Quinoxaline-based polymer dots with ultrabright red to near-infrared fluorescence for in vivo biological imaging

Article abstract of DOI:10.1021/jacs.5b06710

This article describes the design and synthesis of quinoxaline-based semiconducting polymer dots (Pdots) that exhibit near-infrared fluorescence, ultrahigh brightness, large Stokes shifts, and excellent cellular targeting capability. We also introduced fluorine atoms and long alkyl chains into polymer backbones and systematically investigated their effect on the fluorescence quantum yields of Pdots. These new series of quinoxaline-based Pdots have a fluorescence quantum yield as high as 47% with a Stokes shift larger than 150 nm. Single-particle analysis reveals that the average per-particle brightness of the Pdots is at least 6 times higher than that of the commercially available quantum dots. We further demonstrated the use of this new class of quinoxaline-based Pdots for effective and specific cellular and subcellular labeling without any noticeable nonspecific binding. Moreover, the cytotoxicity of Pdots were evaluated on HeLa cells and zebrafish embryos to demonstrate their great biocompatibility. By taking advantage of their extreme brightness and minimal cytotoxicity, we performed, for the first time, in vivo microangiography imaging on living zebrafish embryos using Pdots. These quinoxaline-based NIR-fluorescent Pdots are anticipated to find broad use in a variety of in vitro and in vivo biological research.

Full text of DOI:10.1021/jacs.5b06710

Article
pubs.acs.org/JACS
Quinoxaline-Based Polymer Dots with Ultrabright Red to Near-
Infrared Fluorescence for In Vivo Biological Imaging
Hong-Yi Liu,† Pei-Jing Wu,† Shih-Yu Kuo,† Chuan-Pin Chen,† En-Hao Chang,† Chang-Yi Wu,‡
and Yang-Hsiang Chan*,†
†Department of Chemistry, National Sun Yat-sen University, 70 Lien Hai Road, Kaohsiung, Taiwan 80424
‡Department of Biological Sciences, National Sun Yat-sen University, 70 Lien Hai Road, Kaohsiung, Taiwan 80424
S
* Supporting Information
ABSTRACT: This article describes the design and synthesis
of quinoxaline-based semiconducting polymer dots (Pdots)
that exhibit near-infrared fluorescence, ultrahigh brightness,
large Stokes shifts, and excellent cellular targeting capability.
We also introduced fluorine atoms and long alkyl chains into
polymer backbones and systematically investigated their effect
on the fluorescence quantum yields of Pdots. These new series
of quinoxaline-based Pdots have a fluorescence quantum yield
as high as 47% with a Stokes shift larger than 150 nm. Single-
particle analysis reveals that the average per-particle brightness
of the Pdots is at least 6 times higher than that of the
commercially available quantum dots. We further demonstrated the use of this new class of quinoxaline-based Pdots for effective
and specific cellular and subcellular labeling without any noticeable nonspecific binding. Moreover, the cytotoxicity of Pdots were
evaluated on HeLa cells and zebrafish embryos to demonstrate their great biocompatibility. By taking advantage of their extreme
brightness and minimal cytotoxicity, we performed, for the first time, in vivo microangiography imaging on living zebrafish
embryos using Pdots. These quinoxaline-based NIR-fluorescent Pdots are anticipated to find broad use in a variety of in vitro and
in vivo biological research.
operative imaging, only a paucity of fluorophores (e.g.,
indocyanine green and methylene blue) have been approved
for clinical or preclinical trial.13
INTRODUCTION
■
There have been considerable efforts in the development of
optical imaging techniques with fluorescence microscopy due to
their high signal-to-noise ratio, excellent spatial and/or
temporal resolution, and noninvasive nature, rendering them
extremely useful in the investigation of biological processes in
live organisms. In recent years, the development of several
super-resolution fluorescence microscopy techniques has
successfully surpassed the diffraction limit of light and these
technologies have been employed to visualize biological
structures with a spatial resolution of 10−30 nm.1−9 Moreover,
the new breakthroughs in fluorescence image-guided sur-
gery10,11 and its first in-human proof-of-concept staging and
debulking surgery for ovarian cancer12,13 have also strongly
highlighted the importance of choosing a photostable,
biocompatible, bright, and tumor-specific fluorescent agent. In
the past few years, intraoperative imaging by use of visible to
invisible near-infrared (NIR) fluorescent light has played an
important role between the gap of preoperative detection and
intraoperative techniques. Particularly for the contrast agents
with emission in the NIR range (700−1400 nm), they have
been widely exploited in bioimaging and clinical diagnosis. It is
because NIR light can travel several millimeters (up to
centimeters) through blood or tissues, and minimal scattering
or autofluorescence of biological species is observed in the NIR
spectrum.14,15 Among these NIR contrast agents for intra-
As compared to conventional organic dyes, nanoparticle-
based imaging agents (e.g., dye-doped silica/polymeric nano-
particles16−20 or quantum dots21−23) exhibit much higher
brightness and thereby can be used to improve the temporal
resolution in bioimaging. Nevertheless, several concerns such as
leakage of the doped dyes from the silica/polymeric matrix24,25
and the potential release of the toxic inorganic compounds
(e.g., cadmium)26−28 might largely restrict their biological use
and clinical implementation.
Very recently, semiconducting polymer nanoparticles
(Pdots) have emerged as a new type of highly fluorescent
probes that possess extraordinary fluorescence brightness,
superior photostability, high emission rates, and minimal
toxicity to cells and tissues.29−47 More importantly, Pdot-
based NIR fluorescent agents offer significant advantages over
conventional small fluorophores because most NIR fluorescent
dyes are subjected to poor solubility in aqueous solutions, fast
photobleaching, low fluorescence brightness, and small Stokes
shift. Although great efforts have been exerted to develop NIR
fluorescent Pdots, various challenges have impeded the
Received: June 29, 2015
© XXXX American Chemical Society
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progress, especially for the severe aggregation-caused emission
quenching of the rigid and flat π−π stacking of the conjugated
scaffolds of NIR-emitting polymers upon condensation into a
Pdot form. Several strategies have been explored to tackle this
self-quenching problem. One approach is to encapsulate NIR
dyes or polymers into the Pdot matrix, resulting in efficient
energy transfer from Pdot to the doped dyes or polymers.48−50
However, the leakage of the dopant molecules from the Pdot
matrix remains a serious issue in many applications that require
long-term monitoring.51 To address the leaking issue, our
group have successfully developed a facile method by capping
the dye-embedded Pdots with polydiacetylenes.52−54 Another
strategy is the direct synthesis of NIR-emitting polymers by
incorporating NIR-fluorescing chromophores into the semi-
conducting polymer backbone. To date, however, only a very
small molar fraction of NIR chromophores (usually <3%) could
be incorporated into the Pdot matrix due to the important issue
of self-quenching at higher molar concentrations.55,56 Very
recently, we have designed and synthesized a series of
dithienylbenzoselenadiazole (DBS)-based copolymers to form
NIR fluorescent Pdots where ∼10% of DBS units could be
incorporated once the alkyl substitution was introduced onto
the DBS monomer to alleviate self-quenching phenomenon.57
However, the relatively low molar percentage of NIR emitters
inside the polymer could cause diminished photostability of the
resulting Pdots if a stronger laser excitation is used for
prolonged periods of time.57 Besides, this type of Pdots is
unable to provide high brightness once excited at the
absorption maximum of the NIR monomers owing to their
low absorption.
