JOURNAL OF NANOSCIENCE LETTERS
Influences of size of silica particles on the cellular endocytosis,
exocytosis and cell activity of HepG2 cells
Ling Hu, Zhengwei Mao, Yuying Zhang, Changyou Gao*
MOE Key Laboratory of Macromolecular Synthesis and Functionalization,
Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China
*
Author for correspondence: Changyou Gao, email: [email protected]
Received 25 Nov 2010; Accepted 5 Jan 2011; Available Online 12 Jan 2011
Abstract
Internalization of nanoparticles into live cells closely correlates with their potential applications,
functions and cytotoxicity. In this study, fluorescein isothiocyanate (FITC) or rhodamine B isothiocyanate (RITC)
doped silica nanoparticles with four different diameters were prepared. Their average size and distribution was
measured by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The optical property
and zeta potential in serum containing medium of the FITC-SiO2 nanoparticles were detected, revealing that they
had good stability and negative surface charge of similar value. Flow cytometry found that the uptake behavior of
the FITC-SiO2 nanoparticles was dependent on their size and concentration as well as culture time. The particles
mainly distributed inside the cytoplasms and endosomes, and on the cell membranes. Transportation of the silica
nanoparticles into the cells was energy dependent via a clathrin-mediated endocytosis pathway, which was proved
by an obvious decrease of the cellular uptake under a low temperature incubation, and NaN3, sucrose and
amandatine-HCl treatments. The impact of silica nanoparticles on the HepG2 cells was assayed in terms of
cytotoxicity and cell cycle as well, revealing that they had little influence on cytotoxicity even at a high particle
concentration of 680 μg/ml, and no influence on the DNA synthesis at a particle dosage of 80 μg/ml for 24 h.
Cellular exocytosis of the FITC-SiO2 nanoparticles was confirmed after removal of the free particles from the
culture medium, which was governed by the particle size and pretreatment time too.
Keywords: Silica; Nanoparticles; Cellular uptake; Endocytosis; Pathway; Cytotoxicity; Cell cycle
1. Introduction
With the rapid development of
nanotechnology, many kinds of nanomaterials
have been and are being used in fields of
industry and scientific researches. Especially, a
wide range of engineered nanoparticles, ranging
from 1-1000 nm, have been proposed to be used
in nanomedicine due to their unique physical and
chemical characteristics [1]. As a non-metal
oxide, silica (SiO2) nanoparticles have been
widely used industrially as chemical mechanical
polishing, additives for drugs, cosmetics, printer
toners and varnishes [2], Recently, the use of
silica nanoparticles has been extended to the
biomedical and biotechnological fields, such as
biosensors for simultaneous assay of glucose,
lactate, L-glutamate, and hypoxanthine levels in
rat striatum [3], biomarkers for cellular imaging
[4], cancer therapy [5], DNA and drug delivery
[6-8] and enzyme immobilization [9]. Moreover,
silicone dioxide is also a major component
constituting the sandstorm, which is influencing
many areas of the world nowadays. Therefore,
the public unavoidably exposes to the
environment of silica particles, whose effect on
the biological system is thereby becoming a
critical issue.
Compared to other nanomaterials, the
versatile silica nanoparticles stand out as an ideal
platform to build up multimodal nanoparticles as
well. These silica nanoparticles can be doped
with desired fluorophores (visible or near
infrared region) to generate fluorescent
nanoparticles [10-12] which are useful for
sensing, identifying and tracking intracellular
structures [13-16] and local conditions such as
pH [17] and redox potential [18]. Compared to
those polymer-based nanoparticles, the silica
nanoparticles show less aggregation, little dye
leakage and are chemically inert and physically
stable [19, 20]. A large number of dye molecules
can be incorporated inside a single silica particle
under appropriate synthetic conditions and the
silica matrix provides an effective barrier
keeping the trapped dye from the surrounding
environment, minimizing both photobleaching
and photodegradation [21]. Moreover, the silica
nanoparticles can be incorporated with different
bioactive molecules (such as enzymes, protein,
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peptides and chemotherapeutic drugs) following
standard conjugation protocols to achieve
specific biological functions [22-24]. The
flexible chemistry with the silica nanoparticles
provides
versatile
routes
for
surface
modifications as well. For example, hydroxyl,
amino, thiol and carboxyl groups can be
introduced onto the particle surface with silane
reagents
[25-28],
adding
other
new
functionalities such as probes for magnetic
resonance (MR)/radio imaging, therapeutic and
targeting molecules [29]. All these features make
the silica nanoparticles excellent labeling
reagents for biological applications [30-32] with
various targets under different conditions, from
the single-molecular level to human body
applications, and from in vitro diagnosis to in
vivo real-time imaging [33, 34].
Although many concerns have been
paid to the mesoporous silica nanoparticles in
terms of cell-particle interactions [35-42], very
few attempts are made to explore the interactions
of non-porous silica nanoparticles with cells in
terms of endocytosis, exocytosis, pathways of
endocytosis and cytotoxicity etc [2, 43, 44].
Because the silica particles could be found
everywhere in our life, it is important to be
aware of their potential effects on human body.
Therefore, basic research on the interactions of
silica particles with cells should be performed,
which can in turn guide their future applications
in biology and medicine. In this paper,
fluorescently labeled silica nanoparticles with
different sizes are synthesized and their
interactions with HepG2 cells are studied in
terms of dynamic cellular uptake, exocytosis and
cell activity. The pathways of the cellular uptake,
the cytotoxicity and the potential influence on
cell cycle brought by the uptake are assayed too.
Since the liver is a major organ which deals with
waste, here the HepG2 cells, a kind of liver
source cell line, are chosen in order to disclose
the interaction between the particles and liver
cells.
