Biocompatible nanoparticles with aggregation-induced emission characteristics as far-red/near-infrared fluorescent bioprobes for in vitro and in vivo imaging applications

Article abstract of DOI:10.1002/adfm.201102191

Light emission of 2-(2,6-bis((E)-4-(diphenylamino)styryl)-4H-pyran-4- ylidene)malononitrile (TPA-DCM) is weakened by aggregate formation. Attaching tetraphenylethene (TPE) units as terminals to TPA-DCM dramatically changes its emission behavior: the resulting fluorogen, 2-(2,6-bis((E)-4-(phenyl(4′- (1,2,2-triphenylvinyl)-[1,1′-biphenyl]-4-yl)amino)styryl) -4H-pyran-4-ylidene)malononitrile (TPE-TPA-DCM), is more emissive in the aggregate state, showing the novel phenomenon of aggregation-induced emission (AIE). Formulation of TPE-TPA-DCM using bovine serum albumin (BSA) as the polymer matrix yields uniformly sized protein nanoparticles (NPs) with high brightness and low cytotoxicity. Applications of the fluorogen-loaded BSA NPs for in vitro and in vivo far-red/near-infrared (FR/NIR) bioimaging are successfully demonstrated using MCF-7 breast-cancer cells and a murine hepatoma-22 (H₂₂)-tumor-bearing mouse model, respectively. The AIE-active fluorogen-loaded BSA NPs show an excellent cancer cell uptake and a prominent tumor-targeting ability in vivo due to the enhanced permeability and retention effect. Encapsulation of far-red/near-infrared luminogens with aggregation-induced emission (AIE) characteristics in a bovine serum albumin (BSA) matrix yields nanoparticles (NPs) with uniform size, high brightness, and low cytotoxicity. Applications of these AIE-active NPs for in vitro and in vivo fluorescence imaging are demonstrated using MCF-7 breast-cancer cells and a murine hepatic H₂₂-tumor-bearing mouse model, respectively. Copyright

Full text of DOI:10.1002/adfm.201102191

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Biocompatible Nanoparticles with Aggregation-Induced
Emission Characteristics as Far-Red/Near-Infrared Fluorescent
Bioprobes for In Vitro and In Vivo Imaging Applications
Wei Qin, Dan Ding, Jianzhao Liu, Wang Zhang Yuan, Yong Hu, Bin Liu,*
and Ben Zhong Tang*
increasing attention due to their capa-
bility of overcoming the interferences of
optical absorption, light scattering and
autofluorescence of biological media.[2] To
date, a large variety of materials, including
organic dyes,[3] fluorescent proteins[4] and
inorganic quantum dots (QDs),[5] has been
extensively studied for the purpose of FR/
NIR fluorescence imaging. Organic dyes
and fluorescent proteins, however, suffer
from limited molar absorptivity and low
photobleaching thresholds,[6] while inor-
ganic QDs are highly cytotoxic in an oxida-
tive environment:[7] this has greatly limited
the scope of their in vitro and in vivo
applications. Exploration of novel FR/NIR
fluorescent probes with a high biological
Light emission of 2-(2,6-bis((E)-4-(diphenylamino)styryl)-4H-pyran-4-ylidene)
malononitrile (TPA-DCM) is weakened by aggregate formation. Attaching
tetraphenylethene (TPE) units as terminals to TPA-DCM dramatically changes
its emission behavior: the resulting fluorogen, 2-(2,6-bis((E)-4-(phenyl(4′-
(1,2,2-triphenylvinyl)-[1,1′-biphenyl]-4-yl)amino)styryl)-4H-pyran-4-ylidene)
malononitrile (TPE-TPA-DCM), is more emissive in the aggregate state,
showing the novel phenomenon of aggregation-induced emission (AIE). For-
mulation of TPE-TPA-DCM using bovine serum albumin (BSA) as the polymer
matrix yields uniformly sized protein nanoparticles (NPs) with high bright-
ness and low cytotoxicity. Applications of the fluorogen-loaded BSA NPs for
in vitro and in vivo far-red/near-infrared (FR/NIR) bioimaging are successfully
demonstrated using MCF-7 breast-cancer cells and a murine hepatoma-22
(H22)-tumor-bearing mouse model, respectively. The AIE-active fluorogen-
loaded BSA NPs show an excellent cancer cell uptake and a prominent tumor-
targeting ability in vivo due to the enhanced permeability and retention effect.
compatibility,
strong
photobleaching
resistance, and efficient light emission is
highly desirable for live-animal imaging.
Fluorescent nanoparticles (NPs), such as
organic-fluorophore-loaded NPs, have recently emerged as a new
generation of nanoprobes for bioimaging. They show such advan-
tages as synthetic versatility, low cytotoxicity, high photostability
and facile surface functionalization for specific targeting.[8]
For practical applications, brightly emissive NPs are desirable
for high-contrast imaging. In the ideal case, the brightness of
fluorophore-doped NPs should be proportional to the number of
encapsulated dye molecules. However, at high loading contents,
π-conjugated fluorophores are prone to aggregation. Aggregate
formation often quenches light emission due to π–π stacking
and other non-radiative pathways, a common photophysical phe-
nomenon notoriously known as aggregation-caused quenching
(ACQ). The ACQ effect has been a thorny hurdle in the fabri-
cation of NPs with high brightness.[9] We have recently devel-
oped a novel class of organic luminogens with an extraordinary
aggregation-induced-emission (AIE) feature,[10,11] which is exactly
opposite to the above-mentioned ACQ system. These lumino-
gens, with tetraphenylethene (TPE) being a typical example, are
non-emissive in dilute solution but are induced to luminesce
intensely when aggregated through a mechanism of the restric-
tion of intramolecular rotation.[11] The development of these
unique AIE luminogens sweeps away our concerns of emission
quenching induced by a high fluorophore loading in NPs.
