Article

www.acsnano.org

Ultrasmall Ferrite Nanoparticles Synthesized

via Dynamic Simultaneous Thermal
Decomposition for High-Performance and
Multifunctional T1 Magnetic Resonance
Imaging Contrast Agent
Huan Zhang,†,⊥ Li Li,‡,⊥ Xiao Li Liu,† Ju Jiao,§,⊥ Cheng-Teng Ng,∥ Jia Bao Yi,Δ Yan E Luo,†
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Boon-Huat Bay,∥ Ling Yun Zhao,# Ming Li Peng,† Ning Gu,¶ and Hai Ming Fan*,†
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†
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of the Ministry of Education, College of Chemistry and
Materials Science, Northwest University, Xi’an, Shaanxi 710069, China
‡
State Key Laboratory of Oncology in South China, Imaging Diagnosis and Interventional Center, Sun Yat-sen University Cancer
Center, Guangzhou 510060, China
§
Department of Nuclear Medicine, The Third Affiliated Hospital of Sun Yat-sen University, 600 Tianhe Road, Guangzhou,
Guangdong 510630, China
∥
Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, 4 Medical Drive, MD10, 117594,
Singapore
Δ
School of Materials Science and Engineering, University of New South Wales, Kensington, NSW 2052,
Australia
#
State Key Laboratory of New Ceramics and Fine Processing, Key Laboratory of Advanced Materials, School of Material Science &
Engineering, Tsinghua University, Beijing 100084, China
¶
State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science and
Medical Engineering, Southeast University, Nanjing 210096, China
*
S Supporting Information

ABSTRACT: Large-scale synthesis of monodisperse ultrasmall

metal ferrite nanoparticles as well as understanding the
correlations between chemical composition and MR signal
enhancement is critical for developing next-generation, ultra-
sensitive T1 magnetic resonance imaging (MRI) nanoprobes.
Herein, taking ultrasmall MnFe2O4 nanoparticles (UMFNPs) as a
model system, we report a general dynamic simultaneous thermal
decomposition (DSTD) strategy for controllable synthesis of
monodisperse ultrasmall metal ferrite nanoparticles with sizes
smaller than 4 nm. The comparison study revealed that the DSTD
using the iron-eruciate paired with a metal-oleate precursor
enabled a nucleation-doping process, which is crucial for particle
size and distribution control of ultrasmall metal ferrite nano-
particles. The principle of DSTD synthesis has been further confirmed by synthesizing NiFe2O4 and CoFe2O4 nano-
particles with well-controlled sizes of ∼3 nm. More significantly, the success in DSTD synthesis allows us to tune both
MR and biochemical properties of magnetic iron oxide nanoprobes by adjusting their chemical composition. Beneficial
from the Mn2+ dopant, the synthesized UMFNPs exhibited the highest r1 relaxivity (up to 8.43 mM−1 s−1) among the
ferrite nanoparticles with similar sizes reported so far and demonstrated a multifunctional T1 MR nanoprobe for in vivo
high-resolution blood pool and liver-specific MRI simultaneously. Our study provides a general strategy to synthesize
continued...

Received: November 14, 2016

Accepted: April 3, 2017
Published: April 3, 2017

© 2017 American Chemical Society 3614 DOI: 10.1021/acsnano.6b07684

ACS Nano 2017, 11, 3614−3631
ACS Nano Article

ultrasmall multicomponent magnetic nanoparticles, which offers possibilities for the chemical design of a highly sensitive ultrasmall
magnetic nanoparticle based T1 MRI probe for various clinical diagnosis applications.

KEYWORDS: ultrasmall ferrite nanoparticles, dynamic simultaneous thermal decomposition, T1 MR contrast agent,
chemical composition effect, magnetic resonance imaging, liver-specific MRI,

