Synthesis and in vitro assessment of a bifunctional closomer probe for fluorine (19F) magnetic resonance and optical bimodal cellular imaging

Article abstract of DOI:10.1039/c4cc02126f

The design, synthesis and in vitro assessment of a bifunctional imaging probe for dual fluorine (¹⁹F) magnetic resonance spectroscopy ( ¹⁹F-MRS) and fluorescence detection is reported. Eleven copies of 3,5-bis(trifluoromethyl)phenyl and a single copy of a sulforhodamine-B were covalently attached to a closo-B₁₂^2--core via suitable linkers. The ¹⁹F-MRS and fluorescence imaging shows that, this novel bimodal imaging probe was readily taken up by the cells in vitro after co-incubation. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc02126f

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Synthesis and in vitro assessment of a bifunctional
closomer probe for fluorine (19F) magnetic
resonance and optical bimodal cellular imaging†
Cite this: Chem. Commun., 2014,
50, 5793
Received 21st March 2014,
Accepted 15th April 2014
Lalit N. Goswami, Aslam A. Khan, Satish S. Jalisatgi and M. Frederick Hawthorne*
DOI: 10.1039/c4cc02126f
www.rsc.org/chemcomm
The design, synthesis and in vitro assessment of a bifunctional imaging nephrogenic systemic fibrosis (NSF) when using Gd3+ in
probe for dual fluorine (19F) magnetic resonance spectroscopy (19F-MRS) patients suffering from renal disfunction or failure.8 Recently
and fluorescence detection is reported. Eleven copies of 3,5-bis(trifluoro- the 19F nuclide has gained popularity among researchers as a
methyl)phenyl and a single copy of a sulforhodamine-B were covalently valuable clinical imaging modality.9,10
1
attached to a closo-B122À-core via suitable linkers. The 19F-MRS and
The NMR sensitivity of 19F is 0.83 relative to H, has a 100%
fluorescence imaging shows that, this novel bimodal imaging probe was natural isotopic abundance ratio, has a large chemical shift range
readily taken up by the cells in vitro after co-incubation.
(300 ppm), and 19F MRI probes are often more stable (covalent)
than the Gd3+ based chelates (non-covalent) used in 1H MRI. Also,
The use of multiple imaging techniques in conjunction with one the human body itself provides a negligible endogenous 19F MRI
another has begun to gain popularity propelled by the development signal. One general challenge in the development of MRI/optical
of hybrid scanners with multiple imaging capabilities (PET-CT, bimodal probes is the difference in the detection sensitivity
SPECT-CT, Optical-CT, MRI-PET, MRI-Optical and other).1 These between MRI (low sensitivity) and optical imaging (high sensitivity).
multimodal imaging systems can be used to diagnose and image A much higher concentration of the MRI probe is required
cancerous lesions in the early stages of disease progression and can compared to the optical counterpart. This problem can be
effectively change the clinical outcome of the treatment regime. addressed with the use of a multifunctional dendritic core such
They are thus believed to be the future of medical imaging. Nuclear as a per-hydroxylated icosahedral [closo-B12(OH)12]2À ion (1). Each
imaging (PET and SPECT) is a well-developed area in medicine and B–OH vertex of this ion can be attached to pre-determined payloads
therefore significant research has been focused on designing to generate attractive molecular scaffolds, termed as ‘‘Closomers’’
bimodal imaging probes combining nuclear imaging with radio (Fig. 1). The synthesis of a bimodal imaging probe requires
(CT, X-ray) or MR or optical imaging capabilities.2 However, due to the presence of heterobifunctionalized linker arms on the same
the drawbacks associated with the nuclear imaging probes (i.e. scaffold to permit concurrent attachment of differing payloads on a
ionizing radiation, half-life and special handling), an alternative
imaging modality with comparable sensitivity is desirable. The
combination of MRI and optical imaging can be advantageous
and very logical. MRI can provide detailed anatomic imaging, while
optical imaging can provide the molecular imaging during image
guided procedures.
1H and 19F provide very sensitive nuclei for MRI.3,4 While
there are several dual function 1H MRI/optical probes that have
been developed,5 there are only a few examples of dual 19F MRI/
optical imaging probes.6,7 The clinically used 1H MRI probes
are dominated by Gd3+-chelates. However there is a high risk of
International Institute of Nano and Molecular Medicine, School of Medicine,
University of Missouri, Columbia, Missouri 65211-3450, USA.
E-mail: hawthornem@missouri.edu
Fig. 1 Schematic representation of organic synthesis on an icosahedral
closo-B12 surface.
† Electronic supplementary information (ESI) available: Experimental procedures
2À
and characterization details. See DOI: 10.1039/c4cc02126f
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single core using orthogonal chemistries. To this end, we have
developed a synthetic methodology to differentiate a single B–OH
vertex of [closo-B12(OH)12]2À to produce [closo-B12(OR)(OH)11]2À 11
.
The synthetic strategy has been successfully utilized towards the
preparation of avb3 integrin-targeted closomer for high performance
MRI applications.12 This methodology permits the covalent
attachment of a single optical probe, while the remaining 11
vertices can carry payloads of 19F nuclei, (Fig. 1).
This combination can increase the effectiveness of a bimodal
19F MRI/optical imaging probe by providing high fluorous content
per molecule. Herein, we describe the synthesis and in vitro
