Robust Red Organic Nanoparticles for In Vivo Fluorescence Imaging of Cancer Cell Progression in Xenografted Zebrafish

Article abstract of DOI:10.1002/adfm.201701418

Bright and red-emissive organic nanoparticles (NPs) are demonstrated as promising for in vivo fluorescence imaging. However, most red organic dyes show greatly weakened or quenched emission in the aggregated state. In this work, a robust red fluorophore (

Full text of DOI:10.1002/adfm.201701418

FULL PAPER
Bioimaging
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Robust Red Organic Nanoparticles for In Vivo Fluorescence
Imaging of Cancer Cell Progression in Xenografted Zebrafish
Gengwei Lin, Purnima Naresh Manghnani, Duo Mao, Cathleen Teh, Yinghao Li,
Zujin Zhao,* Bin Liu,* and Ben Zhong Tang*
for disease diagnosis and therapy.[1] The
Bright and red-emissive organic nanoparticles (NPs) are demonstrated as
promising for in vivo fluorescence imaging. However, most red organic dyes
show greatly weakened or quenched emission in the aggregated state. In
this work, a robust red fluorophore (t-BPITBT-TPE) with strong aggregate-
state photoluminescence and good biocompatibility is presented. The NPs
comprised of t-BPITBT-TPE aggregates encapsulated within 1,2-distearoyl-sn-
glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol) (DSPE-
mPEG) micelles exhibit a photoluminescence peak at 660 nm with a high
fluorescence quantum yield of 32% in aqueous media. The NPs can be
facilely charged by using the same polymeric matrix with different terminal
groups, e.g., methoxy (DSPE-mPEG), amine (DSPE-PEG-NH2), or carboxy-
methyl (DSPE-PEG-COOH) groups. The biocompatibility, toxicity, circula-
tion, and biodistribution of the NPs are assessed using the zebrafish model
through whole embryo soaking and intravenous delivery. Furthermore, HeLa
and MCF-7 cells tagged with t-BPITBT-TPE in DSPE-PEG-NH2-TAT polymer
NPs are xenografted into zebrafish larvae to successfully track the cancer cell
proliferation and metastasis, demonstrating that these new NPs are efficient
cancer cell trackers. In addition, the NPs also show good in vivo imaging
ability toward 4T1 tumors in xenografted BALB/c mice.
quality and resolution of fluorescence
imaging is highly dependent on the con-
trast agents for which strong photolumi-
nescence (PL), high photostability, good
biocompatibility, and excellent physical
stability are desirable properties.[1b,2] Fluo-
rescent organic nanoparticles (NPs) built
with emissive organic aggregates and
biocompatible polymeric matrices enjoy
lower cytotoxicity and higher structural
variety relative to inorganic quantum
dots.[3] They also have better photostability
than fluorescent proteins[4] and small-
molecule dyes.[5] Through selection or
modification of the polymeric matrices,
emissive organic NPs with excellent prop-
erties, such as good water dispersibility
and stability, feasible surface charge regu-
lation, high target selectivity, and other
designated functionalities, can be facilely
realized.[3b,6] Therefore, they are consid-
ered to be excellent labeling reagents for
fluorescence bioimaging applications.
The light-emissive molecules determine
the optical property of organic NPs. In comparison with other
visible light-emitting fluorophores, red fluorescent molecules
are more desirable in fluorescence imaging applications
because of their minimized autofluorescence interference,
lowered damage to living cells, and increased penetration
depth.[1a,7] In order to construct red organic fluorophores,
extended π-conjugation and/or electronic donor–acceptor
(D–A) structures with strong dipole moments are generally
involved in the molecular design.[8] However, the large and
planar π-conjugated system often results in strong π–π stacking
interactions between molecules, while D–A structures can also
lead to serious intermolecular dipole–dipole interactions. Both
processes can increase the nonradiative decay rate, leading to
decreased fluorescence quantum yield (ΦF) in the aggregated
state.[7b,8b,c] Therefore, many red fluorescent molecules suffer
from aggregation-caused quenching, which is an obstacle in
their practical applications. In this regard, luminescent mate-
rials with aggregation-induced emission (AIE) attributes have
shown great potentials in bioimaging applications due to their
intensified emission in the aggregated state.[9] Based on the
understanding of AIE mechanism, diverse new luminogens
with AIE characteristics (AIEgens) have been developed over
the past decade.[6a,10] The integration of AIE-active units and
1. Introduction
Noninvasive live animal fluorescence imaging has emerged as
a promising technique to provide valuable in vivo information
G. Lin, Y. Li, Prof. Z. Zhao, Prof. B. Z. Tang
State Key Laboratory of Luminescent Materials and Devices
South China University of Technology
Guangzhou 510640, China
E-mail: mszjzhao@scut.edu.cn; tangbenz@ust.hk
P. N. Manghnani, Dr. D. Mao, Prof. B. Liu
Department of Chemical and Biomolecular Engineering
National University of Singapore
4 Engineering Drive 4, Singapore 117585, Singapore
E-mail: cheliub@nus.edu.sg
Dr. C. Teh
Institute of Molecular and Cell Biology
Biopolis, Singapore 138673, Singapore
Prof. B. Z. Tang
Department of Chemistry
Hong Kong Branch of Chinese National Engineering
Research Center for Tissue Restoration and Reconstruction
The Hong Kong University of Science and Technology
Clear Water Bay, Kowloon, Hong Kong, China
DOI: 10.1002/adfm.201701418
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other functional moieties has been rationalized to be a straight-