T ernary compound nanoparticles with superior compo-

sition-tunable properties have long been of scientific and
technological interest.1−6 In particular, the metal ferrite
nanoparticle MFe2O4 (M = Mn, Co, Ni, etc.) represents an
important class of magnetic ternary compound nanoparticles,
which have found wide biomedical applications, mostly as highly
sensitive magnetic resonance imaging (MRI) nanoprobes for
in vivo and noninvasive detection of clinically important biological
targets.2,7−10 Recent advances in this field have revealed that the
ultrasmall magnetite nanoparticles with sizes smaller than 5 nm
can exhibit remarkable and counterintuitive T1 MR enhancement
due to the strong size-related effects,11,12 in stark contrast to the
conventional superparamagnetic iron oxide based contrast agent
(CA) such as Feridex, which is the representative T2 contrast
agent. Despite its apparent advantages over the commercial
gadolinium-based T1 MRI contrast agent, results from currently
available ultrasmall ferrite nanoparticle-based T1 MRI probes
have not been optimal. Artificially molecular engineering of
chemical composition in magnetic ferrite nanoparticles has been
frequently employed to optimize their application perform-
ance.2,7,13 For example, the MR relaxation and biochemical
properties of magnetic MFe2O4 nanoparticle can be altered by
adjusting the chemical indentity of M2+.2,7 However, synthesis of
ultrasmall metal ferrite nanoparticles with well-controlled size
and composition simultaneously remains a challenging task due
to the complex dynamic nature of the nucleation and growth Figure 1. Diagram for DSTD and DNSTD synthesis of manganese
process in the multicomponent system. For instance, currently ferrite nanoparticles.
reported MFe2O4 nanoparticles synthesized by hydrolytic/
nonhydrolytic process either have large particle size or show monomer, and the burst of nucleation, while stage II describes
poor monodispersity and crystal quality, which hampers their the diffusion-limited growth. The temperature continuously
applications as ultrasensitive T1 MR contrast agents.14−22 increases during the heat-up process until reaching the final
Herein, taking monodisperse ultrasmall MnFe2O4 nanoparticles reactive temperature. For the DNSTD process, one metal
(UMFNPs) as a model system, we report a dynamic simul- precursor will be dissociated first due to the large difference in
taneous thermal decomposition strategy to couple the multi- decomposition kinetics of the two metal precursors, and the
component chemical doping process with the nucleation process, rapid accumulation of a single-metal-containing monomer
which allows us to achieve good control in particle size and results in the formation of a non-dopant-containing nucleus in
distribution of ultrasmall metal ferrite nanoparticles for highly stage I. Upon the decomposition of another metal precursor in
sensitive and multifunctional T1 MR contrast agents. stage II, these nuclei serve as the host to grow ternary MnFe2O4
Co-thermal decomposition of mixed metal precursors to nanoparticles. In order to achieve high-quality nanoparticles, the
prepare monodisperse magnetic MFe2O4 nanoparticles through final reaction temperature should be sufficiently high to activate
the heating-up process has been well-established17−20,23,24 and solid−solid ion diffusion.27 As a result of the high reaction
has also been applied to synthesize other ternary compound temperature and growth-doping process,28 the obtained
nanoparticles.25 However, due to the intricate growth dynamics MnFe2O4 nanoparticles via the DNSTD process usually have
arising from the thermal decomposition process of mixed sizes larger than 6 nm and show a strong T2 MR signal.12,13
precursors,26 the control of size, morphology, composition, and Mixing the dopant and host at stage I may further reduce the
crystal quality is thus quite difficult. In terms of precursor particle size and improve the quality of the nanoparticle.
decomposition behavior, two different scenarios, dynamic The DSTD is thus defined as a special co-thermal decomposition
nonsimultaneous thermal decomposition (DNSTD) and dynamic reaction where the precursors have an approximate on-site
simultaneous thermal decomposition (DSTD) processes, com- dissociation temperature (the offset within ΔT), and a multiple-
monly occur under certain synthetic conditions. Figure 1 metal-containing monomer can be formed before the nucleation.
schematically illustrates the growth dynamics of DSTD and Unlike the hot-injection process, in which nucleation- or growth-
DNSTD synthesis of MnFe2O4 using a modified LaMer diagram. doping can be decoupled by altering the reaction temperature,28
We presume that the intermediate species generated by the the regulation and control of growth dynamics for DNSTD/
dynamic dissociation of the metal complexes act as monomer. DSTD is realized by tuning the thermal stability of the pre-
Stage I depicts the generation of monomer by on-site dis- cursors. In this context, seeking suitable precursors is of utmost
sociation of molecule precursors, the accumulation of the importance for successfully achieving the DSTD process.
3615 DOI: 10.1021/acsnano.6b07684
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However, variations of on-site dissociation temperatures of
currently available metal precursors such as iron/manganese
acetylactonate and carboxylate complexes, are usually too large to
be paired. With due credit to Song and Zhang for demonstrating
the feasibility of artificial engineering of the precursor thermal
stability for the synthesis of high-quality MnFe2O4 nano-
particles,20 we were inspired to consider the possibility of
DSTD synthesis of utlrasmall metal ferrite nanoparticles by
devising green and inexpensive precursors.
Thermal stability of widely used precursors of coordination
compounds in a given metal cation can be easily modulated by
altering the ligands.20,29 Unfortunately, such modification may
also affect the growth kinetics of the nanoparticle by diverse
ligand effects,30 leading to derivation of an uncertain product and
complicated optimization of reactive parameters mediated by
trial and error. Hence, tailoring the molecular structure of
currently available coordination compound precursors to a slight
extent is a viable solution to reduce possible side effects. Among
the various coordination compound precursors used for the
preparation of metal ferrite nanoparticles, metal-oleates are
favored because of their relative nontoxicity and usability.
Furthermore, the formation kinetics of the binary ferrite (Fe3O4)
nanoparticles with high monodispersity and tunable size
obtained by iron-oleate precursors in the heating up process
has been well-studied,19,23 which may provide clues on the
understanding of the growth dynamics of metal ferrite
nanoparticles in the DSTD process. The current issue for
DSTD synthesis of UMFNPs is the distinct difference between
the on-site dissociation temperature of metal-oleate precursors,
which is 215 °C for iron-oleate and 311 °C for Mn-oleate.
Therefore, we prepared Fe-eruciate, a new type of coordination
compound precursor with improved thermal stability, where a
short-chain ligand of oleate (18-carbon chain) is substituted with
long-chain eruciate (22-carbon chain) (Figures S1−S4). Both
thermogravimetric analysis (TGA) and temperature-dependent
FTIR spectra revealed that Fe-eruciate decomposes at around
326 °C, which is very close to that of Mn-oleate (Figure S5).
By using this green Fe-eruciate precursor, we were able to achieve
a DSTD process and attain ultimate control of the UMFNPs with
regard to uniformity of size, single crystallinity, and stoichiom-
etry. The size of UMFNPs can be easily tuned from 2 nm to
4 nm. A comparison study of DSTD and DNSTD synthesis of
MnFe2O4 nanoparticles revealed that the growth behavior of
UMFNPs in the DSTD process was analogous to that of binary
iron oxide nanoparticles using single precursors, while the
DNSTD process in the same conditions may prevent effective
nucleation and growth separation, resulting in nonuniform
particles. As a proof of concept, the Fe-eruciate precursor-
promoted DSTD has been successfully applied to prepare other
ultrasmall ferrite nanoparticles such as CoFe2O4 and NiFe2O4
with sizes of 3 nm. Moreover, the success in DSTD synthesis of
ultrasmall metal ferrite nanoparticles allows us to investigate
the correlations between the composition and MR T1 signal
enhancement in such a system. The molecularly engineered
UMFNPs have exhibited the lowest r2/r1 ratio (2.49) at 3 T that
has been reported thus far for ultrasmall ferrite nanoparticles,
as well as a large r1 relaxivity (up to 8.43 mM−1 s−1) that is
2.09 times higher than a commercial gadolinium complex based
contrast agent (Omniscan). In vivo MRI imaging carried out
on a rat model verified that the UMFNPs could be used as
ultrasensitive and multifunctional T1 MR contrast agents for
high-resolution MR imaging of blood pools and liver. The study
aims to provide a general DSTD strategy for synthesizing
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ultrasmall metal ferrite nanoparticles in a controllable manner,
which undoubtedly offers the possibilities for designing highly
sensitive ultrasmall magnetic nanoparticle based T1 MRI probes
for various clinical diagnosis applications.
RESULTS AND DISCUSSION
Synthesis of the UMFNPs. Recent studies on ultrasmall
ferrite nanoparticle-based T1 MR contrast agents have revealed
that the MR signal enhancement is highly dependent on the
particle size and uniformity, as well as composition and surface
chemistry of the nanoparticles.31−33 The ability to synthesize
metal ferrite nanoparticles with extremely small size, narrow
size distribution, and stoichiometric composition is thus vital
for high-performance MRI applications. Among various ferrite
nanoparticles, the UMFNPs are an ideal model system to
investigate the controllable growth mechanism because the T1
relaxivity of ultrasmall magnetic nanoparticles may be signifi-
cantly enhanced by molecular engineering of the Mn dopant.34
Herein, the UMFNPs were synthesized by DSTD of mixed iron-
eruciate and manganese-oleate complexes in the presence of
oleic acid and oleyl alcohol in benzyl ether or 1-octadecene.
The oleyl alcohol is used to further reduce the on-site decompo-
sition temperature of the precursors, facilitating the formation of
small-sized nanoparticles.31 By varying the ratio of oleyl alcohol
to oleic acid and the final reaction temperature, the particle size
could be finely tuned from 2 nm to 3.9 nm. Detailed reaction
parameters are summarized in Table S2. The low-magnification
TEM images of as-prepared UMFNPs with average sizes of
2, 3, and 3.9 nm are shown in Figure 2a−c, demonstrating that
UMFNPs could be synthesized in a large scale. Corresponding
high-magnification TEM images (Figure 2d−f) demonstrate
that the UMFNPs were fairly uniform in size with a narrow
distribution (Figure S8a−c). The lattice fringes shown in the
high-resolution TEM images (Figure 2g−i) are an indication of
the high crystallinity of the UMFNPs. The observed lattice
spacings of 2.53, 2.45, and 3.01 Å are for 2, 3, and 3.9 nm sized
UMFNPs, correspond to the d spacings of the (311), (222), and
(220) lattice planes, respectively. Inductively coupled plasma−
atomic emission spectroscopy (ICP-AES) reveals that the
Mn/Fe ratios of all UMFNPs are about 0.5, which is consistent
with their stoichiometric ratios. The powder X-ray diffraction
(XRD) patterns of as-prepared UMFNPs (Figure 3a) were in
good accord with the standard spinel MnFe2O4 powder diffrac-
tion data (JCPDS card no. 10-0319), and no other secondary
phases such as manganese oxide or ferrous oxide could be traced.
The broadening of diffraction peaks with decreased particle size
is due to the small crystallite size.35 The mean sizes estimated on
the basis of the strongest (311) peaks using the Debye−Scherrer
equation were 2.07, 2.98, and 4.29 nm, which are basically
consistent with the observations under TEM imaging. Figure 3b
and c show the Fe 2p and Mn 2p X-ray photoelectron
spectroscopy (XPS) of the UMFNPs. The core level binding
energies at 711.6 and 724.7 eV for 3.9 nm UMFNPs, 712.7 and
725.8 eV for 3.0 nm UMFNPs, and 712.5 and 726.1 eV for
2.0 nm UMFNPs are attributed to the characteristic doublets of
Fe 2p3/2 and Fe 2p1/2 of Fe3+, respectively. The binding energies
of Mn 2p3/2 and Mn 2p1/2 were 642.4 and 653.7 eV for 3.9 nm
UMFNPs, 642.9 and 654.7 eV for 3.0 nm UMFNPs, and
642.9 and 654.7 eV for 2.0 nm UMFNPs, respectively. All the
peaks from the Fe 2p and Mn 2p spectra are basically in
good agreement with previously reported values for Fe3+ and
Mn2+.36−38 The slight shifts toward high energy can be found for
2 and 3 nm UMFNPs with respect to that for 3.9 nm UMFNPs,
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Figure 2. TEM and high-resolution TEM images of UMFNPs: (a, d, g) 2 nm UMFNPs, (b, e, h) 3 nm UMFNPs, and (c, f, i) 3.9 nm UMFNPs.
which could possibly be due to a significantly increased surface
state in small-sized particles.39
We posited that the DSTD process is essential for the synthesis
of highly monodisperse UMFNPs, although the exact mecha-
nism remains to be elucidated. Hence, we first investigated the
dynamic thermal decomposition behaviors of the coordination
compound precursors by using temperature-programmed FTIR
in the presence of oleyl alcohol. Figure 4 shows the FTIR spectra
of Fe-eruciate, manganese-oleate, and iron-oleate complexes at
various temperatures, respectively. The characteristic bands
for the iron-eruciate complex appeared at 1590, 1531, and
1445 cm−1 (also found in Figure S4a). The bands at 1590 and
1531 cm−1 arose from the asymmetric vibration of the carbo-
xylate group, while the band at 1445 cm−1 is attributed to the
carboxylate symmetrical stretching vibration.40 Mn-oleate and
Fe-oleate complexes showed three similar vibration bands. While
the bands at 1611 and 1553 cm−1 (Mn-oleate) and 1595 and
1526 cm−1 (Fe-oleate) are assigned to the asymmetrical vibra-
tion of carboxylate, the bands at 1425 cm−1 (Mn-oleate) and
1443 cm−1 (Fe-oleate) are attributed to the symmetrical vibra-
tion of carboxylate.40 Noticeably, the intensity of asymmetrical
vibration of carboxylate for all complexes decreased with an
elevated temperature. This vibration band for the Fe-oleate
complex finally disappeared at a temperature of about 190 °C
(Figure 4c), but remained visible for both Fe-eruciate and
Mn-oleate complexes until the temperature was higher than
220 °C (Figure 4d). The vanishing of the band is a characteristic
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of the breaking of bonds in the carboxylate group and dis-
sociation of these complexes. The approximate dissociation
temperature for Fe-eruciate and Mn-oleate complexes suggests
that the formation of UMFNPs could undergo a DSTD process,
while the DNSTD process may take place when co-thermal
decomposition of Fe-oleate and Mn-oleate complexes occurs due
to the large difference (>20 °C) in the on-site dissociation
temperature.
A comparison study of the DSTD and DNSTD process was
thus performed to further understand the dynamic nature in
the nucleation and growth of the UMFNPs. The DSTD and
DNSTD processes were established using Fe-eruciate/
Mn-oleate complexes and Fe-oleate/Mn-oleate complexes as
the precursors, respectively. The initial Mn/Fe ratio of the mixed
precursors is 0.5, and the reaction parameters are the same
as those for the synthesis of 3 nm UMFNPs. The reaction
temperature rising profile for DSTD and DNSTD is shown in
Figure 5a. The final reaction temperature in this study was
265 °C, and it took about 48 min to reach this temperature.
The temporal evolution of particle size, size distribution, and
composition ratio for DSTD and DNSTD growth is presented in
Figure 5b−d. The representative TEM images of the products
obtained at 35, 39, 48, and 78 min during the DSTD and
DNSTD process are shown in Figure 5e−l. For a better under-
standing, the whole nanoparticle growth process has been
divided into three regimes marked as dashed lines. Regime I
starts from the beginning of the heat-up process and ends at a
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Figure 3. (a) XRD patterns of the different sized UMFNPs. (b) Fe 2p and (c) Mn 2p XPS spectra of the UMFNPs.

