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Nanoparticles enhance brain delivery of blood-brain barrier-impermeable probes for in vivo optical and magnetic resonance imaging

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Nanoparticles enhance brain delivery of blood-brain barrier-impermeable probes for in vivo optical and magnetic resonance imaging

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Several imaging modalities are suitable for in vivo molecular neuroimaging, but the blood-brain barrier (BBB) limits their utility by preventing brain delivery of most targeted molecular probes. We prepared biodegradable nanocarrier systems made up of poly(n-butyl cyanoacrylate) dextran polymers coated with polysorbate 80 (PBCA nanoparticles) to deliver BBB-impermeable molecular imaging probes into the brain for targeted molecular neuroimaging. We demonstrate that PBCA nanoparticles allow in vivo targeting of BBB-impermeable contrast agents and staining reagents for electron microscopy, optical imaging (multiphoton), and whole brain magnetic resonance imaging (MRI), facilitating molecular studies ranging from individual synapses to the entire brain. PBCA nanoparticles can deliver BBB-impermeable targeted fluorophores of a wide range of sizes: from 500-Da targeted polar molecules to 150,000-Da tagged immunoglobulins into the brain of living mice. The utility of this approach is demonstrated by (i) development of a "Nissl stain" contrast agent for cellular imaging, (ii) visualization of amyloid plaques in vivo in a mouse model of Alzheimer's disease using (traditionally) non-BBB-permeable reagents that detect plaques, and (iii) delivery of gadolinium-based contrast agents into the brain of mice for in vivo whole brain MRI. Four-dimensional real-time two-photon and MR imaging reveal that brain penetration of PBCA nanoparticles occurs rapidly with a time constant of ∼18 min. PBCA nanoparticles do not induce nonspecific BBB disruption, but collaborate with plasma apolipoprotein E to facilitate BBB crossing. Collectively, these findings highlight the potential of using biodegradable nanocarrier systems to deliver BBB-impermeable targeted molecular probes into the brain for diagnostic neuroimaging.
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Nanoparticles enhance brain delivery of bloodbrain
barrier-impermeable probes for in vivo optical and
magnetic resonance imaging
Robert M. Kofe
a,b
, Christian T. Farrar
c
, Laiq-Jan Saidi
a
, Christopher M. William
a
, Bradley T. Hyman
a
,
and Tara L. Spires-Jones
a,1
a
Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129;
b
Harvard Biophysics Program, Harvard
University, Boston, MA 02115; and
c
Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and
Harvard Medical School, Charlestown, MA 02129
Edited by William Klunk, University of Pittsburgh, Pittsburgh, PA, and accepted by the Editorial Board October 12, 2011 (received for review July 18, 2011)
Several imaging modalities are suitable for in vivo molecular
neuroimaging, but the bloodbrain barrier (BBB) limits their utility
by preventing brain delivery of most targeted molecular probes. We
prepared biodegradable nanocarrier systems made up of poly(n-bu-
tyl cyanoacrylate) dextran polymers coated with polysorbate 80
(PBCA nanoparticles) to deliver BBB-impermeable molecular imag-
ing probes into the brain for targeted molecular neuroimaging. We
demonstratethat PBCA nanoparticles allow in vivo targeting of BBB-
impermeable contrast agents and staining reagents for electron mi-
croscopy, optical imaging (multiphoton), and whole brain magnetic
resonance imaging (MRI), facilitating molecular studies ranging
from individual synapses to the entire brain. PBCA nanoparticles
can deliver BBB-impermeable targeted uorophores of a wide range
of sizes: from 500-Da targeted polar molecules to 150,000-Da tagged
immunoglobulins into the brain of living mice. The utility of this
approach is demonstrated by (i) development of a Nissl staincon-
trast agent for cellular imaging, (ii) visualization of amyloid plaques
in vivo in a mouse model of Alzheimers disease using (traditionally)
nonBBB-permeable reagents that detect plaques, and (iii)delivery
of gadolinium-based contrast agents into the brain of mice for
in vivo whole brain MRI. Four-dimensional real-time two-photon
and MR imaging reveal that brain penetration of PBCA nanoparticles
occurs rapidly with a time constant of 18 min. PBCA nanoparticles
do not induce nonspecic BBB disruption, but collaborate with
plasma apolipoprotein E to facilitate BBB crossing. Collectively,
these ndings highlight the potential of using biodegradable nano-
carrier systems to deliver BBB-impermeable targeted molecular
probes into the brain for diagnostic neuroimaging.
in vivo multiphoton imaging
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transgenic mice
The ability to image structure and function in the brain using
tools as diverse as multiphoton uorescence imaging and
magnetic resonance imaging (MRI) hold the promise of providing
insight into physiology and pathophysiological conditions, but are
greatly limited by the ability to deliver contrast agents with molec-
ular specicity across the bloodbrain barrier (BBB). Although
several molecular imaging contrast agents targeted to structures of
interest have been developed for research and clinical applications,
only a small fraction of them cross the BBB, including amyloid
binding dyes that required many years to develop (14). This leads
to surprising gaps in the ability to monitor cells and cellular pro-
cesses in the central nervous system (CNS), requiring skull burr
holes and topical application to visualize agents ranging from sim-
ple molecular Nissl stains to highly specic antibodies (57). Mul-
tiple approaches have been developed to nonspecically disrupt the
BBB to allow BBB-impermeable targeted molecular imaging pro-
bes entrance into brain parenchyma, but these approaches induce
uncontrolled neuronal injuries as well as allow circulating toxins
and neuroactive agents to get into the brain (8). There is therefore
a growing need to devise effective and safe ways to selectively
deliver BBB-impermeable targeted molecular imaging probes into
the brain for in vivo diagnostic molecular neuroimaging.