forward strategy to create efficient aggregate-state luminescent
materials, and has furnished numerous bright fluorescent con-
trast agents for sensing, imaging, and therapy applications.[11]
Among the various animal models used to evaluate these
NPs, zebrafish is an attractive model for toxicity, biocompat-
ibility, biodistribution, immune response, teratogenicity, and
long-term effect studies.[12] Zebrafish is a vertebrate model
whose 99% embryonic essential genes are conserved in
humans.[13] The ease of obtaining zebrafish embryos from fer-
tile adults, the small embryo size, and their optical transpar-
ency make it a compatible model for assessment of fluorescent
dyes using standard imaging microscope.[14] Novel fluorescent
dyes can be assessed for desirable properties such as photosta-
bility and brightness of fluorescent signals detected through
living tissues during whole embryo imaging.[15] In addition, as
human oncogenes and tumor suppressor genes have homologs
in zebrafish, many neoplastic signaling pathways between
humans and zebrafish are conserved.[16] Zebrafish model is
hence ideal for monitoring of in vivo tumor growth, angiogen-
esis, and metastasis.[17] What is of particular significance is that
zebrafish larvae have immature adaptive immune system until
one month post-fertilization.[18] As a result, human cancer cells
can survive and metastasize in zebrafish.[19]
In this contribution, we report two efficient red fluorophores
PITBT-TPE and t-BPITBT-TPE with a D–A structure (Scheme 1),
in which benzothiadiazole segment serves as electron-
withdrawing moiety (A), while 1-phenyl-2-(thiophen-2-yl)-1H-
phenanthro[9,10-d]imidazole segment functions as electron-
donating moiety (D). Considering that strong D–A interactions
often decrease the ΦF value in the aggregated state, tetrapheny-
lethene (TPE), a typical AIE-active unit, is incorporated to yield
PITBT-TPE. As the 9,10-phenanthroimidazole fragment is a
large flat structure, a bulky tert-butyl group is further introduced
into PITBT-TPE to minimize the π–π interaction and form a
new red fluorophore of t-BPITBT-TPE. t-BPITBT-TPE was fur-
ther encapsulated into polymeric micelles with different surface
charges to form organic NPs stable in aqueous media. The gen-
erated NPs were used to analyze the behavior of t-BPITBT-TPE
and the surface charge from different polymeric encapsulating
matrices, DSPE-mPEG, DSPE-PEG-NH2, DSPE-PEG-COOH,
and DSPE-PEG-NH2-TAT on the health of zebrafish larvae and
the circulation of NPs in zebrafish. The toxicity, biodistribu-
tion and degradation of the different NPs were analyzed in the
zebrafish larvae through transdermal and intravenous delivery.
Furthermore, highly invasive metastatic cervical cancer cells
(HeLa) and nonmetastatic breast cancer cells (MCF-7) were
tagged with the t-BPITBT-TPE fluorescent NPs and were xeno-
grafted into the zebrafish yolk sac to track for cancer cell prolif-
eration and metastasis over 5 d. The duration of assessment was
limited to a week as development and activation of the immune
response would lead to tumor cell death or xenograft lethality.[20]
Finally, the fluorescence imaging ability of NPs toward 4T1
tumors in xenografted BALB/c mice was evaluated and the NP
distribution in the major organs of mice was studied.
2. Results and Discussion
The synthetic approaches toward PITBT-TPE and t-BPITBT-TPE
are illustrated in Scheme 2. PITBT-TPE and t-BPITBT-TPE were
obtained through Suzuki reaction between the intermediate 4
or 5 and 6 using Pd(PPh3)4 as the catalyst, where 4[21] and 5[22]
were prepared according to the synthetic processes published
previously. The detailed synthesis and characterization results
for both are provided in the Experimental Section.
The single crystals of both PITBT-TPE and t-BPITBT-TPE
were obtained for structure analysis. They were grown from
dichloromethane/methanol and dichloromethane/ethanol mix-
tures, respectively, and analyzed by X-ray diffraction crystallo-
graphy. As depicted in Figure 1, the TPE moiety in PITBT-TPE
adopts a twisted conformation to prevent π–π stacking inter-
actions, but the 2,1,3-benzothiadiazole (BT), thiophene (Th) and
9,10-phenanthroimidazole (PI) fragments are aligned almost in
the same plane, which is in favor of π–π stacking interactions
in the aggregated state. The torsion angles between BT, Th,
and PI are only 1.42° and 3.11°, respectively. However, they are
increased to 3.85° and 9.37°, respectively, in t-BPITBT-TPE. The
more twisted conformation can alleviate the intermolecular π−π
stacking interactions to some degree. Due to the incorporation
of tert-butyl, the molecular packing pattern of t-BPITBT-TPE
differs from that of PITBT-TPE (Figure 2A). The crystal struc-
tures of PITBT-TPE and t-BPITBT-TPE belong to monoclinic
and triclinic crystal systems, respectively. Figure 2B illustrates
the position relationship of PITBT-TPE or t-BPITBT-TPE mol-
ecule with two face-to-face adjacent molecules in the crystals.
As for PITBT-TPE, the BT and Th fragments are partially face-
to-face with the Th and BT fragments of one of the adjacent
molecules, respectively. The shortest distance from Th ring to
the plane of BT of the adjacent molecule is 3.43 Å, indicative
of π–π stacking interaction between the molecules. The BT and
Th fragments are also faced to the PI fragment of the other
S
S
N
N
N
N
N
N
N
N
S
S
PITBT-TPE
t-BPITBT-TPE
em,film = 640 nm
F,film = 49%
em,film = 641 nm
F,film = 24%
Scheme 1. Chemical structures of PITBT-TPE and t-BPITBT-TPE.
