Colloids and Surfaces A: Physicochemical and Engineering Aspects 679 (2023) 132594

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Colloids and Surfaces A: Physicochemical and

Engineering Aspects
journal homepage: www.elsevier.com/locate/colsurfa

Amino-grafted water-soluble ferrimagnetic iron oxide nanoparticles with

ultra-high relaxivity for dual-modal magnetic resonance imaging
Haiyang Jia a, b, *, 1, Fen Zhang c, 1, Chen Zhu a, Jiawei Sun d, Xiao Xie b, **
a
School of Physics and New Energy, Xuzhou University of Technology, Xuzhou 221018, China
b
Key Lab of MEMS of Ministry of Education, Southeast University, Nanjing 210096, China
c
School of Food and Bioengineering, Xuzhou University of Technology, Xuzhou 221018, China
d
College of Electronic and Information Engineering, Suzhou University of Science and Technology, Suzhou 215009, China

G R A P H I C A L A B S T R A C T

Using the deep eutectic solvent as an electrolyte, amino-grafted water-soluble magnetically responsive maghemite nanocrystals are synthesized by
organic electrolysis. The nanoparticles are stably dispersed in water, and the dispersion delivers ultra-high relaxivity. As a contrast agent, the amino-
grafted magnetic nanocrystal is capable of dual-modal magnetic resonance imaging with significant resolution.

A R T I C L E I N F O A B S T R A C T

Keywords: Highly stable dispersion is an important precondition for functionalized magnetic nanoparticles used as a
Iron oxides contrast agent. Amino-grafted ferromagnetic γ-Fe2O3 nanoparticles are synthesized by deep-eutectic-solvent
Magnetic nanoparticles electrolysis for magnetic resonance imaging. The statistical naked scale of the particle is 8.2 nm with a
Contrast agents
magnetization of 71.9 emu g− 1. An ultra-high Zeta potential of + 50 mV boosts the dispersion stable for more
Magnetic resonance imaging
than 600 days, which is unprecedented. The 26.4 nm hydrated nanoparticles exhibit high biocompatibility after
being efficiently ingested by cells in vitro. Both longitudinal relaxation efficiency of 179.7 mM− 1 s− 1 and
transverse relaxivity of 497.8 mM− 1 s− 1 are among the highest demonstrated in recent years. Whether in vitro or
in vivo, the imaging mode changes from T1 to T2 with the increase of iron equivalent. At doses 0.12 and 0.91 mg
Fe per kg living body, the nanoparticles are capable of prominently weighted imaging of T1 and T2 of the liver,
respectively. The nanocrystals can significantly distinguish between normal tissue and the tumor site of the liver
as well. Findings demonstrate that the highly stable dispersion of amino-grafted ferrimagnetic nanoparticles is
capable of dual-modal magnetic resonance imaging with high relaxation efficiency and significant resolution.

1. Introduction indispensable technology for medical imaging and disease diagnosis,

especially for visualizing soft tissue and organs [1]. MR contrast agents
Magnetic resonance imaging (MRI) has been regarded as an could enhance the contrast of imaging by affecting the relaxation of

* Corresponding author at: School of Physics and New Energy, Xuzhou University of Technology, Xuzhou 221018, China.
** Corresponding author.
E-mail addresses: jsz@xzit.edu.cn (H. Jia), xxie@seu.edu.cn (X. Xie).
1
H. Jia and F. Zhang contributed equally to this work.

