Fluorescent nanorods based on 9,10-distyrylanthracene (DSA) derivatives for efficient and long-term bioimaging

Article abstract of DOI:10.1039/c9tb02883h

Fluorescent nanoparticles based on 9,10-distyrylanthracene (DSA) derivatives (4,4′-((1E,1′E)-anthracene-9,10-diylbis(ethene-2,1-diyl))bis(N,N-dimethylaniline) (NDSA) and 4,4′-((1E,1′E)-anthracene-9,10-diylbis(ethene-2,1-diyl))dibenzonitrile (CNDSA)) were prepared using an ultrasound aided nanoprecipitation method. The morphologies of the fluorescent nanoparticles could be controlled by adjusting the external ultrasonication time. NDSA or CNDSA could form spherical nanodots (NDSA NDs, CNDSA NDs) in a THF-H2O mixture with an 80% or 70% water fraction when the ultrasonication time was 30 s. When the ultrasonication time was prolonged to 10 min, NDSA and CNDSA could assemble into nanorods (NDSA NRs, CNDSA NRs). Meanwhile, the sizes of NDSA NRs and CNDSA NRs could be controlled by adjusting the water content in the mixture. As the water fraction was increased from 60% to 80%, the sizes of NDSA and CNDSA nanorods or nanodots reduced from 238.4 nm to 140.3 nm, and 482 nm to 198.4 nm, respectively. When the water fraction was up to 90%, irregular morphologies of NDSA and CNDSA could be observed. The nanoparticles exhibited intense fluorescence emission, good anti-photobleaching properties, as well as excellent stability and biocompatibility. In vitro cell imaging experiments indicated that the nanorods prepared by this simple method had the potential to be used for efficient and noninvasive long-term bioimaging. This journal is

Full text of DOI:10.1039/c9tb02883h

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PAPER
Wei Wei, Guanghui Ma et al.
Macrophage responses to the physical burden of cell-sized
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ARTICLE
Fluorescent Nanorods Based on 9,10-distyrylanthracene (DSA)
Derivatives for efficient and long-term Bioimaging
Wenkun Han, a Yangyang Du, b Meiyu Song, b Kaiju Sun, b Bin Xu, a Fei Yan*b and Wenjing Tian*a
Received 00th January 20xx,
Accepted 00th January 20xx
The fluorescent nanoparticles based on 9,10-distyrylanthracene (DSA) derivatives (4,4'-((1E,1'E)-anthracene-9,10-
DOI: 10.1039/x0xx00000x
diylbis(ethene-2,1-diyl))bis(N,N-dimethylaniline)
(NDSA)
and
4,4'-((1E,1'E)-anthracene-9,10-diylbis(ethene-2,1-
diyl))dibenzonitrile (CNDSA)) were prepared by ultrasound aided nanoprecipitation method. The morphologies of the
fluorescent nanoparticles could be controlled by the external ultrasound time. NDSA or CNDSA could form spherical
nanodots (NDSA NDs, CNDSA NDs) in THF-H2O mixtures with 80% or 70% water fraction when the ultrasound time was 30
s. When the ultrasound time was prolonged to 10 min, NDSA and CNDSA could assemble into nanorods (NDSA NRs, CNDSA
NRs). Meanwhile, the size of NDSA NRs and CNDSA NRs could be controlled by the water content in the mixture. As the
water fraction increased from 60% to 80%, the size of NDSA and CNDSA nanorods or nanodots reduced from 238.4 nm to
140.3 nm, and 482 nm to 198.4 nm, respectively. When the water fraction was up to 90%, irregular morphology of NDSA
and CNDSA could be observed. The nanoparticles exhibited intense fluorescent emission, good anti-photobleaching
property, excellent stability and biocompatibility. In vitro cell imaging experiments indicated the potential for efficient and
noninvasive long-term bioimaging of the nanorods prepared by the simple method.
