Magnetoluminescent agents for dual MRI and time-gated fluorescence imaging

Article abstract of DOI:10.1002/ejic.201200045

The synthesis and properties of two luminescent iron oxide nanoparticles for dual imaging by MRI and time-gated confocal microscopy are presented. These magnetoplasmonic agents consist of macrocyclic terbium complexes conjugated onto magnetite nanoparticles by a polyethylene glycol linker. Two macrocyclic terbium complexes were investigated: Tb-DOTAm-(Phen)₃ and Tb-DOTAm-(acetylene)₃. Both complexes display the characteristic long luminescence lifetimes of lanthanides (0.8 ms and 1.3 ms, respectively), rendering them ideal for time-gated luminescence spectroscopy and confocal imaging - a feature currently unexplored in multimodal imaging agents. Additionally, the 5 ± 1 nm magnetite nanoparticles, with their permeable PEG coating and stable dopamide anchor, render the two constructs efficient MRI contrast agents with longitudinal (r₁) and transverse (r₂) relaxivities of 9.8 mM^-1_Fe s^-1 and 98 mM ^-1_Fe s^-1 at 60 MHz and 37 °C. The synthesis and characterization of Fe₃O₄@Tb-DOTAm-(acetylene) ₃ and Fe₃O₄@Tb-DOTAm-(Phen)₃, two magnetoluminescent agents for dual imaging by MRI and confocal imaging are presented. The nanoparticles are characterized by high transverse and longitudinal relaxivities and long luminescence lifetimes ideal for time-gated spectroscopy. Copyright

Full text of DOI:10.1002/ejic.201200045

FULL PAPER
DOI: 10.1002/ejic.201200045
Magnetoluminescent Agents for Dual MRI and Time-Gated Fluorescence
Imaging
Eric D. Smolensky,[a] Yue Zhou,[a] and Valérie C. Pierre*[a]
Keywords: Luminescence / Lanthanides / Nanoparticles / MRI / Time-gated spectroscopy
The synthesis and properties of two luminescent iron oxide
nanoparticles for dual imaging by MRI and time-gated confo-
cal microscopy are presented. These magnetoplasmonic
agents consist of macrocyclic terbium complexes conjugated
onto magnetite nanoparticles by a polyethylene glycol linker.
Two macrocyclic terbium complexes were investigated: Tb-
DOTAm-(Phen)3 and Tb-DOTAm-(acetylene)3. Both com-
plexes display the characteristic long luminescence lifetimes
of lanthanides (0.8 ms and 1.3 ms, respectively), rendering
them ideal for time-gated luminescence spectroscopy and
confocal imaging feature currently unexplored in
–
a
multimodal imaging agents. Additionally, the 5Ϯ1 nm mag-
netite nanoparticles, with their permeable PEG coating and
stable dopamide anchor, render the two constructs efficient
MRI contrast agents with longitudinal (r1) and transverse (r2)
–1
–1
–1
–1
relaxivities of 9.8 m
37 °C.
M
s
and 98 m
M
s
Fe
at 60 MHz and
Fe
atives of MIONs have already been reported. Their struc-
tures can be divided into two general classes: amalga-
mates[3] and core-shell structures.[4] Amalgamates consist of
MIONs and fluorescent probes, most commonly organic
fluorophores[3a,3d] or quantum dots,[3b,3c,3e] mixed into large
clusters typically ranging between 500 nm and 5 μm in size
and held together by a silica matrix. These amalgams pres-
ent several notable disadvantages: their large size limits
their cellular uptake,[5] and silica-coated nanoparticles have
shown significant cellular toxicity.[3a,3d,3e,6] Alternatively,
one can design magnetoluminescent agents with a core-shell
structure by directly coating single MIONs with or-
ganic[3b,3c,4a] or inorganic[4c,7] fluorophores. Although this
second approach necessitates lengthier syntheses, the re-
sulting agents demonstrate improved relaxivities (r2 from 70
Introduction
Magnetic Resonance Imaging (MRI) is plagued by two
shortcomings that contrast agents have had little success to
alleviate despite advances in their design over the last few
decades: poor spatial resolution and poor sensitivity.
Whether gadolinium or particulate-based, accumulation of
millimolar concentrations of contrast agents are still needed
in a tissue in order to obtain sufficient contrast. In view of
these limitations, researchers have recently sought to com-
bine the three-dimensional and in vivo aspects of MRI with
imaging techniques that are complementary in terms of
both resolution and sensitivity. Confocal imaging, one of
the most widely used imaging techniques in biology, offers
nm resolution with techniques such as PALM[1] and
STORM[2] but is limited to cell cultures and tissue biopsies.
Employing these two imaging platforms concomitantly of-
fers several advantages but first requires the development
of multimodal imaging agents that would behave both as
MRI contrast agents and luminescent probes, so-called
magnetoluminescent imaging agents.