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Scheme 2. Synthetic routes to PITBT-TPE and t-BPITBT-TPE.
adjacent molecule in part, with the shortest distance of 3.42 and
3.31 Å from BT and Th to the PI plane. All the distances indi-
cated in Figure 2B suggest the existence of π−π stacking inter-
actions between adjacent PITBT-TPE molecules. However, the
face-to-face interactions are significantly weakened in adjacent
t-BPITBT-TPE molecules, which are witnessed by the dimin-
ished interaction area between BT and Th, and the increased
distance between Th and the PI plane to 3.41 Å. These results
demonstrate that as compared to PITBT-TPE, the π−π stacking
interactions between t-BPITBT-TPE molecules are substantially
weakened, which should favor improved emission efficiency in
the aggregated state for the latter.
THF solution shows intense PL peaking at 598 nm, with a high
ΦF value of 84%. t-BPITBT-TPE exhibits similar absorption, PL
and ΦF in dilute THF solution (Figure 3; Table 1), indicating
that the incorporation of tert-butyl exerts little impact on the
photophysical property of a dispersed molecule. This finding
is consistent with the computational results as PITBT-TPE and
t-BPITBT-TPE possess almost the same molecular conforma-
tions and identical spatial distributions of the lowest unoccu-
pied molecular orbitals and the highest occupied molecular
orbitals (Figure S1, Supporting Information). Due to the
strong D−A interaction, t-BPITBT-TPE also shows strong solva-
tochromic effect (Figure S2, Supporting Information). The
PL maximum of t-BPITBT-TPE in nonpolar cyclohexane is
559 nm, which is significantly red-shifted to 623 nm in polar
N,N-dimethylformamide. The AIE property of t-BPITBT-TPE
was studied in dimethyl sulfoxide (DMSO)–ethanol mixtures
as an example (Figure S3, Supporting Information). As the
ethanol content was increased, a poor solvent of t-BPITBT-TPE,
the t-BPITBT-TPE molecules start to form aggregate, and the
PL intensity of t-BPITBT-TPE is enhanced gradually, thanks
to the AIE effect of TPE unit. But when the ethanol content is
too high (>70 vol%), the PL intensity becomes lowered slightly,
due to the strong dipole–dipole interactions in the highly con-
densed phase. In films, because of the strong π–π stacking
interactions between PITBT-TPE molecules, the PL maximum
of PITBT-TPE is red-shifted to 641 nm and the ΦF is reduced to
24%. On the other hand, t-BPITBT-TPE has a PL maximum at
640 nm with a higher ΦF of 49% in films. Although the ΦF value
of t-BPITBT-TPE in film is lower than that in THF solution, the
AIE effect of TPE unit indeed offsets the emission quenching
of dipole-dipole interactions, and the bulky tert-butyl group
also efficiently mitigates the intermolecular π–π stacking inter-
actions in the condensed phase to ensure a high ΦF value.
To further understand the difference in PL behaviors, we
measured the time-resolved fluorescence spectra of PITBT-TPE
and t-BPITBT-TPE (Figure S4, Supporting Information) under
ambient conditions. Their fluorescence lifetimes as well as cal-
culated radiative and nonradiative decay rates are summarized
in Table 1. Both PITBT-TPE and t-BPITBT-TPE show almost
the same fluorescence lifetimes and radiative and nonradiative
decay rates in THF, which is consistent with their PL results in
PITBT-TPE and t-BPITBT-TPE exhibit strong absorption
maxima at 476 and 477 nm, respectively, in THF solution
(10 × 10−6 m) with a molar absorptivity close to 3 × 104 −1 cm−1
m
(Figure 3A). The molecularly dispersed PITBT-TPE in dilute
Figure 1. Crystal structures of PITBT-TPE (CCDC 1502965) and t-BPITBT-
TPE (CCDC 1502964).
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Figure 2. Illustration of A) crystal packing and B) intermolecular π–π stacking interactions of PITBT-TPE and t-BPITBT-TPE. The solvent molecules
and hydrogen atoms are omitted for clarity.
Figure 3. A) Absorption spectra in THF solutions and B) normalized photoluminescence spectra in THF solutions and films of PITBT-TPE and
t-BPITBT-TPE.
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Table 1. Photophysical properties of PITBT-TPE and t-BPITBT-TPE.
e)
krd)
[107 s−1
knr
a)
c)
Compound
λabs
λem
[nm]
ΦF
[%]
τ
[ns]
]
[107 s−1
]
[nm]
THF
598
600
Filmb)
641
THF
84
Filmb)
24
THF
5.64
5.65
Filmb)
4.99
THF
14.9
14.9
Filmb)
4.81
THF
Filmb)
15.2
PITBT-TPE
476
477
2.84
2.83
t-BPITBT-TPE
640
84
49
5.09
9.63
10.0
a)Measured in THF (10 × 10−6 m); b)Spin-coated film; c)Absolute fluorescence quantum yield determined by a calibrated integrating sphere; d)kr = ΦF/τ; e)knr = (1 − ΦF)/τ.
THF. For both molecules, the radiative decay rates (kr) in films
are lower than those in THF, while the nonradiative decay rates
(knr) are higher, resulting in decreased ΦF values in films. As
compared to PITBT-TPE, t-BPITBT-TPE has a smaller knr but an
approximately twice larger kr in films, giving rise to its greatly
increased ΦF in the film state. The red PL and high solid-state
ΦF value make t-BPITBT-TPE a more promising fluorophore
for fluorescence imaging.
