Environment-responsive Nanophores for Therapy and Treatment Monitoring via Molecular MRI Quenching

Abstract

The effective delivery of therapeutics to disease sites significantly contributes to drug efficacy, toxicity and clearance. Here we demonstrate that clinically approved iron oxide nanoparticles (Ferumoxytol) can be utilized to carry one or multiple drugs. These so called 'nanophores' retain their cargo within their polymeric coating through weak electrostatic interactions and release it in slightly acidic conditions (pH 6.8 and below). The loading of drugs increases the nanophores' transverse T2 and longitudinal T1 nuclear magnetic resonance (NMR) proton relaxation times, which is proportional to amount of carried cargo. Chemotherapy with translational nanophores is more effective than the free drug in vitro and in vivo, without subjecting the drugs or the carrier nanoparticle to any chemical modification. Evaluation of cargo incorporation and payload levels in vitro and in vivo can be assessed via benchtop magnetic relaxometers, common NMR instruments or magnetic resonance imaging scanners.

Full text

Environment-responsive Nanophores for Therapy and Treatment
Monitoring via Molecular MRI Quenching
Charalambos Kaittanis‡, Travis M. Shaffer‡, Anuja Ogirala‡, Santimukul Santra¶, J. Manuel
Perez¶, Gabriela Chiosis‡, Yueming Li‡, Lee Josephson#, and Jan Grimm*,‡
‡Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center,
1275 York Ave, New York, NY 10065, United States
¶NanoScience Technology Center, University of Central Florida, 12424 Research Pkwy, Suite
400, Orlando, FL 32826, United States
#Center for Translational Nuclear Medicine and Molecular Imaging, Massachusetts General
Hospital, Building 149, 13th Str, Charlestown, MA 02129, United States
Abstract
The effective delivery of therapeutics to disease sites significantly contributes to drug efficacy,
toxicity and clearance. Here we demonstrate that clinically approved iron oxide nanoparticles
(Ferumoxytol) can be utilized to carry one or multiple drugs. These so called ‘nanophores’ retain
their cargo within their polymeric coating through weak electrostatic interactions and release it in
slightly acidic conditions (pH 6.8 and below). The loading of drugs increases the nanophores’
transverse T2 and longitudinal T1 NMR proton relaxation times, which is proportional to amount
of carried cargo. Chemotherapy with translational nanophores is more effective than the free drug
in vitro and in vivo, without subjecting the drugs or the carrier nanoparticle to any chemical
modification. Evaluation of cargo incorporation and payload levels in vitro and in vivo can be
assessed via benchtop magnetic relaxometers, common NMR instruments or MRI scanners.
Introduction
The effective delivery of therapeutics is critical for treatment. Drugs must stay in circulation
for adequate time, avoiding clearance by the liver and kidneys and achieve sufficiently high
accumulation in the site of the disease, in order to maximize therapeutic efficacy and
minimize side-effects. Towards this goal, researchers have utilized innovative strategies,
*Corresponding Author: To whom correspondence should be addressed. Jan Grimm: Tel. 646-888-3095, Fax. 646-888-3059,
grimmj@mskcc.org. Program in Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, 1275 York Ave.,
New York, NY 10065, USA.
Author contributions
C.K. and J.G. contributed the original idea. C.K., J.M.P, L.J. and J.G. designed the experiments. J.G. obtained funding and supervised
the project. C.K., T.M.S, A.O. and S.S. performed the experiments. Y.L. and G.C. provided materials, and all authors contributed to
discussions regarding the research. C.K. and J.G. wrote the manuscript.
Additional information
The authors declare no competing financial interests. Supplementary information accompanies this paper at www.nature.com/
ncomms. Reprints and permission information is available online at http://www.nature.com/reprints. Correspondence and requests for
materials should be addressed to J.G.
NIH Public Access
Author Manuscript
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Published in final edited form as:
Nat Commun. ; 5: 3384. doi:10.1038/ncomms4384.
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including the modification of drugs like Doxorubicin with polymers and targeting moieties,
in order to achieve delivery to tumors via the enhanced permeability and retention (EPR)
effect or the targeting of overexpressed surface markers.1–4 Apart from receptors involved in
signal transduction, nutrient receptors, such as the folate or transferrin receptor, were
targeted for the delivery of chemotherapies.5–7 Furthermore, in order to determine the
distribution of these molecular constructs, tracers or fluorochromes were covalently
conjugated to them.8–11 However, since the parent drugs have undergone modifications,
including addition of new bonds, functional groups and entire molecules to either achieve
targeting or monitoring, these therapeutic agents face extensive scrutiny from regulatory
bodies.12–14
An appealing alternative is the encapsulation of therapeutics within nanoparticles that
provide aqueous stability and longer circulation times, without subjecting the drug to any
chemical modification.15–17 For instance, liposomal formulations of chemotherapeutics and
antifungals, like Doxorubicin (Doxil) and Amphotericin B (AmBisome), are in clinical use
and provide improved pharmacokinetics and ability to deliver high loads of drugs with
otherwise poor aqueous solubility. Drug delivery occurs upon fusion with the plasma
membrane or action of lipases.18, 19 Alternatives to liposomes are nanoparticles that consist
of polymers, like poly (lactic-co-glycolic) acid (PLGA) and hyperbranched polyesters
(HBPE), which can be hydrolyzed in vivo by enzymes, like esterases, and acidic
conditions.20–22 These nanoparticles delivered drugs, like Taxotere, in cultured cells and
animal models. The encapsulation process resulted in loading of the drugs within the
nanoparticle’s cavity and allowed the use of the nanoparticle’s surface functional groups for
further bioconjugation of targeting moieties. However, although targeted nanoparticles can
be developed for both drug delivery and imaging via clinical diagnostic modalities, these
nanoparticles have not been investigated by regulatory agencies.23
Iron oxide nanoparticles (IONP) formulations have been used as contrast agents for
Magnetic Resonance Imaging (MRI). Currently, Ferumoxytol (Feraheme®) is used in the
clinic for the treatment of iron deficiency. These nanoparticles consist of iron and a
carbohydrate (dextran) and are well tolerated, without any side-effects and toxicity.