rigid NIR-emitting derivatives usually results in the fact that the
fluorescence quantum yields of the polymers decrease
dramatically with increasing concentrations of NIR units. As a
trade-off, therefore, only a small molar ratio (∼3%) of NIR
chromophores can be incorporated into the polymers in order
to obtain a high quantum yield. Because the fluorescence
brightness is calculated from the product of the peak absorption
cross-section and the fluorescence quantum yield, the low
molar concentration of NIR chromophores would cause low
fluorescence brightness if the polymer was excited at the
absorption peak of the NIR monomers. As such, the polymers
can be excited only at the absorption of the energy donor (i.e.,
fluorene, λmaxabs < 400 nm). Here we intend to circumvent this
issue by the employment of bulky quinoxaline monomers with
various substituents because we expect the steric hindrance
from the fairly large quinoxaline derivatives can efficiently
eliminate the phenomenon of aggregation-caused quenching.
Specifically, we first synthesized a series of quinoxaline-based
monomers and then polymerized with fluorene via palladium-
catalyzed Suzuki coupling to form a series of copolymers a
shown in Figure 1. Here we fixed the molar percentage of
To address the above-mentioned challenges, we here
describe the design and synthesis of quinoxaline-based Pdots
that show bright NIR emissions and can be readily excited by a
conventional 488- or 532 nm laser. More importantly, the
molar fraction of NIR chromophores (i.e., quinoxaline)
incorporated inside the semiconducting polymers can be as
high as 50% with a quantum yield up to 47%. Additionally, we
directly functionalized carboxyl groups on the side chains of the
polymers for further biomolecular conjugation and specific
cellular labeling. Single-particle brightness analysis reveals that
the per-particle brightness of the quinoxaline-based Pdots is
about 6-8 times brighter than that of quantum dots. We also
performed in vivo microangiography imaging on living
zebrafish. The high quantum yield, large absorption cross-
section in the visible region, and large Stokes shifts of these
quinoxaline-based Pdots are promising in a broad range of in
vitro/vivo bioimaging and analytical detection.
Figure 1. Chemical structures of a series of quinoxaline-containing
polymers.
quinoxaline emitters at 50% so that the resulting copolymers
abs
can be directly excited at the quinoxaline monomers (λmax
=
450−600 nm, vide infra), which is more favorable for many
biological applications.
The general synthetic routes for the monomers and polymers
were illustrated in Scheme 1. Compound 2 and 6 were
synthesized according to previously reported literatures with
modified procedures.58−60 Fluorinated compound 8 was then
synthesized by reacting compound 2 with 6. Monomer 10 was
obtained by use of Stille coupling from compound 7 and 8,
followed the bromination using NBS. Monomers 11−13 were
synthesized according to the reported literature with modified
procedures (see Supporting Information).58,61,62 Finally, Suzuki
polymerization between fluorene derivative 14 and monomers
10, 11, 12, and 13 with Pd(PPh3)4 catalyst gave quinoxaline-
containing conjugated copolymers PF-TC6FQ, PF-TTFQ, PF-
TFQ, and PF-TQ, respectively. We also directly functionalized
the conjugated copolymers with carboxylate groups on the
substituents of polyfluorene to prevent the leaking problem
which is frequently confronted by other functionalization
approaches such as surface encapsulation or polymer
coprecipitation startegies.51 For instance, we incorporated
RESULTS AND DISCUSSION
■
Our aim was to synthesize quinoxaline-based Pdots with
ultrabright red to NIR emission and investigate how the long
alkyl chains on the thiophene rings as well as the fluorine atoms
on the quinoxaline moieties affect their optical properties. We
further functionalized the polymers with carboxylate groups for
in vitro specific cellular labeling and in vivo biological imaging.
Design and Synthesis of Quinoxaline-Containing
Copolymers with NIR Fluorescence. In an effort to develop
NIR fluorescent semiconducting polymers, we exploited the
donor−acceptor concept in which strong electron-donating
units and strong electron-accepting moieties (i.e., NIR-emitting
units) were integrated into the polymer backbone to synthesize
the low bandgap copolymers. Unfortunately, the serious
aggregation-induced fluorescence quenching of the flat and
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Scheme 1. Synthetic Routes of Quinoxaline Comonomers
and Copolymers
Scheme 2. (A) Preparation of Carboxyl-Functionalized
Quinoxaline-Based Pdots and Subsequent Bioconjugation
a
for Specific Celluar Labeling; (B) Hydrodynamic Diameter
of PF-TC6FQ-COOH Pdots Measured by Dynamic Light
Scattering (Left) and their Corresponding Transmission
b
Electron Microscopy Images (Right); (C) Gel
Electrophoresis of Bare Pdots, Streptavidin-Conjugated
Pdots, and Folate-Functionalized Pdots
a
The conjugated polymer (PF-TC6FQ-COOH as an example in this
scheme) was dissolved in THF and then nanoprecipitated in water
under vigorous sonication to form Pdots. The carboxylic-acid-
functionalized Pdots were then conjugated to streptavidin or folic
acid through EDC-catalyzed coupling for specific cellular targeting.
b
Scale bar is 100 nm.
compound 15, a carboxyl-functionalized fluorene moiety, into
the conjugated backbone of PF-TC6FQ-BOC polymer and
then removed the protection group (tert-butyl) by using
trifluoroacetic acid to obtain PF-TC6FQ-COOH (Scheme 1).
It is worth mentioning that we integrated only 5% carboxyl
groups within the polymer backbone because Chiu’s group has
demonstrated that the incorporation of carboxyl groups with a
low density can form stable and bright Pdots without
nonspecific adsorption toward biomolecules.63 The carboxylate
functional polymers (e.g., PF-TC6FQ-COOH) can thereby be
conjugated with biomolecules for biological applications.
Photophysical Properties of Quinoxaline-Based
Pdots. The preparation of Pdots and the subsequent surface
functionalization was illustrated in Scheme 2A. The average
diameter of Pdots determined by dynamic light scattering
(DLS) and transmission electron microscopy (TEM) was 22
nm (Scheme 2B). Scheme 2C show the result of gel
electrophoresis in which bare PF-TC6FQ-COOH Pdots
moved faster than both streptavidin-modified and folate-
functionalized Pdots. This result can be ascribed to the reduced
negative charged on the Pdot surfaces because of the
consumption of the carboxyl groups after streptavidin or folate
conjugation. The absorption and emission spectra of four
different types of quinoxaline-containing Pdots are shown in
Figure 2. The absorption peaks at 350−430 nm and 450−600
Figure 2. (A) Absorption spectra of PF-TC6FQ, PF-TFQ, PF-TTFQ,
PF-TQ Pdots series in water. The inset shows the photograph of the
Pdot solutions (from left to right: PF-TC6FQ, PF-TFQ, PF-TTFQ,
and PF-TQ Pdots). (B) Emission spectra of PF-TC6FQ, PF-TFQ,
PF-TTFQ, PF-TQ Pdots series in water. The inset shows the
photograph of the Pdot solutions under 365 nm UV light illumination
(from left to right: PF-TC6FQ, PF-TFQ, PF-TTFQ, and PF-TQ
Pdots).
