DNA-TiO2 nanoconjugates labeled with magnetic resonance contrast agents

Article abstract of DOI:10.1021/ja0772389

Recent efforts have shown that nanoscale materials, specifically, metal-based nanoparticles, hold particular promise for the development of multifunctional imaging probes. These new materials provide the means to chaperone and concentrate both drugs and c

Full text of DOI:10.1021/ja0772389

Published on Web 11/30/2007
DNA-TiO2 Nanoconjugates Labeled with Magnetic Resonance Contrast
Agents
Paul J. Endres,† Tatjana Paunesku,‡ Stefan Vogt,§ Thomas J. Meade,*,†,‡ and Gayle E. Woloschak*,‡
Departments of Chemistry, Biochemistry and Molecular and Cell Biology, Neurobiology and Physiology, Radiology,
and Radiation Oncology, Northwestern UniVersity, 2145 Sheridan Road, EVanston, Illinois 60208, and Experimental
Facilities DiVision, Argonne National Laboratory, 9700 South Cass AVenue, Argonne, Illinois 60439
Received September 21, 2007; E-mail: tmeade@northwestern.edu; g-woloschak@northwestern.edu
Scheme 1. (A) Synthetic Route to 1; A Dopamine-Modified MR
Contrast Agent (DOPA-DO3A) and (B) Functionalization of TiO2
Nanoparticles with 1
Recent efforts to develop nanoscale materials for use as bio-
compatible delivery scaffolds for drugs and imaging agents have
produced significant advances in the area of multifunctional probes.1
These new materials provide the means to chaperone and concen-
trate both drugs and contrast agents in specific organs, tissues, and
cells.2 Primarily, superparamagnetic (SPIO) nanoparticles, known
for their ability to enhance signal contrast in T2-weighted MR
images, have dominated reports employing this approach.3,4 These
agents typically result in negative contrast enhancement, and
occasionally, their inherent magnetic susceptibility leads to back-
ground artifacts.3 Conversely, T1 contrast agents do not suffer from
this phenomenon. However, there are very few examples of
paramagnetic (T1)-labeled nanoparticles that combine therapeutics
with an MR reporter.5
In conjunction, our recent work shows that TiO2 nanoparticles
decorated with DNA oligonucleotides function in a targeted and
therapeutic capacity.6,7 Targeting is accomplished via oligonucle-
otide hybridization to an intracellular organelle’s matching DNA
sequence, and therapeutic activity is elicited by light-induced
scission of the nanoparticle-bound DNA. Considering these proper-
ties, attachment of a T1 contrast agent to such a targeted nanoparticle
would allow the visualization of certain genomic sequences in cells
and tissues via MRI. Knowing that cancer is a result of DNA
mutations, one can envision that a therapeutic MR probe targeting
only mutant oncogene sequences would have a significant impact
on the therapeutic and diagnostic communities. As a result, we have
functionalized DNA-labeled nanoconjugates with a Gd(III)-based
contrast agent to produce a biocompatible, therapeutically active
delivery scaffold that is detectable by routine T1-weighted MR
imaging.7
The Gd(III)-labeled nanoparticles were prepared from previously
reported TiO2-oligonucleotide nanoconjugates.6,7 The TiO2 nano-
particles (3-5 nm) are surface modified by exploiting their selective
reactivity to ortho-substituted enediol ligands (such as dopamine).8
These reactions form semiconducting, photocatalytically cleavable
bonds which allow for the surface conjugation of dopamine-
modified DNA. This nanoparticle-bound DNA retains its subcellular
targeting capabilities while remaining stable enough to allow in
vivo compartmental accumulation before photocatalytic cleavage
is induced.7
Following characterization (HPLC, ESI-MS, and EA), the
dopamine-modified contrast agent (1) was reacted with TiO2
nanoparticles in the presence and absence of surface-conjugated
oligonucleotides (see Supporting Information, S6-S9). Coupling
1 to the nanoparticle was initially detected by a distinct color change
resulting from a red shift in nanoparticle absorption between the
unmodified nanoparticles (white) to the modified nanoparticles
(orange/brown).8 In addition, formation of the DOPA-DO3A
nanoparticles was confirmed by UV and IR spectroscopy (see
Supporting Information, S10 and S11). The modified nanoparticles
revealed a characteristic rise in UV light absorption toward higher
energies (200-360 nm) that has been attributed to semiconducting
nanocomposites.8,9 Similarly, the modified nanoconjugates showed
significant (20% increase) IR absorption over control nanoparticles
in the carbon-carbon double bond and aromatic stretching frequen-
cies of 1540-1680 cm-1
.