Therefore, we investigated whether Ferumoxytol can serve as a magnetic drug carrier
suitable to carry several hydrophobic drugs after facile loading through co-incubation and
improve their therapeutic efficacy, without further modification of either the nanoparticle or
drugs. Such a drug delivery system promises to move faster to the clinic, since it is based on
an already clinically approved vehicle (Feraheme®) and simply takes up the drug without
chemical reactions, therefore the drug-loaded particles were aptly termed nanophores.
Additionally, developing a facile method to load different drugs on a common delivery
platform has the unique potential of being readily adopted in clinic. We also hypothesized
that the drug loading could be monitored through magnetic resonance imaging, since
IONP’s magnetic properties have been previously used in sensitive assays. These assays rely
on the nanoparticles’ ability to affect the proton nuclear magnetic resonance (NMR) signal
of the surrounding water molecules. Specifically, IONP’s primarily affect the transverse
(spin-spin; T2) relaxation time of bulk water protons, facilitating sensitive quantification and
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imaging with compact relaxometers, NMR or magnetic resonance imaging (MRI)
instruments.24–30
Herein, we report that magnetic nanophores can be a self-reporting delivery vehicle for
many chemotherapeutics. When the cargo is loaded, the nanoparticle’s magnetic properties
are affected, with the relaxation times T2 and T1 increasing. Diffusion MRI, which allows
the measurement of water diffusion in a given voxel through the apparent diffusion
coefficient (ADC), reveals that the cargo hinders water’s diffusion within the nanoparticles’
coating as probable reason for the induced changes in relaxivity. After establishing this
optical-independent method to characterize drug-loaded nanophores, we show that drug-
loaded Ferumoxytol deliver a more efficient therapy than free drugs in vitro and most
importantly in vivo, for the rational inhibition of select oncogenic pathways.
Clinical iron oxide nanoparticles as drug nanophores
Considering the need for translational drug delivery platforms and the already existing
clinical use of Ferumoxytol, we first investigated whether Ferumoxytol could be used as a
nanophore for the delivery of drugs. Hence, we encapsulated therapeutics and fluorophores
within Ferumoxytol, using the solvent diffusion method, in order to facilitate the facile
entrapment of hydrophobic molecules within Ferumoxytol’s carboxymethyl dextran coating.
After dialyzing the nanoparticles in order to remove any free cargo from the solution, we
determined that Ferumoxytol could carry different amounts of the fluorescent Taxol analog
Flutax1 (MW: 1337), without affecting the nanoparticles’ physical properties (Fig. 1a,
Supplementary Fig. 1a–b). The encapsulation process had a minimum of 80% loading
efficiency for Flutax1, as determined through UV-vis spectroscopy after dialysis of the
Flutax1-loaded nanophores in order to remove free cargo, suggesting that large amounts of
payload could be entrapped within Ferumoxytol’s coating (Fig. 1b). Furthermore, we
demonstrated that Ferumoxytol could also carry smaller compounds, such as the near-
infrared fluorophore DiR and the chemotherapeutic Doxorubicin, with molecular weights of
1013 and 580 respectively. Dynamic light scattering (DLS) analysis revealed that drug
loading did not affect the average diameter of Ferumoxytol, indicating that the cargo and the
encapsulation process do not affect the nanoparticles’ size (Fig. 1c). Apart from the clinical
Ferumoxytol, these compounds were effectively loaded in in-house synthesized
nanoparticles, such as poly(acrylic acid)-coated and aminated IONP, with the mean diameter
of the loaded nanoparticles being similar to that of the unloaded (vehicle) nanoparticles
(Supplementary Fig. 1c–d), indicating that the loading capability is not unique to
Ferumoxytol. Since serum stability is a key parameter in drug delivery, we utilized DiR-
loaded Ferumoxytol and determined that the nanoparticles’ near-infrared fluorescence did
not significantly change after prolonged incubation in serum (Fig. 1d), with the formulation
being uniformly suspended and lacking any signs of aggregation (Supplementary Fig. 1e).
The serum stability of cargo-loaded Ferumoxytol prompted us to study whether the cargo
could be retained at physiological conditions but released upon sensing the reduced pH in
many tumors. This feature is ideal for cancers that exhibit decreased interstitial pH, due to
upregulated glycolysis as a result of signaling and metabolic alterations.31 In order to
examine the use of Ferumoxytol as a microenvironment-responsive drug delivery system,
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we studied the retention of Doxorubicin by Ferumoxytol at physiological and lower pH
levels as seen in tumors. We employed a dialysis chamber to separate the nanoparticles from
the potentially released drug. Since Doxorubicin is fluorescent,11 we monitored the
fluorescence emission of Doxorubicin-loaded Ferumoxytol, as well as the presence of free
drug outside the dialysis chamber via HPLC-based spectrophotometry. We determined that
while Ferumoxytol retained Doxorubicin at pH 7.2, it released the drug at slightly lower pH
(Fig. 1e–f).
We next hypothesized that the release of cargo at lower pH was facilitated due to disruption
of the weak electrostatic interactions that mediate the association of the drug with the
nanoparticles’ polymeric coating by the increased positive protons. Changes in the
protonation of the polymer’s side chains disturb hydrogen bonding and van der Waals
interaction between Ferumoxytol’s coating and the cargo, thus triggering release at lower
pH. Although Doxorubicin can form amine salts that improve its water solubility, this occurs
at significantly lower pH, with concomitant changes in the solution’s color (from orange-red
at neutral pH to yellow-orange at acidic pH) that weren’t observed at the pH levels of our
study, as the solutions retained their orange-red appearance. To confirm that the loading of
the cargo into Ferumoxytol is due to weak electrostatic interactions, we attempted to load
the nanoparticles with Doxorubicin in solutions with varying ionic strength. Fluorescence
spectroscopy studies showed that at salt concentrations of 2 M and above almost no
Doxorubicin was loaded into the nanoparticles (Supplementary Fig. 2a), with little changes
in the nanoparticle size (Supplementary Fig. 2b). We also demonstrated that the cargo
retention process is reversible, as demonstrated in pH-based release studies, where after
unloading of Doxorubicin at pH 6.8, the same nanoparticle preparation was re-loaded with
the fluorophore DiR at pH 7.4 (Supplementary Fig. 2c).
