Mesoporous particles
Size Effect on Cell Uptake in Well-Suspended, Uniform
Mesoporous Silica Nanoparticles**
Fang Lu, Si-Han Wu, Yann Hung, and Chung-Yuan Mou*
The problem of size effect on cell uptake of nanoparticles is a
currently important issue in the field of nanobiology. Particle
size is an important parameter in designing suitable celltracking and drug-carrier nanoparticle systems,[1,2] because it
determines the mechanism and rate of cell uptake of a
nanoparticle and its ability to permeate through tissue. The
investigation of particle size effects will impact on all
applications of nanoparticles in biomedicine. It has been
found that particle size can affect the efficiency and pathway of
cellular uptake by influencing the adhesion of the particles and
their interaction with cells.[3] Vallhov et al. reported that
particle size is an important factor that affects the immune
response of human dendritic cells.[4] The cell uptake of
liposomes,[5] quantum dots,[6] and polymeric,[7–10] gold,[11] and
silica nanoparticles[12] has been found to be size-dependent.
However, many factors, such as surface chemistry, charge, and
size, will affect the uptake.
Up to now, previous investigations on size effects in cell
uptake did not include careful control of the charge of the
nanoparticles, so the reported size effect was not clear in
physicochemical terms. On the other hand, theoretical models
based on membrane elasticity and ligand–receptor interaction
appeared to provide insights into the dynamics of endocytosis
of nanoparticles.[13–15] To help with the synergism of
theoretical and experimental investigations, experimental
studies of cell uptake of nanoparticles with uniformity of
zeta potential and surface functionality are much desired for
elucidating the effect of size on cell uptake. However, wellsuspended inorganic nanoparticles (in the range of 30–280 nm)
with controlled surface potential and monodisperse size
distribution are synthetically challenging. Herein, we report
the synthesis of a monodisperse mesoporous silica nanoparticle (MSN) suspension and the study of the effect of
nanoparticle size on cell uptake. MSNs are favored because
we will show that tight control of particle size and charge can
be achieved simultaneously.
[] Dr. F. Lu, S.-H. Wu, Dr. Y. Hung, Prof. C.-Y. Mou
Department of Chemistry and Center for Condensed Matter Sciences
National Taiwan University
Taipei 106 (Taiwan)
E-mail: cymou@ntu.edu.tw
[] This research was supported by a grant from the National Research
Council, Taiwan (NSC-95-2752-M-002-004-PAE).
: Supporting Information is available on the WWW under http://
www.small-journal.com or from the author.
DOI: 10.1002/smll.200900005
small 2009, x, No. x, 1–6
Mesoporous silica materials have been used in many fields
in recent years due to their large surface area, large pore
volume, and facile surface modification potential.[16] They
have many traditional applications, such as catalysis[17] and
chromatography.[18] New biomedical applications in cell
imaging,[19] diagnosis and bioanalysis,[20] and drug/gene/
protein delivery[21] have recently gained attention. For
biomedical applications, the control of pore size, morphology,
and particle size of ordered mesoporous silica is important.[22]
Compared with bulk mesoporous silica materials, which are
above a micrometer in size, nanometer-sized mesoporous
silica particles (30 to 500 nm) have additional properties, such
as fast mass transport, effective adhesion to substrates, and
good suspension in solution.
Small particles with diameters less than 100 nm often have
a poorly ordered mesostructure.[23] Few papers have reported
the formation of well-ordered mesoporous silica materials
with a diameter less than 100 nm. Imai, Ostafin, and coworkers separately described their success in the synthesis of
aggregates of well-ordered mesoporous silica particles with
diameters less than 100 nm.[24] However, interparticle aggregation and polydispersity in the reported samples are great
handicaps for their biological application in general, and for
correctly evaluating the size effect of MSNs on cellular uptake
in particular. It is still a challenge to prepare well-ordered,
discrete mesoporous nanoparticles with uniform size distribution that could form stable colloidal suspensions.
We recently reported the synthesis of dye-functionalized
well-suspended MSNs 110 nm in size that could be internalized into biological cells with high efficiency.[25] It would
be interesting to find out the size dependence of cell uptake.
