Biotin-decorated fluorescent silica nanoparticles with aggregation-induced emission characteristics: Fabrication, cytotoxicity and biological applications

Article abstract of DOI:10.1039/c2tb00155a

Biotin-decorated fluorescent silica nanoparticles (FSNPs) were successfully fabricated by a sol-gel reaction of silole-functionalized siloxane followed by a sequential reaction with tetraethoxysilane, (3-aminopropyl)triethoxysilane and biotin. The FSNPs were uniformly sized, spherical in shape and monodispersed. While their silole precursor was non-emissive in solution, the suspension of the FSNPs emitted strong green light upon photoexcitation due to the aggregation-induced emission characteristics of the silole aggregates in the hybrid nanoparticles. Morphology study and cell viability, trypan blue exclusion, Annexin V-FITC/PI apoptosis and ROS generation assays showed that the FSNPs showed low toxicity to living cells. The FSNPs worked as fluorescent visualizers for selective imaging of the cytoplasm of tumor cells with over-expressed biotin receptors. The fluorescent nanoparticles were lastingly retained inside the living cells, thus enabling long-term tumor cell tracking over multiple passages and quantitative analysis of tumor cell migration. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2tb00155a

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Materials Chemistry B
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Biotin-decorated fluorescent silica nanoparticles with
aggregation-induced emission characteristics:
Cite this: J. Mater. Chem. B, 2013, 1,
676
fabrication, cytotoxicity and biological applications†
Min Li,ab Jacky W. Y. Lam,b Faisal Mahtab,b Sijie Chen,b Weijie Zhang,a Yuning Hong,b
a
a
b
*
*
Jun Xiong, Qichang Zheng and Ben Zhong Tang
Biotin-decorated fluorescent silica nanoparticles (FSNPs) were successfully fabricated by a sol–gel reaction
of silole-functionalized siloxane followed by a sequential reaction with tetraethoxysilane, (3-aminopropyl)
triethoxysilane and biotin. The FSNPs were uniformly sized, spherical in shape and monodispersed. While
their silole precursor was non-emissive in solution, the suspension of the FSNPs emitted strong green light
upon photoexcitation due to the aggregation-induced emission characteristics of the silole aggregates in
the hybrid nanoparticles. Morphology study and cell viability, trypan blue exclusion, Annexin V-FITC/PI
apoptosis and ROS generation assays showed that the FSNPs showed low toxicity to living cells. The
FSNPs worked as fluorescent visualizers for selective imaging of the cytoplasm of tumor cells with over-
expressed biotin receptors. The fluorescent nanoparticles were lastingly retained inside the living cells,
thus enabling long-term tumor cell tracking over multiple passages and quantitative analysis of tumor
cell migration.
Received 22nd September 2012
Accepted 15th November 2012
DOI: 10.1039/c2tb00155a
www.rsc.org/MaterialsB
become weakly emissive or non-luminescent in the solid state
due to the strong p–p stacking interactions, which promote the
Introduction
Fluorescent silica nanoparticles (FSNPs) are promising mate-
rials for bioanalysis and bioimaging as well as for disease
diagnosis and therapy because of their characteristic features
including biocompatibility, inertness, hydrophilicity, size
tuning, ease of surface modication, etc.1 Since hundreds of
uorescent molecules are encapsulated in a single nano-
particle, the detection sensitivity of FSNPs is much higher than
that of direct uorophore labeling. To date, FSNPs have been
used widely in DNA microarrays,2 immunouorescence tech-
nique,3 analogue luminescence detection,4 tissue imaging,5 cell
imaging6 and especially tumor cell imaging.1b,5,7 As silica
nanoparticles have been found to exhibit no remarkably toxic
effect on living cells,8 they thus possess the potential prospect in
tumor cell imaging for early diagnosis and cell tracking.
formation of detrimental species such as excimers or exci-
plexes.10 The weak emission offered by the FSNPs has resulted
in poor sensitivity in uorescence sensory systems, especially in
bioassays of trace amounts of biomolecules. The sensitivity
cannot be enhanced by using higher uorophore concentra-
tions due to the aggregation-caused quenching (ACQ) effect.
The development of inorganic quantum dots can surmount
these disadvantages but create new problems such as difficult
synthesis, limited variety and high cytotoxicity.11
We observed a novel phenomenon of aggregation-induced
emission (AIE) that is exactly opposite to the ACQ effect in a
series of propeller-like molecules. Instead of quenching,
aggregate formation has boosted their uorescence quantum
yields by more than 300-fold, turning them from weak uo-
rophores in the solution state to strong emitters in the solid
state.10,12 The restriction of intramolecular rotation in the
aggregated state has been proposed as the main cause for the
AIE effect.10,13 Because of their efficient solid-state emission, AIE
luminogens are thus ideal uorophores for the fabrication of
FSNPs. In our previous work, we succeeded in creating FSNPs
hybridized with AIE luminogens by a one-step surfactant free
sol–gel process. These highly emissive FSNPs pose good
colloidally stability and are non-toxic to living cells, thus
making them promising uorescent visualizers for intracellular
imaging.14 By modifying the fabrication procedure, silica
nanoparticles with both strong light emission and high
magnetization are prepared, which can function as selective cell
Conventional dyes have been used for the fabrication of
FSNPs.9 These uorophores emit strongly in solutions but
aDepartment of Hepatobiliary Surgery, Union Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan 430022, China. E-mail:
qc_zheng@mail.hust.edu.cn; Fax: +86-27-8535-1676; Tel: +86-27-8535-1623
bDepartment of Chemistry, Institute for Advanced Study, State Key Laboratory of
Molecular Neuroscience, Institute of Molecular Functional Materials and Division of
Biomedical Engineering, The Hong Kong University of Science and Technology, Clear
Water Bay, Kowloon, Hong Kong, China. E-mail: tangbenz@ust.hk; Fax: +852-2358-
1594; Tel: +852-2358-7375
† Electronic supplementary information (ESI) available: IR spectra, SEM images,
TGA thermograms, uorescent photographs, HE staining, TEM images, XPS and
EDX patterns. See DOI: 10.1039/c2tb00155a
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imaging bioprobes and immobilize bovine serum albumin generated FSNP-1-NH2 with numerous amino groups decorated
efficiently.15
on the surface, enabling it to undergo amidation reaction with
To widen the applicability of the AIE-loaded silica nano- biotin in the presence of 1,3-dicyclohexylcarbodiimide (DCC)
particles, their selectivity should be further improved. It is and 4-(dimethylamino)pyridine (DMAP) to furnish FSNP-1-
known that conjugation of targeting ligands such as proteins, biotin.
