Binding and Release of FeIII Complexes from Glucan Particles for the Delivery of T1 MRI Contrast Agents

Article abstract of DOI:10.1002/cmdc.202000003

Yeast-derived β-glucan particles (GPs) are a class of microcarriers under development for the delivery of drugs and imaging agents to immune-system cells for theranostic approaches. However, the encapsulation of hydrophilic imaging agents in the porous GPs is challenging. Here, we show that the unique coordination chemistry of Fe^III-based macrocyclic T₁ MRI contrast agents permits facile encapsulation in GPs. Remarkably, GPs labeled with the simple Fe^III complexes are stable under physiologically relevant conditions, despite the absence of amphiphilic groups. In contrast to the free Fe^III coordination complex, the labeled Fe^III-GPs have lowered T₁ relaxivity and act as a silenced form of the contrast agent. Addition of a fluorescent tag to the Fe^III complex produces a bimodal agent to further enable tracking of the nanoparticles and to monitor release. Treatment of the iron-labeled GPs with a maltol chelator or with mildly acidic conditions releases the intact iron complex and restores enhanced T₁ relaxation of the water protons.

Full text of DOI:10.1002/cmdc.202000003

Accepted Article
Title: Binding and release of Fe(III) complexes from glucan particles for
delivery of T1 MRI contrast agents
Authors: Akanksha Patel, Didar Asik, Eric M Snyder, Alexandra E
Delillo, Paul J Cullen, and Janet Ruth Morrow
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.202000003
Link to VoR: http://dx.doi.org/10.1002/cmdc.202000003
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Binding and release of Fe(III) complexes from glucan particles for
delivery of T1 MRI contrast agents.
Akanksha Patel,a Didar Asik,a Eric M. Snyder,a Alexandra E. Dilillo,a Paul J. Cullen,b and Janet R.
Morrowa*
[a]
[b]
Dr. J. R. Morrow, A. Patel, D. Asik, E. M. Snyder, A. E. Dilillo
Department of Chemistry
University at Buffalo
State University of New York, Amherst, NY 14260
E-mail: jmorrow@buffalo.edu
P. J. Cullen
Department of Biology
University at Buffalo
State University of New York, Amherst, NY 14260
Supporting information for this article is given via a link at the end of the document
Abstract: Yeast derived β-glucan particles (GPs) are a class of
microcarriers under development for the delivery of drugs and imaging
agents to immune system cells for theranostic approaches. However,
encapsulation of hydrophilic imaging agents in the porous GPs is
challenging. Here, we show that the unique coordination chemistry of
Fe(III)-based macrocyclic T1 MRI contrast agents permits facile
encapsulation in GPs. Remarkably, the GPs labeled with the simple
Fe(III) complexes are stable under physiologically relevant conditions,
despite the absence of amphiphilic groups. In contrast to the free
Fe(III) coordination complex, the labeled Fe(III)-GPs have lowered T1
relaxivity and act as a silenced form of the contrast agent. Addition of
a fluorescent tag to the Fe(III) complex produces a bimodal agent to
further enable tracking of the nanoparticles and to monitor release.
Treatment of the iron-labelled GPs with maltol chelator or with mildly
acidic conditions releases the intact iron complex and restores
enhanced T1 relaxation of the water protons.
In order to identify inflammation for diagnostic purposes,
encapsulation of an imaging agent may permit tracking of the GP
carrier. For example, dyes have been encapsulated in GPs to
6
enable fluorescence imaging of the particles.5, There is also
recent interest in using GPs as nature-inspired microcarriers for
delivering MRI contrast agents that specifically target
macrophages.3, 5-8
The delivery of both drugs and imaging agents would
facilitate the development of theranostic applications of GPs.
However, incorporation of low molecular weight drugs or imaging
agents for delivery is complicated by the negative charge and
porous nature of GPs. To circumvent the high porosity of the
particles, the small molecules may be encapsulated into
nanoparticles or liposomes first, and then loaded inside the GP.5,
9, 10
Release of the liposome encapsulated dye is facilitated by
heating the GPs.7 Another method to promote successful
entrapment is to add long hydrophobic alkyl chains on the dye or
on the MRI contrast agent.6 However, simpler systems that could
deliver small molecule contrast agents without added
hydrocarbon chains or without lipids have advantages, especially
for triggered release in the absence of heat.
Introduction
Our laboratory has produced high spin Fe(III) macrocyclic
complexes that function as effective T1 MRI contrast agents in
vivo as potential alternatives to lanthanide agents.11 The solution
speciation and water interactions of the Fe(III) complex are
distinctly different than those of typical Gd(III) agents, yet their
relaxivity is competitive with standard Gd(III) contrast agents. The
bound water in the Fe(III) macrocyclic complexes does not
exchange rapidly on the NMR time scale, rather the complexes
produce relaxivity through strong second-sphere interactions with
water protons. Here we show that one of these complexes
interacts so strongly with GPs; there is no need for an amphiphilic
tail. Fe(TOB) and its bimodal analogue Fe(TOD) (Scheme 1)
remain in the hydrated glucan particles without liposomes or
chemical modification in an interaction that may involve
coordination bonds or electrostatic interactions of the positively
charged complex with the negatively charged β-glucan particles.
Moreover, this interaction with the GPs is readily
Yeast derived β-glucan particles (GPs) are well-known vehicles
for transport and delivery of a wide range of drugs with different
physicochemical properties including charged macromolecules
and lipophilic drugs.1 These particles contain hollow shells that
are prepared by using a series of chemical extractions of
Saccharomyces cerevisiae following destruction of the yeast by
heat. The GP are highly porous, and ellipsoidal in shape with
approximate dimensions between 2-4 microns.2 One advantage
of using GPs as drug carriers is that β-1,3-D-glucan polymers, the
major component of GPs, have dectin-1 receptor targeting
properties. The dectin-1 receptor is expressed in several types of
immune cells that play a key role in anti-fungal immunity. Thus,
GPs are promising delivery vehicles for targeting immune cells
including monocytes and macrophages, as blood-circulating cells
of the immune system that have a crucial role in acute and chronic
inflammation. Targeting of macrophages has potential for
delivering drugs to sites of inflammation that are characteristic of
disease states including cancer or atherosclerosis.3,4
1
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increased r1 relaxation rates due to longer rotational correlation
times. The r2 relaxivity values for the Fe(III) complexes are
between 2-3 times greater than the r1 values.
