Chemical Engineering Journal 360 (2019) 289–298

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Chemical Engineering Journal

journal homepage: www.elsevier.com/locate/cej

Nanostructure-enhanced water interaction to increase the dual-mode MR T

contrast performance of gadolinium-doped iron oxide nanoclusters
Yuanchun Sia,d,1, Guilong Zhangc,1, Dan Wangd, Cheng Zhangb, Chi Yanga, Guo Baia,
Junchao Qiane, Qiaoer Chend, Zhiyuan Zhanga, Zhengyan Wuc, Yunsheng Xub, , Duohong Zoua,
⁎ ⁎

a
Department of Oral Surgery, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, National Clinical
Research Center of Stomatology, Shanghai 200001, People’s Republic of China
b
Department of Dermatovenereology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, People’s Republic of China
c
Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, People’s Republic of China
d
Department of Dental Implant Center, Stomatologic Hospital & College, Key Laboratory of Oral Diseases Research of Anhui Province, Anhui Medical University, Hefei
230032, People’s Republic of China
e
Hefei Cancer Hospital, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, People’s Republic of China

HIGHLIGHTS GRAPHICAL ABSTRACT

• High performance dual-mode contrast

agent (GdIONC) was developed.
• The nested porous structure dramati-
cally enhanced water-interaction
around GdIONC.
• High specific surface area increased
the number of water molecules around
GdIONC.
• Hydrogen bonds effectively prolonged
the rotational correlation time of
proton.
• Oxide vacancies sharply enhanced the
water residence time and exchange
efficiency.

ARTICLE INFO ABSTRACT

Keywords: Rational structure design benefits the development of new classes of contrast agents (CAs) with excellent
Magnetic resonance imaging magnetic resonance imaging performance. In this work, hydrogenated silica with a net nanostructure (HSiO2)
Water molecules was fabricated and used to modify gadolinium-doped iron oxide nanoclusters (GdIONCs) to form a core-shell
Hydrogen bond nanoplatform (HSiO2@GdIONC) with enhanced T1 and T2 contrast ability. In this nanoplatform, the HSiO2 shell
Oxygen vacancy
showed a strong binding capacity for water molecules because of the presence of hydrogen bonds, oxygen
Contrast enhancement
vacancies, and high specific surface areas, and the strong binding capacity significantly improved the spin-spin
(T2) and spin-lattice (T1) imaging of the GdIONC core. In addition, the T1 relaxation rate of the GdIONC core
dramatically increased from 30.8 mM−1 s−1 to 38.2 mM−1 s−1 after being coated with the HSiO2 shell, and the
r2 to r1 ratio decreased from 10.9 to 8.3, which is an appropriate ratio (r2/r1: 5–10) for dual-mode contrast. Cell
and animal experiments suggested that HSiO2@GdIONC exhibited a better T1- and T2-weighted MR imaging
effect than the bare GdIONC core, confirming that this strategy for modifying GdIONCs is a beneficial and
promising approach for obtaining highly efficient dual-mode CAs.

⁎
Corresponding authors.
E-mail addresses: xuyunsheng1018@163.com (Y. Xu), zouduohong@126.com (D. Zou).
1
Co-first authors.