To improve the water dispersibility of t-BPITBT-TPE for bioap-
plications, fluorescent NPs were fabricated by encapsulating the
hydrophobic t-BPITBT-TPE in polymeric micelles via nanopre-
cipitation following the reported technique.[23] 1,2-Distearoyl-sn-
glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol) (DSPE-PEG2000) block copolymers bearing hydrophobic
and hydrophilic domains with different terminal functional
groups allow spontaneous formation of micelles in aqueous
media. This yields NPs born with methoxy (DSPE-mPEG),
amine (DSPE-PEG-NH2), or carboxymethyl (DSPE-PEG-
COOH) groups (Scheme 3). Each surface modification gen-
erates different charges, thereby altering the behavior of the
NPs. It has been shown that the amine groups impart positive
charges and the carboxylic acid and methoxy groups impart
negative charges to the uncharged PEG surface.[24] The cell pen-
etrating peptide TAT, when conjugated to the surface of NPs,
can alter the surface charge to positive value.[25]
30 nm with a polydispersity index of less than 0.2, confirming
the uniformity of the NPs produced (Figure 4A). The spherical
nanoparticle morphology was also confirmed by using TEM
(Figure S5, Supporting Information). The optical properties
of the NPs, such as absorption and PL spectra, were found
to be similar to each other and not significantly affected by
the matrix. They show absorption and emission maxima at
about 480 and 660 nm (Figure 4B), with a high ΦF value of
≈32% in water relative to 4-(dicyanomethylene)-2-methyl-6-[4-
(dimethylaminostyryl)-4H-pyran] (ΦF = 43% in methanol). As
expected, the carboxyl and methoxy groups impart negative
charges to the NPs, resulting in net surface charge values of
−45.82 2.48 and −32.97 0.9 mV, respectively. The amine
group imparts a relatively positive charge to yield a net surface
charge value of −20.78 2.22 mV.
The initial toxicity response of wild-type zebrafish to the dif-
ferent surface properties of NPs was assessed using whole larvae
soaking. The zebrafish embryos were collected after crossing the
wild type parents and incubating the embryos in egg water with
1-phenyl-2-thiourea (PTU) at 28.5 °C and 0.4% CO2 for 3 d. The
biocompatibility of the different NPs was assessed by soaking
the larvae 4 days postfertilization (dpf) with the NPs at different
concentrations for 24 h (Figure 5B). The methodology further
estimates the initial safe working dose for each type of NPs.
Based on the initial toxicity evaluation, a TAT peptide was
further conjugated to the surface of DSPE-PEG-NH2 NPs since
they exhibited the least toxicity to zebrafish larvae. To facilitate
All the NPs were subjected to size measurement using
dynamic light scattering (DLS). The NPs have sizes of around
Scheme 3. Schematic illustration of the nanoprecipitation of t-BPITBT-TPE using DSPE-PEG block copolymers as the encapsulation matrices.
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Figure 4. A) DLS measurements for the different NPs. B) Representative absorption and PL spectra of the NPs.
easy conjugation, a mixture of DSPE-PEG-NH2 and DSPE-PEG-
maleimide were used for the NP formation, which was followed
by click reaction between a thiol functionalized TAT peptide and
the surface maleimide group (Scheme 3). The TAT conjugation
yielded NPs with a net positive surface charge value of 27 0.65
mV and a hydrodynamic size of 30 nm. All the NPs were purified
and concentrated by centrifugation filtration to yield a final NP
stock at a concentration of 640 µg mL−1 based on t-BPITBT-TPE.
The NPs with NH2 and NH2-TAT surface functionalization
were found to be nontoxic at the highest dose of 320 µg mL−1
(Figure 5B). The zebrafish larvae show no adverse side effects
and continue to grow post treatment. However, the larvae upon
shell surrounding the dye aggregate determines the net surface

charge of the NPs and the increased toxicity of COOH and

OCH3 should be attributed to the relatively higher negative
surface charges. This shows that the surface functionalization
of the NPs carrying t-BPITBT-TPE aggregates affects the initial
particle behavior and toxicity.
The healthy zebrafish larvae after soaking were imaged
to characterize the uptake. The images revealed low uptake
through the skin for all the negatively charged NPs. In the case
of COOH and OCH3 functionalization, due to their toxicity at
high concentrations, the larvae incubated with 80 µg mL−1 of
NPs were imaged to show undetectable fluorescence. Upon
incubation with 320 µg mL−1 of the NH2 functionalized NPs,
the larvae failed to show any significant uptake through the
skin due to the net negative surface charge, which is electrosta-
tistically repelled by the negatively charged cell membrane.
However, for the NH2-TAT NPs (320 µg mL−1), they were taken
up through the skin after 24 h of soaking. This substantiates

treatment with the negatively charged NPs, i.e., with COOH

and OCH3 surface groups, show poor survival at the dose of
320 µg mL−1 (Figure 5A). As the concentration of these NPs is
sequentially reduced, the survival improves. Since the core of
the NPs is the same, the toxic response to the different NPs can
be attributed to the different polymeric matrices. The polymeric
Figure 5. A) Percentage of embryo survival for whole embryo soaking in 320, 160, and 80 µg mL−1 NPs based on t-BPITBT-TPE concentration (n = 17).
B) Sequential dilution of stock starting at 320 µg mL−1 t-BPITBT-TPE for NH2-TAT NPs.