peptides and aptamers on the surface of nanoparticles can
enhance their binding affinity and facilitate receptor-mediated
internalization,16 thus enabling selective targeting and efficient
intracellular uptake. Biotin (vitamin B7 or vitamin H), a
member of the vitamin family for growth promotion at the
cellular level, is one of the most common tumor recognition
moieties because tumor cells require extra biotin to sustain
their rapid growth and compared to the normal cells, biotin-
specic receptors are thus generally over-expressed on their
surface.17 Thus, biotin has attracted particular interest in
cancer-targeting drug carrier and uorescent nanoparticles for
specic tumor cell targeting. For example, Panyam and
Structural characterization
We rst characterized FSNP-1-biotin by IR spectroscopy. The IR
spectrum of FSNP-1-biotin is given in Fig. S1 in the ESI;† for
comparison; the spectra of FSNP-1-NH2 and biotin are also
provided in the same gure. The Si–OH, Si–O and N–H
stretching vibrations of FSNP-1-NH2 occurred at 951, 1707 and
3321 cmꢀ1, respectively. Aer biotin modication, the absorp-
tion at 3321 cmꢀ1 was enhanced and new peaks associated with
C]O stretching vibrations emerged at 1638 and 1707 cmꢀ1 in
FSNP-1-biotin, revealing that biotin was covalently graed on
the surface of FSNP-1-NH2 through amidation reaction. Anal-
ysis by transmission electron microscopy (TEM) showed that
both FSNP-1-NH2 and FSNP-1-biotin were spherical in shape
and uniform in diameters with narrow size distributions
(Fig. 1). Compared to FSNP-1-NH2, FSNP-1-biotin possessed a
much rougher surface. The average particle size of FSNP-1-NH2
was measured to be 48.19 ꢁ 2.82 nm, while that of FSNP-1-
biotin was slightly larger (50.50 ꢁ 2.91 nm). Similarly, the
images from a scanning electron microscope (SEM) given in
Fig. S2† showed that the surface morphology of FSNP-1-NH2
altered little aer the biotin conjugation. To realize the
composition of the nanoparticles, X-ray photoelectron spec-
troscopy (XPS) and energy dispersive X-ray spectroscopy (EDX)
were carried out and the results are summarized in Table S1.†
XPS analyses revealed no sulfur atom on the surface of FSNP-1-
NH2, while a trace amount of sulfur element (0.3%) was
detected in FSNP-1-biotin. Similar results were also obtained
from the EDX analysis, proving that biotin is successfully
conjugated on the surface of FSNP-1-NH2. The thermal stability
of the FSNPs was investigated by thermogravimetric analysis
(TGA). As shown in Fig. S3,† FSNP-1-NH2 possessed high
thermal stability and started to degrade at a temperature of
ꢂ300 ꢃC. Even when heated up to 800 ꢃC, more than 70% of its
colleagues prepared
a biotin-functionalized drug delivery
carrier, which signicantly improved the therapeutic efficacy of
tumors.18 Kwon also modied gold nanoparticles with biotin
and rhodamine B to interact selectively with cancer cells for
diagnosis and therapy.17d However, examples of AIE-active silica
nanoparticles functionalized with biotin have not been
described.
In this paper, we aim to synthesize such nanoparticles for
specic living tumor cell imaging. A series of experiments had
been carried out to evaluate the toxicity or biocompatibility of
the synthesized nanoparticles. The biotin-decorated FSNPs
emitted strong photoluminescence (PL) under UV excitation
and interacted selectively with tumor cells with over-expressed
biotin receptors and internalized into cytoplasmic regions. They
can stay inside the live cells for long periods of time, thus
enabling long term cell tracing over multiple passages as well as
analysis of tumor cell migration quantitatively.
Results and discussion
Fabrication of biotin-decorated FSNPs
With a view to fabricate FSNPs with strong light emissions in
the solid state and biotin moieties on the surface for specic
cancer cell targeting, we synthesized
a silole-containing
siloxane (1) by stirring a dimethylsulfoxide (DMSO) solution of 2
and (3-aminopropyl)triethoxysilane (APS) at room temperature
for 24 h (Scheme 1). The sol–gel reaction of 1 followed by
reaction with tetraethoxysilane afforded FSNP-1 with a core–
shell structure.14,15 Addition of APS into the reaction mixture
Fig. 1 TEM images of (A–C) FSNP-1-NH2 and (D–F) FSNP-1-biotin at different
magnifications.
Scheme 1 Fabrication of biotin-decorated fluorescent silica nanoparticles.
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weight was retained. FSNP-1-biotin was also thermally quite
stable and degraded at a similar temperature with high
residual yield at 800 ꢃC. Since biotin decomposed completely at
650 ꢃC, the amount of biotin graed on FSNP-1-biotin could be
calculated as the weight difference between the thermograms
of FSNP-1-NH2 and FSNP-1-biotin at this temperature and was
equal to 6.92 wt%.
