticles as carrier for BBB-
permeable molecules is a promising field, due to almost endless
possibilities of chemical functionalization and formulation of
the GNPs. The introduction of molecular imaging reporters
(PET in this case) expands their potential for in vivo imaging of
brains with uncompromised BBB. In addition, the high
molecular weight of the gold GNPs (50−300 kDa) allows a
straightforward separation of the radiolabeled GNPs from free
radioisotopes and small ligands and a convenient purification
via centrifugal filtration rather than HPLC.
CONCLUSION
■
We have prepared and labeled novel targeted and nontargeted
GNPs (68Ga-GNPs) based on 2 nm sized gold glyconano-
particles carrying the positron emitter 68Ga for in vivo PET
imaging. The nontargeted GNPs are coated with glucose, the
brain’s principal nutrient, and a NOTA derivative as gallium
chelator. Two related neuropeptides were conjugated to the
previous GNPs as targeting ligands for improving brain uptake.
Depending on the nature of the ligands attached to the gold
surface, a different biodistribution profile was observed. We
have shown that targeted GNPs bearing a Leu-enkephalin
peptide (Enk) improve brain accumulation (0.020 0.0050%
ID/g) nearly 3-fold compared to nontargeted GNPs (0.0073
0.0024%ID/g). To our knowledge, this is the first report of
454
dx.doi.org/10.1021/ja411096m | J. Am. Chem. Soc. 2014, 136, 449−457

Journal of the American Chemical Society
Article
68Ga-labeled gold nanoparticles that take advantage of the
remarkable chemical versatility of gold nanoparticles as a
scaffold for the attachment of a diversity of molecules in a
controlled way. The novel radioactive 68Ga-GNPs have enabled
the quantification of BBB permeability in rodents with intact
brains. The results here obtained also indicate that manipu-
lation of the molecules coating GNPs may result in GNPs with
more favorable properties to cross the blood−brain barrier.
Metal chelators others than NOTA can be easily introduced on
the GNPs expanding significantly the utilization of PET as an
ultrasensitive imaging technique for the pharmacokinetic
evaluation of new chemical entities.
adjusted to 3.5 with 10% aqueous NaOH solution (75 μL). From a 1
μg/μL solution of nanoparticles in ultrapure water (Type I water, ISO
3696), 100−200 μL were added to the reaction vial, and the
temperature was raised to 70 °C and incubated for 30 min. The
reaction mixture was filtered by centrifugation (30k, 13400 rpm) for 6
min. The nanoparticles were washed three times by centrifugal filtering
(30k, 13400 rpm, 5 min) with HEPES (0.1 M, pH 7.1) and once with
0.9% saline solution. GNPs were then suspended in 0.9% saline, and
the amount of radioactivity was counted in a gamma counter (2470
Wizard, Perkin-Elmer). Simultaneously, all fractions corresponding to
successive washings were also measured. The incorporation yield was
calculated as the ratio between the amount of radioactivity in the
fraction containing the GNPs and the sum of all fractions
corresponding to the washings.
Animal Experiments. Animals were cared for and handled in
accordance with the Guidelines for Accommodation and Care of
Animals (European Convention for the Protection of Vertebrate
Animals Used for Experimental and Other Scientific Purposes).
Internal guidelines and experimental procedures were approved by
local authorities. Male, 7−8 weeks of age Sprague−Dawley rats (body
mass 180−250 g) were used. Three animals per GNP were submitted
to PET/CT studies followed by dissection and counting; 10 more
animals were subjected to biodistribution studies using two different
GNPs (n = 5 per GNP) via dissection and tissue counting with
previous animal perfusion for complete blood removal.
Image Acquisition. PET studies were performed using an
eXploreVista-CT small animal PET/CT system (General Electric
Healthcare). For each GNP, three animals were submitted to whole
body (WB) scans to assess the biodistribution pattern. In all cases, rats
were anesthetized with a mixture of 3−4% isoflurane in O2 for
induction and reduced to 1−1.5% for maintenance in the camera by a
nose cone to maintain regular breathing at a frequency of 60 10
breaths/minute monitored by a pressure sensor (SA Instrument Inc.,
NY). Respiration and body temperature of the animals were
monitored throughout the scan. The temperature, measured rectally,
EXPERIMENTAL SECTION
■
Materials and Methods. All chemicals were purchased as reagent
grade from Sigma-Aldrich and were used without further purification.