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Additional long-term toxicity evaluation was performed for
the NH2 and NH2-TAT NPs since they showed most prom-
ising biocompatibility through soaking. Both NPs were injected
separately and intravenously into 5 dpf wild type larvae retro-
orbitally. The intravenous retro-orbital injection was performed
using pneumatic microinjection in continuous mode. The
larvae were injected with NPs at 640, 320, and 160 µg mL−1
based on t-BPITBT-TPE concentration to observe the inten-
sity change with concentration. The maximum viable dose for
intravenous injection is higher than that observed in whole
larva soaking since intravenously injected dose is distributed
throughout systemic circulation which is more benign than
continuous exposure in the whole embryo soaking approach.
The larvae were imaged postinjection with various concentra-
tions of NPs and a clear difference in intensity was observed
mainly in the circulation and in the trunk region (Figure 7).
To understand biodistribution of the NPs, both NH2 and
NH2-TAT NPs (320 µg mL−1) were injected separately to the
embryos and the fluorescence was tracked for up to 7 d. It was
found that the zebrafish larvae were healthy and did not develop
any edema or cardiotoxicity. Although some of the NPs were
in circulation for up to 2 d but most were taken up by cells in
the first day itself. The difference in NP uptake was clearly dis-
cernable in the trunk region of the larva. The fluorescent signal
in the trunk for the NH2 NPs shows distribution and uptake
through the tissue, and some of the NPs permeate through to
the skin as well (Figure 8A). This suggests that the NPs per-
meate from the vasculature and are taken up unbiasedly. On
Figure 6. Confocal images of larvae showing normal development post
24 h of whole larval soaking with A) the NH2 NPs and B) the NH2-TAT
NPs. The scale bar is 200 µm.
the claim that TAT loaded NPs have faster uptake due to
better electrostatic interaction and enhanced cell-penetrating
capability (Figure 6).
Figure 8. Embryos with A) NH2 NPs and B) NH2-TAT NPs at 320 µg mL−1
based on the t-BPITBT-TPE concentration postinjection, 1 d after injec-
tion and 2 d after injection. Dashed box is demarcating the caudal vein
tissue and the scale bar is 200 µm.
Figure 7. Embryos postinjection of A) NH2 NPs and B) NH2-TAT NPs at
640, 320, and 160 µg mL−1 based on t-BPITBT-TPE concentration. The
scale bar is 200 µm.
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Figure 9. Photostability assay of t-BPITBT-TPE in NH2-TAT NPs in A,B) HeLa cells and C,D) zebrafish larvae under continuous confocal laser scanning
at 488 nm excitation. Scale in (A) depicts 20 µm and scale in (C) depicts 200 µm.
the contrary, the NH2-TAT NPs in the trunk region show locali-
zation in the caudal hematopoietic tissue and caudal vein tissue
near the tail (Figure 8B). This is due to the quicker uptake of
the NPs in circulation, offering them less time to penetrate
through the vasculature. The NH2-TAT NPs can hence be used
to ensure a quicker uptake in tissue around the vasculature,
owing to its positive charges.
performed for both stained cell lines for 5 d in order to confirm
the presence of fluorescent signal after multiple cell division
cycles. As illustrated in Figure S6 (Supporting Information),
the cells were found to still fluoresce up until the fifth day,
indicating the good long-term tracing ability of these NPs. The
MTT assay was performed to evaluate the toxicity of the NPs to
the HeLa and MCF-7 cells and was found to be of low toxicity
even up to 60 µg mL−1 based on t-BPITBT-TPE concentration.
The stained HeLa and MCF-7 cells were xenografted into the
yolk sac of zebrafish larvae (1 dpf) to track in vivo cancer cell
growth over time. The yolk sac is considered to be the optimal
site for transplantation since it is nutrient rich and acellular.[27]
The HeLa cells stained with t-BPITBT-TPE NPs (30 µg mL−1)
were suspended in a solution containing polyvinylpyrrolidone
and fetal bovine serum (3 × 106 cells mL−1) and microinjected
into zebrafish larvae. Each successfully xenotransplanted
zebrafish was individually tracked for five days to trace the dye
integrity over multiple cancer cell proliferations and analyze the
metastasis potential of injected cells in vivo.
The excellent biocompatibility of the NH2-TAT NPs and the
effective cell uptake in zebrafish larvae inspired us to apply
them for xenotransplantation of cancer cell lines to assess its
potential as a quantitative long term cell tracker that gauge
metastatic potential from different cancer cells. The photosta-
bility of the NPs was assessed in vitro and in vivo by subjecting
HeLa cells and zebrafish larvae tagged with t-BPITBT-TPE in
NH2-TAT NPs to continuous laser ablation for 60 min. The
fluorescent signals in vitro and in vivo were found to be stable
(Figure 9), indicating excellent photostability of the NPs.