The pentadentate macrocyclic ligands of Fe(TOB) and
Fe(TOD) leave an open coordinate site for water or for other
ligands to form ternary complexes. This led us to search for a
ligand that would bind sufficiently strongly to the available
coordination sites to more easily trigger release of the macrocyclic
Fe(III) complexes from GPs.14-17 These ligands should ideally
bind to the complex without removing the iron, but would allow us
to sequester the contrast agent. Moreover, we searched for
ligands that would produce a new electronic absorbance band in
the visible region of the spectrum to monitor complex formation.
UV-vis screening experiments were done with commercially
available ligands which are known to bind the Fe(III) cation.17-20
Our focus was on bidentate ligands that would form ternary
complexes, presumably by replacing the water ligand and forming
a seven-coordinate Fe(III) center.
Several bidentate ligands, when titrated into solutions of
Fe(TOB), produced a new visible electronic absorbance which is
characteristic of ligand to metal to charge transfer (LMCT) bands
for Fe(III) complexes.14, 15, 21 Within the group of bidentate ligands,
ligands having β-hydroxyl ketones displayed promising binding
characteristics. The broad CT band from 400-600 nm imparts a
red color to the adduct solution (Figure S1). Catechol, deferiprone,
and maltol were among those studied, but only maltol produced a
suitably strong interaction as shown by UV-vis spectroscopy
titrations (Figure S15, S16 and S1). Hinokitiol interaction with
Fe(TOB) was strong enough to sequester iron out of the Fe(TOB)
complex over time, whereas catechol appeared to only weakly
bind to Fe(TOB) (Figure S18, S15). Deferiprone bound to
Fe(TOB), but more weakly than did maltol (Figure S17, S2). In
contrast, other bidentate chelators with oxygen donors including
lactate, bis-phosphonate, salicylate, or phalate did not produce a
new absorbance in the visible region (Figure S20-S23).
Scheme 1. Fe(III) complexes shown with predominate species at
neutral pH.
reversed by treatment with mild acid or with a bidentate chelator
to trigger the release the contrast agent. The goal of this study
was to explore GPs as biocompatible delivery vehicles for T1
Fe(III) based MRI contrast agents and to develop chemical
triggers for the release of intact iron complexes.
Results and Discussion
Iron complexes and ternary interactions.
Three Fe(III)
macrocyclic complexes, Fe(TOB), Fe(TOD) and Fe(TOT), were
chosen to study as contrast agents to encapsulate in GPs
(Scheme 1). All three complexes are unusual examples of
cationic contrast agents, whereas Gd(III) based T1 MRI contrast
agents are typically anionic or neutral.12 The cationic nature of
the complexes is supported by pH potentiometry studies of
Fe(TOB) and Fe(TOT) complexes that are consistent with a single
deprotonated hydroxypropyl pendent. The proposed form of the
coordination complexes and their species at neutral pH are shown
in Scheme 1.11-13 These complexes are resistant towards
dissociation in 100 mM acid, and in solutions containing 25 mM
hydrogen carbonate and 0.4 mM hydrogen phosphate at neutral
pH.11, 13 Fe(TOD), which was prepared here as a bimodal agent,
has similar solution properties. Fe(TOD) contains a fluorescent
dansyl group to better visualize the encapsulation of the complex
in yeast cell wall particles, and for future cell uptake studies. The
macrocycle was synthesized by appending dansyl to the non-
coordinating benzyl pendent, followed by alkylation of the
macrocycle (Scheme S1). Fe(TOD) has an effective magnetic
moment in solution of 5.73, similar to that reported for Fe(TOB),
Fe(TOBA) and Fe(TOT) and consistent with high spin Fe(III)
complexes.11, 13 Moreover, most of the 1H NMR resonances of
these complexes are broadened into the baseline, consistent with
strong relaxivity of the Fe(III) center.
Titration of maltol into a solution of Fe(TOB) was fit to a 1:1
binding isotherm to give a dissociation constant of 5.5 x 10 -5 (Fig
S2). The absorbance of the adduct remained unchanged in
solutions maintained at 37 °C with 25 mM hydrogen carbonate,
0.4 mM hydrogen phosphate at neutral pH, or in PBS buffer.
There was no detectable dissociation promoted by 1 mM ZnCl2 at
pH 7.2 over 12 h (Fig S8). In contrast to Fe(TOB) alone which
requires a sugar stabilizer, meglumine, to keep the complex in
solution at high millimolar concentrations at pH 7.2,11 the Fe(TOB)
adduct with maltol has excellent solubility in water even at
physiological pH. This suggests that maltol forms a strong ternary
complex with Fe(TOB).
The relaxivity (r1 or r2 in mM-1s-1) measured over the
concentration range of 100 to 400 µM is given for Fe(TOB) in both
neutral and acidic solutions in Table 1 and for other Fe(III)
complexes at neutral pH. The T1 relaxivity of Fe(TOD) is lower
than that of Fe(TOB), and closer to that of a related complex
which contains a carboxylate in the para-position, Fe(TOBA),
shown in Scheme 1. The lowered relaxivity of the Fe(TOBA)
complex compared to Fe(TOB) is not fully understood.11
Differences in relaxivity may be due to an electronic effect on the
benzyl group and Fe(III) center that modulates interactions with
water protons, or possibly to subtle differences in speciation.
Here too with Fe(TOD), the relaxivity is lowered, despite the larger
size of the molecule that would normally be expected to produce
Table 1. Relaxivity values for Fe(III) complexes measured at 4.7 T, 20 mM
HEPES pH 7.2, 100 mM NaCl at 37 oC.
µeff (BM)
r1 (s·mM)-1
pH 7
r2 (s·mM)-1
pH 7
r1 (s·mM)-1
pH 4.5
Complex
Fe(TOB)[a]
5.90
5.60
5.73
6.03
5.30
2.30 ± 0.10
1.70 ± 0.10
1.55 ± 0.01
2.52 ± 0.07
0.54 ± 0.01
4.47 ± 1.07
5.05 ± 0.48
5.55 ± 0.11
4.18 ± 0.35
0.81 ± 0.08
1.54 ± 0.01
Fe(TOBA)[a]
Fe(TOD)
Fe(TOB):Maltol
Fe(Maltol)3
[a] pH 7 data for Fe(TOB) and Fe(TOBA) taken from reference 10.
[b] with Meglumine.