The cargo affects the nanophores’ magnetic properties
While the utilized agents so far were fluorescent this is not true for the majority of
pharmaceuticals. We reasoned that having a facile non-optical method to characterize
nanophore loading might have important pharmaceutical implications, especially when the
drug is not fluorescent. Furthermore, such a method could provide an option to
noninvasively monitor drug delivery in vivo. We evaluated therefore, if the loading of cargo
into IONP-based nanophores would change their relaxivity due to displacement of water
molecules from IONP’s vicinity. We utilized a benchtop relaxometer and loaded
Ferumoxytol with the fluorescent taxol derivative ([Flutax1]Ferumoxytol=30 μM),
Doxorubicin ([Doxorubicin]Ferumoxytol=828 μM) and DiR ([DiR]Ferumoxytol=920 μM),
observing cargo-modulated alterations in the T2 and T1 signal (Fig. 2a–b). Interestingly, we
observed that as the drug content in the nanophores increased (with the particle
concentration being constant), the T1 and T2 relaxation times rose also over those of the
unloaded nanoparticles (T1= 402±7 ms; T2 = 121±2 ms; mean±s.e.m; n=3) (Fig. 2c). This
demonstrates the use of this method for the quantification of non-fluorescent compounds
loaded in IONP with simple relaxometers, NMR instruments or MR imagers
(Supplementary Fig. 3a–b). Since T2 and T1 are inversely proportional to a contrast agent’s
spin-spin (r2) and spin-lattice (r1) relaxivities, Ferumoxytol’s r2 and r1 decreased after
addition of the cargo (Fig. 2d–e). To demonstrate that the loading of non-fluorescent drugs
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can be monitored through the changes in IONP’s magnetic properties, we encapsulated a
variety of cancer chemotherapeutics and observed similar changes in r2 and r1 (Fig. 2d–e,
Supplementary Table 1; [Drug]Ferumoxytol=100 μM). We further determined that the changes
in the magnetic properties depended on the drug’s DMSO solubility (Fig. 2e), where
compounds that were highly soluble in DMSO caused large changes in Ferumoxytol’s
relaxivity, likely due to enhanced retention by the nanoparticles’ coating. This showed that
the hydrophobicity of the drug was an important factor next to its size for its capability to be
loaded into the nanophores. Relaxivity changes were also observable when Ferumoxytol
was simultaneously loaded with two drugs at the same time, such as the androgen receptor
antagonist MDV3100 and the PI3K inhibitor BEZ235 ([MDV3100]Ferumoxytol=250μM and
[BEZ235]Ferumoxytol=75 μM) (Fig. 2d–e). These drugs were chosen together rationally since
the androgen receptor pathway interacts with the PI3K cascade in prostate cancer,
suggesting that combinatorial approaches targeting both pathways will result in a more
effective therapy.32 Notably, although the extent of cargo loading had an effect on T2 and
T1, it did not affect T2*, which was solely dependent on the iron and therefore particle
concentration. Loading and unloading of Ferumoxytol can therefore be evaluated via the T2
and T1 parameters, while the required information on the relative nanophores concentration
(to exclude that changes in T2 or T12 are barely due to different nanophore
concentration)can be provided through the T2* signal (Fig. 2g; Supplementary Fig. 4a–d).
Additionally, we tested drug loading in non-clinical IONP preparations, such as poly(acrylic
acid)-coated nanoparticles, and observed that the cargo affected their magnetic properties
similar to what was seen in Ferumoxytol, indicating that the effect of cargo on the
nanoparticles’ magnetic properties is shared among polymer-coated IONP (Supplementary
Fig. 5a–f).
Using MR to monitor cargo release from the nanophores
Similar to the prior optical measurements, incubation of Doxorubicin-loaded Ferumoxytol at
physiological conditions for 24 hours did not reveal any major changes in the T2, T1 to
indicate loss of cargo over time (Supplementary Fig. 6a–c). However, changes in T2 and T1
were observed at the slightly acidic pH of 6.8. Again employing a dialysis chamber to
separate the nanoparticles from any released drug, we incubated Doxorubicin-carrying
Ferumoxytol in 1X PBS adjusted to pH 6.8 and 6.0. Rapid decreases in T2 and T1 were
again observed in these mildly acidic conditions (Fig. 3a–b), which were in accordance with
loss of nanoparticle-associated Doxorubicin fluorescence due to release of the drug to the
dialysis’ free fraction (Fig. 1e–f). In control studies, unloaded Ferumoxytol at pH 6.8 and
below did not exhibit any changes in T2 and T1 (Fig. 3c–d), indicating that the observed
changes in relaxation times of the loaded Ferumoxytol were attributed to cargo release and
not due to the pH directly affecting the nanoparticles’ magnetic properties. To confirm that
these changes were mediated by cargo release and not aggregation of particles, we
performed dynamic light scattering-based size measurements (DLS). Results indicated that
nanoparticle size and distribution were constant throughout the experiment, with the
nanoparticles being stable after 2 h at pH 6.0 and both Doxorubicin-loaded and unloaded
Ferumoxytol showing the same size distribution profiles (Fig. 3e–f). This demonstrates that
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the effect of cargo encapsulation on IONP is reversible, since payload release facilitates
recovery of their magnetic properties, leading to T2 and T1 decreases.
Next, we investigated the long-term stability of drug-loaded nanophores in serum. DiR-
carrying Ferumoxytol was stable for up to 8 days in sterile serum (Supplementary Fig. 7a–
b), with no changes in the relaxation times. However, Doxorubicin- and Flutax1-loaded
nanoparticles released their payload with different kinetic profiles, as indicated by changes
in the relaxation times (Supplementary Fig. 7c–f). Specifically, Doxorubicin was completely
released within 4 days, which resulted in restoration of relaxation times (Supplementary Fig.
7c–d). Flutax1 demonstrated sustained release in serum during the ten-day duration of the
study with ~50% of the drug released within 7 days (Supplementary Fig. 7e–f). We attribute
these release patterns to the different chemical characteristics of each payload. Doxorubicin
may be quickly released, due to lower hydrogen bonding formation between its amino end
and the polymer’s carboxyl and hydroxyl groups that results in its overall weaker association
with the nanoparticles. On the other hand, Flutax1 has multiple carbonyl and methyl groups
that facilitate its tighter association with Ferumoxytol’s coating, as well as multiple
segments that favor hydrophobic interactions.
Intracellular cargo release de-quenches the nanophores
We next investigated whether cargo-loaded nanophores could deliver drugs to cancer cells
in vitro. Incubation of the prostate cancer cell line LNCaP with Doxorubicin-loaded
Ferumoxytol for 48 h resulted in significant drug uptake, as indicated by the enhanced
cellular fluorescence due to the presence of Doxorubicin in fluorescence microscopy (Fig.