NO2AtBu-N-(aminoethyl)ethanamide was purchased from CheM-
atech (France). UV−vis spectra were measured on a Beckman Coulter
DU 800 spectrometer. Infrared spectra (IR) were recorded from 4000
to 500 cm−1 with a JASCO FT/IR 410 model spectrometer: solids
1
were pressed into a KBr plate. H NMR and 13C NMR spectra were
recorded on a Bruker AVANCE (500 MHz) spectrometer. Mass
spectra were carried out with an Esquire 6000 ESIIon Trap from
Bruker Daltonics. High-resolution mass spectra (HR-MS) were
obtained using the matrix-assisted laser desorption/ionization
(MALDI) technique with a 4700 Proteomics Analyzer (Applied
Biosystems) with MALDI-time-of-flight (TOF) configuration. TEM
analysis was carried out in a Philips JEOL JEM-2100F working at 200
kV. The number of gold atoms of the GNPs was deduced from the
core size mesasured in TEM and estimated according to a previous
work.63 House distilled water was further purified using a Milli-Q
reagent grade water system (Millipore). 68Ga (T1/2 = 68 min, β+ =
89%, EC = 11%) was obtained as 68GaCl3 from an iThemba 68Ge/68Ga
generator system (IDB Holland bv, Netherlands) with a nominal 68Ge
activity of 740 MBq. The generator was eluted with 0.6 M HCl
solution (6 mL) and the eluate was collected in five fractions (1.5, 0.5,
0.5, 0.5, 3 mL, respectively). Fractions 2−4 contained the highest 68Ga
concentration typically within the range of 74−111 MBq/0.5 mL and
were used without further purification. Organ activity was measured in
an automatic gamma counter (2470 Wizard, Perkin-Elmer). The
glucose conjugate was prepared as previously described.41 The
synthesis and characterization of NOTA derivatives is described in
the Supporting Information.
General Procedure for Direct Synthesis of Gold GNPs. A
0.025 M solution of HAuCl4 (1 equiv) in Milli-Q water was added to a
0.012 M solution of thiolated ligand (3 equiv) in MeOH. If more than
one type of ligand was used a combined amount of 3 equiv was used
with respect to HAuCl4. A freshly prepared 1 M solution of NaBH4
was added in portions to the mixture, and immediately a black
precipitate was formed. The reaction was left on a shaker board (200
rpm) for 2 h at room temperature after which the shaking was stopped
and the precipitate was allowed to sediment. The supernatant was
taken off using a pipet, and the nanoparticles were washed once with
MeOH before they were dissolved in H2O and purified by dialysis
(MWCO = 10k) for 3 days, changing the water twice per day. The
glyconanoparticles were characterized and then lyophilized and stored.
General Procedure for Preparation of Gold GNPs by Ligand
Place Exchange Reaction (LPE). The GNP (1 equiv with respect to
glucoside) was dissolved in Milli-Q water, the thiol-ending function-
alized peptide or 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)
derivative (1 equiv) was added, and the mixture was shaken (180 rpm)
for 2 days at room temperature under Ar atmosphere. After this time,
the nanoparticles were separated from the solution by centrifugal
filtration (MWCO 10000, 10 min). The GNPs were then in a similar
fashion washed three times with Milli-Q water and lyophilized.
General Procedure for the Synthesis of 68Ga-Labeled GNPs.
To a vial containing HEPES (42 mg, 0.176 mmol) was added 400 μL
of 68Ga (68GaCl3 in 0.6 N HCl, 2−3 mCi/0.5 mL), and the pH was
was maintained at 37
1 °C using a water heating blanket
(Homeothermic Blanket Control Unit, Bruker, Germany). For
administration of the GNPs, the tail vein was catheterized with a 24-
gauge catheter and 3.7−22.2 MBq of GNPs (150−300 μL, 100−200
μg) were injected in tandem with the start of the PET dynamic
acquisition.
