Facile non-hydrothermal synthesis of oligosaccharide coated sub-5 nm magnetic iron oxide nanoparticles with dual MRI contrast enhancement effects

Article abstract of DOI:10.1039/c4tb00811a

Ultrafine sub-5 nm magnetic iron oxide nanoparticles coated with oligosaccharides (SIO) with dual T₁-T₂ weighted contrast enhancing effects and fast clearance have been developed as magnetic resonance imaging (MRI) contrast agents. Excellent water solubility, biocompatibility and high stability of such sub-5 nm SIO nanoparticles were achieved by using the in situ polymerization coating method, which enables glucose to form oligosaccharides directly on the surface of hydrophobic iron oxide nanocrystals. Reported ultrafine SIO nanoparticles exhibit a longitudinal relaxivity (r₁) of 4.1 mM^-1 s^-1 and a r ₁/r₂ ratio of 0.25 at 3 T (clinical field strength), rendering improved T₁ or brighter contrast enhancement in T₁-weighted MRI in addition to typical T₂ or darkening contrast of conventional iron oxide nanoparticles. Such dual contrast effects can be demonstrated by liver imaging with T₂ darkening contrast in the liver parenchyma but T₁ bright contrast in the hepatic vasculature. More importantly, this new class of ultrafine sub-5 nm iron oxide nanoparticles showed much faster body clearance than those with larger sizes, promising better safety for clinical applications.

Full text of DOI:10.1039/c4tb00811a

Volume 2 Number 33 7 September 2014 Pages 5311–5482
Journal of
Materials Chemistry B
Materials for biology and medicine
www.rsc.org/MaterialsB
ISSN 2050-750X
PAPER
Hui Mao et al.
Facile non-hydrothermal synthesis of oligosaccharide coated sub-5 nm
magnetic iron oxide nanoparticles with dual MRI contrast enhancement
effects

Journal of
Materials Chemistry B
View Article Online
View Journal | View Issue
PAPER
Facile non-hydrothermal synthesis of
oligosaccharide coated sub-5 nm magnetic iron
oxide nanoparticles with dual MRI contrast
enhancement effects†
Cite this: J. Mater. Chem. B, 2014, 2,
344
5
a
a
b
a
c
a
Jing Huang, Liya Wang, Xiaodong Zhong, Yuancheng Li, Lily Yang and Hui Mao*
1 2
Ultrafine sub-5 nm magnetic iron oxide nanoparticles coated with oligosaccharides (SIO) with dual T –T
weighted contrast enhancing effects and fast clearance have been developed as magnetic resonance
imaging (MRI) contrast agents. Excellent water solubility, biocompatibility and high stability of such sub-5
nm SIO nanoparticles were achieved by using the “in situ polymerization” coating method, which
enables glucose to form oligosaccharides directly on the surface of hydrophobic iron oxide nanocrystals.
ꢀ1
ꢀ1
Reported ultrafine SIO nanoparticles exhibit a longitudinal relaxivity (r
of 0.25 at 3 T (clinical field strength), rendering improved T or “brighter” contrast enhancement in
-weighted MRI in addition to typical or “darkening” contrast of conventional iron oxide
nanoparticles. Such dual contrast effects can be demonstrated by liver imaging with T “darkening”
contrast in the liver parenchyma but T “bright” contrast in the hepatic vasculature. More importantly, this
1
) of 4.1 mM
s
1 2
and a r /r ratio
1
T
1
T
2
Received 19th May 2014
Accepted 20th June 2014
2
1
DOI: 10.1039/c4tb00811a
new class of ultrafine sub-5 nm iron oxide nanoparticles showed much faster body clearance than those
www.rsc.org/MaterialsB
with larger sizes, promising better safety for clinical applications.
5
nm, due to their relatively lower r
area, which allows the exchange of more water molecules
Iron oxide nanoparticles (IONPs), also known as super- between the inner and outer layers of the particle surface.
paramagnetic iron oxide (SPIO) nanoparticles, have been
To date, various forms of IONPs have been developed for
2
relaxivity and larger surface
1
. Introduction
extensively investigated as magnetic resonance imaging (MRI) MRI applications, of which only two agents (Feridex®,
contrast agents in both clinical applications (e.g. liver, lymph Resovist®) formulated with dextran coating have been approved
12
node imaging) and preclinical investigations with animal by FDA for clinical uses. Both have an average core size over
5,13
models (e.g. cell tracking and biomarker targeted molecular 5 nm and an overall size of 60–150 nm. Particles with such
1
–4
imaging). Typical IONPs predominantly increase the trans- overall size are rapidly trapped in the organs of the reticuloen-
verse relaxation rate (R or 1/T ), together with signal dephasing dothelial system (RES) and can take several weeks or even
2
2
1
4
caused by the perturbed local eld, leading to signal drops in T2 months to be degraded and cleared from the body. Slow
or T * weighted MR imaging. However, T or T
*-weighted MRI clearance not only causes concern about long term side effects
with IONPs is oen interfered by image artefact and co-found T
2
2
2
of such IONPs but also limits them from being used repeatedly
in longitudinal imaging studies. Moreover, the larger overall
2
1
5,6
effects from other signal sources. Therefore, using “bright” T
1
contrast agents that increase the longitudinal relaxation rate size prevents IONPs from maintaining T contrast enhancement
15,16
1 1
(R or 1/T
) and enhance the signal intensity is more desirable properties.
for easier and better detection of abnormalities. Previous
In order to synthesise IONPs below 5 nm, especially mono-
studies have shown that the longitudinal r and transverse r
dispersed IONPs with controlled sizes because of their size-
1
2
17–20
relaxivities of IONPs are dependent on the particle size and the sensitive magnetic properties,
thermal decomposition is the
7–11
surface coating properties.
