Folic acid modified superparamagnetic iron oxide nanocomposites for targeted hepatic carcinoma MR imaging

Article abstract of DOI:10.1039/c3ra45878d

A novel targeted MRI contrast agent for tumor cells and tumor over-expressing affinity receptor was synthesized and characterized. Dopamine (DA) was used to present functional molecules on the surface of superparamagnetic iron oxide nanoparticles (SPIONs)

Full text of DOI:10.1039/c3ra45878d

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PAPER
Folic acid modified superparamagnetic iron oxide
nanocomposites for targeted hepatic carcinoma
MR imaging†
Cite this: RSC Adv., 2014, 4, 7483
ab
c
a
c
b
Zhongling Wang, Jing Zhu, Yinyin Chen, Kaiming Geng, Nong Qian,
d
a
c
a
a
c
Liang Cheng, Ziwei Lu, Yue Pan, Liang Guo,* Yonggang Li* and Hongwei Gu*
A novel targeted MRI contrast agent for tumor cells and tumor over-expressing affinity receptor was
synthesized and characterized. Dopamine (DA) was used to present functional molecules on the surface
of superparamagnetic iron oxide nanoparticles (SPIONs), and dextran was conjugated with the folic acid
(
2
FA) that forms stable nanocomposites. The T values of the targeted and non-targeted nanoparticles at
ꢀ ꢀ1
1
3
.0 T were 10.9 ms and 11.8 ms, respectively. The T
2
relaxivity values (r
4.7 s mM , respectively. The results of the competitive inhibition test suggest that the SPION-DA-
signal intensity
of hepatic carcinoma cells (Bel 7402) incubated with the folate targeting nanocomposites decreased
significantly. In contrast, the T signal did not show an obvious decrease for cells treated with the non-
targeting nanocomposites. In the in vivo study, the T signal decreased significantly 18 hours after
injection of the folate targeting contrast agent. In contrast, the maximum intensity of the non-targeting
group appeared 0.5–2 hours after injection and the T signal intensities recovered gradually 4 hours after
2
) were 91.7 s mM and
ꢀ1
ꢀ1
8
dextran-FA uptake is associated with folate receptor binding. In the in vitro study, the T
2
2
2
Received 16th October 2013
Accepted 19th November 2013
2
DOI: 10.1039/c3ra45878d
injection. Our results indicated that FA targeting SPIONs have the ability for use as a novel targeting MRI
contrast agent and have a better targeting tropism to the Bel 7402 cells and tumor.
www.rsc.org/advances
their recognition by macrophages of the mononuclear phagocyte
system (MPS).
1
. Introduction
In recent years, magnetic resonance imaging (MRI) has become
SPIONs can be coated with lipids to improve their biocompat-
one of the most useful diagnostic techniques in providing ibility and enhance their circulation half-life in the blood stream.
noninvasive and real time detection of diseases such as tumors Several approaches have been reported, using materials such as
1–3
etc. Various contrast agents have been applied to improve the dextran, poly(ethylene glycol) (PEG) or polyvinylpyrrolidone as
9–14
quality of MRI. Among these contrast agents, superparamagnetic coatings materials.
One way to increase the concentration of
iron oxide nanoparticles (SPIONs) have been intensively studied SPIONs to targeted tumors is to conjugate these nanoparticles with
as promising MRI probes because of their nontoxicity, biocom- targeting molecules that have a high affinity to target tumors. One
patibility, large surface area and uniform physical properties promising candidate is folic acid since its receptor is overexpressed
4–8
15–22
such as good magnetic properties.
However, traditional on a variety of tumors of diverse origins.
SPIONs lack targeting abilities and also would be rapidly elimi-
In this article, we report a general strategy that uses dopa-
nated from the blood stream aer injection which is due to mine as the anchor to link folic acid on the surface of the
23,24
SPIONs for hepatic carcinoma MR imaging.