4a). Similarly, high cell-associated fluorescence was seen in LNCaP cells incubated for 48
hours with nanophores carrying the near-infrared fluorophore DiR, compared with empty
control nanoparticles (Fig. 4b). Inhibition of the endocytic process with sodium azide and 2-
deoxyglucose prevented Ferumoxytol’s uptake and led to nominal fluorescence, which
demonstrated that Ferumoxytol was taken up via the functional endocytic machinery and not
through passive means. Additionally, we found that the unloading of DiR within the cells
recovered Ferumoxytol’s superparamagnetic properties, which approached the r2 and r1
relaxivities of the parent empty nanoparticles (Fig. 4c–d). It is likely that some of the cargo
might have still been retained within Ferumoxytol, consequently affecting its properties and
preventing r2 and r1 to fully regain the relaxivity of the unloaded nanoparticles. Since
Ferumoxytol undergoes rapid lysosomal degradation and release of iron cations, the
observed changes in relaxivity are most likely attributed to cargo release, and not due to
nanoparticle aggregation within the endocytic vesicles. Our findings demonstrate that
measurement of nanoparticle relaxation can be used for the sensitive characterization of
non-fluorescent payloads carried by IONP utilizing portable relaxometers,33, 34 NMR
analyzers and MRI.
The cargo affects nanophores’ water accessibility
To elucidate the cargo’s effect on the relaxivity, we reasoned that once the payload
intercalates non-covalently within the nanophores’ polymeric coating, it might obstruct the
free diffusion of water to the vicinity of their magnetic core. This may reduce the
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nanoparticles’ capability to affect the relaxation time of the bulk water and consequently
result in higher relaxation times (Fig. 5a). To study whether the cargo limits the accessibility
of water molecules or exerts an effect by itself, we evaluated DiR-loaded IONP in increasing
concentrations of deuterium oxide (D2O) instead of H2O. Deuterium has a different
magnetic moment from hydrogen, allowing its use in the identification of the role of water
and its hydrogen protons, via nuclear magnetic resonance methods. If the drug would exert
an intrinsic effect then the changes in relaxivity would not be affected as much by the
Deuterium. However, with increasing amount of D2O, the T2 and T1 signal of DiR-loaded
IONP solutions decreased, indicating that the changes in relaxation times are due to the
cargo reducing the access of the bulk water molecules rather than an effect exerted by the
drug itself (Fig. 5b–c, Supplementary Fig. 8a–b). Since the relaxation of water by iron oxide
nanoparticles arises from the water molecules diffusing near the nanoparticles35, 36, we
utilized diffusion-weighted MRI, in order to confirm with an additional method that the
cargo impairs the diffusion of water molecules within the nanophores’ coating. The apparent
diffusion coefficient map revealed that the Flutax1- and Doxorubicin-loaded Ferumoxytol
had lower diffusion coefficients than the unloaded nanoparticles (vehicle)at the same
particle concentration (Fig. 5d–f). This supported our hypothesis that the changes in
relaxation times is due to the cargo reducing the diffusion of water within the nanoparticles
rather then the cargo itself exerting a direct effect.
Previous studies have utilized the target-induced clustering of IONP in sensitive assays for
the detection of numerous biomolecules and targets.24, 29, 30 Specifically, it was
demonstrated by others and us that the nanoparticles form extensive supramolecular
assemblies in the presence of their target.30, 37, 38 The formation of these assemblies
containing multiple nanoparticles was predominantly associated with T2 decreases but no
reported effect on T1.39 While we did not observe any size changes to indicate clustering
(Fig. 1c, Supplementary Fig. 1a), we nevertheless sought to confirm that the mechanism
described herein is not due to aggregation-induced changes. We utilized Concanavalin A
(Con A), a protein with high affinity towards carbohydrates,40, 41 to facilitate the clustering
of the carbohydrate-coated (carboxymethyl dextran) Ferumoxytol. As expected, addition of
the Con A to Ferumoxytol induced decrease in the solution’s T2 but little increase in the T1
(Supplementary Fig. 9a–b), with nanoparticle aggregation confirmed with DLS
(Supplementary Fig. 9c). Moreover, when excess dextran was used to obtain larger clusters
([Dextran]=2.5 mg mL−1), the T1 increased, but the T2 decreased (Supplementary Fig. 9a–
c) due to Ferumoxytol’s Con A-induced clustering and did not increased as seen secondary
to drug loading. Therefore, these results demonstrate that the effect of cargo on the
nanoparticles, such as after drug loading, is novel, and not based on nanoparticle clustering.
This likely explains why previous studies have not identified the effect of clustering on
T1,39 as the extent of the aggregation state is critical.
Improved therapy efficacy with cargo-carrying nanophores
Finally, we examined whether Ferumoxytol-based nanophores could efficiently deliver
chemotherapeutics to cancer cells and cause cytotoxicity. Due to the aberrant signaling of
many pathways in cancer, combinatorial therapies ideally require the successful
administration of several chemotherapeutics at the same time. Towards this direction, we
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utilized Ferumoxytol as a drug delivery vehicle for combinatorial therapy in prostate cancer,
in order to concurrently inhibit the crosstalk between the androgen receptor pathway and the
PI3K cascade and prevent resistance to chemotherapy32. We simultaneously encapsulated
BEZ235 – a PI3K inhibitor – together with the androgen receptor antagonist MDV3100 in
order to deliver both drugs to prostate cancer cells. Dose-response studies revealed that
nanophores loaded with both BEZ235 and MDV3100 had an IC50 of 10 nM (BEZ235) and
0.2 μM (MDV3100) for the human prostate adenocarcinoma cells LNCaP cultured in media
with charcoal-stripped serum, where the serum was depleted of any androgens (Fig. 6a).