Cytotoxicity
To nd useful biological applications, a luminogen should
neither inhibit nor promote the growth of living cells. To eval-
uate the toxicity of FSNP-1-biotin, we rst studied the
morphology change of HeLa and mouse broblast NIH 3T3 cells
cultured in the presence of FSNP-1-biotin. As shown in Fig. S5,†
only a small amount of cells showed vacuole shrinkage and
chromatin condensation, suggesting that FSNP-1-biotin
possessed good biocompatibility. A cell viability assay was also
carried out at nanoparticle concentrations of 0.1, 1, 10, 100 and
200 mg mLꢀ1 for 48 h using a WST-8 cell counting kit. The cell
viability decreased with an increase in the nanoparticle
concentration. Compared with the control group, the nano-
particles had no discernible deleterious effects on the viability
of both HeLa and 3T3 cells (P > 0.05) at concentrations below
100 mg mLꢀ1 (Fig. 3A). At 100 mg mLꢀ1, the viability decreased to
81.60% and 85.55% for HeLa cells and 3T3 cells (P < 0.05),
respectively. To further verify the cytotoxicity of FSNP-1-biotin,
the cell survival was examined by the trypan blue exclusion
assay.8a The viable cells exclude the trypan blue dye and are not
stained to identify living cells. At a nanoparticle concentration
of 100 mg mLꢀ1, the cell survival of HeLa cells and 3T3 cells was
reduced to 88.49% and 86.74% (P < 0.05), respectively, which
echoed in the foregoing data (Fig. 3B). Consequently, the cells
were exposed to up to 80 mg mLꢀ1 of FSNP-1-biotin for the
experiments aerward in accordance with the preliminary
cytotoxicity effect and requisite nanoparticle concentration for
tumor cell imaging.
Light emission
The PL spectra of a solution of 1 and suspensions of FSNP-1-
NH2 and FSNP-1-biotin in ethanol are displayed in Fig. 2. Nearly
no uorescence signals were recorded when the ethanol solu-
tion of 1 was photoexcited. In the solution state, the multiple
peripheral phenyl rings in the isolated molecules of 1 undergo
active intramolecular rotation, which effectively annihilates
their excited states and hence renders the luminogen non-
emissive.10 When the molecules of 1 are covalently incorporated
into and aggregate in the silica networks of FSNP-1-NH2 and
FSNP-1-biotin, strong PL spectra peaked at ꢂ490 nm are
recorded. Evidently, 1, similar to its congener 2, is AIE-active.
The rigid silica network largely restricts the intramolecular
rotations of the luminogen. This blocks the nonradiative
relaxation channels and populates the radiative excitons,10,12a,19
thus making the FSNPs highly luminescent. Under the same
measurement conditions, the PL intensities of FSNP-1-NH2 and
FSNP-1-biotin were 95- and 87-folds higher than that of 1 in an
ethanol solution, respectively. The photographs of a solution of
1 and the suspensions of FSNP-1-NH2 and FSNP-1-biotin taken
under UV exposure from a hand-held UV lamp are shown as an
inset in Fig. 2. While intense light was emitted from FSNP-1-
NH2 and FSNP-1-biotin, the solution of 1 was invisible under the
UV illumination. This visual observation further substantiates
that the intramolecular rotation of 1 is restricted by its covalent
melding with the silica matrix. The light emission was very
stable, with no detectable change in the PL spectrum aer
FSNP-1-biotin was put on shelves for 6 months without pro-
tecting from light and air (Fig. S4†).
Cell apoptosis is an important parameter for the toxicity of
nanomaterials. Nanoparticles could induce apoptosis through
Fig. 3 Cytotoxicity of FSNP-1-biotin on HeLa and 3T3 cells evaluated by (A) CCK
8 and (B) trypan blue exclusion assays. The data were analyzed by one-way
analysis of variance followed by Dunnett's test and the mean value ꢁ standard
deviation was reported. * Means P < 0.05 when compared with the control (0 mg
mLꢀ1) and indicates an obvious toxicity.
Fig. 2 PL spectra of 1, FSNP-1-NH2 and FSNP-1-biotin in ethanol solutions.
Nanoparticle concentration: 200 mg mLꢀ1; excitation wavelength: 370 nm. Inset:
photographs of ethanol solutions of 1, FSNP-1-NH2 and FSNP-1-biotin taken
under 365 nm UV irradiation from a hand-held UV lamp.
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many kinds of pathways. The apoptotis-related proteins, such dichlorouorescein. Herein, the HeLa and 3T3 cells were
as Bcl-2, Bax, CytC and Caspase-3, were up-regulated when treated with various concentrations of FSNP-1-biotin for 24 h.
HepG2 cells were exposed to silica nanoparticles.20 Herein, the Aerwards, they were washed with a buffer solution and incu-
quantication of apoptosis of HeLa and 3T3 cells was studied by bated with DCFH-DA for 20 min. The uorescence from the cells
ow cytometry (FCM) with Annexin V-FITC/propidium iodide was then immediately measured by a FCM. The ROS generation
(PI) double staining assay at different concentrations of FSNP-1- and hence the uorescence from the solution became higher
biotin. The cells stained with Annexin V-FITC alone represent when HeLa and 3T3 cells were cultured with an increasing
early apoptosis, while those labelled with PI demonstrate amount of FSNP-1-biotin nanoparticles (Fig. 5). The difference
necrosis. The late apoptotic cells are stained with both uo- in the ROS generation between the control and treated cells was
rescent dyes. As depicted in Fig. 4, FSNP-1-biotin could induce small at a nanoparticle concentration of 40 mg mLꢀ1 but was
apoptosis in a dose-dependent manner. Compared with the obvious at 80 mg mLꢀ1 (Fig. 5C). However, when compared to
control group, the number of apoptotic cells increased with the positive (Rosup) control, the extent of ROS generation was
increasing nanoparticle concentration. In the presence of 80 mg not large at such nanoparticle concentrations. On the other
mLꢀ1 of nanoparticles, obvious apoptosis was observed in both hand, the intuitive uorescent images shown in Fig. 5D also
HeLa and 3T3 cells (P < 0.05).