Magnetoluminescent imaging agents are achieved by
conjugating, either in a nanomaterial or a discrete small
molecule, a luminescent probe with either a gadolinium or
MION-based contrast agent. Given the superior longitudi-
nal and especially transverse relaxivities of Magnetic Iron
Oxide Nanoparticles (MIONs), we reasoned that improved
MRI sensitivity would be achieved with the use of magne-
tite nanoparticles. In this regard, several luminescent deriv-
to 150 mm–1 s–1) and cellular uptake.
Fe
We have sought to improve on this latter design in two
ways: (1) by replacing the silica coating with polyethylene
glycol; and (2) by employing luminescent lanthanide probes
(Figure 1). The use of a PEG coating in lieu of a silica one
offers several advantages. First silica nanoparticles have
shown significant cellular toxicity, with cell viability begin-
ning to decrease at about 50–200 μg/mL of silica,[3a,3d,3e,6]
whereas PEG-coated MIONs have not.[4b,6] Second, the
substantial silica coating reduces the saturation magnetiza-
tion, MS, of the magnetite core[8] and consequently also the
relaxivity of the contrast agent. Polyethylene glycol coat-
ings, on the other hand, have little effect on both MS and
relaxivities if the anchoring group is chosen wisely.[9] Dopa-
mide-PEG-coated MIONs are characterized with r2 of
110 mm–1 s–1, almost double that of dextran-coated com-
[a] Department of Chemistry, University of Minnesota
207 Pleasant Street SE, Minneapolis, MN 55455, USA
Fax: +1-612-626-7541
Fe
mercial USPIOs (Ultra Small Particles of Iron Oxide) of
E-mail: pierre@umn.edu
similar size.[9,10] The clinical USPIO Sinerem®, which also
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E. D. Smolensky, Y. Zhou, V. C. Pierre
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comprises magnetite cores 4–5 nm in diameter but is instead ported by Parker to be an efficient antenna for Tb lumines-
coated with a 20 nm thick dextran layer, displays a signifi- cence.[14]
cantly lower r2 of 53.1 mm–1 s–1.[10] The water permeability
Fe
of the shorter PEG coating, as opposed to the impenetra-
bility of the thick silica layer, further increases the relaxivity
of the agent.
Results and Discussion
Synthesis
The two magnetoluminescent probes Fe3O4@Tb-
DOTAm-(acetylene)3 (1) and Fe3O4@Tb-DOTAm-(Phen)3
(2) were synthesized according to Scheme 1. The magnetic
iron oxide nanoparticles were synthesized by high-tempera-
ture thermal decomposition of Fe(acac)3 in the presence of
oleylamine and oleic acid, according to the procedure devel-
oped by Wang et al.[15] Importantly, this procedure yields
relatively monodisperse nanoparticles of magnetite (Fe3O4)
with high saturation magnetization (MS), typically around
78 Am2/kg, which are stable in organic solvents for ex-
tended periods of time (months).[15] The synthesized nano-
particles are faceted in morphology (Figure 2a), with a dia-
meter of 5.0Ϯ1.3 nm, as measured by TEM, but their high
stability, and more importantly their high MS, render them
Figure 1. Design and chemical structure of magnetoluminescent
probes for dual MRI and time-gated confocal imaging Fe3O4@Tb-
DOTAm-(acetylene)3 (1) and Fe3O4@Tb-DOTAm-(Phen)3 (2).
Particles are fully coated (simplified for clarity).
With respect to the fluorophore, the use of luminescent
lanthanide complexes presents several significant advan-
tages over their organic and transition-based analogues.[11]
First, their emission spectra are characterized by very nar-
row bands with little overlap – facilitating the quantitative
visualization of multiple analytes simultaneously. Second,
their sensitized emission gives rise to large Stokes shift
(Ͼ 100 nm), such that concentration-dependent self-absorp-
tion problems do not arise. As a result, the responses of
luminescent lanthanide probes are linear over a wide range
of concentrations. Third, and most important, they present
long-lived luminescence with lifetimes in the millisecond
range, orders of magnitude longer than the autofluoresc-
ence of biological samples or that of organic or transition-
metal-based dyes. Lanthanides are thus ideal for time-gated
imaging – a technique, which employs a delay, or gate, be-
tween the excitation pulse and the luminescence detection,
thereby enabling the background luminescence of the media
to decay to zero before measuring that of the probe. This
technique thus allows for significantly more sensitive and
precise detection of luminescence in biological samples.[12]
Our magnetoluminescent probes for dual MRI and time-
gated confocal imaging were thus designed to contain three
different features: a magnetite nanocrystal, which gives the
assemblies their high relaxivity; a PEG coating coordinated
to the core by dopamide ligands, which solidly anchors the
protective polymer coating on the core while maintaining
the magnetization saturation and relaxivity of the latter;
and a terbium complex directly conjugated to the PEG,
which gives the assembly its long-lived luminescence.