It is conceivable that r
1
relaxivity preferred method. However, the subsequent surface modica-
can be preserved for ultrasmall IONPs with a core size below tion to transfer and stabilize IONPs into aqueous physiological
conditions is a critical procedure for IONPs applied in diag-
a
nostic imaging. Previous reports have shown that the relaxiv-
ities that determine MRI contrast enhancement are dependent
on the surface properties of IONP-based contrast agents, such
Department of Radiology and Imaging Sciences, Emory University School of Medicine,
Atlanta, Georgia 30322, USA. E-mail: hmao@emory.edu
b
MR R&D Collaborations, Siemens Healthcare, Atlanta, Georgia 30322, USA
c
Department of Surgery, Emory University School of Medicine, Atlanta, Georgia 30322, as the thickness, the hydrophilicity, and the anchoring groups
USA
Electronic supplementary information (ESI) available: DOI: 10.1039/c4tb00811a
21–24
of the coating layer.
Especially for IONPs below 5 nm, their
†
5344 | J. Mater. Chem. B, 2014, 2, 5344–5351
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
ꢁ
strong tendency to aggregate makes them difficult to be stabi- was heated to 120 C, and kept at this temperature for 2 hours.
lized in aqueous media. Traditional surface coating with poly- Aer cooling down to room temperature, the product was
mers of high molecular weight, e.g. dextran, polyethylene glycol precipitated by adding ethanol. The precipitant was washed and
25–28
(PEG), poly(methacrylic acid) (PMAA),
is not effective in centrifuged several times. The nal product was collected and
stabilizing sub-5 nm IONPs as they are “patchy” and less redispersed in distilled water for other characterization and
uniform with tangling chains and inter-molecular steric repul- applications.
sion. The imperfect surface coating may lead to instability and
loss of magnetism, while the subsequent formation of IONP Characterization of SIO nanoparticles
1
clusters results in the loss of T contrast enhancement proper-
ties. Therefore, a new surface modication strategy is needed to
ensure the water solubility and stability of IONPs smaller than
The morphology and size of SIO nanoparticles were studied
using a transmission electron microscope (TEM, Hitachi H-
7500, accelerating voltage 75 kV). Typically, TEM samples are
5
nm, and to preserve the T -weighting contrast effect.
1
prepared by dropping diluted nanoparticle solutions on the
carbon coated copper grid and air-drying. The hydrodynamic
size and surface charges of nanoparticles in the aqueous solu-
tion were evaluated using a dynamic light scattering (DLS)
instrument (Malvern Zeta Sizer Nano S-90) equipped with a
Here we report a new class of ultrane oligosaccharide
coated iron oxide nanoparticles (SIO-3, average core size of
.5 nm) prepared by in situ polymerization of glucose on the
particle surface. The reported sub-5 nm SIO-3 is highly stable in
the aqueous solution and exhibits an improved r /r ratio over
3
1
2
22 mW He–Ne laser operating at 632.8 nm. The structural
IONPs with larger overall sizes, leading to an excellent T MRI
1
analysis of SIO nanoparticles was carried out by powder X-ray
diffraction (XRD, Bruker D8 DIFFRAC powder diffractometer, Co
Ka). For studying the coating of nanoparticles, Fourier trans-
form infrared spectroscopy (FTIR) spectra were collected on a
PerkinElmer Spectrum 100 FT-IR spectrometer (Bucks, UK). UV-
vis absorption spectra were obtained with a scanning spectro-
photometer (Shimadzu UV-2401PC) with a slit width of 1.0 nm.
contrast enhancement and a novel dual T –T contrast effect for
1
2
new applications. In addition, SIO-3 showed shortened body
clearance time with partial renal secretion compared with
IONPs with larger sizes, therefore, promising to address the
lasting concern of possible long term toxicity associated with
IONPs.
Measurement of relaxation times and calculation of
relaxivities
2
. Experimental section
Synthesis of hydrophobic iron oxide nanoparticles (IONPs)
To evaluate MRI contrast enhancement capability, SIO solu-
tions with different concentrations were examined with a 3T
MRI scanner (Magnetom Tim Trio, Siemens Medical Solutions,
Erlangen, Germany) using T - and T -weighted fast spin echo
1 2
sequences, an inversion recovery turbo spin echo sequence and
The hydrophobic iron oxide nanoparticles were synthesized by
thermo-decomposition. Briey, iron(III) oleate was rst
20
prepared by a modied published method. Typically, 4.04 g of
ferric nitride (10 mmol) and 9.13 g of sodium oleate (30 mmol)
were dissolved in the solvent mixed with 40 mL distilled water,
a multi-echo T -weighted spin echo sequence. A commercial T1
2
50 mL hexane and 10 mL absolute ethanol. The mixture of iron
contrast enhancement agent Multihance® (Gd-BOPTA) was
used for comparing the MRI contrast enhancement effect. Each
sample was prepared with Fe or Gd concentrations varying from
oleate was stirred at room temperature for 4 hours, and then
kept still overnight. The resulting red-brownish hexane layer
was used as the iron source for thermo-decomposition. In a
typical reaction, 5 mL of the iron oleate was mixed with 5 mL of
1
0.004 to 40 mM. To measure the longitudinal relaxation time T ,
an inversion recovery turbo spin echo (TSE) sequence with an
echo train length (ETL) of 3, an echo time (TE) of 13 ms and a
repetition time (TR) of 1500 ms was used to obtain images at
different inversion times (TI) of 23, 46, 92, 184, 368, 650, 850,
1
-octadecene at room temperature, and degassed with ultrahigh
ꢁ
argon for 20 min. Aer evaporating hexane at 70 C, the reaction
mixture was heated to 320 C with a heating rate of 0.6 C s
ꢁ
ꢁ
ꢀ1
.