responses and behaviours largely.38-42 In general, the
morphology of organic nanoparticles prepared by common
reprecipitation method is spherical.43-45 While some fluorescent
Introduction
Bioimaging as a powerful tool in biological research has
attracted much more attention because it can provide a unique
approach to visualize the morphological details of cells or
tissues for cancer diagnosis, and easily understand the
biological processes, metabolism and pharmacokinetics.1-6 As a
major class of bioimaging techniques, fluorescence imaging
possesses indispensable and versatile features including real-
time and non-invasive tracking, high spatial and temporal
resolution, free of radiation, abundant signals, high contrast,
fast response, etc.7-21 Among various fluorescent probes,
organic nanorods can be obtained by modified nano-
precipitation methods. For example, Li et al. prepared
biocompatible host/guest tetracene/pentacene nanorods using
a reprecipitation method for highly effective upconversion
photodynamic therapy;46 Wang et al. constructed a nanorod
fluorescent probe by the coordinating self-assembly of copper
perylenetetracarbonate, which can light up both cancer cells
and periphery normal cells with different fluorescent emission
to recognize the normal cells selectively;47 Fan et al. prepared a
serials of Zn porphyrin nanorods through a surfactant induced
micelle encapsulation method, exhibiting enhanced
photocatalytic degradation of the exemplary pollutant methyl
orange (MO) under visible light irradiation.48 However, the
above mentioned nanorods often show weak emission or no
emission due to the aggregation-caused quenching (ACQ)
effect, resulting in the limitation of their applications in
bioimaging.49-54
To avoid fluorescence quenching of organic dyes in aggregation
state, fluorogens with aggregation-induced emission (AIEgens)
were introduced in preparing nanorods with enhanced
fluorescent property.55-64 For instance, Liu et al. prepared a
series of AIEgens based fluorescent polymeric nanorods using a
modified nano-precipitation method by prolonging the
ultrasound sonication time to 120 min at 80°C with the help of
a biocompatible lipid–PEG block copolymer for enhanced
cancer targeting and imaging.65 Xie and Xu et al. prepared
different rod-like nanorods by assembling of poly(ethylene
glycol)-block-poly(L-lactic acid) (PEG5k-PLA10k) or poly(ethylene
organic
fluorescent
nanoparticles
with
excellent
biocompatibility, multifunctionality, and ease of fabrication and
modification have been successfully used in tumor targeting
and imaging, drug delivery and therapy.
fluorescent nanoparticles are fabricated through encapsulating
organic dyes by matrices (like polymers, silicon dioxide, etc.),
which could largely improve the optical properties and water
solubility of organic dyes.32-37 As we know, the size, and
especially the morphologies of nanoparticles can not only
influence their chemical and physical properties, but also affect
the pathway of their endocytosis, biodistribution, biological
22-31
As usual, organic
a. State Key Laboratory of Supramolecular Structure and Materials, College of
Chemistry, Jilin University, Changchun 130012, China.
b. State Key Laboratory of Inorganic Synthesis and Preparative Chemistry,
International Joint Research Laboratory of Nano-Micro Architecture Chemistry
(NMAC), International Research Center for Chemistry-Medicine Joint Innovation,
College of Chemistry, Jilin University, Changchun 130012, China.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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glycol)-block-poly(caprolactone) (PEG5k-PCL10k
)
with 9,10-
Results and discussion
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DOI: 10.1039/C9TB02883H
distyrylanthracene (DSA) for noninvasive long-term imaging.66
Our group prepared 2,3-bis(4′-(diphenylamino)-[1,1′-biphenyl]-
4-yl) fumaronitrile (BDBF) nanorods with high fluorescence
quantum yield (QY) and 1O2 generation ability through
reprecipitation method for image-guided photodynamic
therapy (PDT).67 Although the fluorescent nanorods based on
organic dyes, especially AIEgens were prepared by modified
nano-precipitation method and applied in bioimaging and PDT,
there still exists some problems.68 First, the use of surfactants
or polymers will result in tedious preparation process and high
cost.69,70 Second, the varied factors in preparation process, such
as solvents,71 PH value,72 concentration,73 as well as the
temperature74 will lead to uncontrolled morphology of
nanorods. Third, for the sonication aided precipitation
methods, overlong ultrasound time will enhance the
temperature of preparative environment,65 resulting in the
uneasy uncontrolled morphology and size of nanorods.