https://doi.org/10.1016/j.cej.2018.11.219
Received 6 September 2018; Received in revised form 18 November 2018; Accepted 29 November 2018
Available online 30 November 2018
1385-8947/ © 2018 Elsevier B.V. All rights reserved.
Y. Si et al.
1. Introduction
Contrast agents (CAs) disturb the magnetic properties of sur-
rounding water protons and are used in magnetic resonance imaging
(MRI) to enhance disease diagnostic capabilities [1–3]. Currently, there
are two main types of MRI CAs, paramagnetic Gd compounds and su-
perparamagnetic iron oxide nanoparticles (SPIOs) [4–6]. Paramagnetic
Gd compounds affect the spin-lattice (T1) contrast of water protons
[7,8], whereas SPIOs shorten the spin-spin relaxation time (T2) of water
molecules and are used for T2 imaging [9,10]. However, a single MR
contrast process has some inherent drawbacks, for example: (1) dis-
turbances originating from calcification, bleeding, or metal deposits in
T2 imaging [11–13] and (2) insufficient signal intensity in disease tissue
for T1 imaging [14,15]. These drawbacks are difficulty in discrimina-
tion between normal tissue and disease tissue and may seriously limit
accurate interpretation for the diagnosis image. Therefore, developing
multifunctional imaging modes using a one-scanner device to obtain
more accurate diagnosis information is necessary.
Currently, several types of dual-mode CAs have been established,
such as Gd-chelate-grafting SPIOs [16], Gd-compound coatings on
magnetic cores [17], or Gd-doped SPIO nanoparticles (GdIONs)
[8,18,19]. In addition, according to previous reports [20–22], the spin-
lattice relaxation process of Gd compounds outside a magnetic core can
be significantly disturbed by the magnetic field originating from SPIOs
because of the strong magnetic coupling and high susceptibility effect
of T1 materials, which severely affect the T1 MRI performance. Never-
theless, Gd ions inside SPIOs (i.e., Gd-doped SPIOs) have parallel spin
ordering in the same direction as the magnetic field induced by the
SPIOs [17,23], which could effectively prevent this disturbance of the
magnetic core. In addition, the spin order of Gd(III) located inside
GdIONs can generate a local magnetic field with the same direction as
the magnetic core, which dramatically increases the local magnetic
field intensity and inhomogeneity and further improves the T1 and T2
imaging performance [24]. Recently, Gao et al. synthesized engineered
Scheme 1. Schematic of the synthetic procedure for HSiO2@GdIONC and the interaction mechanism between the HSiO2 shell and water molecules.
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magnetic nanoplates with Gd-doped Fe3O4 and found that the T1 and T2
relaxation rates gradually decreased as the size increased [24,25].
Meanwhile, Park et al. further explored the magnetic properties of these
nanoplates and found that the extent of Gd doping directly affected the
dual-mode contrast ability [26]. However, the prepared GdIONs are
usually hydrophobic and require multistep modification procedures to
obtain a hydrophilic surface that can enter the body. These processes
are costly and time consuming; therefore, a method to manufacture
hydrophilic GdIONs is needed.
In our previous work, we developed a Gd-doped iron oxide na-
nocluster (GdIONC) with good water solubility and biocompatibility
and higher T1 and T2 relaxation rates than bare SPIOs [27]. However,
the r2/r1 ratio of the GdIONCs was greater than 10, implying that its
dual-mode functions for in vivo imaging would be severely limited
[28,29]. Therefore, a method that can significantly enhance the T1 re-
laxation rate and not decrease the T2 relaxation rate is needed to op-
timize the r2/r1 value. Currently, the strategies for enhancing the T1
relaxation rate mainly include increasing the number of bound water
molecules [30], optimizing the water residence time [31], and
prolonging the rotational correlation time [32]. Therefore, designing
nanostructures that bind a large number of water molecules and en-
hance the contact efficiency between water protons and magnetic cores
might be a good route for improving the r2/r1 value.
Herein, we optimized the previous method used to prepare
GdIONCs, and the new GdIONCs presented higher T1 and T2 relaxation
rates. Meanwhile, the GIONCs were further modified using hydro-
genated silica (HSiO2) and formed a nested core-shell nanostructure
(HSiO2@GdIONC). In addition, as shown in Scheme 1, the nested core-
shell nanostructure shows many advantages, including (1) a higher
porosity and specific surface area, which cause the distribution of water
molecules around the magnetic core to increase; (2) the formation of
numerous hydrogen bonds between the silanol (–Si–OH) groups and
water molecules, which can prolong the rotational correlation time of
the water protons; and (3) the abundant oxide vacancies in
Y. Si et al.
hydrogenated silica, which significantly enhances the water residence
time and exchange efficiency between the magnetic core and the water
protons. Based on the aforementioned advantages, the r1 relaxivity of
HSiO2@GdIONC was dramatically enhanced, and the HSiO2 shell pre-