Biodegradable nanoparticles (NPs) are promising carrier sys-
tems for brain delivery of exogenous molecules (9). Among the
various brain delivery nanocarrier systems devised over the years,
polysorbate 80-coated poly(n-butyl cyanoacrylate) (PBCA) NPs
show tremendous promise. PBCA NPs have been explored to
deliver a number of BBB-impermeable drugs including growth
factors, antiepileptics, and chemotherapeutic agents into the brain
of animals with varying success (1014). Most studies demon-
strating use of PBCA NPs to deliver exogenous agents into the
brain, however, rely on secondary assessment of drug efcacy or
postmortem examination of brain tissue to ascertain BBB crossing;
direct real-time visualization of PBCA nanocarriers crossing the
BBB in living animals was previously unexplored, making it dif-
cult to determine whether this nanoparticulate approach could be
used for live in vivo diagnostic imaging of dynamic CNS processes
in living animals.
In this work, we investigate the potential of using PBCA NPs to
deliver BBB-impermeable molecular imaging contrast agents and
biologics into the brain of mice for in vivo multiphoton microscopy
and MRI. First, we use PBCA NPs to deliver three well-known
BBB-impermeable uorophores for in vivo imaging: one for im-
aging nuclei in the brain of wild-type mice and another two for
imaging neuropathological changes of Alzheimers disease (AD)
in transgenic mouse models. Second, using four-dimensional (4D)
real-time two-photon in vivo microscopy, we explore the kinetics
of PBCA NP-mediated delivery of one of these uorophores in the
brain of living intact animals. Third, we demonstrate that PBCA
NPs can be used to deliver not only uorophores into the brain for
optical imaging, but therapeutic antibodies and MR contrast
agents as well. Finally, we explore the mechanism by which PBCA
NPs cross the BBB and show that PBCA NPs do not non-
specically disrupt the BBB, but instead use endogenous lipidated
apolipoprotein E particles to facilitate BBB crossing. From the
scale of nanometer gold particles detected by transmission elec-
tron microscopy (TEM) postmortem, through micrometer reso-
lution using in vivo two-photon microscopy in animals with a
craniotomy, all of the way to MR imaging in completely intact
animals, we were able to observe the movement of compounds
bound to NPs into the brain parenchyma without a global
Author contributions: R.M.K., B.T.H., and T.L.S.-J. designed research; R.M.K., C.T.F., L.-J.S.,
C.M.W.,and T.L.S.-J. performed research; R.M.K. and C.T.F. analyzed data; andR.M.K., B.T.H.,
and T.L.S.-J. wrote the paper.
The authors declare no conict of interest.
This article is a PNAS Direct Submission. W.K. is a guest editor invited by the Editorial
Board.
1
To whom correspondence should be addressed. E-mail: tspires@partners.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1111405108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1111405108 PNAS Early Edition
|
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NEUROSCIENCE
disruption of the BBB. Collectively, our results demonstrate that
PBCA NPs can be used as effective nanocarriers to enhance de-
livery of BBB-impermeable targeted molecular imaging probes
for in vivo optical and MR imaging of the brain.
Results
To determine whether PBCA NPs coated with polysorbate 80 can
effectively deliver molecular imaging probes into the brain of living
animals, we synthesized PBCA NPs encapsulated with different
molecular imaging uorophores (Fig. S1 for characterization),
administered them i.v. into mice, and imaged the rst several
hundred micrometers of cortex in vivo using intravital multiphoton
microscopy through a craniotomy sealed with a glass coverslip. We
rst explored nanoparticle-assisted brain delivery of a small polar
DNA-binding dye bis-benzimide (Hoechst 33258, 531.86 Da M
r
;
Fig. S2 for structure). Previously, we have conrmed this dye as
useful for monitoring neuronal nuclei in vivo despite its tendency
to photobleach, but it previously required topical application to
the brain because it does not cross the BBB, precluding longitu-
dinal imaging (15). We conrmed that Hoechst 33258 alone dis-
solved in saline does not cross the BBB when administered i.v.
(Fig. 1Aand Movie S1). On the contrary, in vivo two-photon mi-
croscopy of cortical layers III of living anesthetized mice i.v.
injected with Hoechst 33258 encapsulated in polysorbate 80-
coated PBCA NPs reveals robust Hoechst uorescence in a pat-
tern of neuronal/glial nuclei beginning 30 min after injection,
peaking at 24 h, and persisting for 2 d (n= 8 mice) (Fig. 1Band
Movie S1). Postmortem imaging of brain slices of animals not
subjected to cranial window surgery revealed robust nuclear
staining throughout the brain in cohorts injected with Hoechst
adsorbed onto PBCA NPs, but not those injected with Hoechst
alone (Fig. S3), suggesting that PBCA NP-mediated delivery of
Hoechst into the brain is not an artifact of cranial window surgery
or imaging. PBCA NPs did not induce toxic histopathological
changes or overt physical distress in injected mice (Fig. S4).