Therefore, it is necessary to explore a simple and cheap method
to prepare nanorods with controllable morphology and size.
Herein, we developed fluorescent nanoparticles based on DSA
derivatives through modified nano-precipitation method with
the help of sonication (Scheme 1). The morphology of
nanoparticles could be controlled by the ultrasonic time and the
size of nanorods could be reduced as the water contents
increased. These nanoparticles exhibited excellent stability and
biocompatible. In vitro experiments showed that these
nanoparticles could be well endocytosed by A549 cells, and
NDSA NRs and CNDSA NRs exhibited the potential noninvasive
long-term imaging. The method to fabricate nanorods based on
DSA derivatives provides a simple strategy to design efficient
AIEgen nanorods for bioimaging.
Synthesis and characterization of NDSA and CNDSA
9,10-Bis(p-dimethylaminostyryl)-anthracene (NDSA) and 4,4’-
(1E,1’E)-2,2’-(anthracene-9,10-diyl)
bis(ethene-2,1-
diyl)dibenzonitrile (CNDSA) were successfully prepared
according the literature,75,76 and the synthesis routes as well as
chemical structures of NDSA and CNDSA were exhibited in Fig.
S1. The chemical structures and molecular weights of NDSA and
CNDSA were fully characterized using proton nuclear magnetic
resonance
(1H
NMR)
and
matrix-assisted
laser
desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) (Fig. S2-5). The standard calibration curve
technique was used to calibrate the concentrations of NDSA and
CNDSA in nanostructures through calculating the main
absorbance of NDSA at 437 nm and CNDSA at 421 nm by UV/Vis
absorption spectra in THF/H2O = 4:1 (Fig. S6).
We carefully measured the AIE or aggregation enhanced
emission (AEE) character of NDSA and CNDSA in THF–H2O
mixture at water volume fraction (fw). As shown in Fig. S7,
there was weak fluorescence emission when NDSA was in pure
THF. Until fw is larger than 50%, the fluorescence emission
intensity of NDSA is evidently enhanced, but when fw was
more than 80%, there was a decrease in fluorescence emission
intensity due to the intramolecular charge transfer (ICT) of
NDSA in high polar solvent. These results manifested NDSA
exhibited the classical AIE and ICT character.77-80
Compared to NDSA, CNDSA showed quite strong yellow green
fluorescence emission in THF, and the fluorescence intensity
gradually reduced when fw increased owing to the ICT effect of
CNDSA in more polar environment. The fluorescence emission
has slightly hypochromatic shift because the aggregates of
CNDSA was formed when increasing the concentration of
water. As the concentration of water was up to 60%, CNDSA
aggregated more closely (resulting in a further hypochromatic
shifted emission from 564 nm to 540 nm), which greatly
restricted the intramolecular motion and activated the AEE
process leading to an increase of the fluorescence intensity.
There was also a further reduce in fluorescence intensity with
the increase of water content due to the promoted ICT
process, and the more closely aggregates of CNDSA led to the
blue-shifted emission (530 nm) again. These results
manifested CNDSA showed AEE and ICT character. 81-85
Preparation and characterization of NDSA NRs and CNDSA NRs
NDSA NRs, CNDSA NRs, NDSA NDs and CNDSA NDs were
prepared by modified nano-reprecipitation method. In brief,
NDSA in THF (2 ml) was quickly added into deionized water (8
mL) with sonication. When the mixture solution was then
followed by 30 s sonication under an ultrasonic tip, NDSA NDs
were obtained after the evaporation of THF. And after
prolonging ultrasonic time to 10 min, NDSA NRs were obtained.