vented the loss of r2 relaxivity of the GdIONC core, optimizing the r2/r1
ratio and improving the T1/T2 dual-mode in vivo imaging. In addition,
the HSiO2 shell significantly improved the biocompatibility of the
magnetic core, which promotes the in vivo application of
HSiO2@GdIONC. Therefore, designing nanostructures is a promising
route for improving dual-mode in vivo MR imaging.
2. Experimental section
2.1. Materials
All chemical reagents were used as received without further pur-
ification. Fe(acac)3 (98%), Gd(acac)3 (99%), triethanolamine (TEA,
98%), tetraethylorthosilicate (TEOS, 99%), N-hydroxysuccinimide
(NHS), and N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC)
were purchased from Aladdin Co. (Shanghai, China). Ethylene glycol
(EG, 99%), diethylene glycol (DEG, 99%), polyvinyl pyrrolidone (PVP),
and sodium borohydride (NaBH4, 96%) were purchased from
Sinopharm Co. (Shanghai, China). CCK-8 was obtained from Dojindo
(Japan), and an Annexin V-FITC Apoptosis Detection Kit was purchased
from BD Biosciences (San Jose, CA, USA).
2.2. Synthesis of GdIONCs
Fe(acac)3 (0.2 g) and Gd(acac)3 (0.12 g) were added to a mixed
solution of EG (10 mL) and DEG (20 mL), and the mixture was stirred
for 30 min at 80 °C. Then, PVP (1 g) was added to the resultant solution
and continuously stirred for 30 min. Subsequently, TEA (3 mL) was
added to the solution over 30 min with stirring to form a homogeneous
and transparent orange solution. Finally, the obtained solution was
poured into Teflon-lined stainless-steel autoclaves and kept at 230 °C
for 72 h. The black product was collected by centrifugation and washed
with ethanol and distilled water at least three times.
2.3. Synthesis of SiO2@GdIONC
The core-shell SiO2@GdIONC was synthesized via a classical Stöber
method. Briefly, GdIONC (50 mg) was ultrasonically dispersed into a
mixed solution of distilled water (20 mL) and ethanol (140 mL).
Subsequently, ammonium hydroxide (2 mL) was added to the resultant
solution and stirred at 250 rpm/min for 30 min. After that, the solution
containing TEOS (1.5 mL) and ethanol (8.5 mL) was added dropwise to
the mixture and continuously stirred for 30 h. Finally, SiO2@GdIONC
was collected via magnetic separation and washed three times with
distilled water.
2.4. Preparation of HSiO2@GdIONC
SiO2@GdIONC (100 mg) was dispersed in distilled water (20 mL)
via sonication, and an ice-cold aqueous solution of NaBH4 (8 mg/mL,
10 mL) was subsequently poured into the resultant solution. The mixed
solution was then quickly transferred into Teflon-lined stainless-steel
autoclaves and allowed to react for 48 h at 80 °C. Afterwards, the ob-
tained particles were collected via centrifugation at 12,000 rpm and
etched for 12 h via repeating the aforementioned steps. Finally, the
obtained product was collected and washed at least three times using
distilled water and ethanol.
2.5. MRI experiment
The contrast ability of HSiO2@GdIONC was explored using a 3.0 T
MRI scanner. For in vitro or in-cell phantom MR experiments, a series of
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inversion-prepared fast spin-echo images were acquired for the long-
itudinal relaxation time (T1) measurement. This series was identical in
all aspects (TR 6000 ms, effective TE 5.6 ms, BW 25 kHz, slice thickness
1 mm, matrix 96 × 96, 1 average) except for the 20 different inversion
times (TIs) that were linearly varied from 10 to 2500 ms. The transverse
relaxation (T2) time was measured using a multiecho spin echo se-
quence with a repetition time (TR) of 2500 ms and 20 echoes with echo
times (TE) ranging from 10 to 200 ms.
2.6. Cell culture and cytotoxicity assay
HeLa cell lines were purchased from the Institute of Biochemistry
and Cell Biology of the Chinese Academy of Sciences and cultivated
using a DMEM/high-glucose medium containing fetal bovine serum
(10%), penicillin G sodium (100 U/mL) and streptomycin sulfate
(100 μg/mL) at 37 °C in a humidified incubator with 5% CO2.
Subsequently, HeLa cells were seeded into 96-well plates and adjusted
to a density of 1 × 104 cells/well. HSiO2@GdIONC with a wide range of
concentrations was used to incubate HeLa cells for 24. Afterwards, the
culture medium was discarded and the cells were washed with PBS and
then treated with 200 μL of 10% CCK-8 for 2 h. The viability of the cells
was measured at a wavelength of 450 nm with a Fluostar Optima mi-
croplate reader (BMG Labtechnologies, Germany).
2.7. Confocal laser scanning microscopy (CLSM) observations
HeLa cells were seeded on 10 mm2 glass coverslips, placed in 24-
well plates (5 × 104 cells/well) and incubated with FITC-labeled
HSiO2@GdIONC. After that, the HeLa cells were stained with Hoechst
33342 for 0.5 h at 37 °C in the dark. The cells were washed twice using
PBS and fixed with 4% paraformaldehyde for 30 min. Finally, the cells
were mounted onto glass slides and visualized using a confocal mi-
croscope (Zeiss LSM710 NLO, Germany).
2.8. T1-T2 dual-mode in vivo MRI investigation
The mice used in the experiment were treated in accordance with
the Ethics Committee Guidelines of Hefei Institutes of Physical Science,