Next, we explored the use of PBCA NPs as nanocarriers for
delivering targeted probes for imaging neuropathological lesions
of a neurodegenerative disease in living mice. Alzheimers disease
is characterized histopathologically by the deposition of neuritic
plaques made up of amyloid-beta (Aβ) peptides and neurobril-
lary tangles (NFTs) composed of hyperphosphorylated tau protein
(16). Signicant progress has been made in developing imaging
modalities for visualizing amyloid plaques in vivo with BBB-per-
meable agents including Pittsburgh compound B and methoxy
XO4 (1, 3). However, synthesizing additional BBB-permeable
high-afnity contrast agents that specically stain NFTs or soluble
oligomeric Aβ, which are believed to be the most toxic species in
the brain of AD patients (1720), remains elusive. We synthesized
PBCA NPs using dextran (70,000 Da M
r
) covalently conjugated to
Texas red (TX-red-Dx), a molecular imaging dye that binds senile
plaques ex vivo, but is routinely used to generate uorescence
angiograms during in vivo intravital imaging due to its long
vascular half-life and BBB impermeability. We conrm that TX-
red-Dx alone did not cross the BBB in 6-mo-old AD transgenic
mice (n= 6, APP
swe
/PS1
deltaE9
) and remained in blood vessels
even 2 h following i.v. injection (Fig. 2Aand Movie S2). Nano-
particulate integration of TX-red-Dx and polysorbate-80 coating,
however, allowed it to cross the BBB, labeling cerebral amyloid
angiopathy (CAA), amyloid plaques, and glial/neuronal cell
bodies (n= 6 mice; Fig. 2 Band Cand Movie S2) 2 h after i.v.
administration. NP-conjugated TX-red-Dx moved across the
BBB to stain CNS cell bodies, CAA, and plaques, resulting in
a dramatic 45% decrease in uorescence intensity in blood
vessels within 1 h after i.v. injection compared with only a 5%
decrease in uorescence intensity upon injecting TX-red-Dx un-
incorporated into PBCA NPs.
To further explore the potential of using PBCA NPs to deliver
targeted molecular imaging dyes that are noncovalently adsorbed
onto nanoparticles for imaging AD neuropathological changes
in vivo, we synthesized NPs loaded with Trypan blue (M
r
872.9 Da),
a plaque-binding red uorescing diazo dye that is well documented
not to cross the BBB (21, 22). We rst conrmed through confocal
imaging of postmortem brain slices that i.v. administration of
Trypan blue adsorbed onto PBCA NPs crosses the BBB and spe-
cically labels senile plaques in the brain of APP/PS1 mice (n=5)
after 2 h of injection, but not Trypan blue administered alone in
saline (Fig. S5). Trypan blue uorescence is not dependent on
target binding, making it particularly suitable for kinetic studies.
We therefore studied the kinetics of PBCA NP-mediated Trypan
blue delivery into the brain of APP/PS1 mice using in vivo 4D two-
photon microscopy and found that the uorophore had a circulat-
ing half-life of 60.6 ±8.2 min when adsorbed onto PBCA NPs (Fig.
3). Trypan blue uorescence signal in amyloid plaques was rst
detectable above noise within 10 min after injection and increased
progressively, peaking at 2 h following i.v. administration of
PBCA NPs with penetrating and plaque-binding time constants of
18.0 ±2.3 and 59.6 ±6.9 min, respectively (kinetics follow Boltz-
manns model equation) (Fig. 3Band Movie S3). Because in vivo
two-photon microscopy only allows visualization of tissue <400 μm
deep, we also carried out postmortem analysis of brain slices after
kinetic studies and conrmed that amyloid plaques throughout
cortical and subcortical regions of APP/PS1 mice are robustly
stained with Trypan blue (Fig. 3Cand Fig. S5). Counterstaining
these postmortem sections with thioavin S, a well-established
amyloid stain, reveals that 100% of thioavin S (ThioS)-positive
plaques were labeled with Trypan blue from the PBCA NP in-
jection (Fig. S5). This is a robust staining of Alzheimer pathology
with PBCA NP-bound dye. Collectively, these data indicate that
PBCA NPs can effectively deliver small BBB-impermeable tar-
geted optical probes for in vivo neuroimaging.
We next sought to determine whether PBCA NPs can deliver
therapeutic/imaging biological agents such as antibodies into the
brain after peripheral injection. Thus, we coated PBCA-NPs with
Alexa-488conjugated anti-Aβantibody (6E10), i.v. injected them
into APP/PS1 mice, and used in vivo multiphoton microscopy to
determine whether the antibodies cross the BBB. We found that
Alexa-488conjugated 6E10 antibodies cross the BBB upon ad-
sorption onto PBCA NPs and stain amyloid plaques in the brains
of APP/PS1 mice (n= 5) beginning at 15 min after injection and
peaking at 2 h (Fig. 4). Aβ-antibody binding to senile plaques in the
brain was conrmed postmortem by immunohistochemical sec-
ondary labeling with Cy3-conjugated antimouse IgG (Fig. 4).
Control experiments in which Alexa-488labeled anti-Aβanti-
bodies were i.v. injected without adsorption onto PBCA NPs
showed no Alexa-488 signal in plaques after2 h of imaging (Fig. 4).
Postmortem immunostaining with a different anti-Aβantibody
(R1282) raised in rabbit and costaining with antimouse-488 to
ensure all 6E10 PBCA NP signal was detected showed that 92.2%
of R1282-stained plaques were also labeled by 6E10 (Fig. 4 FH).
The 7.8% of plaques that were not labeled by the 6E10 PBCA NPs
Fig. 1. PBCA NPs deliver BBB-impermeable uorophores into mouse brain.