2
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Scheme 2. Fe(TOB)-Maltol ternary complex
Several methods were used to further characterize the
Fe(TOB)-maltol adduct. A high resolution ESI-FT-ICR mass
spectrum of a solution with a 1:1 ratio of Fe(TOB) to maltol
showed a peak for the Fe(TOB)(maltol) adduct (Fig S7). Moreover,
1
a H NMR titration of Fe(TOB) into a 5 mM solution of maltol at
pH 7.2 showed the disappearance of the maltol proton
resonances (Fig. S9). The disappearance of the maltol
Figure 2. Comparison of 10 mM Fe(TOB):Maltol (1:1 equivalence), 10 mM
Fe(TOB), 10 mM Fe(DTPA) and 10 mM Fe-CDTA 17O NMR resonance
broadening at pH 6.0 as a function of temperature. Fe(CDTA) was run at pH
5.6.
resonances are consistent with binding of maltol to
a
paramagnetic high spin Fe(III) center. Furthermore, if the maltol
were to remove Fe(III) from Fe(TOB), the Fe(maltol)3 complex
would result. Synthesis of this complex and analysis by 1H NMR
spectroscopy shows two very broad proton resonances at 42 and
75 ppm (Fig S12), which is clearly not what we observe in maltol
titrations of Fe(TOB) (Fig S13). Solutions of Fe(TOB)-maltol that
we do not know that exact nature of the coordination mode of
maltol to Fe(TOB), it seems reasonable that the ligand would be
bidentate. Consistent with this postulate, titration of Fe(TOT), a
closed coordination complex, with maltol did not produce a UV-
vis absorbance peak characteristic of a ternary complex (Figure
S6). This comparison suggests that a displaceable water ligand
on the Fe(III) complex is required for maltol binding.
Labeling and release of Fe(TOB) in glucan particles.
The GPs were labelled by stirring the particles in an aqueous
solution of Fe(TOB) for 16 h. The resulting particles were
collected as a pellet and were visibly orange-brown in color due
to Fe(TOB) loading (Figure S24). Fe(TOB) loading was found to
be directly proportional to the concentration of iron complex in
incubating solutions as shown by ICP-MS analysis of the labelled
particles, (Figure 1). Moreover, a series of Z-spectra recorded on
the Fe(TOB) labelled GPs also showed a correlation with
Fe(TOB) labelling. In these experiments, a pre-saturation pulse is
used to magnetically saturate protons that may chemically
exchange with bulk water. The frequency of the pre-saturation
pulse is varied to produce a plot of the water proton intensity
were monitored for 12
h
by 1H NMR spectroscopy were
unchanged over this time period, suggesting that the ternary
adduct persisted. (Fig S14).
Other solution characterization included variable
temperature 17O NMR studies. There is little broadening of the 17
O
NMR resonance in the presence of Fe(TOB)-maltol (Figure 2).
This suggests that the adduct, like Fe(TOB), does not have a
inner-sphere water ligand that exchanges rapidly on the NMR
time scale.11, 22 For comparison, Fe(DTPA) which lacks an inner-
sphere water and Fe(CDTA) which has a bound water are shown.
The 17O NMR data for Fe(TOB) and Fe(TOB)(maltol) much more
closely resemble that of Fe(DTPA) which lacks an inner-sphere
water ligand.
Relaxivity measurements show a slight increase in r1
relaxivity for the adduct compared to Fe(TOB) (Table 1). All in all,
the data suggest that the maltol is bound strongly to the Fe(III)
center as a bidentate ligand. Based on our data, we favour a
ternary complex that has a directly bound maltol ligand, consistent
with the observed LMCT band of the Fe(TOB)(maltol) adduct and
the lack of maltol 1H resonances observed in NMR titrations upon
addition of Fe(TOB) (Fig S1, S3, S10). The maltol likely binds
directly to the Fe(TOB) through displacement of water to give a
seven coordinate complex (Scheme 2), analogous to a seven-
coordinate Fe(III) macrocyclic complex reported recently.23 While
(Mz/Mo) as
a function of pre-saturation pulse frequency.
Encapsulation of paramagnetic complexes into liposomes or cells
produces broadened Z-spectra that are slightly asymmetric in
shape. The asymmetry is attributed to the paramagnetic shift of
the interior water protons and exchange of the protons to give a
chemical exchange saturation (CEST) effect (Fig S25).24, 25 We
observed a broadened peak, consistent with increased T2 effects
from incorporation of the complex into the GP and a slight
asymmetry characteristic of a CEST effect from magnetically
shifted protons. The same broadened Z-spectra were observed
even after 4 days of incubation in phosphate buffered saline at
room temperature (Fig S26). This suggests the glucan particles
are stable and will not release Fe(TOB) without an external
stressor. Further experiments described below show that the
water proton relaxivity of solutions containing the labelled GP did
not change over time, supporting the robust nature of the
Fe(TOB)-labelled GPs. SEM characterization showed mild
morphology changes in the surface of the GP (Fig S29). The
unlabeled GP appeared shrunken and dehydrated, but the
labeled version was round, similar to literature reports of
nanoparticle loaded GPs.5
For comparison, GPs were treated with Fe(TOT), as an
example of a cationic complex that lacks water ligands and would
not be expected to bind to the GPs through a coordination bond.
Figure 1. Total Fe content in (a) unlabeled, Fe(TOB) and Fe(TOT) labeled
GP measured by ICP-MS. Mean ± SD is reported.
3
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To further study the nature of small molecule dye
interactions with GPs, we attempted to encapsulate dansyl alone
or rhodamine-B in the GP. Interestingly, the dansyl dye could not
be visualized upon treatment with GP by confocal microscopy.
This suggests that either the dye was not encapsulated, or it
passed through the large pores. By contrast, the cationic
rhodamine-B dye was encapsulated and visualized in GPs by
fluorescence microscopy (Figure S37), although the long
exposure times we used suggested low dye loading. Time
dependent studies suggested that rhodamine-B was partially
released from the initial time points up to 2 h, which is attributed
to loosely bound dye, perhaps at the surface. However, after 2 h,
no further release was observed up to 24 h (Figure S36).
Microscopy of the rhodamine-B loaded GPs also showed little
leaching of the dye. This suggests that cationic dyes may be
encapsulated into the GPs without liposomes or amphiphilic tails.
Figure 3. Fluorescence confocal microscopy images of glucan particles. (TOP)
Fe(TOD) labeled GP (middle) Dansyl chloride labeled GP and (Bottom) Control
GP. Excitation (405 nm) emission (520-560 nm). All examples show
fluorescence with 5 µM scale.
To study the potential of the Fe(TOB) loaded GP for MRI
studies, R1 rate constants were measured on labelled and
unlabelled glucan particles suspended in collagen on a 4.7 Tesla
MRI scanner (Figure S30). The R1 relaxation rate constants for
the Fe(TOB) labeled particles (3.03 ± 0.03 s-1) were only
marginally higher than the control GPs (3.45 ± 0.03 s-1). The
apparent quenching of R1 relaxation rates of Fe(TOB) in GPs
suggests a disruption of the second sphere water interaction
which is the major contributor of the r1 relaxivity of Fe(TOB).11 In
addition, the hydrophobic nature of the β-1,3-D-glucan polymer
may inhibit water exchange. The increase in the R1 relaxation rate
constant for water protons in the sample, however, corresponds
to the number of GPs per volume in a collagen matrix (Figure 4,
Table S1). Dividing the relaxation rate constants by the iron
content of the particles gave an r1 of 0.21 mM-1s-1 (Figure S31).