Chinese Academy of Sciences. The mice were anesthetized with iso-
flurane (2.5% induction, 0.5–1.5% maintenance) in air/O2 (2:1) for the
duration of the scan. The animals were placed in a prone position on a
specially designed cradle and inserted into the magnet. The respiratory
rate and body temperature were monitored throughout the experiment
with a physiologic monitoring unit (model 1030; SA Instruments, Inc.,
Stony Brook, NY, USA). For the duration of the experiment, the animal’s
body temperature was maintained at 36.5 °C with a homemade heating
pad.
T1- and T2-weighted MR images were acquired along the transverse
orientation at 15, 30, 60, 90, and 120 min postinjection. For the mouse
studies, the following acquisition parameters for T1 imaging were
chosen: repetition time (TR) = 370 ms, echo time (TE) = 11.6 ms, field
of view (FOV) = 40 mm × 40 mm, matrix size = 192 × 192, slice
thickness = 1 mm (12 slices, gap = 0), 1 average, and bandwidth
(BW) = 50 kHz. The T2-weighted imaging parameters were set as fol-
lows: fast spin echo, TR = 400.0 ms, TE = 10.0 ms, field of
view = 40 mm × 40 mm and matrix size = 128 × 128, slice thick-
ness = 1 mm (12 slices, gap = 0), 1 average, and BW = 50 kHz.
Throughout the scanning sessions, pulse oximeter triggering was used
for the MRI acquisition to reduce artifacts arising from respiratory
movement.
2.9. Characterization
The particle size distribution of the samples was measured using a
dynamic light scattering (DLS) detector (Nanotrac Wave II, Microtrac
Co., USA). The morphology and structure of the samples were observed
Y. Si et al.
with an H-800 transmission electron microscope (JEM-ARM200F, JEOL
Co., Japan). The crystal structure and interaction of the samples were
analyzed by X-ray diffraction (XRD) (TTR-III, Rigaku Co., Japan) and
FT-IR spectroscopy (iS10, Nicolet Co., USA). The thermogravimetric
analysis (TGA) of the samples was carried out with a thermogravimetric
analyzer (Q5000IR, TA Instruments Co., USA). The magnetic properties
of the samples were characterized with a superconducting quantum
interference device (SQUID) magnetometer (Bruker Co., Germany).
3. Results and discussion
3.1. Synthesis and characterization of HSiO2@GIONC
As shown in Scheme 1, HSiO2@GIONCs were prepared via three
steps as follows. First, GdIONCs were synthesized via the solvothermal
reaction of Fe(acac)3 and Gd(acac)3 complexes that contained PVP as a
template. Second, the core-shell structure SiO2@GdIONC was prepared
using GIONCs as the core and TEOS as the silica source via a classical
Stöber method [33]. Finally, the core-shell-structured SiO2@GdIONC
was further treated using a NaBH4 solution to form the nested
HSiO2@GdIONC. A number of water molecules could be distributed
around the magnetic core because of the high porosity of the HSiO2
shell and the presence of hydrogen bonds and oxygen vacancies.
The TEM images show that the GdIONCs have a loose structure
compared to that of previous Fe3O4 nanoclusters [34], which might be
because the enhanced lattice distortion results in disordered self-as-
sembly of tiny crystals (Fig. 1a). The interplanar distances of GdIONC
was analyzed via high resolution transmission electronic microscopy
(HRTEM) as shown in Fig. S2. The distance between two adjacent
planes of originated Fe3O4 nanocrystal was measured to be 0.252 nm,
corresponding to the (311) plane. However, for GdIONC, the distance
between these adjacent planes increased to 0.262 nm and the crystal
Fig. 1. TEM images of (a) GdIONC, (c) SiO2@GdIONC, and (d) HSiO2@GdIONC; (b) high-resolution TEM image of GdIONC.
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lattice became blurry in comparison with bare Fe3O4, suggesting the
increase of the interplanar spacing and the lattice distortion.
In addition, for SiO2@GdIONC, an obvious core-shell structure ap-
peared and the thickness of the SiO2 shell could be adjusted via con-
trolling the hydrolysis of TEOS (Fig. 1c). After treatment with NaBH4,
the dense shell became significantly looser and formed the nested core-
shell structure, indicating that HSiO2@GdIONC was successfully pre-
pared by the Ostwald ripening process (Fig. 1d). Interestingly, the size
of HSiO2@GdIONC increased after the treatment, which might be due
to expansion of the HSiO2 shell. Moreover, plenty of pore channels
appeared in the HSiO2@GdIONC; thus, more water molecules could
enter the pore channels and increase the water distribution around the
magnetic core. Besides, the content of Gd, Fe, and Si in HSiO2@GdIONC
was also analyzed via ICP-MS and the result was as follows: Fe: Gd:
Si = 0.1684: 0.0824: 0.683. The analysis of the particle size distribu-
tion showed that the GdIONCs possess a narrow peak, and their size was
approximately 160 nm (Fig. S1d), which suggested that the GdIONCs
were well dispersed in the solution. In addition, compared to the bare
magnetic core, the SiO2@GdIONCswere larger (∼310 nm) because of
the presence of the silica shell. Furthermore, the size of
HSiO2@GdIONC further increased after hydrogenation, indicating that
a nest-like shell was successfully formed. These results were consistent