Herein, we report a simple method for tailoring the size of
well-ordered and dispersed MSNs by adjusting the pH of the
reaction medium, which leads to a series of MSNs with
uniform size ranging from 30 to 280 nm. The nanoparticles
are well suspended in solution. We also note that this range
of particle sizes is less than that of the leaky pores of blood
vessels supplying tumor cells. The physical properties of the
MSNs were characterized with various methods, such as
nitrogen adsorption–desorption, transmission electron microscopy (TEM), X-ray powder diffraction (XRD), zeta
potential, and dynamic light scattering (DLS). Then, we
studied the particle size effect on the cell uptake efficiency
with human cervical cancer cells (HeLa cells). Cellular
uptake of MSNs of various sizes was investigated by confocal
laser scanning microscopy (CLSM). The uptake efficiencies
were determined by inductively coupled plasma mass
spectrometry (ICP-MS) analysis of total silicon content.
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The cytotoxicity of the MSNs was examined by MTT assay
(see the Experimental Section).
Well-suspended uniform MSNs were prepared by a twostep method under dilute tetraethyl orthosilicate (TEOS) and
low surfactant concentration conditions in NH4OH solution.
Dilute TEOS (0.2 M, in ethanol) was added to the ammonia
solution containing cetyltrimethylammonium bromide
(CTAB) as the first step. A clear and homogeneous solution
was formed. Then an ethanolic solution of TEOS (1.0 M) and
the fluorescent dye N-1-(3-trimethoxysilylpropyl)-N0 fluoresceyl thiourea (FITC-APTMS, see Experimental Section) was
added, and a precipitate started to form as the reaction
proceeded. The time at which the precipitate appears is highly
dependent on the pH of the reaction mixture: a lower pH value
needs a longer precipitation time. The dye-functionalized
MSNs were formed by adding FITC-APTMS before ethanolic
TEOS (1.0 M) was added in the second step. Our extraction
procedure allows complete removal of surfactant while
retaining covalently bonded dye. This results in a stable
suspension of MSNs. On the other hand, calcination of the
product would lead to a nonsuspendible aggregate due to
interparticle dehydration of surface silanol groups (see
Supporting Information, Figure S1). Previous methods[24] of
making size-controlled MSNs were based on calcination and
are thus unsuitable for biological applications. The prepared
MSNs are termed MSN-x where x is the pH of the reaction
solution in the preparation process.
TEM images (Figure 1) show that the particle size of the
MSNs can be modulated by controlling the pH of the reaction
solution. A decrease in particle size is observed when the pH is
decreased from 11.52 to 10.86 by reducing the amount of
ammonium hydroxide. Under these reaction conditions, the
highest pH (11.52) resulted in particles with the largest size,
approximately 280 nm (Figure 1a). Fast Fourier transform
(FFT) analysis of local-area electron diffraction (inset of
Figure 1a) clearly shows that the silica nanoparticles have a
regular two-dimensional (2D) hexagonal symmetry structure.
As the pH decreased to 11.38, 11.32, and 11.00, the average
particle size changed to 170 (Figure 1b), 110 (Figure 1c, f), and
50 nm (Figure 1d), respectively, while the regular hexagonally
ordered pore structure remained. When the lowest pH (10.86)
was employed, the diameter decreased to approximately
30 nm (Figure 1e), but the pore structure was not as well
ordered; instead, a wormlike mesostructure was formed.
Similar results were reported by Bein and co-workers; they
decreased the amount of organic base used (triethanolamine)
to obtain a steady decrease in particle size. However, the
wormhole-type pore assembly dominated the silica nanoparticles.[23a]
The hydrodynamic diameters of the MSNs (Figure 2) were
measured by using DLS. Except for the 30-nm sample, the
observed hydrodynamic diameters are just slightly larger than
those observed in the corresponding TEM images. This is
understandable because hydrodynamic diameters are generally larger than the core particle sizes observed by TEM.
Figure 2 also shows the same trend of a steady decrease in
particle size as the pH decreases from 11.52 to 11.00, like the
TEM images. This shows that these MSNs suspend very well in
solution, and no interparticle aggregation occurs. However,
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Figure 1. TEM images of mesoporous silica with different average
sizes: a) 280 nm; inset: FFT analysis of the TEM image; b) 170,
c) 110, d) 50, e) 30 nm. f) High-resolution TEM image of a single particle
in (c).
Figure 2. DLS measurements of MSNs synthesized with decreasing pH
values from 11.52 to 10.86.