The oxidative stress-mediated pathway is demonstrated as
one of the apoptotic mechanisms due to the induction of
nanomaterials.21 Nanoparticles can induce production of
intracellular reactive oxygen species (ROS), which can change
the permeability of a mitochondrial membrane, damage the
ultrastructure of mitochondria21a and then trigger secondary
damage effects such as mitochondrial dysfunction and DNA
damage.22 The ROS assay was based on the peroxide-dependent
oxidation of DCFH-DA to form a uorescent compound named
Fig. 4 Apoptosis of HeLa and 3T3 cells in the presence of FSNP-1-biotin for 48 h.
The cytograms given in (A) showed the fluorescence from the cells measured
using the Annexin V-FITC/PI assay at different nanoparticle concentrations.
According to the emission intensity from the fluorescence dye (“+” ¼ high, “ꢀ” ¼
low), four regions, named Q1 (Annexin V-FITC ꢀ, PI +), Q2 (Annexin V-FITC +, PI +),
Q3 (Annexin V-FITC ꢀ, PI ꢀ) and Q4 (Annexin V-FITC +, PI ꢀ) were divided, which
represented the necrotic, late apoptotic, living and early apoptotic cells, respec-
tively. (B) Quantitative analysis of cell apoptosis from three independent experi-
ments. The total apoptotic cells (Q2 + Q4) were shown in the histograms,
analyzed by Dunnett's test and compared with the control (0 mg mLꢀ1). * Means
P < 0.05 and indicates that the total number of apoptotic cells is significantly
different from that of control.
Fig. 5 Intracellular ROS generation of HeLa and 3T3 cells in the presence of
FSNP-1-biotin analyzed by the DCFH-A assay for 24 h. (A and B) FCM histograms
of HeLa and 3T3 cells, respectively, in the absence and presence of different
nanoparticle concentrations. (C) The data were analyzed by one-way analysis of
variance followed by Dunnett's test, compared with a negative (0 mg mLꢀ1) or
positive (Rosup) control and reported as mean value ꢁ standard deviation. *
Means P < 0.05 when compared with the negative control. ** Means P < 0.05
when compared with the positive control. (D) Fluorescent images of HeLa and 3T3
cells associated with ROS generation in the absence and presence of FSNP-1-
biotin.
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indicated that the nanoparticles induced ROS generation when triggers cell internalization into intracytoplasmic vesicles or the
the cells were exposed to a high concentration of FSNP-1-biotin. formation of clathrin-coated vesicles. The nanoparticles may be
further processed in vacuoles and endosomes, which are then
eventually released to the cytoplasm. On the other hand, the
nanoparticles without biotin coating enter the cells mainly by
caveolae-dependent endocytosis, whose rate, specicity and
affinity are much lower than those of clathrin-dependent
endocytosis.23 The uptake of FSNP-1-biotin nanoparticles by
LO2 cells is low due to the absence of biotin receptors on their
surface. Moreover, they, unlike HeLa and BEL-7402 cells,
require a small amount of biotin for proliferation, thus result-
ing in low nanoparticle uptake and weak uorescence.
To prove the existence of ligand–receptor interactions or the
occurrence of receptor-mediated endocytosis, uorescence
imaging of tumor cells by FSNP-1-biotin was carried out in the
presence of free biotin. As shown by the images given in Fig. 7,
the uorescence from the HeLa and BEL-7402 cells was mark-
edly decreased when they were pretreated with a 10 mg mLꢀ1
biotin solution prior to staining. This result suggests that the
free biotin molecules competitively bind to the biotin receptors
on the surface of tumor cells and interdict the receptor-medi-
ated endocytosis of FSNP-1-biotin. Evidently, bio-conjugation of
FSNP-1-NH2 with biotin molecules has enhanced the targeting
efficiency and endocytosis of the resulting FSNP-1-biotin.
The uptake of nanoparticles by cells involves several
different pathways such as nonspecic diffusion, phagocytosis
and receptor-mediated or uid phase endocytosis.24 To conrm
the uptake and distribution of FSNP-1-biotin, we analyzed the
HeLa cells treated with FSNP-1-biotin for 12 h by TEM. The
nanoparticles were distinguished based on their diameters and
spherical shape. As displayed in Fig. S6,† the nanoparticles were
Tumor cell targeting and long-term cell tracking
In our previous study, we found that FSNPs hybridized with AIE
luminogens can be utilized as uorescent visualizers for intra-
cellular imaging.14 Would FSNP-1-biotin show similar or even
better performance? To test this, the targeting property of FSNP-
1-biotin was investigated using HeLa and BEL-7402 cells, which
are typical cell lines for cervical carcinoma and hepatocellular
carcinoma, respectively, with over-expressed biotin receptors.
Normal liver cells LO2 containing less-expressed receptors were
also used in the investigation for the purpose of comparison.
Aer 3 h staining, strong PL was emitted from the HeLa and
BEL-7402 cells, while dim uorescence was observed from LO2
cells (Fig. 6). This phenomenon should be attributed to
receptor-mediated endocytosis.23 For tumor cells with over-
expressed biotin receptors on the surface, FSNP-1-biotin binds
the cell membrane via ligand–receptor interaction (Scheme 2).
The interaction of the nanoparticles with the biotin receptor
Fig. 6 Bright-field, fluorescence and overlapping images of (A1–A3) HeLa, (B1–
B3) BEL-7402 and (C1–C3) LO2 cells stained with FSNP-1-biotin for 3 h.
Fig. 7 Bright-field, fluorescence and overlapping images of (A and B) HeLa and
(C and D) BEL-7402 cells stained with FSNP-1-biotin in the (A and C) absence and
(B and D) presence of free biotin.
Scheme 2 Schematic illustration of tumor cell targeting and imaging by FSNP-
1-biotin.
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internalized into cells via receptor-mediated endocytosis just as
depicted in Scheme 2. This facilitates their uptake and inter-
nalization in the cells (Fig. S6A†). The nanoparticles were then
released into the cytoplasm (Fig. S6B†) and distributed as iso-
lated species or clusters (Fig. S6C†). No nanoparticles were
found in the nucleus, which is consistent with the literature
results, and it was presumably due to the large size of the
nanoparticles.25
In our previous work, we found that a cytophilic uorescent
bioprobe fabricated from 1-decyl-1-methyl-2,5-bis{4-[(N,N-
diethylamino)methyl]phenyl}-3,4-diphenylsilole could track
living cells for four passages, while almost no PL signals were
detected in cells labelled with MitoTracker Green FM, a
commercial dye, aer two generations.26 As an excellent tumor
targeting probe, FSNP-1-biotin should also possess the property
of high photostability and long-term tracking. Thus, the uo-
rescence from the HeLa and BEL-7402 cells stained with FSNP-
1-biotin was investigated in a continuous manner of cell
culture. The tumor cells were observed aer incubation with
40 mg mLꢀ1 nanoparticles for 24 h. When 70–80% conuence
was reached, the cells were trypsinized, counted and sub-
cultured at a density of 2 ꢄ 105 cells per well into a 6-well plate.
Generally, the cells grow into another generation within 1 day.
Although the PL from the cells became weaker along the
passage due to the division of the nanoparticles accompanied
by cellular ssion, they were still visible even aer 5 day culture
(Fig. 8). Evidently, FSNP-1-biotin is a superb long-term cell
tracer. No uorescence was observed in the cell nucleus, which
was consistent with the TEM analysis. We prolonged the culture
to 7 days. Although dim uorescence was still observed from the
cells, their morphology was hard to be discernible (Fig. S7†).
Thus, the retention time of the nanoparticles inside the cell is
approximately one week.
Fig. 9 Migration of FSNP-1-biotin-labeled HeLa cells treated with 20 or 5%
serum for 24 h. (A) Quantitative analysis of cell migration at different serum
concentrations using HeLa cells labeled with FSNP-1-biotin. The data are
expressed as mean value ꢁ standard deviation from three independent experi-
ments. * Means P < 0.05 and indicates significant difference when compared to
cells treated with 20% serum. (B) Optical images of migrated HeLa cells stained by
the conventional crystal violet.
passages. With such regard, we explored the possibility of using
FSNP-1-biotin to track the migration of tumor cells. Briey,
different concentrations of serum were loaded to the medium in
the lower compartment. FSNP-1-biotin labelled HeLa cells were
then introduced into the upper compartment of a cell. Because
the HeLa cells require additional nutrients to maintain their
rapid proliferation, they are thus induced to move from the
upper compartment to the lower one. The migrated cells were
collected by trypsin and the uorescence of the cell suspension
was then measured. As shown in Fig. 9A, the uorescence
became stronger with an increase in the serum concentration.
Since the PL intensity is associated with the number of labelled
cells, this makes quantitative analysis possible. Same result was
achieved by the conventional crystal violet staining method
(Fig. 9B) but the uorescence-based technique was more
objective and accurate.
Tumor cell migration
Conclusions
Cell migration is an important pathological process in tumor
cells and reects their ability of tissue invasiveness and
metastasis.27 In the above discussion, we found that FSNP-1-
biotin could selectively stain tumor cells with over-expressed
biotin receptors and enable long-term cell tracing over multiple
In summary, biotin-decorated uorescent silica nanoparticles
were successfully fabricated by a surfactant-free sol–gel reaction
of silole-functionalized siloxane followed by esterication
reaction with biotin. The structure of FSNP-1-biotin was inves-
tigated by IR, TEM, SEM and XPS analyses. The FSNP-1-biotin
was uniformly sized, spherical in shape and monodispersed.
Although their silole precursor was non-emissive, the FSNP-1-
biotin emitted strong green light upon photoexcitation, as a
result of the aggregation-induced emission characteristics of
the silole aggregates in the hybrid nanoparticles. Morphology
study and cell viability, trypan blue exclusion, Annexin V-FITC/
PI apoptosis and ROS generation assays showed that FSNP-1-
biotin possessed low toxicity to living cells. The FSNP-1-biotin
can be utilized to selectively image the cytoplasm of tumor cells
with over-expressed biotin receptors. They can stay inside the
living cells for a long period of time, thus enabling long term
tumor cell tracing over multiple passages and quantitative
analysis of tumor cell migration. These attributes make these
AIE-active, low cytotoxic, strongly uorescent and photostable
silica nanoparticles promising for an array of biomedical
applications.
Fig. 8 Fluorescent images of (A1–A3) HeLa and (B1–B3) BEL-7402 cells stained
with FSNP-1-biotin at different times.
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in open air. The elementary particle size was estimated by
measuring the diameter of 100 particles. The bioconjugation of
biotin on FSNP-1-NH2 was investigated by thermogravimetric
analysis (TGA). PL spectra were recorded on a Perkin-Elmer LS
50B spectrouorometer (Perkin-Elmer Corporation, USA) with
Xenon discharge lamp excitation.
Experimental section
Materials
Tetraethoxysilane (TEOS), (3-aminopropyl)triethoxysilane (APS),
dimethylsulfoxide
(DMSO),
1,3-dicyclohexylcarbodiimide
(DCC), 4-(dimethylamino)pyridine (DMAP), biotin and other
reagents were all purchased from Aldrich and used as received.
Silole–APS adduct 1 was prepared according to the reported
experimental procedures.14 Human cervical cancer HeLa cells,
human hepatoma BEL-7402 cells, normal liver LO2 cells and
mouse broblast NIH 3T3 cells were purchased from Cell
Resource Center, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences (China). Culture medium and
fetal bovine serum (FBS) were purchased from Hyclone Labs
(Logan, USA). A WST cell counting kit was purchased from
Dojindo Laboratories Co (Kumamoto, Japan). Penicillin–strep-
tomycin mixture, trypan blue exclusion assay kit, trypsin and
crystal violet were bought from Sigma-Aldrich (Saint Louis,
USA). An Annexin V-FITC Apoptosis Detection Kit was obtained
from KeyGEN BioTECH (Nanjing, China). 2,7-Dichlorohydro-
uorescein diacetate (DCFH-DA) and hematoxylin and eosin
(HE) staining assay kit were obtained from Beyotime Institute of
Biotechnology (Jiangsu, China).
Cell culture
HeLa cells were cultured in minimum essential medium. BEL-
7402 cells, LO2 cells and NIH 3T3 cells were grown in an RPMI
1640 medium supplemented with 10% FBS, 100 U mLꢀ1 peni-
cillin G and 100 mg mLꢀ1 streptomycin in a 5% CO2, 90%
ꢃ
relative humidity incubator at 37 C. Suspensions of the nano-
particles were dispersed in 0.01 M phosphate buffer solution
(PBS) by an ultrasonic bath Model SB5200 (Shanghai Branson,
China), diluted to various working concentrations, and then
added to cells immediately. Every experiment was performed in
triplicate.
Hematoxylin and eosin (HE) staining
The morphology change of cells incubated with FSNP-1-biotin
was investigated by HE staining. The HeLa cells and 3T3 cells
were seeded into a 12-well plate overnight, incubated with
nanoparticles at a concentration of 0, 40 or 80 mg mLꢀ1 for 48 h,
xed with 4% paraformaldehyde and then stained with hema-
toxylin and eosin. Hematoxylin mainly stains the chromatin in
nucleus and ribosome in cytoplasm into blue and eosin mainly
stains components in cytoplasm into pink or red. The cellular
morphology change was observed under an Olympus CKX41
phase contrast microscope (Olympus, Japan).
Preparation of FSNP-1-biotin
A silole–APS conjugate (1) was synthesized by stirring a mixture
of 6 mmol of 1,1-dimethyl-2,5-bis[4-(2-bromoethoxy)phenyl]-3,4-
diphenylsilole (2) and 16 mmol of APS in 50 mL of DMSO over-
night according to the procedure published in our previous
paper.15 Then, FSNP-1 was fabricated by a two step sol–gel
reaction.14 Briey, 1 was added to a mixture of ethanol (64 mL),
ammonium hydroxide (1.28 mL) and distilled water (7.8 mL).
The solution was then stirred at room temperature for 1 h to
prepare the silole–silica nanocores, aer which a mixture of
2 mL TEOS and 8 mL ethanol was slowly added. The reaction
mixture was stirred at room temperature for 3 h to coat the
nanocores with silica shells. FSNP-1-NH2 was prepared by stir-
ring a mixture of FSNP-1 and APS at room temperature for
additional 24 h. The nanoparticles were centrifuged and
washed with ethanol and water. Finally, the FSNP-1-biotin was
fabricated by stirring a mixture of FSNP-1-NH2 and biotin at
room temperature overnight in the presence of DCC and DMAP.
The nanoparticles were washed with deionized water and
ethanol to get rid of unwanted substances and dispersed in
deionized water or ethanol for further experiments.
Cell viability assay
The viability of cells treated with FSNP-1-biotin was measured
using a WST-8 cell counting kit (CCK-8) according to the sup-
plier's instruction. Cells were seeded into a 96-well plate at a
density of 8000 cells per well and exposed to various concen-
trations of nanoparticles for 48 h. 10 mL of a CCK-8 solution was
added into each well and the cells were incubated for additional
2 h at 37 ꢃC. The optical density was measured on a microplate
reader (Thermo, USA) using a test wavelength of 490 nm and a
reference wavelength of 630 nm.
Trypan blue exclusion assay
To further verify the cell survival rate, a trypan blue exclusion
assay was performed. The viable cells exclude trypan blue dye
and are not stained to identify living cells. Aer being exposed
to the nanoparticles for 48 h, the cells were harvested and
scored as alive or dead using trypan blue according to the
manufacturer's instruction. The number of viable cells was
counted using a conventional hemocytometer. The rate of
viability was derived by comparing with the negative control.
Characterization
The IR spectra were recorded on a Perkin-Elmer 16 PC FT-IR
spectrophotometer. The chemical compositions of the nano-
particles were determined by XPS and EDX analyses. Their
morphologies and sizes were investigated using JOEL 2010 TEM
and JOEL 6700F SEM (JEOL Ltd Tokyo, Japan) at accelerating
voltages of 200 and 5 kV, respectively. Samples were prepared by
drop-casting a dilute suspension of the nanoparticles onto
Annexin V-FITC/PI apoptosis assay
copper 400-mesh carrier grids covered with carbon-coated for- The quantication of apoptosis induced by FSNP-1-biotin in
mvar lms and the solvent was evaporated at room temperature HeLa cells and 3T3 cells was analyzed by ow cytometry (FCM)
682 | J. Mater. Chem. B, 2013, 1, 676–684
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Paper
Journal of Materials Chemistry B
with an Annexin V-FITC/PI double staining assay. Briey, aer
incubation with various concentrations of nanoparticles for
48 h, the cells were harvested with an EDTA-free trypsin solution
and then treated with 5 mL Annexin V-FITC and 5 mL PI for at
least 10 min at room temperature in the dark. Immediate
analysis was performed using FCM (BD Biosciences, USA). For
each sample, 1 ꢄ 104 cells were measured.
Tumor cell migration
The HeLa cells labelled with FSNP-1-biotin were utilized for the
cell migration assay. Briey, the cells were incubated with 40 mg
mLꢀ1 FSNP-1-biotin for 24 h, aer which they were harvested,
washed twice with 0.01 M PBS and resuspended in a serum-free
medium at a density of 5 ꢄ 105 mLꢀ1. Aer addition of 600 mL
culture medium with 20% or 5% serum to each well of the 24-
well plate, 200 mL of the cell suspension was seeded into a
millicell (8 mm pores). The cells were incubated for 24 h at 37 ꢃC
and 5% CO2. Subsequently, the cell culture inserts were trans-
ferred into a fresh 24-well plate containing 500 mL of a pre-
warmed trypsin–EDTA solution per well for 10 min at 37 ꢃC.