with the TEM analysis.
Subsequently, the composition and functional groups of the parti-
cles were measured using FT-IR spectroscopy (Fig. 2a). In the spectrum
of the GdIONCs, the peaks at 580 cm−1 were ascribed to the Fe–O
stretching vibration [35,36] and the peaks at 1090, 1409, and
1597 cm−1 were attributed to C–N stretching vibrations and C–H and
N–H bending vibrations, respectively, suggesting that PVP might play
an important role in the assembly of the GdIONC crystals. In addition,
the spectrum of SiO2@GdIONC had an obvious new peak at 1093 cm−1,
which was assigned to the Si–O stretching vibration [37,38], indicating
Y. Si et al.
that silica successfully coated the GdIONCs to form a core-shell struc-
ture. The peak intensity at 1634 cm−1 significantly increased, implying
that many silanol groups formed in the HSiO2@GdIONC. Meanwhile,
the peak of Si–O dramatically shifted from 1093 to 1000 cm−1 because
the Si–O–Si bond changed to –Si–OH, which resulted in the formation of
numerous oxygen vacancies in the HSiO2 shell. On the basis of this
analysis, the dense silica shell might transform into silanol linkages
with oxygen vacancies, indicating that the nested core-shell structure
was created by silanol linkages.
In addition, XRD analysis showed that GdIONC had a spinel struc-
ture and no new peak appeared in comparison with bare Fe3O4, sug-
gesting that the crystal structure of GdIONC were different from the
Gd2O3 simply embedded or coated in Fe3O4 (Fig. 2b). Moreover, the
diffraction peaks of GdIONC significantly shifted to the left, which
could be attributed to the increase of interplanar spacing owing to Gd
ions doping. Meanwhile, it could be seen that the peak intensity of
GdIONC dramatically weakened, and the half peak width of GdIONC
enlarged, which could be attributed to the enhancement of lattice dis-
tortion. Additionally, the XRD pattern of SiO2@GdIONC exhibited a
typical broad peak of amorphous silica, and the broad peak was sig-
nificantly weaker for HSiO2@GdIONC. This result illustrates that the
silica shell successfully encapsulated the GdIONC core and was effec-
tively hydrogenated by NaBH4. The nitrogen adsorption-desorption
isotherms and pore size distributions of SiO2@GdIONC and
HSiO2@GdIONC were subsequently measured to explore the porosity
variation before and after NaNH4 treatment.
As shown in Fig. 2d, the porosity and specific surface area of
SiO2@GdIONC were very low, whereas those of HSiO2@GdIONC
sharply increased, suggesting that numerous pore channels and the
nest-like shell formed after the borohydride reduction treatment. In
addition, HSiO2@GdIONC displayed a wide range of pore sizes from 1.2
to 6.7 nm, and the pore content was more than five times that of
Fig. 2. (a) FT-IR spectra and (b) XRD patterns of GdIONC, SiO2@GdIONC, and HSiO2@GdIONC; (c) TG curves and (d) N2 adsorption-desorption isotherms of
SiO2@GdIONC and HSiO2@GdIONC (inset: corresponding pore size distribution).
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Chemical Engineering Journal 360 (2019) 289–298
SiO2@GdIONC. These results indicate that abundant water molecules
could enter porous HSiO2@GdIONC, enhancing the interactions be-
tween the protons and the core. Afterwards, the water-retention rate of
HSiO2@GdIONC was investigated by TGA (Fig. 2c). The weight loss of
SiO2@GdIONC and HSiO2@GdIONC occurred in three steps: free water
bound by physical absorption (< 200 °C), water bound by hydrogen-
bond interactions (200–550 °C), and water bound by oxygen-vacancy
interactions (> 550 °C). For SiO2@GdIONC, the contents of physically
absorbed free water and water bound by hydrogen bonds were 8.3%
and 3.3%, respectively, and the water bound by oxygen-vacancy in-
teractions was difficult to observe. Nevertheless, the free and bound
water contents of HSiO2@GdIONC due to physical absorption, hy-
drogen bonds, and oxygen vacancies appeared to dramatically increase
and were 12.45%, 5.83%, and 2.52%, respectively. These results fur-
ther confirmed that the HSiO2 shell promotes water distribution around
the GdIONC core and significantly enhances the T1 MRI contrast ability.
XPS was used to investigate the compositions and interactions of the
samples. As shown in Fig. 3a, C, N, Gd, Fe, and O peaks were observed for
the GdIONCs, indicating that Gd ions were successfully doped into Fe3O4
and that PVP plays an important role in the GdIONCs. In the spectrum of
SiO2@GdIONC, the Gd and Fe peaks disappeared because the dense SiO2
shell was thick enough to cover the GdIONC signal. On the other hand, for
HSiO2@GdIONC, relatively weak Fe and Gd peaks appeared (Fig. 3b, c),
implying that a loose HSiO2 shell formed. Remarkably, the Si 2p peak for
SiO2@GdIONC significantly shifted from 103.39 to 102.24 eV (Fig. 3d).
This shift was attributed to the Si structure transformation, i.e., from a
dense silica layer to nested silanol linkages.
3.2. Magnetic properties and MRI performance
The field-dependent magnetization (M−H) curves showed that all
samples had obvious coercivity and remanence at 3 K and presented
Y. Si et al. Chemical Engineering Journal 360 (2019) 289–298