The r2 relaxativity based on iron concentration was much higher
(3.45 ± 0.27) mM-1s-1 which is expected from dominance of T2
processes observed in paramagnetic particles at higher field
strengths.6 In comparison, Gd(III) labeled GPs have r1 relaxivities
of 22.3 mM-1s-1 at low magnetic fields (0.5 T), but r1 decreases
several fold at higher field strengths (7 T). Similarly, the r2 values
for the Gd(III) labeled GPs are an order of magnitude higher than
r1 at 7 T.6 However, the Gd(III) labelled GPs have amphiphilic tails
that anchor the complexes into the GPs. The Gd(III) complexes in
these labeled GPs cannot be easily released. The goal in our
studies, was to release the Fe(III) contrast agent.
However, this closed coordination complex might adhere to GPs
due to cation charge of the complex. Fe(TOT) was indeed
encapsulated in GPs, but only at a fraction (25%) of the amount
of Fe(TOB) as shown in Figure 1 (right). This shows that the open
coordination sphere of Fe(TOB) that permits binding to additional
ligands also increases loading into GPs.
The Fe(TOD) complex was treated with GPs in order to
utilize the fluorescent tag. The dansyl group of Fe(TOD) showed
a fluorescence emission peak at 560 nm upon excitation at 340
nm (Figure S33). Confocal fluorescence microscopy of Fe(TOD)
treated GPs suggests encapsulation of the Fe(TOD) complex in
the GP (Figure 3). However, due to the long UV excitation of
dansyl fluorophore, some auto fluorescence is observed in the
control GP. In order to gauge the stability towards release of the
Fe(III) complex in media, the labeled GPs were incubated in
solution for 26 h and the fluorescence in solution was measured.
(Figure S35). Between initial time points and 2 h, there was a
small increase in fluorescence intensity, which we attribute to
release of surface bound complex. However, from 2 h to 26 h
there was little change in fluorescence intensity of the solution
above the GPs, consistent with the robust nature of the Fe(TOD)
labeled particles.
Maltol treatment of the Fe(TOB) labeled GPs released the
Fe(TOB) from the GPs as shown by Z-spectra and ICP-MS
analysis of the yeast cell wall particles (Figure S27, Figure 5b).
These studies show that essentially all of the Fe(TOB) was
removed upon treatment with excess maltol after 3 h. Moreover,
maltol treatment of Fe(TOB)-loaded GPs increased the R1 rate
constant for proton relaxation of the solution in contact with the
particles (Figure 5a). Mildly acidic conditions (pH 4.5) also
produced an increase in the proton relaxivity (Figure 5a). Acid
triggered release of the Fe(III) complex bodes well for freeing the
iron complexes in acidic phagosomes,26-28 whereas maltol may
provide a means of adding a chemical agent to trigger release.
The use of maltol as a triggering agent is promising based
on its low toxicity in cell lines and in mice. For example, treatment
with maltol is used to reduce nephrotoxicity, an off-target effect of
cisplatin through reduction of oxidative stress, lipid peroxidation
and apoptosis in HEK293 cell line.29 The anti-inflammatory and
anti-oxidant properties of maltol have been shown to prevent
Figure 4. Relaxation rate constants of Fe(TOB) labeled GP
suspended in collagen at 37 oC in 4.7 T MRI scanner.
4
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Figure 5.(a) R1 relaxation rate constants for control GP-media and Fe(TOB) labeled GP-media at various pH values after 3 h incubation measured on a 9.4
T (400 MHz) NMR spectrometer. (b) Total Fe content in unlabeled and Fe(TOB) labelled GP (red) and total Fe content after 10 mM Maltol treatment for 3 h
at 30 oC as measured by ICP-MS. Mean values are reported with ± 1% SD.
alcohol induced redox stress both in cell lines and in mice models
31
even at high doses of 50-100 mg/kg.30,
These studies are
promising for the application of maltol as a chemical trigger to
release Fe(TOB) from the glucan particles.
Experimental Section
Instrumentation. A Varian Inova 500 MHz NMR spectrometer equipped
with FTS Systems TC-84 Kinetics Air Jet Temperature Controller was used
to collect Z-spectra and 1H NMR spectra. Fourier transform ion cyclotron
resonance mass spectrometry (FT-ICR-MS) with 12T Bruker SolariXR 12
Hybrid was used to collect high resolution mass spectral data. Absorbance
Conclusions
The unusual properties of cationic Fe(III) T1 MRI contrast
agents studied here facilitate labelling of GPs without further
functionalization of the complexes or incorporation into liposomes
as necessary for most dyes or contrast agents.4,5,9 We propose
that the cationic nature of the complexes enables this tight
encapsulation in GPs, which are known to be negatively charged.2,
spectra were collected using
a
Beckman-Coulter DU 800 UV-vis
Peltier temperature controller.
Spectrophotometer equipped with
a
Fluorescence microscopy was done on Zeiss Axioplan2 microscope. T1
imaging was performed on a 4.7 Tesla MRI scanner (ParaVision 3.0.2,
Bruker Biospin, Billerica MA) with 35 mm Bruker single channel RF coil.
Temperature was maintained at 37 oC during imaging using an MR-
compatible heating system (SA Instruments, Stony Brook, NY). The
concentration of Fe in yeast cell wall particles was determined by using
Thermo X-Series 2 inductively coupled plasma mass spectrometry (ICP-
MS). Confocal imaging was done on Lieca DMIR2 inverted fluorescence
microscope equipped with a QImaging Retiga EXi CCD camera.
Materials. 3-Hydroxy-2-methyl-4H-pyran-4-one, 3-Hydroxy-1,2-dimethyl-
4(1H)-pyridone, 5-(Dimethylamino)naphthalene-1-sulfonyl chloride and 4-
(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer was
purchased from Alfa Aesar. Nitric acid at 65-70% with greater than
≥99.999% purity (trace metals basis) was obtained from BeanTown
Chemical. 100 ppm Fe standard solutions were purchased from Inorganic
Ventures. Bovine collagen (3 mg/mL) was obtained from Advanced
Biomatrix. [Fe(TOB)Cl]Cl and Fe(TOT)Cl2 were prepared as reported.11,41
The TOT ligand was synthesized according to a procedure reported in the
literature.35
5, 9
Further, Fe(TOB) shows higher loading than the Fe(TOT)
complex suggesting additional coordination interactions with the
GPs. The Fe(III) complex must be released from the GPs,
however, to produce enhanced T1 water proton relaxivity. As we
show here, both mildly acidic conditions or a bidentate chelator
trigger the release of the Fe(TOB) or the Fe(TOD) complex from
the GPs.