https://doi.org/10.1016/j.colsurfa.2023.132594
Received 23 August 2023; Received in revised form 13 October 2023; Accepted 14 October 2023
Available online 16 October 2023
0927-7757/© 2023 Elsevier B.V. All rights reserved.
H. Jia et al.
Fig. 1. (a-c) Schematic diagram using amino-grafted water-soluble ferrimagnetic iron oxide (AFIO, synthesis by the DES-electrolysis) nanoparticles as a contrast
agent for MRI. (d) The presentation of AFIO (inner diameter of glass dish: 150 mm). (e) Two times gradient concentration of AFIO aqueous dispersion on the 600th
day (volume of glass bottle: 10 mL). (f) High-resolution TEM image of an AFIO nanoparticle (scale bar: 2 nm). (g) Statistical particle size distribution of AFIO based
on TEM observations (count: > 1000).
hydrogen protons around them [2,3]. Over 60 million MRI procedures
are implemented each year worldwide, and about a third part of patients
need the aid of a contrast agent [1,4]. The gadolinium-based contrast
agents (GBCAs) were the most widely used MRI contrast agents in
clinics, and they have been approved by the U.S. Food and Drug
Administration (FDA) and clinically used for about 30 years [1,5].
GBCAs belonged to positive (T1) contrast agents, they could shorten the
longitudinal relaxation (related to energy) time of protons and then
exhibit a brighter image [2]. Although GBCAs have been verified safe to
use for most procedures, risks still suffered objectively for a small pro­
portion of patients. For patients with renal function impairment, neph­
rogenic systemic fibrosis resulting from leached free gadolinium ions
was confirmed [5–7]. The gadolinium deposition in bone tissue and
accumulation in the brain of a normal individual was also proven,
especially for those after repeated exposure to GBCAs diagnosis [6–8]. In
addition, the relatively short retention time in vivo (half-life of 0.9–3.0
h) of GBCAs sometimes could not meet the time required for
high-resolution MRI [5,6].
Magnetic responsive nanoparticles could shorten the transverse
relaxation (related to phase) time of surrounding hydrogen protons, and
then be used as a T2-weighted negative contrast agent resulting in darker
images. Studies also found that nanoparticles as small as a certain size
could be used as the positive contrast agent under T1 weighting [2,8,9].
Iron oxide nanoparticles in the body were predominantly captured by
the reticuloendothelial system. They were metabolized into free iron
and stored in the liver and bone marrow, where they were used to
construct the hemoglobin by red blood cells [3,10]. Compared with
gadolinium, iron is one of the essential elements in living organisms, and
participates in many biological processes. In a rodent model, an equiv­
alent intake of 100 mg Fe per kg weight did not cause adverse effects,
and it was not fatal even an injection of 600 mg Fe per kg [10]. In recent
years, many studies have been devoted to the development of iron
oxide-based contrast agents (IOCAs). The first IOCA (ferumoxides, Fer­
idex®) was approved by FDA in 1996. Since that, dozens of analogs have
been developed and tested in clinical trials, pending approval, or
approval for marketing [3,10]. In consideration of deviations in the
diagnosis, several commercially available IOCAs were discontinued by
the supervision department [3,11,12]. The deviation was mainly false
positive, which was attributed to the dark signal of T2-weighted and
variation of the physicochemical properties of IOCAs. Due to the
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Colloids and Surfaces A: Physicochemical and Engineering Aspects 679 (2023) 132594
interference of background signals on dark signals, the false positive
could be found in weak-intensity areas such as bleeding, calcification,
and metal deposition [4,9]. For the latter, particle size, surface charge,
and surface coating of batch products were difficult to control within an
envisaged range [11,12]. The development of IOCAs was facing chal­
lenges, which also posted higher expectations for the preparation of