Fig. 3. (a) XPS full spectra, (b) Fe 2p spectra, (c) Gd 4d spectra, and (d) Si 2p spectra of the samples.

typical ferromagnetic behavior (Fig. 4a) because of the low-tempera- showing typical superparamagnetic behavior. Notably, the saturated
ture-limited thermal randomization of the magnetic domains [39]. The magnetization of SiO2@GdIONC (6.4 emu/g) slightly increased in
coercivity and remanence of the samples disappeared at 300 K (Fig. 4b), comparison with that of GdIONC (22.1 emu/g), and HSiO2@GdIONC

Fig. 4. Hysteresis loops of the samples at (a) 3 K and (b) 300 K; (c) the temperature dependence of the ZFC and FC magnetization curves for the different samples.

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Y. Si et al.
exhibited a moderate magnetization value (10.2 emu/g). In addition,
the temperature-dependent magnetization curves gave the blocking
temperature (TB) of the samples with an applied magnetic field of 100
Oe (Fig. 4c). The blocking temperature of the GdIONCs did not vary
before and after modification, indicating that the silica shell did not
affect the superparamagnetism of the GdIONC core. In addition, all of
the samples showed obvious Gd signal peaks at the critical temperature,
further confirming successful doping of Gd ions.
According to the previously reported [40,41], iron oxide nano-
particles (IONs) which was less than 3 nm in size showed excellent
paramagnetism and contributed to T1 imaging. Nevertheless, significant
ferromagnetism appeared with the size of IONs increasing, suggesting
that IONs could possess good T2 contrast ability and hardly contribute
T1 MRI contrast. Unlike IONs, Gd ions had seven single-electron spins
and showed very strong paramagntism, thereby Gd ions could effec-
tively contribute to T1 imaging. However, the local magnetic field in-
duced by the spins of Gd ions under external magnet was still very low
in comparison with that of IONs, thereby the T2 contribution originated
from Gd ions was negligible. Therefore, for GdIONC, SiO2@GdIONC,
and HSiO2@GdIONC, the r1 value was calculated according to Gd
concentration, and the r2 value was calculated according to Fe con-
centration (Fig. 5a). The GdIONCs exhibited higher r1 (30.8 mM−1 s−1)
and r2 (320 mM−1 s−1) values than those reported in previous studies;
these values show that the GdIONCs could be an excellent dual-mode
CA. However, the r2/r1 ratio (10.5) for the GdIONCs exceeded 10, in-
dicating that the T1 and T2 contrast would interfere with each other and
that the dual-mode in vivo imaging performance would be inhibited.
After the addition of the silica shell coating, the r1 value of
SiO2@GdIONC dramatically decreased to 3.1 mM−1 s−1 and the r2/r1
significantly increased to 100, mainly because the dense SiO2 shell
prevented contact between the water molecules and the GdIONC.
Nevertheless, an ultrahigh r1 value (38.3 mM−1 s−1) was obtained after
hydrogenation of the silica shell because the nested mesoporous
structure contained a number of water molecules around the GdIONC
core (Fig. 5d). Moreover, the r2 value of HSiO2@GdIONC had almost no
Fig. 5. (a) Longitudinal and (b) transverse relaxation rates of the samples using 3.0 T; (c) MR map images of HeLa cells treated with HSiO2@GdIONC; (d) schematic
of the distribution of water molecules in GdIONCs with different structures.
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change (Fig. 5b); thus, a moderate r2/r1 ratio (8.3) that is beneficial for
dual-mode MR imaging was obtained. Meanwhile, the etching degree of
SiO2@GdIONC was investigated via adjusting reaction time. As shown
in Fig. S3, the porosity and specific surface area of HSiO2@GdIONC
gradually increased with etching time increasing at the beginning stage,
which indicated that the etching degree of HSiO2@GdIONC was sig-
nificantly enhanced and the amount of oxygen vacancies increased.
Interestingly, the r1 value of HSiO2@GdIONC also gradually increased