2. Experimental
2.1. Materials
Fluorescein isothiocyanate (FITC),
rhodamine
B
isothiocyanate
(RITC),
amandatine-HCl, genestein, propidium iodide
(PI) and RNase A (DNase-free) solution were all
purchased from Sigma-Aldrich. Lyso Tracker
Green
DND-26
(Invigrogen,
Molecular
ProbesTM) was used to stain the lysosomes in
cells, which gives fluorescence by excitation at
488 nm. Tetraethyl orthosilicate (TEOS) and
sodium azide (NaN3) were purchased from
Beijing chemical reagents company (Beijing,
China). 3-Aminopropyl triethoxysilane (APS)
was purchased from Taizhou chemical plant
(Hangzhou, China) and used after vacuum
distillation. Human liver cancer cells (HepG2)
were maintained in Dulbecco’s Modified Eagles
Medium (DMEM, Gibco) supplemented with
100 U/ml penicillin, 100 μg/ml streptomycin and
10% fetal bovine serum (FBS, Sijiqing Co. Ltd.,
Hangzhou, China). The cells were incubated at
37 °C in a humidified atmosphere containing 5%
CO2 and used at an appropriate degree of
confluence. All other reagents were analytical
grade and used without further purification.
Endotoxins free (<0.05 EU/ml) Millipore water
with a resistivity of 18.2 M was used throughout
the experiments.
2.2. Particle preparation
FITC-modified APS was prepared from
a reaction between the amino group of APS and
isothiocyanate of FITC under a nitrogen flow
using a standard Schlenk line technique [45, 46].
In a typical synthesis process, 0.28 ml TEOS and
1 ml FITC-modified APS solution (in ethanol,
the weight ratio of TEOS/APS was 0.26/0.0094)
were separately injected into a mixture of 42.4
ml ethanol and 18.8 ml water under 30 oC.
Condensation reaction was immediately initiated
by addition of ammonia solution (0.8 ml 30 wt%
of NH3.H2O) [47]. Another 2.52 ml TEOS was
injected into the flask after 1 h. 5 h later, the
FITC labeled silica nanoparticles (FITC-SiO2)
were obtained by repeated centrifugation and
washing with ethanol, and then freeze-dried. The
FITC-SiO2 with different diameters were
obtained by changing the weight ratio of
TEOS/FTIC-APS, the volume ratios of the
alcohol (or isopropanol for larger silica
particles), water and ammonia solution [48]. The
RITC labeled silica particles with same
diameters of the FITC-SiO2 particles were
similarly prepared by using RITC modified APS.
2.3. Characterization
Morphology
of
the
FITC-SiO2
nanoparticles
was
characterized
by
a
transmission electron microscope (TEM, JEOL
JEM-200) with an acceleration voltage of 100
kV. Briefly, aqueous dispersion of the particles
was drop-cast onto a carbon coated copper grid,
and air dried. The average particle size and size
distribution was also measured by dynamic light
scattering (DLS, Beckman Coulter, USA) after
the dried particles were re-suspended in
phosphate buffered saline (PBS, pH 7.4) and
DMEM/10% FBS under sonication, respectively.
Zeta potential of the particles was detected by a
zeta potential analyzer using an aqueous dip cell
in the automatic mode (Zetasizer 3000, Malvern
Instruments, Southborough, MA). The samples
were prepared by mixing the particle suspension
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in PBS with equal volume of 2 DMEM solution
or 2 DMEM/20% FBS solution, resulting in a
same medium as the cell incubation. The pH
value of all the pre-tested solutions was adjusted
to 7.4. Optical property of the FITC-SiO2
particles was detected at room temperature with
a fluorescence spectrometer (LS55, Perkin Elmer
precisely) after they were dispersed in PBS under
sonication. The ratio of the fluorescence intensity
for these particles was obtained by comparing
the integrated areas of the emission peaks and
was further used for normalization of the flow
cytometry data.
2.4. Cell culture and uptake/exocytosis of the
fluorescent silica nanoparticles
The cells were seeded onto a 24-well
culture plate (Corning, New York, USA) at a
density of 10×104 cells per well with 1 ml
culture medium (DMEM containing 10% FBS,
supplemented with 100 U/ml of penicillin and
100 μg/ml of streptomycin) and were cultured at
37 oC in 5% CO2 for 24 h to allow their
attachment. The particles of different diameters
were added into the cell culture medium at given
concentrations (10 μg/ml - 640 μg/ml) and coincubated with the cells for 24 h to explore the
correlation of cellular uptake and particle
concentration. For the cellular uptake dynamic
analysis, at given time intervals the HepG2 cells
were carefully washed 3 times with PBS, and
then detached by 0.25% trypsin and resuspended in PBS after centrifugation and
washing in PBS for several times. For the
exocytosis process, the particles of different
diameters with a final concentration of 80 μg/ml
were first added into culture medium at 37 oC
and co-incubation with the HepG2 cells for 12 h.
The cells were then carefully washed 3 times
with PBS to remove the free particles. This time
was set as the starting point of the exocytosis
process. After 1 ml fresh cell culture medium
was added into each well, the cells were
continually cultured until the pre-determined
time intervals. The cells were carefully washed
with PBS, trypsinized, re-suspended in PBS. The
particles-free cultured cells were used as
negative controls.
The cell suspensions were introduced
into a FACSCalibur flow cytometry (BD
Bioscience) with a 488-nm argon ion laser for
the quantitative study of the cellular
uptake/exocytosis
of
the
FITC-SiO2
nanoparticles. The Cellquest software (BD
Bioscience) was used to analyze the FACS data
if not otherwise stated. The mean fluorescence
intensity per cell was averaged from at least
10,000 cells. The cellular uptake/exocytosis
caused by the particle internalization was
assessed using the difference between the mean
fluorescence intensity of the treated cells to that
of the controls. After normalized by the relative
fluorescence intensity ratio of these particles
respectively, the fluorescence intensity per cell
was directly correlated to the uptake/exocytosis
amount of the particles. The cellular uptake
efficiency is defined as the ratio of the uptaking
cells to the total cells, and the cellular exocytosis
fraction is defined as the ratio of the removed to
the uptaken particles of the cells.
2.5. Distribution of the fluorescent silica
nanoparticles in HepG2 cells
To monitor the dynamic cellular uptake
and distribution of the fluorescent silica
nanoparticles with different diameters, all the
cells were cultured on glass slides (the slides
were pre-laid on the bottom of the wells in a 24
well culture plate) in the presence of 40 μg/ml
particles at 37 oC. After co-incubation for 1 h
and 18 h, the free particles were aspirated and
the cells were carefully washed with PBS. The
cells were then stained by diluted Lyso Tracker
Green solution in PBS for 10~15 min, followed
by thorough washing with PBS. The images
were acquired using confocal laser scanning
microscopy (CLSM, LSM510, Zeiss). Section
thickness was manually set smaller than 0.7 μm
and located in the middle plane of the cells.
2.6. Morphology of HepG2 cells after cellular
uptake
To observe the cell morphology under
scanning electron microscopy (SEM), the cells
were carefully washed with PBS and fixed with
2.5% glutaraldehyde (GA) in PBS at 4 oC for 48
h after co-cultured with the FITC-SiO2
nanoparticles of different diameters for 18 h.
After removal of the excessive GA, the cells
were dehydrated with a graded series of ethanol,
and treated with acetone and isoamyl acetate.
After dried by the critical point drying method,
the cells were sputter coated with an ultrathin
gold layer and observed under SEM (Cambridge
stereoscan 260 and FEI SIRION-100).
2.7. Cellular uptake inhibition
Various well characterized inhibiting
drugs can induce certain degree of block in
specific steps in the cellular uptake pathways and
were chosen as the inhibitors to study the
mechanism of the cellular uptake. A suitable
concentration of each additive was chosen for
efficacy. These inhibitive additives, including
sodium azide, sucrose, amandatine-HCl and
genestein, were injected into the cell culture
medium prior to addition of the FITC-SiO2
nanoparticles at a final concentration of 50 mM,
200 mM, 5 mM and 0.1 mM, respectively [49,
50]. The fluorescent silica nanoparticles with
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different diameters were then immediately added
into each culture well with a final concentration
of 80 μg/ml and were co-incubated at 37 oC. The
cells without additives were also co-incubated
with these silica nanoparticles at 4 oC. After 4 h
incubation, the cells were carefully washed with
PBS, trypsinized, re-suspended and introduced
into a flow cytometry. The data were analyzed as
mentioned above.
2.10. Statistical analysis
Data were obtained from 3 parallel
experiments and are expressed as mean ±
standard deviation (S.D.). Each experiment was
repeated for 3 times to check the reproducibility
if not otherwise stated. Statistical analysis was
performed by two-tailed Student’s t-tests
between two groups. The significant level was
set as p<0.05.
2.8. Toxicity of the fluorescent silica
nanoparticles
The cell toxicity was measured by a
methylthiazoletetrazolium (MTT) method which
is a simple non-radioactive colorimetric assay
[51]. Briefly, 200 μl cell suspension was seeded
onto each well of a 96-well polystyrene plate,
with a final cell density of 1×104 /well. After 24
h, 20 μl particle suspension was added into the
culture medium with a final concentration of 190
μg/ml and 680 μg/ml, respectively. The culture
medium was changed every 2 d with the fresh
culture
medium
containing
the
same
concentration of FITC-SiO2 nanoparticles. 20 μl
5 mg/ml MTT was added into each well and
incubated for 4 h at 37 oC before measurement.
After the culture medium was aspirated, the dark
blue formazan crystals were dissolved with
dimethyl sulfoxide. The absorbance of the
supernatant was recorded at 570 nm by a
microplate reader (Bio-Rad model 550). The
cells initially cultured with the particles and then
cultured with MTT-free medium were used as
the blank references (The optical density was set
as 0). The cells cultured with particles-free
culture medium were set as controls. The data
were analyzed with 3 parallel experiments and
were expressed as mean ± standard deviation.
3. Results
2.9. Cell cycle analysis [52]
HepG2 cells were seeded onto a 24-well
plate at a density of 10×104 cells per well in 1 ml
complete culture medium. The FITC-SiO2
nanoparticles of different diameters with a final
concentration of 80 μg/ml were separately cocultured with the cells at 37 oC for 24 h. The
cells were carefully washed 3 times with PBS,
harvested by trypsinization, washed twice with
PBS and then re-suspended and fixed in 75%
ice-cold ethanol under -20 °C overnight. After
washed with PBS for 1 time, the cells were resuspended in cold PI stain buffer solution (20
μg/ml PI and 100 μg/ml DNase-free RNase A in
PBS) for 30 min. The samples were analyzed by
flow cytometry under a 543-nm argon ion laser.
By statistically analyzing the DNA content in at
least 20,000 cells, the DNA content distribution
which is representative of the cell cycle
distribution was obtained. Untreated HepG2 cells
were used as negative controls.
3.1. Preparation and characterization of FITCSiO2 particles
To study the uptake of the silica
nanoparticles with the equipments such as flow
cytometry and CLSM et al, fluorescent labeling
with good enough stability is necessary. In this
work, FITC or RITC was covalently conjugated
to APS, which was then reacted with TEOS to
or
RITC-SiO2
obtain
the
FITC-SiO2
nanoparticles. By variation of the fabrication
conditions, the fluorescently tagged SiO2
particles with different sizes were fabricated, as
typically shown in Figure 1 for the FITC-SiO2
particles. They showed a regular spherical shape
and smooth surface, except for the smallest ones
(Figure 1a). The mean diameter of each type of
the particles was obtained by measuring about 50
particles under TEM. Table 1 summarizes the
data of the four FITC-SiO2 particles with
diameters of 60.3 nm (S60), 177.8 nm (S180),
368.6 nm (S370) and 592.1 nm (S600),
respectively.
The sizes of the particles were also
measured by DLS in PBS and DMEM/10% FBS,
which are basically used to treat the particles for
cell culture. Figure 2a shows that only 1 peak
was recorded for each type of the particles
regardless of the type of the solvents. Moreover,
all the particles showed almost the same average
diameters as that characterized by TEM. Slight
overlapping between the S60 and S180, S370
and S600 could be observed, but the degree was
not significant. In DMEM/10% FBS, the average
diameters of all the particles and the
polydispersity indexes were increased to some
extent, implying that slight particle aggregation
had occurred. It is understandable that the small
particles have large surface area and tend to
aggregate to reduce the surface energy,
especially under the existence of proteins which
decrease the surface charge intensity (Figure 2b)
and may bind between the particles.
Nevertheless, the aggregation was not significant
and no serious overlapping of two types of
particles was observed. Therefore, the colloidal
dispersity and stability of the particles are good
enough for cell uptake evaluation in further
experiments.
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(a)
(b)
(d)
(c)
Figure 1. TEM images of FITC-SiO2 nanoparticles with different size. (a) S60, (b) S180, (c) S370, (d) S600.
The zeta potentials of these silica
nanoparticles were measured in DMEM and
DMEM/10% FBS medium, at which conditions
the cells were cultured. Figure 2b shows that all
the particle surfaces were negatively charged,
which is resulted from the abundant -OH groups
on the particle surfaces. The absolute value of
the zeta potentials were decreased to some extent
when the silica nanoparticles were dispersed in
the DMEM/10% FBS medium, revealing the
adsorption of serum proteins which are less
negatively charged at physiological conditions (5.2±1.1 mV). Therefore, the initial surfaces with
different charging degrees became almost
identical due to the coverage of the proteins.
The fluorescence property of these
silica particles was quantitatively measured in
PBS (Figure 2c), from which the relative
fluorescent intensity of the silica particles was
calculated as 1.2 : 4.9 : 2.7 : 1.0 for S60, S180,
S370 and S600, respectively. Since the
fluorescent dye was doped inside the particles,
the fluorescent intensity of the particles was very
stable and not sensitive to the environment at
least for a week (over 95% of the fluorescent
signals were remained, data not shown).
Together with the relatively narrower
distribution (Table 1), these particles can thus be
used to study the influences of particle size on
the cellular performance since the difference in
their surface chemistry in the culture medium
can be neglected.
3.2. Cellular uptake of fluorescent silica
nanoparticles
Figure
3a
and
3b
displayed
representative FACS assessments of the control
and the particles-ingested HepG2 cells. Cells of
the M2 group were defined as the cells uptaking
nanoparticles (internalized or tightly adsorbed on
the cell surfaces) because they showed
fluorescent signals originated from the silica
Table 1. Diameters of FITC-SiO2 nanoparticles measured by TEM and DLS in different medium.
Measuring method
Particles size (nm)
TEM
S60
60.3 ±3.7
S180
177.8±6
S370
368.6±13.8
S600
592.1±18.4
DLS in PBS
74.5 (0.13)a
153.9 (0.16)
346.4 (0.19)
578.4 (0.21)
DLS in
DMEM/10% FBS
102.3 (0.16)
196.1 (0.18)
407.1 (0.25)
662.7 (0.33)
\
a
Data in the parentheses represent the polydispersity index.
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40
S370
S180
30
S600
S60
20
10
-10
-15
-20
-25
0
200
400
600
800
1000
S600
S370
-5
(a)
0
S180
S60
0
In PBS
In DMEM/10% FBS
Zeta potential (mV)
Number fraction percentage (%)
JOURNAL OF NANOSCIENCE LETTERS
In DMEM
In DMEM/10% FBS
(b)
-30
1200
Particle size (nm)
Fluorescence intensity
800
S60
S180
S370
S600
700
600
500
400
300
200
100
Figure 2
0
500
(c)
510
520
530
540
550
Wavelength (nm)
Figure 2. (a) Size distribution and (b) zeta potentials of FITC-SiO2 nanoparticles measured in PBS and
DMEM/10% FBS by DLS. (c) Fluorescence spectra of FITC-SiO2 nanoparticles with a concentration of 12.2
μg/ml for S180 and 24.4 μg/ml for all other particles.
nanoparticles. Cellular uptake (endocytosis) of
the particles follows the general process
identified before, in which the particles are
enclosed by a portion of the cell membranes and
brought into the cells in a membrane-bound
vesicle [53]. In the first study of the cellular
uptake of the FITC-SiO2 particles, the influence
of particle concentration was assessed at a coincubation time of 24 h (Figure 3c). The amount
of internalized particles showed a positive
correlation with the concentration regardless of
the particle size, but their absolute amount and
alteration patterns were different. Initially, the
cellular uptake was increased slowly at a lower
particle concentration, and then increased rapidly
until ~320 g/ml. For the smaller particles such
as S60 and S180, further increase of uptake was
not observed, but for the S600 particles the
uptake was still increased until 640 g/ml
detected so far (p<0.01). Figure 3c also shows
that the uptake was increased along with the
particle size at a given concentration, especially
above 160 g/ml.
Secondly, the dynamics of cellular
uptake was monitored for a 24 h culture period
by using HepG2 cells (Figure 3d) with 40 g/ml
particles. It shows that the uptake monotonously
increased along with the prolongation of the
culture time, except for the S60 particles which
were saturated at 3 h (Figure 3d, p<0.05).
Moreover, decrease of the uptake amount for the
S60 particles was found in the following
experimental time (3 h to 24 h, p<0.05). The
cellular uptake efficiency by the HepG2 cells
was found to be 80.1%, 99.6%, 97.6% and
88.6% for the S60, S180, S370 and S600,
respectively, indicating that almost all the cells
had ingested the S180 and S370 particles. These
results reveal that all the particles can be
effectively internalized or bound onto the cell
surface regardless of the particle size, but the
uptake amount and uptake efficiency are really
size dependent.
3.3. Distribution of fluorescent silica
nanoparticles in HepG2 cells
Cellular uptake and distribution of the
silica nanoparticles was further studied by
CLSM (Figure 4). Here the lysosomes of the
cells were stained with green color, while the
RITC-SiO2 particles with red color were used.
Overlay of the green and red colors produced
yellow, indicating the co-localization of the
particles and lysosomes. At the first 1 h, some
particles adhered onto the cell membranes but
were hardly found inside the cytoplasms (Figure
4a-d). This result is consistent with the FCM
characterization, in which the fluorescence
intensity per cell was quite low at the first 1 h,
since these adhered particles might be partially
removed during the treatment of the cells. When
the co-culture time was extended to 18 h, many
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(b)
600
140
S60
S180
S600
500
400
300
200
100
(c)
0
0
100
200
300
400
500
600
700
Mean fluorescence intensity per cell
Mean fluorescence intensity per cell
(a)
S60
S180
S370
S600
120
100
80
60
40
20
(d)
0
0
5
Concentation(g/ml)
10
15
20
25
Incubaiton time (h)
Figure 3
Figure 3. Representative FACS pictures of control cells (a) and cells uptaking FITC-SiO2 nanoparticles (b).
M1 and M2 represent the particle-free and particles-ingested cells, respectively. Uptake of the FITC-SiO2
nanoparticles by HepG2 cells as a function of (c) concentration with a culture time of 24 h and (d) culture
time with a particle concentration of 40 g/ml. Data were measured by flow cytometry and averaged to each
cell. * Significant difference (p<0.05) from respective control.
particles formed red fluorescent aggregates and
accumulated both on the membranes and inside
the cells. No particles were found inside the
nuclei (Figure 4e-h). The existing yellow color,
which was most prominent in the S370 sample,
reveals that some of the particles resided in the
lysosomes. Therefore, the fluorescent silica
S370
S180
S60
(b)
(a)
S60
nanoparticles with a negative surface can
distribute on the cell membranes, in the
cytoplasms and lysosomes. In this sense, the
FCM collects the overall fluorescent signals,
including that from the particles adhering on the
cell membranes.
S180
S600
(c)
S370
(d)
S600
(e)
(f)
(g)
(h)
4 18 h during the uptake process of FITC-SiO2
Figure 4. CLSM images taken at (a-d) 1 hFigure
and (e-h)
nanoparticles by HepG2. (a), (e) S60; (b), (f) S180; (c), (g) S370 and (d), (h) S600.
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(a)
(b)
S60
S370
(d)
S180
(c)
S600
(e)
Figure 5. SEM images to show cell morphology (a) before and after cellular uptake of (b) S60, (c) S180, (d)
S370 and (e) S600 for 18 h, respectively.
3.4. Morphology of HepG2 cells after cellular
uptake
After the fluorescent silica particles
were incubated with HepG2 cells for 1 h, very
few ones adsorbed onto the HepG2 cells and the
cell morphology was not obviously changed in
comparison with the particles-free cells (Figure
5a). After 18 h, a relative large number of silica
particles attached onto the cell membranes in a
format of clusters for the smaller ones such as
S60 and S180 (Figure 5b,c). However, there
were many S370 and S600 particles which singly
adhered onto the cell surfaces (Figure 5d,e).
nanoparticles
There are several possible uptake
pathways for the nanoparticle internalization,
including fluid-phase endocytosis, adsorptive
endocytosis and receptor-mediated endocytosis.
In the receptor-mediated endocytosis, certain
ligands on the particle surfaces are needed to
bind to the corresponding receptors on the cell
surface [54]. The mechanism for cellular uptake
can be mainly divided into four categories:
clathrin-mediated
endocytosis,
caveolaedependent endocytosis, phagocytosis and
micropinocytosis [55]. Since the FITC-SiO2
nanoparticles could be efficiently internalized
into the HepG2 cells, some pharmacological
3.5. Uptake pathways of the fluorescent silica
200
control
o
4C
NaN3
140
120
Mean fluorescenc intensity per cell
Mean fluorescence intensity per cell
160
100
80
60
40
20
0
S60
S370
S180
S600
Control
Sucrose
Amandatine-HCl
Genestein
180
160
140
120
#
#
100
#
80
#
60
40
20
0
S60
S180
(a)
S370
S600
(b)
Figure 6. Uptake of the FITC-SiO2 nanoparticles by HepG2 cells under different treatments. The cells were
incubated with 80 g/ml FITC-SiO2 nanoparticles for 4 h under (a) 4 oC or the existence of 50 mM NaN3, and
(b) with the existence of 200 mM sucrose, 5 mM amandatine-HCl and 0.1 mM genestein, respectively. # No
significant difference (p > 0.05) from respective control.
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JOURNAL OF NANOSCIENCE LETTERS
120
100
Cell viability
(% untreated control cells)
Cell viability
(% untreated control cells)
100
80
60
S60
S180
S370
S600
40
20
0
80
60
S60
S180
S370
S600
40
20
0
1
2
3
4
1
Incubation time (d)
2
3
4
Incubation time (d)
(a)
(b)
Figure 7. Viability of HepG2 cells as a function of incubation time in the presence of (a) 190 μg/ml and (b)
680 μg/ml SiO2 nanoparticles. The data were normalized to that of the particle-free control cells.
inhibitors were chosen to explore the uptake
mechanism in this study. First, the endocytosis is
an energy-dependent process [36], indicating an
inhibition of cellular uptake under low
temperature or ATP-depleted (adenosinetriphosphate) environment [56]. In order to
determine the silica nanoparticle internalization
via endocytosis, HepG2 cells with 80 g/ml
particles were co-cultured under 4 oC or in the
medium having NaN3, each for 4 h. As shown in
Figure 6a, a significant decrease of the
fluorescence intensity (p<0.01) was observed at
both conditions, leaving a value of only 10%20% of control regardless of the particle size. For
a further study of the existing pathway during
cellular endocytosis, sucrose, amandatine-HCl
and genestein were chosen as inhibitors for
different kinds of endocytosis. It is known that
sucrose and amandatine-HCl can inhibit the
clathrin-mediated endocytosis by disrupting the
formation of clathrin-coated vesicles [57] and
preventing the budding of clathrin-coated pits
[58], respectively, and genestein has an ability to
block tyrosine kinases of the Src-family involved
in caveolae-mediated uptake [59, 60]. Figure 6b
shows that uptake of the particles by the HepG2
cells was weakened and mostly inhibited after
sucrose and amandatine-HCl treatments,
respectively (p<0.05). The percentage of cellular
uptake was decreased to 46.8%, 56.2%, 41.3%
and 41.1% after the sucrose treatment and
significantly to 1.4%, 6.4%, 5.9% and 16.2%
after amandatine-HCl pre-treatment for S60,
S180, S370 and S600, respectively (p<0.01). By
contrast, the cellular uptake was not significantly
influenced (p>0.05) by the genestein treatment.
These results disclose the mechanism of clathrinmediated endocytosis for the cellular uptake of
the silica nanoparticles. Since the similar results
Journal of Nanoscience Letters | Volume 1 | Issue 1 | 2011
were obtained for the four samples, this
mechanism can be applied universally to the
silica nanoparticles with the diameter ranging
from 60 nm to 600 nm.