magnetic iron oxide, including the controllability of physicochemical
properties and the preparation process [5]. Moreover, magnetic nano­
particles should also be endowed with sufficiently high relaxation effi­
ciencies to enhance the resolution and reduce the injection dose.
Chemical co-precipitation was commonly used for the mass pro­
duction of hydrophilic magnetic iron oxide nanoparticles, including
commercially available IOCAs [4]. However, it suffered from control­
lability in terms of process and product characteristics. In recent years,
another method of thermal decomposition aroused great research
enthusiasm. Specifically, magnetic iron oxide nanoparticles were
formed by the thermal decomposition of iron-based organic precursors
(e.g. Fe-oleate, Fe-acetylacetonate, Fe-pentacarbonyl) in
high-boiling-point nonpolar solvents at high a temperature (>200 ◦ C).
The greatest feature of the process lay in the excellent control ability of
the particle size of monodisperse particles. The fly in the ointment was
that the resulting primary magnetic particles were usually hydrophobic,
implying these particles could not be directly used in biological medi­
cine. The remedy was to carry out surface hydrophilic modifications
based on organic ligand exchange, but procedures were generally
complex and costly [1,4,5,13–16].
In the face of challenges, deep eutectic solvent electrolysis (DES-
electrolysis) was proposed by our group and then applied in the syn­
thesis of hectogram-level water-soluble magnetic responsive iron oxide
nanoparticles [17–19]. The synthesized monodisperse nanoparticles
were in situ loaded with hydrophilic amino groups and endowed with
high saturation magnetization. Those particles could be efficiently
dispersed in water with high Zeta potential and exhibited highly
dispersed stability [19]. It was expected that the aqueous dispersion
could be used as a contrast agent for MRI. In the present work, cellular
uptake and cytotoxicity tests of the nanoparticles were performed first.
The relaxation efficiency of the dispersion was then quantified in vitro.
Finally, the in vivo MRI would be validated and evaluated using the
dispersion as a simulated contrast agent.
H. Jia et al.
Fig. 2. Confocal laser scanning microscope images of HepG2 cells incubated with AFIO (a1 - a3) and FITC@AFIO (b1 - b3) for 2 h, respectively. Fluorescence (a1, b1),
optical (a2, b2), and merge (a3, b3) images of cultured HepG2 cells. Scale bar: 50 µm.
2. Results and discussion
2.1. Synthesis and characterizations
The schematic diagram using amino-grafted water-soluble ferri­
magnetic iron oxide (AFIO) nanoparticles as a contrast agent for mag­
netic resonance imaging was exhibited in Fig. 1a-c. The magnetic
nanoparticles were synthesized by DES-electrolysis [19]. Briefly, by
using the DES composed of quaternary ammonium and urea as an
electrolyte, electrolytic synthesis was carried out by employing iron
flakes as anode and cathode. In the electrolysis process, iron atoms at the
interface were oxidized to ferric irons and then diffused into the elec­
trolyte. Subsequently, these ions combined with the reactive oxygen
species generated by the decomposition of the electrolyte to form iron
oxide nanocrystals. Concomitantly, hydrophilic amino/amine groups
produced by the DES decomposition were grafted on the nanocrystals
[19]. Detailed synthesis procedures can be found in the Supporting In­
formation. Based on the analyses of X-ray diffraction (Fig. S1) and X-ray
photoelectron spectroscopy (Fig. S2), the synthesized sample was
identified to be amino-loaded maghemite (γ-Fe2O3) [19].
The ethanol-washed but undried black AFIO was shown in Fig. 1d,
which could be highly dispersed in water. As exhibited in Fig. 1e and
Fig. S3, the highly transparent aqueous dispersions on the 60th and
600th day indicated an excellent dispersing state. Absolute values of the
Zeta potential of the dispersion (pH ≈ 5.5) on the 60th and 600th day
were 52.2 and 49.7 mV, respectively, such stability was unprecedented