The resistance of Fe(TOB) to acid and retention of r1
relaxivity under acidic conditions makes Fe(TOB) an excellent
candidate for imaging acidic vesicles found in cells such as
macrophages.27, 28 Thus, an enhanced MRI signal is anticipated
upon maltol-mediated or acid mediated release of the Fe(III)
complex. Conveniently, β-glucan particles selectively interact with
dectin-1 receptors on the macrophages. This unique interaction
has been extensively investigated in the literature for in-vivo and
ex-vivo labelling of macrophages with drugs and imaging agents.8,
10, 32, 33
Studies are underway to determine optimal conditions for
the uptake and release of Fe(TOB) labeled particles in
macrophages. 4, 34
Binding of Maltol with Fe(TOB)
5
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UV-Vis characterization. Solutions were prepared that contained 500 µM
Fe(TOB) complex in 20 mM HEPES and 100 mM NaCl. A solution of maltol
solution was added to the Fe(TOB) solution and monitored by using UV-
vis spectroscopy. The change in absorbance at 480 nm was plotted
against Fe(TOB) concentration in the cuvette to obtain the binding
isotherms. The dissociation constants were determined by fitting the
binding isotherms to binding models using graphpad prism 7.
Glucan particles and Fe(TOB) labeling
Isolation of yeast glucan particles. Glucan particles were prepared as
6,
7
described.3,
Briefly, Saccharomyces cerevisiae of the Sigma1278b
strain background was used.37 The specific strain (PC538) is a typical wild-
type (WT) strain with the following genotype: MATa ste4 FUS1-HIS3
FUS1-lacZ ura3-52,38 and was used for all experiments in the study. Cells
were grown for 3 days in 250 mL YEPD media to about 10 g. The cells
were washed and resuspended in 1 M NaOH and heated for 1 h at 80 oC.
The insoluble residue was collected by centrifugation at 4000 rpm for 15
min. To the pellet was added 1 M HCl to adjust the pH to <5. The resulting
solution was heated at 55 oC for 1 h. The insoluble residue was again
collected by centrifugation at 4000 rpm for 15 min. The resulting pellet was
washed twice with isopropanol, acetone and ethanol respectively. The
porous yeast cell wall particles were collected by centrifugation and
allowed to dry for 16 h at 25 oC.
For the kinetic inertness study, a 1:1 solution of 500 µM FeTOB and
Maltol was prepared containing 20 mM HEPES and 100 mM NaCl. For
extinction coefficient measurement 1:1 solution of FeTOB and Maltol was
prepared at native pH. The stock solution was diluted to 3 different
concentrations with 20 mM HEPES and 100 mM NaCl and UV-Vis
response was observed. The change in absorbance at 480 nm was plotted
against the adduct concentration. The resulting graph was fit to linear
regression passing through the origin. The slope of the fit was used to
obtain extinction coefficient in the 1 cm path length cuvettes.
Characterization of the Fe(TOB)-maltol adduct by NMR spectroscopy.
Samples were prepared by dissolving 5 mM Maltol and 3.0 mM TMPS as
an internal standard in D2O and 1H NMR spectra were recorded. For each
experiment, Fe(TOB) was titrated into the maltol sample, the pH was
adjusted to ~7.2 using NaOD and 1H NMR spectra was recorded. All
solutions were incubated for 10 min between the measurements. The
average integration of paramagnetic peaks relative to the internal standard
was used to determine the concentration of Fe(TOB)-Maltol adduct. For
further NMR spectroscopy studies, a 1:1 solution of 10 mM FeTOB and
Maltol was prepared and pH was adjusted to ~7.2. 1H NMR spectra were
recorded every 15 min to study the stability of the adduct.
Fe(TOB) labeling of glucan particles. The glucan particles were
hydrated for 16 h at 25 oC in 1X PBS. 10 mM FeTOB solution was prepared
and added to the hydrated yeast cell wall particles. The solution was stirred
at 25 oC for 1 h. The labeled particles were harvested by centrifugation
followed by 3 washes in 1X PBS wash. The resulting labeled particles were
suspended in 1X PBS for further studies.
Z-spectra measurements on Fe(TOB) labeled glucan particles. All
samples were resuspended in 1X PBS for analysis. Z-spectra have been
acquired in a range of ± 50ppm by acquiring a total of 101 data points
(steps of 1 ppm) with B1 = 12 µT at 37 oC.
Scanning Electron Microscopy (SEM) on glucan particles. Labeled
and unlabeled glucan particles were concentrated by syringe filtration by a
0.2 micron Whatman nucleopore polycarbonate filter paper with 1 mL
syringe (GE Whatman, catalog #889-78084, Maidstone, UK).37 Cells were
rinsed with buffer by syringe, treated with 100 % ethanol by syringe and
incubated for 15 min. The filter paper was removed from the holder, placed
in a petri dish and treated with hydroxymethyldiazane (HMDS). Samples
were placed at 4 oC for 16 h and imaged the following day.
Monitoring ternary Fe(TOB)-maltol adduct by UV-Vis spectroscopy.
Samples were prepared by combining 500 µM Fe(TOB) complex and 500
µM Maltol. The absorbance peak at 482 nm was recorded over a period of
12 h at 37 oC. Control samples contained complex with 20 mM HEPES
and 100 mM NaCl. For stability in PBS, samples were incubated in 1X PBS.
The anion stability was determined in the presence of 0.40 mM hydrogen
phosphate and 25 mM hydrogen carbonate solution at neutral pH.
Solutions containing 1 mM HCl were used for acid stability studies. Zn(II)
displacement assays contained 1 mM Zn(II) in 20 mM HEPES buffer and
100 mM NaCl.
17O NMR measurements. Samples were prepared in water with 1% H217O.
The chemical shifts as well as the line width at half-height of the symmetric
water peak were determined in the absence and in the presence of metal
complex at variable temperatures at pH 7. The line width at half-height of
the signal in absence and in presence of metal complex at variable
temperatures was used to calculate the transverse relaxation times using
Swift-Connick equations, as described in literature.35 1/T2 was calculated
by subtracting the full width at half maximum (FWHM) of the 17O resonance
with Fe(III) complex from that in the absence of complex at pH 6.5.