3.6. Toxicity of SiO2 particles
Cytotoxicity of the FITC-SiO2 particles
was tested using a MTT assay. At a lower
particle concentration, i.e. 190 μg/ml, all the
cells could normally proliferate and maintain
>80% viability to that of the control cells during
the 4 d culture (Figure 7a). In particular, the cells
cultured with S60 even showed higher viability
than the control after day 3 (p<0.05). When the
concentration of the silica nanoparticles was
improved to 680 μg/ml, relatively lower viability
was recorded for all the particles, in particular
for the larger ones (Figure 7b). Especially at day
1, the cells cultured with S370 and S600 showed
obviously lower viability than those cultured
with S60 and S170 (p<0.05). Nevertheless, for
all the samples the cell viability could still
increase along with the culture time as that of the
control. These results confirm that uptake of the
silica particles brings rather low toxicity to the
HepG2 cells.
3.7. Cell cycle analysis
Cell cycle is a series of events which
can induce the cell division and duplication. In
eukaryotes, the cell cycle is consist of two
periods including interphase (gap1 (G1),
synthesis (S) and gap2 (G2)) and mitosis (M)
phase [54]. The cell cycle analysis can reflect
whether the cellular uptake will influence the
proliferation of live cells during their co-culture.
For this study, at least 20,000 cells were
collected to analyze the cell cycle status which
was classified into G1, S and G2 phases. Figure
8 displays a representative FACS-histogram and
9
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1800
2400
60
Number
90
R1
600
30
SSC-H
Debris
Aggregates
Dip G1
Dip G2
Dip S
1200
120
G1
G2
0
0
S
0
30
60
90
120
0
30
60
90
120
150
Channels (FL2-A)
FSC-H
(a)
(b)
Figure 8. A representative (a) FACS-histogram and (b) a picture of cell cycle assessment obtained from the
FACS data. The data was analyzed by Modfit software.
a picture of cell cycle analysis by the Modfit
software. Table 2 summarizes the relative
percentage of the phases for the cells coincubated with the FITC-SiO2 particles of
different sizes. No significant difference was
found for all the samples, revealing that cellular
uptake of the FITC-SiO2 nanoparticles does not
induce adverse effect on the cell cycle. This is
consistent with the cytotoxicity assay.
3.8.
Exocytosis
of
fluorescent
silica
nanoparticles
The nanoparticles may suffer from the
enzymes degradation or exocytosis after cellular
uptake. In the exocytosis process, the materials
are packaged in secretary vesicles inside the cell
and then the vesicles fuse with the plasma
membrane and finally open to the exterior space
[61]. For this study, the FITC-SiO2 particles
were firstly co-incubated with the HepG2 cells
for 12 h, and then with particles-free medium for
another 12 h. Figure 9a shows that the
fluorescence intensity was monotonously
decreased for all the samples, suggesting that the
initially internalized particles were cleaned out
steadily during this culture period. To better
quantify and thereby compare the ability of
particle clearance, the data in Figure 9a were
transformed to exocytosis percentage and shown
in Figure 9b. Clearance of the particles during
the first few hours was more prominent for all
the samples, with final exocytosis percentages of
63%, 67%, 58% and 38% for S60, S180, S370
and S600, respectively, revealing that smaller
particles are more easily cleaned out. Moreover,
clearance of the particles depends on the preincubation time as well. For example, if the preincubation time was extended to 24 h, the
exocytosis percentages dropped to 50% and 10%
for S60 and S600 even after a 24 h culture,
respectively.
4. Discussion
The silica nanoparticles are widely used
in industry, scientific research and more recently
biological field along with the development of
nanotechnology. People are inevitably exposed
to an environment with silica nanoparticles.
These tiny particles can enter the human body
through skin, lung or intestinal tract and may
deposit in several organs and act on living cells
at the nano-level [62-64]. Therefore, it is of high
significance and urgency to study the
Table 2. Influence of cellular uptake of FITC-SiO2 nanoparticles on cell cycle of HepG2.
Sample
control
S60
S180
S370
S600
Cell cycle distribution
G1%
73.1
74.8
75.3
76.9
76
S%
18
15.7
15
14.6
15.6
G2%
8.9
9.5
9.7
8.5
8.4
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125
Fraction of nanoparticles exocytosed
Mean Fluorescence Intensity per cell
JOURNAL OF NANOSCIENCE LETTERS
S60
S180
S370
S600
100
75
50
25
0
0
2
4
6
8
10
12
0.7
0.6
0.5
0.4
0.3
S60
S180
S370
S600
0.2
0.1
0.0
0
2
4
6
8
10
12
Exocytosis time (h)
Exocytosis time (h)
(b)
(a)
Figure 9. Time dependent exocytosis of FITC-SiO2 nanoparticles from HepG2. (a) mean fluorescence
intensity per cell, and (b) fraction of exocytosed particles.
interactions of silica nanoparticles with cells
from the biological and ecological point of view.
It is known that the cellular uptake and
the process of cellular delivery are influenced by
various factors such as the physiochemical
properties of the nanoparticles (chemical
composition, size, shape and surface charge), the
concentration of the nanoparticles, the incubation
time, and the cell types, etc [35, 56, 65-67]. In
the biological applications, the particle size is an
important parameter when they are used as
fluorescent probes or drug carriers [68, 69]. It
has been confirmed that the particle size plays a
key role in their adhesion to and interaction with
biology cells. For example, the cellular uptake of
both inorganic (gold [56], quantum dots [70],
silica [37, 38, 71]) and organic (liposomes [72],
polymer [73-76]) nanoparticles are sizedependent. However, other results showed that
the extent of particle uptake is indirectly
proportional to the particle size [77]. The particle
size can also affect the pathway of cellular
uptake [78, 79] and the cytotoxicity [39, 71, 80].
Moreover, the body circulation time and
biodistribution of the nanoparticles are also
influenced by their sizes [81-83]. However, there
is lack of universal disciplines describing the
size effect on the interactions of nanoparticles
with cells, which is therefore of practical
importance to be explored.
In this regard, the FITC-SiO2
nanoparticles with four different diameters were
synthesized (Figure 1, Table 1). The mono
dispersed silica nanoparticles could adsorb serum
proteins and eventually had a similar surface
charge in the culture medium (Figure 2b), so that
the influence of surface chemistry can be ruled
out. They showed a slight larger aggregation in
DMEM/10% FBS too (Figure 2a), presumably
due to the protein adsorption and weakening of
surface charge and thereby electrostatic
repulsion. Since the FITC or RITC was doped
inside the particles, the dye became stable and
not sensitive to the environment. The fluorescent
intensity of four size particles had been
normalized in further quantitative comparison of
the cellular uptake study (Figure 2c).