In vivo two-photon imaging of the brain of wild-type mice reveals that PBCA
NPs coated with polysorbate 80 efciently deliver BBB-impermeable optical
imaging uorophores into the brain of living anesthetized mice. Hoechst
alone dissolved in PBS does not cross the BBB after i.v. injection into mice (A),
but upon adsorbing Hoechst onto PBCA NPs and coating with polysorbate
80, Hoechst crosses the BBB and stains nuclei in the brain beginning at 30
min and persisting over 24 h (B). (Scale bar, 15 μm.)
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were small, with a diameter of <5μm. This is slightly less robust
than the 100% staining efciency observed with Trypan blue
PBCA NPs but still an impressive efciency for a large biological
molecule crossing the BBB. Together, these data suggest that
PBCA NPs are not only effective at brain delivery of small targeted
optical imaging dyes, but large biologic imaging/therapeutic anti-
bodies as well.
Because optical imaging techniques such as multiphoton mi-
croscopy are generally very sensitive and can detect very weak
uorescence signals in scattering tissue, it is unclear whether
PBCA NPs can be used to deliver sufcient amounts of targeted
molecular imaging contrast agents for clinically relevant imaging
technologies such as MRI, which requires substantial amounts of
contrast agents to enter the brain for contrast enhancement. We
therefore asked whether MRI contrast agents could be adsorbed
onto PBCA NPs and delivered into the brain for in vivo neuro-
anatomical MR imaging. To address this question, we adsorbed
gadobutrol (Gadovist)a 605-Da MRI gadolinium (Gd)-based
contrast agent routinely used in humans for imaging anatomical
lesionsonto PBCA NPs and administered them into wild-type
mice for neuroanatomical MRI. Moreover, we used inductively
coupled plasma-mass spectroscopy (ICP-MS) to quantify the
amount of gadolinium adsorbed onto PBCA NPs and delivered
into the brain so that we could precisely determine the efciency of
BBB transport. We found that 2.5 h after i.v. injecting gadobutrol
adsorbed onto PBCA NPs (10% adsorption efciency), 5.34% of
the injected nanoparticle-loaded gadolinium per gram of tissue
remained in the brain compared with only 0.009% injected dose of
free gadolinium per gram of tissue when gadobutrol is injected
without PBCA NPs; this corresponds to several hundred fold in-
crease in delivery of gadobutrol into the brain when PBCA NPs are
used. ICP-MS measurements at longer time points conrmed that
PBCA NP-loaded gadolinium was almost completely cleared from
the brain within 24 h after injection (Table S1).
After conrming that substantial amounts of gadolinium get
into the brain, we carried out serial T1-weighted gradient-echo
imaging of the brain of injected mice over a 2-h time period to
assess the kinetic properties of gadobutrol adsorbed PBCA NPs
entering the brain upon i.v. administration. We nd that PBCA
NPs deliver gadobutrol into the brain with a brain parenchyma
penetrating time constant of 19.5 min (kinetics follow mono-
exponential t), which is very similar to that observed by in vivo
two-photon microscopy using Trypan blue-loaded PBCA NPs
(Fig. 5). We note that because the MRI signal depends on both the
R1 relaxation rate (which is effected by the Gd concentration) as
well as the experimental repetition time (TR), the signicantly
increased Gd concentration translates into a signal increase of only
1.5 fold for a TR of 600 ms (Fig. 5C). Whereas some of the ob-
served signal increase in the MRI will be attributable to Gd in the
blood due to the 2-h half-life of the PBCA nanoparticles, it will
likely be a small contribution. First, the brain blood volume is only
3%. Second, because the gadobutrol is sequestered inside the
PBCA nanoparticle, the Gd relaxivity will depend on the water
exchange across the dextran-coated PBCA nanoparticle surface
and the encapsulated gadobutrol will likely be less effective at
relaxing the bulk vascular water than free gadobutrol. This hy-
pothesis is supported by the observed slow increase over time in
the brain MRI signal (Fig. 5C) and the slow decrease over time of
the T1 relaxation time (Fig. 5D), which can only be attributed to a
slowly increasing Gd concentration. Because the Gd concentration
in the blood vessels decreases by 50% over the 2-h time period,
the slowly decreasing T1 and increasing MRI signal can only be
explained by a slow increase in the tissue Gd concentration. These
data suggest the PBCA NPs enhance delivery of a clinically ap-
proved MRI contrast agent to the brain toan extent that is suitable
for MR imaging, indicating the possibility of delivering targeted
MRI contrast agents into the brain.
To more carefully characterize the cellular location of exoge-
nous molecules delivered into the brain using PBCA NPs, we
adsorbed 10 nm gold particles onto PBCA NPs and used TEM
to image the brain after injection of PCBA NPs coated with
10 nm gold particles. We observed gold in endothelial cell cyto-
plasm, basal lamina, neuropil structures including synapses, neu-
ronal soma, and glial soma, consistent with the multiphoton
Fig. 2. Texas red dextran covalently linked to PBCA NPs
crosses BBB and labels neuropathological changes of Alz-
heimers disease. In vivo two-photon imaging of the brain of
living mice (APP
swe
/PS1
deltaE9
) show that BBB-impermeable
uorophores covalently conjugated to PBCA NPs enter the
brain and stain senile plaques, neuropathological lesions of
AD. Administering Texas red dextran (70 kDa M
r
) alone
dissolved in PBS does not cross the BBB, remaining in blood
vessels after 2 h following i.v. injection (A). On the contrary,
covalently incorporating Texas red into PBCA NPs and
coating NPs with polysorbate 80 allows them to cross the
BBB and stain amyloid plaques (arrow), cerebral amyloid
angiopathy, and neuronal/glial cell bodies (Band C).