Figure 4. Temperature-programmed Fourier transform infrared spectroscopy (FTIR) of (a) iron-eruciate complex, (b) manganese-oleate
complex, and (c) iron-oleate complex in the presence of oleyl alcohol. The asymmetric vibration of the carboxylate group is marked by red
arrows. (d) Plot of the temperature dependence of the peak intensity of the asymmetric vibration of the carboxylate group in the FTIR spectra.

reaction time of 39 min, at which the reaction temperature
reaches the dissociation temperature of Fe-eruciate/Mn-oleate
complex precursors (220 °C). Regime 2 covers the time slot from
39 to 78 min, during which the reaction is terminated for real
UMFNP growth by rapid cooling. Regime 3 is the additional
aging process from 78 to 168 min.
As shown in Figure 5b, for the DSTD process, no nanoparticle
was formed in regime I (Figure 5e), as the reaction temperature
was below the dissociation temperature of Fe-eruciate/
Mn-oleate complexes. In regime II, the particle size was observed
to rapidly increase from 1.68 nm (39 min, Figure 5f) to 2.58 nm
(Figure 5g), and then the particle size slowly increased to 3 nm
from 48 to 78 min. The slow increase in particle size can still be
3618
observed in regime III. A similar trend in particle size variation
has been observed for the DNSTD process except that the
nanoparticle was formed in regime I (Figure 5i), which is earlier
than the DSTD process. The particle sizes in the DNSTD
process were always much larger than that in the DSTD process
at a designated reaction time. The size distribution for the DSTD
process was “focused” from 17.31% to 15.68% and then
maintained throughout regime II (Figure 5c), before gradually
increasing up to 30% in regime III. In contrast, the size distri-
bution observed in the DNSTD process started out with a
standard deviation of 30.49%, which is much larger than that
observed in the DSTD process. There was an initial slow decrease
from 30.49% to 28.53%, before a sudden jump to 35.36% at
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Figure 5. Comparison study of DSTD and DNSTD synthesis of manganese ferrite nanoparticles. (a) Temperature profile, (b) particle size,
(c) standard deviation, and (d) Mn/Fe ratio evolution in the DSTD and DNSTD processes. TEM images of the representative products taken at
reaction times of (e and i) 35 min, (f and j) 39 min, (g and k) 48 min, and (h and l) 78 min during DSTD and DNSTD processes.