However, administration of both drugs in their free forms had IC50 values of 75 nM
(BEZ235) and 1.5 μM (MDV3100). The unloaded nanophores did not affect the cells’
viability, which demonstrated that the drug delivery vehicle lacked any intrinsic cytotoxic
properties. To confirm that the nanophores could facilitate the cargo’s tumor delivery, we
used nanophores loaded with the near-infrared fluorophore DiR and performed reflectance
fluorescence imaging. Twenty-four hours after injection, there was enhanced fluorescence in
the tumors, showing that the nanophores targeted to the tumors with no observable signal
from other organs (Fig. 6b). In vivo therapy studies with nude male mice bearing PC3-
derived prostate cancer xenografts (Fig. 6c–d) and nude female mice with breast cancer
BT20 tumors (Fig. 6e) revealed that the drug-loaded nanophores were considerably more
effective than the free drugs, following iv administration. Notably, delivery of bortezomib
with nanophores achieved enhanced tumor regression, while the free drug was ineffective
and marginally suppressing tumor growth when compared to the tumors volume of control
(DMSO-treated) animals (Fig. 6f). Prostate and breast tumors treated with doxorubicin-
carrying nanoparticles significantly regressed, as opposed to the free drug that only achieved
tumor control at its initial pre-treatment size and vehicle (DMSO) therapy that resulted in
continuous tumor growth (Fig. 6g–h).
Biodistribution studies with radiolabeled PU-H71 (131I-PU-H71) showed that the
nanophores substantially improved the delivery of the drug to the tumors, serving as
efficient, long-circulating delivery vehicles (Fig. 6i–j). We selected this Hsp90 inhibitor for
these studies, because this drug contains as iodine atom, which can be substituted with
radioactive iodine, such as 131I, without altering the drug’s structure and intermolecular
interactions. Twenty-four hours post iv administration the amount of drug at the tumors
more than doubled thanks to the nanophore-based delivery. Apart from higher tumor uptake,
the nanophores allowed the drug to stay longer in circulation, as indicated by the higher
levels of radiolabered compound in blood, lungs and spleen. This allows a larger amount of
chemotherapeutic to be released at the tumor, without the need of higher dosages or more
frequent drug administration. These results also suggested that the nanophores are cleared
through the hepatic route, similar to other nanoparticles of the same size. Taken together,
these findings demonstrate that encapsulation of drugs within the nanophores enhances the
therapeutics’ bioavailability, preventing their nonspecific association with proteins and
lipids, while delivering them within tumors and cells at effective dosages, thus vastly
improving the efficacy of the utilized drugs over their free administration.
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Monitoring drug release in vivo with MRI
Lastly, monitoring of in vivo drug release was achieved by examining changes in the T2 and
T2* relaxation times via MRI (Fig. 7a–d). Our imaging studies showed that 2 and 4 h after
iv administration, the tumor T2 signal of animals treated with drug-loaded FH was higher
than that of mice treated with empty nanoparticles (Fig. 7a and 7c). At the same time, there
were no significant differences in the tumor T2* of animals treated with drug-loaded or
empty nanoparticles, indicating similar particle concentrations in the tumor. However, as
expected the presence of the nanophores at the tumor caused decrease in T2* compared to
the pre-administration (0 h) reading (Fig. 7b and 7d). Twenty four hours after nanophore
administration, there were no differences between the tumor T2 and T2* of animals treated
with Doxorubicin-loaded Ferumoxytol or empty nanophores, demonstrating that release of
the drug occurred in vivo due to the vehicle’s fast drug release kinetics (Fig. 1e–f, Fig. 3a–
b).
Discussion
Here, we demonstrated the use of clinical nanophores as drug carriers, in a process that
relies on weak electrostatic interactions and preserves both the drugs’ and nanoparticles’
structure, while enhancing their aqueous stability and bioavailability. We showed that
Ferumoxytol is a diverse drug delivery platform, accommodating payloads with a wide
range of molecular weights. As the cargo incorporation causes significant changes in the
nanophores’ magnetic properties, the loading of non-fluorescent agents – otherwise difficult
to monitor – can be evaluated via magnetic relaxation, thus providing a drug delivery
platform self-reporting the drug delivery and release through the changes in their MR signal
properties. Importantly, the therapeutic efficacy was significantly improved over the free
drug using nanophores. Therefore, this novel integrated drug delivery and monitoring
strategy, which employs clinically approved agents in a non-altered form, is suitable to
improve chemotherapy of cancer significantly and will be an integral part of understanding
the dynamics, risk assessment and approval of nanoparticle-based drug delivery.12
Methods
Materials
All chemicals were of analytical grade, unless otherwise stated. Ferrous and ferric chloride
(FeCl2•4H2O and FeCl3•6H2O) were from Fluka and deuterium oxide (D2O) was from
Acros. Poly(acrylic acid) (PAA, MW 1.8 kDa), ammonium hydroxide, hydrochloric acid
and dimethyl sulfoxide (DMSO) were from Sigma-Aldrich. Dextran (10 kDa) was acquired
from Pharmacosmos, while Concanavalin A (Con A) was bought from Sigma-Aldrich.
Payload included the following compounds: Alendronate (MW: 325) from Sigma Aldrich,
AZD8055 (MW: 466) from Selleck Chemicals, BEZ235 (MW: 470) from Cayman
Chemicals, BKM120 (MW: 580) was a gift from Professor Lewis Cantley (Harvard Medical
School, Beth Israel Deaconess Medical Center), Dasatinib (MW: 488) from Selleck
Chemicals, DiR (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo-tricarbocyanine iodide, MW:
1013) from Invitrogen, doxorubicin (Adriamycin, MW: 580) from Selleck Chemicals,
Flutax1 – a fluorescent taxol analog (MW: 1337) – from Tocris Bioscience, FR230 (MW:
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687) was provided by Dr. Horst Kessler (Technische Universität München), GSI-34 (MW:
534) was provided by Dr. Yueming Li (MSKCC), Lapatinib ditosylate (MW: 925) was
purchased from Selleck Chemicals, MDV3100 (MW: 464) was provided by Professor
Charles Sawyers (MSKCC), Bortezomib (MW: 384) was purchased from Selleck Chemicals
and PU-H71 (MW: 512) was provided by Dr. Gabriela Chiosis (MSKCC). Stocks of these
chemicals were prepared in DMSO, and stored at −20 °C according to the suppliers’
instructions. Commercial IONP preparations were obtained from Micromod
Partikeltechnologie GmbH (Rostock, Germany; NH2-nanomag-D-spio) and AMAG
Pharmaceuticals (Lexington, MA; Ferumoxytol).