The reaction time was adjusted to control the size of IONPs,
which was about 5 min for IONPs with a core size of 3.5 nm, and
reheated approximately 10, 15, 20, and 30 min for IONPs with
1100, and 1400 ms, respectively. To measure the transverse
relaxation time T , a multi-echo spin echo sequence was used
2
with a TR of 2400 ms and 15 TEs, starting at 11 ms with
increments of 11 ms. The signal intensity (SI) of each region-of-
interest (ROI) at different TIs or TEs was measured for samples
of each concentration.
4.8, 9.9, 15.6, and 19.9 nm core size. Aer cooling down to room
temperature, ethanol was added to precipitate the nano-
particles. The products were collected by centrifugation, and
washed with hexane and ethanol several times.
MRI of mice administered with SIO nanoparticles
Synthesis of oligosaccharide coated iron oxide (SIO)
nanoparticles
All animal experiments were conducted following a protocol
approved by Institutional Animal Care and Use Committee
Oligosaccharide coating was introduced on the hydrophobic (IACUC). BALB/c mice were anesthetized by intraperitoneal
ꢀ1
IONPs by in situ-polymerization. Briey, the oleic acid coated injection of a ketamine–xylazine mixture (95 : 5 mg kg ). The
IONPs were redispersed in chloroform aer being puried by saline diluted SIO-3 solution was intravenously administered at
centrifugation, and carefully added dropwise into the preheated a dosage of 2.5 and 10 mg Fe per kg of mouse body weight. For
glucose solution in dimethylformamide (DMF). The mixture comparison, Gd-BOPTA and SIO-20 (core size of 20 nm) were
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B, 2014, 2, 5344–5351 | 5345

View Article Online
Journal of Materials Chemistry B
injected at the dosage of 2.5 mg kg and 0.2 mmol kg
Paper
ꢀ1
ꢀ1
,
decomposition conditions. The hydrophobic IONPs were highly
respectively. Fat suppressed T -weighted spin echo images were uniform with diameters of 3.5 (IO-3), 4.8 (IO-5), 9.9 (IO-10), 15.6
1
obtained to investigate the contrast changes in different organs (IO-15), and 19.9 nm (IO-20) respectively, as revealed by TEM
and anatomic structures, such as the liver, kidney and iliac images (Fig. 1 and S1†). In this work, the hydrophobic IO
artery, at different time points. The imaging parameters nanoparticles were mixed with glucose solution in DMF, and
included: TR ¼ 724 ms, TE ¼ 10 ms, matrix ¼ 320 ꢂ 134, eld of heated to allow the in situ-polymerization of glucose on the
2
view (FOV) ¼ 120 ꢂ 60 mm , ip angle ¼ 70, and slice thickness particle surface. A thin oligosaccharide coating layer was
¼
1.00 mm. The signal-to-noise ratio (SNR) was calculated formed, rendering water soluble nanoparticles. The core sizes
according to the equation: SNR ¼ SImean/SDnoise. The relative showed no signicant changes before and aer the surface
contrast enhancement at different time points was dened as modication (Fig. 1c, S1e–h†). To evaluate the hydrodynamic
the signal decrease DSNR ¼ (SNR
ꢀ SNRpost)/SNRpre. The diameters of these oligosaccharide coated IONPs in aqueous
pre
contrast-to-noise ratio between the liver parenchyma and solution, DLS measurement was performed. The hydrodynamic
vasculature was calculated as CNR ¼ (SNRpost(vasculature)
ꢀ
sizes are 7.3, 9.5, 11.5, 15.7, and 20.9 nm for SIO-3, 5, 10, 15, and
20, respectively (Fig. S2†), which are slightly larger than the TEM
core sizes due to the addition of hydrophilic oligosaccharide
coating layers. The hydrodynamic size of 7.3 nm measured in
SIO-3 suggests the thinnest coating layer among those IONPs
with core size below 5 nm, which may play a signicant role in
SNRpost(liver parenchyma))/SNRpre(liver parenchyma).