1. Introduction
The emergence of non-invasive, live-animal fluorescence
imaging technology has opened new avenues for the devel-
opment of cancer diagnosis and therapeutics.[1] Fluores-
cence imaging probes with intense emission in the far-red/
near-infrared (FR/NIR) region (>650 nm) are attracting
W. Qin, Dr. J. Liu, Dr. W. Z. Yuan, Prof. B. Z. Tang
Department of Chemistry
The Hong Kong University of Science & Technology
Clear Water Bay, Kowloon
Hong Kong, China
E-mail: tangbenz@ust.hk
Dr. D. Ding, Prof. B. Liu
Department of Chemical and Biomolecular Engineering
National University of Singapore, 117576, Singapore
E-mail: cheliub@nus.edu.sg
Y. Hu
National Laboratory of Solid-State Microstructure
Department of Material Science and Engineering
Nanjing University
Nanjing 210093, China
Prof. B. Liu, Prof. B. Z. Tang
Institute of Materials Research Engineering
3 Research Link, 117602, Singapore
The AIE fluorogens that have been developed so far are largely
blue and green emitters. Although a fluorescence quantum yield
(ΦF) of almost unity has been realized in some AIE systems in
DOI: 10.1002/adfm.201102191
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the aggregate state,[11] they are not ideally suited to bioimaging
applications due to their light emission in the short-wavelength
region. Many red emitters (e.g., 2-(4H-pyran-4-ylidene)malononi-
trile (DCM)) are known, but they are unfortunately not so emis-
sive in the solid state due to the involved ACQ process. We
recently developed a structural design strategy for transforming
conventional ACQ chromophores to efficient solid emitters by
covalent integration with AIE luminogens.[12] In general, the
resultant adducts inherit the AIE feature and display a red-shifted
emission, in comparison with those of their ACQ parents, due to
the extension in the electronic conjugation. As FR/NIR chromo-
phores generally possess planar molecular structures with a weak
emission in the solid state, our new strategy should enable us to
develop efficient FR/NIR emitters by simply modifying the con-
ventional ACQ chromophores with AIE fluorogens. As a proof
of concept, we chose a triphenylamine (TPA)-modified DCM
derivative, 2-(2,6-bis((E)-4-(diphenylamino)styryl)-4H-pyran-4-yli-
dene)malononitrile (TPA-DCM; Scheme 1), which shows poor
FR emission in the aggregate state, as a model ACQ chromo-
phore. Two TPE units were introduced to the periphery of the
TPA-DCM core to generate a 2-(2,6-bis((E)-4-(phenyl(4′-(1,2,2-
triphenylvinyl)-[1,1′-biphenyl]-4-yl)amino)styryl)-4H-pyran-4-yli-
dene)malononitrile (TPE-TPA-DCM) adduct that is AIE active,
emitting bright FR/NIR light in the aggregate state. Its excitation
and emission profiles match well with commercial laser sources
and fluorescence-imaging systems, making it promising for bio-
imaging applications.
In this contribution, we report the synthesis of TPE-TPA-DCM,
the fabrication of its NPs, and the application of the NPs for in
vitro and in vivo imaging. Bovine serum albumin (BSA) was
selected as the polymer matrix for formulating NPs loaded with
TPE-TPA-DCM because albumin is biocompatible, non-antigenic
and clinically utilized.[13] In addition, BSA-based NPs have been
reported to accumulate preferentially in tumors through the
enhanced permeability and retention (EPR) effect, known as “pas-
sive” targeting.[14] As there have been almost no previous reports
on the application of AIE-active NPs in live-animal fluorescence
imaging via intravenous administration, the successful demon-
stration of the AIE-active fluorogen-loaded BSA NPs for FR/NIR
imaging of targeted tumors in living bodies should inspire more
exciting research in this emerging field for cancer diagnosis.
NC
CN
NC
CN
O
NC
CN
Ar−CHO, piperidine
CH3CN, reflux
Ac2O, reflux
O
O
Ar
O
N
Ar
Br
1
2
Ar =
N
TPA-DCM
Br-TPA-DCM
B(OH)2
Pd(PPh3)4
Br-TPA-DCM
TPE-TPA-DCM
+
K3PO4, THF/H2O
3
Scheme 2. Synthetic routes to TPA-DCM and TPE-TPA-DCM.