In-house preparation of Iron Oxide Nanoparticles
Poly(acrylic acid)-coated IONP were prepared with the alkaline precipitation method,
involving the rapid mixing of ferrous and ferric chloride in ammonium hydroxide that was
followed by addition of the polymer solution.42 To remove excess reagents and byproducts,
the nanoparticles were washed, concentrated and reconstituted in pH 7.4 phosphate buffered
saline (PBS), with a KrosFlo Research II TFF system that was equipped with a 10 kDa
column (Spectrum). The nanoparticles were stored at 4 °C until further use, without any
precipitation being observed, similar to the aminated nanoparticles obtained from
Micromod, which were used without any additional preparation. Ferumoxytol was subjected
to magnetic separation using a 1X PBS-equilibrated LS25 MACS column (Miltenyi,
Cambridge, MA), in order to isolate IONP with good magnetic properties from any free
polymer in the solution. Subsequently, Ferumoxytol was stored at 4 °C until further use.
Drug loading into Nanophores
Encapsulation of the molecular payload was achieved using a modified solvent-diffusion-
based protocol, facilitating the entrapment of hydrophobic molecules within IONP’s
polymeric coating. In general, the nanoparticles (25 μL of either PAA-IONP or NH2-
nanomag-D-spio, 30 μL of Ferumoxytol) were resuspended in 500 μL distilled water,
whereas the cargo was diluted to the desired concentration in DMSO (final volume of
payload solution was 100 μL). The fluorophore or drug payload solution was added
dropwise to the nanoparticle solution under vortexing (1000 rpm) at room temperature,
without any visible precipitation. Subsequently, the preparation was subjected to dialysis in
a small-volume dialysis chamber (MWCO 3000, Fisher) against 1X PBS. The cargo-
carrying IONP were subsequently stored in the dark at 4 °C, until further use.
Nanophore characterization
The size of the nanophores was determined through dynamic light scattering (DLS) (Nano-
ZS, Malvern, Westborough, MA). The same instrument was used in nanoparticle surface
charge measurement (ζ potential), whereas to determine Ferumoxytol’s nanoparticle
concentration the NS500 instrument was utilized (NanoSight, Duxbury, MA). Magnetic
relaxation measurements, including r1 and r2 relaxivities, were determined with a 0.47T
mq20 NMR analyzer (Minispec, Bruker, Billerica, MA). For T2 measurements a CPMG
pulse-echo train with a 1.5 ms interpulse spacing was used, whereas the T1 sequence varied
the interpulse spacing from 5 ms up to 8500 ms. The preparations’ iron concentration was
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determined spectrophotometrically as previously reported,43 using a SpectraMax M5
instrument (Molecular Devices, Sunnyvale, CA). Briefly, the nanoparticles were subjected
to acid digestion, and subsequent conversion of all iron ions to iron(III). A standard curve
was created based on the absorbance at 410 nm of solutions of known concentration of
FeCl3 in the digesting solution. Fluorescence emission measurements were performed using
the SpectraMax M5, as well as an Odyssey near-infrared imaging station (LI-COR
Biosciences, Lincoln, NE), equipped with two solid-state lasers for excitation at 685 and 785
nm. To determine the cargo load of each preparation, the following molar extinction
coefficients were used: ε Doxorubicin=11500 M−1 cm−1 at 480 nm, εFlutax1=52000 M−1 cm−1
at 495 nm and εDiR =270000 M−1 cm−1 at 748 nm. For all other cargo, we quantified the
amount of drug-loaded into the nanoparticles using HPLC and standard curves with known
amounts of the corresponding drug. We first induced release of the cargo by incubating the
loaded nanoparticles in a 2M NaCl solution for 30 min, followed by spin filtration (MWCO
5,000) to collect the cargo-containing solution. Stability experiments were performed in pH-
adjusted phosphate buffered saline, whereas serum experiments were performed at 37 °C,
using fetal bovine serum obtained from Gemini Bio-products. The sterile serum lacking any
nanoparticles had a T2 of 600 ± 10 ms and T1 of 1700 ± 30 ms (mean±s.e.m.; n=3), which
remained unaltered during the experiments. The clustering of Ferumoxytol ([Fe]=20 μg
mL−1) was studied in the presence of Con A ([Con A]final=50 μg mL−1) using dynamic light
scattering and the Minispec. Release of doxorubicin from drug-loaded Ferumoxytol was
performed using a dynamic dialysis setup, as previously described.22 A dialysis chamber
was utilized (MWCO 3000, Fisher), containing doxorubicin-loaded Ferumoxytol in either
pH 7.2 or pH 6.8 1X PBS. The nanoparticles were dialyzed against the corresponding pH-
adjusted buffer at room temperature and under constant stirring (150 rpm), where at regular
time intervals aliquots from the external aqueous milieu of the device were collected for
further analysis. The collected samples were analyzed via a Beckman Coulter HPLC
instrument, equipped with a C18 reverse phase column and set to monitor doxorubicin’s
absorbance at 480 nm mixing An animal MRI from Bruker Biospin (Billerica, MA)
operating at 4.7T and a 35-mm radiofrequency coil were used to image phantoms of the
nanoparticle preparations that were spotted on a microplate.
In vitro drug release from loaded nanophores
LNCaP cells were grown to confluence, on a 12-well poly(lysine)-coated plate in 10% FBS-
containing RPMI medium at 37 °C, 5% CO2. The medium was aspirated, and the cells were
supplemented with 1 mL fresh media, plus 50 μL of either empty (vehicle), Doxorubicin-
loaded Ferumoxytol or DiR-loaded Ferumoxytol. After 48h, the cells treated with
Doxorubicin-loaded Ferumoxytol were examined under a Nikon Eclipse TiE fluorescence
microscope, in order to determine the nanoparticle uptake. Likewise, following 48h-long
incubation at 37 °C, 5% CO2, the cells treated with vehicle and DiR-loaded nanoparticles
were trypsinized and subjected to centrifugation at 1000 rpm for 6 min. The resulting pellets
were then resuspended in 400 μL 1X PBS and aliquoted in two eppendorf tubes for
fluorescence emission and magnetic relaxation measurements, using the near-infrared
imager (LI-COR) and the benchtop relaxometer (Bruker). Studies of inhibition of
nanoparticle uptake were performed at 37 °C, 5% CO2 in the presence of sodium azide (10
mM) and 2-deoxyglucose (50 mM), as well as at 4 °C, with either way inhibiting active
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endocytosis. For near-infrared fluorescence, excitation was achieved at 785 nm, with
emission recorded at 800 nm; with the instrument settings set as follows: focus offset = 4
mm, intensity = 0.5 and resolution = 169 μm. The iron content of the cell pellets was
determined as described above, with untreated samples of equal cell numbers serving as
control.