Body clearance of SIO nanoparticles in mice
The clearance of nanoparticles was evaluated by both chemical
analysis of iron contents from the collected organs or tissues
preserving the T contrast enhancing effect due to less restraints
1
and ROI analysis of T
2
-weighted MRI and T
2
relaxometry
21,24,27,28,30
in water exchange between the inner and outer layers.
mapping of live animals, which allows for time dependent
changes of iron concentrations at the specic organ in the same
animal. SIO-3, SIO-20 and SHP20, which are commercially
available amphiphilic polymer coated SPIO nanoparticles (core
size 20 nm from Ocean NanoTech, LLC), were intravenously
administered into BALB/c mice (n ¼ 3) at a dosage of 2.5 mg
Moreover, the small hydrodynamic size indicates the single
dispersion of SIO-3 in the aqueous solution, preventing the T
2
23
effect caused by the aggregation. The oligosaccharide coated
particles were stable and highly dispersed in the aqueous
solution at room temperature for at least 2 months, showing no
aggregation (Fig. S3†).
ꢀ
1
kg
2
mouse weight. For MRI monitoring, T -weighted MR
The powder X-ray diffraction (XRD) patterns of IO-3 and SIO-
images of the mice were acquired on a 3T MRI scanner before
and aer administration of nanoparticle contrast agents using a
volumetric wrist coil. The imaging parameters included: TR ¼
3
are shown in Fig. 2a. Broadened diffraction peaks were
observed for both samples due to the ultrane nano-sized
crystals. The broadened diffraction peaks became clearer aer
applying the coating, due to the rearrangement of the canted
3
710 ms, TE ¼ 12–180 ms, matrix ¼ 256 ꢂ 128, eld of
2
ꢁ
view (FOV) ¼ 120 ꢂ 60 mm , ip angle ¼ 180 and slice thick-
31
surface during the heating process. However, the grain size
ness ¼ 1 mm. Colorized T maps were then generated as
2
changed little according to the half width of the diffraction
peaks. Both of the XRD peaks of IONPs before and aer coating
were assigned to the spinal magnetite or maghemite structure.
The formation of oligosaccharide coating on the surface of
IONPs was further conrmed by Fourier-transform infrared
described in the ESI.† ROIs with the same areas were drawn in
2
the liver and spleen at the same T maps. The relative contrast
enhancement at different time points was calculated to show
the average signal changes. The organs (liver, spleen, kidney,
lungs, heart, and muscle) and blood samples were collected at
10 min, 1 day, 1 week, 2 weeks and 3 weeks aer injection. For
chemical analysis of tissue iron, the phenanthroline colori-
metric method was used to determine the iron concentration in
29
3
organs aer the organs were digested in concentrated HNO .
In addition, Prussian blue staining was performed for the major
organ slices following a standard protocol. Briey, frozen
tissues mounted in an optimal cutting temperature compound
(OCT) were sliced in 8 mm thickness, xed with 4% paraf-
ormalin for 10 min, then soaked into the working solution
composed of 10% potassium ferrocyanide(II) trihydrate and
ꢁ
2
0% HCl solution (v : v ¼ 1 : 1) at 37 C for 4 hours. Aer being
washed with PBS, the slices were counterstained with nuclear
fast red for 5 min. Blue dots representing the remaining IONPs
in organs were investigated with a light microscope.
3
. Results and discussion
Fig. 1 (a) A low magnification and (b) high magnification TEM image of
hydrophobic IONPs of 3.5 nm size (IO-3). The inset of (a) is the size
distribution after measuring 100 particles. (c) TEM image of hydrophilic
IONPs with different diameters were prepared by thermal
decomposition of ferric oleate through
adjusting IONPs coated with oligosaccharides (SIO-3).
5346 | J. Mater. Chem. B, 2014, 2, 5344–5351
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
reaction by absorbing water molecules produced during the
38
polymerization. Evidently, glycosylation under the same
reaction conditions, but in different solvents, e.g., octadecene
(ODE), diethylene glycol (DEG), and dimethyl sulfoxide (DMSO),
did not yield such sufficient oligosaccharides (Fig. S4†).
The MRI contrast enhancement effects of SIOs were inves-
tigated at the clinically relevant magnetic eld (3 T). Fig. 3a and
b show T - and T -weighted MR images of the SIO aqueous
1 2
solutions with different Fe concentrations. SIO-3 exhibits the
highest T1 contrast enhancement, while SIO-20 exhibits the
highest T contrast enhancement. This observation is expected,
2
as SIO-3 has the highest surface-to-volume ratio due to the
ultrane size. For nanoparticulate contrast agents, the T
1
contrast enhancement is believed to be majorly contributed by
the inner-sphere relaxivity, which comes from the direct coor-
dination between water molecules and magnetic ions on the
5,27,39
particle surface.
A high surface-to-volume ratio in combi-
nation with a thin hydrated coating layer of oligosaccharides for
SIO-3 would facilitate the water molecule interaction with the
inner layer.
Fig. 2 (a) Powder XRD pattern and (b) FTIR spectra of IO-3 and SIO-3.
(
c) Monitoring of the reaction stage by FL emission. The insets of (c) are
photographs of the reaction mixture collected at different reaction
times under normal light (left) and 365 nm UV light (right). (d) Solvent
phase transition of IONPs from organic phase hexane to the aqueous
solution over time.
Although transverse relaxivity r
2
of IONPs has been exten-
sively studied, and could be predicted theoretically, limited
analysis was done with longitudinal relaxivities r of IONPs.