Figure 3a shows that along with the
increase of particle concentration, the amount of
uptaken particles was monotonously increased
regardless of the particle size, which is consistent
with the results reported previously [56]. It is
easy to understand that if the particle
concentration is too low in the bulk, the particles
would have lower opportunity to meet cells and
thus the cellular uptake is relatively hard to
happen. Higher uptake could be achieved by
increase of the particle concentration in the
culture medium to some extent. Above this
value, the cellular uptake does not increase
again. At such as case, it is conceivable that the
bottom of the culture well could be overspread
with the nanoparticles and thereby the further
added particles are not able to contact with the
cell membranes. Since the mass of a larger
particle is (rlarge/rsmall)3 times of the smaller one,
e.g. 960 times for the S600 to S60, for a similar
extent of overspreading on the culture well it is
quite reasonable that the larger particles are
uptaken with larger amount and a higher
saturation concentration. However, the number
of larger particle is much smaller, e.g. the HepG2
took about 1% S600 compared with S60 at a
feeding particle concentration of 640 g/ml.
The dynamic cellular uptake of the
silica nanoparticles in 24 h showed the sizedependent feature too. The uptaken amount
monotonously increased until 24 h, except for
the S60 which decreased after 3 h. A similar
decrease was also found for the PLGA
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JOURNAL OF NANOSCIENCE LETTERS
internalization [67]. The reason is not clear so
far. One possible explanation is that the cells
prefer to eat relative larger particles.
Consequently, exocytosis of the particles might
happen as observed in Figure 8, leading to a
decrease of the particle amount inside the cells.
Another possible reason is that smaller particles
precipitate slower and/or have larger surface area
with certain mass, leading to a lower
concentration of particles which the cells can
“feel”. Figure 3b,c shows also that the dynamic
cellular uptake lasted for a relatively long time,
which is also reported previously [56,73]. A
further study by co-incubation of HepG2 with
S180 and S370 for 48 h found that the cellular
uptake was increased by a factor of 13.9% and
4.9% compared with that at 24 h, respectively.
This is not a big increase compared with the
earlier stage before 24 h. The slope of the curves
in Figure 3b,c can be regarded as the rate of
cellular uptake. For the initial 3 h, the HepG2
cells took up the silica nanoparticles in a order of
rate of S60 > S370 (≈ S600) > S180. The uptake
rate had no obvious difference among S180,
S370 and S600 from 3 h-24 h. Jin et al reported
that 23 nm silica nanoparticles penetrated 81% of
A549 cells, whereas the 85 nm ones penetrated
only 16% over 1 h, demonstrating the cell
membrane penetration of silica nanoparticles is
size-dependent with a faster rate for smaller
particles [71]. Chithrani et al also found that the
cellular uptake of Au nanoparticles significantly
increased in the first 2 h, and slowed down to
reach a plateau at 4-7 h. The saturation time for
cellular uptake and uptake rate of Au
nanoparticles is size-dependent too, with an
uptake half-life of 2.10 h, 1.90 h and 2.24 h with
a rate of 622, 1294 and 417 nanoparticles per
hour for the 14 nm, 50 nm and 74 nm Au
nanoparticles, respectively [56].
The cellular uptake amount was sizedependent. A recent communication compared
the size influence on cellular uptake of the
mesoporous silica nanoparticles (MSN) of
different sizes too. The amount of these particles
were uptaken by HeLa cells in the order of 50
nm>30 nm>110 nm>280 nm>170 nm [37].
Chithrani et al reported that the Au nanospheres
of 50 nm could be internalized most by HeLa
cells among others with a diameter ranging from
14 nm - 100 nm [56]. According to Osaki’s
report, 50 nm ‘‘glycovirus’’ entered more
efficiently into cells via receptor-mediated
endocytosis than smaller ones (15 nm and 5 nm)
[70]. Win et al compared the cellular uptake of
polystyrene (PS) nanoparticles with different
diameters, showing that the PS nanoparticles
internalized Caco-2 cells in an order of amount
of 100 nm>200 nm>500 nm>1000 nm> 50 nm
[73]. Gaumet et al found that the cellular uptake
of 100 nm poly(lactic-co-glycolic acid) (PLGA)
nanoparticles into Caco-2 cells is most efficient
[84]. Jin et al confirmed that the cellular uptake
of carbon nanotubes with the length of 320 nm
and 430 nm is more pronounced than 660 nm
and 130 nm for NIH-3T3 cells [85]. These
reports reveal that the size influence varies
greatly and even contradicts from case to case,
which might be the results of chemical and
structural differences. Furthermore, the cellular
uptake efficiency is slightly affected by the
particle size too. The results showed that more
percentage of HepG2 cells internalized S180 and
S370 for a 24 h co-cultivation.
It is known that the cellular uptake can
take place by the adsorptive and receptor
mediated endocytosis pathways. Generally,
smaller particles (0.2 μm) and larger ones (>0.5
μm) are endocytosed via pinocytosis and
phagocytosis, respectively [54]. In more detail,
the clathrin-mediated endocytosis is mediated by
small vesicles with a diameter of 100-150 nm,
the caveolae dependent endocytosis forms small
and flask-shaped pits in a diameter of 50-100
nm, and the micropinocytosis usually occurs
from highly ruffled regions of the plasma
membrane to form 0.5-2 µm vesicles in
diameter. In the phagocytosis the cells can bind
and internalize particulate matter larger than 0.75
µm [79]. In the present study, the FITC-SiO2
nanoparticles were designed negatively charged
and not decorated with any specific ligands. As a
result they were all internalized in HepG2 cells
by nonspecific endocytosis. However, the
particle size showed no influence on the
distribution and uptake mechanism of the silica
nanoparticles with a diameter ranging from 60
nm to 600 nm in HepG2 cells (Figure 4-6).