Two cell lines of different proliferative and metastatic phe-
notype, HeLa and MCF-7, were selected for the study. HeLa
cells are known to possess aggressive tumorigenic proper-
ties while MCF-7 cells are known to be relatively mild in their
behavior.[26] The cellular uptake and cytotoxicity evaluation
results are shown in Figure 10. The confocal images show sig-
nificant internalization of the NH2-TAT NPs into the cytoplasm
of the cells to give bright red fluorescence. Cell tracking was
To compare the behaviors of two different cancer cell lines,
grafts of identical size were selected. The grafts selected were
of small size (50–100 cells) so that the metastatic phenotype
could be captured. In Figure 11A, individually tracked zebrafish
xenografted with HeLa cells shows cancer cell proliferation and
metastasis at 5 d post transplantation. In comparison, MCF-7
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Figure 10. A) Confocal images of HeLa and MCF-7 cells stained with 30 µg mL−1 of NH2-TAT NPs based on t-BPITBT-TPE concentration. The scale bar
is 50 µm. B) MTT assay depicting the viability of HeLa and MCF-7 cells stained with different concentrations of NH2-TAT NPs.
cancer cells do not proliferate as aggressively. Metastasis of the
cells from the site of injection occurs through transport routes
like the circulation network[28] and hence is analogous to the
cancer cell metastasis in human. The HeLa cells are capable
of detaching from the tumor and migrating through the circu-
lation down to the caudal vasculature as seen in Figure 11B.
Figure 11. A) Tumor graft progression over 5 d in zebrafish larvae xenografted with fluorescent HeLa and MCF-7 cells; inset showing grayscale images
of cancer cells. B) Fluorescent image of HeLa cell migration post dissemination. The scale bar is 200 µm.
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was readily realized just by a tert-butyl group,
which had been found to apparently impact
the molecular arrangement in the crystalline
state. A detailed toxicity and biodistribution
profile in the zebrafish model was established
for t-BPITBT-TPE-based NPs with different
encapsulating matrices. The NPs with highly
negative surface charges were found to be
significantly toxic to the zebrafish embryos
through transdermal permeation. As the sur-
face charge increased to more positive, the
toxicity decreased. The NP formulations with
least toxicity were chosen to be injected into
the zebrafish larvae intravenously. Through
this delivery method, the long term toxicity
and uptake of the NPs was evaluated. The
difference in uptake from the vasculature
and ability to penetrate tissue of positively
and negatively charged NPs was demon-
strated. The NPs decorated with TAT peptide
showed efficient cellular uptake and were
used to stain HeLa and MCF-7 cells which
were xenografted in zebrafish. The NH2-TAT
NPs enabled long term tracing of the prolif-
eration and metastasis of HeLa and MCF-7
cells in zebrafish. Since a large number of
zebrafish embryos can be transplanted with
biocompatible fluorescent NP tagged cancer
cells, the same strategy can further be used
Figure 12. A) Accumulation of t-BPITBT NH2-TAT NPs in tumor xenograft at different time
postintravenous injection, and B) distribution of the NPs in different organ tissues.
No such metastatic behavior was observed in zebrafish larvae
xenografted with MCF-7 cells (Figure S7, Supporting Informa-
tion). These results reveal that the NH2-TAT NPs enable the
quantitative tracking of dynamic cancer cell proliferation and
metastasis.
in zebrafish model for drug screening. In addition, NH2-TAT
NPs also showed good imaging ability toward 4T1 tumors in
xenografted mice. These results demonstrate that organic nano-
particles with strong red fluorescence and various functional
groups on surface could be promising contrast reagents for bio-
logical applications.
To demonstrate that t-BPITBT-TPE NPs are also suitable for
mice imaging, BALB/c mice xenografted with 4T1 tumors were
injected with 1 mg mL−1 of NH2-TAT NPs intravenously. The
results showed that the NPs accumulated in the tumor over a
period of 7 h, and the fluorescent signals in the tumor gradually
intensified over time and achieved the highest brightness at 7 h
postintravenous injection of NPs (Figure 12A), demonstrating
good in vivo imaging ability of NH2-TAT NPs toward 4T1
tumors. The mice were sacrificed after 7 h and the NP distribu-
tion was investigated in the different organs, including heart,
liver, spleen, kidney, lung, intestine, and stomach, resected and
imaged immediately using a Maestro optical imaging system.
As displayed in Figure 12B, the NPs flowed in the circulatory
system of mice, and predominantly accumulated in liver, which
was ascribed to the metabolic function of animals.
4. Experimental Section
Synthesis
of
4-(5-(1-(4-(Tert-Butyl)Phenyl)-1H-Phenanthro[9,10-
d]Imidazol-2-yl)Thiophen-2-yl)-7-(4-(1,2,2-Triphenylvinyl)Phenyl)-
Benzo[c][1,2,5]Thiadiazole (t-BPITBT-TPE): Into a mixture of 4b (563 mg,
1.1 mmol), 5 (411 mg, 1.0 mmol), 6 (388 mg, 1.0 mmol), Pd(PPh3)4
(70 mg, 0.06 mmol), and Na2CO3 (530 mg, 5.0 mmol), 40 mL of
THF and 10 mL of deionized water were added. The reaction mixture
was stirred and refluxed for 18 h under nitrogen atmosphere. After
cooling to room temperature, the mixture was poured into water
and extracted with dichloromethane for three times. Before solvent
evaporation, the combined organic extracts were dried over anhydrous
magnesium sulfate. The crude product was purified by silica-gel column
chromatography using petroleum ether/dichloromethane as eluent.
t-BPITBT-TPE was obtained as red solid in 22% yield (142 mg). 1H NMR
(500 MHz, CD2Cl2), δ (ppm): 8.91 (d, J = 7.6 Hz, 1H), 8.72 (dd, J =
20.5, 8.3 Hz, 2H), 7.91 (d, J = 4.1 Hz, 1H), 7.88 (d, J = 7.5 Hz, 1H),
7.81–7.76 (m, 5H), 7.69–7.66 (m, 2H), 7.63–7.60 (m, 2H), 7.53–7.50 (m,
1H), 7.30–7.26 (m, 1H), 7.23–7.10 (m, 16H), 7.07–7.03 (m, 3H), 1.54
(s, 9H). 13C NMR (126 MHz, CD2Cl2), δ (ppm): 155.01, 154.14, 153.00,
146.06, 144.40, 144.17, 144.13, 144.11, 141.96, 141.36, 140.90, 135.75,
135.50, 132.86, 131.81, 131.70, 131.63, 131.59, 129.50, 128.98, 128.81,
128.71, 128.23, 128.14, 128.04, 127.75, 127.02, 126.93, 126.87, 126.18,
126.12, 125.83, 125.51, 124.39, 123.58, 123.19, 121.18, 35.53, 31.66.