100 mL of the trypsin–EDTA solution was then loaded into the
quartz cell and the uorescence from the solution at 490 nm
was measured using a Perkin-Elmer LS55 spectrouorometer
(Perkin-Elmer Corporation, USA) at an excitation wavelength of
370 nm. The conventional method was performed according to
the supplier's instruction with modication. Similarly, 200 mL of
the cell suspension without pre-treatment with FSNP-1-biotin at
a density of 5 ꢄ 105 mLꢀ1 was seeded into a millicell. The cells
were induced to migrate by the serum medium in the lower
compartment. Aer removal of non-migrated cells in the upper
compartment, the cells were stained with 0.1% crystal violet and
observed under an optical microscope (Olympus, Japan).
Intracellular reactive oxygen species assay
Detection of intracellular reactive oxygen species (ROS) was
based on the peroxide-dependent oxidation of DCFH-DA to
form a uorescent compound named dichlorouorescein
(DCF). Aer treatment with various concentrations of FSNP-1-
biotin or in the absence of nanoparticles as a negative control
for 24 h, the cells were washed three times with PBS and
ꢃ
incubated with 10 mM DCFH-DA at 37 C for 20 min. The cells
incubated with 50 mg Lꢀ1 of a Rosup solution for 30 min were
treated as the positive control. The uorescence of the cells was
immediately measured using FCM (BD Biosciences, USA). For
each sample, 1 ꢄ 104 cells were collected. The visual image of
the ROS generation in cells was taken on an Olympus BX41
inverted uorescence microscope (Olympus, Japan) at an exci-
tation wavelength of 488 nm and an emission wavelength of
525 nm.
Tumor cell targeting
Statistical analysis
HeLa, BEL-7402 and LO2 cells were seeded on a round cover slip
mounted onto a 6-well plate overnight. The living cells were
incubated with a serum-free medium containing FSNP-1-biotin
at a specic concentration (40 mg mLꢀ1) with or without 10 mg
mLꢀ1 of a biotin solution for 3 h. The cells were then washed
three times with 0.01 M PBS and imaged under a Nikon A1
confocal laser scanning microscope (CLSM, Nikon Corporation,
Japan) at an excitation wavelength of 405 nm.
Data are expressed as mean ꢁ standard deviation. Statistical
analysis was performed using one-way analysis of variance
(ANOVA) with PASW Statistics 18.0 (Chicago, USA). The level of
signicance for all the comparisons was P < 0.05.
Acknowledgements
The authors thank the support from the Programs for Science
and Technology Development of Wuhan City, Hubei Province
(201260523172-8), the Research Project Competition of HKUST
(RPC11SC09 and RPC10SC13), the Research Grants Council of
Hong Kong (604711, 603509, HKUST2/CRF/10 and
N_HKUST620/11), the National Science Foundation of China
(20974028), the Innovation and Technology Commission
(ITCPP/17-9), and the University Grants Committee of Hong
Kong (AoE/P-03/08).
Cellular uptake of FSNP-1-biotin
The cellular uptake and distribution of FSNP-1-biotin in cells
were analyzed by TEM (FEI Corporation, Netherlands) accord-
ing to a modied procedure.28 Briey, HeLa cells were treated
with 40 mg mLꢀ1 of FSNP-1-biotin for 12 h. Aerwards, the cells
were washed three times with 0.01 M PBS to get rid of the
unbound nanoparticles and xed with 2.5% glutaraldehyde
buffered in 0.01 M PBS for 1 h at room temperature. Fixed cells
were washed three times with 0.01 M PBS and collected in a
centrifuge tube, and then post-xed in 1% osmium tetroxide
for 1 h at room temperature. The sample was dehydrated by
ethanol solutions at different concentrations (40, 50, 70, 80, 90,
95 and 100%), treated with propylene oxide and then
embedded in Spurr's resin by inltration with a series of
mixtures of resin and propylene oxide (ratios of propylene
oxide to resin: 1 : 1, 1 : 2 and 1 : 3). The resin blocks were
hardened at 70 ꢃC overnight. Ultrathin sections with a
dimension of 70 nm were cut using glass knives and then
stained with uranyl acetate and lead citrate prior to analysis
under Tecnai G2 20 TEM (FEI Corporation, Netherlands)
operating at 200 kV.
References
1 (a) L. Wang, K. M. Wang, S. Santra, X. J. Zhao, L. R. Hilliard,
J. E. Smith, J. R. Wu and W. H. Tan, Anal. Chem., 2006, 78,
646–654; (b) S. Santra, Methods Mol. Biol., 2010, 624, 151–162.
2 X. Zhou and J. Zhou, Anal. Chem., 2004, 76, 5302–5312.
3 W. Lian, S. A. Litherland, H. Badrane, W. Tan, D. Wu,
H. V. Baker, P. A. Gulig, D. V. Lim and S. Jin, Anal.
Biochem., 2004, 334, 135–144.
4 L. M. Rossi, L. Shi, F. H. Quina and Z. Rosenzweig, Langmuir,
2005, 21, 4277–4280.
5 S. Santra, H. Yang, D. Dutta, J. T. Stanley, P. H. Holloway,
W. Tan, B. M. Moudgil and R. A. Mericle, Chem. Commun.,
2004, 2810–2811.
This journal is ª The Royal Society of Chemistry 2013
J. Mater. Chem. B, 2013, 1, 676–684 | 683

View Article Online
Journal of Materials Chemistry B
Paper
6 I. Sokolov and S. Naik, Small, 2008, 4, 934–939.
7 (a) J. M. Rosenholm, A. Meinander, E. Peuhu, R. Niemi,
Y. J. Chuang, C. T. Chen and C. C. Lin, Adv. Funct. Mater.,
2010, 20, 3948–3958.