Quantication of uorescence intensity in these experi-
ments revealed a 10-fold increase of TX-red-Dx signal in the
brain parenchyma and robust staining of amyloid plaques
in vivo upon delivering the uorophore using PBCA NPs
compared with TX-red-Dx injected alone (D). (Scale bars in B
and C,20μm.)
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observations of widespread distribution of nanoparticles coated
with uorophores (Fig. S6). Gold particles were not observed in
the brain of an animal injected with gold particles not adsorbed
onto PBCA NPs. This nding conrms at an ultrastructural sub-
cellular resolution that PBCA NPs effectively deliver exogenous
BBB-impermeable molecules into the brain parenchyma.
Having demonstrated that PBCA NPs can effectively deliver
molecular imaging dyes and biologic probes into the brain, we next
wanted to determine the mechanism by which they cross the BBB.
On the basis of size (200 nm mean hydrodynamic diameter), it is
very unlikely that PBCA NPs cross the BBB through passive par-
acellular diffusion. To determine whether PBCA NPs non-
specically disrupt the BBB, as has been previously reported in a
porcine in vitro model (23), we carried out three control experi-
ments. First, we imaged the brains of living animals injected with
two different dyes using in vivo multiphoton microscopyrst
with Hoechst adsorbed onto PBCA NPs, and then with Texas red
dextran (70 kDa M
r
) in saline without NPs i.v. injected 30 min
afterward. We found that Hoechst adsorbed onto PBCA NPs
crossed the BBB and stained neuronal and glial nuclei in vivo,
whereas Texas-red-Dx remained in cerebral blood vessels without
evidence of substantial leakage (n=5;Fig.6A). In the inverse
experiment, injection of Texas-red-Dx incorporated in PBCA NPs
followed 30 min later by unbound 1 mM Hoechst-labeled plaques
and neurons in APP/PS1 transgenic brain without any Hoechst
labeling in the parenchyma (Fig. 6A,Inset). Second, we used dy-
namic contrast enhanced (DCE) MRI to visualize the brains of
living intact wild-type mice to test whether PBCA NPs induce BBB
leakage of gadolinium-dithylenetiamine penta-acetic acid (Gd-
DTPA, 545.6 Da M
r
), a BBB-impermeable MR contrast agent
used to assess cerebral vascular integrity. DCE-MRI sequences
were performed 30 min following i.v. administration of PBCA NPs
loaded with Hoechst (n= 3 mice). DCE MR images revealed
strong Gd-DTPA signal enhancement in blood vessels and muscle
tissue within minutes after injection (Fig. 6 Band C), but no in-
crease in Gd-DTPAinduced signal intensity changes in the brain.
Third, we examined the BBB of animals that had been injected
with nanoparticles coated with 10 nm colloidal gold using TEM
and found that the BBB appears structurally completely normal
(Fig. 6D). Together these results show that PBCA NPs cross the
BBB without inducing nonspecic disruption of the BBB in the
intact brain in contrast to data derived from an in vitro model of
the BBB (23).
We next wanted to identify molecular candidates that may be
involved in transporting PBCA NPs across the BBB. It has been
hypothesized that biodegradable nanoparticles adsorb apolipo-
protein E (apoE) particles in plasma onto their surfaces and cross
the BBB via receptor-mediated transcytosis (24). To determine
whether or not apoE is necessary for PBCA NPs to cross the BBB,
we i.v. injected Hoechst-encapsulated PBCA NPs into APOE
knockout (APOE
/
) mice and then used multiphoton microscopy
to image the brain in vivo. We nd the Hoechst-loaded poly-
sorbate-80 coated PBCA NPs do not cross the BBB in APOE
/
mice (n=3;Fig.6E). However, preincubating Hoechst-loaded
PBCA NPs in lipidated apoE particles for 2 h at 25 °C before
injecting the NPs i.v. into APOE
/
mice allowed them to cross the
BBB, releasing Hoechst to robustly stain neuronal/glial nuclei
Fig. 3. Trypan blue delivered to brain of AD transgenic mouse using PBCA
NPs. Four-dimensional in vivo multiphoton microscopy of the brain of living
anesthetized AD transgenic mice (APP
swe
/PS1
deltaE9
) shows that PBCA NPs
coated with polysorbate-80 deliver Trypan blue into the brain. Snapshots of
the brains of AD transgenic mice injected with NPs loaded with Trypan blue
obtained at the indicated times via in vivo two-photon microscopy are
shown (A). Quantitative analysis of the kinetics of NP-mediated delivery of
Trypan blue into the brain reveals an increase in Trypan blue uorescence in
plaques that follow a sigmoidal Boltzmann equation with time constants
t
o
= 18 min, corresponding to BBB crossing, and Δτ = 60 min, corresponding
plaque labeling. The half-life of Trypan blue adsorbed onto NPs in the vessels
is 61 min (B). Postmortem histological analysis reveals that Trypan blue de-
livered to the brain using NPs stains plaques throughout both cortical and
subcortical regions of the brain.
Fig. 4. PBCA NPs deliver uorescently labeled Aβantibodies into mouse
brain. Aβantibodies (6E10) conjugated to Alexa-488 were adsorbed onto
PBCA NPs and injected i.v. into 6-mo-old APP/PS1 mice. In vivo two-photon
imaging of brains was carried out 2 h after injecting PBCA NPs loaded with
6E10Alexa-488. Administrating free 6E10Alexa-488 antibodies i.v. and
imaging the brain revealed no staining of senile plaques (A). Upon injecting
PBCA NPs loaded with 6E10Alexa-488, some uorescence signal was
detected in amyloid plaques (arrow) within the brain of APP/PS1 mice (B).