2. Results and Discussion
2.1. Synthesis and Characterization of TPE-TPA-DCM
TPA-DCM and TPE-TPA-DCM were prepared according to
the synthetic routes shown in Scheme 2. 2-(2,6-Dimethyl-
4H-pyran-4-ylidene)malononitrile (2) was prepared in a 73%
yield from 2,6-dimethyl-4-pyrone (1) according to the synthetic
procedures published by Wood.[15] Knoevenagel condensation
reactions of 2 with TPA-containing aldehydes[16] gave TPA-DCM
and Br-TPA-DCM adducts in yields of over 70%. TPE-TPA-DCM
was obtained in a 60% yield by Suzuki coupling between the Br-
TPA-DCM and 4-(1,2,2-triphenylvinyl)phenylboronic acid (3),[17]
using tetrakis(triphenylphosphane)palladium(0) (Pd(PPh3)4) as
a catalyst, under basic conditions.
The TPE-TPA-DCM was isolated by column chromatography
followed by recrystallization. The purified product was charac-
terized using standard spectroscopic methods. Its numerical
spectral data are summarized in the Experimental Section and
examples of its spectra (Figure S1–S3, Supporting Information)
are shown. The coupling constant of its vinyl protons in the
1H-NMR spectrum was 16 Hz, proving that it possesses a trans
conformation. Formation of the trans isomer was favored in the
reaction due to the thermodynamic stability of the trans con-
formation and the steric hindrance hampering the formation
of the cis structure. The NMR spectroscopy peaks of the
minor cis isomer were absent, possibly because any cis isomer
was removed by the recrystallization process during product
purification.
TPA-DCM is composed of two TPA donor (D) units and
one DCM acceptor (A) unit, with the latter being a well-known
ACQ chromophore. The absorption spectrum of its tetrahydro-
furan (THF) solution has a peak at ≈487 nm, and it has a molar
absorptivity of 7.1 × 104 L mol−1 cm−1. Such D-A fluorophores
often show the photophysical phenomenon named twisted
intramolecular charge transfer (TICT), which is featured with
a red-shifted emission color and a decreased emission inten-
sity with increasing solvent polarity.[18] We measured the
photoluminescence (PL) spectra of TPA-DCM in THF/water
mixtures, with different water fractions (fw), which enabled fine-
Scheme 1. Chemical structures of TPA-DCM and TPE-TPA-DCM.
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800
fw (vol %)
A
B
0
10
20
30
40
50
60
70
80
90
600
400
200
0
TICT
ACQ
550
600
650
700
750
800
0
30
60
90
Wavelength (nm)
Water fraction (vol %)
Figure 1. a) PL spectra of TPA-DCM in THF/water mixtures with different water fractions (fw). b) Plot of peak intensity of TPA-DCM versus water frac-
tion in the aqueous mixtures. [TPA-DCM] = 10 × 10−6 m; λex = 480 nm.
tuning of the solvent polarity and the extent of solute aggrega-
tion. The pure THF solution of TPA-DCM had an intense red
fluorescence with an emission maximum at 620 nm, which
was dramatically quenched when a small amount of water was
added, as evidenced by its weak emission in the THF/water
mixtures with fw ≤ 50 vol% (Figure 1). Meanwhile, the emission
maximum was bathochromically shifted to ≈670 nm. This is a
typical TICT effect arising from the increased solvent polarity.
When more water (fw > 50 vol%) was added, the TPA-DCM
molecules formed nanoaggregates. Although a hydrophobic
environment was created inside the nanoaggregates and the
TICT effect should be alleviated,[18] the fluorescence did not
recover because of the prevailing ACQ effect.
TPE is a paradigm of AIE luminogens. According to our
recently developed structural-design principle,[12] attaching TPE
units to TPA-DCM should endow the resultant adduct, TPE-
TPA-DCM, with an AIE attribute, whilst retaining the TICT
features of its parent form, TPA-DCM.[18] This proved to be the
case. As shown in Figure 2, the TPE-TPA-DCM exhibited an
emission maximum at 633 nm in THF, which was red-shifted
13 nm from that of TPA-DCM. With a gradual addition of water
into the THF, the emission of the TPE-TPA-DCM was dramati-
cally weakened and the emission color was bathochromically
shifted due to the increase in the solvent polarity and the trans-
formation to the TICT state. The light emission was invigorated
from fw ≈ 50 vol% and was intensified with a further increase
800
0
fw (vol %)
A
B
0
10
20
30
40
50
60
70
80
90
600
90
50
AIE
TICT
400
200
0
10
40
650
Wavelength (nm)
0
30
60
90
550
600
700
750
800
Water fraction (vol %)
Figure 2. a) PL spectra of TPE-TPA-DCM in THF/water mixtures with different water fractions (fw). b) Plot of peak intensity of TPE-TPA-DCM versus
water fraction in the aqueous mixtures. [TPE-TPA-DCM] = 10 × 10−6 m; λex = 480 nm.
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increased with an increase in the fluorogen
feeding. The EE of the fluorogen was >85 wt%
when the TPE-TPA-DCM feeding ratio was
<1 wt%, while a decrease in the EE was
observed when the fluorogen feeding ratio
was increased to >1 wt%. The average size
of the pure BSA NPs without AIE-fluorogen
encapsulation was 97.1 nm, with a narrow
size distribution or polydispersity (PDI =
0.065). The average size of the BSA NPs
increased from 98.8 nm to 148.1 nm when
the fluorogen loading was increased from
0.25 wt% to 3.07 wt%. In comparison, the
average size of the bare TPE-TPA-DCM NPs
prepared from an aqueous mixture with fw
=
90 vol% (cf., Figure 2) was measured to be
307.3 nm by laser-light scattering (LLS) with
a broad size distribution (PDI = 0.279).