and has never been reported [19]. It was believed that nanoparticle
dispersion was highly stable in drug delivery when the absolute value
was greater than 30 mV [20]. As exhibited in Fig. S4, the statistical
hydration diameter was determined to be 26.4 and 30.3 nm with the
polydispersity index of 0.012 and 0.017 at the above time points,
respectively [19]. The excellent dispersibility and stability were not only
attributed to the good compatibility between grafted hydrophilic groups
and water but also closely related to the monodisperse characteristic of
nanoparticles. The AFIO was also dispersed in the other two solvents of
the phosphate-buffered saline (PBS, pH = 7.4) buffer and the Tris-HCl
buffer (pH = 7.4) to evaluate the extensiveness of the dispersion.
Compared with the acidic aqueous dispersion, the Zeta potential in the
buffer of PBS and Tris-HCl changed from positive to negative. The
reversal of charge was due to the substitution of the active ions in the
dispersion solvent from positively charged hydrogen ions (pH ≈ 5.5) to
negatively charged hydroxide ions (pH = 7.4). The absolute value of the
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Colloids and Surfaces A: Physicochemical and Engineering Aspects 679 (2023) 132594
Zeta potential was dropped from ca. 50 mV in the acidic aqueous
dispersion to 33 mV in the Tris-HCl buffer and 19 mV in the PBS buffer.
The decrease in the absolute value of the Zeta was by reason of the low
concentration of hydroxide ions (pH = 7.4), which suggested that the
stability of the dispersion was weakened according to the
Derjaguin-Landau-Verway-Overbeek theory of colloidal dispersions
[20]. From the Zeta perspective, it meant that the Tris-HCl buffer-based
dispersion was still suitable for the in vivo injection (greater than
30 mV) [20]. The dynamic light scattering statistical particle size in the
two buffers was exhibited in Fig. S5. As the decrease of the absolute
value of the Zeta potential of the dispersion, the hydrated particle size in
the dispersion increased from 26 to 30 nm in the acidic aqueous
dispersion to 36 nm in the Tris-HCl buffer and 87 nm in the PBS buffer. It
suggested that the particles were still stably dispersed in the Tris-HCl
buffer, but would be agglomerated due to the induction of cations in
the PBS buffer (e.g. sodium and potassium ions). A high-resolution TEM
record was exhibited in Fig. 1 f, the apparent lattice demonstrated a high
degree of crystallinity. TEM images of the particles on the 60th and
600th day are shown in Fig. S6, indicating the dispersibility of the
particles and the stability of the dispersion. The TEM particle size dis­
tribution was plotted in Fig. 1 g, and the average diameter of the naked
particles was statistically determined to be 8.2 nm (Fig. 1 g). The
magnetization curve was shown in Fig. S7, the embedded partially
enlarged view illustrated the ferrimagnetic property. The saturation
magnetization, remnant magnetization, and coercivity were determined
to be 71.9 emu g− 1, 5.4 emu g− 1, and 32.3 Oe, respectively. Analyses
suggested that the AFIO had the potential to be used as a contrast agent
in magnetic resonance imaging.
2.2. Cellular uptake and cytotoxicity
For the AFIO nanocrystals to be used as a candidate IOCA, cellular
uptake, and cytotoxicity should be examined in advance [14,21,22].
Confocal laser scanning microscopy (CLSM) was used to observe the
cellular uptake of nanocrystals. The composite of fluorescein 5(6)-iso­
thiocyanate (FITC) labeled on the nanoparticle (FITC@AFIO) was con­
structed (see supporting information for details) and then confirmed by
the UV–visible spectroscopy (Fig. S8). Two peaks were observed around
455 and 476 nm for FITC aqueous solution. No absorbance peak was
found at the wavelength between 440 and 500 nm of the AFIO disper­
sion [23]. Two peaks were located at 458 and 476 nm for the mixture of
FITC and AFIO (FITC&AFIO), and they were regarded as the absorption
H. Jia et al. Colloids and Surfaces A: Physicochemical and Engineering Aspects 679 (2023) 132594