Postmortem analysis of the brain of PBCA NPs injected animals reveals ro-
bust amyloid plaque staining (C), both with the labeled 6E10Alexa-488
(green) and with a secondary antimouse IgG antibody labeled with Cy3 (red)
(D). Of note, a subset of diffuse plaques was neither seen in vivo nor post-
mortem with 6E10488, but was clearly seen upon addition of the Cy3 sec-
ondary antibody (arrowhead in D). Merge of Cand Dis shown in E.
Immunostaining with a different Aβantibody, R1282,and secondary anti-
rabbit Cy3 and antimouse-488 (to detect all 6E10 injected with NPs), shows
that the vast majority of plaques (92.2%) are labeled by 6E10 (FH). Large
plaques are very strongly labeled, whereas smaller plaques occasionally had
only weak 6E10 staining (arrow) and 7.8% of plaques were unlabeled with
6E10. These unlabeled plaques were all <5μm in diameter (circle). (Scale bars
in Band E,50μm; in H,20μm.)
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(Fig. 6E). These ndings support the hypothesis that PBCA NPs
cross the BBB through a mechanism that involves the adsorption
of apoE from plasma, making receptor-mediated transcytosis
through vascular endothelial cells the likely mechanism by which
PBCA NPs cross the BBB.
Discussion
Breakthroughs in molecular imaging continue to revolutionize
medicine and biological research, yet one area that has been
challenging involves imaging molecular targets in the brain in real-
time, in part due to difculties in developing contrast agents with
target specicity that also cross the BBB. Enhancing delivery of
BBB-impermeable contrast agents and molecular imaging probes
therefore has the potential to greatly increase the utility of imaging
techniques for visualizing specic targets in the brain. We in-
vestigated the use of nanocarrier systems, specically PBCA NPs
coated with polysorbate 80, for delivering BBB-impermeable
molecular imaging contrast agents into the brain. We demon-
strated that (i) PBCA NPs can efciently and safely deliver BBB-
impermeable optical molecular imaging uorophores into the
brain in mice to allow in vivo imaging of neuronal and glial nuclei;
(ii) PBCA NPs can serve as nanocarriers for transporting BBB-
impermeable targeted uorophores for imaging neuropathologi-
cal changes in a neurodegenerative disease, Alzheimers; (iii)
PBCA NPs can be used to deliver biologics such as antibodies into
the brain; and (iv) PBCA NPs can deliver sufcient amounts of
BBB-impermeable molecular contrast agents to allow imaging of
the brain using clinically relevant techniques such as MRI with
improved signal-to-noise ratio. Using 4D multiphoton microscopy,
DCE-MRI, and ICP-MS, we carefully studied the kinetics of
PBCA NPs-mediated delivery of BBB-impermeable molecules
into the brain and found that NPs deliver >5% of nanoparticle-
adsorbed administered dose of BBB-impermeable molecular
probes into the brain with a time constant of 18 min. We further
show that PBCA NPs do not induce nonspecic disruption of the
BBB, but cross the BBB using a mechanism that requires endog-
enous apoE, suggesting that PBCA NPs are a safe and viable ap-
proach for delivering BBB-impermeable molecules into the brain.
Several nonnanotechnology-based methods have been in-
vestigated to noninvasively deliver BBB-impermeable molecules
into the brain. Approaches involving nonspecically disrupting
the BBB using hypertonic osmotic agents, bradykinins, and
alkylglycerols have all been demonstrated to be effective at en-
hancing delivery of BBB-impermeable drugs into the brain (2530).
Focused ultrasound beams (FUSB), electromagnetic radiation
(EMR), and recently focused pulsed laser disruption of vessel walls
have also been used to disrupt the BBB to enhance delivery of u-
orophores into the brain with some success (22, 3134). However,
all of these chemical and mechanical approaches nonspecically
disrupt BBB integrity; thus, their use for molecular imaging is lim-
ited because they increase the inux of toxins into the brain and
inuenceCNS biology in ways that cannot be adequately controlled.
The use of nanotechnology to transport exogenous molecules
into the brain provides a simple and effective alternative approach
Fig. 5. PBCA NPs enhance delivery of MRI contrast agent into the brain.
Serial signal intensity measurements of T1 gradient-echo MR images of wild-
type mouse brain before and after i.v. administration of polysorbate 80-
coated PBCA NPs loaded with gadobutrol reveal contrast enhancement of
brain parenchyma over time. Whereas gadobutrol enters muscles almost
instantaneously upon delivery with nanoparticles, the kinetics of gadobutrol
signal in the brain followed a monoexponential model with a time constant
of 19 min (A). T1 decreased by 25% over 2 h with kinetics that followed
a monoexponential decay [T
1
= 1074 + 386*exp(t/τ); time constant τ= 29.7
min] (B). Sample T1 gradient-echo images showing an increase in gadobutrol
signal in the brain over time is also shown (C).