Dynamic images (20 frames: 4 × 2, 4 × 4, 3 × 10, 3 × 20, 3 × 30
min) were acquired in four bed positions in the 400−700 keV energy
window, with a total acquisition time of 234 min; after each PET scan,
CT acquisitions were also performed (140 μA intensity, 40 kV
voltage), providing anatomical information as well as the attenuation
map for the later image reconstruction. After finalizing the image
acquisition, animals were sacrificed, and brain, lungs, kidneys, spleen,
liver, heart, and intestine were quickly removed and measured in an
automatic gamma counter (2470 Wizard, Perkin-Elmer). Blood
samples (0.5 mL) were also taken and measured in the gamma
counter.
Image Analysis. Dynamic acquisitions were reconstructed
(random, scatter, decay and CT-based attenuation corrected) with
filtered back projection (FBP) using a Ramp filter with a cut-off
frequency of 1 Hz. PET images were analyzed using PMOD image
analysis software (PMOD Technologies Ltd., Zurich, Switzerland).
̈
Volumes of interest (VOIs) were manually drawn (regions of interest
(ROIs) in the Y-axis of the animal were added up to VOIs) for brain,
liver, kidneys, lungs, heart, spleen, intestine, stomach, and bladder
using the CT images as anatomical reference. VOIs were then
transferred to the PET images, and the concentration of radioactivity
was obtained for each organ and time frame as cps/cm3. Values were
transformed into real activity (Bq/cm3). Finally, injected dose
normalization was applied to data to obtain the percentage of injected
dose per organ. After determination of the time activity curves, frames
at t > 54 min (corresponding to the plateau) were averaged and the
same quantification process was applied.
Dissection Method with Perfusion. Ten animals (5 per GNP)
were submitted to biodistribution studies using perfusion and further
455
dx.doi.org/10.1021/ja411096m | J. Am. Chem. Soc. 2014, 136, 449−457

Journal of the American Chemical Society
Article
dissection and gamma counting. Rats were anesthetized with a mixture
of 3−4% isoflurane in O2 for induction and reduced to 1−1.5% for
maintenance by a nose cone to maintain regular breathing at a
frequency of 60 10 breaths/minute monitored by a pressure sensor
(SA Instrument Inc., NY). Respiration and body temperature of the
animals were monitored throughout the study. The temperature,
measured rectally, was maintained at 37 1 °C using a water heating
blanket (Homeothermic Blanket Control Unit; Bruker, Germany). For
administration of the GNPs, the tail vein was catheterized with a 24-
gauge catheter and 3.7−22.2 MBq of GNPs (150−300 μL, 100−200
μg) were injected. Two hundred forty minutes after administration,
and without recovery from anesthesia, animals were perfused using
saline solution. The brain was quickly removed, rinsed with deionized
water, and measured in an automatic gamma counter (2470 Wizard,
Perkin-Elmer).
(16) Yan, H.; Wang, L.; Wang, J.; Weng, X.; Lei, H.; Wang, X.; Jiang,
L.; Zhu, J.; Lu, W.; Wei, X.; Li, C. ACS Nano 2012, 6, 410−420.
(17) Koffie, R. M.; Farrar, C. T.; Saidi, L-.J.; William, C. M.; Hyman,
B. T.; Spires-Jones, T. L. Proc. Natl. Acac. Sci. 2011, 108, 18837−
18842.
(18) Veiseh, O.; Sun, C.; Fang, C.; Bhattarai, N.; Gunn, J.; Kievit, F.;
Du, K.; Pullar, B.; Lee, D.; Ellenbogen, R. G.; Olson, J.; Zhang, M.
Cancer Res. 2009, 69, 6200−6207.
(19) Hwang, D. W.; Ko, H. Y.; Kim, S.-K.; Kim, D.; Lee, D. S.; Kim,
S. Chem.Eur. J. 2009, 15, 9387−9393.
(20) Guerrero, S.; Araya, E.; Fiedler, J. L.; Arias, J. I.; Adura, C.;
Albericio, F.; Giralt, E.; Arias, J. L.; Fernan
Nanomedicine 2010, 5 (6), 897−913.
́
dez, M. S.; Kogan, M. J.
(21) Guerrero, S.; Herance, J. R.; Rojas, S.; Mena, J. F.; Gispert, J. D.;
Acosta, G. A.; Albericio, F.; Kogan, M. J. Bioconjugate Chem. 2012, 23,
399−408.