Fig. 3. In vitro cytotoxicity tests of AFIO nanoparticles based on the MTT assay. Viability of the MCF-7 cell under concentration (a, b) and time (c) gradient. Viability
of the HepG2 cell under concentration (d, e) and time (f) gradient. Data were collected from three independent experiments. Unit of AFIO dose: μg Fe per mL culture
medium; error bar: ± s.d. (n = 3).

Fig. 4. A set of bright-field (the first row) and FDA/PI co-staining fluorescent images (from the second to the fourth row: live, dead, and merge) of HepG2 cells after
treatment for 24 h. Live and dead cells were stained green and red, respectively. Scale bar: 200 µm. The leftmost numbers were AFIO dose values, and the unit was μg
Fe per mL culture medium.

peak of FITC. For the dispersion of FITC@AFIO, only one peak centered
on 484 nm was detected, implying FITC was labeled on the particle [23].
CLSM images of the human hepatocellular carcinomas (HepG2) cells
after 2 h incubation with AFIO and FITC@AFIO were shown in
Fig. 2a1-a3 and Fig. 2b1-b3, respectively. The bright green fluorescence
displayed covering each cell. Lots of uniformly distributed green spots of
labeled AFIO nanoparticles were detected in the cells not only
confirming the effective cellular uptake of these nanocrystals but also
indirectly demonstrating these nanoparticles were well dispersed in the
medium.
Human breast cancer (MCF-7) and human liver cancer (HepG2) cell
lines were employed to evaluate the cytotoxicity by the MTT colorimetry
assay [22,23]. In terms of concentration gradient, when the AFIO dosage
increased from 0.002 to 2 μg Fe per mL medium, the viability of MCF-7