The prepared processes of CNDSA NRs and CNDSA NDs were
similar with NDSA NRs. Briefly, CNDSA in THF (3 ml) was quickly
added into deionized water (7 mL) to form mixture solution,
then mixture solution was under 30 s or 10 min ultrasonic time,
respectively. CNDSA NDs and CNDSA NRs were obtained after
Scheme 1 Schematic of nanodots and nanorods based on DSA derivatives for
efficient and long-term Bioimaging.
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Fig. 2 Normalized UV/Vis absorption and fluorescence spectra of (a) NDSA
NDs, (b) NDSA NRs, (c) CNDSA NDs, (d) CNDSA NRs in water.
Fig. 1 Size distributions of (a) NDSA NRs, (b) NDSA NDs, (c) CNDSA NRs, (d)
CNDSA NDs under 10 min sonication.
protein on the surface of the nanorods. The zeta potential of
NDSA NRs and CNDSA NRs in physiological condition remained
unchanged after 24 h incubation, indicating the excellent
physiological stabilities. NDSA NDs and CNDSA NDs also showed
good stability under physiological condition through detecting
their size distribution and zeta potentials under physiological
condition.
evaporating excessive THF. The preparation method of CNDSA
NRs was similar with NDSA NRs, only the water content was
70%. The prepared processes of NDSA NDs and CNDSA NDs
were similar with NDSA NRs and CNDSA NRs, only the sonication
time was 30 s. The size distributions and morphologies of these
prepared nanorods and nanodots were carefully characterized
by dynamic light scattering (DLS) and transmission electron
microscopy (TEM). As shown in Fig. 1, NDSA NRs possessed
181.6 nm in hydrodynamic diameter with a unimodal size
distribution (polydispersity index (PDI) = 0.137), and CNDSA NRs
were about 244.6 nm corresponding to the PDI of 0.116.
Meanwhile, the zeta potentials of the nanorods were -21.3 mv
for NDSA NRs and -17.8 mv for CNDSA NRs, indicating these
nanorods had good stability and dispersibility in physiological
conditions. NDSA NDs and CNDSA NDs present fine size
dispersions (PDI=0.216 and 0.167) in aqueous solution with a
relatively different sizes of 140.3 nm and 189.0 nm,
respectively. TEM images showed NDSA NRs and CNDSA NRs
exhibited smooth rod-like morphology, while NDSA NDs and
CNDSA NDs were spherical but not homogeneous (Fig. S8). The
TEM and DLS results of these nanorods were different from
those of the nanodots. However, no crystal lattice and
diffraction patterns of NDSA NRs, CNDSA NRs NDSA NDs and
CNDSA NDs were observed by selected area electron diffraction
(SAED), manifesting both nanorods and nanodots are
amorphous (Fig. S9). And powder X-ray diffraction (PXRD)
analysis further proved that these nanorods and nanodots were
amorphous (Fig. S10).65,66
The UV/Vis absorption, fluorescence spectra, fluorescent
lifetimes as well as fluorescence quantum yields (QYs) of these
nanostructures in aqueous solution were carefully
characterized. As displayed in Fig. 2, NDSA NRs showed the main
absorption peak at 456 nm, and bright yellow fluorescence
emission at 554 nm under UV light excitation with a QY of
11.9%. CNDSA NRs showed brightly yellow green fluorescence
emission at 542 nm with a QY of 7.9%, and the absorption peak
at 453 nm in aqueous solution. The optical properties of
nanodots exhibited a slight difference from their corresponding
nanorods. The main absorption of NDSA NDs were at 454 nm
with yellow fluorescence emission at 552 nm with a QY of 9.1%,
and CNDSA NDs showed emission peak and the main absorption
at 537 nm and 438 nm with a QY of 6.9%. NDSA NRs and NDSA
NDs had the resembled fluorescence lifetime (1.04 ns for NDSA
NRs, 1.16 ns for NDSA NDs), and the average fluorescence
lifetimes of CNDSA NRs and CNDSA NDs in water were 1.42 ns
and 1.40 ns (Fig. S15).
The absorbance and fluorescence spectra of the nanostructures
in aqueous solution under the irradiation of ultraviolet lamp
whose power intensity is 0.3 mW/cm2 measured by using aLPE-
1C laser powermeter (Wuke Photoelectric Technique, Beijing,
China) for different time were recorded to compare their anti-
photobleaching properties.86,87 As shown in Fig. 3, the
absorbance values of NDSA NRs CNDSA NRs were little decrease
upon continuous ultraviolet lamp irradiation for 80 min and
stayed about 95% of their initial value, while the values of
fluorescence emission were reduced to 69.3% and 71.7% of the
original value. The photostability test of NDSA NDs and CNDSA
NDs were collected under the same irradiation condition with
nanorods. The fluorescence intensities of NDSA NDs and CNDSA
NDs exhibited great decrease to 41.2% and 26.4% compared to
original value, illustrating the poor photostability in contrast to
The stabilities of these nanostructures were scrupulously
evaluated by monitoring the size distribution and zeta
potentials under physiological condition. As shown in Fig. S11-
13, the sizes of NDSA NRs and CNDSA NRs, stored in Roswell
Park Memorial Institute-1640 (RPMI-1640) with or without 10%
fetal calf serum (FBS) and 1% penicillin/streptomycin, showed
slight increased after incubation for 24 h at 37°C, and NDSA NRs
and CNDSA NRs were still transparent without obvious
aggregates or precipitates (Fig. S14). The zeta potentials of
NDSA NRs and CNDSA NRs sharply increased when they were
added in physiological condition due to the aggregation of
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NDSA NRs and CNDSA NRs. The above-mentioned spectral Morphology and size control of NDSA and CNDSA DRVsiew Article Online
DOI: 10.1039/C9TB02883H
results indicated that NDSA NRs and CNDSA NRs possess favor
physical and optical stability, which is appropriate for long-term
bioimaging.
As we know, the different water contents have more effects on
the morphology and size during the self-assembly process of
small organic molecules. We conscientiously investigated the
morphologies of NDSA in THF-H2O mixture at different water
contents (fw) by TEM. The same concentrations of NDSA in
various water fraction systems (fw = 60%, 70%, 80% and 90%)
were prepared. After 24 h aging time, these samples were
recorded by TEM. As shown in Fig. 4, the large sizes of rod-like
structures were found when the water fraction was 60%. As fw
raised to 70%, 80% and 90%, the rod-like structures vanished,
and the spherical structures appeared. Also, we could observe
the morphology of NDSA changed from the marginal irregular
sphere to nanoparticles. The water contents could not only
Fig. 3 The fluorescence intensity of (a) NDSA NRs, (b) NDSA NDs, (c) CNDSA NRs and (d) CNDSA NDs under continuous ultraviolet light for different time. The
absorbance intensity of (e) NDSA NRs, (f) NDSA NDs, (g) CNDSA NRs, (h) CNDSA NDs under ultraviolet light for different time. The power intensity of the UV
lamp is 0.3 mW/cm2.
change the morphology of NDSA, but also influence the size of Meanwhile, we also measured morphology and size of NDSA
NDSA. As shown in Fig. 4, the size of NDSA in THF-H2O mixture nanostructures with different NDSA concentrations in the same
would be decreased as water increased.