around 39 min in regime I (Figure 5c). The size distribution in
the DNSTD process was “refocused” to 29.14% in regime II and
then gradually increased to 38.78% in regime III. In comparison
with nearly spherical nanoparticles obtained in the DSTD
process, the nanoparticles generated by the DNSTD process
were nonuniform and irregularly shaped with a relatively large
size distribution, as shown in Figure 5i−l. The variation of
Mn/Fe ratio in the nanoparticles during the growth was also
determined analytically for each aliquot using ICP-AES as shown
in Figure 5d. As for the DSTD process, the Mn/Fe ratios of the
3619
initial products obtained at 39 min were higher than 0.42,
indicating the high Mn/Fe ratio in the nucleus. This ratio rapidly
decreased from 0.42 to 0.16 in regime II and increased slightly in
regime III. But for the DNSTD process, the Mn/Fe ratios of the
initial products were quite low (0.06), suggesting that the nucleus
contained mainly iron oxide. It then escalated to 0.19 at 39 min of
regime I, followed by a rapid decrease to 0.11, and was main-
tained over the entire regime II. Similar to the DSTD process, a
slight increase in the Mn/Fe ratio during the aging process was
observed.
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On the basis of the above experimental results, it is clear that
the metal ferrite nanoparticles synthesized through DSTD and
DNSTD will undergo different nucleation and growth kinetics,
where DSTD is crucial for the synthesis of high-quality
UMFNPs. As illustrated in Figure 5, after rapid nucleation in
the DSTD process, the fast growth of the nanoparticles and the
narrowing of their size distribution occurred simultaneously.
This behavior is essentially the same as that previously observed
in the thermal decomposition synthesis of Fe3O4 binary
ferrites using single Fe-oleate precursors,19,23 which suggests
the effective separation of nucleation and growth in the DSTD
process. Further evidence can be found in the evolution of the
composition ratio. The high Mn/Fe ratio in the nucleus indicates
that the dual-metal mixed monomer was formed before the
nucleation. Moreover, the subsequent decrease of the Mn/Fe
ratio in the growth stage could be ascribed to the faster
decomposition rate of the Fe-eruciate complex than the
Mn-oleate complex (Figure S5d). Benefiting from both the
effective separation of nucleation and growth and the diffusion-
limited growth, highly monodisperse UMFNPs could be
synthesized via the DSTD process by lowering the reaction
temperature and shortening the growth time simultaneously.
As for the DNSTD process, the extremely low initial Mn/Fe ratio
implies that the nucleation of the iron oxide host occurred first.
The sudden increase in both particle size and size distribution at
39 min (220 °C) accompanied by a rapidly increased Mn/Fe
ratio suggests explicitly that the growth-doping process was
induced by the subsequent decomposition of the Mn-oleate
complex. This late decomposition leads to narrowing of the size
distribution with an inevitable increase in particle size to some
degree, similar to that of “size refocusing” observed in the second
Figure 6. TEM images of (a) ultrasmall nickel ferrite nanoparticles and (b) ultrasmall cobalt ferrite nanoparticles prepared by the DSTD process
with the XRD pattern of (c) nickel ferrite nanoparticles and (d) cobalt ferrite nanoparticles.
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hot-injection process.41 Unfortunately, the multistep sequential
decomposition in this case can prevent effective separation of
nucleation and growth, as evidenced by the irregular shape and
broad size distribution of the obtained nanoparticles. Although
such defects may be largely eliminated in the growth of large
nanoparticles by elaborately designed synthetic conditions such
as reducing the temperature rising rate and elevating the final
reaction temperature,42 our results clearly show its limitation in
the synthesis of highly monodisperse UMFNPs. In addition, the
variation in particle size and size distribution during the aging
process is an indication that nanoparticle growth in both DSTD
and DNSTD undergoes a classic Ostwald ripening process.43
The elongated reaction time will not affect the Mn/Fe ratio
significantly, as the slight increase possibly originated from the
uncompleted decomposition of the Mn-oleate complex. It would
appear that the findings obtained from the comparison study
indicate that the control of particle size and size distribution of
UMFNPs in DSTD follow the same rules constructed for binary
iron oxide nanoparticles; however, due to the different decom-
position kinetics of the two precursors, the appropriate optimization
of their initial ratio is necessary for the synthesis of stoichiometric
UMFNPs.
To demonstrate its generality, the principle of DSTD has been
applied to synthesize other ultrasmall ferrite nanoparticles such
as NiFe2O4 and CoFe2O4. The Ni-oleate and Co-oleate
complexes were paired with an Fe-eruciate complex for DSTD
synthesis because of their approximate thermal decomposition
temperatures (Figure S7, Ni-oleate 324.7 °C and Co-oleate
340.9 °C). Figure 6a and b show the TEM images of the
as-prepared ultrasmall NiFe2O4 and CoFe2O4 nanoparticles.
As shown in Figure 6a and b, the particles sizes were 3.1 and 3.4 nm
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with quite small size distribution (Figure S9), respectively. 25K T

The XRD patterns of the as-prepared ultrasmall ferrite nano-
particles are shown in Figure 6c and d, which are in good
agreement with the standard cubic spinel NiFe2O4 and CoFe2O4
powder diffraction data (JCPDS card nos. 10-0325 and 22-1086).
These results indicate that highly monodisperse ultrasmall
NiFe2O4 and MnFe2O4 nanoparticles can be achieved by the
DSTD process. Moreover, the successful synthesis of other
ultrasmall ferrite nanoparticles not only suggests that the DSTD
is a relatively robust method for the synthesis of ultrasmall
ternary compound nanoparticles but also demonstrates the high
versatility of the newly designed Fe-eruciate complex for the
synthesis of various ultrasmall magnetic nanoparticles.
Magnetic Characterizations of the UMFNPs. The
magnetic properties of the UMFNPs were measured by using a
superconducting quantum interference device (SQUID) mag-
netometer. The hysteresis loops of the UMFNPs are shown in
Figure 7a. The saturation magnetization (Ms) for 2, 3, and 3.9 nm
sized particles is 18.59, 24.91, and 29.18 emu g−1, respectively.
The largely reduced magnetic moment of the UMFNPs com-
pared to the bulk counterpart (∼80 emu g−1) can be attributed to
the reduction in the volume magnetic anisotropy and the surface
spin canting induced by their extremely small particle size.31
The coercivity and remanence of the UMFNPs are negligible at
room temperature (Figure 7b), indicating their superparamag-
netic behavior. The temperature-dependent magnetization
curves after zero-field cooling and field cooling with an applied
magnetic field of 20 Oe for these nanoparticles are shown in
Figure 7c. The blocking temperatures (TB) of the 2, 3, and 3.9 nm
sized particles were measured to be 6, 12.5, and 15 K, respectively
(Figure 7c). The effective magnetic anisotropy constant (Keff)
was calculated by the equation Keff = VB B , where kB represents
the Boltzmann’s constant and V, the volume of a single nano-
crystal.19 As shown in Figure 7d, the Keff is found to increase with
the decreasing particle size, similar to early observations for
nanoscale ferrimagnetic particles.19,44 Since the significantly
increased surface to volume ratio and surface spin disorder,
surface anisotropy will play a leading role over volume mag-
netic anisotropy in the effective magnetic anisotropy in the
UMFNPs.31,45,46 The smaller the particle, the higher the Keff and
the larger the surface spin disorder. Because the T1 MR contrast
effect is the result of protons of water molecules interacting with
the electron spins of the contrast agent, the phenomenon that
occurs in UMFNPs is believed to significantly promote the T1
MR effect by increasing the surface atoms with unpaired
electrons and at the same time suppress the T2 MR effect by
decreasing the magnetic moment.
Surface Modification and Colloidal Stability of the
UMFNPs. The as-synthesized UMFNPs are hydrophobic and
hence do not dissolve in aqueous media. As such, it is essential to
carry out appropriate surface modification of the UMFNPs to
disperse them in an aqueous medium for MRI application.
Herein, phosphorylated mPEG is used for surface modification
by ligand exchange. As a result of the high surface energy of the
UMFNPs, aggregation or dissociation may occur during the
ligand exchange process in a harsh chemical environment.47
However, the ligand exchange with phosphorylated mPEG
was very successful in our situation. The obtained hydrophilic
UMFNPs show very good uniformity and unchanged particle
size, as observed in Figure 8d−f. The measured hydrodynamic
size was around 7.81 ± 1.72, 8.79 ± 1.06, and 12.50 ± 1.24 nm for
2, 3, and 3.9 nm UMFNPs in deionized (DI) water, respectively.

Figure 7. (a) Hysteresis loop of the UMFNPs with diameters of 2 nm (black ■), 3 nm (red ●), and 3.9 nm (blue ▲) at room temperature.
(b) Magnified hysteresis loops of 2 nm (black ■), 3 nm (red ●), and 3.9 nm (blue ▲) sized UMFNPs. (c) Temperature-dependent magnetization
curves for the manganese ferrite nanoparticles measured after zero-field cooling (ZFC) and field cooling (FC) with diameters of 2, 3, and 3.9 nm
under an applied magnetic field of 20 Oe. For the sake of presentation, the magnetization data were normalized with respect to the value at the
maximum of ZFC magnetization, M(TB), for individual samples. (d) Size dependence of the blocking temperature TB and the effective magnetic
anisotropy constant Keff of the UMFNPs obtained from the temperature-dependent magnetization curve.

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Figure 8. Hydrodynamic diameters of the phosphorylated mPEG-modified UMFNPs of (a) 2 nm, (b) 3 nm, and (c) 3.9 nm in aqueous solution.
The inset is a photograph of the aqueous UMFNP dispersions. TEM images showing modified UMFNPs of (d) 2 nm, (e) 3 nm, and (f) 3.9 nm.
Hydrodynamic size variation of the modified UMFNPs of (g) 2 nm, (h) 3 nm, and (i) 3.9 nm as a function of time upon incubation in water, 10%
FBS, normal saline, and 0.2, 0.4, and 1 M NaCl solution.