Scheme 3. Schematic illustration of the fabrication of BSA NPs loaded with TPE-TPA-DCM.
in fw. Meanwhile, the emission maximum was gradually red-
shifted to ≈660 nm when fw reached 90 vol%. These data verify
our anticipation that TPE-TPA-DCM is a luminogen with both
TICT and AIE characteristics.
Transmission electron microscopy (TEM) and field-emis-
sion scanning electron microscopy (FE-SEM) images of the
fluorogen-loaded BSA NPs with a 0.86 wt% loading of TPE-
TPA-DCM are shown in Figure 3 as examples. The images
indicate that the AIE-active fluorogen-loaded NPs had a
spherical shape and a smooth surface with an almost uni-
form size of around 90 nm. This size is smaller than that
obtained from the LLS measurement (124.7 nm) due to a
shrinkage of the BSA NPs in the ultra-dry state under the
high vacuum in the TEM and FE-SEM chambers.
Figure 4 shows the absorption and emission spectra of the
AIE-active fluorogen-loaded BSA NPs with a 0.86 wt% fluor-
ogen loading and of the bare TPE-TPA-DCM NPs suspended
in water. The fluorogen-loaded BSA NPs show two absorp-
tion maxima at 360 and 505 nm, while those of the bare TPE-
TPA-DCM NPs are slightly blue-shifted, appearing at 359 and
497 nm. The emission maximum of the fluorogen-loaded BSA
NPs is located at 668 nm, similar to that of the bare TPE-TPA-
DCM NPs in water. The emission intensity of the fluorogen-
loaded BSA NPs increased almost linearly with increasing
fluorogen loading within the studied range (Figure 5). The ΦF
values of the fluorogen-loaded BSA NPs in water were meas-
ured using Rhodamine 6G in ethanol as the standard. The
ΦF initially increased rapidly and then slowly with increasing
fluorogen loading. It eventually reached a value of ≈12% at a
fluorogen loading of 3.07 wt%.
2.2. Fabrication and Characterization of BSA NPs Loaded
with TPE-TPA-DCM
The procedure for the preparation of the fluorogen-loaded
BSA NPs is illustrated in Scheme 3. Upon addition of the
TPE-TPA-DCM solution in THF to the aqueous solution of BSA,
the TPE-TPA-DCM molecules aggregate and entangle with the
hydrophobic domains of the BSA chains. The BSA gradually
phase-separates, accompanying its hybridization with the hydro-
phobic fluorogen. The fluorogen-loaded BSA NPs form instantly
upon sonication. The BSA matrix is knitted together by glutar-
aldehyde, an amine-reactive homo-bifunctional cross-linker.
The THF is then removed and the cross-linked NPs are further
purified by filtration through a 0.45 μm microfilter, followed by
washing with Milli-Q water. The zeta potential of the purified
NPs was −29 mV in aqueous suspension, suggesting that the
NPs are stabilized by outer layers of ionized carboxylic groups.
Table 1 shows the encapsulation efficiencies (EE) and average
sizes of the AIE-active fluorogen-loaded NPs prepared at dif-
ferent feeding ratios of TPE-TPA-DCM. The fluorogen loading
Table 1. Characteristics of the BSA NPs loaded with TPE-TPA-DCM.
TPE-TPA-DCM feeding ratioa)
[wt%]
TPE-TPA-DCM loading ratiob)
[wt%]
Encapsulation efficiency
[wt%]
Sizec) [nm] (PDI)d)
0
0
97.1 (0.065)
98.8 (0.089)
124.8 (0.125)
124.7 (0.110)
141.3 (0.180)
148.1 (0.161)
0.25
0.5
1.0
2.5
5.0
0.25
0.49
0.86
1.87
3.07
100
98.7
85.6
74.8
61.4
a)The ratio of the weight of TPE-TPA-DCM to that of BSA in the feed mixture; b)The ratio of the weight of the loaded TPE-TPA-DCM to that of the BSA matrix in the NPs;
c)Average diameter of the NPs measured by laser light scattering (LLS); d)Polydispersity index (PDI).
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12.5
11.5
10.5
9.5
1000
800
600
400
200
Figure 3. a,b) TEM (a) and FE-SEM (b) images of the AIE-active fluor-
ogen-loaded BSA NPs.
8.5
7.5
0
3
Nanomaterials with an average diameter of ≈100 nm have
been reported to show prolonged blood circulation in vivo,
resulting in their accumulation in tumors through the EPR
process.[19] In the following in vitro and in vivo imaging experi-
ments, AIE-active fluorogen-loaded BSA NPs with a TPE-
TPA-DCM loading ratio of 0.86 wt% were used, due to their
proper particle size, relatively high EE and good quantum yield.
0
1
2
Loading ratio (wt %)
Figure 5. Changes in the quantum yield and PL intensity with the weight
ratio of TPE-TPA-DCM in the fluorogen-loaded BSA NPs.
Figure 6B shows a CLSM image of MCF-7 cells after incubation
with bare TPE-TPA-DCM NPs (0.4 × 10−6 m) for 2 h at 37 °C.