Macrocyclic, DOTA-type terbium complexes were chosen
due to their hydrophilicity and their high thermodynamic
and kinetic stability.[13] Lanthanide luminescence requires
sensitization by a nearby antenna for practical applica-
tion.[11] Two antennae were investigated: acetylenes and 4-
Scheme 1. Synthesis of Fe3O4@Tb-DOTAm-(acetylene)3 (1) and
Fe3O4@Tb-DOTAm-(Phen)3 (2). Reagents and conditions: (a) tert-
butyl 2-bromoacetate, Cs2CO3, CH3CN, 25 °C, 6 h; (b) 2-chloro-N-
(prop-2-yn-1-yl)acetamide, Cs2CO3, CH3CN, 60 °C, 12 h; (c) TFA,
CH2Cl2, 25 °C, 12 h; (d) Fe3O4@DopPEG-NH2, HATU, DIPEA,
H2O/DMA (9:1), 25 °C, 2 h; (e) TbCl3, H2O pH = 7, 70 °C, 2 h;
(f) 4-(azidomethyl)phenanthridine, CuI ascorbate, H2O/DMA (3:1),
methylphenanthridine. Phenanthridine was previously re- 25 °C, 15 h.
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Magnetoluminescent Agents for Dual MRI and Time-Gated Fluorescence Imaging
ideal for applications as MRI contrast agents. Both the lon-
The hydrophilic polymer, Dop-PEG-NH2, which con-
gitudinal and transverse relaxivities of particulate contrast tains both a catechol group and an amine for further func-
agents are directly proportional to their saturation magne- tionalization, was synthesized as previously reported.[18]
A
tization.[16]
2000 MW polyethylene glycol was chosen for coating as this
hydrophilic polymer has previously been reported to be
long enough to ensure complete dispersity of the MION in
water without aggregation.[19] The polymer was grafted on
the magnetite nanoparticles according to a ligand exchange
protocol, which has previously been reported to maintain
the magnetism of the magnetite core while limiting particle
aggregation and increasing dispersibility.[9] Importantly, in
the resulting nanoparticle-PEG conjugate, Fe3O4@Dop-
PEG-NH2, the catechol stabilizes the iron oxide surface, but
the free amine remains available for further functionaliza-
tion. Notably, when functionalized by a stable dopamide
anchor, iron oxide nanoparticles coated with polyethylene
Figure 2. TEM of (a) Fe3O4@Dop-PEG-NH2, (b) Fe3O4@Tb-
DOTAm-(acetylene)3 (1), and (c) Fe3O4@Tb-DOTAm-(Phen)3 (2).
To anchor a luminescent component to the particle sur- glycol show remarkable stability (months) in phosphate
face, terbium complexes are directly conjugated to a poly- buffer saline as evidenced by both dynamic light scattering
ethylene glycol polymer terminated with a dopamide chela- (no aggregation) and constant longitudinal and transverse
tor. The role of the dopamide is to anchor the luminescent relaxivities.[9]
coating solidly on the iron oxide core without affecting the
Advantageously, the resulting functionalized nanopar-
saturation magnetization of the latter. The synthesis of the ticles, Fe3O4@Dop-PEG-NH2, can readily and rapidly be
assembly first involves that of the DOTAm-(acetylene)3 purified by two successive filtrations. A first filtration using
moiety, followed by its conjugation to PEG-coated iron ox- a microfilterfuge with 2 μm pores separates any unstable
ide nanoparticles. Subsequent formation of the terbium MION aggregate. The filtrate is then recovered and the
complex and grafting on the phenanthridine antennas nanoparticles resuspended in water. A second filtration
yields the two magnetoluminescent agents (Scheme 1).
using a much smaller filter with a 10 kDA MW cut-off
It should be noted that, consequently, our design em- readily separates any excess polymer that is not function-
ploys two metals, which are hard acids (iron and terbium), alized on the nanoparticles. The functionalized MIONs are
and two different chelators, which are also hard bases [cate- recovered in the small volume of residue. These very small
chol and macrocyclic poly(amino carboxylate)]. The selec- pore filters, commonly used in protein purification, are also
tivity of each chelate for its respective metal (catechol for very efficient at purifying nanoparticles after performing
iron and DOTA for terbium) is achieved in two ways. First, any surface chemistry. In fact, this purification technique
catechol ligands have previously been demonstrated to have has several advantages over the most widespread centrifu-
higher affinity for the harder FeIII than LnIII [17]
Moreover, gation method: it is more rapid, gentler, and higher-yield-
.
carboxylate ligands are readily displaced by catechol-ter- ing. Indeed, purification of nanoparticles by pellet forma-
minated polymers.[9] The selectivity is also achieved by the tion by centrifugation has two drawbacks. First, it often
synthetic route followed. Functionalization of the nanopar- results in irreversible aggregation of nanoparticles, and
ticles with dopamide-PEG prior to conjugating the macro- therefore subsequently in significantly less stable disper-
cycle enables the iron oxide core to effectively behave as a sions. Second, in the opposite case, when nanoparticles are
protective group for the catechol moiety, thereby preventing initially very well suspended, the aggressive centrifugation
it from binding nonselectively the lanthanide ion later. In- at high RPM and long periods of time required to separate
deed, an alternative synthetic route, which first involves ini- the nanoparticles result in significant loss of material.