19
1
Size, coating, crystallinity, and composition of nanoparticle
cores are considered to be important factors in maintaining the
spectroscopy (FTIR) (Fig. 2b). The characteristic bands of oleic
T effect, according to the Solomon–Bloembergen–Morgan
1
ꢀ
1
40
acid, including C–H stretching (2923, 2852 cm ), CH
(
2
bending (SBM) theory. It is well recognized that both r
1
and r increase
2
ꢀ
1
ꢀ1
1457, 1375 cm ) and C]O stretching (1540 cm ), became with the increasing size however with different proportions, as
1
0
weakened aer being replaced by oligosaccharides on the
r
2
has a stronger size dependent effect than r
and r of SIO showed size dependency (Fig. 3c and d).
the presence of aromatic structures of oligosaccharides on the The increased r with increased size could be ascribed to size
dependent magnetic susceptibility (Fig. S5†). On the other
It should be noted that the temperature we used for in situ hand, r of SIOs kept increasing until a maximum was reached
1
.
In our case,
ꢀ
1
surface. The emerged sharp C]C band at 1653 cm indicated both r
1
2
2
32,33
particle surface.
1
polymerization of glucose on the surface of nanoparticles is at the particle size around 10 nm. The similar trends were
ꢁ
much lower (ꢃ120 C) than that of the established hydro-
thermal methods used to synthesize carbonized materials from
32,34–36
glucose.
When the formation of oligosaccharides was
monitored by UV and uorescent spectroscopy, a turquoise

uorescent signal with UV excitation at l ¼ 365 nm was
observed (Fig. 2c) aer 0.5 h reaction. This signal can be
ascribed to the aromatic groups derived from the intermolec-
ular dehydration and aldol condensation during glycosyla-
32,35,36
tion.
At this time, oleic acid capped IONPs began to
transfer into the aqueous phase as the formation of oligosac-
charide coating took place. However, at this early stage, the
hydrophilic oligosaccharides were insufficient to stabilize the
particles in the aqueous solution, resulting in a light yellow
turbid suspension (Fig. 2d). In order to keep the small size of
the whole particle, oligosaccharide coating was controlled to be
minimal, but sufficient to stabilize the particles. When the
IONPs were transferred completely and dispersed into aqueous
solution (i.e. reaction time is 2.0 h, yielded brownish trans-
parent solution), the reaction was terminated immediately.
Parallel experiments indicated the pivotal role of DMF solvent
in the formation of oligosaccharide coating, together with the
Fig. 3 (a) T
1
- and (b) T
2
-weighted MR images of SIO solutions with
and (d) r value
37
possible catalytic effect from cationic iron. DMF provides an
different concentrations, and the corresponding (c) r
1
2
alkaline condition for the glycosylation and facilitates the changes with particle size.
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B, 2014, 2, 5344–5351 | 5347

View Article Online
Journal of Materials Chemistry B
Paper
observed for the ultrasmall IONPs coated with PEG, CTAB, and is to attenuate r
2
while attempting to increase r
1
. For example,
10,19,41,42
2+
3+
DEG.
Such size dependency on r1 is attributed to the magnetic cations with unpaired electrons (e.g. Mn , Gd ) have
monodispersed size, together with a compact and highly been introduced into iron oxide nanostructures to increase r ,
1
6,42,44
hydrophilic coating, resulting in good dispersity in solvent thus to realize the positive contrast enhancement.
without aggregation. For larger particles (>10 nm), longitudinal Regardless of the metal toxicity, Gd-doping may be considered
6
relaxivity r
1
decreased with the increased size, which is attrib- as a more effective way because of the slighter increase of r
may allow the IONP concentration to be
SI enhancement, which is oen compro-
To further evaluate the contrast enhancement efficiency and mised at higher IONP concentrations.
behaviour of SIOs, we computed a signal intensity prole of a With good biocompatibilities (i.e. non-toxic to cells up to 200
2
.
uted to the locked particle magnetic moment on anisotropy Moreover, reducing r
axes, thus relaxivity is dominated by Curie relaxation.
2
19,43
increased for the T
1
ꢀ
1
contrast agent (Fig. 4a and b) using the equation describing mg mL , Fig. S6†), the T contrast enhancement of SIO-3 in vivo
1
signal intensity (SI) evolution from the T -weighted spin echo was investigated. MRI was performed on a 3T MRI scanner for
1
sequence. Given the same Fe concentration (i.e., 1 mM) and mice intravenously injected with contrast agents. SIO-3 showed
image acquisition parameters (i.e., TR ¼ 500 ms and TE ¼ 12 excellent positive T
ms) typically used for T -weighted spin echo MRI, the highest T and highly vascularised organs, e.g. the heart, kidney and spleen
contrast, i.e., brightest signal, for a given r can be only obtained (Fig. 5 and 6). In comparison, T contrast enhancement is not
when r reaches zero. Furthermore, SI is more sensitive to the obvious in the group injected with SIO-20. Interestingly, SIO-3,
1
contrast enhancement in the vasculature
1
1
1
1
2
ꢀ
1
change in r than in r for the contrast agents with an r larger particularly at the higher dosage (10 mg kg ), led to a “dual”
than 4.5 mM
2
1
1
ꢀ
1
ꢀ1
s . For example, although SIO-3 has a lower r1, T –T contrast effect in the liver as shown in the T -weighted MR
1 2 1
it has a much higher T enhancement efficiency than SIO-20 images (Fig. 5). Both “darkening” T2 contrast in the liver
1
because of a sharper reduction in r
2
.
parenchyma and “bright” T
ratio may were observed at the same time. The darkening T
1
contrast enhancement properties of the magnetic the liver parenchyma is caused by the uptake of SIO-3 by Kupffer
1
contrast in the hepatic vasculature
Previous studies have suggested that the r
1
/r
2
2
contrast in
dictate the T
nanoprobes.