3. Conclusion
In summary, robust red fluorophores BPITBT-TPE and
t-BPITBT-TPE with high solid-state fluorescence efficiencies
were designed and synthesized. The introduction of AIE-active
TPE could effectively alleviate the emission quenching of the
molecules with electronic D–A structures in the aggregated
state. In addition, solid-state fluorescence efficiency modulation
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HRMS (C61H44N4S2): m/z 896.3014 [M+, calcd 896.3007]. Anal. Calcd for
C61H44N4S2: C, 81.66; H, 4.94; N, 6.24. Found: C, 81.55; H, 5.10; N, 6.15%.
The synthetic procedure to PITBT-TPE was analogous to that of
t-BPITBT-TPE. Red solid, yield 18%. 1H NMR (500 MHz, CD2Cl2), δ
(TMS, ppm): 9.02 (br, 1H), 8.81 (d, 1H, J = 9.0 Hz), 8.77 (d, 1H, J =
8.5 Hz), 7.99−7.95 (m, 2H), 7.89−7.83 (m, 6H), 7.77−7.75 (m, 4H),
7.60−7.57 (m, 1H), 7.34−7.31 (m, 1H), 7.25−7.10 (m, 19H). The 13C
NMR data are not available due to its limited solubility in common
deuterated solvents, such as CDCl3 and CD2Cl2. HRMS (C57H36N4S2):
m/z 840.2393 (M+, calcd 840.2381). Anal. Calcd for C57H36N4S2: C,
81.40; H, 4.31; N, 6.66. Found: C, 80.97; H, 4.34; N, 6.57%.
buffered saline (PBS) buffer twice. The NH2-TAT NPs in DMEM at a
concentration 30 µg mL−1 (based on t-BPITBT-TPE mass concentration)
were added into the well plate. After 2 h of incubation, the cells were
washed with 1× PBS buffer three times and following were fixed with
75% ethanol for 20 min. The cells were further washed with 1× PBS
buffer two times. The cells were then mounted and studied with confocal
laser scanning microscope (Zeiss LSM 800, Jena, Germany).
The toxicity of NH2-TAT NPs to HeLa and MCF-7 cells was
characterized with MTT assay. HeLa and MCF-7 cells were cultured
for 24 h in the 96 well plates (Costar, IL, USA) at a density of
5 × 104 cells mL−1. Different concentrations of NPs suspension were
used to replace the cell medium. The cells were cultured for another
4 h. Subsequently, 10 µL of freshly prepared MTT solution with
a concentration of 5 mg mL−1 was added into each well. Post 3 h of
incubation, MTT medium solution was removed. 100 µL of DMSO was
added into each well to dissolve the formed precipitates. A microplate
reader (Genios Tecan) was used to monitor the absorbance at 570 nm
of MTT. The cell viability was calculated by the ratio of the absolute
absorbance of the cells treated with NPs suspension to that of the cells
only cultured in culture medium.
HeLa and MCF-7 Cell Preparation and Xenotransplantation: HeLa
and MCF-7 cells were stained with t-BPITBT-TPE NPs encapsulated
in DSPE-PEG-NH2-TAT matrix. Cells were cultured with DMEM at
37 °C in a humidified environment with 5% CO2. The DMEM media
contained 10% of fetal bovine serum and 1% of penicillin streptomycin.
Before experiments, the cells were precultured until confluence was
reached. The cells were treated with 30 µg mL−1 of t-BPITBT-TPE NPs
in DMEM media for 2 h. Subsequently, the adherent cells were washed
with 1× PBS buffer twice, trypsinized and resuspended in media. The
cells were transferred to 1.5 mL Eppendorf tubes and centrifuged
5 min, at 1500 rpm. Cells were resuspended in 0.5 mL of solution
containing 2.5% polyvinylpyrrolidone (PVP-40) in fetal calf serum to
obtain a concentration of 3 × 106 cells mL−1. 1 dpf zebrafish embryos
were dechorionated and immobilized by mounting in a mixture 1%
low melting agarose and 5% methyl cellulose. Using a manual injector
(Harvard apparatus), the cell suspension was loaded into an injection
needle (O.D. 1.0 mm, I.D. 0.75 mm). Cells were now injected in 1 dpf
wild type zebrafish embryos. After injection, embryos were incubated
for 1 h at 25 °C and checked for cell presence. All other fish were
incubated at 32.5 °C for the following days and imaged using Carl Zeiss
LSM 510 Meta microscope.