4
H. Jia et al.
Fig. 5. Fitting lines of 1/T2 (a) and 1/T1 (b) against iron concentration in aqueous dispersions. T1- (c) and T2-weighted (d) MR images of aqueous dispersions.
Magnetic field: 1.0 T.
decreased from 99.7% to 96.3% after 24 h incubation (Fig. 3a). For
HepG2, the viability was weakly reduced from 99.8% to 97.6% with the
same drug administration, as plotted in Fig. 3d. Even under incubation
at the concentration of 20 μg Fe per mL for 48 h, the survival rate of
MCF-7 and HepG2 cells was still as high as 94.9% (Figs. 3b) and 94.3%
(Fig. 3e), respectively. In terms of time-dependent, when the AFIO
stimulation amount was 20 μg Fe mL− 1 medium, the survival rate of
MCF-7 cells after incubation for 2, 10, and 30 h were 99.3%, 98.1%, and
96.6%, respectively (Fig. 3c). For HepG2 cells, the viability at the
above-mentioned time was 99.6%, 99.2%, and 97.8%, respectively
(Fig. 3f). Even under high-dose injury for a long time, both cell lines
showed super-high viability in MTT assays, indicating the high
biocompatibility of the AFIO nanoparticles [23]. Further cytotoxic test
of cells was carried out by the fluorescein diacetate/propidium iodide
(FDA/PI) double staining method. As shown in Fig. 4 and Fig. S9, no
dead cell (stained red) was observed even HepG2 cells were stimulated
by the AFIO dose of 100 μg Fe mL− 1 medium for 24 h, demonstrating
AFIO nanoparticles were noncytotoxic.
2.3. In vitro magnetic resonance relaxivity
The r1 and r2 relaxivities of aqueous dispersion were examined by
using a 1-T MR scanner, and the test liquid was the acidic aqueous
dispersion. It was generally believed that the sedimentation and ori­
ented drift of particles in dispersions would decrease the relaxation ef­
ficiency value [24]. During the scanning, the colloidal system of
magnetic nanoparticles had to be assisted by stabilizers (e.g. agarose) to
ensure stability, even after those particles had undergone surface mod­
ifications [4,14,23–26]. In our case, the stability of the dispersion was so
excellent that it did not require any auxiliary substance to prevent
sedimentation and oriented drift in the magnetic field during the mea­
surement. From the linear fitting of 1/T2 (Fig. 5a) and 1/T1 (Fig. 5b)
against Fe concentration, the r2 and r1 values were calculated to be
497.8 and 179.7 mM− 1 s− 1, respectively. Compared with magnetic iron
oxide synthesized by traditional methods and then modified by hydro­
philic substances (as listed in Table S1), the AFIO nanoparticles pre­
pared by DES-electrolysis delivered one of the highest r2 and r1 relaxivity
[14,28–35]. These values were also 4- to 10-fold higher than that of
commercial IOCAs [27,36,37]. Generally, when the value of r2/r1 was in
the range of 1–2 and greater than 10, materials could be used for T1 and
T2 MRI, respectively [38]. In our case, the ratio of 2.77 implied that the
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Colloids and Surfaces A: Physicochemical and Engineering Aspects 679 (2023) 132594
AFIO had the potential for use as a dual-modal contrast agent [2,39].
The T1- and T2-weighted MR images of the AFIO dispersion are shown in
Figs. 5c and 5d, respectively. In the T1 weighting, as the iron content in
the dispersion increased, the image gradually brightened and reached
the brightest state at the concentration of 40.66 μM Fe. Then, as the iron
concentration continued to rise, the scanned image darkened until it was
completely dark (812.5 μM Fe). Under the T2 weighting course, the MR
image changed monotonously. With the increase of iron concentration,
images were gradually darkened to complete darkness. When the iron
content in the dispersion reached 40.66 μM, the image was almost
completely dark. Thus, this concentration was considered to be the
cut-off point of discrimination.
The AFIO delivered excellent relaxation efficiency, which was
completely induced by its desirable physicochemical properties. On one
hand, the particles had a high magnetization, and the efficiency value
was usually positively correlated with the magnetization [14,28,40]. In
addition, the in situ grafted amino/amine groups conveyed an important
contribution [40,41]. Specifically, the disturbance and diffusion of
water molecules around particles were affected by their surface coat­
ings. Fewer, thinner, and more uniform hydrophilic coatings could make
more water molecules easier to approach and contact the magnetic core.
In this way, more hydrogen protons would be significantly affected by
the induced magnetic field derived from the magnetically responsive
particles, thus resulting in a shorter relaxation time and a higher
relaxivity [32,35,40–42]. Compared to iron oxide nanoparticles with
tedious surface coatings, the AFIO particles were only in situ loaded with
amino groups. Coupled with excellent water solubility and high satu­