5,16
An increased maximum SI in T
/r ratio as shown in aer intracellular clustering of SIO-3. On the other hand, the
Fig. 4c. It has been theoretically studied that the r /r ratios are bright T contrast in the vasculature is attributed to SIO-3 highly
1 2 2
-weighted MR cells which results in a dominant T -effect due to the r increase
45
images was observed with the increasing r
1 2
1
2
1
monotonically increased against the translational diffusion dispersed in the blood pool. This “dual” contrast effect of SIO-3
24
time sD, which is related to the radius of IONPs, water improves the sensitivity and image clarity for visualizing the
permeability of the coating layer and the coating thickness. morphology of the liver parenchyma and the structure of
Unsurprisingly, SIO-3 has the highest r
compared to the counterparts in larger sizes (Fig. 4d).
Since most nanoparticulate T contrast agents reported so
1 2
/r ratio of 0.25
1
ꢀ1
ꢀ1
far have an r
1
larger than 4.5 mM
s
but also substantially
16
high r2, one alternative strategy for future development of
magnetic nanoparticle based T -weighted MRI contrast agents
1
Fig. 4 (a) Prediction of SI in T
1
-weighted MR images determined by r
1
1
Fig. 5 Fat-suppressed T -weighted MR images of a mice before and
ꢀ1
and r , and (b) the top view. (c) The maximum SI of each SIO solution after administration of SIO-3 at a dosage of 10 mg kg . Positive
2
related to the r
sizes.
1
/r
2
ratio. (d) r
1
/r
2
ratio changes with hydrodynamic contrast enhancement was observed in the heart, spleen (red dotted
circles), kidney (green dashed circles), and liver vessels.
5348 | J. Mater. Chem. B, 2014, 2, 5344–5351
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
1 2
Fig. 7 Signal and contrast changes over time in (a) T - and (b) T -
weighted MR images of the bladder of a mouse received SIO-3 reveal
the excretion of SIO-3 by the kidney. Slow filling of SIO-3 into the
bladder resulted in slow and gradual extension of the brightening T
signal (a) and darkening T signal (b).
1
2
Fig. 6 Percentage of signal changes in major mouse organs (n ¼ 3)
after administration of contrast agents, (a) heart, (b) kidney, (c) spleen,
(
d) liver, (e) vessels. (f) Contrast changes between the liver parenchyma
and vessels (n ¼ 3); (*P < 0.05, **P < 0.01, ***P < 0.001; t-test).
Fig. 8 Clearance studies of intravenously administered SIO nano-
particles at 2.5 mg Fe per kg in BALB/c mice. (a) Pseudocolored T
2
-
maps following the clearance of nanoparticles in the liver (L) and
spleen (S). The corresponding signal change of the (b) liver and (c)
hepatic vasculature, which cannot be achieved by either SIO-20
or Gd-BOPTA alone (Fig. 6f). Therefore, it potentially provides spleen in T -weighted MR images (n ¼ 3).
2
the capability of circumscribing a liver mass or detection of very
small liver lesion with information from both size/volume and
tumor vasculature at the same time using only one contrast
pre-injection level in two weeks aer injection of SIO-3.
However, only 60% was recovered in animals that received SIO-
0, suggesting a much faster clearance of SIO-3 from the liver.
Consistent with in vivo MRI observation, the ex vivo bio-
distribution data based on iron concentration analysis also
revealed the faster clearance of SIO-3 than SIO-20 in the liver
and spleen (Fig. S7†). Furthermore, the oligosaccharide coated
IONPs (SIO-20) showed faster clearance in the liver and spleen,
compared with the conventional bi-block copolymer-coated
IONPs (SHP20).
agent instead of generating “double” contrast by sequentially
injections of both IONPs and Gd-DTPA as explored
2
46,47
previously.
contrast enhancement in the kidney by SIO-3 may also
T
1
offer potential applications of imaging renal functions, espe-
cially in patients suffering from NSF and who are vulnerable to
Gd toxicity. The accumulation of SIO-3 in the kidney, but not
SIO-20, suggested possible renal clearance of the sub-5 nm
48,49
IONPs due to the smaller hydrodynamic size than 8 nm.
Both T and T -weighted images of mice administrated with
1
2
SIO-3 showed the gradual changes of MRI signals in the bladder
over time, indicating the excretion of SIO-3 from the kidney to
the bladder at a time of 1 hour aer administration (Fig. 7) and
4. Conclusions
the stability of the nanoparticles in the blood stream upon In summary, a new class of highly stable and biocompatible
ltration and secretion by the kidney. Such MRI signal changes oligosaccharide coated sub-5 nm ultrane IONPs has been
were not observed in the bladders of those animals receiving developed for improving T contrast enhancement in MRI. The

1
larger size IONPs, such as SIO-20.