J. E. Eriksson, C. Sahlgren and M. Linden, ACS Nano, 2009, 17 (a) K. Na, T. Bum Lee, K. H. Park, E. K. Shin, Y. B. Lee and
3, 197–206; (b) X. Le Guevel, B. Hotzer, G. Jung and
M. Schneider, J. Mater. Chem., 2011, 21, 2974–2981; (c)
C. P. Tsai, C. Y. Chen, Y. Hung, F. H. Chang and
C. Y. Mou, J. Mater. Chem., 2009, 19, 5737–5743.
8 (a) Y. Jin, S. Kannan, M. Wu and J. X. Zhao, Chem. Res.
Toxicol., 2007, 20, 1126–1133; (b) E. J. Park and K. Park,
Toxicol. Lett., 2009, 184, 18–25.
9 H. Ow, D. R. Larson, M. Srivastava, B. A. Baird, W. W. Webb
and U. Wiesner, Nano Lett., 2005, 5, 113–117.
10 (a) Y. Hong, J. W. Y. Lam and B. Z. Tang, Chem. Commun.,
H. K. Choi, Eur. J. Pharm. Sci., 2003, 18, 165–173; (b)
W. Yang, Y. Cheng, T. Xu, X. Wang and L. P. Wen, Eur. J.
Med. Chem., 2009, 44, 862–868; (c) S. Chen, X. Zhao,
J. Chen, L. Kuznetsova, S. S. Wong and I. Ojima,
Bioconjugate Chem., 2010, 21, 979–987; (d) D. N. Heo,
D. H. Yang, H. J. Moon, J. B. Lee, M. S. Bae, S. C. Lee,
W. J. Lee, I. C. Sun and I. K. Kwon, Biomaterials, 2012, 33,
856–866.
18 Y. B. Patil, S. K. Swaminathan, T. Sadhukha, L. Ma and
J. Panyam, Biomaterials, 2010, 31, 358–365.
2009, 4332–4353; (b) Y. Hong, J. W. Y. Lam and B. Z. Tang, 19 Z. Zhao, Z. Wang, P. Lu, C. Y. Chan, D. Liu, J. W. Y. Lam,
Chem. Soc. Rev., 2011, 40, 5361–5388.
11 X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose,
H. H. Sung, I. D. Williams, Y. Ma and B. Z. Tang, Angew.
Chem., Int. Ed., 2009, 48, 7608–7611.
J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir and S. Weiss, 20 L. Sun, Y. Li, X. Liu, M. Jin, L. Zhang, Z. Du, C. Guo, P. Huang
Science, 2005, 307, 538–544. and Z. Sun, Toxicol. In Vitro, 2011, 25, 1619–1629.
12 (a) J. Luo, Z. Xie, J. W. Y. Lam, L. Cheng, H. Chen, C. Qiu, 21 (a) E. J. Park, J. Yi, K. H. Chung, D. Y. Ryu, J. Choi and
H. S. Kwok, X. Zhan, Y. Liu, D. Zhu and B. Z. Tang, Chem.
Commun., 2001, 1740–1741; (b) Q. L. Zhao, K. Li,
S. J. Chen, A. J. Qin, D. Ding, S. Zhang, Y. Liu, B. Liu,
K. Park, Toxicol. Lett., 2008, 180, 222–229; (b) M. Ahamed,
R. Posgai, T. J. Gorey, M. Nielsen, S. M. Hussain and
J. J. Rowe, Toxicol. Appl. Pharmacol., 2010, 242, 263–269.
J. Z. Sun and B. Tang, J. Mater. Chem., 2012, 22, 15128–15135. 22 C. F. Jones and D. W. Grainger, Adv. Drug Delivery Rev., 2009,
13 Z. Li, Y. Dong, B. Mi, Y. Tang, M. Haussler, H. Tong, 61, 438–456.
J. W. Y. Lam, Y. Ren, H. H. Sung, K. S. Wong, P. Gao, 23 M. Mahmoudi, K. Azadmanesh, M. A. Shokrgozar,
I. D. Williams, H. S. Kwok and B. Z. Tang, J. Phys. Chem. B,
2005, 109, 10061–10066.
W. S. Journeay and S. Laurent, Chem. Rev., 2011, 111,
3407–3432.
14 M. Faisal, Y. Hong, J. Liu, Y. Yu, J. W. Y. Lam, A. Qin, P. Lu 24 M. D. Chavanpatil, A. Khdair and J. Panyam, J. Nanosci.
and B. Z. Tang, Chem.–Eur. J., 2010, 16, 4266–4272. Nanotechnol., 2006, 6, 2651–2663.
15 F. Mahtab, Y. Yu, J. W. Y. Lam, J. Z. Liu, B. Zhang, P. Lu, 25 Z. Chu, Y. Huang, Q. Tao and Q. Li, Nanoscale, 2011, 3, 3291–
X. X. Zhang and B. Z. Tang, Adv. Funct. Mater., 2011, 21,
1733–1740.
16 (a) K. Y. Pu and B. Liu, Adv. Funct. Mater., 2011, 21, 3408–
3299.
26 Y. Yu, C. Feng, Y. N. Hong, J. Z. Liu, S. J. Chen, K. M. Ng,
K. Q. Luo and B. Z. Tang, Adv. Mater., 2011, 23, 3298–3302.
3423; (b) F. M. Kievit and M. Zhang, Adv. Mater., 2011, 23, 27 J. Condeelis and J. W. Pollard, Cell, 2006, 124, 263–266.
H217–H247; (c) T. M. Allen, Nat. Rev. Cancer, 2002, 2, 750– 28 A. Nan, X. Bai, S. J. Son, S. B. Lee and H. Ghandehari, Nano
763; (d) C. H. Lai, C. Y. Lin, H. T. Wu, H. S. Chan,
Lett., 2008, 8, 2150–2154.
684 | J. Mater. Chem. B, 2013, 1, 676–684
This journal is ª The Royal Society of Chemistry 2013