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Figure 3. a) XRD patterns of MSNs prepared at pH in the range of
11.52–10.86. b) N2 adsorption–desorption isotherms of various MSNs.
particles obtained at pH 10.86 differ substantially in their TEM
and hydrodynamic diameters. The average hydrodynamic
diameter of MSN-10.86 is 130 nm, much bigger than the 30 nm
seen in the TEM image. This result is attributed to some
aggregation of the MSNs in solution.
Figure 3a provides XRD patterns for various sizes of MSNs
formed at different pH values. All the patterns show an intense
XRD peak and three weaker peaks (except for MSN-10.86),
which is characteristic of a 2D hexagonally ordered structure.
Dye-loaded FITC-MSNs have essentially the same XRD
patterns (see Supporting Information, Figure S2). The
excellent structural order of the MSNs is rather unusual for
nanoparticles of mesoporous silica of such small sizes. The
structural order is much better than that in previous reports,[23]
in which a single broad (100) peak was generally obtained in
the XRD pattern. The structural order of our MSNs is so good
that faceted hexagon-shaped MSNs are often observed, as
shown in Figure 1f. In fact, it is probably the good structural
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order of the MSNs that leads to the uniformity in size
distribution.
Figure 3b presents the N2 adsorption–desorption isotherms for different MSNs. The samples exhibited type IV
isotherms, as expected for mesoporous silica with highly
uniform cylindrical pores. Filling of the framework-confined
mesopores occurred at P/P0 ¼ 0.3–0.5. Each sample also
displayed an additional capillary condensation of N2 at high
relative pressures (P/P0 > 0.90), a signature of a high degree of
textural porosity.[26] The smaller particle size synthesized
under lower pH values affords a higher level of textural
mesoporosity. Different MSNs display similar pore size
distribution curves with a pore size of about 2.5 nm by the
Barrett–Joyner–Halenda (BJH) method (see Supporting
Information, Figure S3). Similar behavior was observed for
FITC-MSN-x. The N2 adsorption–desorption isotherms and
pore size distributions of FITC-MSN-x are shown in Figures S4
and S5 in the Supporting Information.
It is known that the rate of hydrolysis of TEOS and the rate
of condensation of Si–OH to form siloxane bonds are the two
key steps in controlling the morphology of silica structures.[27]
The study by Aelion et al. on the influence of electrolyte
concentration on the hydrolysis of TEOS showed that the
hydrolysis rate increases linearly with the concentration of
OH in basic medium.[28] Often a slow hydrolysis rate would
lead to a long delay time in forming the silica precipitate. On
the other hand, the pH dependence of the silica condensation
rate is not monotonic. Below pH 8.0 it is an increasing function
of pH, reaching a maximum at pH 8.4. Above the maximum,
the silica condensation rate decreases as the pH increases
because the silicates are increasingly negatively charged. In
our system, more OH ions were present at higher
concentrations of ammonium hydroxide. Hence, the hydrolysis rate of TEOS at higher pH is faster, thus leading to more
primary silicate species in the solution. These primary species
self-assemble with surfactants to form micelle–silicate assemblies. Then, nuclei of the new 2D hexagonal phase form when
enough condensation occurs. In the meantime, the pH
decreases as hydrolysis progresses, and more primary silicate
species condense to the 2D nuclei which in turn grow larger.
Finally, although the growth process accelerates, the materials
are limited resulting in a finite size. In the nucleation step, the
silica condensation is rate-limiting. At higher pH values, there
are smaller numbers of nuclei, which therefore results in larger
MSNs. Our design of the two-step process is essential for a
uniform particle size distribution. The prehydrolyzed silicates
in the first step facilitate simultaneous growth from seeds upon
the addition of a large amount of TEOS in the second step, to
give uniform particles. The half-width of the particle size
distribution is about 10% of the particle size.
We also measured the surface charge of FITC-MSNs with
various sizes, because the surface charge could affect the
ability of MSNs to internalize cells and to escape endosomal
entrapment.[29] Figure 4 shows the zeta potential versus pH of
an aqueous solution of FITC-MSNs with various sizes. All
samples showed similar zeta potential/pH curve behavior
between pH 5 and 9. Isoelectric points (IEPs) at pH 6.5–6.9
were observed for all samples. This indicates that FITC-MSNs
with various sizes have a similar surface charge under most pH
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Figure 4. Zeta potential of FITC-MSNs prepared at a pH ranging from
11.52 to 10.86.
conditions except for the very high pH situation (>8.5, see
Figure 4). We also note that for pure silica, the IEP should be
around 3. Our use of an amine-functionalized group in FITCAPTMS makes the IEP rise to near 7. This is an advantage in
fact because at the physiologically interesting region of pH 7
our MSN is nearly neutral in charge, which will be more useful
in drug delivery.