nm are attributed to polyfluorene segments and quinoxaline
units, respectively. The insets in Figure 2A and 2B represent
the photographs of Pdot solutions under white light and 365
nm UV light, respectively (from left to right: PF-TC6FQ, PF-
TFQ, PF-TTFQ, and PF-TQ Pdots). We found that the
introduction of thiophene moieties on the quinoxaline
monomer (PF-TFQ vs PF-TTFQ) led to the redshift of
both the absorption and emission spectra because of the
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−1
increased conjugation system. On the other hand, the
incorporation of two electron-withdrawing fluorine atoms
blueshifted the absorption and fluorescence peaks (PF-TFQ
vs PF-TQ), probably due to the enhanced charge transfer
inside the polymer backbone.64,65 Moreover, the alkyl
substitution on the thiophene rings slightly blueshifted the
absorption and fluorescence peaks (PF-TC6FQ vs PF-TFQ)
owing to the reduced conjugation length caused by kinking or
bending of the polymer chain. Their photophysical properties
in Pdot form were summarized in Table 1.
τ = (KR + KNR
)
(2)
Take PF-TC6FQ Pdots as an example, the PF-TC6FQ
Pdots have a fluorescence radiative decay rate of 3.2 × 108 s−1,
which is comparable to other types of Pdots.44,52,56
Single-Particle Fluorescence Brightness Comparison.
As a new type of fluorescent probe, single-particle brightness is
one of the important properties for Pdots to be evaluated.66
The fluorescence brightness can be estimated by the product of
peak absorption cross section and the fluorescence quantum
yield, in which the absorption cross-section refers to the light-
harvesting ability. Table 2 summarizes the optical properties of
quinoxaline-containing Pdot aqueous solutions in comparison
to several NIR dyes and quantum dots. Here we take PF-
TC6FQ as an example because of its high quantum yield. The
per-particle absorption cross sections (σ) of the Pdots were
obtained from the analysis of the UV−visible absorbance at
known Pdot concentrations (Beer’s Law) and the absolute
quantum yields (Φ) were measured by use of an integrating
sphere unit. For example, the per-particle absorption cross-
section of PF-TC6FQ Pdots (21 nm) at 493 nm was
determined to be 2.00 × 10−13 cm2. Because the final
brightness of Pdots is the product of the absorption cross-
section and the fluorescence quantum yield, the brightness of
PF-TC6FQ Pdots was calculated to be 9.20 × 10−13 as shown
in Table 2. The results show that PF-TC6FQ Pdots possess the
highest fluorescence brightness, which is around 3 orders of
magnitude higher than conventional organic dyes or 3−5 times
brighter than that of Qdots655 and Qdots705 quantum dots
(purchased from Invitrogen), when excited at their absorption
maxima. Additionally, we found that PF-TC6FQ Pdots are
calculated to be ∼8 times brighter than Qdots655/Qdots705
nanocrystals under 488 nm laser excitation (values are shown in
the brackets in Table 2). We further performed single-particle
imaging to experimentally compare the single-particle bright-
ness between PF-TC6FQ Pdots and Qdots655 under identical
excitation (488 nm) and acquisition conditions. As shown in
Figure 3 where the intensity histograms were collected by
analyzing hundreds of nanoparticles for each samples, we found
that the average per-particle brightness of PF-TC6FQ Pdots
were 6−7 times higher than that of Qdots655, consistent with
the calculated results. The photostability of Pdots were also
evaluated as compared to Cy5.5 and it can be clearly observed
that Pdots appeared to be extraordinarily photostable (Figure
S2).
Table 1. Summary of Optical Properties of Quinoxaline-
Based Copolymer Series in Pdot Form in Water
a
b
c
d
abs
em
copolymers λmax (nm) λmax (nm) size (nm) Φ (%) τ (ns)
PF-TC6FQ
PF-TFQ
PF-TTFQ
PF-TQ
362, 493
379, 511
392, 531
389, 529
652
664
695
712
21
23
28
27
47
11
8
1.46
0.68
0.89
1.08
9
a
b
c
Absorption maximum. Fluorescence maximum. Quantum yield.
Fluorescence lifetime.
d
The quantum yields of PF-TC6FQ, PF-TFQ, PF-TTFQ,
and PF-TQ in aqueous solutions were determined to be 47, 11,
9, and 8%, respectively. It can be clearly observed that the
incorporation of thiophene moieties on the quinoxaline
monomer (PF-TFQ vs PF-TTFQ) eliminated the fluorescence
quantum yield (11 to 9%) although both the absorption and
the emission maxima were modestly red-shifted (20−30 nm).
However, the introduction of fluorine atoms (PF-TFQ vs PF-
TQ) only slightly raised the quantum yield. Most importantly,
the integration of the hexyl alkyl chains into the thiophene rings
(e.g., PF-TC6FQ) drastically increased the quantum yield (11
to 47%), suggesting that the extent of aggregation-cased
quenching was effectively prohibited. It should be noted that
the molar percentage of quinoxaline for all copolymers was
fixed at 50%, indicating that the resulting Pdots could be
directly excited by a commonly used 488 or 532 nm laser rather
than a 405 nm laser to facilitate energy from polyfluorene to
quinoxaline.
Additionally, we used a time-correlated single-photon
counting module system to determine the fluorescence lifetime
(τ) of this series of quinoxaline-based Pdots (Figure S1). The
fluorescence radiative decay rate constant (KR) and non-
radiative rate constant (KNR) containing all possible non-
radiative decay pathways can also be estimated from the
combination of the following two equations:
Bioconjugation and Specific Cellular Labeling with
PF-TC6FQ-COOH Pdots. We next employed these quinoxa-
line-based Pdots for both cell-surface and subcellular labeling to
demonstrate their potential in biological applications. In one
experiment, subcellular microtubule labeling inside HeLa cells
Φ = KR/(KR + KNR
)
(1)
Table 2. Photophysical Data of Quinoxaline-Based Pdots in Water Compared with Other Water-Soluble Typical NIR Dyes
e
abs
em
fluorescent probes λmax (nm) λmax (nm) fwhm (nm)
εmax (M−1 cm−1
)
σ (cm2)
size (nm) Φ (%) brightness (σ × Φ) (cm2)
a
ATTO740
740
764
694
798
651
705
652
712
43
1.20 × 105
1.95 × 105
1.20 × 105
5.70 (2.90) × 106
8.30 (3.00) × 106
5.23 (5.20) × 107
1.32 × 108
4.58 × 10−16
−
10
23
2.5
91
82
46
9
4.58 × 10−15
1.71 × 10−14
b
Cy 5.5
674
44
7.45 × 10−16
−
−
b
NIR7.0−2
Qdots655
777
50
4.58 × 10−16
1.15 × 10−15
c
405 (488)
405 (488)
493 (488)
529
30
2.18 (1.11) × 10−14
3.17 (1.15) × 10−14
2.00 (1.99) × 10−13
5.04 × 10−13
11
14
21
27
1.98 (1.01) × 10−12
2.60 (0.94) × 10−12
9.20 (9.15) × 10−12
4.54 × 10−12
c
Qdots705
66
d
PF-TC6FQ
129
148
d
PF-TQ
a
b
c
d
e
Data from ATTO-TEC catalog. Data from ref 73. Data from Invitrogen (Life Technologies). Pdots in deionized water. Full width at half-
maximum.