These nanoparticles enter into the HepG2 via the
clathrin-mediated pathway, but the caveolae
independent endocytosis (Figure 6b). The
internalized particles mainly distributed in
lysosomes and cytoplasms, but not in the cell
nuclei (Figure 4). Interestingly, the amandatineHCl reduced endocytosis percentage was
weakened with the increase of particle size,
which has also been observed previously by
Rejman et al [86]. Lu et al reported that the
fluorescent MSN with the diameters of 170 nm,
110 nm, 50 nm and 30 nm are accumulated in
the perinuclear region of HeLa cells, but cannot
penetrate the nucleus [37]. However, Chen et al
found that 70 nm silica nanoparticles can
translocate into the nucleus of epithelial cells
[87]. The pathway for the cellular uptake has
been well documented too. Witasp et al found
that uptake of the MSN with diameters of 378
nm and 2.4 μm by macrophages is an active,
energy-dependent process through both the
endocytosis and phagocytosis pathways [71].
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Chung et al showed that the cellular uptake of
110 nm MSN by human mesenchymal stem cells
and 3T3-L1 cells is via the clathrin-mediated
endocytosis and actin-phagocytosis pathways
[35]. Xing et al proved that the RITC-SiO2
nanoparticles can be transported into HeLa cells
in part through adsorptive endocytosis and in
part through fluid-phase endocytosis [88]. It is
assumed that the efficient nonspecific uptake of
MSN is partially due to their strong affinity to
clathrin-coated vesicles [89].
The cytotoxicity of nanoparticles is of
critical importance for their applications in the
biological field. The nanoparticles are very
reactive in the cellular environment because of
their large surface area per unit mass. It has been
confirmed that the cellular uptake of
nanoparticles can induce a series of responses to
cells to affect the cell behaviors, such as cell
growth,
apoptosis,
adhesion,
migration,
differentiation, survival and tissue organization,
resulting in the cytotoxicity [90, 91]. As a widely
used product, the silica nanoparticles have been
studied in terms of the cytotoxicity [2, 41, 65,
66, 92]. Generally, amorphous silica nano
particles is considered to be safe and accredited
as a food ingredient by FDA. As Brunner et al
reported, the silica nanoparticles are non-toxic to
human mesothelioma cells and mouse embryonic
fibroblast cells under the concentrations from 015 mg/ml for 6 d and 30 mg/ml for 3 d [65].
However, Chang et al observed that the silica
nanoparticles are slightly toxic to cells at high
concentrations (138 μg/ml), especially for
normal human fibroblast cells, by retarding the
cell proliferation and destroying the cell
membrane [66]. Lin et al also demonstrated that
the silica nanoparticles (15 nm and 46 nm)
significantly reduced the viability of human
alveolar epithelial cells [2]. Our previous results
showed also that cellular uptake of the silica
nanoparticles by HepG2 can affect cell adhesion
and migration [92]. In the present study, cellular
uptake of the silica nanoparticles showed low
cytotoxicity to HepG2 cells with slight sizedependency and concentration-dependency. The
MTT absorption of HepG2 cells appeared to be
higher for S60 under a lower particle
concentration (170 μg/ml). This could be
explained by two possible reasons: 1) small
particles may induce higher mitochondrial
activity, and 2) small particles may influence cell
proliferation. Since uptake of the silica
nanoparticles did not affect the DNA synthesis in
HepG2 cells by the cell cycle analysis (Table 2),
the improved MTT absorption is mostly
attributed to higher mitochondrial activity
induced by small particles. One can thereby
conclude that compared with other types of
nanoparticles the silica nanoparticles possess
overall better safety in a cellular level.
The fate of the nanoparticles after the
cellular uptake is a critical issue yet has not been
well disclosed so far. One of the important
events is exocytosis of the nanoparticles, which
is an opposite process to the endocytosis [55, 93,
94]. The exocytosis of nanoparticles can directly
influence their applications in biological system,
especially for those non-degradable or
decomposable particles like SiO2. In the present
study, the average fluorescence intensity per cell
decreased with the post culture time after the free
particles were removed from the culture medium
(Figure 9a), indicating that the particle expulsion
from the cells takes place. There is a balance of
the particle concentration inside the cells and in
the culture medium which governs the
endocytosis and exocytosis. After exchange with
the particle-free culture medium, the particle
concentration inside the cells becomes
instantaneously high, and thereby drives the
cellular exocytosis to such an extent that a new
balance can be established. The influence of cell
proliferation and apoptosis, which reduce the
particle number per cell, can be ruled out since
the total cell number kept unchanged during the
12 h post culture. Figure 9b reveals that the
cellular exocytosis is size-dependent, e.g. larger
particles like S600 are hard to be exocytosed.
This phenomenon was also observed by
Chithrain et al [95], in which the Au
nanoparticles of smaller size could be more
easily removed by HeLa cells with a faster rate
and a larger dosage. Moreover, a longer preincubation time makes the exocytosis of the
silica nanoparticles more difficult. Unlike the
endocytosis, the exocytosis of nanoparticles is
less studied in terms of the disciplines and
mechanisms, and therefore should be paid more
attention in the future study.
5. Conclusions
In summary, FITC or RITC doped SiO2
nanoparticles ranging from 60 nm to 600 nm
were prepared, which showed similar surface
potential (-7~-10 mV) in FBS containing culture
medium. The SiO2 nanoparticles uptaken by the
HepG2 cells increased along with the particle
concentration and particle size. The uptaken
amount for the S180, S370 and S600 was also
monotonously increased along the 24 h culture
time, except for the S60 which was saturated at 3
h. The uptaken amount increased along with the
particle size and reached maximum for S370,
and then decreased significantly. It was also
found that the particles distributed inside the
cytoplasms and lysosomes and on the cell
membranes. Aggregates of smaller particles were
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JOURNAL OF NANOSCIENCE LETTERS
observed on the cell membranes too. The uptake
of the silica nanoparticles was energy dependent
via a clathrin-mediated endocytosis pathway
regardless of the particle size. Although uptake
of the particles especially the larger ones with
high amount (680 μg/ml) brought some extent of
cytotoxicity, no influence was found on the cell
cycle. Upon removal of the free particles from
the culture medium, up to 60% of the
internalized particles with a smaller size (<370
nm) were exocytosed within a 12 h post culture.
These results thus disclose the basic processes
and intrinsic interactions of the silica particles
with cells, and may in turn guide their
applications in a more safe and reasonable way.
14.
15.
16.
17.
18.
19.
Acknowledgment
This work is financially supported by
the Natural Science Foundation of China
(50903069, 50873087) and Zhejiang Provincial
Natural Science
Foundation of
China
(Z4090177).
21.
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