tial formation of the Dop-PEG-Tb-DOTAm-(acetylene)3
Successful functionalization of the MION was assessed
(Tb·6), was unsuccessful. The terbium complex could not by infrared spectroscopy (Figure 3). The sharp C–H
be functionalized on the MION, likely because the dopam- stretching bands at 2919 cm–1 and 2848 cm–1 characteristic
ide was already chelating TbIII
.
of the Fe3O4@oleic acid starting material are indicative of
The DOTAm-(acetylene)3 moiety was synthesized in four a well-ordered monolayer of surfactants. Displacement of
steps. First, a single acetate arm was conjugated on the cy- the original oleic acid coating with polyethylene glycol re-
clen backbone by standard coupling procedure using a large sults in a significant loss of surface order and consequential
excess of cyclen. The acetylene-terminated acetamide arm, loss in the fine structure of the C–H stretching bands. The
2-chloro-N-(prop-2-yn-1-yl)acetamide, was synthesized C–H stretching band at 2882 cm–1, for instance, is broad
separately from chloroacetyl chloride and propargylamine and featureless. More importantly, the appearance of the
in acetonitrile in the presence of a weak base. Three of these C–O stretching band at 1109 cm–1 and disappearance of the
arms were then coupled on the cyclen ring to complete the C=O stretching band at 1560 cm–1 from the oleic acid cor-
acetylene-terminated and tert-butyl-protected DOTAm li- relates with refunctionalization with the PEG ligand. Trans-
gand (4), which was then deprotected under acidic condi- mission electron micrography of the nanoparticles (Fig-
tions to give the mono-acid DOTAm ligand (5).
ure 2) indicates that functionalization does not affect
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E. D. Smolensky, Y. Zhou, V. C. Pierre
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the particle size or their morphology. The hydrophilic viously obtained from the bromomethyl analogue, which
Fe3O4@Dop-PEG-NH2 nanoparticles maintain their was synthesized as previously reported.[20] This time, the
mostly faceted form with a particle size of 5.0Ϯ1.3 nm.
conjugation was monitored by the time-gated excitation
profile of the terbium ion (λemission = 545 nm). Advan-
tageously, the excitation profiles of the two terbium com-
plexes are unique: that of Tb-DOTAm-(acetylene)3 is char-
acterized by a single sharp band centered at 279 nm,
whereas Tb-DOTAm-(Phen)3, like other phenanthridine
lanthanide complexes,[14] has a distinctive pattern of two
bands at 240 nm and 296 nm with a shoulder at 341 nm
(Figure 4). Complete conjugation of the phenanthridine
arms was deemed complete when the bands at 245 nm and
300 nm stopped growing.
Figure 3. Infrared spectra of (a) Fe3O4@oleic acid, (b) Fe3O4@Dop-
PEG-NBOC, (c) Fe3O4@Dop-PEG-NH2, (d) Fe3O4@DOTAm-
(acetylene)3.
The Fe3O4@DOTAm-(acetylene)3 was synthesized by in-
cubating the Fe3O4@Dop-PEG-NH2 with DOTAm-(acetyl-
ene)3 in the presence of the coupling reagent HATU. The
resulting Fe3O4@DOTAm-(acetylene)3 complex could
easily be purified from any uncomplexed DOTAm-(acetyl-
ene)3 by the same filtration techniques as previously men-
tioned. The sample was passed through a 2 μm filter to re- Figure 4. Excitation (gray) and emission (black) spectra of
Fe3O4@Tb-DOTAm-(acetylene)3 (1) [a and b, respectively, dashed
move large clusters, and then the filtrate was passed
through a 10 kDa MW filter to remove unconjugated
lines, DMA/H2O (3:1)] and Fe3O4@Tb-DOTAm-(Phen)3 (2) (c and
d, respectively, solid lines, H2O, pH = 7.0). Experimental condi-
DOTAm-(acetylene)3. Notably, attempts at directly conju-
tions: excitation slit width = 20 nm, emission slit width = 20 nm,
gating the Tb complex, Tb-DOTAm-(acetylene)3, onto the
amine-terminated nanoparticles were unsuccessful, most
likely because coordination to the lanthanide ion reduces
the reactivity of the carboxylate.
The terbium complex was subsequently formed by incub-
ating the nanoparticles with an excess of TbCl3 in water at
time delay = 0.1 ms, T = 20 °C.
Luminescence and Relaxivity
Fe3O4@Tb-DOTAm-(acetylene)3 (1) and Fe3O4@Tb-
slightly basic pH while heating. Formation of the terbium DOTAm-(Phen)3 (2) were designed to behave both as ef-
complex was assessed by monitoring the time-gated lumi- ficient MRI contrast agents and luminescent probes with
nescence of the terbium center [λexcitation = 279 nm, λemission long luminescence lifetimes for time-gated confocal im-
= 545 nm, time-gate = 0.1 ms, DMA/H2O (3:1)]. The reac- aging. Interestingly, both assemblies are luminescent and
tion was deemed complete when the intensity of the terbium display characteristic terbium emission (Figure 4) with long
emission stopped increasing. It should be noted (see later) luminescence lifetimes in the millisecond range (Table 1),
that acetylene groups are surprisingly efficient at sensitizing rendering them ideal probes for time-gated spectroscopy.
the terbium luminescence even in mixed aqueous solutions. Although the ability of phenanthridine chromophores to
The nanoparticles were purified by chromatography on a sensitize terbium emission has been reported by both Par-
NAP-5 column. Size-exclusion NAP-5 columns, although ker[14] and ourselves,[20] the ability of simple acetylenes to
not as effective as MW cut-off filters at separating nanopar- do so, albeit with lower efficiency, is less known.
ticles from larger unfunctionalized polymers, are still par- Fe3O4@Tb-DOTAm-(acetylene)3 (1), however, is not the
ticularly efficient at purifying nanoparticle from small mo- first example of a luminescent lanthanide/acetylene com-
lecules such as small organic moieties, inorganic complexes, plex. Kallistratos and Mündner previously reported that
or excess buffer.
The second magnetoluminescent agent, Fe3O4@Tb-DO- olic acid efficiently sensitize both europium and terbium.[21]
TAm-(Phen)3 (2), was directly synthesized from its acetyl- The efficiency of both nanocomposites to behave as MRI
both propiolic acid and to a greater extent 3-phenylpropi-
ene precursor 1 and an excess of 4-(azidomethyl)phen- contrast agents was evaluated by measuring both their lon-
anthridine using a standard copper(I)-catalyzed Huisgen gitudinal and transverse relaxivities in water at neutral pH,
cycloaddition. The 4-(azidomethyl)phenanthridine was pre- 37 °C and 60 MHz (Table 1). The longitudinal relaxivity of
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Magnetoluminescent Agents for Dual MRI and Time-Gated Fluorescence Imaging
Table 1. Physical properties of magnetoluminescenct nanoparticles.
[b]
[b]
Core diameter[a]
[nm]
r1
r2
Tb τ[c]
[ms]
[mmFe–1 s–1]
[mmFe–1 s–1]
Fe3O4@DopPEG-NBoc
Fe3O4@DopPEG-NH2
Fe3O4@Tb-DOTAm-(acetylene)3
Fe3O4@Tb-DOTAm-(Phen)3
4.9(1.3)
5.0(1.3)
5.1(1.0)
5. 1(1.4)
6.4
6.5
9.8
3.2
115
114
98
n/a
n/a
1.3
0.8
160
[a] Numbers in parentheses represent polydispersity (1 standard deviation). [b] H2O, pH = 7.0, 37 °C, 60 MHz. [c] H2O, pH = 7.0, 25 °C,
solutions were not degassed.
the two magnetoluminescent agents is somewhat affected by small (5 nm) PEG-coated magnetite core renders the nano-
addition of the paramagnetic macrocyclic terbium com- composite powerful MRI contrast agents with superior lon-
plexes. Conjugation of the hydrophilic Tb-DOTAm-(acetyl- gitudinal (r1) and transverse (r2) relaxivities compared to
ene)3 increases r1 by 50%, whereas the more hydrophobic clinical gadolinium-based contrast agents and commercial
Tb-DOTAm-(Phen)3 decreases it by 50%. In either case, at USPIOs, respectively. Both terbium complexes show bright
identical temperature and magnetic field (37 °C and 1.5 T), luminescence with characteristic long luminescence lifetime
the longitudinal relaxivities of the magnetoluminescent in the millisecond range, rendering them ideal for time-
probes remain comparable to that of commercial MIONs gated spectroscopic and microscopic applications for bio-
(Sinerem®: r1 = 9.9 mm–1 s–1) and greater than that of clin- logical and medical samples. Their long lifetimes enable the
Fe
ical gadolinium-based contrast agents (Gd-DOTA, Dota- background autofluorescence of such samples to be readily
rem®: r1 = 2.9 mm–1 s–1).
removed from the luminescent signal of the probe. More-
The transverse relaxivity is also moderately affected by over, the acetylene moieties of Fe3O4@Tb-DOTAm-(acetyl-
addition of the luminescent probes. Fe3O4@Tb-DOTAm- ene)3 (1) further gives the assembly the unique opportunity
(acetylene)3 (1) displays an r2 value of 98 mmFe–1s–1, com- to be readily conjugated to azide-functionalized biomolec-
parable to that of its precursor and almost twice that of the ules such as proteins and oligonucleotides directly in a
commercial Sinerem® (r2 = 53.1 mm–1 s–1). This notable cellular environment by CuI-catalyzed Huisgen cycload-
Fe
and beneficial increase is likely the result of the thinner and dition. The ability to use these magnetoluminescent probes
water-permeable PEG coating used in our construct.[9] to tag and monitor proteins by both MRI and time-gated
Fe3O4@Tb-DOTAm-(Phen)3 (2) displays a notably higher confocal microscopy is currently being investigated.
transverse relaxivity, which, in accordance with the lower
longitudinal relaxivity observed for the assembly, suggests
that some aggregation is taking place in solution.[18] The
phenanthridine antenna does favor energy transfer to the
Experimental Section
General Considerations: Unless otherwise stated, all chemicals were
purchased from commercial suppliers and used without further pu-
rification. Water was distilled and further purified by a Millipore
Simplicity UV system (Resistivity 18ϫ106 Ω). All organic extracts
were dried with anhydrous MgSO4, and solvents were removed with
a rotary evaporator. Flash chromatography was performed on
terbium ion, thereby yielding a brighter probe, but its con-
jugation comes at the detriment of the water dispersibility
of the resulting MIONs.