As expected, conjugation of 1 to the DNA-modified nanocon-
jugates occurs in a linear relationship to the equivalents added. The
success of the conjugation reaction (assessed by the ICP-MS of Ti
and Gd in purified trials of varying [1]:[TiO2 active site] equiva-
lents) increases linearly until the ratio of [1]:[TiO2 active sites]
reaches 1.0. At a 1:1 equivalence ratio, the conjugation reaction
plateaus, giving a value of 4.6 ( 0.2% of functionalized TiO2 active
sites at ratios extending to 5:1. This is most likely due to steric
effects as the available TiO2 active sites may be blocked by the
DNA oligonucleotides and the freely rotating, surface-conjugated
DOPA-DO3A contrast agents.10
To determine the relaxivity of the DOPA-DO3A-TiO2 nano-
conjugates, the slope of the line generated by plotting the inverse
of the T1 relaxation time versus Gd(III) concentration was measured
(see Supporting Information, S12). The nanoconjugates yielded a
relaxivity of 3.5 ( 0.1 mM-1 s-1 per Gd(III) ion, similar to clinical
small molecule contrast agents.11 On the basis of the Ti:Gd ratio
acquired from ICP-MS, each individual nanoparticle has an average
The synthesis of a dopamine-modified contrast agent was
accomplished by coupling the commercially available succinimidyl
ester activated 1,4,7,10-tetraazacyclododecane-1,4,7-tetraacetic acid
to dopamine hydrochloride (Scheme 1). Metalation (with GdCl3)
and purification were carried out under strict pH control as the
catechol in the metalated complex (1) is easily oxidized at pH >5.
† Departments of Chemistry, Biochemistry and Molecular and Cell Biology,
Neurobiology and Physiology, Northwestern University.
‡ Departments of Radiology and Radiation Oncology, Northwestern University.
§ Argonne National Laboratory.
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J. AM. CHEM. SOC. 2007, 129, 15760-15761
10.1021/ja0772389 CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
incubation with the nanoconjugates (Figure 2). Comparison of the
images from Figure 2 reveals that the cells incubated with the
DOPA-DO3A nanoconjugates display a greater contrast enhance-
ment over control cells. Corroborating the nanoconjugate phantom
images (Supporting Information Figure S4) and XRF images (Figure
1), we see that Figure 2 demonstrates the utility of quantitative T1
analysis and XRF to predict viable contrast enhancement via MR
imaging.
In conclusion, we have prepared a Gd(III)-modified DNA-TiO2
semiconducting nanoparticle that is detectable in cells by MR
imaging. The labeled particles appear to be retained at specific
locations inside cells by the conjugated DNA oligonucleotides
hybridizing to intracellular targets, hence creating the first nano-
particle system capable of targeting specific DNA sequences while
being simultaneously detected by MR imaging.7 As a result, we
anticipate that any dopamine-functionalized molecule (e.g., cell-
penetrating peptides for passive cell membrane transport) can be
linked to this modified TiO2 nanoparticle scaffold, allowing
noninvasive monitoring of cells containing target molecules and
removal of target gene sequences pending TiO2 excitation.