The insets of the photographs in Figure 8a−c show that there was
no observable aggregation in the water-dispersed UMFNPs.
Since high colloidal stability of the UMFNPs in various
conditions is essentially mandatory for biomedical applications,
the colloidal stability of the hydrophilic UMFNPs was further
examined by monitoring their hydrodynamic sizes in DI water,
normal saline, different concentrations of NaCl solution, and cell
culture medium containing 10% FBS. The time-dependent size
curves of the UMFNPs were plotted in Figure 8g−i. Preliminary
studies have shown that the UMFNPs exhibited excellent
colloidal stability in all media after a 12-day incubation at room
temperature, as there was no apparent change in size and
polydispersity index (PDI). The results also imply that these
hydrophilic UMFNPs can maintain the good colloidal stability
when they are used as MRI contrast agents under physiological
conditions.
MR Relaxation Properties of the UMFNPs. In order to
assess the MR T1 relaxivity of the UMFNPs, in vitro MRI
measurements were performed using a 3 T MRI scanner.
The samples were dispersed in deionized water with an [Fe+Mn]
concentration from 0.07 to 1 mM determined by ICP-AES
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measurements. Figure 9a shows the qualitative comparison of T1-
weighted MR images of the different sized UMFNPs with respect
to [Fe+Mn] concentration. The T1-weighted MR images of
all the samples tend to be brighter with increasing [Fe+Mn]
concentration, showing that the UMFNPs as positive contrast
agents can effectively reduce the spin−lattice relaxation time of
water protons. Furthermore, small-sized UMFNPs exhibited a
more significant reduction in T1 relaxation time, as shown by the
much brighter images at the designated [Fe+Mn] concentration.
In addition, as the release of Mn2+ from Mn-containing nano-
particles in acid conditions has been reported to brighten the
images in T1-weighted MRI,48,49 we further measured the MR
images of the UMFNPs dispersed in citrate buffer solution
with pH 5.0, which simulates the pH microenvironment of
the endosomes or lysosomes within the cells.49 As shown in
Figure S12, there is no obvious change in brightness after
dispersing the samples in citrate buffer for 1 h, suggesting that the
T1 enhancement effect in our case was due to the UMFNPs
rather than the free Mn2+ released from the particles. In fact, both
T1 and T2 signal intensity of the UMFNPs can maintain their
original values within 12 days in neutral conditions, but obvious
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proton relaxation entails the direct bonding of water molecules to

paramagnetic ions, while the outer-sphere relaxation is linked
with the dynamics and diffusion of nearby water molecules
beyond the directly bonding ones. T1 relaxivity is therefore the
sum of inner-sphere and outer-sphere contributions that can be
given by33,34

1 ⎛1⎞ ⎛1⎞
=⎜ ⎟ +⎜ ⎟
T1 ⎝ T1 ⎠inner sphere ⎝ T1 ⎠outer sphere (1)

⎛1⎞ 128π 2γI2M n ⎛ 1 ⎞3 2

⎜ ⎟ = ⎜ ⎟ Ms
⎝ T1 ⎠outer sphere 405ρ ⎝ 1 + L /a ⎠

τDJA ( 2ωIτD ) (2)