Only a few bare NPs were observed in the cytoplasm with a
weak fluorescence. The internalization of the bare fluorogen
NPs in the cytoplasm was also confirmed by a 3D CLSM image
(Figure S5, Supporting Information). The homogeneous distri-
bution of the AIE-active fluorogen-loaded BSA NPs in the cyto-
plasms, with a stronger fluorescence compared with the bare
TPE-TPA-DCM NPs, suggests that BSA as the encapsulation
matrix efficiently enhanced the intracellular uptake of the for-
mulated NPs. These results manifest that the fluorogen-loaded
BSA NPs are effective FR/NIR fluorescent bioprobes for cel-
lular imaging with a high fluorescence contrast. In addition,
the photostabilities of the AIE-fluorogen-loaded BSA NPs and
fluorescein isothiocyanate in the MCF-7 cancer cells were also
studied under continuous laser scanning upon excitation at
488 nm (Figure 7). The AIE-fluorogen-loaded NPs showed a
decrease of less than 10% in their fluorescence intensity after
continuous laser exposure for 10 min, which was much better
than that of fluorescein isothiocyanate under the same condi-
tions. These data indicate the good photostability of the AIE-
fluorogen-loaded BSA NPs in a biological environment, which
meets the requirements for bioimaging applications.
2.3. In Vitro Cellular Imaging
The application of the fluorogen-loaded BSA NPs for in vitro
cellular imaging was studied using confocal laser scanning
microscopy (CLSM). After incubation with the BSA-NP suspen-
sion (0.4 × 10−6 m TPE-TPA-DCM) for 2 h at 37 °C in the cul-
ture medium, MCF-7 breast-cancer cells were fixed and the cell
nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).
The cells were imaged by CLSM with a 488 nm laser excita-
tion and the fluorescent signals were collected at above 650 nm
for the NPs and 560 nm for DAPI, respectively. As shown in
Figure 6A, an intense red fluorescence was observed in the
cellular cytoplasms and was further confirmed by the corre-
sponding 3D CLSM image (Figure S4, Supporting Information).
The in vitro cytotoxicity of the fluorogen-loaded BSA NPs
against MCF-7 breast-cancer cells was investigated using a
300
400
500
600
700
800
Wavelength (nm)
Figure 6. a,b) CLSM images of MCF-7 breast cancer cells after incuba-
tion with fluorogen-loaded BSA NPs (with a fluorogen loading of 0.86%)
(a) and bare TPE-TPA-DCM NPs (b) for 2 h at 37 °C. [TPE-TPA-DCM] =
0.4 × 10−6 m.
Figure 4. Normalized UV–vis absorption and PL emission spectra of the
fluorogen-loaded BSA NPs (with TPE-TPA-DCM loading of 0.86%; red)
and the bare TPE-TPA-DCM NPs (blue) in water.
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120
100
80
60
40
20
0
100
80
60
40
20
12 h
24 h
48 h
0
0
2
4
6
8
10
0.1
0.4
0.8
Time (min)
Fluorogen concentration (µM)
Figure 7. Photostability comparison between TPE-TPA-DCM-loaded BSA
NPs (red) and fluorescein isothiocyanate (blue) in MCF-7 cancer cells
upon continuous excitation at 488 nm (1.25 mW) for 0 to 10 min. I0 is
the initial fluorescence intensity and I is the fluorescence intensity of the
sample after continuous exposure for designated time intervals.
Figure 8. Metabolic viability of MCF-7 breast cancer cells after incubation
with fluorogen-loaded BSA NPs at various fluorogen concentrations for
12, 24, and 48 h.
AIE-active NPs accumulated in the tumor were highly fluores-
cent, while the second is the “passive” tumor-targeting ability
by the EPR effect, which benefited from the uniform NP size
of ≈100 nm.[19] Although strong fluorescence was also observed
in the abdomen and liver areas of the same mouse at 3 h post-
injection, it had almost completely disappeared after 28 h.
This suggests that the AIE-fluorogen-loaded BSA NPs under-
went uptake by the reticuloendothelial system (RES) organs,
such as the liver and the spleen,[20] followed by facile excre-
tion from the body through the biliary pathway,[21] which was
further confirmed by the clear fluorescent signals observed in
the collected feces. The clearance rate of the NPs within the
tumors, however, was very slow, due to the lack of lymphatic
drainage.[22] At 28 h post-injection, the uptake of the fluorogen-
loaded BSA NPs in the tumor became prominent, in sharp
contrast to the weak fluorescence signals in the other parts of
the mouse body, demonstrating the promise of the NPs as FR/
NIR fluorescent bioprobes for cancer diagnosis. The promi-
nent EPR effect of the AIE-fluorogen-loaded BSA NPs was also
demonstrated using ex vivo fluorescence imaging, which was
performed on the excised tumor tissue, as well as the major
organs, when the mice were sacrificed (Figure 10).
Asacontrol, themiceintravenouslyinjectedwiththebareTPE-
TPA-DCM NPs at the same fluorogen concentration were imaged
and analyzed. As shown in Figure 9B, the fluorescence intensi-
ties in the abdomen and liver areas of the mouse were much
higher than in the tumor tissue at all of the tested time points.