with porosity and oxygen vacancies increasing, further confirming that
the oxygen vacancies and porosity could enhance MRI performance of
the CAs (Fig. S4).
The MR contrast enhancement of HSiO2@GdIONC on HeLa cells
was subsequently investigated (Figs. 5c and S2). In this work, a wide
range of HSiO2@GdIONC concentrations was used to incubate HeLa
cells and the cells were subsequently collected into tubes and subjected
to MRI. The MR images of the HeLa cells treated with HSiO2@GdIONC
were significantly brighter than those without, and the brightness was
dosage dependent. Meanwhile, the T1 relaxation time of HeLa cells
incubated with HSiO2@GdIONCwas determined via measurement of
the MR map images. The T1 and T2 values of HeLa cells incubated with
HSiO2@GdIONC appeared to have a dosage-dependent decrease, im-
plying that abundant HSiO2@GdIONC was internalized into HeLa cells
and endowed HeLa cells with excellent contrast ability.
3.3. Cell viability, uptake, and localization
To assess in vivo application potential, the biocompatibility of
HSiO2@GdIONC must be explored. As shown in Fig. 6a,
HSiO2@GdIONC did not cause a significant decrease in HeLa cell via-
bility, indicating that HSiO2@GdIONC is nontoxic, with good bio-
compatibility. Subsequently, HSiO2@GdIONC was internalized by HeLa
cells and examined via confocal laser scanning microscopy (CLSM).
First, fluorescein isothiocyanate (FITC) was rationally conjugated to
HSiO2@GdIONC to achieve green fluorescence and observe the location
of the particles in living cells. As shown in Fig. 6d, only parts of
Y. Si et al.
HSiO2@GdIONC crossed the cell membrane and entered the cytoplasm
after incubation for 1 h. As the incubation time increased, the fluor-
escent intensity of the HeLa cells gradually increased, indicating that
more particles were internalized by the HeLa cells. Thus, cell uptake of
HSiO2@GdIONC was dependent on time. In addition, the green fluor-
escent intensity of HeLa cells gradually brightened with increasing
HSiO2@GdIONC concentration, suggesting that the intake of
HSiO2@GdIONC by HeLa cells is dose dependent. Subsequently, flow
cytometric measurements were used to quantitatively investigate cell
uptake of HSiO2@GdIONC (Fig. 6b, c). Approximately 47.02% of cells
were labeled with HSiO2@GdIONC at a concentration of 40 μg/mL after
incubation for 1 h. Moreover, as the incubation time was increased, the
number of HeLa cells labeled by HSiO2@GdIONC gradually increased to
83.75% (2 h) and 91.62% (4 h), confirming the time-dependent uptake
ability. In addition, the intake of HeLa cells was also proportional to the
concentration of HSiO2@GdIONC and was 45.76% (20 μg/mL), 66.47%
(40 μg/mL), and 99.98% (80 μg/mL). According to the previous ana-
lysis, HSiO2@GdIONC could be effectively internalized by HeLa cells,
which is helpful for the MR imaging of tumors.
Next, the MRI performance of HSiO2@GdIONC in vivo was studied
using a 9.4 T MR scanner (Fig. 7). Nanoparticle accumulation has been
previously demonstrated to occur in the liver within hepatic Kupffer
cells [42]. Subsequently, we investigated T1 imaging of a region of
interest within the liver both pre- and postinjection (15, 30, 60, 90, and
120 min) of 2.0 mg (Gd)/kg (mouse body weight) GdIONC and
Fig. 6. (a) MTT assay of HeLa cell viability after incubation with different concentrations of HSiO2@GdIONC for 24 h. Flow cytometry histograms show the FITC
fluorescence intensity in HeLa cells treated with (b) HSiO2@GdIONC at a dose of 30 μg/mL for different times and (c) different concentrations of HSiO2@GdIONC for
4 h. (d) CLSM observations of the HeLa cell uptake of FITC-labeled HSiO2@GdIONC.
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Chemical Engineering Journal 360 (2019) 289–298
HSiO2@GdIONC through tail vein injection. The liver region of the
mice (average body weight of 20 g) treated with GdIONC was slightly
brighter (Fig. 7a), suggesting that GdIONC could rapidly accumulate in