ration magnetization, the high relaxivity of r1 and r2 was predictable.
The hydrodynamic diameter of the AFIO was 26–30 nm, meaning
they could well be bound to the cell surface and permeate into cells [1,3,
43,44]. Studies also demonstrated that the amino-grafted nanoparticles
were more readily phagocytosed than those loaded with carboxyl, sul­
fate, and hydroxyl groups [3,45]. Iron oxide nanoparticles beyond
10 nm were ultimately taken up by the macro­
phages/reticuloendothelial system in the liver, spleen, and lymph nodes
[3,46]. Selective phagocytosis was regarded as the basis for in vivo
imaging and clinical diagnosis. The benign lesion and normal hepatic
tissue had a greater tendency to capture the nanoparticles due to their
increased levels of phagocytic cells and blood pools in contrast with the
cancerous lesion [3,46]. Considering the remarkable stability of aqueous
dispersion and ultra-high in vitro relaxivity, the in vivo MRI evaluation
H. Jia et al.
Fig. 6. T1- (a, g) and T2-weighted (b, h) images of Sprague-Dawley rat liver before and after intravenous injecting the AFIO aqueous dispersion. T1- (c, d, i, j) and T2-
weighted (e, f, k, l) ΔSNR data of coronal and axial position after the injection of AFIO. Injection: tail vein; scanning field intensity: 1.0 T; error bar: s.d. (n = 3).
would be performed on the healthy and subcutaneous tumor model rat.
2.4. In vivo magnetic resonance imaging of healthy rat
Considering the high relaxation efficiency of AFIO nanoparticles,
two doses with a distinct gradient were used for the in vivo T1- and T2-
weighted MRI study. All healthy Sprague-Dawley (SD) male mice
weighing about 20 g and aged 6–8 weeks old. With the acidic aqueous
dispersion as a stock solution of the simulated contrast agent, those SD
mice were injected via the tail vein, and then the MRI effect was
examined by using a 1.0 T scanner. When the dispersion was injected,
the AFIO would be captured by the liver tissue cells and then showed
brighter or darker images under an external magnetic field. The signal-
to-noise ratio (ΔSNR) of the coronal (Cor) and transverse (Axi) position
was used to quantitatively analyze the light-dark change in the region of
interest (liver).
The imaging of T1- and T2-weighted with a low dose injection of
0.12 mg Fe per kg of rat body was shown in Figs. 6a and 6b, respectively.
At the dose, imaging images derived from the other two parallel
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Colloids and Surfaces A: Physicochemical and Engineering Aspects 679 (2023) 132594
experiments were shown in Fig. S10 and S11. Under T1 weighting,
significantly brightened images were observed. As exhibited in Figs. 6c
and 6d, within 24 h, the minimum Cor- and Axi-ΔSNR was + 55.9 and
+ 44.5%, respectively. The corresponding maximum ΔSNR was + 66.3
and + 70.2%. These ratios suggested that the contrast was significant
and could be maintained for a long time. In the T2 weighted state, the
degree of image darkening was not significant. As shown in Fig. 6e, the
minimum and maximum Cor-ΔSNR were − 28.0 and − 14.0%, respec­
tively. For the transverse view (Fig. 6f), the Axi-ΔSNR at 0.5, 2, 8, and
24 h were as weak as − 2.1, + 2.4, + 6.8, and − 4.4%, respectively.
Therefore, the AFIO could be used for T1-weighted positive imaging at a
relatively low injection dose.
When the dose was increased to 0.91 mg Fe per kg of rat body, the
displayed MRI images changed dramatically, and the imaging was very
different from that at the low dose. As shown in Figs. 6g and 6h, in both
T1- and T2-weighted modes, the region of interest darkened significantly
within 24 h. At the dose, imaging images collected from the other two
parallel experiments were shown in Fig. S12 and S13. For the coronal
view in the T1 model, the minimum and maximum Cor-ΔSNR was
H. Jia et al.
Fig. 7. (a) T2-weighted images of the liver and subcutaneous tumor sites before and after injecting the AFIO dispersion. The dispersion was first injected through the
tail vein with a dose of 0.45 mg Fe per kg of rat body weight. After 24 h, the tumor location was injected with the dispersion at a dose of 0.15 mg Fe per kg. (b - d)
Analyses of ΔSNR before and after injection. Scanning field intensity: 1.0 T; tumor cell line: HepG2.
− 88.1 and − 85.1%, respectively (Fig. 6i). The corresponding Axi-
ΔSNR collected from the transverse view was − 79.8 ~ − 82.1%
(Fig. 6j). As performed on the T2-weighted pattern, in both coronal
(Fig. 6k) and transverse (Fig. 6l) view, the ΔSNR was darkened ranging
from − 57.5 to − 63.6%. It could be argued that the negative contrast
was striking. The imaging results implied that the AFIO could be used for
T2-weighted negative imaging at the relatively high injection dose.
Overall, the in vivo imaging results were consistent with those ob­
tained by the in vitro MR scanning. That is, the AFIO nanocrystals could