Crystal Data for t-BPITBT-TPE (CCDC 1502964): C61H44N4S2·CH2Cl2,
MW = 982.05, triclinic, P-1, a = 12.9771(7) Å, b = 12.9950(8) Å,
c = 16.5755(9) Å, α = 98.365(2)°, β = 100.440(2)°, γ = 111.832(2)°,
V = 2481.5(2) Å−3, Z = 2, Dc = 1.314 g cm−3, µ = 0.261 mm−1
(MoKα, λ = 0.71073), F(000) = 1024, T = 173(2) K, 2θmax = 25.38°
(98.0%), 22615 measured reflections, 8943 independent reflections
(Rint = 0.0709), goodness of fit (GOF) on F2 = 1.027, R1 = 0.1208, wR2 =
0.1283 (all data), Δe 0.290 and −0.418 eÅ−3
.
Crystal Data for PITBT-TPE (CCDC 1502965): C57H36N4S2·2(CH2Cl2),
MW = 1010.87, monoclinic, P21/n, a = 11.8521(15) Å, b = 26.434(4) Å,
c = 14.763(2) Å, β = 91.886(6)°, V = 4622.9(11) Å−3, Z = 4, Dc = 1.452 g
cm−3, µ = 0.394 mm−1 (MoKα, λ = 0.71073), F(000) = 2088, T = 173(2) K,
2θmax = 24.50° (99.1%), 28671 measured reflections, 7623 independent
reflections (Rint = 0.0875), GOF on F2 = 1.068, R1 = 0.1194, wR2 = 0.1930
(all data), Δe 0.651 and −0.976 eÅ−3
.
Nanoparticle Preparation: The organic NPs were prepared by
 
nanoprecipitation. 200 µL DSPE-PEG2000
( OCH3, COOH, or NH2) in
THF at 4 mg mL−1 was mixed with 800 µL of t-BPITBT-TPE in THF at
2 mg mL−1 to yield a 1 mL solution. The organic solution was injected
rapidly into 10 mL of water under probe sonication at 12 W to ensure
maximum agitation. The solution was stirred overnight to ensure
evaporation of the THF. The solution was then filtered through a 220 nm
syringe filter and concentrated to 640 µg mL−1 stock using a centrifuge
filter.
The NH2-TAT NPs were prepared by mixing 100 µL of DSPE-
PEG2000-NH2, 100 µL of DSPE-PEG2000-maleimide at 4 mg mL−1 in
THF and 800 µL of t-BPITBT-TPE in THF at 2 mg mL−1 to yield a 1 mL
solution. The organic solution was injected rapidly into 10 mL of water
under probe sonication at 12 W to ensure maximum agitation. The
10 mL stock after syringe filtration was stirred with a solution of TAT
peptide in DMSO (10 mg mL−1) for 24 h followed by dialysis (10 kDa
MWCO) for 3 d to ensure complete DMSO removal. The stock was then
concentrated using a centrifuge filter.
Zebrafish Line and Toxicity Test: Four to five pairs of zebrafish were
placed in crossing tanks for spawning overnight. Embryos were
settled to the bottom of the tank, and were collected using a sieve and
transferred to petri dishes for embryo culture. They were screened,
incubated at 27 °C, 0.4% CO2 and grown in egg water (10% NaCl; 1.63%
MgSO4·7H2O; 0.4% CaCl2; 0.3% KCl). After 22 h postfertilization, PTU
was added to prevent melanin formation to yield optically transparent
fish. 3 d postfertilization the embryos were seeded to into 96 well plates
at 1 embryo per well. The embryos were soaked in 100 µL of NPs at
various concentrations for 24 h and were imaged for uptake using
confocal imaging (Carl Zeiss LSM 510 Meta) using 488 nm.
Microinjection: Local in vivo injection of the NPs intravenously was
done using a nitrogen gas injector. The needle used for injection is made
by pulling glass capillary tubes (O.D. 1.0 mm, I.D. 0.75 mm) in a needle
puller which heats to pull the glass tubes into fine needles (20 µm). The
NPs were filled into the needles which were loaded into the nitrogen gas
injector. The injector was operated in continuous mode for the retro-
orbital delivery of the NPs. The fishes were mounted in a mixture of
1% low melting agarose and 5% methyl cellulose for the injection and
confocal imaging.
HeLa and MCF-7 Cell Imaging and Toxicity Analysis: HeLa and MCF-7
cells were cultured at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle
Medium (DMEM). After reaching 70% confluence, the culture medium
was removed. Then the adherent cells were washed with 1× phosphate
Fluorescence Imaging in Xenografted Mice: 4T1 cancer cells (1 × 106)
suspended in 30 µL of saline were injected subcutaneously into the left
back of the BALB/c mice. Tumors were grown until a single aspect was
≈7 mm (approximately two weeks) before fluorescence imaging. BALB/c
mice bearing 4T1 tumors were intravenously injected with t-BPITBT-TPE
NH2-TAT NPs (100 µL, 1 mg mL−1) (n = 5), respectively. Fluorescence
signals were captured after NPs administration. 7 h after NP injection,
mice were sacrificed and different tissues were collected, imaged by the
Maestro system.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
G.L. and P.N.M. contributed equally to this work. The authors
acknowledge the financial support from the National Natural Science
Foundation of China (21673082), the Guangdong Natural Science Funds
for Distinguished Young Scholar (2014A030306035), the Fundamental
Research Funds for the Central Universities (2017ZD001 and 2015ZY013),
NRF Investigatorship (R279-000-444-281), National University of
Singapore (R279-000-482-133), the Natural Science Foundation of
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Technology Commission of Hong Kong (ITC-CNERC14SC01).
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Conflict of Interest
The authors declare no conflict of interest.
Keywords
aggregation-induced emission, cancer cell progression, fluorescence
imaging, nanoparticles, zebrafish
Received: March 17, 2017
Revised: May 4, 2017
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