To investigate the size effect on cell labeling, the uptake of
various sizes of FITC-MSNs by HeLa cells was examined by
CLSM. Merged confocal microscopic images of HeLa cells
(Figure 5) show that FITC-MSNs 170, 110, 50, and 30 nm in
size (Figure 5a–d) were internalized into cells. Inside the HeLa
cells, FITC-MSN-x formed nonuniform green fluorescent
Figure 5. CLSM images of HeLa cells after incubation for 5 h at 37 8C
with FITC-MSNs (100 mg mL1, green) of size a) 170, b) 110, c) 50 and d)
30 nm. The cell skeleton was stained with rhodamine phalloidin (red),
and the cell nucleus with 40 ,6-diamidino-2-phenylindole (DAPI; blue).
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Figure 6. Cell uptake of FITC-MSN-x. The graph shows the mass of
silicon per cell versus size of FITC-MSN-x.
aggregates and accumulated in the perinuclear region, but did
not penetrate the nucleus. To quantitatively determine the
uptake of FITC-MSN-x, HeLa cells were incubated with
FITC-MSNs of various sizes for 5 h in Dulbecco’s modified
Eagle’s medium (DMEM) without serum. After the allowed
time, cells were detached from the Petri dish with trypsin, and
the Si concentration was measured by ICP-MS. The results
show that the mass of silica per cell is related to the size of the
FITC-MSN (Figure 6). Clearly, cellular uptake is highly
particle-size-dependent in the order 50 > 30 > 110 > 280
> 170 nm. The uptake of 50-nm FITC-MSNs was approximately 2.5 times that of 30-nm particles, 4 times that of 110-nm
particles, 20 times that of 170-nm particles, and 11 times that of
280-nm particles. The optimum size of 50 nm for cell uptake
that we have observed is similar to the cell uptake of other
particles recently reported.[6,11,30] Osaki et al. reported
that 50-nm ‘‘glycovirus’’ entered cells via receptor-mediated
endocytosis more efficiently than smaller nanoparticles.[6]
Additionally, Chan and co-workers investigated herceptinfunctionalized gold nanoparticles ranging from 14 to 100 nm,
and also reported that the maximum uptake by cells occurred
at a nanoparticle size of 50 nm.[11,30] To evaluate the in vitro
cytotoxicity of MSNs, cell viability was examined by MTT
assay on the FITC-MSNs of diameter 50 nm (see Supporting
Information, Figure S7). The results show that there is little
cytotoxicity and cell proliferation was not hindered by the
presence of MSNs for loadings of up to 100 mg mL1.
In summary, we have presented a simple method for
controlling the size of well-ordered and dispersed MSNs
ranging from 30 to 280 nm in diameter by adjusting the pH of
the reaction solution. We also examined the influence of
particle size on the uptake of FITC-MSNs by HeLa cells.
Uptake is particle-size-dependent and the maximum uptake
by cells occurs at a nanoparticle size of 50 nm. These findings
suggest that MSNs 50 nm in diameter may be the most suitable
candidate to serve as a carrier for further studies in biological
applications. It is expected that the size effect on cell uptake
would lead to size-dependent biochemical responses.[15] The
detailed downstream cell response, however, needs more
detailed study.
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small 2009, x, No. x, 1–6
Experimental Section
Characterization: The particle size and zeta potential were
respectively measured by photon correlation spectroscopy and
zeta potential measurement employing a Nano ZS90 laser particle
analyzer (Malvern Instruments, UK) at 25 8C. The zeta potential
values were calculated by using the Smoluchowski equation.