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signal from Pdots was observed for negative control samples
(Figure 4D−F) where the experimental conditions were
identical as that in Figure 4A−C except for the absence of
the biotinylated primary antibody. These results indicate this
new class of quinoxaline-based Pdots exhibit highly specific
binding activity with minimal nonspecific adsorption. The
cellular labeling of PF-TQ Pdots was also performed (Figure
S3).
In addition to the above-mentioned specific labeling through
the strong and stable streptavidin−biotin interaction, we also
utilized receptor-mediated endocytosis, a commonly used
strategy for in vivo tumor targeting, to label cancer cells.
Here we functionalized the surface of Pdots with folic acid and
then incubated with SKOV-3 cancer cells with overexpressed
folate receptors. As shown in Figure 5A, red fluorescence was
Figure 3. (A) Single-particle fluorescence image of commercial
Qdots655 (upper graph) and the corresponding histograms showing
the intensity distributions (bottom graph). (B) Single-particle
fluorescence image of PF-TC6FQ Pdots (upper graph) and the
corresponding histograms showing the intensity distributions (bottom
graph). The black curves were obtained by fitting a log-normal
distribution to the histogram, resulting in mean brightness of 993 and
7622 counts for Qdots655 and PF-TC6FQ Pdots, respectively. Both
images were obtained with a 488 nm laser under the same excitation
power and identical detection conditions.
was performed. We first conjugated streptavidin onto the
surface of Pdots via 1-ethyl-3-[3-(dimethylamino)propyl]-
carbodiimide hydrochloride-(EDC)-catalyzed coupling
(Scheme 2A). We then incubated Pdot-streptavidin conjugates
together with biotinylated monoclonal anti-α-tubulin antibody
with HeLa cells so that Pdots could label the microtubules of
HeLa cells. Figure 4A−C shows representative confocal images
of Pdot labeled microtubules in HeLa cells, indicating Pdots
could be specifically targeted onto subcellular structures in the
presence of biotinylated antibody. No noticeable fluorescence
Figure 5. Confocal fluorescence images of SKOV-3 cells labeled by
Pdot-folate conjugates and the flow cytometry results using MCF-7
cells. (A) Blue fluorescence is from nuclear counterstain Hoechst
34580, and red fluorescence is from Pdot-folate conjugates. The right
panel shows fluorescence overlaid of blue and red fluorescence. (B)
Images of negative control samples in which cells were incubated with
bare Pdots without folate functionalization. The scale bars are 30 μm.
(C) Flow cytometry measurements of MCF-7 breast cancer cells
labeled by Pdot-streptavidin conjugates via nonspecific binding
(negative control, no primary biotin antihuman CD326 EpCAM
antibody). (D) Fluorescence intensity distribution of MCF-7 cells
labeled via positive specific targeting using Pdot-streptavidin
conjugates.
clearly observed for Pdot-folate stained SKOV-3 cells, while a
significantly lower red fluorescence signal was found for bare
Pdot (without folate functionalization) treated cells (Figure
5B). The results reveal that folate-functionalized Pdots could be
internalized into SKOV-3 ovarian cancer cells via folate
receptor-mediated endocytosis pathways. We believe this
strategy could be further expanded for in vivo studies on
tumor-bearing mice models if needed.
Figure 4. Two-color confocal microscopy images of microtubules in
HeLa cells labeled with Pdot-streptavidin conjugates. The blue
fluorescence was from nuclear counterstain Hoechst 34580 and the
red fluorescence was from Pdot-streptavidin. (A) Image of nucleus.
(B) Image of microtubules. (C) The overlay of panels (A) and (B).
(D−F) Images of negative control samples where cells were incubated
with Pdot-streptavidin conjugates but in the absence of biotinylated
primary antibody. The scale bars are 30 μm.
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Moreover, we also executed flow cytometry experiments
using Pdot-streptavidin to label membrane proteins on MCF-7
cells. Here, the Pdot-streptavidin conjugates, together with
primary biotin antihuman CD326 EpCAM antibody were
labeled onto the surfaces of live MCF-7 cells. The quinoxaline-
based Pdots exhibit emissions in the NIR, which is beneficial in
many biological studies. Figure 5C−D shows the flow
cytometry results in which an excellent separation between
Pdot-targeted cells (Figure 5D, with primary antibody) and the
negative control/background (Figure 5C, without primary
antibody). These results demonstrate Pdots could be targeted
to MCF-7 cells effectively and specifically with minimal
nonspecific binding, consistent with the results from confocal
microscopy in HeLa cells. Their corresponding confocal
microscopy images were also shown in Figure S4.
In Vivo Cytotoxicity and Microangiography Imaging
in Zebrafish. For biological applications and translation of
these NIR fluorescent Pdots, the cytotoxicity of Pdots is the
major concern. We first conducted ex vivo cytotoxicity
experiments using MTT assay and found minimal cytotoxic
effect of these Pdots (10 nM) on HeLa cells. (Figure S5).
Additionally, we assessed in vivo toxicity by using zebrafish
model as shown in Figure 6A in which the results demonstrate
that the zebrafish could grow normally in the presence of Pdots
(2−4 nM). Both ex vivo and in vivo results suggest that these
Pdots exhibit high biocompatibility and should be suitable for
advanced biological applications. For in vivo biological
applications, zebrafish present an excellent animal model for
several reasons, which can accelerate our understanding in
nanoparticle toxicity and bioimaging.67 For example, zebrafish
are evolutionarily close to human and can produce more than
100 embryos in a single mating. Besides, zebrafish embryos are
optically transparent and can be genetically modified which
allow for the direct observation of biological behaviors. For in
vivo microangiography imaging on zebrafish, we injected PF-
TC6FQ-COOH Pdots into the sinus venosus of the embroys.
As shown in Figure 6B(a−c), we found that the Pdots (red
fluorescence) were immediately carried away with the blood-
stream and distributed in all the vascular system. Figure 6B(d−
f) show representative images of Pdot microangiography in
zebrafish larvae. The blood vessel walls which consisted of
eGFP-expressing endothelial cells exhibited green fluorescence,
while the lumen of the vessels displayed red fluorescence of
Pdots. Although some Pdots inevitably deposited on the
endothelial walls probably due to the hydrophilicity of Pdot
surface, Pdots remained stable in the blood circulation without
observation of leaking even 2 h after injection. In vivo
microangiography imaging on zebrafish using PF-TQ Pdots
was also shown in Figure S6. These Pdots show high
fluorescence brightness and good biocompatibility, which
might be used for further in vivo targeting37 and long-term
observation or tracking.20,68−70
CONCLUSIONS
■
In summary, we have successfully developed a series of
ultrabright NIR-emitting Pdots with large Stokes shifts based
on quinoxaline derivatives. We also found the alkyl substitution
and fluorine atom on thiophene rings in quinoxaline plays an
important role in the interchain interaction at an increased
quinoxaline concentration. These Pdots can be readily excited
by a 488 nm laser or a longer wavelength (e.g., 532 nm) laser
due to the high molar percentage (i.e., 50%) of quinoxaline
emitters inside the polymer backbones. Single-particle bright-
ness measurements indicate that the quinoxaline-based Pdots
can be up to 8 times brighter than Qdots655. Besides, the
covalent fabrication of carboxylic acid groups on the side chains
of fluorene monomers offered the feasibility for further
conjugation and analytical detection. We have demonstrated
the bioconjugation of the carboxyl-functionalized Pdots and
their highly specific binding to both cell surfaces and interiors
without any detectable nonspecific binding. We also performed
in vivo microangiography imaging on zebrafish. We anticipate
this new type of quinoxaline-based NIR-emitting Pdots to find
broad use both in basic biological studies and in bioimaging and
bioanalytical applications.
Figure 6. (A) Biotoxicity assessment of Pdots by zebrafish model.
Time course recording morphology of zebrafish embryos exposed to 2
and 4 nM Pdot solutions in the period of 0−48 h post fertilization
(hpf). (B) Zebrafish microangiography by injection of PF-TC6FQ-
COOH Pdots into 3 days post fertilization Tg(kdrl:eGFP) zebrafish
embryos. The global view of zebrafish vessels and Pdots images is
shown in left panels (a−c), while a close view of the trunk vasculature
(intersegmental vessel, ISV) is shown in the right panels (d−f). Green
emission is from endothelial cells expressing eGFP and red emission is
from Pdots. Scale bars represent 400 μm in a−c and 20 μm in d−f,
respectively.
EXPERIMENTAL SECTION
■
Materials. All of the chemicals were purchased from Sigma-Aldrich
or Alfa Aesar, and used as received unless indicated elsewhere. All
biorelated agents such as streptavidin, anitibody, or medium are
purchased form Invitrogen (Life Technologies). High purity water
(18.2 MΩ·cm) was used throughout the experiment. All 1HNMR and
13CNMR spectra were recorded on a Bruker AV300 spectrometer
(300 MHz).
Synthesis of 1-Bromo-3-hexyloxybenzene, 1..59−61 Potassium
carbonate (3.75 g, 66.8 mmol) was added to the solution of 3-
F
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1
bromophenol (5 g, 28.9 mmol) and 1-bromohexane (7.14 g, 43.3
mmol) in dimethyl foramide (DMF, 50 mL), and the reaction
mixtures were stirred at 90 °C under nitrogen atmosphere overnight.
After that, the solid was filtered out and the solvent was removed by a
rotary evaporator. The crude product was purified by column
chromatography on silica gel using hexane as eluent to afford 3.7 g
(50%) of compound 1 as colorless liquid. 1H NMR (300 MHz,
CDCl3) δ 7.10 (t, J = 8.4 Hz, 1H), 7.05−7.02 (m, 2H), 6.81−6.78
(m,1H), 3.90 (t, J = 6.4 Hz, 2H), 1.75 (m, J = 6.8 Hz, 2H), 1.46−1.39
(m, 2H), 1.36−1.26 (m, 4H), 0.89 (t, J = 6.8 Hz, 3H).13C NMR (75
MHz, CDCl3) δ 159.93, 130.40, 123.47, 122.73, 117.69, 113.49, 68.69,
31.51, 29.07, 25.63, 22.56, 13.98. HRMS (ESI) (M+, C12H17BrO) calcd
256.0463, found 256.0460.
liquid. H NMR (300 MHz, CDCl3) δ 7.23 (m, 2H). HRMS (ESI)
(M+, C6H2Br2F2) calcd 269.8491, found 269.8488.
Synthesis of 1,4-Dibromo-2,3-difluoro-5,6-dinitrobenzene,
5..59−61 5 mL of fuming nitric acid was added dropwise to
trifluoromethanesulfonic acid (100 mL) in an ice bath and stirred
for 30 min. 3.2 g (11.77 mmol) of compound 4 was added to the
mixture in portions over 30 min. After stirring at room temperature for
2 h, the mixture was cooled down to 0 °C and then another 5 mL of
fuming nitric acid was added slowly to react overnight at 70 °C. After
that, the reaction mixture was poured into cold water. The precipitate
was filtered and then recrystallized from ethanol to yield 2.5 g (59%)
of compound 5 as a yellow solid. HRMS (ESI) (M+, C6Br2F2N2O4)
calcd 359.8193, found 359.8196.
Synthesis of 1,2-Bis(3-hexyloxyphenyl)ethane-1,2-dione,
2..59−61 A solution of 1-Bromo-3-hexyloxybenzene, labeled as
Solution A, was prepared as follows: 5 mL of 2.5 M n-butyllithium
(12.5 mmol) in hexanes was added via cannula to 20 mL of anhydrous
THF, previously cooled to −78 °C under nitrogen atmosphere. The
mixture was stirred for 10 min, and then 1.17 mL of 1-Bromo-3-
hexyloxybenzene (2.04 g, 12.5 mmol) was added dropwise. The
mixture was stirred for 150 min, and the temperature was kept at −78
°C. Meanwhile, a solution, labeled as Solution B, was prepared as
follows: In a 250 mL round-bottom flask (equipped with stir bar and a
septum), containing 50 mL of anhydrous THF, was added LiBr (1.09
g, 12.5 mmol, 1 equiv) and CuBr (1.79 g, 12.5 mmol, 1 equiv), the
CuBr and LiBr mixture was stirred until all the salts were dissolved.
The mixture was then cooled to −40 °C or to lower temperatures.
Solution C: 483 μL of oxalyl chloride (714 mg, 5.63 mmol, 0.45 equiv)
was dissolved in 10 mL of anhydrous THF in a 100 mL round-bottom
flask (previously equipped with a septum) and then cooled to −40 °C
or to lower temperatures. Solution A was added via cannula to
Solution B, and the mixture was stirred for 5 min. Finally Solution C
was slowly added to the mixture via cannula. The mixture was kept in a
cold bath for 2 h, allowed to warm up to room temperature. After that,
the reaction was quenched with 100 mL of saturated NH4Cl aqueous
solution and then extracted with ethyl acetate, and dried over
anhydrous MgSO4. The solvent was removed via rotary evaporation,
and was subsequently purified by column chromatography on silica gel
by using ethyl acetate/hexane (1:7, v/v) as eluent to afford 1.1 g
(34%) of compound 2 as yellow liquid. 1H NMR (300 MHz, CDCl3)
δ 7.49 (m, 2H), 7.43 (m, 2H), 7.35 (t, J = 10.4 Hz, 2H), 7.18−7.15
(m, 2H), 3.98 (t, J = 6.4 Hz, 4H), 1.77 (m, 4H), 1.47−1.40 (m, 4H),
1.34−1.29 (m, 8H), 0.88 (t, J = 7.2 Hz, 6H). 13C NMR (75 MHz,
CDCl3) δ 194.52, 159.62, 134.21, 129.95, 122.91, 122.19, 113.62,
68.35, 31.51, 29.05, 25.63, 22.55, 13.98. HRMS (ESI) (M+, C26H34O4)
calcd 410.2457, found 410.2462.
Synthesis of 1,4-dibromo-2,3-difluoro-5,6-diaminobenzene,
6..59−61 To a one-bottomed flask was added 0.35 g (0.97 mmol) of
compound 5, 0.40 g (7.1 mmol) of iron powder, and 12 mL of acetic
acid. The reaction mixture was stirred at 45 °C for 4 h before poured
into 25 mL of cold 5% NaOH solution. The solution was extracted
with ether and then the organic phase was washed with 5% NaHCO3
solution for 5 times. After dried over MgSO4, the solvent was removed
under reduced pressure to afford 0.2 g (68%) of compound 6 as a
1
brown solid. H NMR (300 MHz, CDCl3) δ 3.31 (s, 4H). HRMS
(ESI) (M+, C6H4Br2F2N2) calcd 299.8709, found 299.8708.
Synthesis of Tributyl(4-hexylthiophen-2-yl)stannane, 7.57
A
solution of 3-hexylthiophene (1.5 g, 8.91 mmol) in anhydrous THF
(35 mL) was cooled to −78 °C under nitrogen atmosphere. 6.73 mL
of n-BuLi (1.6 M in hexane) was added dropwise to the solution and
the reaction mixture was allowed to warm up to room temperature and
stirred for 2 h. After that, the solution was cooled to 0 °C and 2.4 mL
(8.91 mmol) of tributyltin chloride was added. After being stirred for 2
h, the mixture was poured into water and diluted with hexane to
extract with brine. The organic phase was dried over MgSO4 and the
solvent was removed under reduced pressure. The resulting yellow oil
was purified by column chromatography on silica gel to afford 2.5 g
(61%) of compound 7 as yellow liquid. 1H NMR (300 MHz, CDCl3)
δ 7.20−7.16 (m, 1H), 7.01−6.92 (m, 1H), 2.69−2.62 (m, 2H), 1.70−
1.49 (m, 8H), 1.41−1.26 (m, 12), 1.21−0.96 (m, 6H), 0.95−0.85
(m,12H). HRMS (ESI) (M+, C22H42SSn) calcd 458.2029, found
458.2021.
Synthesis of 5,8-Dibromo-6,7-difluoro-2,3-bis(3-
hexyloxyphenyl)quinoxaline, 8. A mixture of compound 6 (3.61
g, 10.0 mmol), iron powder (6.70 g, 120.0 mmol), and acetic acid (60
mL) was stirred at 50 °C for 4 h. After filtering, compound 2 (4.10 g,
10.0 mmol) was added to the filtrate, followed by heating under reflux
for 8 h. After that, the reaction mixture was poured into water,
extracted with dichloromethane, and dried with anhydrous MgSO4.
The solvent was then removed via rotary evaporation and
subsequently purified by column chromatography on silica gel using
dichloromethane/hexane (1:7, v/v) as eluent to afford 4 g (59%) of
Synthesis of 2,3-Difluoro-1,4-bis(trimethylsilyl)benzene, 3.
5.7 mL (40.3 mmol) of n-butyllithium (1.6 M in exane) was added to a
solution of diisopropylamine (5.7 mL, 40.3 mmol) in anhydrous THF
(20 mL) at 78 °C under nitrogen atmosphere. After being stirred at 78
°C for 30 min, 1,2-difluorobenzene (1.7 mL, 17.5 mmol) and
chlorotrimethylsilane (4.9 mL, 38.5 mmol) was added to the solution
at a rate which allowed the internal reaction temperature to remain
below 50 °C. The solution was stirred at −78 °C for additional 1 h and
then 1 M H2SO4 solution (10 mL) was added to extract with diethyl
ether for three times. The combined organic layers were washed with
brine, dried over MgSO4, and concentrated under reduced pressure to
afford 3.5 g (77%) of compound 3 as colorless needle-shaped crystals.
1H NMR (300 MHz, CDCl3) δ 7.09 (s, 2H), 0.32 (s, 18H). HRMS
1
compound 8 as a white solid. H NMR (300 MHz, CDCl3) δ 7.24−
7.18 (m, 4H), 7.15−7.12 (m, 2H), 6.94−6.91 (m, 2H), 3.83 (t, J = 6.4
Hz, 4H), 1.70 (m, 4H), 1.42−1.35 (m, 4H), 1.34−1.24 (m, 8H), 0.89
(t, J = 7.2 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ 194.52, 159.62,
134.21, 129.95,122.91, 122.19, 113.62, 68.35, 31.51, 29.05, 25.63,
22.55, 13.98. HRMS (ESI) (M+, C32H34Br2F2N2O2) calcd 674.0955,
found 674.0960.
Synthesis of 5,6-Difuoro-4,7-di(thiophen-2-yl)benzo[c]-
[1,2,5]-thiadiazole, 9. A mixture of compound 7 (0.845 g, 1.85
mmol), compound 8 (0.5 g, 0.74 mmol), and PdCl2(PPh3)2 (51.9 mg,
0.074 mmol) in toluene (30 mL) was heated under reflux for 48 h
under nitrogen atmosphere. After the reaction, the solvent was
removed under reduced pressure and the crude product was
subsequently purified by column chromatography on silica gel to
afford 0.25 g (40%) of compound 9 as an orange solid. 1H NMR (300
MHz, CDCl3) δ 8.03 (d, J = 3.6 Hz, 2H), 7.60 (dd, J = 5.2, 1.2 Hz,
2H), 7.34 (s, 2H), 7.24−7.22 (m, 6H), 6.95−6.92 (m, 2H), 3.89 (t, J =
6.4 Hz, 4H), 1.76−1.71 (m, 4H), 1.45−1.40 (m, 4H), 1.34−1.31 (m,
8H), 0.91 (t, J = 6.8 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ 159.06,
151.33, 139.28, 134.70, 130.88, 130.78, 129.86, 129.20, 126.5, 122.76,
(ESI) (M+, C12H20F2Si2) calcd 258.1072, found 258.1071.
Synthesis of 2,3-Difluoro-1,4-dibromobenzene, 4. To a one-
bottomed flask was added 2.3 mL (43.8 mmol) of bromine and then
cooled to 0 °C. 3.5 g (13.5 mmol) of compound 3 was added
portionwise while maintaining the internal temperature at 20 to 40 °C.
The reaction mixture was stirred at 60 °C for 1 h and then additional
0.4 mL (7.30 mmol) of bromine was added to stir for another 1h. The
reaction mixture was cooled to 0 °C and slowly poured into ice-cold
saturated NaHCO3 solution to extract with diethyl ether. The organic
layer was washed with brine, dried over MgSO4, and concentrated
under reduced pressure to afford 3.2 g (87%) of compound 4 as yellow
G
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117.92, 116.62, 115.71, 68.16, 31.52, 29.10, 25.70, 22.62, 14.01.
MALDI-MS m/z 850.08 (M+).
Bioconjugation and Characterization of Pdots. Bioconjuga-
tion was performed by using the EDC-catalyzed reaction between
carboxylate-functionalized quinoxaline-based Pdots and the respective
amine-containing streptavidin. In a typical bioconjugation reaction, 80
μL of polyethylene glycol (5% w/v PEG, MW 3350) and 80 μL of
concentrated HEPES buffer (1 M) were added to 4 mL of Pdot
solution, resulting in a Pdot solution in 20 mM HEPES buffer with a
pH of 7.3. Then 240 μL of streptavidin (1 mg/mL) was added to the
solution and mixed well on a vortex. After that, 80 μL of freshly
prepared EDC solution (5 mg/mL in Milli-Q water) was added to the
solution, and the mixture was stirred for 4 h at room temperature.
After bionconjugation, 80 μL of BSA (10 wt %) was added to the Pdot
solution and the reaction was continued for another 20 min. A 80-μL
aliquot of Triton X-100 in Milli-Q water (2.5 wt %) was added to the
Pdot-streptavidin mixture. The mixture was then transferred to a
centrifugal ultrafiltration tube (Amicon Ultra-4, MWCO: 100 kDa),
and then concentrated to 0.5 mL by centrifugation. Finally, the Pdot-
streptavidin bioconjugates were purified by gel filtration using
Sephacryl HR-300 gel media.
The average particle size was determined by dynamic light
scattering and transmission electron microscopy (TEM). TEM images
of the synthesized Pdots were acquired using a JEOL 2100
transmission electron microscope at an acceleration voltage of 200
kV. For TEM, a drop of Pdot aqueous solution was placed onto a
carbon-coated grid and allowed to evaporate at room temperature.
The absorption spectra of Pdots were measured using UV−visible
spectroscopy (Dynamica Halo DB20S, Dynamica Scientific). The
fluorescence spectra were collected using a Hitachi F-7000 fluorometer
(Hitachi, Tokyo, Japan) under 450 nm excitation. Absolute
fluorescence quantum yield of Pdots was determined by using an
integrating sphere unit of Hitachi F-7000 fluorescence spectropho-
tometer.
Cell Culture and Labeling. The cervical cancer cell line HeLa and
breast cancer cell line MCF-7 were ordered from Food Industry
Research and Development Institute (Taiwan). Primary cultured HeLa
cells were grown in Dulbecco’s Modified Eagle Medium (cat. no.
11885, Invitrogen) supplemented with 10% fetal bovine serum (FBS)
and 1% penicillin-streptomycin solution (5000 units/mL penicillin G,
50 μg/mL streptomycin sulfate in 0.85% NaCl) at 37 °C with 5% CO2
humidified atmosphere. MCF-7 cells were cultured at 37 °C, 5% CO2
in RPMI 1640 medium supplemented with 10% FBS and 1%
penicillin-streptomycin solution. The cells were precultured in a T-25
flask and allowed to grow for 5−7 days prior to experiments until
∼80% confluence was reached. To prepare cell suspensions, the
adherent cancer cells were quickly rinsed with media and then
incubated in 0.8 mL of trypsin-ethylenediaminetetraacetic (EDTA)
solution (0.25 w/v % trypsin, 0.25 g/L EDTA) at 37 °C for 3 min.
The cell suspension solution was then centrifuged at 1000 rpm for 5
min to precipitate the cells to the bottom of the tube. After taking out
the upper media, the cells was rinsed and resuspended in 5 mL of
culture media. Approximately tens of thousands cells were split onto a
glass-bottomed culture dish and allowed to grow for 12 h before Pdot
labeling. Prior to fluorescence imaging, the cells were rinsed with PBS
buffer to remove any nonspecifically bound Pdots on the cell surface.
For cell labeling experiments, BlockAid blocking buffer was
purchased from Invitrogen (Eugene, OR, USA). For labeling cell-
surface markers with IgG, a million MCF-7 cells in 100-μL labeling
buffer (1× PBS, 2 mM EDTA, 1% BSA) was incubated with 0.3 μL of
0.5 mg/mL primary biotin antihuman CD326 EpCAM antibody
(BioLegend, San Diego, CA, USA) on a rotary shaker in the dark and
at room temperature for 30 min, followed by a washing step using
labeling buffer. Then the cells were incubated with 1.5 nM Pdot-
streptavidin conjugates in BlockAid buffer for 30 min on a shaker in
the dark and at room temperature, followed by two washing steps with
labeling buffer. Prior to cell incubation, Pdot solutions were sonicated
for 3 min in order to disperse any potential aggregates. Negative
controls were obtained by incubating cells with Pdot-streptavidin
conjugates in the absence of primary biotinylated-antibody. Cell
fixation was performed by dissolving the cell pellet obtained by
Synthesis of 5,8-Bis(5-bromothiophen-2-yl)-6,7-difluoro-
2,3-bis(3-hexyloxyphenyl)quinoxaline, 10. Compound 9 (156
mg, 0.18 mmol) was dissolved in 8 mL of DMF under nitrogen
atmosphere and then n-bromosuccinimide (75 mg, 0.42 mmol) was
added into the mixture in one portion. The mixture was stirred in the
dark for 12 h and then the mixture was poured into water, diluted with
CH2Cl2, and extracted with brine. The organic extract was separated
ad dried over anhydrous MgSO4. The solvent was removed via rotary
evaporation and subsequently purified by column chromatography on
1
silica gel to afford 111 mg (61%) of compound 10 as a red solid. H
NMR (300 MHz, CDCl3) δ 7.76 (d, J = 4.0 Hz, 2H), 7.50−7.49 (m,
2H), 7.19 (t, J = 7.6 Hz, 2H), 7.14 (d, J = 4.4 Hz, 2H), 7.06−7.04 (m,
2H), 6.98−6.95 (m, 2H), 4.02 (t, J = 6.8 Hz, 4H), 1.82−1.75 (m, 4H),
1.50−1.44 (m, 4H), 1.37−1.31 (m, 8H), 0.92−0.88 (m, 6H). 13C
NMR (75 MHz, CDCl3) δ 159.41, 151.67, 138.75, 133.91, 132.41,
130.90, 129.37, 129.11, 122.90. MALDI-MS m/z 1006.24 (M+).
General Procedures of Polymerization.57 Suzuki coupling
reaction was used to synthesize the copolymers as shown in Scheme
1. In a 100 mL flask, monomer 10 or 11 or 12 or 13 (0.45 mmol-0.5
mmol), monomer 14 (0.5 mmol), and monomer 15 (0−0.05 mmol)
were dissolved in 10 mL of toluene, and then 4−6 mg (0.02 mmol) of
tetra-n-butylammonium bromide (Bu4NBr) and 3 mL of Na2CO3 (2
M) was added. The mixture solution was purged with nitrogen for 1 h.
After that, the mixture solution was degassed and refilled with N2
(repeated 4 times) before and after the addition of Pd(PPh3)4 (7 mg,
0.006 mmol). The reactants were stirred at 100 °C for 48 h and then
50 mg of phenylboronic acid dissolved in 1 mL of THF was added.
After 2 h, 0.5 mL of bromobenzene was added and further stirred for 3
h. The mixture was poured into 120 mL of methanol. The precipitate
was filtered, washed with methanol and acetone to remove monomers,
small oligomers, and inorganic salts. The crude product was dissolved
in CH2Cl2 and then extracted with brine for 3 times. The organic
extract was separated, dried over MgSO4, and the solvent was removed
under reduced pressure. The crude polymers were reprecipitated in
CHCl3/methanol and washed with acetone. Finally, the product was
collected by filtration to afford 120−150 mg of polymer PF-TC6FQ,
PF-TTFQ, PF-TFQ, or PF-TQ. GPC: PF-TC6FQ Mn: 25995, Mw:
44627, PDI: 1.72; PF-TFQ Mn: 13347, Mw: 31026, PDI: 2.32; PF-
TTFQ Mn: 12199, Mw: 51200, PDI: 4.19; PF-TQ Mn: 8779, Mw:
28415, PDI: 3.24.
Synthesis of Folate-PEG-NH2.71 Folic acid (260 mg, 0.58
mmol)) was completely dissolved in 15 mL of DMSO under
ultrasonication. Dicyclhexylcarbodiimide (74 mg, 0.65 mmol) and
N-hydroxysuccinimide (134 mg, 0.65 mmol) was then added and the
mixture was stirred in the dark for 12 h. After the reaction, the
precipitate was filtered out using a hydrophilic 0.2 μm PTFE
membrane filter and the filtrate was added into the acetone-ether
(3:7) mixture solution. The solid was collected by centrifugation and
washed with acetone and ether several times to obtain 511 mg of
Folate-NHS ester as a yellow solid, which was used immediately for
the next step. MALDI-MS m/z 539.91 (M+). Folate-NHS ester (511
mg, 0.95 mmol) was dissolved fully in 15 mL of DMSO. 2.1 mL of
3,3′-oxybis(ethyleneoxy)bis(propylamine) and 102 mL of pyridine was
added to the solution in sequence. The mixture was stirred in the dark
for 8 h and then 2−3 mL of acetonitrile was added to solution to
obtain a yellow precipitate. The precipitate was collected and washed
with acetone to yield 148 mg (48%) of Folate-PEG-NH2. MALDI-MS
m/z 644.79 (M+). Folate-PEG-NH2 can be dissolved in DMSO and
stored at 4 °C as a stock solution, while Folate-PEG-NH2 solid should
be stored at −20 °C.
Preparation of Quinoxaline-Based Pdots. Typically, 200 μL of
copolymer solution (1 mg/mL in THF) and 0−20 μL of PS−PEG-
COOH (2 mg/mL in THF) were added into 5 mL of THF. This
mixture solution was then quickly injected into 10 mL of water under
vigorous sonication. After that, THF was removed by purging with
nitrogen on a 96 °C hot plate for 60 min. The resulting Pdot solution
was filtered through a 0.2 μm cellulose membrane filter to remove any
aggregates formed during Pdot preparation.
H
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centrifugation in 500 μL of fixing buffer (1× PBS, 2 mM EDTA, 1%
BSA, 1% PFA).
Notes
The authors declare no competing financial interest.
For microtubule-labeling experiments, tens of thousands of HeLa
cells were plated on a 22 × 22 mm glass coverslip (Biogenesis,
Taiwan) and cultured until the density reached 60−70% confluence.
The cells were fixed with 4% paraformaldehyde for 15 min,
permeabilized with 0.5% Triton-X 100 in PBS buffer for 15 min,
and then blocked in BlockAid blocking buffer for another 40 min. The
fixed and blocked HeLa cells were subsequently incubated with 2 μg/
mL biotinylated monoclonal anti-α-tubulin antibody (BioLegend, San
Diego, CA, USA) for 60 min, and then Pdot-streptavidin conjugates
for 40 min. The Pdot labeled cells were then counterstained with
Hoechst 34580 for 15 min and imaged immediately on a fluorescence
confocal microscope (Nikon D-Eclipse C1).
ACKNOWLEDGMENTS
■
We would like to thank the Ministry of Science (103-2113-M-
110-004-MY2) and National Sun Yat-sen University. We also
gratefully acknowledge support from Prof. Chao-Ming Chiang,
Prof. Wei-Lung Tseng, Prof. Chin-Hsing Chou, and Dr. Ying-
Hsiao Chen from National Taiwan University. We especially
thank Dr. Fangmao Ye for the measurements of single-particle
fluorescence brightness.
REFERENCES
■
Flow cytometry measurements were performed on fresh samples
with 106 cells/0.5 mL and prepared following the procedure previously
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described. The flow cytometer CyFlow SL (Partec, Munster,
̈
Germany) was used: Cells flowing in the detection chamber were
hit with a 488 nm laser beam, fluorescence emission were collected
and filtered by a 590 long-pass, and all signals were detected by PMT
arrays. Representative populations of cells were chosen by selection of
an appropriate gate. Detection of cell fluorescence was continued until
at least 104 events had been collected in the active gate.
Cell Imaging. The fluorescence spectra of Pdot-tagged cells were
acquired with a fluorescence confocal microscope (Nikon D-Eclipse
C1) under ambient conditions (24 2 °C). The confocal fluorescence
images were collected using a diode laser at 488 nm (∼15 mW) as the
excitation source and an integration time of 1.6 μs/pixel. A CF1 Plan
Fluor 40x (N. A. 0.75, W.D. 0.66 mm) objective was utilized for
imaging and spectral data acquisition; the laser was focused to a spot
size of ∼7 μm2. The blue fluorescence was collected by filtering
through a 450/35 band-pass (λex = 408 nm) while the red fluorescence
was collected by filtering through a 570 long-pass (λex = 488 nm).
MTT Assay. The cellular cytotoxicity of the Pdots was examined on
HeLa cells. The number of viable cells was determined using the MTT
assay with 3-(4,5-dimethylthiazole-2-yl)-2,5-phenyltetrazolium bro-
mide. HeLa cells were first seeded in each well of a 24-well culture
plate and then incubated with various concentrations of Pdots (100
pM, 200 pM, and 400 pM) for 6, 12, and 24 h. After that, 20 μL (5
mg/mL) of MTT aqueous solution was added to each well and the
cells were further incubated for 4 h at 37 °C to deoxidize MTT. The
medium was then washed out and 300 μL of DMSO was added into
each well to dissolve formazam crystals. Absorbance was measured by
a BioTek ELx800 microplate reader at 570 nm, while the cells cultured
with the pure medium (e.g., without Pdots) served as controls.
In Vivo Imaging on Zebrafish. The transgenic zebrafish,
Tg(kdrl:eGFP)la116 expressing eGFP in the endothelial cells,72 were
maintained at 28 °C and bred under standard conditions with approval
from National SunYat-sen University Animal Care Committee. For
angiography imaging, 37 nL Pdots (125 nM) in 20 mM HEPES buffer
was injected into the sinus venosus of the anaesthetized zebrafish
embryos 3 days post fertilization (dpf) with 5% (v/v) tricaine (Sigma).
The injected embryos were recovered for 30 min, immobilized in 1.5%
low melting point agarose (invitrogen), and then imaged immediately
using a fluorescence confocal microscope (Nikon D-Eclipse C1). The
green fluorescence was collected by filtering through a 515/30 band-
pass (λex = 488 nm) while the red fluorescence was collected by
filtering through a 570 long-pass (λex = 488 nm).
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.5b06710.
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S
(25) Peng, H.-s.; Stolwijk, J. A.; Sun, L.-N.; Wegener, J.; Wolfbeis, O.
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AUTHOR INFORMATION
Corresponding Author
*yhchan@mail.nsysu.edu.tw
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DOI: 10.1021/jacs.5b06710
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