A DTTA-ligated uridine-quantum dot conjugate as a bimodal contrast agent for cellular imaging

Article abstract of DOI:10.1039/c2cc17555j

A uridine-quantum dot conjugate, a contrast agent for multimodal imaging, was synthesized. Its T₁ relaxivity was 655 and 571.2 mM^-1 s^-1 per particle at 36 °C in phosphate buffered saline at 60 and 200 MHz, respectively. In vitro multimodal images confirmed its uptake by RAW 264.7 cells. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc17555j

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COMMUNICATION
A DTTA-ligated uridine–quantum dot conjugate as a bimodal contrast
agent for cellular imagingw
a
a
Junwon Park,z Sankarprasad Bhuniya,z Hyunseung Lee,b Young-Woock Noh,c
Yong Taik Lim,c Jong Hwa Jung,d Kwan Soo Hong*bc and Jong Seung Kim*a
Received 3rd December 2011, Accepted 3rd February 2012
DOI: 10.1039/c2cc17555j
A uridine–quantum dot conjugate, a contrast agent for multi-
modal imaging, was synthesized. Its T1 relaxivity was 655 and
571.2 mMꢀ1 sꢀ1 per particle at 36 8C in phosphate buffered
saline at 60 and 200 MHz, respectively. In vitro multimodal
images confirmed its uptake by RAW 264.7 cells.
Multifunctional nanoparticles engineered through nanoscale
integration of multiple discrete components exhibit much
more diverse and tunable optical, electronic, and magnetic
properties than the corresponding bulk or single-component
counterparts.1 These superior properties are potentially useful
in a variety of applications.2 Recently, magnetofluorescent
nanoparticles have attracted significant interest because of
their great potential in diverse biomedical applications such
as drug delivery, magnetic resonance (MR) and fluorescence
(FR) imaging, and therapeutic systems.3 Functionalized nano-
particles have become important for enhancing image contrast
in medical diagnostics and are essential in molecular imaging.4
Multimodal imaging agents have the potential to provide more
than one signal from a biological sample, and thus, they enhance
the visualization of biological processes. A number of multimodal
probes with both paramagnetic and fluorescence signatures
have been developed for cellular imaging and imaging in develop-
mental biology, as MRI and optical techniques are excellent
complementary imaging methods.5–8 Previously we reported that
DTTA-ligated uridine based amphiphilic MRI CAs are stable
against biologically abundant Zn2+ and comparable with DTPA
analogues Magnevists/Omniscans. This transmetallation stability
also well reflected with the order of thermodynamic stability of the
complexes.9 Here, we report a new uridine-based bimodal contrast
agent in conjunction with quantum dots (QDs) (Scheme 1).
The QDs were employed as optical contrast agents because of
a Department of Chemistry, Korea University, Seoul 136-701, Korea.
E-mail: jongskim@korea.ac.kr; Fax: +82-2-3290-3121
Scheme 1 Synthetic routes of (a) 6-Gd3+ and (b) 6-Gd3+-QD.
b Division of MR Research, Korea Basic Science Institute,
Cheongwon 363-883, Korea
their high brightness, long-term photo stability, and narrow,
tunable emission spectrum.10
c Graduate School of Analytical Science and Technology,
Chungnam National University, Daejeon 305-764, Korea
d Department of Chemistry and Research Institute of Natural Science,
Gyeongsang National University, Jinju 660-701, Korea
w Electronic supplementary information (ESI) available: Experimental
details, synthesis, spectroscopic data, and NMR data. See DOI:
10.1039/c2cc17555j
The Gd3+ complex was synthesized as shown in Scheme 1.
N-Alkylation of 50-aminodeoxyuridine (2) with N,N-bis[(tert-
butoxycarbonyl)methyl]-2-bromoethylamine in the presence of
KHCO3 at room temperature gave 3. Further reaction of 3 with
lipoic acid gave compound 4 in 45% yield. Subsequently,
z These authors contributed equally to this work.
c
3218 Chem. Commun., 2012, 48, 3218–3220
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S–S bond reduction of 4 by NaBH4 followed by BOC deprotec-
tion afforded 6 where a diethylene-triaminotetraacetic acid
(DTTA) group acted as the binding site for Gd3+. The ligand
6 was complexed with GdCl3 in ultrapure water with a pH of
6.5 to yield an anionic complex (6-Gd3+) (Scheme 1(a)). Next,
the 6-Gd3+ was attached to CdSe/ZnS QDs (lmax: 655 nm) by a
ligand exchange protocol reported previously.11
QDs (1.0 mL, 1.0 mM, Invetrogen) in decane were added to
an aqueous ethanol solution of 6-Gd3+ (100 mg, 90 mmol) and
stirred for 10 h at 80 1C. After cooling the reaction mixture to
room temperature, filtering the precipitates from the solution,
and washing with cold ethanol, 6-Gd3+-QDs were obtained as
pale yellow solids. This new multimodal contrast agent has core
Cd–Se QDs incorporated in the centre with the paramagnetic
uridine-based Gd3+ complex (6-Gd3+) coated on the surface to
create the potential for bimodal MRI and fluorescence imaging
in biological systems.
Fig. 2 T1 relaxivity measurement of the 6-Gd3+-QD. Gd-concentration
dependence of the measured relaxation rates (R1) of the 6-Gd3+-QD are
shown and are compared with those of clinically available T1 contrast