Small-angle powder XRD patterns were obtained by using a Rigaku
Rotaflex diffractometer with CuKa radiation (l ¼ 0.154 nm). N2
adsorption–desorption isotherms were obtained at 196 8C on a
Micromeritics ASAP 2010 sorptometer by static adsorption
procedures. Samples were degassed at 100 8C and 103 Torr for
a minimum of 12 h prior to analysis. Brunauer–Emmett–Teller
(BET) surface areas were calculated from the linear part of the BET
plot according to IUPAC recommendations. Pore size distribution
was estimated from the adsorption branch of the isotherm by the
BJH method. TEM images were obtained on a JEOL 100CX
microscope with a CeB6 filament and an accelerating voltage of
100 kV.
Preparation of MSNs: MSNs of different sizes were prepared by
a two-step preparation method under dilute TEOS and low
surfactant concentration conditions with NH4OH as catalyst. First,
CTAB was dissolved in NH4OH (pH ¼ 11.52, 11.38, 11.32, 11.00,
10.86, separately) at 40 or 50 8C, and dilute TEOS (0.2 M, in
ethanol) was added with stirring. The stirring was continued for
5 h, and then more ethanolic TEOS (1.0 M) was added with
vigorous stirring for 1 h. The solution was then aged at 40 or 50 8C
for 24 h. Samples were collected by centrifugation at 18 000 rpm
for 20 min, washed, and redispersed with deionized water and
ethanol several times. The surfactant templates were removed by
extraction in acidic ethanol (1 g of HCl in 50 mL ethanol, 24 h)
rather than by calcination, which would result in aggregation. The
dye-functionalized MSNs were synthesized by the same process,
except that FITC-APTMS was added before more ethanolic TEOS
(1.0 M) was introduced in the second step. FITC-APTMS was formed
by stirring fluorescein isothiocyanate (FITC) in a solution of 3aminopropyltrimethoxysilane (APTMS) in ethanol in the dark for
24 h.
Cell uptake: HeLa cells were seeded at 2 106 cells in a 10-cm
Petri dish and were cultured in DMEM containing 10% fetal bovine
serum and penicillin/streptomycin at 37 8C in 5% CO2 and 95%
air. After 24 h of cell attachment, the cells were treated with FITCMSNs (100 mg mL1) of various sizes for 5 h at 37 8C in serum-free
medium. Then, the cells were washed with phosphate-buffered
saline (PBS) three times for further testing.
CLSM: HeLa cells were seeded on Lab-Tek chambered cover
glasses (Nalge Nunc International, Napervile, IL, USA) and were
cultured in DMEM as above. Treated cells were then washed
several times with PBS and fixed with 4% paraformaldehyde at
room temperature for 10 min. The cells were washed with PBS
three times and incubated with 0.2% Triton X-100 and then 3%
bovine serum albumin in PBS for 5 and 30 min, respectively.
Rhodamine phalloidin was used for staining the filamentous actin
skeleton at room temperature for 20 min. The nucleus was stained
with DAPI (2 mg mL1) in H2O for 5 min. The samples were
observed by a confocal spectral microscopy imaging system (Leica
TCS SP5).
ICP-MS detection of FITC-MSN uptake: HeLa cells were seeded
at 2 106 cells in a 10-cm Petri dish and were cultured as
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described above. The cells were washed with PBS three times
and trypsinized. After centrifugation, the cell pellet was washed
once. The cells were resuspended in water and were centrifuged
again. After the cells were dried overnight, 48–51 wt% HF (100 mL)
was added to allow dissolution of the MSNs with ultrasound.
Then, 2 wt% HNO3 (9.9 mL) in aqueous solution was added to
allow dissolution of the cells with ultrasound. These clear acidic
solutions were diluted for further testing. The mass of FITC-MSNs
in the HeLa cells was measured by detecting the silicon
concentration with ICP-MS.
MTT assay: 1 105 HeLa cells per well were seeded in 24-well
plates 24 h before proliferation assays. After incubation with
different amounts of FITC-MSNs in suspension in serum-free
medium for 5 h, the cells were allowed to grow in regular growth
medium for 24 h followed by incubation with fresh serum-free
medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 0.5 mg mL1) for 4 h at 37 8C for the
proliferation assay. The amount of dark blue formazan dye
generated by the live cells was proportional to the number of
live cells, and the absorbance at 570 nm was measured by using a
microplate reader (Bio-Rad, model 680). Cell numbers were
determined from a standard plot of known cell numbers versus
the corresponding optical density.
Keywords:
cell uptake . mesoporous materials . nanoparticles .
particle size . silica
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Received: January 5, 2009
Published online:
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