Although it is not the focus of this investigation, it
should be noted that the two tetramide complexes, Tb-
DOTAm-(acetylene)3 and Tb-DOTAm-(Phen)3, and their
respective assemblies should also function as PARACEST
agents (Paramagnetic Chemical Exchange Saturation
Transfer).[22] This class of complexes presents the two key
advantages for CEST-type MRI experiments: their water
exchange rates are typically slow,[23] and the two exchangea-
ble proton sites (amide NH and coordinated H2O) are fre-
quency shifted well away from the bulk water NMR fre-
quency.[22] As such presaturation of the magnetization of
the complex’s NH or H2O protons efficiently transfers over
to that of bulk water, with a resulting decrease in magne-
tization of the latter.
1
Merck silica gel (40–7 mesh). H NMR spectra were recorded with
a Varian 500 or with a Varian 300 at 300 MHz; the residual solvent
peak was used as an internal reference. Data for 1H NMR are
recorded as follows: chemical shift (δ, ppm), multiplicity (s, singlet;
d, doublet, t, triplet; q, quartet; sept, septet; br., broad; m, mul-
tiplet), coupling constant (Hz), integration. Mass spectra (LR =
low resolution; HR = high resolution; ESI-MS = electrospray ion-
ization mass spectrometry) were recorded with a Bruker BioTOF
II at the Mass Spectrometry Facility of the Department of Chemis-
try at the University of Minnesota, Twin-Cities. UV/Vis data was
obtained with a Cary Bio 100 UV/Vis spectrophotometer. Data
was collected over the range of 200–800 nm. Fluorescence data was
acquired with a Varian Cary Eclipse fluorescence spectrophotome-
ter using a quartz cell with a path length of 10 mm, excitation slit
width of 10 nm and emission slit width of 20 nm at T = 20 °C.
Solutions were not degassed prior to measurement of their lumines-
cence spectra and lifetimes. pH measurements were taken using a
Thermo Orion 3 Benchtop pH meter. TEM images were collected
with a JOEL JEM1210 at 120 kV. Solid-state infrared spectra were
recorded with a Thermo Nicolet 6700 FTIR using an ATR adapter.
Conclusions
Fe3O4@Tb-DOTAm-(acetylene)3 (1) and Fe3O4@Tb-
DOTAm-(Phen)3 (2), two magnetoluminescent assemblies,
show promises for dual imaging by Magnetic Resonance
Imaging and time-gated luminescence spectroscopy. The Samples were dried and placed on the surface of a germanium
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E. D. Smolensky, Y. Zhou, V. C. Pierre
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crystal, and FTIR-ATR (attenuated total reflectance) spectra were
collected between 700 cm–1 and 3700 cm–1 at a resolution of 2 cm–1.
the product was further dried under high vacuum overnight. The
amine 3 was obtained as a light yellow oil (142 mg, 33.3%). 1H
NMR (300 MHz, CD2Cl2, 25 °C): δ = 5.33 (s, 3 H), 3.34 (m, 4 H),
2.79 (m, 14 H), 1.44 (s, 9 H) ppm. ESI-MS: m/z = 287.1 [M + H+]
(calcd. 286.2).
Relaxivity: Longitudinal (T1) and transverse (T2) relaxation times
of the nanoparticles in mQ water were measured with a Bruker
Minispec mq60 NMR analyzer at 60 MHz and 37 °C according to
the inverse recovery sequence and the Carr-Purcell-Meiboom-Gill
sequence, respectively. The total concentration of iron of each sam-
ple was determined by Equation (1). Briefly, 5 μL of each probe
was suspended in 200 μL of HNO3 and 100 μL of mQ water. Each
sample was heated at 110 °C overnight, after which T1 of each solu-
tion was measured. The resulting concentration of iron was calcu-
lated from a calibration plot created from ICP-OES analysis of
solutions of FeCl3 in HNO3/mQ water (2:1). For each probe, the
longitudinal (r1) and transverse (r2) relaxivities were fitted to Equa-
tion (1).