Dextran is also
introduced to improve the hydrophilicity and biocompatibility
and also to prolong the circulation half-life of the folic acid
modied SPIONs (Fig. 1). The resulting nanocomposites, that
is, the SPIONs conjugated with dopamine, dextran and folic
acid (SPION-DA-dextran-FA) were characterized by transmission
electron microscopy (TEM), Fourier transform infrared spec-
a
Department of Radiology, The First Affiliated Hospital of Soochow University,
Soochow University, Suzhou, Jiangsu, 215006, China. E-mail: liyonggang224@163.
com; ilguoliang@sohu.com
b
Department of Imaging, The Second People's Hospital of Changzhou, Nanjing Medical
University, Changzhou, Jiangsu, 213003, China
c
Key Laboratory of Organic Synthesis of Jiangsu Province, College of Chemical,
Chemical Engineering and Materials Science, Soochow University, Suzhou, Jiangsu, troscopy (FTIR) and X-ray powder diffraction (XRD). In vitro
2
15123, China. E-mail: hongwei@suda.edu.cn; Fax: +86-65880905; Tel: +86-
experiments were performed using Bel 7402 cells, a human
hepatic carcinoma cell line which over-expresses surface
receptors for folic acid. And in vivo MR imaging was also per-
formed. The aim of this study was to study the targeting func-
tion of FA-conjugated SPION-DA-dextran to hepatoma cells via
6
5880905
d
Functional Nano & So Materials Laboratory (FUNSOM), Soochow University,
Jiangsu, Suzhou 215123, China
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c3ra45878d
1
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Activated dextran was synthesized following a literature
26
method. Firstly, a dextran solution was prepared in DMSO
30 mM). Next, ve amounts of disuccinimidyl carbonate (DSC)
(
dissolved in DMSO were added to the dextran solution with
stirring. Five amounts of 4-dimethylamino pyridine (DMAP) in
DMSO were then slowly added, and the solution was stirred for
6
h. Dextran was precipitated by a slow addition of acetone, and
washed with acetone and DMSO using the precipitation–redis-
persion protocol. The activated dextran powder was dried and
ꢁ
stored at 4 C.
The method for conjugating the Fe O nanoparticles to
3
4
activated dextran was as follows: activated dextran (0.5 g) was
dissolved in 30 mL of DMSO and then the DA-functionalized
Fe O nanoparticles dissolved in 10 mL DMSO were added
3 4
slowly to the stirring solution of the above mixture. The mixture
was stirred for 24 h at room temperature under nitrogen and
then ve amounts of 1,6-hexanediamine were added. Aer that,
the mixture was washed with ethanol three times, and the
product was re-dispersed in ethanol.
The N-hydroxysuccinimide ester of folic acid (NHS-folate) was
27
prepared by a known procedure. Folic acid (1 g, 2.26 mmol) was
Fig. 1 (a) The chemical structure and (b) schematic representation of dissolved in 20 mL of dry dimethylformamide (DMF) to which
SPION-DA-dextran-folate acid (FA).
0.31 g (1.52 mmol) of dicyclohexylcarbodiimide (DCC) and
.257 g (2.26 mmol) of NHS were added. The reaction mixture
was stirred for 12 h at room temperature in the dark. The
the FA receptor and to asses the feasibility for surveillance of byproduct, dicyclohexylurea, was ltered off, and 100 mL of 30%
0
tumor targeting with MRI.
acetone in diethyl ether was added with stirring. A yellow
precipitate formed and was collected on a sintered glass
crucible; aer washing with acetone and ether several times, the
material was used immediately for the next step in the synthesis.
2
. Materials and methods
The method for conjugating the Fe
folate was as follows: NHS-folate (0.5 g) was dissolved in 50 mL
Iron oxide nanoparticles were synthesized using a reported of dry pyridine and then the Fe nanoparticles were added
3 4
O nanoparticles to
2.1 Synthesis of iron oxide nanoparticles
3 4
O
23,25
procedure:
FeCl $6H
O (2.7 g, 10 mmol) and sodium oleate slowly to the stirring solution of the above mixture. The mixture
3 2
ꢁ
(
9.125 g, 30 mmol) were added to a mixture of ethanol (20 mL), was stirred for 24 h at 25 C under nitrogen, the mixture was
deionized water (15 mL) and hexane (35 mL). The mixture was reacting in dark, and collected by centrifugation at 6000 rpm.
ꢁ
reuxed at 70 C for 4 h; the upper reddish brown hexane solu- Aer washed with ethanol and hexane (1/5, v/v) three times, the
tion containing the iron–oleate complex was then separated, and product was re-dispersed in ethanol. The SPION-DA-dextran-FA
washed three times with deionized water (10 mL) in a separatory was obtained.
funnel. The hexane was then evaporated in a rotary evaporator to
yield a dark reddish brown, oily iron–oleate complex. Using a
standard Schlenk line, the iron oleate complex (9 g, 10 mmol)
2.2 Characterization
was dissolved in 25 g of 1-octadecene; oleic acid (1.41 g, 5 mmol) The average core size, size distribution and morphology were
or sodium oleate (1.52 g, 5 mmol) was then added. The mixture examined using a FEI TecnaiG20 transmission electron micro-
ꢁ
ꢁ
ꢀ1
was heated to 320 C at a ramp of 3–5 C min for 30 min under scope (TEM) at a voltage of 200 KV. Fourier transform infrared
argon. The resulting black nanocrystal solution was cooled to spectroscopy (FTIR) measurements were recorded on a ProStar
room temperature, and 2-propanol (50 mL) was added to LC240 Fourier Transform Spectrophotometer. The UV-Vis spec-
precipitate the magnetic nanoparticles. Aer centrifugation, the troscopy of the nanoparticles was taken on a Beckman Coutler
nanoparticles were washed with hexane and ethanol three times, DU-800 UV-Vis spectrometer (Beckman Coutler, Fullerton, CA)
and then redispersed in hexane or toluene.
Dopamine hydrochloride (200 mg) was dissolved in dimethyl loop was measured with a Quantum Design SQUID MPMS XL-7
sulfoxide (DMSO) (20 mL), and then Fe (100 mg) in 10 mL magnetometer at an applied eld (H) of 9 kOe at 298 K.
DMSO was added. The mixture was stirred overnight at room The average core size and structural properties of IONP were
temperature. The modied Fe O nanoparticles were precipi- characterized by X-ray powder diffraction (XRD) using a D/Max-III
with a 10 mm optical path length quartz cuvette. The hysteresis
3 4
O
3
4
tated by adding ethanol, and collected by centrifugation at 6000 C X-ray diffractometer with monochromatized Au Ka radiation.
rpm. Aer washed with ethanol and hexane (5/1, v/v) three
2
The T values of the SPION-DA-dextran-FA and IONP-DA-
times, the product was re-dispersed in ethanol.
dextran nanoparticles were measured at 3.0 T MRI Scanner
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2
GE Signa HDx, USA.) at room temperature. The T relaxation 2.6 In vitro cell cytotoxicity analysis
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(
times were measured using the following parameters: MR
The Bel 7402 cell line was used to perform an MTT (methyl
thiazolyl tetrazolium) test to determine the in vitro cell cyto-
toxicity of SPION-DA-dextran-FA and SPION-DA-dextran. An
imaging was performed with a 3.0 T MR. T -weighted images
2
were acquired using the following parameters: TR/TE, 1000 ms/
1
2
08 ms; slice thickness, 1 mm; slice spacing, 1 mm; matrix,
4
amount of 5 ꢂ 10 Bel 7402 cells were seeded on a 96 well plate
ꢀ
1
ꢀ1
56 ꢂ 256; FOV, 8 cm ꢂ 8 cm. The relaxivities r
2
(s mM )
in folate-free DMEM 1640 medium and then incubated for 24 h
were obtained from the tting of the 1/T
2
versus plots.
ꢁ
at 37 C under 5% CO
2
. Aer incubating the Bel 7402 cells for
1
8 h with the media containing SPION-DA-dextran-FA or SPION-
ꢀ
1
2.3 Cell culture and animal model
DA-dextran at Fe concentration of 40 mg mL , the supernatant
was removed and the cells were washed three times with PBS.
Absorption values were measured and recorded every day. For
the MTT assay, 20 mL MTT stock solution (5 mg mL ) was
added to each well, Aer incubating for 4 h, the medium was
then removed and each well was treated with 150 mL dimethyl
sulfoxide (DMSO) with pipetting, for 10 min. Cell viability was
assessed by absorbance which was measured at 490 nm using a
microplate reader.
Bel 7402, a human hepatic carcinoma cell line which stably
express surface receptors for folic acid, was obtained from the
Shanghai Academy of Life Sciences. Bel 7402 cells were cultured
ꢀ1
ꢁ
at 37 C in folate-free DMEM 1640 medium (Gibco Co., Ltd USA)
ꢀ1
ꢀ1
supplemented with 50 mg mL penicillin, 50 mg mL strep-
tomycin and 10% fetal bovine and maintained in a humidied
contained 5% CO atmosphere.
2
Nude mice (20 g) were purchased from the Shanghai Labo-
ratory Animal Center and the animal experiments were per-
formed in accordance with the institute guide lines. 12 nude
2
.7 In vitro MR imaging
7
mice were injected with Bel 7402 cells via 0.2 mL (4 ꢂ 10 cells)
6
Bel 7402 cells (1 ꢂ 10 ) were washed three times with PBS and
incubated with SPION-DA-dextran-FA and SPION-DA-dextran, in
a folate-free DMEM 1640 medium for 2 h, respectively, at Fe
concentrations of 0, 5, 10, 20, 40 and 80 mg mL . Aer diges-
tion with 0.25% trypsin, the mixture was centrifuged at
folate-free DMEM 1640 medium suspension under the le
armpit. All animals were maintained on a folate-free diet for
5
weeks, the mice were housed in polycarbonate microisolator
ꢀ1
cages and the tumor sizes were monitored per week. Tumors
reaching a diameter of 1.0–1.2 cm aer 8 weeks were used for in
vivo MR imaging.
1
200 rpm for 4 min and resuspended in 0.5 mL of 1% agarose in
an Eppendorf tube. MR imaging was performed with a 3.0 T MR.
-weighted images were acquired using the following param-
eters: TR 4240 ms; TE108 ms; slice thickness, 1 mm; slice
Bel 7402 cells at a density of 1 ꢂ 10 cells per dish were washed, spacing, 1 mm; matrix, 256 ꢂ 256; FOV, 8 cm ꢂ 8 cm. The T
trypsinized aer incubation for 1 h with SPION-DA-dextran-FA signal intensities were measured within the region of interest.
T
2
2.4 Prussian blue staining
6
2
ꢀ1
and SPION-DA-dextran at Fe concentrations of 40 mg mL in a
ꢁ
folate-free DMEM 1640 medium at 37 C. Then cells were
2
.8 In vivo MR imaging
washed with phosphate-buffered saline (PBS) three times and
ꢁ
Mice were anesthetized with 0.04 mL of 3% sodium pentobar-
bital by intraperitoneal injection. Tumor-bearing nude mice
were divided into the experimental group (n ¼ 6) and control
group (n ¼ 6) randomly. They were injected with an SPION-DA-
dextran-FA solution and SPION-DA-dextran (0.2 mL) via tail vein
respectively. The MR imaging of mice was performed using a

xed with 4% glutaraldehyde for 20 min, and incubated at 37 C
with 2 mL Prussian blue solution comprising equal volumes of a
% hydrochloric acid aqueous solution and 2% potassium fer-
2
rocyanide(II) trihydrate for 30 min. Aer staining with 0.5%
neutral red for 3 min, the iron staining was nally observed
using microscope.
For histopathological analysis, liver samples were quickly
obtained and xed in a 10% neutral buffered formaldehyde
solution, embedded in paraffin and sectioned. The 5 mm thick
sections were stained with hematoxylin and eosin (HE), Mas-
son's trichrome for collagen ber and Prussian blue for iron.
3
.0 T MR imager and a high resolution animal coil. All
samples were measured by a T -weighted spin-echo sequence
2
(
1
TR/TE ¼ 4000/108 ms; slice thickness, 2 mm; slice spacing,
mm; matrix, 256 ꢂ 256; FOV, 8 cm ꢂ 8 cm). Tumor and
muscle signal variation was observed aer the injection of
contrast agent at 0.5, 1, 2, 4, 8, 12, 18, and 24 hours and CNR
was calculated at different time points.
2.5 Competitive inhibition experiment
ꢀ1
Under experimental Fe concentrations (40 mg mL ), cells that
2.9 Statistical analysis
were pre-treated with large amounts of free folic acid of
ꢀ1
different concentrations (0.5, 1.0, 1.5, 2.0 mmol L ) for 1 h All data are expressed as mean ꢃ standard deviation (SD).
before the targeting nanocomposite was added to the medium. Statistical analyses of the MRI signal intensity were performed
MR imaging was performed with a 3.0 T MR. T -weighted with univariate Analysis of Variance, and a binary comparison
2
images were acquired using the following parameters: TR 4240 between the mean were performed with the Student–Newman–
ms; TE108 ms; slice thickness, 1 mm; slice spacing, 1 mm; Keuls (SNK) and LSD test. The analyses were performed using
matrix, 256 ꢂ 256; FOV, 8 cm ꢂ 8 cm. The T
2
signal intensities SPSS 17.0 soware. We considered a difference to be statistically
signicant if the p value was less than 0.05.
were measured within the region of interest.
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3. Results and discussion
3.1 Preparation and characterization of nanoparticles
Nanoparticles were usually coated with a layer of biocompatible
polymer such as polyethylene glycol (PEG), dextran, or den-
drimers for surface modication. In the present research, we
used a series of dextran molecules (MW ¼ 40 000) coupled with
28
dopamine. We followed the procedure published previously.
Aer ligand exchange, the nanocomposites were easily
dispersed in water forming a clear solution. Then we used a
targeting ligand, folate to functionalize the nanocomposites.
These nanocomposites are stable in water and no aggregates
were observed for several days.
The FTIR spectra of unmodied SPION and SPION-DA,
SPION-DA-dextran and SPION-DA-dextran-FA are shown in
Fig. 2. The IR spectra of Fig. 2d exhibits peaks at about the
ꢀ
1
ꢀ1
1
600–1610 cm and 1680–1690 cm ranges, which are char-
Fig. 3 The UV-vis spectra of (a) SPION (Fe
dextran; (c) FA; (d) SPION-DA-dextran-FA.
O
3 4
); (b) SPION-DA-
5,29
acteristic of the FTIR spectra of folic acid.
C–OH stretch vibrations of the dextran in the 1180–1200 cm
In addition, the
ꢀ
1
range are present in Fig. 2d. These results indicated that the
folic acid had been coated successfully on the surface of the
magnetic nanoparticles in the presence of dextran.
The UV-Vis spectra of SPION, SPION-DA-dextran, FA, SPION-
DA-dextran-FA are shown in Fig. 3. The folic acid content (line c)
can be obtained from the absorbance of 286 nm, which can be
attributed to folic acid only. Since there is no absorption peaks
at 286 nm for SPION (line a) and SPION-DA-dextran (line b), the
absorbance of SPION-DA-dextran-FA at 286 nm conrms that
the SPIONs are surface-modied with folic acid.
Fig. 6 shows the TEM image of the as-prepared SPIONs. It
can be found that the SPIONs are uniform with a diameter of
about 14 nm. The dynamic light scattering (DLS) measurements
showed that diameter was around 96 nm and 89 nm for SPION-
DA-dextran-FA and SPION-DA-dextran, respectively (Table S1†).
2
The T values of the SPION-DA-dextran-FA and SPION-DA-
dextran nanoparticles at 3.0 T were 10.9 ms and 11.8 ms,
ꢀ1 ꢀ1
respectively. Their T
2
relaxivity values (r
2
) were 91.7 mM
s
ꢀ1 ꢀ1
and 84.7 mM
s , respectively (Table S1†).
Fig. 4 shows the X-ray powder diffraction (XRD) patterns of
SPION-DA-dextran-FA. From the XRD data, a series of charac-
teristic peaks of Fe O still exist aer modication with
3
.2 Cytotoxicity study of SPION-DA-dextran-FA and SPION-
3
4
DA-dextran
DA-dextran-FA, compared with the JCPDS standard card. The
XRD data shows that the crystalline Fe O nanoparticles were
An MTT test assay using the (FR+) Bel 7402 cell line was carried
out to analyze the potential cytotoxicity of SPION-DA-dextran
and SPION-DA-dextran-FA. Cell viability was measured for 3
days. Fig. 7 shows that these modied SPION do not inhibit the
3
4
not changed aer surface modication.