ASSOCIATED CONTENT
* Supporting Information
(22) Yim, Y. S.; Choi, J.-s.; Kim, G. T.; Kim, C. H.; Shin, T.-H.; Kim,
D. G.; Cheon, J. Chem. Commun. 2012, 48, 61−63.
■
S
(23) Qiao, R.; Jia, Q.; Huvel, S.; Xia, R.; Liu, T.; Gao, F.; Galla, H.-J.;
̈
Supplemental figures, synthetic schemes, experimental proce-
dures, characterization of all new compounds including GNPs,
TEM images and size distribution histograms, and radio
labeling as well as control experiments. This material is available
free of charge via the Internet at http://pubs.acs.org.
Gao, M. ACS Nano 2012, 6, 3304−3310.
(24) Rana, S.; Bajaj, A.; Mout, R.; Rotello, V. M. Adv. Drug Delivery
Rev. 2012, 64, 200−216.
(25) García, I.; Marradi, M.; Penades
792.
(26) Marradi, M.; Chiodo, F.; García, I.; Penades
́
, S. Nanomedicine 2010, 5, 777−
́
, S. Chem. Soc. Rev.
AUTHOR INFORMATION
Corresponding Author
spenades@cicbiomagune.es
2013, 42, 4728−4745.
■
́
(27) Reichardt, N. C.; Martín-Lomas, M.; Penades, S. Chem Soc. Rev.
2013, 42, 4358−4376.
(28) (a) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164−
1167. (b) Fyrner, T.; Lee, H. H.; Mangone, A.; Ekblad, T.; Pettitt, M.
E.; Callow, M. E.; Conlan, S. L.; Mutton, R.; Clare, A. S.; Konradsson,
P.; Liedberg, B.; Ederth, T. Langmuir 2011, 27, 15034−15047.
(29) Van Rooy, I.; Cakir-Tascioglu, S.; Hennink, W. E.; Storm, G.;
Schiffelers, R. M.; Mastrobattista, R. Pharm. Res. 2011, 28, 456−471.
(30) Nicolazzo, J. A.; Charman, S. A.; Charman, W. N. J. Pharm.
Pharmacol. 2006, 58, 281−293.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Spanish Ministry of Science
(Grant Nos. CENIT AMIT CEN20101014 and CT2011-
27268) and the Basque Department of Industry (Grant No.
ETORTEK).
(31) Bilsky, E. J.; Egleton, R. D.; Mitchell, S. A.; Palian, M. M.; Davis,
P.; Huber, J. D.; Jones, H.; Yamamura, H. I.; Janders, J.; Davis, T. P.;
Porreca, F.; Hruby, V. J.; Polt, R. J. Med. Chem. 2000, 43, 2586−2590.
(32) Polt, R.; Porreca, F.; Szabo, L. Z.; Bilsky, E. J.; Davis, P.;
Abbruscato, T. J.; Davis, T. P.; Harvath, R.; Yamamura, H. I.; Hruby,
V. J. Proc. Natl. Acac. Sci. U.S.A. 1994, 91 (15), 7114−7118.
(33) Witt, K. A.; Davis, T. P. AAPS J. 2006, 8, E76−E88.
(34) Szeto, H. H.; Lovelace, J. L.; Fridland, G.; Soong, Y.; Fasolo, J.;
Wu, D.; Desiderio, D. M.; Schiller, P. W. J. Pharmacol. Exp. Ther. 2001,
298, 57−61.
REFERENCES
■
(1) Pardridge, W. M. J. Drug. Target 2010, 18 (3), 157−167.
(2) Nunes, A.; Al-Jamal, K. T.; Kostarelos, K. J. Controlled Release
2012, 161, 290−306.
(3) Miller, G. Science 2002, 297, 1116−1118.
(4) Praveen, B.; Alex, B.; Maiken, N. Neurobiol. Dis. 2004, 16, 1−13.
(5) Gabathuler, R. Neurobiol. Dis. 2010, 37, 48−57.
(6) Orive, G.; Anitua, E.; Pedraz, J. L.; Emerich, D. F. Nat. Rev.
Neurosci. 2009, 10, 682−692.
(7) Wong, H. L.; Chattopadhyay, N.; Wu, X. Y.; Bendayan, R. Adv.
Drug Delivery Rev. 2010, 62, 503−517.
(35) Riss, P. J.; Kroll, C.; Nagel, V.; Rosch, F. Bioorg. Med. Chem. Lett.