agent, Omniscans, and 6-Gd3+ at 200 MHz and 36 1C.
concentration (Fig. 2). The measured r1 value of 6-Gd3+-QD
at 200 MHz was 10.2 ꢁ 0.1 mMꢀ1 sꢀ1 per Gd3+ ion
(11.7 ꢁ 0.1 mMꢀ1 sꢀ1 at 60 MHz, data not shown), which is
2.5-fold higher than the r1 values of clinically used T1 CA
Omniscans (Gd-DTPA-BMA; Amersham, USA) and the
free ligand 6-Gd3+ (r1 values of Omniscans and 6-Gd3+ are
B4.2 mMꢀ1 sꢀ1). The relaxivity of 6-Gd3+-QD per QD particle
was 571.2 mMꢀ1 sꢀ1 at 200 MHz, which was significantly higher
than those reported in the case of QD-Gd3+ nano-particles.14 It
is assumed that this significantly higher r1 value of 6-Gd3+-QD is
because of restricted molecular tumbling and reduced global
rotational motion due to the conjugation of the complex on the
QD surface, which enhances the r1 of each complex.15 These
results indicate that the high T1 relaxivity per particle in cellular
imaging application after in vivo or in vitro labeling of specific
cells with 6-Gd3+-QD could facilitate highly sensitive MRI cell
tracking with minimal cell labeling.
To demonstrate the application of the 6-Gd3+-QD as a
sensitive MR/FR cellular imaging probe, we first investigated cell
biocompatibility of the nano-constructed 6-Gd3+-QD using a
murine macrophage cell line (RAW 264.7). As shown in Fig. 3,
we observed by comparing with the control that labeling of RAW
264.7 cells with 6-Gd3+-QD and incubation for 24 h does not
significantly influence the cell viability at Gd3+ concentrations
lower than 11.2 mM. No significant 6-Gd3+-QD cytotoxicity was
observed in RAW 264.7 cells at Gd3+ concentrations lower than
11.2 mM after labeling in culture media for 24 h.
To confirm the binding of 6-Gd3+ on QD surface, the UV-Vis
absorption spectra of 6-Gd3+ were measured before and after
QD attachment (Fig. S8, ESIw).12 Based on the UV-Vis absorp-
tion spectroscopy results, the number of 6-Gd3+ moieties per
QD particle was estimated to be approximately 56 ꢁ 6. The
fluorescent property of QD remains unaltered even in conjunc-
tion with 6-Gd3+ (Fig. S9–S11, ESIw). In addition, we obtained
TEM images of 6-Gd3+-QD, which also verified that 6-Gd3+
s
are attached to the QD surface as shown in Fig. 1. Furthermore,
electron energy loss spectroscopy (EELS),13 which can provide
elemental and chemical information with high spatial resolution,
was utilized to characterize 6-Gd3+-QD and the results are
shown in Fig. 1(B) and Fig. S12 (ESIw). The 6-Gd3+-QD
contains the elements C, O, Cd, and Gd. This proves that
6-Gd3+ is homogenously attached to the external surface of the
QD. By the dynamic light scattering (DLS) method (Fig. 1(A)),
the average diameter of the 6-Gd3+-QD was found to be
ca. 160 nm, which is in good agreement with the TEM results.
To verify the ability of the 6-Gd3+-QD to act as an MRI
contrast agent, we measured the T1 relaxivity (r1) of 6-Gd3+-QD
in phosphate buffered saline. The T1 relaxivity (r1) is calculated
from the linear fitting of the measured R1 (=1/T1) data vs. Gd
Intracellular delivery of 6-Gd3+-QD to RAW 264.7 cells
was easily investigated by fluorescence microscopy (Fig. 4) and
flow cytometry (Fig. S13, ESIw), and the extent of labeled
6-Gd3+-QD could be measured on the basis of the fluorescence
intensity from QDs.
Fig. 1 DLS and TEM characterization of 6-Gd3+-QD. (A) DLS of
6-Gd3+-QD, (B) TEM images of 6-Gd3+-QD: (a) TEM image of
6-Gd3+-QD, (b) C component, (c) O component, (d) Cd component,
and (e) Gd component. Scale bars: 50 nm.
Fig. 3 Measurement of cytotoxicity of 6-Gd3+-QD in RAW 264.7
cells.
c
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In summary, a bimodal fluorescent MR contrast agent,
6-Gd3+-QD, has been developed by conjugation of CdSe/ZnS
QDs with a uridine-based paramagnetic complex (6-Gd3+).
The relaxivities of 6-Gd3+-QD are 655 and 572 mMꢀ1 sꢀ1 per
particle at 60 and 200 MHz, respectively. 6-Gd3+-QD can
smoothly penetrate the cell surface and can be delivered into
the intracellular regions of RAW 264.7 cells. Further, 6-Gd3+-QD
facilitates high-performance MR and fluorescence imaging. Since
this QD-conjugated T1 contrast agent easily labeled macrophages,
which are phagocytic cells and since it had low cytotoxicity,
6-Gd3+-QD as a bimodal MR/FR cellular imaging nanoprobe
can be used to probe similar phagocyte cells such as T cells,
B cells, neutrophils, granulocytes, and dendritic cells.
Fig. 4 Fluorescence microscopic images of RAW 264.7 cells labeled
with 6-Gd3+-QD. Bright-field microscopy images (A, C, E, G) and
fluorescence microscopic images (B, D, F, H) with blue (DAPI for
nucleus) and red (for 6-Gd3+-QD) are shown to be dependent on
QD concentration in cell culture media. Images were acquired using
490/20 nm excitation and fluorescent emission windows of 617/73 nm
(red). Scale bar: 10 mm.
This work was supported by a grant from the CRI project
(No. 2011-0000420) (JSK), (2011-0029263) (KSH), Korea
Basic Science Institute (T31403) (KSH) and WCU project
(R32-2008-000-20003-0) (JHJ) of Korea.
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3220 Chem. Commun., 2012, 48, 3218–3220
This journal is The Royal Society of Chemistry 2012