⎛1⎞ qPM
⎜ ⎟ =
⎝ T1 ⎠inner sphere T1M + τM
(3)
Figure 9. (a) T1-weighted phantom images of the 2, 3, and 3.9 nm
sized UMFNPs. (b) Plot of 1/T1 over [Fe+Mn] concentration of the where γI is the proton gyromagnetic ratio and Mn and ρ are the
UMFNPs of 2, 3, and 3.9 nm. The slope indicates the specific molar mass and density of the UMFNPs. Ms and a are the
relaxivity (r1). saturation magnetization and the radius of the UMFNPs,
respectively. D is the diffusion coefficient of water molecules, L
changes will be observed after 12 h in acidic conditions probably is the thickness of an impermeable surface coating, τD is the
due to the degradation of UMFNPs (Figures S15 and S16). The translational diffusion time (τD = r2/D), r is the effective radius
r1 relaxivity of the UMFNPs was extracted from the measured T1 of the particles (r = a + L), and JA represents Ayant’s s
data and plotted in Figure 9b. The measured r1 relaxivities were pectral density function, which is related to Z = 2ωIτD . PM is
8.43, 8.23, and 6.98 mM−1 s−1 for the 2, 3, and 3.9 nm UMFNPs, the mole fraction of surface metal ions, q is the number of
respectively. The MR relaxivity comparison of the UMFNPs, water molecules bound per metal ion (i.e., the hydration
3 nm γ-Fe2O3 nanoparticles, 6 nm MnFe2O4 nanoparticles, 7 nm number), and τM is the residence lifetime of the bound water.
MnO nanoparticles, and clinical contrast agent Omniscan is The “m” subscript refers to the shift or relaxation rate of the
presented in Table 1. The highest r1 values of the UMFNPs are solvent molecule in the inner-sphere. T1M is the relaxation time
of the bound water.
Table 1. Comparison of Relaxivity of UMFNPs, 3 nm γ-Fe2O3, In terms of the boundary condition associated with a high
6 nm MnFe2O4, 7 nm MnO, and Clinical Omniscan at 3 T magnetic field, the outer-sphere mechanism is usually assumed to
dominate proton relaxation for superparamagnetic nanoparticle
Ms at 300 K (emu r1 (mM−1 r2 (mM−1 based contrast agent systems, while the contribution from the
sample g−1) s−1) s−1) r2/r1
inner-sphere mechanism is omitted. In this case, r1 is propor-
2 nm UMFNPs 18.59 8.43 21.02 2.49
tional to the square of Ms and τDJA ( 2ωIτD ) according to eq 2,
3 nm UMFNPs 24.91 8.23 21.97 2.67
3.9 nm UMFNPs 29.18 6.98 27.08 3.88 where τDJA ( 2ωIτD ) monotonically increases when the particle
3 nm γ-Fe2O3 31 4.96 28.59 5.76 size is less than 4 nm (Table S4). Therefore, the UMFNPs with
6 nm MnFe2O4 50 1.39 64.08 46.10 larger particle size and higher Ms should have shown a larger r1
7 nm MnO 1.57 7.82 4.98 theoretically. But quite to the contrary, as shown in Table 1, the
Omniscan 4.04 4.19 1.04 larger sized UMFNPs gave rise to smaller r1 values, which
strongly suggests that the outer-sphere mechanism alone cannot
double those of 3 nm γ-Fe2O3 and commercial Omniscan, 5.4 explain our experimental observations because some important
times higher than that of 7 nm MnO nanoparticles, and 6 times contributions are missing. More evidence can be found in the r1
higher than that of the large-sized MnFe2O4 (6 nm), for which comparison of the same sized UMFNPs and γ-Fe2O3. Both of
the T2 MR effect is dominant. Notably, this r1 value is the highest them have the same τDJA ( 2ωIτD ) and the Ms of the γ-Fe2O3
measured among the ferrite nanoparticles with similar size nanoparticles is even slight higher than that of the UMFNPs;
reported so far, where the r2/r1 ratio is the smallest one as shown however, the UMFNPs exhibit a higher r1 value than γ-Fe2O3
in Table S3. As compared to γ-Fe2O3 nanoparticles with the same nanoparticles. In fact, the UMFNPs can be treated as a core/shell
size and surface modification, it can be easily deduced that the structure comprising a ferromagnetic core and paramagnetic
large r1 value of the UMFNPs is attributed to the doping of shell (spin disorder layer), where the thickness of the spin dis-
Mn2+ ions. Nevertheless, the mechanism of T1 enhancement by order layer for manganese iron nanoparticles is about 0.95 nm.50
molecular engineering of ultrasmall ferrite nanoparticles remains With the dramatically decreasing size, the paramagnetic to
unclear. ferromagnetic composition ratio in the UMFNPs is significantly
According to theoretical considerations, the relaxation enhance- increased.31 Therefore, they can be conceived to some extent as
ment of a contrast agent is known generally to follow the “pseudo-macromolecules”, in which the inner-sphere contribu-
inner-sphere and outer-sphere mechanisms.34 The inner-sphere tion cannot be ignored.
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Along this line of thought, the enhanced r1 with the decreasing
size in the UMFNPs could be due to the increased inner-sphere
contributions originating from the increased amount of para-
magnetic Fe3+ and Mn2+ ions on the particle surface. The high r1
value of the UMFNPs compared to γ-Fe2O3 nanoparticles can
also be explained in terms of the inner-sphere mechanism. As
described by eq 3, the r1 value is determined by qPM, T1M, and τM.
The qPM is comparable for the UMFNPs and γ-Fe2O3 because of
their same size. The T1M (Supporting Information part 5) is also
the same as that of the Mn2+ and Fe3+, which have equal
total electron spins.51 However, the τM for Mn(H2O)62+ was
4.3 × 104 ns, which is 2 orders of magnitude smaller than that of
Fe(H2O)63+ (5.3 × 106 ns),34 suggesting that UMFNPs rather
than γ-Fe2O3 will have a superior r1 relaxivity. Clearly, the
artificial Mn2+ doping in ultrasmall iron oxide nanoparticles could
effectively expedite spin−lattice relaxation and increase inner-
sphere contribution by altering the residence lifetime of the
bound water (τM). This is distinctly different from the early
doping engineering of magnetic nanoparticles for ultrasensitive
T2 MR contrast agents, where high Ms and large r2 relaxivity are
pursued.2,7 It is to be noted that the outer-sphere mechanism
may still be the dominant contribution to transverse spin−echo
relaxation of the UMFNPs, not only because the r2 value in the
UMFNPs increases with the increasing particle size and Ms but
also because their r2/r1 ratio monotonically increases against τD,
which are well in agreement with the prediction based on the
outer-sphere mechanism. Our results indicate that both outer-
sphere and inner-sphere proton relaxation contributions are
responsible for the experimental relaxation enhancement in the
UMFNP system. More significantly, the results also reveal that
the strategy of metallic doping in ultrasmall iron oxide
nanoparticles could modulate r1 and r2 relaxivity independently
on some level by adjusting outer-sphere and inner-sphere contri-
butions separately, which provides the possibility to develop the
innovative nanoparticle contrast agent with desirable r1 and r2
relaxivity for specific MRI diagnostics.
In Vivo Contrast-Enhanced MRI. In vivo MRI was carried
out for Sprague−Dawley (SD) rats in the clinical 3.0 T Siemens
Trio MRI scanner using the 3d-flash and a turbo spin echo (TSE)
sequence for imaging the blood vessels and liver, respectively.
Due to the good colloid stability and the high r1 relaxitivity, the 3 nm
UMFNPs were employed as a model system for contrast-enhanced
Figure 10. In vivo UMFNP-enhanced MR images obtained using the 3d-FLASH sequence.
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MRI for angiography and liver imaging with a dosage of 2.5 mg
[Fe+Mn]/kg body weight (0.045 mmol [Fe+Mn]/kg body
weight). The clinical MR angiography contrast agent Omniscan
(gadodiamide) and liver-specific contrast agent Primovist
(Gd-EOB-DTPA) were set as controls for imaging blood vessels
and the liver with dosages of 0.2 and 0.025 mmol [Gd]/kg body
weight, respectively. All the contrast agents were intravenously
administrated.
Figure 10 shows the T1-weighted MR images acquired at
preinjection and postinjection (20 min) of the UMFNPs and
Omniscan. Quite notably, the blood vessels and liver were
brightened in the T1-weighted MR images after the injection of
the UMFNPs, demonstrating that the UMFNPs can effectively
shorten the spin−lattice relaxation time of water protons in the
circulating system. The prominent vascular details including the
external jugular vein, axillary vein, internal jugular vein, sub-
clavian vein, superior vena cava, and aortic arch can be distinctly
distinguished, which indicates that the resolution of the image is
very high, such that even the 0.47 mm veins were able to be
accurately imaged. In contrast, for Omniscan-enhanced images
with a dosage 4.4-fold higher than the UMFNPs, the essential
vascular details were ambiguous. Figure 11a shows UMFNPs-
enhanced MR images acquired at 0, 0.5, 1, 5, 10, 20, 30, 60,
120, and 180 min, respectively. The intense signal of the blood
vessels can be maintained for 3 h on contrast-enhanced magnetic
resonance angiography, while the Omniscan-enhanced images
showed weak contrast in vascular nets and the vascular nets can
be depicted only within 20 min (Figure S17). Such long-term
blood pool imaging achieved by UMFNPs is ascribed to their
optimal particle size, which is neither very large to prevent the
uptake by the reticuloendothelial system52 nor so small as to be
rapidly excreted through the kidney.53 Moreover, because the
[Fe+Mn] content of the aorta is almost the same as that of
the blank aorta (Figure S19), there is no noticeable uptake by
endothelium cells in the blood vessels. As such, it is rather the
relatively long-term blood circulation of the UMFNPs than
endothelium cell uptake that is responsible for the increased
signal duration. The contrast-enhancement ratios of MR signal