2-Chloro-N-(prop-2-yn-1-yl)acetamide: The acetamide was synthe-
sized as previously reported.[24] Briefly, chloroacetyl chloride
(134 mg, 1.20 mmol) was added dropwise to a solution of propar-
gylamine (55 mg, 1.0 mmol) in CH2Cl2 (10 mL). N,N-Diisopropyle-
thylamine (156 mg, 1.20 mmol) was added dropwise to the reaction
mixture, which turned yellow. The reaction mixture was stirred for
30 min, and the solvents were removed under reduced pressure. The
crude product was purified by flash chromatography on silica elut-
ing with a gradient of 100% CH2Cl2 to 97% CH2Cl2/3% CH3OH.
The solvents were removed under reduced pressure yielding the
acetamide as an off-white solid (45 mg, 69%). 1H NMR (300 MHz,
CD2Cl2, 25 °C): δ = 4.07 (m, 4 H), 2.33 (t, J = 2.4 Hz, 1 H) ppm.
tert-Butyl
2-{4,7,10-Tris[2-oxo-2-(prop-2-yn-1-ylamino)ethyl]-
1,4,7,10-tetraazacyclododecan-1-yl}acetate (4): The amine
3
(1)
(272 mg, 0.950 mmol) was dissolved in acetonitrile (10 mL). 2-
Chloro-N-(prop-2-yn-1-yl)acetamide (500 mg, 3.81 mmol) was
added to the reaction mixture followed by Cs2CO3 (1.24 g,
3.81 mmol), which was stirred overnight at 60 °C. The solvent was
removed under reduced pressure and the crude product purified by
flash chromatography on silica eluting with a gradient of 100%
CH2Cl to 85% CH2Cl2/14% CH3OH/1% NH4OH yielding the
product (70 mg, 27%). 1H NMR (300 MHz, CD2Cl2, 25 °C): δ =
4.12 (m, 6 H), 2.8 (m, 27 H), 1.47 (s, 9 H) ppm. ESI-MS: m/z =
571.2 [M+] (calcd. 571.3).
Fe3O4@Oleic Acid: Oleic acid coated magnetite nanoparticles were
synthesized from Fe(acac)3, oleic acid and oleylamine according to
a procedure developed by Wang et al.[15] The sample was kept as a
hexane suspension, and no precipitation or change in relaxivity was
evident over the course of months.
Dop-PEG-NBoc: The protected amine was synthesized from NHS-
PEG-Boc (Jenkem) in 71% yield as previously reported[18] with suc-
cessful synthesis verified by 1H NMR and LR MS. 1H NMR
(300 MHz, CDCl3, 25 °C): δ = 6.81 (d, J = 8 Hz, 1 H), 6.75 (s, 1
H), 6.57 (d, J = 8 Hz, 1 H), 3.94 (s, 2 H), 3.61 (m, 167 H), 2.71 (t,
J = 7 Hz, 2 H), 1.44 (s, 9 H) ppm. ESI-MS: (m/z) = 1191 [M + H+]
(calcd. 1192).
2-{4,7,10-Tris[2-oxo-2-(prop-2-yn-1-ylamino)ethyl]-1,4,7,10-tetra-
azacyclododecan-1-yl}acetic Acid (5): The acetate
4 (70 mg,
0.12 mmol) was dissolved in CHCl3 (10 mL). Trifluoroacetic acid
(5 mL) was added to the solution, which was stirred at room temp.
overnight. The solvents were removed under reduced pressure. The
crude product was redissolved in methanol (10 mL) and the solvent
removed under reduced pressure. Successive methanol washes were
repeated for a total of three times. The acid 5 was obtained as a
Fe3O4@Dop-PEG-NBoc: Fe3O4@Oleic acid (2 mg) was suspended
in THF (2 mL). Separately, Dop-PEG-NBoc (20 mg) was dissolved
in mQ water, and the pH was adjusted to 10 with KOH(aq) (1.0 m).
The two solutions were mixed together and stirred at room temp.
for 12 h. The brown suspension was filtered with a microfilterfuge
(2 μm) to remove any unstable particulate clusters. The filtrate was
redispersed in mQ water and filtered through a 10 kDa MW cut-
off filter to remove excess unfunctionalized polymer. The residual
brown suspension of pure Fe3O4@Dop-PEG-NBoc in water, was
adjusted to a final [Fe]total = 1.15 mm. See Figure 3 for infrared
spectra.
1
yellow oil (70 mg, quant.). H NMR (300 MHz, CDCl3, 25 °C): δ
= 4.03 (s, 1 H), 4.00 (s, 2 H), 3.42 (m, 24 H) 2.63 (m, 2 H) ppm.
ESI-MS: m/z = 516.3 [M + H+], 538.3 [M + Na+] (calcd. 515.3).
Fe3O4@Dop-PEG-DOTAm-(acetylene)3 (6): The acid 5 (5 mg,
0.03 mmol) was dissolved in N,N-dimethylacetamide (DMA).
HATU [O-(7-azabenzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium
hexafluorophosphate, 11 mg, 0.03 mmol] was added to the reaction
mixture, which was stirred at room temp. for 10 min. N,N-Diisopro-
pylethylamine (5.0 μL, 0.027 mmol) was added to the reaction mix-
ture, followed by Fe3O4@Dop-PEG-NH2 (50 μL, 0.38 mm [Fe]total).