T1/T2 Phantom Relaxivity measurements. Samples with variable
concentrations (100, 200 and 400 µM) of 1:1 stoichiometric amounts of
Fe(TOB) and Maltol in 20 mM HEPES, 100 mM NaCl and 35 g/L HSA at
pH 7.4 were studied. T1/T2 relaxivity values were determined on a 4.7 Tesla
MRI system as reported previously.36 Briefly, T1 relaxation rates of serial
dilutions were measured using an inversion-recovery, balanced steady-
state free precession (bSSFP) acquisition with the following parameters:
TE/TR=1.5/3.0 ms, flip angle=30°, inv. repetition time=10 s, segments=8,
frames=100. T2 relaxation rates were measured using a multi-echo, Carr-
Purcell-Meiboom-Gill (CPMG) sequence with a fixed TR of 4200 ms and
TE times ranging from 20-1200 ms in 20 ms increments. The relaxation
rate of each sample was calculated using non-linear regression analysis
within MATLAB (MathWorks, Natick MA) and relaxivities were then
calculated by linear regression (concentration vs. relaxation rate).
T1 measurements of glucan particles and data analysis. Labeled and
unlabeled glucan particles were washed three times with 1X PBS. The
particles were resuspended in 3D collagen gel using the referenced
protocol39 in 5 mm borosilicate NMR sample tubes.42 The relaxation rates
of the particles were measured on 4.7 T animal MRI at 37 oC using the
same protocol used for phantom measurements mentioned above. The
relaxation rate of each sample was calculated using non-linear regression
analysis in MATLAB.
Determination of Fe(TOB) loading in glucan particles. The
concentration of Fe in the glucan particles was determined by inductively
coupled plasma mass spectrometry (ICP-MS) (Thermo X-Series 2). After
the internalization experiments, the glucan particles with and without
Fe(TOB) complex were collected in 200 µL mili-Q (Milipore) water. Glucan
solutions (100 µL) were digested with metal free nitric acid (900 µL) (65-
70%). After digestion for 3 d, the samples were diluted to 2% HNO3, 30 ppb
cobalt standard solution was added in 10 mL mili-Q (Milipore) water and
analyzed by ICP-MS. For internal standards, cobalt and indium standard
solutions were used.
Statistical analysis. Results were expressed as mean value ± standard
deviation (SD). Statistical analyses were performed using one-way
ANOVA analysis followed by Tukey's multiple comparisons test by using
GraphPad Prism 8. A P value of less than 0.005 was regarded as
significant for cell uptake and viability studies, P value of less than 0.5 was
regarded as significant for T1 measurements on cells.
Procedure for synthesis of [Fe(TOD)]Cl2.
6
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10.1002/cmdc.202000003
ChemMedChem
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N-(2-aminoethyl)-5-(dimethylamino)naphthalene-1-sulfonamide (1).
The compound was synthesized according to a procedure reported in the
literature.40
added under an synthetic dextrose (SD) agar pad to avoid sample
dehydration during the experiment.41
Maltol removal of Fe(TOB) bound to glucan particles. To the Fe(TOB)
labeled glucan particles, 10 mM Maltol solution was added. The particles
were incubated for 3 h at 25 oC. After the incubation the particles were
harvested through centrifugation followed by 3 times 1X PBS wash. Both
supernatant and pellet was saved for ICP-MS analysis.
4-(bromomethyl)-N-(2-((5-(dimethylamino)naphthalene)-1-
sulfonamido)ethyl)benzamide
(2).
1-(4-(bromomethyl)phenyl)-2-
chloroethan-1-one (0.28 mg, 0.95 mmol) was dissolved in THF and stirred
at room temperature to form a homogenous solution. Compound (1) (0.22
mg, 0.95 mmol) was dissolved separately in THF and added slowly to the
stirring solution. Immediate precipitation was observed upon addition.
However, the reaction was stirred overnight till the solution turned clear.
Solvent was removed under vacuum. The pure product was obtained using
column chromatography with 20% MeOH in chloroform. The product was
obtained as off-white powder in 60% yield. 1H NMR (500 MHz, CDCl3) δ
8.75 (1H, br), 8.38 (1H, br), 8.23 (1H, d, 10 Hz), 8.07 (1H, d, 10 Hz), 7.66
(2H, d, 10 Hz), 7.57 (3H, br, s), 7.51 (3H, br, s), 4.51 (2H, s), 3.53 (2H, t,
5 Hz), 3.17 (6H, s), 2.99 (2H, t, 5 Hz). 13C NMR (75 MHz, CD3OD) δ 167.8
(1C, s), 151.9 (1C, s), 142.5 (1C, s), 133.7 (1C, s), 130.6 (2C, s), 129.8
(2C, s), 129.2 (2C, s), 129.1 (1C, s), 128.6 (2C, s), 127.5 (2C, s), 127.4
(2C, s), 45.8 (2C, s), 43.3 (1C, s), 39.8 (1C, s), 32.3 (2C, s). FT-ICR-MS
of [C22H24BrN3O3S+H]+ calculated: 490.07945 found: 490.08010.
Z-spectra measurements on Fe(TOB) labeled glucan particles. All the
samples were suspended in 1X PBS for the analysis. Z-spectra were
acquired in a range of ± 50 ppm by acquiring a total of 101 data points
(steps of 1 ppm) with B1 = 12 µT at 37 oC.
T1 measurement on media. The Fe(TOB) labeled glucan particles were
incubated in 1X PBS, acetate buffer pH 4.5, 10 mM Maltol in 1X PBS and
10 mM Maltol in acetate buffer at pH 4.5 at 37 oC. Aliquots were taken at
3 h and 24 h, and media was collected through centrifugation. T1 values of
water protons were measure on Varian 400 MHz NMR spectrometer using
an inversion-recovery pulse sequence.
Determination of Fe(TOB) loading in glucan particles using ICPMS.
Concentration of Fe in the glucan particles was determined by using
inductively coupled plasma mass spectrometry (ICP-MS) (Thermo X-
Series 2). After the incubation, the glucan particles with and without
Fe(TOB) followed by Maltol treatment were collected in 200 µL milli-Q
(Millipore) water. GP solution (100 µL) were digested with metal free nitric
acid (900 µL) (65-70%). After a three-day digestion process, the samples
were diluted to 2% HNO3, 30 ppb cobalt standard solution in 10 mL mili-Q
(Milipore) water and analyzed by ICP-MS. Cobalt and indium standard
solutions were used as internal standards.