intensity ΔSI (ΔSI = |SIpost − SIpre|/SIpre) for the UMFNPs in the
region of interest of the subclavian vein were extracted and
compared with that of Omniscan. As shown in Figure 11b, the
ΔSIUMFNPs was always much larger than ΔSIomniscan. At 30 s, the
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Figure 11. (a) Representative UMFNP-enhanced MR angiography images using the 3d-flash MR sequence acquired at 0, 0.5, 1, 5, 10, 20, 30, 60,
120, and 180 min after intravenous administration of 3 nm UMFNPs. (b) Enhancement ratio of the MR signal ΔSI of the subclavian vein after
being contrast-enhanced by UMFNPs and Omniscan.
ΔSIUMFNPs reached its maximum of 426%, which was 2.6-fold
higher than ΔSIomniscan, before decreasing to 178% at 180 min,
while ΔSIomniscan was dramatically down to zero. The high
contrast-enhancement ratio for the UMFNPs indicates that the
UMFNPs have potential to be a superior T1 contrast agent
for MR angiography, which is clinically important to detect
myocardial infarction, kidney failure, atherosclerotic plaques,
thrombosis, and tumoral angiogenesis.54−60
In addition to high-resolution MR angiography, the high
contrast-enhancement in the liver by the UMFNPs has also
attracted much interest. To further investigate its ability for liver-
specific MRI, UMFNP-enhanced MR images of the liver were
acquired using the TSE sequence, where the clinical Primovist
was used as the control. Figure 12a shows the transverse-plane MR
images of liver acquired at 0, 10, 20, 30, 45, 60, 120, and 180 min
Figure 12. (a) T1-weighted MR images of transverse planes of the
liver using the TSE sequence acquired at 0, 10, 20, 30, 45, 60, 120,
and 180 min after intravenous administration of 3 nm UMFNPs.
(b) Enhancement ratio of MR signal ΔSI of the liver after contrast-
enhanced by UMFNPs and Primovist.
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after the injection of UMFNPs. The significantly brightened MR
image suggests the effective accumulation of UMFNPs in the
liver. The contrast-enhancement ratio ΔSI of liver images by the
UMFNPs and Primovist are shown in Figure 12b. The highest
ΔSIUMFNPs of 70.3% appeared at 10 min, which was almost
3 times higher than that of 3.3 nm Fe3O4.61 Over the entire
imaging time window, the ΔSIUMFNPs was always higher than
ΔSIPrimovist. Since the Mn2+ ion can be selectively taken up by
hepatocytes via the vitamin B6 transporter,62 it is reasonable to
assume that the UMFNP is a hepatocyte-specific contrast agent.
This assumption is further confirmed by the in vivo UMFNP
distribution in liver tissues, where many UMFNPs were found in
the hepatocytes (Figure S20). In contrast, Resovist is localized
mostly in Kupffer cells. Although the exact mechanism still needs
further investigation, we believe that the presence of a large
number of Mn2+ ions on the surface of the UMFNPs plays an
important role in hepatocyte-specific uptake. All results obtained
from the in vivo MRI verify that the UMFNPs are a highly
sensitive and multifunctional T1 MR contrast agent for high-
resolution imaging of the blood pool and liver.
Pharmacokinetics, Biodistribution, and Excretion of
the UMFNPs. To investigate the in vivo behavior of the
UMFNPs, the pharmacokinetics, biodistribution, and excretion
of the 3 nm UMFNPs were then carried out with the same dosage
(2.5 mg [Fe+Mn]/kg body) used for contrast-enhanced MRI.
Inductively coupled plasma−mass spectrometry (ICP-MS) was
used to quantify the related metal elements in the rats’ blood and
tissue at designed time points after a single tail vein injection.
Figure 13a shows the normalized [Fe+Mn] concentration−time
curve in blood for the UMFNPs after a single intravenous (i.v.)
injection. This curve shows a single-exponential disposition;
therefore, the pharmacokinetic parameter was determined with a
one-compartment pharmacokinetic model63 and the blood
elimination half-life (t1/2) is about 0.31 h. Figure 13b shows
the in vivo biodistribution of the UMFNPs at 24 h after i.v.
injection. The results revealed that the UMFNPs mainly
accumulated in the liver, spleen, and kidney. A similar bio-
distribution has also been found in 2 and 3.9 nm UMFNPs
(Figure S21). The [Fe+Mn] concentration in both feces and
urine of SD rats was also measured to examine the excretion
pathway of the UMFNPs. Quantitative analysis showed that
more than 27.04% of the UMFNPs was excreted from the body
via the renal clearance pathway within 24 h and up to 30.33%
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Figure 13. Pharmacokinetics, biodistribution, and excretion of 3 nm UMFNPs in rats. (a) Time−[Fe+Mn] concentration curves of blood and
(b) biodistribution pattern at 24 h after i.v. injection of the UMFNPs. Percentage of (c) renal and (d) hepatobiliary excreted [Fe+Mn] versus time
after i.v. injection of the UMFNPs. Data are reported as mean ± standard deviation (n = 3).
within 60 h (Figure 13c), indicating that the UMFNPs could be
efficiently renally cleared from the body. About 22.42% of
UMFNPs were excreted via the hepatobiliary pathway within
24 h and 67.40% of the UMFNPs were excreted within 60 h
(Figure 13d). This efficient renal clearance of the UMFNPs is
ascribed to their small hydrodynamic size (8.79 nm, slightly
larger than the filtration-size threshold of glomerular capillary
walls) and neutral hydrophilic mPEG coating.63,64 The rapid
excretion of the UMFNPs within 60 h is of value to minimize
systematic toxicity. In addition, the histological assessment of the
excised heart, liver, lung, kidney, spleen, and brain was also
performed to evaluate the acute systemic toxicity and the
subacute systemic toxicity after intravenous exposure to the
UMFNPs (Figures S22 and S23). According to the standard of
biological evaluation of medical device testing for systemic
toxicity (ISO 10993-11:2006), the acute systemic toxicity and
the subacute systemic toxicity were tested at 24 h and 14 d,
respectively. As shown in Figures S22 and S23, there were no
obvious pathological changes in these organs, indicating the safe
use of the UMFNPs for potential clinical trials.
In Vitro Cytotoxicity Assay. Since cytotoxicity is an
important issue for the clinical application of nanoparticle-
based contrast agents, an in vitro cell cytotoxicity assay was thus
carried out to assess the toxicity profiles of the UMFNPs using
standard cytotoxicity tests. Figure 14 shows the viability of Chang
liver cells, HepG2 liver cancer cells, and endothelium cells after
24 h of incubation with the UMFNPs at 37 °C. All UMFNP
samples show insignificant toxicity with a cell viability of more
than 80% at an Fe concentration of less than 100 μg/mL for
Chang liver cells and endothelium cells. According to the relative
growth rate and toxicity grade conversion,65 the toxicity of the
UMFNPs is classified as grade 1, which is safe for the Chang liver
cells and endothelium cells. For HepG2 cells, a slight decrease in cell
viability was observed for smaller UMFNPs (2 and 3 nm samples)
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at a high Fe concentration (>50 μg/mL). At a low Fe concen-
tration, there was no obvious change in the viability of the
HepG2 cells in comparison with that of Chang liver cells and
endothelium cells. We further investigated the cell localization of
the UMFNPs, which is critical for their cytotoxicity. The uptake
of the UMFNPs by Chang liver cells was visualized by TEM and
is shown in Figure 15. According to the TEM observations, the
small UMFNPs (the 2 and 3 nm samples) were observed to
localize in the cytosol, cytoplasmic vesicles, and the nucleus
(Figure 15a−d), while the large UMFNPs (the 3.9 nm samples)
were mainly distributed in cytoplasmic vesicles and the cytosol
(Figure 15e and f). It is not surprising that such small nano-
particles can enter the nucleus, as their hydrodynamic sizes
(7.81 and 8.79 nm) are smaller than the nuclear pore size
exclusion limit of ∼9 nm.66 A similar phenomenon has been
observed for ultrasmall Au nanoparticles.67,68 We speculate that
the increased toxicity of the small UMFNPs may be associated
with their nuclear internalization; hence, their cytotoxicity may
be largely reduced by loading them onto a relatively large and
biocompatible carrier for practical applications in the future.
CONCLUSIONS
In summary, we have developed a general DSTD strategy for
synthesis of ultrasmall metal ferrite nanoparticles with a well-
controlled size of <4 nm using UMFNPs as a model system.
The new iron-eruciate complex was paired with a metal-oleate
complex as the precursors to attain DSTD synthesis. The com-
parison study revealed that the DSTD facilitated a nucleation-
doping process, which is crucial for particle size and distribu-
tion control of ultrasmall ferrite magnetic nanoparticles, while
the DNSTD in the same conditions prevented effective nuclea-
tion and growth separation, resulting in nonuniform nanoparticles.
The principle of Fe-eruciate precursor promoted DSTD has
been further confirmed by synthesizing NiFe2O4 and CoFe2O4
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Figure 14. Cell viability of 2, 3, and 3.9 nm sized UMFNPs co-incubated with Chang liver cells (a−c), HepG2 liver cancer cells (d−f), and
endothelium cells (g−i) using standard MTS colorimetric assays. The one-way ANOVA with post hoc test method was used for analysis. Error
bars = standard error of the mean; *p < 0.05; **p < 0.01; ***p < 0.001.
nanoparticles with sizes of ∼3 nm. In addition, the UMFNP as a
good ultrasmall magnetic ferrite nanocrystalline model system gives
a clear relationship between chemical composition and T1 MR