This is due to the fact that the bare TPE-TPA-DCM NPs had a
relatively large average particle size (≈300 nm), most not being
able to escape RES uptake.[19,23] As a consequence, accumula-
tion of the bare fluorogen NPs in the tumors was limited and
the tumor imaging showed poor fluorescence contrast. In com-
parison, H22-tumor-bearing mice treated with saline were also
imaged and analyzed under the same experimental conditions
as those for Figure 9A,B (Figure S7, Supporting Information).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
cell-viability assay. Figure 8 shows the cell viability after incu-
bation with the NP suspension at fluorogen concentrations of
0.1 × 10−6, 0.4 × 10−6, and 0.8 × 10−6 m for 12, 24, and 48 h,
respectively. Cell viabilities of more than 95% were observed
for all the fluorogen concentrations within the tested periods of
time, indicating that the AIE-active fluorogen-loaded BSA NPs
had low cytotoxicity or good biocompatibility. The low cytotox-
icity makes the NPs promising for bioimaging applications and
superior to QDs, which are well-known for their concentration-
dependent cytotoxicity.[7b]
2.4. In Vivo Imaging on a Tumor-Bearing Mouse Model
The fluorogen-loaded BSA NPs were also examined for
in vivo FR/NIR bioimaging applications, employing the
non-invasive live-animal fluorescence imaging technique.
Murine hepatoma-22 (H22) -transplanted tumor-bearing ICR
mice were used as model animals, which were established
by subcutaneously inoculating the H22 cancer cells into the
left axillae of the mice. After intravenous injection with
the fluorogen-loaded BSA NPs, the H22-tumor-bearing mice
were imaged using a Maestro EX in vivo fluorescence-imaging
system. The mouse autofluorescence was removed by spectral
unmixing using the Maestro software (Figure S6, Supporting
Information). Figure 9A shows the time-dependent in vivo dis-
tribution profile and tumor accumulation of the AIE-fluorogen-
loaded BSA NPs in the H22-tumor-bearing mice. Clear tumor
delineations with intense fluorescence were observed in
the left axilla of the mouse at all of the imaging times, indi-
cating an accumulation of the fluorogen-loaded BSA NPs in
the tumor tissue. The capability of the fluorogen-loaded BSA
NPs to illuminate the tumor tissue selectively with a high con-
trast may be associated with two factors. The first is that the
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Time
3 h
8 h
28 h
A
Figure 10. Ex vivo fluorescence imaging on tumor tissue and major
organs of mice treated with AIE-fluorogen-loaded BSA NPs, which were
sacrificed at 24 h post-injection. The tissue autofluorescence was removed
by spectral unmixing software.
for tumor tissues from mice treated with the fluorogen-loaded
BSA NPs and the bare TPE-TPA-DCM NPs. The average flu-
orescence intensities of the tumors stained by the fluorogen-
loaded BSA NPs were nearly twice as high as those for the bare
TPE-TPA-DCM NPs at all of the imaging time points, (i.e., 3,
8, and 28 h). Clearly, the BSA formulation favors enhanced
accumulation of the AIE-active fluorogen-loaded BSA NPs in
tumors, resulting in clear differentiation of the tumor from
other tissues.
B
1500
Dye-loaded BSA NPs
Bare fluorogen NPs
1000
500
0
3. Conclusions
In summary, we have synthesized TPE-TPA-DCM, fabricated
its BSA NPs, and explored their in vivo and in vitro bioimaging
applications. The TPE-TPA-DCM possesses both TICT and AIE
features, and its BSA-formulated NPs show efficient FR/NIR
fluorescence with low cytotoxicity, uniform size and spherical
morphology. As compared with the bare TPE-TPA-DCM NPs,
the fluorogen-loaded BSA NPs show an increased cancer-cell
uptake and a prominent tumor-targeting capability by the EPR
effect on the H22-tumor-bearing mouse model, clearly demon-
strating the great potential of the BSA-formulated AIE-active
NPs as an FR/NIR fluorescent probe for in vitro and in vivo
imaging. This study thus opens up a new avenue for the devel-
opment of AIE-active, biocompatible, NP-based probes for
cancer imaging and diagnostics.
3
8
28
C
Time (h)
Figure 9. a,b) In vivo non-invasive fluorescence imaging of H22-tumor-
bearing mice after intravenous injection of fluorogen-loaded BSA NPs
(with TPE-TPA-DCM loading of 0.86 wt%) (a) and bare TPE-TPA-DCM
NPs (b) at the same fluorogen concentration. The white circles mark the
tumor sites. c) Average PL intensities for the tumor tissues from the mice
treated with the fluorogen-loaded BSA NPs and the bare fluorogen NPs
at different times.
4. Experimental Section
No fluorescence signals were observed after the removal of
the mouse autofluorescence, which indicates that the fluores-
cence signals (red) in Figure 9A,B were indeed from the TPE-
TPA-DCM-loaded BSA NPs and the bare TPE-TPA-DCM NPs,
respectively. Figure 9C summarizes the semiquantitative anal-
ysis data of the average TPE-TPA-DCM fluorescence intensities
Materials: The malononitrile derivative, 2,[15] the TPE derivative, 3,[17]
4-(diphenylamino)benzaldehyde[16] and 4-((4-bromophenyl)(phenyl)
amino)benzaldehyde[16] were prepared according to the reported
experimental procedures. BSA, glutaraldehyde, penicillin-streptomycin
solution, trypsin-ethylenediaminetetra-acetic acid (EDTA) solution, MTT
and DAPI were purchased from Sigma–Aldrich (St. Louis, USA). The
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fetal bovine serum (FBS) was purchased from Gibco (Lige Technologies,
Switzerland). Acetonitrile was distilled over P2O5. THF was distilled from
sodium benzophenone ketyl under dry nitrogen immediately prior to use.