studies of the first bifunctional imaging probe derived from an
icosahedral closo-B122À-core for applications in bimodal cellular
imaging (19F MRI and optical).
In this novel design, the entire fluorous payload is attached to the
central closo-B122À-core via carbamate linkages (B–OCO–NH–) using
tetraethyleneglycol (TEG) linkers and thereby generates a single
19F NMR signal for all 66 19F nuclei, a much desired criteria for
successful 19F-MRS/MRI (Scheme 1). First, the 4-nitrophenyl
carbonate closomer 2 was prepared according to the reported
literature procedure.11 Next, 2 was reacted with the amine
terminated 3,5-bis(trifluoromethyl)benzoic acid derivative S3
(ESI). The resulting 11-fold carbamate closomer 3 was purified by
size-exclusion column chromatography on Lipophilic Sephadex
LH-20 using MeOH as eluent and characterized by NMR and
HRMS analysis (ESI). Finally, the synthesis of bifunctional closomer
19F-B12-FL was accomplished by attaching the alkyne terminated
sulforhodamine-B derivative S5 (ESI) to the azide functionality of
the lone vertex of closomer 3 via an azide–alkyne click reaction. The
19F-B12-FL was purified by size-exclusion column chromatography
on Lipophilic Sephadex LH-20 using MeOH as the eluent and was
characterized by NMR and HRMS analysis.
Fig. 2 Time dependent uptake of 19F-B12-FL by human A549 cells. Figure
shows 19F NMR signal intensity of cell lysates post incubation of 100 mM of
19F-B12-FL at various time-points. (A) 5 min, (B) 30 min, (C) 1 h, (D) 3 h.
time for maximum cellular uptake of 19F-B12-FL. The A549 cells
(human lung cancer cell line) were co-incubated with a 100 mM
solution of 19F-B12-FL for various time points. Cellular uptake of
19F-B12-FL was then investigated using 19F NMR (Fig. 2) and
fluorescence spectroscopy (Fig. S1, ESI†) of the lysed cell pellets.
In 19F NMR spectroscopy of the lysed cell pellets, the uptake of
19F-B12-FL was observed by a characteristic peak at À61 ppm
(referenced to peak for TFA at À76 ppm). The intracellular
concentration of 19F-B12-FL reached a maxima after 1 h of
incubation, although there was no significant difference in cell
uptake between 30 min and 1 h.
Next, the dose dependent cellular uptake of 19F-B12-FL was
determined. Cells (A549) were co-incubated with various con-
centrations (5–80 mM) of 19F-B12-FL for a period of 1 h. Cells
were lysed and the lysates were analyzed using fluorescence
(Fig. 3) and 19F NMR spectroscopy (Fig. 4). As expected, the
fluorescence spectroscopy showed cellular uptake of 19F-B12-FL
even at the lowest dose of 5 mM. Even though the sensitivity of
19F MRI is low as compared to optical imaging techniques, a
very high payload of 19F nuclei (total 66) in 19F-B12-FL was able
to display the cellular uptake of the 19F-B12-FL as a single peak
at À61 ppm (Fig. 4). A linear relationship between incubation
concentration and cellular uptake of 19F-B12-FL was found via
both fluorescence and by 19F NMR spectroscopy, demonstrating
the utility of 19F-B12-FL as a bimodal imaging probe.
The cell labeling experiments were carried out by performing
a time dependent cellular uptake study to determine the average
The cell viability MTT assay of 19F-B12-FL was examined on
four different cell lines including both human and mouse cancer
cell lines. Of all four cell lines, EMT-6 mouse breast cancer cells,
Scheme 1 Synthesis of 19F-B12-FL. Reagents and conditions: (i) S3, ACN,
RT, 3 days, ion exchange with Na+, 84%. (ii) S5, CuI, DIPEA, ACN–THF, RT,
12 h, 91%.
Fig. 3 Dose dependent labeling of human A549 cells with bimodal probe
19F-B12-FL. Figure shows emission spectra of cell lysates 1 h post incubation
of 19F-B12-FL at various concentrations.
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Fig. 4 Dose dependent labeling of human A549 cells with bimodal probe
19F-B12-FL. Figure shows 19F NMR signal intensity of cell lysates 1 h post
incubation of 19F-B12-FL at various concentrations. (A) TFA reference,
À76 ppm, (B) 19F-B12-FL reference, À61 ppm, (C–G) cells incubated with
5, 10, 20, 40, 80 mM of 19F-B12-FL respectively, À61 ppm.
Fig. 6 Fluorescence microscopy images of human T47D cells labeled
with bimodal probe 19F-B12-FL and lysotracker green. Panels A–D show
intracellular localization of 10 mM of 19F-B12-FL 1 h post incubation. Panels:
(A) red, 19F-B12-FL. (B) Green, lysotracker green. (C) Light. (D) Merge.
19F MRI. The in vitro assessment shows that 19F-B12-FL
is nontoxic to cells and is readily taken up by the cells after
co-incubation for a short period of time (o1 h). The in vitro cell
localization studies show that the probes localize preferentially
in lysosomes. These in vitro results highlight the potential of
19F-B12-FL as a cellular imaging agent and warrant the further
evaluation in vivo for toxicity, bio-distribution and imaging
capabilities. Nevertheless, in conjugation with target specific
ligands this bifunctional imaging platform would further open
up the possibility of imaging (19F-MR and fluorescence) of
cancerous lesions and other abnormalities in vivo.
Authors thank Brett Meers for mass spectrometry analysis
and Andrew Muelleman for technical assistance.
Fig. 5 Cell viability assessment of bimodal probe 19F-B12-FL by MTT assay
at various concentrations (0.1–100 mM) in four different cell lines.
Notes and references
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starting point (B–O–), the 19F-B12-FL generates a single 19F NMR
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This journal is ©The Royal Society of Chemistry 2014
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