Fig. 6. PBCA NPs do not induce nonspecic BBB disruption but require apoE
to cross BBB. Injecting PBCA NPs loaded with Hoechst and then injecting
Texas red dextran dissolved in PBS (not on NPs) 30 min afterward and im-
aging the brain through a cranial window using in vivo two-photon mi-
croscopy reveals robust Hoechst staining of nuclei without leakage of Texas
red dextran into the brain (A). Conversely, injecting Texas red dextran in-
corporated into PBCA NPs followed 30 min later by unbound Hoechst
resulted in plaque and intraneuronal labeling with Texas red and no pa-
renchymal Hoechst labeling (A,Inset). Dynamic contrast enhanced MRI se-
quence consisting of 100 repetitions of T1-weighted gradient-echo sequence
with a temporal resolution of 4.8 s in the brains of mice injected with Gd-
DTPA 30 min after PBCA NPs loaded with Hoechst were i.v. injected reveals
robust increase of Gd-DTPA signal intensity in muscle but not the brain,
suggesting that PBCA NPs do not induce nonspecic disruption of the BBB (B
and C). In animals injected with nanoparticles, TEM conrms an intact BBB
with normal endothelial cells (e), basal lamina (bl), and astrocytic processes
(AsP) surrounding capillaries (cap) (D). Unlike wild-type mice, APOE knock-
out mice i.v. injected with polysorbate 80-coated PBCA NPs loaded with
Hoechst did not have robust nuclei staining with Hoechst >2 h after NP in-
jection (E). Preincubating Hoechst-loaded PBCA NPs with astrocyte-secreted
apoE particles for 2 h and then i.v. administering NPs into APOE knockout
mice allowed Hoechst to cross the BBB, robustly staining neuronal and glial
nuclei (E). Images in Erepresent a z-projection of a stack of seven images.
[Scale bar, 100 μm(A), 20 μm(A,Inset), 500 nm (D).]
Kofeetal. PNAS Early Edition
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5of6
NEUROSCIENCE
(35). Most previous studies using nanoparticulate delivery systems
for getting BBB-impermeable compounds into the brain have fo-
cused on drug delivery. For instance, multiple studies have shown
that PBCA NPs can be used to deliver a variety of small polar drugs
into the brain (1013, 35, 36). In all of these studies, however, the
level of brain delivery of exogenous molecules was not directly vi-
sualized inrealtime,butinstead indirect reporters such as assessing
drug effects were used to evaluate BBB crossing. We took advan-
tage of advanced molecular imaging techniques to directly dem-
onstrate that PBCA NPs can be used to deliver targeted BBB-
impermeable contrast agents into the brain in sufcient amounts to
allow in vivo optical imaging of cellular and neuropathological
structures and BBB penetration ofMR contrast agents inthe brain
of living mice. We characterize the kinetics of PBCA NPs-mediated
BBB crossing as well as the amount of exogenous molecules de-
livered to the brain upon loading onto PBCA NPs and also dem-
onstrate that PBCA NPs require adsorption of plasma apoE to
cross the BBB. Future studies will be able to apply this technology
to a wide variety of imaging modalities that will be useful in animal
and human brain including near infrared imaging and using tar-
geted MR probes. We anticipate that these ndings will provide
a technique to allow advances in in vivo neuroimaging at the cel-
lular and systems level by helping to overcome the challenges posed
to contrast agent and ligand development by the BBB.
Materials and Methods
Preparation and Characterization of Nanoparticles. All protocols for preparing
and characterizing poly(N-butyl cyanoacrylate) nanoparticles are outlined in
SI Materials and Methods.
Transmission Electron Microscopy. All details for TEM experiments are as
outlined in SI Materials and Methods.
Animal Surgeries and Imaging. Detailed surgery and imaging protocols are
outlined in SI Materials and Methods.
Inductively Coupled Plasma-Mass Spectroscopy. Detailed procedures for ICP-
MS are outlined in SI Materials and Methods.
Magnetic Resonance Imaging Experiments. Dynamic contrast enhanced MRI
and serial T1-weighted MRI experimental protocols are as detailed in SI
Materials and Methods.
ACKNOWLEDGMENTS. We thank Dr. Marian DiFiglia for use of the trans-
mission electron microscope and Dr. Peter Caravan for the use of the ICP-MS.
This work was supported by National Institutes of Health (NIH) Grants K99
AG033670, K25 AG029415, K08 NS069811, P50 AG005134, AG12406, and
AG08487, and a Coins for Alzheimers Research Trust award. R.M.K. is sup-
ported by the Harvard Biophysics and Medical Scientist Training Programs
(NIH T32 GM07753) and the Paul and Daisy Soros Foundation. L.-J.S. is sup-
ported by the Foundation Family Klee Young Scientist Award.
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www.pnas.org/cgi/doi/10.1073/pnas.1111405108 Kofeetal.
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Alzheimer disease (AD) is an illness that can only be diagnosed With certainty with postmortem examination of brain tissue. Tissue samples from afflicted patients show neuronal loss, neurofibrillary tangles (NFTs), and amyloid-beta plaques. An imaging technique that permitted in vivo detection of NFTs or amyloid-beta plaques would be extremely valuable. For example, chronic imaging of senile plaques would provide a readout of the efficacy of experimental therapeutics aimed at removing these neuropathologic lesions. This review discusses the available techniques for imaging amyloid-beta deposits in the intact brain, including magnetic resonance imaging, positron emission tomography, single photon emission computed tomography, and multiphoton microscopy. A variety of agents that target amyloid-beta deposits specifically have been developed using one or several of these imaging modalities. The difficulty in developing these tools lies in the need for the agents to cross the blood-brain barrier while recognizing amyloid-beta with high sensitivity and specificity. This review describes the progress in developing reagents suitable for in vivo imaging of senile plaques.