̈
2008, 18, 5364−5367.
(36) Cartier, R.; Kaufner, L.; Paulke, B. R.; Wustneck, R.;
Pietschmann, S.; Michel, R.; Bruhn, H.; Pison, U. Nanotechnology
2007, 18, 195102−195114.
(8) Malakoutikhah, M.; Teixido,
2011, 50, 7998−8014.
́
M.; Giralt, E. Angew. Chem., Int. Ed.
(37) Kim, J. S.; Kim, Y. H.; Kim, J. H.; Kang, K. W.; Tae, E. L.; Youn,
H.; Kim, D.; Kim, S- K.; Kwon, J.-T.; Cho, M.-H.; Lee, Y.-S.; Jeong, J.
M.; Chung, J-H; Lee, D. S. Nanomedicine 2012, 7 (2), 219−229.
(38) Lee, Y.-K.; J, M. J.; Hoigebazar, L.; Yang Bo, Y.; Lee, Y.-S.; Lee,
B. C.; Youn, H.; Lee, D. Soo.; Chung, J-K; Lee, M. C. J. Nucl. Med.
2012, 53, 1462−1470.
(39) Locatelli, E.; Gil, L.; Israel, L. L.; Passoni, L.; Naddaka, M.;
Pucci, A.; Reese, T.; Gomez-Vallejo, V.; Milani, P.; Matteoli, M.; Llop,
J.; Lellouche, J. P.; Franchini, M. C. Int. J. Nanomed. 2012, 7, 6021−
6033.
(9) Serwer, L. P.; James, C. D. Adv. Drug Delivery Rev. 2012, 64,
590−597.
(10) Huang, R.; Ke, W.; Liu, Y.; Jiang, C.; Pei, Y. Biomaterials 2008,
29, 238−246.
(11) Michaelis, K.; Hoffmann, M. M.; Dreis, S.; Herbert, E.;
Alyautdin, R. N.; Michaelis, M.; Kreuter, J.; Langer, K. J. Pharmacol.
Exp. Ther. 2006, 317, 1246−1253.
(12) Huwyler, J.; Wu, D.; Pardridge, W. M. Proc. Natl. Acac. Sci.
U.S.A. 1996, 93, 14164−14169.
(40) Clark, E. T.; Martell, A. E. Inorg. Chim. Acta 1991, 181, 273−
(13) Schwarze, S. R.; Ho, A.; Vocero-Akbani, A.; Dowdy, S. F. Science
1999, 285 (3), 1569−1572.
280.
́
(41) Martínez-Avila, O.; Hijazi, K.; Marradi, M.; Clavel, C.; Campion,
C.; Kelly, C.; Penades
, S. Chem.Eur. J. 2009, 15, 9874−9888.
(42) Barrientos, A. G.; de la Fuente, J. M.; Rojas, T. C.; Fernan
A.; Penades
(14) Li, J.; Feng, L.; Fan, L.; Zha, Y.; Guo, L.; Zhang, Q.; Chen, J.;
Pang, Z.; Wang, Y.; Jiang, X.; Yang, V. C.; Wen, L. Biomaterials 2011,
32 (21), 4943−4950.
(15) Kumar, P.; Wu, H.; McBride, J. L.; Jung, K.-E.; Kim, M. H.;
Davidson, B. L.; Lee, S. K.; Shankar, P.; Manjunath, N. Nature 2007,
448, 39−43.
́
́
dez,
́
, S. Chem.Eur. J. 2003, 9, 1909−1921.
(43) Costantino, L.; Gandolfi, F.; Tosi, G.; Rivasi, F.; Vandelli, M. A.;
Forni, F. J. Controlled Release 2005, 108, 84−96.
456
dx.doi.org/10.1021/ja411096m | J. Am. Chem. Soc. 2014, 136, 449−457

Journal of the American Chemical Society
Article
(44) Tosi, G.; Fano, R. A.; Bondioli, L.; Badiali, L.; Benassi, R.; Rivasi,
F.; Ruozi, B.; Forni, F.; Vandelli, M. A. Nanomedicine 2011, 6 (3),
423−436.
(45) Pert, C. B.; Pert, A.; Chang, J. K.; Fong, B. T. W. Science 1976,
194, 330−332.
(46) Vogel, Z.; Alstein, M. Prog. Biochem. Pharmocol. 1980, 16, 49−
59.
(47) Barrientos, A. G.; de la Fuente, J. M.; Jimen
́
ez, M.; Solís, D.;
Canada, D.; Martín-Lomas, M.; Penades
1474−1478.
́
, S. Carbohydr. Res. 2009, 344,
̃
(48) Hostetler, M. J.; Templeton, A. C.; Murray, R. W. Langmuir
1999, 15, 3782−3789.
(49) Velikyan, I.; Maecke, H.; Langstrom, B. Bioconjugate Chem.
2008, 19, 569−573.
(50) Slack, J. D.; Kanke, M.; Simmons, G. H; DeLuca, P. P. J. Pharm.
Sci. 1981, 6, 660−664.
(51) (a) Litzinger, D. C.; Buiting, A. M.; van Rooijen, N.; Huang, L.
Biochim. Biophys. Acta 1994, 1190 (1), 99−107. (b) Herant, M.;
Heinrich, V.; Dembo, M. J. Cell. Sci. 2006, 119, 1903−1913.
(52) Per
A.; Calvo-Fernan
ACS Nano 2013, 7 (4), 3498−3505.
́
ez-Campana, C.; Gom
́
ez-Vallejo, V.; Puigivila, M.; Martin,
̃
́
dez, T.; Moya, S. E.; Ziolo, R. F.; Reese, T.; Llop, J.
(53) Sun, G.; Hagooly, A.; Xu, J.; Nystrom, A. M.; Li, Z.; Rossin, R.;
Moore, D. A.; Wooley, K. L.; Welch, M. J. Biomacromolecules 2008, 9,
1997−2006.
(54) Semmler-Behnke, M.; Kreyling, W. G.; Lipka, J.; Fertsch, S.;
Wenk, A.; Takenaka, S.; Schmid, G.; Brandau, W. Small 2008, 4 (12),
2108−2111.
(55) (a) De Jong, W. H.; Hagens, W. I.; Krystek, P.; Burger, M. C.;
Sips, A. J. A. M.; Geertsma, R. E. Biomaterials 2008, 29, 1912−1919.
(b) Sonavanea, G.; Tomodaa, K.; Makino, K. Colloids Surf., B 2008, 66,
274−280. (c) Lasagna-Reeves, C.; Gonzalez-Romero, D.; Barria, M.
A.; Olmedo, I.; Clos, A.; Sadagopa Ramanujam, V. M.; Urayama, A.;
Vergara, L.; Kogan, M. J.; Soto, C. Biochem. Biophys. Res. Commun.
2010, 393, 649−655.
(56) Sa,
́ ́
A. D.; Prata, M. I. M.; Geraldes, C. F. G. C.; Andre, J. P. J.
Inorg. Biochem. 2010, 104, 1051−1062.
(57) Khlebtsov, N.; Dykman, L. Chem. Soc. Rev. 2011, 40, 1647−
1671.
(58) Longmire, M.; Choyke, P L.; Kobayashi, H. Nanomedicine 2008,
3, 703−717.
(59) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.;
Torchilin, V.; Langer, R. Science 1994, 263, 1600−1603.
(60) Lockman, P. R.; Oyewumi, M. O.; Koziara, J. M.; Roder, K. E.;
Mumper, R. J.; Allen, D. D. J. Controlled Release 2003, 93 (3), 271−
282.
(61) Xie, Y.; Ye, L.; Zhang, X.; Cui, W.; Lou, J.; Nagai, T.; Hou, X. J.
Controlled Release 2005, 105 (1−2), 106−119.
(62) Ke, W.; Shao, K.; Huang, R.; Han, L.; Liu, Y.; Li, J.; Kuang, Y.;
Ye, L.; Lou, J.; Jiang, C. Biomaterials 2009, 30 (36), 6976−6985.
(63) Hostetler, M. J.; Wingate, J. E.; Zhong, C.-J.; Harris, J. E.;
Vachet, R. W.; Clark, M. R.; Londono, J. D.; Green, S. J.; Stokes, J. J.;
Wignall, G. D.; Glish, G. L.; Porter, M. D.; Evans, N. D.; Murray, R. W.
Langmuir 1998, 14, 17−30.
457
dx.doi.org/10.1021/ja411096m | J. Am. Chem. Soc. 2014, 136, 449−457