Milli-Q water was supplied by a Milli-Q Plus System (Millipore Corp.,
Breford, USA). The MCF-7 breast-cancer cells were obtained from the
American Type Culture Collection. The murine hepatic H22 cancer cells
were purchased from the Shanghai Institute of Cell Biology (Shanghai,
China). Male ICR mice (6–8 weeks old) were provided by the animal
center of Drum-Tower Hospital (Nanjing, China).
prepare the bare TPE-TPA-DCM NPs, 60 μL of a THF solution of the
fluorogen (0.5 mg mL−1) were added into 3 mL of a water/THF (9:1 v/v)
mixture, followed by sonication of the fluorogen mixture for 60 s at
an 18 W output. The emulsion was then stirred at room temperature
overnight to evaporate the THF solvent.
Cell Culture: The MCF-7 breast-cancer cells and the murine hepatic
H22 cancer cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% fetal bovine serum and 1% penicillin
streptomycin at a constant temperature of 37 °C in a humidified
environment containing 5% CO2. Prior to the imaging experiments, the
cells were precultured until confluence was reached.
Characterization: The 1H- and 13C-NMR spectra were recorded using
a Bruker AV 300 spectrometer, in CDCl3, using tetramethylsilane (TMS)
(δ = 0) as an internal reference. The high-resolution mass spectra
(HRMS) were recorded using a GCT premier CAB048 mass spectrometer
operating in matrix-assisted laser desorption ionization time-of-flight
(MALDI-TOF) mode. The absorption spectra were recorded using a
Shimadzu UV-1700 spectrometer. The emission spectra were recorded
using a Perkin–Elmer LS 55 spectrofluorometer. The average particle
size and the size distribution of the NPs were measured by LLS, using
a 90Plus particle-size analyzer (Brookhaven Instruments Co., USA)
at a fixed angle of 90° and at room temperature. The zeta potential of
the NPs was measured using a Brookhaven ZetaPlus zeta-potential
analyzer at room temperature. The morphology of the NPs was studied
using FE-SEM (JSM-6700F, JEOL, Japan) at an accelerating voltage of
10 kV. The samples were fixed on a stub with double-sided sticky tape
and then coated with a platinum layer using an autofine coater (JEOL,
Tokyo, Japan) for 60 s in a vacuum at a current intensity of 10 mA. The
morphology of the NPs was also investigated by TEM (JEM-2010F, JEOL,
Japan).
Synthesis of TPE-TPA-DCM: The fluorogen was prepared according
to Scheme 1. Pd(PPh3)4 (36 mg) was added into a stirred mixture
of 336 mg (0.4 mmol) of Br-TPA-DCM, 526 mg (1.4 mmol) of 3 and
1060 mg of K3PO4 (5 mmol) in 50 mL of THF and 8 mL of water under
nitrogen. The mixture was heated to 70 °C for 36 h. After cooling to room
temperature, the solution was extracted with dichloromethane (100 mL)
twice, washed with water and dried over Na2SO4. After filtration and
solvent evaporation under reduced pressure, the product was purified
by silica-gel column chromatography using hexane/dichloromethane as
the eluent. TPE-TPA-DCM was obtained in a 60% yield (322 mg) as a red
powder after recrystallization from a mixture of chloroform/isopropyl
alcohol. 1H NMR (300 MHz, CDCl3, δ): 7.51–7.40 (m, 10H), 7.35–7.29
(m, 8H), 7.17–7.01 (m, 48H), 6.63 (s, 2H; pyran H), 6.60 (d, J = 16 Hz,
2H; pyran –CH=); 13C NMR (75 MHz, CDCl3, δ): 159.39, 156.53, 150.50,
147.26, 146.54, 144.41, 144.39, 144.37, 143.40, 141.83, 141.15, 138.59,
138.07, 136.97, 132.55, 132.03, 130.29, 129.75, 128.34, 127.16, 126.44,
126.27, 126.14, 125.08, 122.50, 116.51, 116.35, 107.07, 58.96; HRMS
(MALDI-TOF, m/z): [M]+ calcd for C100H70N4O, 1343.5583; found,
1343.5820. Anal. calcd for C100H70N4O: C 89.39, H 5.25, N 4.17; found:
C 89.66, H 5.23, N, 4.22.