the liver. Moreover, the contrast signals of the liver gradually increased
and reached a maximum at 90 min postinjection. However, for
HSiO2@GdIONC, the liver region of the imaged mouse was dramati-
cally brighter and showed a stronger T1 contrast enhancement com-
pared with that of bare GdIONC, suggesting that HSiO2@GdIONC has a
better liver T1 contrast ability than GdIONC. Moreover, the maximum
contrast enhancement appeared at 60 min postinjection, implying that
the HSiO2 shell promotes the metabolism of particles in the liver. Si-
milar results were observed in the T2 mouse liver images: the liver re-
gions of mice injected with HSiO2@GdIONC exhibited darker images
than those injected with bare GdIONC. These results suggest that
HSiO2@GdIONC has a better dual-mode MRI contrast ability than
GdIONC, further confirming that the HSiO2 shell with a nested meso-
porous structure could effectively improve the dual-mode in vivo
imaging performance of the GdIONC core. The analysis of the MRI
signal-to-noise ratio change (ΔSNR) in the liver region indicated that
the maximal ΔSNR appeared at 60 min postinjection of
HSiO2@GdIONC and was 91.8% and 61.2% for the T1 and T2 imaging,
respectively (Fig. 7b, c). However, the liver ΔSNR of mice injected with
GdIONC was 41.2% and 51.6% for T1 and T2 imaging, respectively,
possibly because the high r2/r1 ratio of GdIONC limits the T1-T2 dual-
mode imaging ability in vivo. On the basis of the aforementioned
Y. Si et al.
Fig. 7. T1-T2 dual-modal liver imaging of HSiO2@GdIONC. (a) T2- and T1-weighted MR images (9.4 T) of mice liver (white arrows) acquired preinjection and 15, 30,
60, 90, and 120 min postinjection of GdIONC and HSiO2@GdIONC with a dose of 2.0 mg(GIONC)/kg (mouse body weight); (b) MRI signal changes in the liver of (b)
T2- and (c) T1-weighted images at transverse planes (mice number: n = 3, data represent mean ± standard deviation).
results, the design of the nested shell structure is a promising approach
to decrease the r2/r1 ratio and avoid a disturbance of the T1 and T2
signals of the GdIONC core. Thus, HSiO2@GdIONC has potential for
application as a liver MRI CA to accurately diagnose liver lesions.
4. Conclusion
In summary, an efficient strategy was introduced to develop a high-
performance dual-mode MRI CA via modifying the nanostructure and
increasing the number of water interactions. HSiO2@GdIONC has a
nested, mesoporous core structure and can effectively bind abundant
water molecules via physical absorption and hydrogen-bond and
oxygen-vacancy interactions, dramatically improving the T1 and T2
contrast ability. In addition, compared with that of bare GdIONC
(30.2 mM−1 s−1), the T1 relaxation rate of HSiO2@GdIONC sig-
nificantly increased to 38.2 mM−1 s−1 and the T2 relaxation rate did
not decrease. Therefore, the r2/r1 ratio of HSiO2@GdIONC decreased
from 10.5 to 8.3, effectively preventing the disturbance of T1 and T2
imaging and allowing HSiO2@GdIONC to exhibit an optimal dual-mode
contrast ability. Based on the results of the cell and animal experiments,
HSiO2@GdIONC showed better T1- and T2-weighted MR contrast in the
liver region than bare GdIONC, further confirming that
HSiO2@GdIONC possesses a better dual-mode contrast ability.
Therefore, the strategy of nanostructure-enhanced water interaction is a
beneficial and promising approach to obtaining highly efficient dual-
mode CAs.
Acknowledgements
The authors acknowledge financial support from the National
Natural Science Foundation of China (Nos. 21407151, 81771531,
81571395), the Youth Innovation Promotion Association of Chinese
Academy of Sciences (No. 2015385), the Science and Technology Major
297
Chemical Engineering Journal 360 (2019) 289–298
Project of Anhui Province (No.17030701051), and the Natural Science
Foundation of Anhui Province (No.1808085MB38).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.cej.2018.11.219.
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