be used for T1-weighted MRI at a low dose, and could be employed as a
T2-weighted contrast agent at a high dose. These findings indirectly
demonstrated that AFIO nanoparticles could be diffused in the animal
body with the blood flow, and directly proved that the nanoparticles had
a long blood circulation time [8]. It was worth noting that the so-called
high dose was only one percent of the iron intake which could adversely
affect the body [9]. A slight regret of this study was that the time interval
between injection and capture of iron oxide nanoparticles by the liver
was not well defined. Although a 24-hour continuous contrast capability
had been maintained, the half-life of the particles in the liver and even
the corresponding metabolites were still uncertain. In-depth studies of
these concerns will be carried out in the future.
2.5. In vivo magnetic resonance imaging of tumor model rat
Given the r2 value was significantly greater than that of r1, the tumor
model rat was further employed for the in vivo T2-weighted MRI study.
The protocol would help to verify whether the AFIO could distinguish
between normal and tumorous liver tissue. Using the acidic aqueous
dispersion as a stock solution, the scanned images of coronal (Cor) and
transverse (Axi) positions are shown in Fig. 7a. The injection equivalent
was 0.45 mg Fe per kg of rat body weight, and the striking negative
contrast effect could be maintained for at least 24 h. At the 2, 8, and 24 h
after the injection, the Cor-ΔSNR of the liver was − 77.7, − 71.2, and
− 74.2%, respectively (Fig. 7b). Comparatively, the scanned images of
the subcutaneous tumor location showed weak contrasts. It was attrib­
uted to the phagocytic ability of liver tumor cells being weaker than that
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Colloids and Surfaces A: Physicochemical and Engineering Aspects 679 (2023) 132594
of normal liver cells. Within 24 h, the Cor-ΔSNR absolute value of the
tumor was not greater than 33.0% (Fig. 7c). 24 h after the intravenous
injection, the AFIO dispersion was directly injected into the tumor site,
in the dose was 0.15 mg Fe per kg body weight. In a short time (e.g.
10 min), the recorded image was significantly darkened. Specifically,
the Cor-ΔSNR of the tumor position changed from − 25.3 to − 59.3%
(Fig. 7c), and the Axi-ΔSNR changed from + 11.8 to − 66.7% (Fig. 7d).
Besides, the signal collected from the liver darkened further, and the
Cor-ΔSNR changed from − 74.2 to − 83.6%. These findings demon­
strated the excellent and durable T2-weighted MRI capability of the
AFIO. Although the AFIO lacked targeted imaging capability for liver
tumors, the tumor sites could be distinguished from the normal portion
in the T2-weighted mode, thus promoting the diagnosis of liver tumors.
3. Conclusions
AFIO nanoparticles were synthesized by DES-electrolysis and used as
a contrast agent for MRI. The magnetization of the particles was
71.9 emu g− 1 with a statistical TEM size of 8.2 nm. The 26.4 nm hy­
drated particles exhibited high biocompatibility after being efficiently
ingested by cells in vitro. Benefiting from good compatibility between
grafted hydrophilic groups and water, nanoparticles were stably
dispersed for over 600 days. The dispersion exhibited ultra-high trans­
verse and longitudinal relaxivity with r2 and r1 of 497.8 and
179.7 mM− 1 s− 1, respectively. With the increase of iron equivalent, the
imaging mode changed from T1 to T2. At low and high doses, the
nanoparticles were capable of prominently weighted imaging of T1 and
T2 of the liver, respectively. Although the AFIO lacked targeted imaging
capability for liver tumors, it delivered a distinct contrast between the
normal tissue and tumorous location. Findings demonstrated that the
AFIO could be used as a dual-modality MRI contrast agent.
CRediT authorship contribution statement
Haiyang Jia: Conceptualization, Methodology, Formal analysis,
Investigation, Writing – original draft, Writing – review & editing. Fen
H. Jia et al.
Zhang: Methodology, Investigation, Writing – review & editing. Chen
Zhu: Investigation, Writing – review & editing. Jiawei Sun: Investiga­
tion. Xiao Xie: Conceptualization, Methodology, Supervision, Writing –
review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data Availability
Data will be made available on request.
Acknowledgements
This work was supported by the Natural Science Foundation of the
Jiangsu Higher Education Institutions of China (22KJB430010), the
Xuzhou Key Research and Development Project (KC22304), and the
National Natural Science Foundation of China (22204138). This work
was also supported by the open research fund of Key Laboratory of
MEMS of Ministry of Education, Southeast University.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.colsurfa.2023.132594.
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