4-((4,7-bis((S)-2-hydroxypropyl)-1,4,7-triazonan-1-yl)methyl)-N-(2-((5-
(dimethylamino)naphthalene)-1-sulfonamido)ethyl)benzamide (TOD).
Compound (3) (0.10 mg, 0.50 mmol) was dissolved in ACN and stirred at
room temperature. Compound (2) (0.28 mg, 0.56 mmol) was dissolved in
acetonitrile separately and added slowly to the stirring solution. The
solution was stirred at room temperature for 4 h. After 4 h the solution was
centrifuged to remove salts and the supernatant was collected. Acetonitrile
was removed from this solution under vacuum. A minimum amount of
ethanol was added to the resultant solid to dissolve it. Diethyl ether was
added to the solution to precipitate the product as a yellow sticky solid in
30% yield. 1H NMR (500 MHz, CD3OD) δ 8.50 (1H, t, 10 Hz), 8.39 (1H, d,
9Hz), 8.31 (1H, d, 10 Hz), 8.23 (2H, d, 10 Hz), 8.19 (1H, d, 5 Hz), 8.10 (1H,
t, 10 Hz), 7.96 (2H, d, 5Hz), 7.71 (1H, t, 10 Hz), 7.52 (1H, d, 10 Hz), 7.50
(1H, d, 5 Hz), 3.54-3.47 (6H, br, s), 3.19-3.09 (6H, br, s), 2.86 (3H, s), 2.84
(2H, s), 2.80-2.70 (6H, m), 2.68-2.62 (6H, m), 1.13 (3H, s), 1.12 (3H, s).
13C NMR (75 MHz, CDCl3) δ 167.7 (1C, s), 135.0 (1C, s), 134.5 (1C, s),
133.0 (1C, s), 130.4 (2C, s), 129.7 (1C, s), 129.4 (1C, s), 129.0 (1C, s),
127.3 (2C, s), 116.4 (1C, s), 115.2 (1C, s), 63.67 (2C, s), 63.38 (1C, s),
58.31 (2C, s), 49.56 – 45.50 (6C, m), 45.33 (1C, s), 42.98 (1C, s), 39.88
(2C, s), 21.02 (2C, s). FT-ICR-MS of [C29H51N8O7S + H]+ calculated:
655.35959 found: 655.35975.
Determination of dissociation of Fe(TOD) and rhodamine-B labeled
glucan particles using Fluorescence spectroscopy. Fe(TOD) and
rhodamine-B labeled glucan particles were incubated for 26 h at 37 oC. 2
mM Maltol was added to all the samples after 24 h and incubated at 37 oC
for 2 h. The particles were centrifuged and aliquots were drawn from the
supernatant at various time intervals for fluorescence spectroscopy. The
Fe(TOD) samples were excited at λex = 340 nm and Rhodamine-B samples
were excited at λex = 555 nm. The rhodamine-B labeled glucan particles
were collected at the end for fluorescence microscopy.
Acknowledgements
JRM acknowledges the National Science Foundation (CHE-
1710224). The authors would like to thank Chemistry Instrument
Center (CIC), University at Buffalo. This work utilized ICP-MS and
FTMS that was purchased with funding from a NSF Major
Research Instrumentation Program (NSF CHE-0959565) and
National Institutes of Health (S10 RR029517). The confocal
imaging studies were funded by National Science Foundation
Major Research Foundation grant # DBI 0923133. We thank Peter
Bush for providing assistance with electron microscopy.
[Fe(TOD)]Cl2. TOD ligand (0.12 g, 0.17 mmol) was dissolved in ethanol.
The mixture was stirred for 30 min and 5.0 mL ethanolic solution of FeCl2
(0.035 g, 0.17 mmol) was added dropwise to the reaction mixture. No
attempts were made to exclude O2 in air from solutions. A precipitate was
formed immediately upon addition of the FeCl2 solution, but the reaction
mixture was stirred overnight at room temperature. The precipitated
complex was filtered and washed with diethyl ether (10 mL x 3).
[Fe(TOD)]Cl2 was collected as a yellow-orange solid with yields of 60%.
Effective magnetic moment as measured by Evans method was 5.73. FT-
ICR-MS of [Fe(TOD)]+ calculated: 708.27796 found: 708.27508.
Fluorescence spectroscopy. Fe(TOD) samples were dissolved in 1X
PBS with milli-Q water in 5 mm quartz cuvette. The samples were excited
at the optimal wavelength for the dansyl fluorophore in the complex
(Dansyl λex = 340 nm).
Keywords: Molecular imaging, β-glucan microparticles,
biocompatible delivery, bimodal imaging agent, chelator mediated
release, acid mediated release, targeted delivery, Imaging agent
design.
Microscopy characterization. Yeast cells were washed three times in
water before preparing the samples for imaging by fluorescence
microscopy. Fluorescence imaging was done using RITC (Rhodamine
Isothiocyanate) channel with exposure 1.5 s on a Zeiss Axioplan2
fluorescence microscope. For confocal microscopy the samples were
[1]
M. Chandana, D. Manasi, R. K. Jagat, K. S. Sanjeeb, Curr. Drug Delivery
2011, 8(1), 45-58.
7
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10.1002/cmdc.202000003
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FULL PAPER
[2]
T. K. Upadhyay, N. Fatima, D. Sharma, V. Saravanakumar, R. Sharma.
[22] E. M. Gale, J. Zhu, P. Caravan, J. Am. Chem. Soc. 2013, 135(49), 18600-
18608.
EXCLI Journal 2017, 16, 210-228.
[3]
Curr. Mol. Imaging 2015, 4(1), 29-34.
[4] K. R. Peterson, M. A. Cottam, A. J. Kennedy, A. H. Hasty, Trends
Pharmacol. Sci. 2018, 39(6), 536-546.
[5] T. Ren, J. Gou, W. Sun, X. Tao, X. Tan, P. Wang, Y. Zhang, H. He, T.
Yin, X. Tang, Mol. Pharmaceutics 2018, 15(7), 2870-2882.
[6] S. Figueiredo, J. N. Moreira, C. F. G. C. Geraldes, S. Rizzitelli, S. Aime,
E. Terreno, Chem. Commun. 2011, 47(38), 10635-10637.
[7] F. Garello, R. Stefania, S. Aime, E. Terreno, D. Delli Castelli, Mol.
Pharmaceutics 2014, 11(10), 3760-3765.
[8] E. R. Soto, A. C. Caras, L. C. Kut, M. K. Castle, G. R. Ostroff, J. Drug
Delivery 2012, 2012, 13
C. Cirizzi, W. Dastru, D. D. Castelli, V. Menchise, S. Aime, E. Terreno,
[23] C. J.Bond, G. E. Sokolow, M. R. Crawley, P. J. Burns, J. M. Cox, R.