Figure 1. Two-dimensional XRF maps of (A) transfection of PC12 cells
with DNA-DOPA-DO3A-modified nanoconjugates; (B) control, non-
transfected PC12 cells. Phosphorus is red, titanium is green, and gadolinium
is blue. The scale bars represent 10 µm. Note that each image is scaled to
its respective maximum value (displayed at the upper right corner and given
in µg/cm2).
Acknowledgment. We thank Keith W. MacRenaris for helpful
discussions, P. N. Venkatasubramanian at the Center for Basic MR
Research (CBMRR) for assistance with MR image acquisition, and
Drs. Aiguo Wu, Mohammed Aslam, and Vinayak Dravid for
assistance and materials concerning nanoparticle synthesis. This
research was supported by the National Institutes of Health under
Grant Numbers 1 R01 EB005866-01 and R01 EB0021000, and the
National Cancer Institute under Grant Numbers 5 U54 CA90810
and R01 CA107467. Use of the Advanced Photon Source was
supported by the U.S. Department of Energy, Office of Science,
Office of Basic Energy Sciences, under Contract Number W-31-
109-ENG-38. The MR images were acquired on a 14.1 T-WB
imaging spectrometer operated by the CBMRR under NIH/NCRR
Grant Number 1 S10 RR13880-01.
Figure 2. T1-weighted MR images of (A) control PC3M cells (T1 ) 3527
( 48 ms); (B) PC3M cells incubated with 0.001 mM DNA-DOPA-DO3A
nanoparticles with 1.8% 1:TiO2 active site coverage (T1 ) 2178 ( 88 ms);
(C) PC3M cells incubated with 0.001 mM DNA-DOPA-DO3A nano-
particles with 4.4% 1:TiO2 active site coverage (T1 ) 2356 ( 100 ms).
The scale bar represents 0.5 mm (at 14.1 T within a FOV of 20 mm and a
slice thickness of 0.5 mm). T1 values were confirmed to be statistically
different via student t tests at a 95% confidence level (Supporting
Information Table S4).
relaxivity of 61.0 ( 1.7 mM-1 s-1. In order to assess the viability
of 1 to enhance the MR image contrast of the TiO2 nanoparticles,
DOPA-DO3A-TiO2 nanoconjugates at varying [1]:[TiO2 active
site] ratios were gravity packed in glass capillary tubes and imaged.
As expected, the TiO2 nanoconjugates functionalized with 1 display
a brighter signal than the control nanoparticles (see Supporting
Information Figure S4).
Supporting Information Available: Experimental details. This
material is available free of charge via the Internet at http://pubs.acs.org.
The cellular distribution and single cell association of the
DOPA-DO3A-TiO2 nanoconjugates were evaluated by X-ray
fluorescence (XRF).12 XRF data, when deconvoluted and standard-
ized, provides high-resolution (0.3 × 0.3 µm), two-dimensional
images that can be employed to map locations and total elemental
concentrations in a desired subcellular region of interest.6 Therefore,
this imaging modality allows direct visualization of the optically
undetectable nanoconjugates while simultaneously providing cellular
outlines and contrast agent location (phosphorus and gadolinium,
respectively).12,13
A DNA oligonucleotide targeted to the mitochondrial genome
was used to functionalize the nanoparticles. The targeting sequence
was specific for the sense strand of a NADH dehydrogenase 2
(ND2) mitochondrial gene present in the rat PC12 cell line: 5′-
carboxy dT-cacgacaccttagcaccaacttac (ND2s).7 Presence of nano-
conjugates in the cytoplasmic but not the nuclear regions suggests
localization in either the targeted organelle, mitochondria, or
possibly endosomes (this oligonucleotide has previously shown
sequence specificity in this cell line).7 Colocalization of the Ti and
Gd fluorescence signals reveals that the DOPA-DO3A-TiO2
nanoconjugates are biologically stable and that their presence inside
cells is responsible for the increase in MR image intensity versus
control untreated cells (Figure 1).12
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To assess the biocompatibility and MR image contrast of the
modified nanoparticles in vitro, PC3M cells were imaged after
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