To further examine the body clearance of SIO-3 accumulated ization of glucose on the particle surface. The resulting ultrane
in RES organs, we used MRI to follow the change of T “dark- SIOs exhibit excellent stability and colloidal properties in
ening” contrast and T relaxometry mapping in the liver and physiological medium with improved T1 MRI contrast
stability of such sub-5 nm SIOs is achieved by in situ polymer-
2
2
spleen (Fig. 8), which was shown to be correlated with the iron enhancing effects, thus providing potential blood pool MRI
concentration in the tissue. The results showed almost contrast agents with longer blood half-time than small mole-
complete recovery of T values of the liver and spleen to the cule contrast agents. Furthermore, dual T –T dual contrast
2
1
2
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B, 2014, 2, 5344–5351 | 5349

View Article Online
Journal of Materials Chemistry B
Paper
observed in liver imaging provides a new capability of simul- 18 Q. L. Vuong, J. F. Berret, J. Fresnais, Y. Gossuin and
taneous imaging of the liver parenchyma and lesion as well as O. Sandre, Adv. Healthcare Mater., 2012, 1, 502–512.
the vasculature using one single agent in clinical applications. 19 E. D. Smolensky, H.-Y. E. Park, Y. Zhou, G. A. Rolla,
More importantly, sub-5 nm SIO showed faster clearance from
the RES than that of IONPs with the larger size, and can be also
M. Marjanska, M. Botta and V. C. Pierre, J. Mater. Chem. B,
2013, 1, 2818–2828.
secreted from the kidney, thus may potentially address the long 20 J. Park, K. J. An, Y. S. Hwang, J. G. Park, H. J. Noh, J. Y. Kim,
term toxicity concerns associated with translating such material
to clinic imaging.
J. H. Park, N. M. Hwang and T. Hyeon, Nat. Mater., 2004, 3,
891–895.
2
2
1 U. I. Tromsdorf, O. T. Bruns, S. C. Salmen, U. Beisiegel and
H. Weller, Nano Lett., 2009, 9, 4434–4440.
2 J. Nam, N. Won, J. Bang, H. Jin, J. Park, S. Jung, S. Jung,
Y. Park and S. Kim, Adv. Drug Delivery Rev., 2013, 65, 622–
Acknowledgements
This work is supported in parts by the Cancer Nanotechnology
Platform Project (CNPP) grant (U01CA151810-02 to HM and LY)
and a research grant (R01CA154846-02 to HM and LY) from
National Institutes of Health.
6
48.
3 A. Roch, R. N. Muller and P. Gillis, J. Chem. Phys., 1999, 110,
403–5411.
2
2
2
5
4 J. Zeng, L. Jing, Y. Hou, M. Jiao, R. Qiao, Q. Jia, C. Liu,
F. Fang, H. Lei and M. Gao, Adv. Mater., 2014, 26, 2694–2698.
5 K. E. Kellar, D. K. Fujii, W. H. H. Gunther, K. Briley-Saebo,
A. Bjornerud, M. Spiller and S. H. Koenig, J. Magn. Reson.
Imag., 2000, 11, 488–494.
Notes and references
1
2
3
4
B. Y. S. Kim, J. T. Rutka and W. C. W. Chan, N. Engl. J. Med.,
010, 363, 2434–2443.
A. J. Cole, V. C. Yang and A. E. David, Trends Biotechnol.,
011, 29, 323–332.
E. T. Ahrens and J. W. M. Bulte, Nat. Rev. Immunol., 2013,
55–763.
2
26 L. Sandiford, A. Phinikaridou, A. Protti, L. K. Meszaros,
X. Cui, Y. Yan, G. Frodsham, P. A. Williamson,
N. Gaddum, R. M. Botnar, P. J. Blower, M. A. Green and
R. T. M. de Rosales, ACS Nano, 2013, 7, 500–512.
27 B. H. Kim, N. Lee, H. Kim, K. An, Y. I. Park, Y. Choi, K. Shin,
Y. Lee, S. G. Kwon, H. B. Na, J. G. Park, T. Y. Ahn, Y. W. Kim,
W. K. Moon, S. H. Choi and T. Hyeon, J. Am. Chem. Soc.,
2011, 133, 12624–12631.
2
7
D. Ghosh, Y. Lee, S. Thomas, A. G. Kohli, D. S. Yun,
A. M. Belcher and K. A. Kelly, Nat. Nanotechnol., 2012, 7,
677–682.
5
6
7
H. B. Na, I. C. Song and T. Hyeon, Adv. Mater., 2009, 21, 28 Z. Li, P. W. Yi, Q. Sun, H. Lei, H. L. Zhao, Z. H. Zhu,
2
133–2148.
S. C. Smith, M. B. Lan and G. Q. Lu, Adv. Funct. Mater.,
2012, 22, 2387–2393.
Z. Zhou, D. Huang, J. Bao, Q. Chen, G. Liu, Z. Chen, X. Chen
and J. Gao, Adv. Mater., 2012, 24, 6223–6228.
29 R. C. Atkins, J. Chem. Educ., 1975, 52, 550.
J. H. Lee, Y. M. Huh, Y. W. Jun, J. W. Seo, J. T. Jang, 30 L. Shen, J. Bao, D. Wang, Y. Wang, Z. Chen, L. Ren, X. Zhou,
H. T. Song, S. Kim, E. J. Cho, H. G. Yoon, J.-S. Suh and
J. Cheon, Nat. Med., 2007, 13, 95–99.
H. Duan, M. Kuang, X. Wang, Y. A. Wang, H. Mao and S. Nie,
J. Phys. Chem. C, 2008, 112, 8127–8131.
X. Ke, M. Chen and A. Yang, Nanoscale, 2013, 5, 2133–2141.
31 R. Costo, V. Bello, C. Robic, M. Port, J. F. Marco,
M. P. Morales and S. Veintemillas-Verdaguer, Langmuir,
2012, 28, 178–185.