Cell viability and in vivo studies
LNCaP, PC3 and BT20 cells were obtained from ATCC (Manassas, VA), and maintained
according to the supplier’s instructions. LNCaP cells were seeded on black-walled, clear
bottom 96-well plates at a cell density of 10,000 cells per well, supplemented with 100 μL
10% FBS-containing RPMI medium. Controls included cells incubated with unloaded
nanoparticles or DMSO, corresponding to the free drug’s final solvent concentration. Dose-
response curves were obtained after the cells were treated for 48h with corresponding agent.
Subsequently, the old medium was aspirated, and cell viability was assessed via the Alamar
Blue method (Invitrogen). Briefly, the cells were supplemented with 10%-alamar-blue-
containing medium (10% FBS-containing RPMI), followed by 3h incubation in a humidified
incubator (37 °C, 5% CO2) and recording of fluorescence emission (λexc=565 nm, λem=585
nm) with the SpectraMax M5 plate reader. The localization of DiR-loaded nanophores was
assessed with an IVIS 200 in vivo imaging system, equipped with an ICG filter set
(Waltham, MA). Adult, male, nude mice (n=3) with LNCaP xenografts on their flanks were
administered 100 μL of nanophores ([DiR]=400 μM) iv (Radiant efficiencymax= 9.1 ± 0.4 ×
108; mean±s.e.m). Adult, male, nude mice (n=12) that had PC3 tumors on their flanks were
treated on days 0, 2, 6 and 8 with 100 μL of equimolar ([Bortezomib]=0.5 mM)
concentrations of either free (diluted in 5% DMSO-containing 1X PBS) or Ferumoxytol-
encapsulated bortezomib. Control animals were treated with either 5% DMSO-containing
1X PBS or unloaded Ferumoxytol that had the same iron concentration as the loaded
nanophores ([Fe]=0.75 mg mL−1). Adult, male, nude mice (n=10) bearing PC3 tumors and
adult, female, nude mice (n=10) with BT20 tumors on their flanks were treated every other
day (days 0, 2, 4, 6 and 8) with 100 μL (retro-orbital injection) of either doxorubicin alone
or doxorubicin-loaded Ferumoxytol, both at a final doxorubicin concentration of 0.28 mM.
Control animals were treated with 100 μL of 10% DMSO-containing 1X PBS to match the
DMSO content of free Doxorubicin, since the drug was dissolved in DMSO and diluted to
the desired concentration in PBS. Change in tumor volume was defined as the ratio of the
tumor volume on day 10 minus the tumor volume on day 0 divided by the tumor volume of
day 0. For biodistribution studies, we used 20 adult, male, nude mice with PC3 xenografts
on their flanks, and either free or Ferumoxytol-loaded 131I-PU-H71. Following retro-orbital
administration, the mice were euthanized at the designated time points, and the radioactivity
of the collected organs was measured on PerkinElmer’s (Waltham, MA) Wizard2 2480
Automatic Gamma Counter. Change in drug uptake and retention following nanophore
administration (Δ[PU-H71]NP) was calculated as the ratio of nanoparticle-delivered
radiolabeled drug minus the free radiolabeled drug divided by the free radiolabeled drug. An
animal MRI from Bruker Biospin (Billerica, MA) operating at 4.7T and a 35-mm
radiofrequency coil was used to image the mice. Changes in tumor size were evaluated with
a microcaliper, and at the end of the study the mice were euthanized, according to the
MSKCC Institutional Animal Care and Use Committee guidelines.
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Data Analysis
All experiments were performed in triplicate unless otherwise stated, with the results
presented as mean ± s.e.m. The data were analyzed in Prism (GraphPad Software), whereas
the MR images were processed through the OsiriX DICOM viewer.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank Professor Lewis Cantley (Harvard Medical School, Beth Israel Deaconess Medical Center), Professor
Charles Sawyers (MSKCC) and Dr. Horst Kessler (Technische Universität München) for providing
chemotherapeutics; Dr. Jason S. Lewis for providing animals; Dr, Naga Vara Kishore Pillarsetty, Dr. Carl Le, Dr.
Simone Alidori, Dr. Stefan Harmsen, Kuntalkumar Sevak, Dr. Priyanka Shukla and Florian Rechenmacher for
expert technical assistance. This study was supported by the MSKCC Gerstner Young Investigator Award (to J. G.),
Starr Cancer Consortium (to J. G.), the MSKCC Experimental Therapeutics Center (to J. G.), the MSKCC Center
for Molecular Imaging and Nanotechnology (to J. G.), Commonwealth Foundation for Cancer Research (to J. G.),
Mr. William H. and Mrs. Alice Goodwin (to J. G.), the Alex’s Lemonade Stand Foundation (to C. K.) and the NIH
(P30 CA008748-44 S5).
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Figure 1. Ferumoxytol as nanophores for drug delivery
(a) The gradual incorporation of Flutax1 within Ferumoxytol’s coating increased the
nanoparticles’ Flutax1-derived fluorescence emission (mean±s.e.m, n=3), (b) with the
nanoparticle coating capable of accommodating many cargo molecules within it (mean
±s.e.m, n=3). The nanoparticles were first dialyzed to remove any unloaded compound,
followed by fluorescence and DLS measurements. (c) Size distribution of cargo-loaded
Ferumoxytol (vehicle = unloaded Ferumoxytol; means and distributions of three
independent experiments). (d) DiR-loaded Ferumoxytol was stable in sterile fetal bovine
serum, with its fluorescence remaining unaltered (mean±s.e.m, n=3). (e) Ferumoxytol
released Doxorubicin at slightly acidic conditions. The fluorescence emission of the
nanoparticles (λex=485 nm, λem=590 nm, mean±s.e.m, n=3)) that were retained within the
dialysis chamber decreased, due to Doxorubicin’s release to the free fraction found in the
chambers’ exterior. (f) The release of Doxorubicin from Ferumoxytol to the exterior of the
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dialysis chamber was confirmed by recording the drug’s absorbance in the free fraction at
480 nm (mean±s.e.m, n=3).