Mayilmurugan, J. R. Morrow, Inorg. Chem. 2019, 58(13), 8710-8719.
[24] G. Mulas, G. Ferrauto, W. Dastrù, R. Anedda, S. Aime, E. Terreno, Mag.
Reson. Med. 2015, 74(2), 468-473.
[25] E. Terreno, S. Geninatti Crich, S. Belfiore, L. Biancone, C. Cabella, G.
Esposito, A. D. Manazza, S. Aime, Mag. Reson. Med. 2006, 55(3), 491-497.
[26] D. Hirayama, T. Iida, H. Nakase, Int. J. mol. sci. 2017, 19(1), 92.
[27] D. G. Russell, B. C. Vanderven, S. Glennie, H. Mwandumba, R. S.
Heyderman, Nat Rev Immunol 2009, 9(8), 594-600.
[28] Y. K. Oh, R. M. Straubinger, Infec. Immun. 1996, 64(1), 319-325.
[29] X.-J. Mi, J.-G. Hou, Z. Wang, Y. Han, S. Ren, J.-N. Hu, C. Chen, W. Li,
Sci. rep. 2018, 8(1), 15922-15922.
[9]
T. K. Upadhyay, N. Fatima, A. Sharma, D. Sharma, R. Sharma, Artif.
[30] Y. X. Han, Q.; Hu, J.-N.; Han, X.-Y.; Li, W.; Zhao, L.-C. , Nutrients 2015,
7, 682-696.
[31] W. W. Liu, Z.; Hou, J.-G.; Zhou, Y.-D.; He, Y.-F.; Jiang, S.; Wang, Y.-P.;
Ren, S.; Li, W., Molecules 2018, 23, 2120.
[32] S. Figueiredo, J. C. Cutrin, S. Rizzitelli, E. De Luca, J. N. Moreira, C. F.
G. C. Geraldes, S. Aime, E. Terreno, Mol. Imaging Biol. 2013, 15(3), 307-315.
[33] F. Garello, F. Arena, J. C. Cutrin, G. Esposito, L. D'Angeli, F. Cesano, M.
Filippi, S. Figueiredo, E. Terreno, RSC Adv. 2015, 5(43), 34078-34087.
[34] Y. Pei, Y. Yeo, J. Controlled Release 2016, 240, 202-211.
[35] S. M. Abozeid, E. M. Snyder, T. Y. Tittiris, C. M. Steuerwald, A. Y.
Nazarenko, J. R. Morrow, Inorg. Chem. 2018, 57(4), 2085-2095.
[36] S. J. Dorazio, P. B. Tsitovich, K. E. Siters, J. A. Spernyak, J. R. Morrow,
J. Am. Chem. Soc. 2011, 133(36), 14154-14156.
Cells, Nanomed., and Biotechnol. 2019, 47(1), 427-435.
[10] M. Aouadi, G. J. Tesz, S. M. Nicoloro, M. Wang, M. Chouinard, E. Soto,
G. R. Ostroff, M. P. Czech, Nature 2009, 458(7242), 1180-1184.
[11] E. M. Snyder, D. Asik, S. M. Abozeid, A. Burgio, G. Bateman, S. G.
Turowski, J. Spernyak, J. R. Morrow, Angew. Chem.., Int. Ed.,2020, 132(6),
2435-2440.
[12] J. Wahsner, E. M. Gale, A. Rodríguez-Rodríguez, P. Caravan, Chem.
Rev. 2019, 119(2), 957-1057.
[13] D. Asik, R. Smolinski, S. M. Abozeid, T. B. Mitchell, J. A. Spernyak, J. R.
Morrow, Manuscript in preparation.
[14] J. M. Harrington, S. Chittamuru, S. Dhungana, H. K. Jacobs, A. S.
Gopalan, A. L. Crumbliss, Inorg. Chem. 2010, 49(18), 8208-8221.
[15] J. Li, V. Abbate, J. Jurach, G. Zhang, X. Kong, R. C. Hider,
ChemistrySelect 2016, 1(2), 297-300.
[37] C. J. Gimeno, P. O. Ljungdahl, C. A. Styles, G. R. Fink, Cell 1992, 68(6),
1077-1090.
[16] R. J. Abergel, K. N. Raymond, Hemoglobin 2011, 35(3), 276-290.
[17] A. S. Grillo, A. M. SantaMaria, M. D. Kafina, A. G. Cioffi, N. C. Huston,
M. Han, Y. A. Seo, Y. Y. Yien, C. Nardone, A. V. Menon, J. Fan, D. C. Svoboda,
J. B. Anderson, J. D. Hong, B. G. Nicolau, K. Subedi, A. A. Gewirth, M.
Wessling-Resnick, J. Kim, B. H. Paw, M. D. Burke, Science 2017, 356(6338),
608-616.
[38] J. Chow, M. Notaro, A. Prabhakar, S. J. Free, P. J. Cullen, J. Fungi. 2018,
4(3), 93-110.
[39] Y. Farhat, The Protocol Place. September 2012. Accessed on
03/08/2019. <http://protocol-place.com/>
[40] C. Blaszykowski, S. Sheikh, P. Benvenuto, M. Thompson, Langmuir
2012, 28(5), 2318-2322.
[18] Z. D. Liu, R. C. Hider, Coordination Chem. Rev. 2002, 232(1), 151-171.
[19] Y.-S. Sohn, W. Breuer, A. Munnich, Z. I. Cabantchik, Blood 2008, 111(3),
1690-1699.
[20] A. Cilibrizzi, V. Abbate, Y.-L. Chen, Y. Ma, T. Zhou, R. C. Hider, Chem.
Rev. 2018, 118(16), 7657-7701.
[41] D. T. Daniel R. Rines, Jonas F. Dorn, Paul Goodwin, and Peter K. Sorger,
Cold Spring Harb Protoc 2011, 9, 1026-1041.
[42] A. Patel, D. Asik, J. A. Spernyak, P. J. Cullen, J. R. Morrow, Journal of
Inorganic Biochemistry 2019, 201, 110832.
[21] K. H. Thompson, C. A. Barta, C. Orvig, Chemical Society Reviews 2006,
35(6), 545-556.
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Yeast derived β-glucan particles (GPs) are effective microcarriers for targeting immune system cells, but the porosity of the GPs makes it
challenging to load small molecule drugs or imaging agents. Here we show that members of a new class of cationic Fe(III)-based MRI
contrast agent are encapsulated in the GPs in a stable form. Release of contrast agent produces increased T1 proton relaxation as triggered
by mild acid or a bidentate chelator.
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