8
9
J. Huang, L. Wang, R. Lin, A. Y. Wang, L. Yang, M. Kuang, 32 X. M. Sun and Y. D. Li, Angew. Chem., Int. Ed., 2004, 43, 597–
W. Qian and H. Mao, ACS Appl. Mater. Interfaces, 2013, 5,
632–4639.
0 L. Zhang, X. Zhong, L. Wang, H. Chen, Y. A. Wang, J. Yeh,
L. Yang and H. Mao, J. Magn. Reson. Imag., 2011, 33, 194–202. 34 S. Srivastava, R. Awasthi, D. Tripathi, M. K. Rai, V. Agarwal,
1 J. Huang, X. Zhong, L. Wang, L. Yang and H. Mao,
601.
4
33 A. Golon and N. Kuhnert, J. Agric. Food Chem., 2012, 60,
3266–3274.
1
1
1
V. Agrawal, N. S. Gajbhiye and R. K. Gupta, Small, 2012, 8,
1099–1109.
Theranostics, 2012, 2, 86–102.
2 L. H. Reddy, J. L. Arias, J. Nicolas and P. Couvreur, Chem. 35 Y. Hu, X. Gao, L. Yu, Y. Wang, J. Ning, S. Xu and X. W. Lou,
Rev., 2012, 112, 5818–5878. Angew. Chem., Int. Ed., 2013, 52, 5636–5639.
3 I. Hilger and W. A. Kaiser, Nanomedicine, 2012, 7, 1443–1459. 36 M. Sevilla and A. B. Fuertes, Chem.–Eur. J., 2009, 15, 4195–
1
1
4 L. Gu, R. H. Fang, M. J. Sailor and J.-H. Park, ACS Nano, 2012,
, 4947–4954.
4203.
6
37 W. Jiang, X. Zhang, Z. Sun, Y. Fang, F. Li, K. Chen and
C. Huang, J. Magn. Magn. Mater., 2011, 323, 2741–2747.
38 J. Muzart, Tetrahedron, 2009, 65, 8313–8323.
1
1
5 C. Chambon, O. Clement, A. Leblanche, E. Schoumanclaeys
and G. Frija, Magn. Reson. Imaging, 1993, 11, 509–519.
6 S. Laurent, L. V. Elst and R. N. Muller, in The Chemistry of 39 F. Hu and Y. S. Zhao, Nanoscale, 2012, 4, 6235–6243.
Contrast Agents in Medical Magnetic Resonance Imaging, ed. 40 J. S. Ananta, B. Godin, R. Sethi, L. Moriggi, X. Liu, R. E. Serda,
´
´
A. Merbach, L. Helm and E. Toth, John Wiley & Sons, Ltd,
Chichester, UK, 2nd edn, 2013.
R. Krishnamurthy, R. Muthupillai, R. D. Bolskar, L. Helm,
M. Ferrari, L. J. Wilson and P. Decuzzi, Nat. Nanotechnol.,
2010, 5, 815–821.
1
7 Y. W. Jun, J. W. Seo and A. Cheon, Acc. Chem. Res., 2008, 41,
179–189.
5350 | J. Mater. Chem. B, 2014, 2, 5344–5351
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
41 P. Kucheryavy, J. He, V. T. John, P. Maharjan, L. Spinu, 46 J. Ward, J. A. Guthrie, D. J. Scott, J. Atchley, D. Wilson,
G. Z. Goloverda and V. L. Kolesnichenko, Langmuir, 2013,
9, 710–716.
M. H. Davies, J. I. Wyatt and P. J. Robinson, Radiology,
2000, 216, 154–162.
2
42 Z. Li, S. X. Wang, Q. Sun, H. L. Zhao, H. Lei, M. B. Lan, 47 F. Zhang, X. Huang, C. Qian, L. Zhu, N. Hida, G. Niu and
Z. X. Cheng, X. L. Wang, S. X. Dou and G. Q. Lu, Adv.
Healthcare Mater., 2013, 2, 958–964.
X. Chen, Biochem. Biophys. Res. Commun., 2012, 425, 886–
891.
43 S. Laurent, D. Forge, M. Port, A. Roch, C. Robic, L. V. Elst and 48 H. S. Choi, W. Liu, P. Misra, E. Tanaka, J. P. Zimmer, B. I. Ipe,
R. N. Muller, Chem. Rev., 2008, 108, 2064–2110.
4 L. J. Wang, Q. Wu, S. Tang, J. F. Zeng, R. R. Qiao, P. Zhao,
M. G. Bawendi and J. V. Frangioni, Nat. Biotechnol., 2007, 25,
1165–1170.
4
Y. Zhang, F. Q. Hu and M. Y. Gao, RSC Adv., 2013, 3, 49 M. Longmire, P. L. Choyke and H. Kobayashi, Nanomedicine,
3454–23460. 2008, 3, 703–717.
2
45 A. Roch, Y. Gossuin, R. N. Muller and P. Gillis, J. Magn. Magn.
Mater., 2005, 293, 532–539.
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B, 2014, 2, 5344–5351 | 5351