signal enhancement effect. The artificially engineered UMFNPs
exhibited the highest r1 relaxivity (up to 8.43 mM−1 s−1) among the
ferrite nanoparticles with similar sizes reported so far and demon-
strated a multifunctional T1 MR nanoprobe for in vivo high-
resolution blood pool and liver-specific MRI simultaneously.
Moreover, in vivo biodistribution and excretion of the UMFNPs
reveal that UMFNPs are quite safe for a potential clinical contrast
agent. Altogether, our study provides a universal DSTD synthetic
approach for controllable synthesis of ultrasmall metal ferrite
nanocrystals, which may promote the chemical design of highly
sensitive and multfunctional nanoparticle-based T1 MR contrast
agents for various clinical diagnosis applications.
EXPERIMENTAL SECTION
Synthesis of Iron-Eruciate and Manganese-Oleate Com-
plexes. The iron-eruciate complex was formed by the reaction of
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iron chlorides, erucic acid, and sodium hydroxide in a methanol solution.
Briefly, 2.7 g of iron chloride (FeCl3·6H2O, 10 mmol, 99%) and 10.2 g of
erucic acid (30 mmol, Aladdin, 80%) were dissolved in 50 mL of
methanol. Subsequently, 1.2 g of sodium hydroxide (30 mmol, 96%)
dispersed in 100 mL of methanol was added slowly into this solution at
40 °C under magnetic stirring. After the reaction was completed, the
precipitate containing the iron-eruciate complex was washed three times
with deionized water and methanol. After washing, the as-obtained
iron-eruciate complex was dried in a vacuum at 45 °C for 12 h. The
manganese-oleate complex was prepared in a similar procedure, with
substitution of erucic acid by oleic acid.
Synthesis of the UMFNPs. The UMFNPs were prepared by
dynamic simultaneous thermal decomposition of the iron-eruciate
complex and manganese-oleate complex in the presence of oleic acid
and oleyl alcohol in benzyl ether or 1-octadecene. In a typical synthesis
of 3-nm-sized UMFNPs, 2.14 g of iron-eruciate complex (2 mmol),
0.62 g of manganese-oleate complex (1 mmol), 0.57 g of oleic acid
(2 mmol), and 1.61 g of oleyl alcohol (6 mmol) were dissolved in 10 g of
benzyl ether at room temperature. Then, the reaction mixture was
heated to 265 °C at a rate of 5 °C/min and maintained for 30 min under
a constant argon flow. Subsequently, cooling of the reaction mixture to
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Figure 15. TEM images of Chang liver cells incubated with the UMFNPs. Panels (a)−(d) show that the 2 and 3 nm UMFNPs are stored in the
vesicle, cytoplasm, and nucleus of the cell. Panels (e) and (f) show that the 3.9 nm UMFNPs are stored only in the vesicle and cytoplasm of
the cell.
room temperature was done quickly, and 20 mL of ethanol was added to
the solution. Separation of the precipitated nanoparticles was carried out
by centrifugation. Size tuning of the UMFNPs from 2 nm to 3.9 nm was
attained by varying the ratio of oleic acid and oleyl alcohol and the final
reaction temperature in the same procedure.
Surface Modification of the UMFNPs with Phosphorylated
mPEG. First, phosphorylated mPEG was prepared by reaction of
poly(ethylene glycol) methyl ether (mPEG, Mw = 2000 Da) and
phosphoryl chloride (POCl3). In brief, 2 g of mPEG in 15 mL of
tetrahydrofuran (THF) was added dropwisely into a mixture containing
1 mL of POCl3 and 15 mL of tetrahydrofuran in an ice-bath. The
resulting solution was then stirred for 4 h. After that, the products were
precipitated and washed by diethyl ether three times. The as-prepared
phosphorylated mPEG was then dried in vacuo at room temperature.
Ligand exchange was carried out using phosphorylated mPEG.
In a typical experiment, the UMFNPs (5 mg) were transferred into
chloroform (20 mL), followed by the addition of phosphorylated mPEG
(50 mg; Mw = 2000 Da). The mixture was then shaken for 2 h under
ambient conditions. After the reaction was complete, the solvent was
evaporated under a flow of argon and then dried under vacuum for
2 days to obtain a completely dried nanoparticle powder. The dried
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nanoparticles were redispersed into water (5 mL), and the solution was
centrifuged at 3000 rpm for 10 min. The supernatant was collected via
filtration through a 0.22 μm syringe filter and stored for future use.
MR Relaxation Properties Measurements. In vitro MR relaxivity
measurements were then carried out using a 3 T MRI scanner (Siemens
Medical Solutions, Erlangen, Germany) with a head coil. A turbo spin
echo sequence was utilized for measurement of T1 and T2 relaxation
time with concentrations from 0.13 to 1 mM [Fe+Mn] of the UMFNPs
dispersed in deionized water. The T1 measurement parameters were as
follows: TR = 4000 ms, TE = 19 ms, and TI = 25−3500 ms. The T2
parameters were TR = 5000 ms and TE = 13−320 ms.
In Vivo MR Imaging. Female SD rats (with a body weight of about
150 g) were purchased from the animal experiment center of the
Medical College, Sun Yat-sen University, China, and maintained in a
specific pathogen-free environment. 3d-FLASH and TSE images of rats
were acquired using a surface coil (CG-RBC18-H300-AS, Shanghai
Chenguang Medical Technologies Co., Ltd.) on a 3.0 T clinical
Siemens Trio MRI scanner before and after intravenous injection of the
UMFNPs (2.5 mg [Fe+Mn]/kg). T1-weighted images of the rat blood
vessels were acquired at 0.5, 1, 5, 10, 20, 30, 60, 120, and 180 min after
administration of UMFNPs. The imaging parameters of 3d-FLASH
DOI: 10.1021/acsnano.6b07684
ACS Nano 2017, 11, 3614−3631
ACS Nano
sequences are as follows: flip angle = 10, ETL = 1, TR = 4.28 ms, TE =
1.66 ms, field of view (FOV) = 83 × 120 mm2, slice thickness/gap =
2.0 mm/0 mm, and NEX = 2. T1-weighted images of the liver were
acquired at 10, 20, 30, 45, 60, 120, and 180 min after intravenous
administration of UMFNPs. The imaging parameters of TSE sequences
are as follows: flip angle = 120, ETL = 3, TR = 485 ms, TE = 10 ms,
FOV = 40 × 80 mm2, slice thickness/gap = 3.0 mm/0.15 mm, and
NEX = 12. The animal experiments were conducted according to
guidelines that were approved by the Institutional Animal Care and Use
Committee of the Sun Yat-sen University.
Pharmacokinetics, Biodistribution, and the Excretion of the
UMFNPs. The SD rats (with a body weight of about 150−180 g) were
used to study the pharmacokinetics, biodistribution, and the excretion of
the UMFNPs. The rats were purchased from the animal experiment center
of the Medical College, Sun Yat-sen University, China, and maintained in a
specific pathogen-free environment. The animal experiments were
conducted according to guidelines that were approved by the Institutional
Animal Care and Use Committee of the Sun Yat-sen University.
Pharmacokinetics. For the pharmacokinetic study, the SD rats
were injected intravenously with 3 nm UMFNPs (2.5 mg [Fe+Mn]/kg
body weight), and the blood samples were collected from the tail vein at
predetermined time points. The blood pharmacokinetic parameters
of the UMFNPs were analyzed with an ICP mass spectrometer
(PerkinElmer SCIEX, ELAN DRC-e).
Biodistribution. The rats were injected intravenously with 2, 3, and
3.9 nm UMFNPs at the same dose of 2.5 mg [Fe+Mn]/kg body weight and
killed 24 h after injection. Heart, liver, lung, kidney, spleen, and brain were
removed and weighed. Elemental Fe and Mn in the UMFNPs-exposed
groups, respectively, were separately quantified using ICP-MS (Perki-
nElmer SCIEX, ELAN DRC-e). Briefly, samples (about 100 mg of various
organs) were predigested overnight with 5.0 mL of concentrated nitric acid,
mixed with 1.0 mL of 30% hydrogen peroxide, and then digested for 2 h in
open vessels on a hot plate at 150 °C. Finally, the remaining solution (about
0.5 mL) was cooled and diluted to 3.0 mL with 2% HNO3. Uptake of
UMFNPs in various organs was expressed as μg/g tissue weight.
Excretion of the UMFNPs. For excretion of the UMFNPs, the SD
rats were injected with 3 nm UMFNPs (2.5 mg [Fe+Mn]/kg body
weight). The concentrations of Fe and Mn were measured using ICP-MS
(PerkinElmer SCIEX, ELAN DRC-e) at different times after i.v. injection
of 3 nm UMFNPs. Percentage of excreted [Fe+Mn] in feces and urine
was then calculated by subtracting the total injected quantity of [Fe+Mn].
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsnano.6b07684.
Figures of mass spectra, 1H NMR spectra, FTIR, and TGA
results for coordination compound precursors, detailed
experimental procedures, particle size distribution, and
EDX data for synthesis of ultrasmall ferrite nanoparticles,
zeta potential for hydrophilic UMFNPs, measurements of
MRI relaxation parameters for 2, 3, and 3.9 nm UMFNPs,
3 nm γ-Fe2O3, and 6 nm MnFe2O4, calculations of MRI
relaxation parameters for 2, 3, and 3.9 nm UMFNPs,
in vivo MR images enhanced by Omniscan and Primovist,
biodistribution pattern of 2, 3, and 3.9 nm UMFNPs,
in vivo acute systemic toxicity and subacute systemic
toxicity test, detailed experimental procedures of in vitro
cytotoxicity assay and intracellular localization for 2, 3, and
3.9 nm UMFNPs (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail (H. M. Fan): fanhm@nwu.edu.cn.
ORCID
Ning Gu: 0000-0003-0047-337X
3629
Article
Hai Ming Fan: 0000-0002-0091-772X
Author Contributions
⊥
H. Zhang, L. Li, and J. Jiao contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors wish to acknowledge financial support provided
by National Natural Science Foundation of China (Grant Nos.
81571809, 21376192, and 81471711) and Natural Science Foun-
dation of Guangdong China (Grant No. 2014A030311036).
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