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Figure 2. The cargo quenches the nanophores’ magnetic properties
Loading nanophores with cargo increased the T2 (a) and T1 (b)([Fe]=10 μg mL−1; mean
±s.e.m, n=3). The nanoparticles were first dialyzed to remove any unloaded compound,
followed by relaxation measurements. c, The gradual addition of Flutax1within
Ferumoxytol’s coating increased the nanoparticle formulation’s T2 and T1 signal (linear
regression correlation coefficients rT2=0.98 and rT1=0.94; [Fe]=10 μg mL−1;mean±s.e.m,
n=3).d–e, The incorporation of cargo within the nanophores’ coating resulted in changes on
the nanoparticles’ relaxivities (mean±s.e.m, n=3).f, The change in relaxivity (Δr2) was
associated with the drug’s solubility in DMSO (linear regression correlation coefficient
r=0.90; mean±s.e.m, n=3; solubility information was obtained from Selleck Chemicals) g,
MRI phantom images of unloaded and loaded Ferumoxytol, demonstrating that the cargo
does not affect the nanoparticles’ T2* signal, as opposed to its effect on T2 (iron
concentrations: high=10 μg mL−1, medium=6 μg mL−1 and low=4 μg mL−1).
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Figure 3. The nanophores release their cargo at mildly acidic conditions
As Ferumoxytol released Doxorubicin in acidified buffers, the T2 (a) and T1 decreased (b)
([Fe]=10 μg mL−1; mean±s.e.m, n=3). The T2 (c) and T1 (d) of Ferumoxytol (unloaded
nanoparticles) were not affected by the slightly acidic pH ([Fe]=10 μg mL−1; mean±s.e.m,
n=3). e, No changes in the nanoparticle size were observed via DLS during cargo release,
suggesting structural integrity of Ferumoxytol in these conditions (means and distributions
of three independent experiments). f, Stability of unloaded Ferumoxytol at different pH
(means and distributions of three independent experiments). (Middle horizontal line of a
rectangle = the sample’s mean diameter; Upper and lower horizontal lines are the boundaries
of the nanoparticles’ Gaussian distribution.)
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Figure 4. Intracellular cargo delivery with nanophores
a, Composite image of bright field and fluorescence images of LNCaP cells treated for 48 h
with Doxorubicin-loaded Ferumoxytol. Doxorubicin fluorescence is shown in red (Scale bar
= 50 μm). b, Cells were treated with DiR-loaded Ferumoxytol, and after washing and
trypsinization, the harvested cell pellets were imaged with an Odyssey reader at 800 nm to
quantify the uptake of the nanophores. Control cells were treated with unloaded
nanoparticles. Inhibition of endocytosis was performed at 4 °C and with the inhibitors
sodium azide and 2-deoxyglucose (mean±s.e.m, n=3). The cell pellets were subjected to iron
digestion, and revealed that upon release of the cargo the r2 (c) and r1 (d) relaxivities of
DiR-carrying Ferumoxytol were higher than those of the corresponding fully loaded
formulation (mean±s.e.m, n=3).
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Figure 5. The cargo obstructs the access of water in the nanophores’ proximity
a, Schematic representation of the proposed model that suggests that the presence of cargo
within the coating of IONP hinders the diffusion of water molecules, concomitantly
affecting the ability of nanoparticles to efficiently dephase water’s protons. At high D2O
concentrations, the changes on (b) T2 and (c) T1 were abrogated (mean±s.e.m, n=3),
suggesting that the observed increases in T2 and T1 during cargo loading occurred upon
blockage of water molecules by the entrapped cargo ([Fe]PAA IONP=2.5 μg mL−1) rather
than an effect exerted by the payload. d–f, Diffusion-weighted MRI revealed that the
presence of molecular payload within Ferumoxytol’s coating affected the diffusion of water
molecules ([Fe]Ferumoxytol=5 μg mL−1 for all wells; mean±s.e.m, n=6). The cargo’s effect on
ADC correlated with the observed changes in T2 and T1 signal (mean±s.e.m, n=6). (ADC:
apparent diffusion coefficient; linear regression correlation coefficients rT2=0.95 and
rT1=0.92; vehicle: unloaded nanoparticles) (Mean ± SE).
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Figure 6. Nanophores as in vitro and in vivo chemotherapeutic vehicles
a, Cytotoxicity profile of the human prostate cancer cells LNCaP treated with co-
administered free drugs or Ferumoxytol that was loaded with both the PI3 kinase inhibitor
BEZ235 and the anti-androgen MDV3100 (mean±s.e.m, n=3). b, Representative IVIS
images of DiR-loaded nanophores demonstrating the fluorophore’s localization in the
tumors. c–e, Drug-loaded nanophores (FH-Bortezomib or FH-Doxo) efficiently reduced
tumor volume in mice bearing (c–d) human prostate and (e) human breast xenografts (mean
±s.e.m; n=3 per treatment group for the Bortezomib study; for prostate cancer chemotherapy
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with Doxorubicin: nDMSO=3, nDoxo=3, nFH-Doxo=4; for breast cancer chemotherapy with
Doxorubicin: nDMSO=3, nDoxo=3, nFH-Doxo=4;). f–h, The bar graphs depict the change in
tumor volume between day 10 and 0 of the (c–d) treatment regimes. (i) Biodistribution
profiles of the free and nanophore-encapsulated 131I-PU-H71 24 h after administration (n=4
per treatment group). (j) Tumor retention profiles of free and nanophore-encapsulated 131I-
PU-H71 (%Id/g: % injected dose/tissue mass, n2h=3 per treatment group, n8h=3 per
treatment group, n24h=4 per treatment group), with the corresponding net change in drug
delivery and retention achieved with the nanophores (Δ[PU-H71]NP).
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Figure 7. Noninvasive monitoring of nanophore drug release in vivo with MRI
Mice were injected with unloaded (FH) or Doxorubicin-loaded Ferumoxytol (Doxo-FH),
and the tumors (a) T2 and (b) T2* signals were monitored across time (Scale bars = 25 mm).
(c) As Ferumoxytol released doxorubicin in vivo, the nanoparticles’ T2 signal gradually
decreased and eventually reached that of the unloaded nanoparticles (mean±s.e.m; n=3 per
treatment group per time-point). (d) The T2* indicated that the unloaded and loaded
nanoparticles were equally retained at the tumors, as there were no differences in the T2*
signal (mean±s.e.m; n=3 per treatment group per time-point).
Kaittanis et al. Page 24
Nat Commun. Author manuscript; available in PMC 2014 September 04.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript