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Introduction
Gliomas are the most common and mortiferous type of
primary brain tumors[1,2]. The differentiation of central
gliomas from peripheral brain tissue is very crucial for accurate
resection of all diseased tissues. The effectiveness of tumor
resection is severely limited by the poor visual contrast
between gliomas and normal brain tissue by gadolinium
chelate-enhanced magnetic resonance imaging
(MRI)[3]. Superparamagnetic iron oxide nanoparticles (SPIO) have also
been widely used for MRI contrast
enhancement[4_6], and labeled as glioma cells to visualize tumors at real cell-level
resolution by MRI[3,7_9]. Moreover, fluorophores can be
conjugated with SPIO to visualize labeled glioma cells in optical
imaging[10]. However, the low specificity of intracellular uptake
by gliomas limits the extensive use of SPIO. SPIO were also
internalized by reactive astrocytes and
macrophages[3,10,11], which lead to overestimating the extent of gliomas. Therefore,
to achieve specific glioma cell recognition and increased
efficiency of the SPIO intracellular uptake, a novel SPIO MRI
contrast agent for specific targeting gliomas needs to be developed.
Nanoparticles can be internalized into cells generally via
receptor-mediated endocytosis[12]. One strategy to realize
the specific and efficient intracellular uptake of nanoparticles
is to modify the nanoparticles surface with a ligand which is
preferentially and efficiently taken up by target cells via
receptor-mediated endocytosis[13_15]. Chlorotoxin is a small
36-amino acid peptide that was originally isolated from the
giant Israeli scorpion (Leiurus
quinquestriatus) venom[16,17]. Chlorotoxin specific receptor on the surface of glioma cells
was proven to be membrane-bound matrix metalloproteinase-2
(MMP-2), which binds with high affinity to
chlorotoxin[18]. MMP-2 is specifically upregulated in gliomas and other
tumors of neuroectodermal origin, but is not normally
expressed in tumors of non-neuroectodermal origin and
normal brain tissue, including neural
cells[19]. MMP-2 can specifically inhibit the glioma-specific chloride channel which
only exists on glioma cells rather than normal brain tissue
cells, including neural cells. Therefore, chlorotoxin is
capable of binding to gliomas cells with highly specific and
selective targeting characters[20,21]. Despite the specific
targeting role of chlorotoxin nanoparticles
conjugated to 9L gliosarcoma cells having been
confirmed[22], little effort has been made on the basis of glioma cells of human origin.
Investigating the specific targeting role of
chlorotoxin-nanoparticles conjugated to human U251 glioma cells will
set a theoretical foundation for following an in
vivo study. Moreover, the appropriate biocompatibility of
conjugated chlorotoxin nanoparticles was essential for introducing the
conjugated material into clinical practice. Therefore, the
present study paid additional attention on the
biocompati-bility of conjugated chlorotoxin nanoparticles.
In this study, SPIO-fluorescein isothiocynate
(FITC)-chlorotoxin (SPIOFC) was synthesized for improving the
specificity of SPIO for targeting glioma cells. The
biocompatibility of SPIOFC was examined to estimate the value of
its biomedical application. Prussian blue staining and MRI
were utilized to investigate the intracellular uptake of SPIOFC
by human U251-MG and rat C6 glioma cells in order to reflect
the actual specificity of SPIOFC for targeting glioma cells.
Inductively-coupled plasma emission spectroscopy (ICP)
was employed to quantify the intracellular uptake of SPIOFC.
Confocal laser scanning microscopy was employed to
evaluate the optical imaging ability of SPIOFC.
Materials and methods
Newborn Wistar rats (Harbin Medical University
Experimental Animals, Harbin, China) were used in this study. The
experiments were conducted in accordance with the Guide
for the Care and Use of Laboratory Animals as adopted and
promulgated by the Declaration of Helsinki.
Reagents 3-Aminopropyl-triethoxy-silane (APS),
bicin-choninic acid (BCA) kit, FITC,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), Nuclear Fast Red
solution, and gelatin were purchased from Sigma (St Louis, MO,
USA). Chlorotoxin was obtained from AnaSpec (San Jose, CA,
USA). Fetal bovine serum (FBS), Dulbecco's modified Eagle's
medium (DMEM), Dulbecco's Modified Eagle's Medium:
Nutrient Mixture F-12 (DMEM/F-12), and B-27 supplements
were purchased from Gibco (Grand Island, New York, USA).
Synthesis of multifunctional specific targeting contrast
agent for gliomas (SPIOFC) SPIOFC consisted of SPIO,
FITC, and chlorotoxin. Water-soluble SPIO (7±1.6 nm in size)
were prepared via our previously published
approach[23,24]. Fluorescent and magnetic bifunctional core-shell
nanopar-ticles (SPIOF) were prepared by encapsulating SPIO
cores in a FITC dropped silica shell using a sol-gel
approach[25]. Amino-modified SPIOF were obtained by hydrolyzing
APS in SPIOF suspension solution. Then chlorotoxin was covalently
conjugated with the surface amine groups on SPIOF to form
SPIOFC via a standard covalently binding method as described
by Weissleder et al[26]. The morphology of SPIOFC was
investigated by transmission electron microscope (TEM) on a
Philips Tencai 20 electron microscope (Eindhoven,
Nether-lands) at an accelerating voltage of 200 kV. The amount of
chlorotoxin immobilized on the contrast agent was quantified
using a BCA kit for the protein assay.
Cells and cell cultures Human U251-MG glioma and rat
C6 glioma cell lines were kindly provided by Keiji
KAWAMOTO (Department of Neurosurgery, Kansai Medical University, Osaka, Japan), and were routinely cultured at
37 °C in a humidified atmosphere with 5%
CO2 in DMEM supplemented with 10% FBS, 100 kU/L penicillin, and 100
mg/L streptomycin. Cell viability and density were
determined through staining with trypan blue. Cell counts were
obtained using a hemocytometer.
Neural cells were prepared from the cortex of newborn
Wistar rats in accordance with the methods described
previously[27]. In brief, the cortical tissue was carefully freed from
blood vessels and meninges. The tissue was trypsinized for
20 min, carefully disintegrated with a fire-polished pipette,
and washed twice. The cortical cells were cultured in
DMEM/F-12 medium supplemented with 10% FBS, 100 kU/L penicillin,
100 mg/L streptomycin, and 2% B-27 supplements. The
medium was changed every third day.
Cytotoxicity assay by MTT The MTT assay was
performed to evaluate whether SPIO, chlorotoxin, and SPIOFC
impair the viability of glioma cells and neural cells using
corresponding untreated cells as the control. The cells were
seeded onto 96-well plates at a density of
6×103 cells/well for 24 h (U251-MG and C6 glioma cells) and 7 d (neural cells),
respectively. Then SPIO at final concentrations of 5, 10, 25,
50, 75, 100, and 200 mg/L (iron concentrations), chlorotoxin at
concentrations of 0.8, 1.6, 2.4, 3.2, 4.0, 4.8, and 5.6
μmol/L, and SPIOFC at final concentrations of 100 mg/L (iron concentrations)
and 1.6 μmol/L (chlorotoxin concentration), respectively, were
added to the cells and incubated for 24 h. The cells were washed
twice with phosphate-buffered saline (PBS) and replenished
with fresh medium, followed by incubation for a further 48 h.
Then the cells were examined by MTT assay.
Cytochemistry analysis Prussian blue staining was
employed to determine the presence of iron inside the cells.
Untreated cells were seeded onto 6-well plates at a density
of 3×105 cells/well. Twenty four hours after seeding, glioma
cells (human U251-MG and rat C6) were cultured with SPIOFC
and SPIOF at a concentration of 100 mg/L for 1 h. Seven
days after seeding, neural cells were treated with SPIOFC at
a concentration of 100 mg/L for 1 h as the control for
SPIOFC-targeted glioma cells. Following labeling, the samples were
washed twice with cell culture medium and twice with PBS. The
cells were fixed with 4% paraformaldehyde solution and washed
with PBS, followed by incubation in Perl's solution (equal parts
of 6% hydrochloric acid and 2% potassium ferrocyanide) for 30
min. After washing with deionized water, the treated cells were
counterstained with Nuclear Fast Red solution. The specimens
were then mounted and examined under a light microscope.
The percentage of Prussian blue stained-positive cells was
counted under a random field of view (×200) and the procedure
was repeated 20 times for each specimen.
Quantification of nanoparticle intracellular uptake
The intracellular iron concentrations were quantified using ICP
(Perkin_Elmer Optima 5300 DV, Perkin Elmer, Beaconsfield,
UK). After being cultured with the abovementioned
nanoparticles, the cells were washed with PBS, detached,
resuspended, counted, centrifuged down, and the cell pellet
was dissolved in 37% HCl solution at 70_80 °C for 30
min. The samples were diluted to final iron
concentrations of 1.0_4.0 mg/L. Three replicates were measured.
MRI For MRI, the specimens were prepared by
suspending 2×106 cells in 100 μL of 4% gelatin. The cell
suspensions were seeded onto 96-well plates at a volume of
100 μL/well and allowed to solidify at 4 °C. The spaces
surrounding each well were full of deionized water to allow
appropriate image acquisition. MRI was performed with a
1.5-T clinical MR imager (TOSHIBA Visart, Japan) using a
human knee coil. A T2-weighted (TR, 3000 ms; TE, 20 ms)
spin-echo sequence was selected to acquire the magnetic
resonance (MR) images. The spatial resolution parameters were
as follows: an acquisition matrix of 256×192, field of view of
15×15 cm, section thickness of 2.5 mm, and thickness gap of
0.5 mm. The regions of interest of 0.12
cm2 were placed in the center of each sample image to obtain signal intensity
measurements using the provided image quantification tool.
Confocal laser scanning microscopy Confocal laser
scanning microscopy was employed to ensure the optical
imaging ability of SPIOFC for visualizing gliomas. As mentioned
above, 1×105 cells (human U251-MG and rat C6 glioma cells)
were seeded onto coverslips 24 h prior to labeling and
imaging. Then the cells were cultured with SPIOFC for 1 h.
After the cells were labeled by SPIOFC, the coverslips were
washed twice with cell culture medium and several times
with PBS buffer. The confocal images of the labeled cells
were obtained with a Zeiss LSM 510 META confocal laser
scanning microscope (Zeiss, Oberkochen, Germany). A 488
nm Ar ion laser was used to excite the green fluorescence of
FITC (510_525 nm).
Data process and statistical analysis It was difficult to
determine the absolute value of cell viability. Therefore, the
viability of the untreated cells was considered as 100%
control. Then the viability of cells with SPIO, chlorotoxin,
and SPIOFC was expressed as the percentage of absorbance
of the control untreated cells. Data were presented as
mean±SD. ANOVA was used to compare cell viability,
and P<0.05 indicated significant statistical differences in ANOVA.
The Kruskal_Wallis test was used to compare percentages
of Prussian blue stained-positive cells, qualification of
intracellular iron, and T2 signal intensity;
P<0.0125 was considered to be statistically significant.
Results
Characters of SPIOFC The morphology of SPIOFC was
analyzed by TEM as shown in Figure 1. The TEM image
showed that the core-shell structure SPIOFC was comprised
of the core (SPIO) and homogeneous silica shell. The final
diameter of SPIOFC was 80±15.6 nm. The average number of
chlorotoxin molecules per SPIOFC was calculated to be
approximate 50.2 using a BCA kit for the protein assay.
Cytotoxicity assay by MTT The cell viability of the
SPIO-treated cells (human U251-MG and rat C6 glioma cells, and
neural cells) was not statistically less than that of the
corresponding untreated cells. The viability of U251-MG (Figure
2A) and C6 glioma cells (Figure 2B), and neural cells (Figure 2C)
ranged from 92.4%±17.3% to 101.8%±11.0%, 94.7%±23.3%
to 106.8%±16.2%, and 90.0%±14.5% to 123.0%±20.6%,
respectively, whereas the viability of untreated U251-MG
and C6 glioma cells, and neural cells was 100.0%±7.5%,
100.0%±16.5%, and 100.0%±14.4%, respectively. This
result suggests that SPIO at the abovementioned
concentrations have no obvious cytotoxicity on the 3 cell types.
The viability of the untreated U251-MG and C6 glioma
cells was 100.0%± 15.8% and 100.0%±14.7%, respectively.
The cell viability of the chlorotoxin (0.8 and 1.6
μmol/L)-treated glioma cells was 101.3%±17.7%, 98.1%±23.7%, 98.4%±14.4%,
and 94.8%±19.9% for human U251-MG (Figure 3A) and rat
C6 cells (Figure 3B), respectively. This suggested that
chlorotoxin at the abovementioned concentrations did not
impair the glioma cell viability. In contrast, the cell viability
of the chlorotoxin (2.4, 3.2, 4.0, 4.8, and 5.6 μmol/L)-treated
glioma cells ranged from 67.6%±13.6% to 83.8 %±15.1% and
67.7%±14.2% to 80.8%±9.3% for human U251-MG (Figure
3A) and rat C6 cells (Figure 3B), respectively. This
suggested that chlorotoxin at the specific concentrations
impaired glioma cell viability. Moreover, the cell viability of
the chlorotoxin-treated neural cells was not statistically less
than that of the corresponding untreated cells (Figure 3C).
SPIOFC was then synthesized by SPIO at a
concentration of 100 mg/L (iron concentrations) and chlorotoxin at the
concentration of 1.6 μmol/L for the following measurement.
The cell viability of the SPIOFC-treated cells (U251-MG and
rat C6 glioma cells, and neural cells) was not statistically
lower than that of the control untreated cells. The viability
of the SPIOFC-treated U251-MG (Figure 4A) and C6 glioma
cells (Figure 4B), and neural cells (Figure 4C) was 94.6%±11.7%,
93.3%±14.3%, and 126.8%±11.9%, respectively, and the
viability of the untreated U251-MG and C6 glioma cells,
and neural cells was100.0%±12.3%, 100.0%±15.3%,
and 100.0%±9.4%, respectively. This suggests that SPIOFC at
the abovementioned concentration has no deleterious
effects on the aforementioned cells.
Prussian blue staining for the presence of iron
Both SPIOFC and SPIOF were located in the cytoplasm and
became blue spots after Prussian blue staining. The cytoplasm
of the U251-MG and C6 glioma cells cultured with SPIOFC
(Figure 5B, 5D) contained lots of blue particles, whereas
almost no blue spots were observed in the cytoplasm of
corresponding glioma cells cultured with SPIOF (Figure 5A, 5C).
The percentage of Prussian blue stained-positive (Figure
6A) U251-MG and C6 cells cultured with SPIOFC
(98.7%±1.4% and 99.4%±0.8%, respectively) was significantly more than those
of U251-MG and C6 cells cultured with SPIOF
(2.6%±1.7% and 2.4%±1.6%, respectively). This result indicated U251-MG
and C6 cells could specifically and efficiently internalize
SPIOFC rather than SPIOF.
U251-MG and C6 cells cultured with SPIOFC (Figure
5B,5D) took up a substantial amount of SPIOFC, whereas neural
cells cultured with SPIOFC (Figure 5E) took up virtually no
SPIOFC. The percentage of Prussian blue stained-positive
(Figure 6B) U251-MG and C6 cells cultured with
SPIOFC (98.7%±1.4% and 99.4%±0.8%, respectively) was
significantly greater than those of neural cells cultured with SPIOFC
(0%). This result demonstrated that SPIOFC was
specifically and efficiently taken up by U251-MG and C6 glioma
cells rather than neural cells.
Quantification of nanoparticle intracellular uptake
The ICP results were consistent with those obtained through
Prussian blue staining. The iron uptake by U251-MG and C6
cells cultured with SPIOFC (72.5±1.8 and 74.9±2.2 pg/cell,
respectively) was much higher than those by U251-MG and
C6 cells cultured with SPIOF (6.6±1.0 and 7.1±0.8 pg/cell,
respectively; Figure 7A). This result indicated that
U251-MG and C6 cells could specifically and efficiently internalize
SPIOFC rather than SPIOF.
The iron uptake by U251-MG and C6 cells cultured with
SPIOFC (72.5±1.8 and 74.9±2.2 pg/cell, respectively) was
much higher than those by neural cells cultured with SPIOFC
(1.3±0.3 pg/cell; Figure 7B). This result demonstrated that
SPIOFC could specifically and efficiently be internalized by
U251-MG and C6 glioma cells rather than neural cells.
MRI The MRI results were also consistent with those
obtained through Prussian blue staining. The U251-MG and
C6 cells cultured with SPIOFC (Figure 8B, 8D) showed a much
greater negative contrast than the U251-MG and C6 cells
cultured with SPIOF (Figure 8A, 8C). The
T2 signal intensity (Figure 9A) of the U251-MG and C6 cells cultured with
SPIOFC (233.6±25.9 and 211.4±17.2, respectively) was
substantially lower than those of the U251-MG and C6 cells
cultured with SPIOF (2275.3±268.6 and 2342.7±222.4,
respectively). This result also demonstrated that U251-MG
and C6 cells could specifically and efficiently internalize
SPIOFC rather than SPIOF.
The U251-MG and C6 cells cultured with SPIOFC (Figure
8B,8D) displayed a significantly greater negative contrast
than neural cells cultured with SPIOFC (Figure 8E). The
T2 signal intensity (Figure 9B) of the U251-MG and
C6 cells cultured with SPIOFC (233.6±25.9 and 211.4±17.2,
respectively) was much lower than those of neural cells cultured with
SPIOFC (2533.6±199.2). This result also indicated that
SPIOFC was specifically and efficiently taken up by
U251-MG and C6 cells rather than neural cells, and MRI in
connection with SPIOFC had the potential to differentiate gliomas
from normal brain tissue.
Confocal laser scanning microscopy SPIOFC
internalized by U251-MG and C6 cells was observed as a green
particle in confocal laser scanning microscopy (Figure 10). This
suggests that SPIOFC has the ability to act as an optical
imaging probe for visualizing gliomas.
Discussion
The molecular imaging of SPIO-labeled glioma cells has the
potential to achieve precise surgical resection of
gliomas[3,7_10,22] for the improved prognosis of patients. However, SPIO
encountered low specificity for glioma cells and limited
internalization by glioma cells. To modify SPIO with chlorotoxin
might be beneficial for enhanced specificity and efficiency of
SPIO in labeling glioma cells. Moreover, the
biocompatibility of modified SPIOFC is a prerequisite for biomedical application.
The present study was therefore performed to determine: (i)
whether chlorotoxin-conjugated SPIO (SPIOFC) is suitable
for biomedical application; (ii) whether U251-MG and C6
glioma cells can be specifically and efficiently labeled by
SPIOFC; and (iii) whether SPIOFC internalized by cells can
be detected by both MRI and optical imaging. Our results
demonstrated that SPIOFC, a safe contrast agent, was a
specific and efficient targeting contrast agent for gliomas, and
MR and optical imaging could effectively detect
SPIOFC-labeled glioma cells.
Theoretically, excessive iron may be harmful to cell
survival. However, what is considered the safe application range for
SPIO concentrations remains unclear. Our study
investigated the effect of SPIO at concentration of 5, 10, 25, 50, 75,
100, and 200 mg/L on cell viability. The results demonstrated
that SPIO at the abovementioned concentrations did not
impair cell viability. Previous studies demonstrated that
chlorotoxin can inhibit the growth of
gliomas[17,18]. In this study, we found that chlorotoxin at low concentrations
(0.8 and 1.6 μmol/L) did not impair the viability of glioma cells,
whereas chlorotoxin at higher concentrations (2.4, 3.2, 4.0, 4.8,
and 5.6 μmol/L) had cytotoxic effects on glioma cells. These
results were consistent with Deshane et al's
report[18]. Chlorotoxin at all the abovementioned concentrations did
not impair the viability of neural cells. This might due to the
fact that there was no chlorotoxin specific receptor (MMP-2)
on the surface of the neural cells. Therefore, SPIO and
chlorotoxin, utilized for the synthesis of SPIOFC, was
controlled at concentrations of 100 mg/L (iron concentrations)
and 1.6 μmol/L, respectively. The MTT results showed that
SPIOFC at this concentration was safe for cell survival. This
might be due to the fact that SPIO (100 mg/L iron
concen-trations) and chlorotoxin (1.6 μmol/L) did not impair the
viability of glioma and neural cells. The viability of the neural
cells cultured with SPIOFC (100 mg/L) was higher than the
corresponding control groups, which is consistent with
another report[28]. The mechanism for this phenomenon needs
to be further investigated.
In this study, U251-MG and C6 glioma cells took up lots
of SPIOFC, whereas SPIOF had difficulty entering the
cytoplasm of both cells by internalization. Moreover, in
comparison with glioma cells, neural cells virtually did not contain
SPIOFC despite being cocultured with SPIOFC for an
adequate duration. This demonstrates that SPIOFC has high
specificity and efficiency in labeling glioma cells. Chlorotoxin,
a small peptide, should be responsible for the marked
advantage and prospect of SPIOFC in the cell-level differentiation
of gliomas from neural cells. MMP-2, a specific receptor of
chlorotoxin, only exists on the membrane of glioma cells rather
than normal brain tissue cells, including neural cells. In glioma
cells, the close connection between MMP-2 and chlorotoxin
contributes to the transplantation of SPIOFC into
intracellular space across the cellular membrane via
receptor-mediated endocytosis. According to our estimate, it is hard for
SPIOF to be linked to the surface of both glioma and neural
cells. More importantly, the MR and optical imaging of glioma
cells were distinct from those of neural cells after SPIOFC
was used to treat 2 cell types. Thus, MRI and optical
imaging might be a feasible and effective tool for discriminating
gliomas from surrounding normal brain tissue in cell level
during preoperative and intraoperative procedures,
respec-tively.
The preferential uptake of chlorotoxin nanoparticles
conjugated by 9L gliosarcoma cells has been reported using
cardiomyocytes as control cells[22], and our data are
consistent with those observations. However, the present study
investigated the influence of SPIOFC on glioma cells
originated from humans and rats. The species did not affect the
efficacy of SPIOFC in the specific labeling of glioma cells.
The U251-MG cell line is glioma cell line of human origin.
These provide the insight into the future clinical application
of SPIOFC. The biocompatibility of SPIOFC was assessed
in this study, and the encouraging results indicated that
SPIOFC was suitable for biomedical applications. Moreover,
the present study utilized neural cells as the control for glioma
cells, which imitated the histological difference between
central tumors and peripheral normal brain tissue in the setting
of clinical practice. Previous studies employed a 4.7-T or
3.0-T MR imager to assess the ability of SPIO in visualizing
gliomas[3,10,22]. In our study, a clinical 1.5-T MR imager was
selected. As a result, the 1.5-T MR scanner in conjunction
with SPIOFC was sufficient to produce a visual contrast
between gliomas and normal brain tissue. These facilitate the
wide application of SPIOFC in clinical practice. Nevertheless,
the in vivo biodistribution, metabolic dynamics, and
targeting effects of SPIOFC remain to be clarified. Those issues
are currently under investigation in our laboratory.
In conclusion, both SPIOFC and SPIO are safe contrast
agents for glioma and neural cells. However, SPIOFC is more
suitable for the specific and efficient targeting of glioma cells
than SPIO, because chlorotoxin play a critical role in the
specific and efficient transporting of functional SPIO into
glioma cells rather than neural cells. As a result, MRI and
optical imaging in conjunction with SPIOFC in
vitro can
differentiate glioma cells from normal brain tissue cells.
Acknowledgements
We are grateful to professor Keiji KAWAMOTO
(Depart-ment of Neurosurgery, Kansai Medical University, Osaka,
Japan) for providing the glioma cell lines.
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