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Alzheimer disease (AD) is an illness that can only be diagnosed with certainty with postmortem examination of brain tissue. Tissue samples from afflicted patients show neuronal loss, neurofibrillary tangles (NFTs), and amyloid-β plaques. An imaging technique that permitted in vivo detection of NFTs or amyloid-β plaques would be extremely valuable. For example, chronic imaging of senile plaques would provide a readout of the efficacy of experimental therapeutics aimed at removing these neuropathologic lesions. This review discusses the available techniques for imaging amyloid-β deposits in the intact brain, including magnetic resonance imaging, positron emission tomography, single photon emission computed tomography, and multiphoton microscopy. A variety of agents that target amyloid-β deposits specifically have been developed using one or several of these imaging modalities. The difficulty in developing these tools lies in the need for the agents to cross the blood–brain barrier while recognizing amyloid-β with high sensitivity and specificity. This review describes the progress in developing reagents suitable for in vivo imaging of senile plaques.
Poly(butyl cyanoacrylate) nanoparticles coated with polysorbate 80 (Tween 80) enable the transport of a number of drugs across the blood-brain barrier (BBB) into the brain following intravenous injection. These drugs include the hexapeptide endorphin dalargin, the dipeptide kytorphin, loperamide, tubocurarine, doxorubicin, and the NMDAreceptor antagonists MRZ 2 / 576 and MRZ 2 / 596.After binding to the polysorbate-coated particles, dalargin as well as loperamide exhibited a dose-dependent antinociceptive effect after i.v. injection as determined by the tail-flick as well as by the hot plate test. This effect was accompanied by a Straub reaction and was totally inhibited by pretreatment with naloxone, indicating that it is a central effect and not peripheral analgesia. After brain perfusion of rats with tubocurarine bound to the polysorbate-coated nanoparticles epileptic spikes were observable in the EEC of the rats but not with the controls. Other very interesting results were obtained with the NMDA-receptor antagonists MRZ 2 / 576 and MRZ 2 / 596. The very short anticonvulsive response of MRZ 2 / 576 was increased from below 30 to 300 min, and the transport across the BBB of the non-penetrating MRZ 2 / 596 was enabled after i.v. injection. Intravenous injection of polysorbate 80-coated nanoparticles loaded with doxorubicin (5 mg / kg) achieved very high brain levels of 6 g / g brain tissue while all the controls, including uncoated nanoparticles and doxorubicin solutions mixed with polysorbate, did not reach the analytical detection limit of 0.1 g / g. Moreover, experiments with the extremely aggressive glioblastoma 101 / 8 transplanted intracranially showed a long term survival for 6 months of 40 % of the rats after intravenous injection of the polysorbate 80-coated nanoparticle preparation (3 x 1.5 mg / kg). The surviving animals were sacrificed after this time and showed total remission by histological investigation. Untreated controls died within 10 - 20 days, the animals in the six other control groups between 10 - 50 days.The mechanism of the drug transport across the blood-brain barrier with the nanoparticles requires further elucidation. The most likely mechanism at present appears to be endocytotic uptake by the brain capillary endothelial cells followed either by release of the drugs in these cells and diffusion into the brain or by transcytosis. Endocytotic uptake of the polysorbate-coated nanoparticles but not of uncoated particles has been shown with bovine, murine, rat, and human brain capillary endothelial cells. After injection of the nanoparticles, apolipoprotein E (apo E) or apo B adsorption of the particles seems to occur as already shown in vitro, followed by interaction with the LDL receptor and endocytotic uptake. This scenario is rather likely since both apolipoproteins can interact with the LDL-receptor. They may then be taken up like the naturally occurring lipoprotein particles.
Article
In previous studies it was shown that polysorbate 80(PS80)-coated poly(n-butylcyano-acrylate) nanoparticles (PBCA-NP) are able to cross the blood-brain barrier (BBB) in vitro and in vivo. In order to explore and extend the potential applications of PBCA-NP as drug carriers, it is important to ascertain their effect on the BBB. The objective of the present study was to determine the effect of PS80-coated PBCA-NP on the BBB integrity of a porcine in vitro model. This has been investigated by monitoring the development of the transendothelial electrical resistance (TEER) after the addition of PBCA-NP employing impedance spectroscopy. Additionally, the integrity of the BBB in vitro was verified by measuring the passage of the reference substances (14)C-sucrose and FITC-BSA after addition of PBCA-NP. In this study we will show that the application of PS80-coated PBCA-NP leads to a reversible disruption of the barrier after 4h. The observed disruption of the barrier could also be confirmed by (14)C-sucrose and FITC-BSA permeability studies. Comparing the TEER and permeability studies the lowest resistances and maximal values for permeabilities were both observed after 4h. These results indicate that PS80-coated PBCA-NP might be suitable for the use as drug carriers. The reversible disruption also offers the possibility to use these particles as specific opener of the BBB. Instead of incorporating the therapeutic agents into the NP, the drugs may cross the BBB after being applied simultaneously with the PBCA-NP.
Article
Aggregation of amyloid beta peptide into senile plaques and hyperphosphorylated tau protein into neurofibrillary tangles in the brain are the pathological hallmarks of Alzheimer's disease. Despite over a century of research into these lesions, the exact relationship between pathology and neurotoxicity has yet to be fully elucidated. In order to study the formation of plaques and tangles and their effects on the brain, we have applied multiphoton in vivo imaging of transgenic mouse models of Alzheimer's disease. This technique allows longitudinal imaging of pathological aggregation of proteins and the subsequent changes in surrounding neuropil neurodegeneration and recovery after therapeutic interventions.