Fabrication of Fluorogen-Loaded BSA NPs: The BSA NPs loaded with
TPE-TPA-DCM were prepared by a modified desolvation method. The
NPs were prepared with varied feeding ratios ranging from 0.25 to
5 wt%, defined as the ratio of the weight of the fluorogen to that of the
BSA in the feed mixture. In brief, 13 mg of BSA were dissolved in 5 mL of
Milli-Q water. Subsequently, 8 mL of THF (desolvation agent) containing
a predetermined amount of TPE-TPA-DCM were added dropwise into the
aqueous solution of BSA under sonication at room temperature, using a
microtip probe sonicator with an 18 W output (XL2000, Misonix Incorp.,
USA), leading to the formation of the fluorogen-loaded BSA NPs. A small
amount (5 μL) of glutaraldehyde solution (50%) was then added to
cross-link the NPs at room temperature for 4 h. The THF was removed
by rotary evaporation under vacuum. The cross-linked fluorogen-loaded
BSA-NP suspension was filtered through a 0.45 μm microfilter and was
then washed with Milli-Q water. The amounts of the fluorogen aggregates
successfully encapsulated into the BSA NPs were determined from the
absorption spectra with reference to a calibration curve established
from dimethyl sulfoxide (DMSO) solutions of TPE-TPA-DCM. The EE is
defined as the ratio of the amount of the fluorogen aggregates loaded
in the NPs to the total amount of the fluorogen in the feed mixture. To
Cell Imaging: The MCF-7 cells were cultured in a LAB-TEK chamber
(Chambered Coverglass System, Rochester, USA) at 37 °C. After 80%
confluence, the medium was removed and the adherent cells were
washed twice with 1 × phosphate buffered saline (PBS) buffer. The AIE-
active fluorogen-loaded BSA NPs (with a fluorogen loading of 0.86 wt%)
or the bare TPE-TPA-DCM NPs (0.4 × 10−6 m) in FBS-free DMEM were
then added to the chamber. After incubation for 2 h, the cells were
washed three times with 1 × PBS buffer and then fixed with 75% ethanol
for 20 min, followed by further washing, twice with 1 × PBS buffer. The
nuclei were stained by DAPI for 10 min. The cell monolayer was then
washed twice with 1 × PBS buffer and imaged by CLSM (Zeiss LSM 410,
Jena, Germany) using the Olympus Fluoview FV1000 imaging software.
The fluorescent signals from the NPs were collected upon excitation
at 488 nm (1.25 mW) with a 650 nm long-pass barrier filter. To study
the photostability of the AIE-active fluorogen-loaded BSA NPs in a
biological environment, CLSM images of NP-stained MCF-7 cancer cells
were recorded at 2 min intervals under continuous laser scanning at an
excitation wavelength of 488 nm (1.25 mW). The fluorescence intensity
of each image was analyzed using the Image Pro Plus software. The
photostability of the NPs was expressed by the ratio of the fluorescence
intensity, after excitation for a designated time interval, to its initial
value as a function of exposure time. As a control, the photostability of
fluorescein isothiocyanate in the MCF-7 cancer cells was also studied
following the same procedures.
Cytotoxicity of Fluorogen-Loaded BSA NPs: The cytotoxicity of the NPs
against MCF-7 breast-cancer cells was assessed by MTT assay. In brief,
MCF-7 cells were seeded in 96-well plates (Costar, IL, USA) at a density
of 4 × 104 cells mL−1. After 24 h incubation, the cells were exposed to
a series of doses of fluorogen-loaded BSA NPs at 37 °C. To eliminate
the UV-absorption interference of the fluorogen-loaded NPs at 570 nm,
the cells were incubated with the same series of doses of the fluorogen-
loaded NPs as the control. After the designated time intervals, the
sample wells were washed twice with 1 × PBS buffer, and 100 μL of freshly
prepared MTT solution (0.5 mg mL−1) in culture medium were added
into each sample well. The MTT-medium solution was carefully removed
after 3 h incubation in the incubator for the sample wells, whereas
the control wells without the addition of MTT solution were washed
twice with 1 × PBS buffer. DMSO (100 μL) was then added into each
well and the plate was gently shaken for 10 min at room temperature
to dissolve all of the precipitates formed. The absorbance of individual
wells at 570 nm was then monitored using a Tecan GENios Microplate
Reader. The absorbance of MTT in the sample well was determined by
the differentiation between the absorbance of the sample well and that
of the corresponding control well. The cell viability was expressed by the
ratio of the absorbance of MTT in the sample wells to that of the cells
incubated with culture medium only.
In Vivo Real-Time Fluorescence Imaging: All of the animal studies
were performed in compliance with guidelines set by the Animal Care
Committee at Drum-Tower Hospital. An H22-cell suspension containing
5–6 × 106 cells (0.1 mL) was injected subcutaneously into the ICR mice
(average body weight of 25 g) at the left axilla. When the tumor volume
reached a mean size of about 400 mm3, the mice were intravenously
injected with 250 μL of the fluorogen-loaded BSA NPs (with a fluorogen
loading of 0.86 wt%) at an NP concentration of 1 mg mL−1. Similar
experiments were conducted with the bare TPE-TPA-DCM NPs at the
same fluorogen concentration. The mice were anesthetized and placed
on an animal plate heated to 37 °C. The time-dependent biodistribution
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Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
W.Q and D.D contributed equally to this work. The authors are grateful to
support from the Singapore National Research Foundation (R-279-000-323-
281), the Research Grants Council of Hong Kong (603509 and HKUST2/
CRF/10), the Temasek Defence Systems Institute of Singapore (R279-000-
305-592/422/232), the Institute of Materials Research and Engineering of
Singapore (IMRE/11-1C0213), the Innovation and Technology Commission
of Hong Kong (ITP/008/09NP), the University Grants Committee of Hong
Kong (AoE/P-03/08), an NSFC/RGC grant (N_HKUST620/11) and the
National Science Foundation of China (20974028 and 21074051).
Received: September 15, 2011
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Adv. Funct. Mater. 2012, 22, 771–779
2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
779