To determine the magnetic properties of SPION-DA-dextran-
FA, a SQUID magnetometer was used. The SPION-DA-dextran-
FA were found to be superparamagnetic with a magnetization
ꢀ
1
(
M) value of 60 emu g at an applied eld (H) of 9 kOe at 298 K
(Fig. 5). The hysteresis loop demonstrated that the coercivity
and remanence are almost negligible, which is typically a
superparamagnetic behaviour.
Fig. 2 The FTIR spectra of (a) SPION (Fe
O
3 4
); (b) SPION-DA; (c) Fig. 4 The X-ray powder diffraction (XRD) pattern of SPION-DA-
SPION-DA-dextran; (d) SPION-DA-dextran-FA.
dextran-FA.
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Fig. 7 The MTT test of Bel 7402 cells treated with SPION-DA-dextran
ꢀ1
Fig.
5
The SPION-DA-dextran-FA were found to be super- and SPION-DA-dextran-FA (Fe concentration: 40 mg mL ).
ꢀ1
paramagnetic with a magnetization (M) value of 60 emu g at an
applied field (H) of 9 kOe at 298 K. Magnetic hysteresis loops of
SPION-DA-dextran-FA measured at 298 K.
of targeting contrast agents, while no obvious blue areas can be
observed inside the tumor tissues of the control group (Fig. 8c
and d). Obviously, the folate targeted nanocomposites have a
good targeting ability for Bel 7402 cells and hepatic carcinoma.
The Prussian blue staining of cells further evidenced the
selective labeling of the FR-positive cells by SPION-DA-dextran-
FA. Our results demonstrated the effective in vitro targeting of a
specic type of cancer cells using SPION-DA-dextran-FA.
3.4 Competitive inhibition test
Fig. 9 demonstrates the change of the T signal intensities when
2
different concentrations of free folic acid were added to the
culture medium before adding the targeting nanocomposites
(Table S2†). Because of the blocking effect of FA, an increase in
Fig. 6 The TEM image of as-prepared SPION.
ꢀ
1
Bel 7402 cell growth with up to 40 mg mL of Fe. These results
demonstrate that both SPION-DA-dextran and SPION-DA-
dextran-FA are biocompatible and express low cytotoxicity.
3.3 Prussian blue staining
The presence of intracellular SPIONs in hepatoma cells was
shown by Prussian blue staining. As shown in Fig. 8, Prussian
blue staining experiments of in vitro and in vivo study evidenced
that the cell uptake level of the nanocomposites strongly
depended on the targeting ligand. For in vitro study, the cell Fig. 8 Prussian blue staining (ꢂ200) of Bel 7402 cells (1 ꢂ 10 ) after 1 h
experiments showed a much stronger blue appearance in the
6
incubation with folate targeted nanocomposites (a) and non-folate
ꢀ1
targeted nanocomposites (b) at an Fe concentration of 40 mg mL in a
folate-free DMMC 1640 medium. Colocalization of iron staining is
detected in the tumor tissues after 24 h injection of targeting contrast
agents (c). Blue areas are not seen in the tumor after 24 h injection of
cells of the folate targeting nanocomposites group than the
ꢀ
1
control group when the Fe concentration reached 40 mg mL
Fig. 8a and b). For in vivo study, the blue areas in the experi-
(
ment group could be seen in the tumor mass aer 24 h injection non-targeting contrast agents (d).
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the signal intensity, resulting from a lower dose of SPION, was
seen when the free folic acid concentration in the culture
medium was higher. This result suggests that the uptake of
SPION-DA-dextran-FA is associated with the folate receptor
binding.
3.5 In vitro MRI studies
Prior to showing the specicity of the folate targeting nano-
composites for in vivo MRI, the signal intensity of cells on
T2-weighted decreased signicantly with the exaltation of iron
concentration in the SPION-DA-dextran-FA. The rate of the
signal intensity variety was ꢀ89.76% at an iron concentration of
ꢀ1
8
0 mg mL in culture medium. No signicant reduction of the
signal intensity was found in SPION-DA-dextran and the rate
of signal intensity variety was ꢀ37.92% (Fig. 10). The rate of the
signal intensity variety for folate targeting nanoparticles by
the Bel 7402 cells was about 2–3 times higher than that for the
nontargeting nanoparticles. The T2 signal intensity of the
receptor-positive Bel 7402 cells incubated with SPION-DA-
dextran-FA was signicantly different compared with SPION-
DA-dextran (P < 0.01) (Table S3†).
6
Fig. 10 (a) T
2
-weighted images of Bel 7402 cells (1 ꢂ 10 ) after a 2 h
ꢀ
1
treatment with different iron concentrations (0–80 mg mL ) of
SPION-DA-dextran-FA ((b)) and SPION-DA-dextran nanocomposites
In recent years, one of the focuses of research in medical
diagnostics is molecular imaging that can facilitate early diag-
nosis, providing fundamental information on pathological
((a)). (b) Low T
2
signal intensities correlate with a high iron concen-
signal
tration (Targeting nanocomposites), reduction significantly of T
2
processes. The cellular imaging is developing rapidly in this intensities when the iron concentration in culture media was higher
3
0
(non-targeting nanocomposites), change of T2 signal intensities was
not obvious.

eld. SIONPs have been intensively studied for these appli-
cations in biomedical science such as MRI contrast agents for
31
cancer diagnosis and target-drug delivery. In this study, the
results from Fig. 10(a) and (b) indicate that the SPIONs play an
important role in receptor-mediated endocytosis into the
hepatic carcinoma cells. The tumor cells targeting events could
be monitored with 3.0 T MR imaging.
SPION-DA-dextran (Fig. 11c). We tested the feasibility of tar-
geting using an animal (mice) model (n ¼ 12) bearing Bel 7402
tumors. At 0.5 h aer the injection of the folate targeting
contrast agent, the T
nude mice and the T
hours injection of the folate targeting contrast agent, this effect
2
signal slightly decreased in the tumors of
2
signal decreased signicantly aer 18
3.6 In vivo MRI studies
To understand the tumor (FR+) and muscle uptake of folate is persistent up to the 24 h end point (Fig. 11b and d). The
targeting nanoparticles and nontargeting nanoparticles, MR selective accumulation of the folate receptor-targeting nano-
imaging of tumor and muscle were observed at different time particles in this tumor model was further conrmed by Prussian
points aer the injection of SPION-DA-dextran-FA (Fig. 11a) and blue staining of tissue sections (Fig. 8c). We believe that the
persistent tumor enhancement at 18 h postinjection of the
folate-targeting nanoparticles is due to the folate receptor-
binding effect. In addition to prolonged circulation which is a
32,33
prerequisite for tumor accumulation of liposomes,
dextran
is the best basis for a formulation that confers a long half-life in
circulation. However, it may also be due to a passive targeting of
the nanoparticles into the tumor resulting from the leakage of
the agent into the tumor interstitium via capillaries present in
tumors. These results showed that the folate-targeting nano-
particles were able to accumulate in hepatocellular tumors
expressing the FR. In contrast, the maximum intensify of the
nontargeting group appeared in 0.5–2 hours aer injection. The
T
2
signal intensities in the tumor recovered gradually aer 4
6
Fig. 9
T
2
-weighted images of Bel 7402 cells (1 ꢂ 10 ) after a 1 h
hours injection of nontargeting contrast agent (Fig. 11,Table
S4†). We did not detect a SPION-induced T signal intensity
change in the mouse-bearing 24 hours aer the injection of
2
nontargeting nanoparticles. On the other hand, the T signal
incubation with different concentration of free folic acid (0, 0.5, 1.0,
ꢀ
1
2
1
.5, 2.0 mmol L ), followed by treatment with folate targeting nano-
ꢀ
1
composites for 1 h at an iron concentration of 40 mg mL (a). Change
of T signal intensities with different concentrations of free folic acid
were used (b).
2
intensities varied in the muscle 0.5 h aer the injection of the
7488 | RSC Adv., 2014, 4, 7483–7490
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from the New Faculty Start Foundation of Soochow University
(
Q410900413); Y.G.L. acknowledges nancial support from the
National Natural Science Foundation of China (NSFC) (no.
1171394, 81171392), the Natural Science Fund of Jiangsu
8
Province (no. BK2011307) and the Natural Science Fund for
Colleges and Universities in Jiangsu Province (no. 09KJB320016).
Notes and references
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7490 | RSC Adv., 2014, 4, 7483–7490
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