The solution was stirred at room temp. overnight, after which the
sample was filtered through a 10 kDA filter to remove unconju-
gated ligand. The suspension product, Fe3O4@Dop-PEG-DotAm-
(acetylene)3, was adjusted to a final [Fe]total of 19 μm. See Figure 3
for infrared spectra.
Fe3O4@Dop-PEG-NH2: An aqueous suspension of Fe3O4@Dop-
PEG-NBoc was acidified to pH = 1 with HCl(aq) (1.0 m). The sus-
pension was stirred at room temperature for 30 min, then neutral-
ized with NaHCO3 (aq). The brown suspension was filtered with a
microfilterfuge (2 μm) to remove any unstable particulate clusters.
The filtrate was redispersed in mQ water and filtered through a
10 kDA MW cut-off filter to remove any free ligand. The residual
brown suspension, Fe3O4@Dop-PEG-NH2, was adjusted to a final
[Fe] = 0.383 mm. See Figure 3 for infrared spectra.
Fe3O4@Tb-DOTAm-(acetylene)3 (1): A solution of TbCl3 (0.58 mg,
2.2 μmol) in mQ water (190 μL) was added to a suspension of
Fe3O4@DOTAm-(acetylene)3 (50 μL, 19 μm Fe) in mQ water
(1 mL). The pH was adjusted to 8 with NaOH (1.0 m), and the
tert-Butyl 2-(1,4,7,10-Tetraazacyclododecan-1-yl)acetate (3): CsCO3
(766 mg, 2.35 mmol) was added to a solution of cyclen (754 mg,
5.88 mmol) dissolved in acetonitrile (20 mL). tert-Butyl bromoacet-
ate (382 mg, 1.96 mmol) was added slowly over the course of suspension was stirred at 50 °C. The successful formation of the
30 min, and the reaction mixture was stirred at room temp. for 6 h.
The solvents were removed under reduced pressure, and the crude
product was purified by flash chromatography on silica eluting with
a gradient of 100% CH2Cl2 to 80% CH2Cl2/17% CH3OH/3%
NH4OH. The solvents were removed under reduced pressure, and
complex was monitored by time-gated luminescence of the product
(see Figure 4). The suspension was filtered with a microfilterfuge
(2 μm) to remove unstable particulate clusters. The filtrate was then
redispersed in mQ water and purified by NAP-5 column
chromatography eluting with 100% mQ water to remove uncom-
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2012, 2141–2147

Magnetoluminescent Agents for Dual MRI and Time-Gated Fluorescence Imaging
Zhang, Cancer Res. 2009, 69, 6200–6207; b) S. Wan, J. Huang,
plexed terbium. The suspension product, Fe3O3@Tb-DOTAm-
(acetylene)3, was adjusted to a final [Fe] of 6 μm and stored in mQ
water. See Figure 3 for infrared characterization.
M. Guo, H. Zhang, Y. Cao, H. Yan, K. Liu, J. Biomed. Mater.
Res., Part A 2007, 80A, 946–954; c) A. J. Kell, M. L. Barnes,
Z. J. Jakubek, B. Simard, J. Phys. Chem. C 2011, 115, 18412–
18421.
4-(Azidomethyl)phenanthridine:
4-(Bromomethyl)phenanthridine
was synthesized as previously reported.[20] 4-(Bromomethyl)phen-
anthridine (50 mg, 0.18 mmol) was dissolved in acetone (50 mL),
and NaN3 (119 mg, 1.84 mmol) was added. The mixture was heated
to reflux and stirred for 2 h, after which the acetone was removed
under reduced pressure, and the resulting solid was redissolved in
CH2Cl2. The organic phase was extracted with a solution of NaCl
(10% w/v, 3ϫ30 mL). The organic phase was dried with MgSO4,
the solvent was removed under reduced pressure and the crude
product purified by flash chromatography on silica eluting with
100% CH2Cl2. The combined fractions were dried yielding the
product (32 mg, 74%). 1H NMR (300 MHz, CD2Cl2, 25 °C): δ =
9.57 (s, 1 H), 8.76 (m, 2 H), 8.28 (d, J = 7.5 Hz), 8.10 (t, J = 7.5 Hz,
1 H), 7.89 (m, 3 H), 5.30 (s, 2 H) ppm. ESI-MS: m/z = 257.0 [M
+ Na+] (calcd. 257.1).
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Fe3O4@Tb-DOTAm-(Phen)3 (2): A solution of 4-(azidomethyl)-
phenanthridine (0.56 mg, 2.4 μmol) was added to a suspension of
Fe3O4@Tb-DOTAm-(acetylene)3 (50 μL, 6 μm) in DMA (300 μL)
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solution, 2.5 μmol] was added to the reaction mixture, which was
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This work was supported partially by the MRSEC Program of the
National Science Foundation under Award Number DMR-
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Received: January 17, 2012
Published Online: March 13, 2012
Eur. J. Inorg. Chem. 2012, 2141–2147
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
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