THF-H2O mixture (80%) under 10 min ultrasonic. TEM images in
In order to obtain rod-like morphology, we used ultrasound in Fig. S16 showed the morphologies of NDSA nanorods were
the process of nanostructures preparation and varied water similar within a certain range of NDSA concentration (20 – 80
contents simultaneously.88-90 We carefully tested the μM). When the concentration of NDSA increases to 100 μM, the
morphology changes of NDSA in the various water contents rod-like structures changed to anomalous nanorods due to the
under settled ultrasonic condition (Fig. 5). As shown in Fig. 5, solubility of NDSA in THF-H2O mixture. DLS results exhibited the
after 10 min of ultrasound, the uniform NDSA NRs were size of NDSA nanorods has almost no change within the range
observed by TEM with the water contents of 60%, 70% and 80%, of NDSA concentration (20 – 80 μM), but it became large when
indicating the ultrasound could induce morphology changes of the concentration of NDSA increased to 100 μM. In
NDSA nanostructures in THF-H2O mixture. However, when the consequence, the water contents, ultrasonic as well as
water content was up to 90%, NDSA in THF-H2O mixture could ultrasonic time have vital influence on the morphology and size
not form neat nanorods under 10 min ultrasound (Fig. 5d), of NDSA nanorods.
manifesting that water content also influence the morphology
of NDSA nanostructures. It is noted that the size of NDSA NRs
detected by DLS reduced from 283.4 nm to 140.3 nm as the
water contents increased from 60% to 80%, revealing the
content of water had a decisive effect on their sizes. It
demonstrated that the morphology and size of NDSA
nanostructures could be controlled by adjusting ultrasound and
water content.
Considering the suitable size and morphology for biological
applications, we choose NDSA NRs prepared with 80% water
contents for enhanced permeability and retention (EPR) effect
and endocytosis.91--93 The TEM images of NDSA in THF-H2O
mixture with different ultrasonic times were displayed in Fig. 6.
After 30 seconds of ultrasound, the spherical nanodots were
obtained. When the ultrasonic time was 1 min, the spherical
nanodots turned into irregular shapes. As the ultrasonic times
increased, these nanodots gradually transformed into regular
and integrated nanorods, manifesting ultrasound and ultrasonic
time could have a great impact on the morphology of NDSA.
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Fig. 4 TEM images of NDSA particles in THF-H2O mixtures with water
fractions of a) 60%, b) 70%, c) 80%, d) 90%. Scale bars, a) 1 μm; b) 500 nm;
c) 200 nm; d) 200 nm.
Fig. 7 CLSM images of A549 cells incubated with NDSA NRs for 1 h (upper), 2 h
(middle) and 4 h (lower) at 37°C. For each panel, the images from left to right
showed nuclei stained with Hoechst 33258 (blue), NDSA NRs fluorescence in
cells (yellow), overlays of both images, and the images of bright field. Scale
bars, 20 μm.
used for the investigation of the cellular uptake of these
nanostructures by confocal laser scanning microscopy (CLSM).
As shown in Fig. 7, the intracellular fluorescence intensity of
NDSA NRs increased gradually with the incubation time from 1
to 4 h, indicating that NDSA NRs had a sustained cellular uptake
in A549 cells. NDSA NDs, CNDSA NRs as well as CNDSA NDs were
also endocytosed by A549 cells in a time-dependent behaviour
(Fig. S22-24). Flow cytometry analysis showed efficient cellular
uptake of these nanostructures by A549 cells in a time-
dependent endocytosis (Fig. S25). A549 cells were also
incubated with NDSA NRs or NDSA NDs at various
concentrations for 4 h at 37°C, and cellular nuclei were dyed by
blue fluorescent dye Hoechst 33258. From the fluorescent
intensity (Fig. S26) we found a concentration-dependent
manner of nanorods internalized by A549 cells, and this manner
also emerged in relevant NDSA NDs, CNDSA NRs and CNDSA
NDs (Fig. S27-29). Co-localization of internalized these
nanostructures (NDSA NRs, NDSA NDs, CNDSA NRs, CNDSA NDs)
experiments were carried out using lyso-tracker blue. As
presented in Fig. S30-31, the co-localization of the NDSA NRs or
NDSA NDs (yellow) with the endosome (blue) generated a white
fluorescence in the merged images, and the similar white
fluorescence merged images were also obtained through co-
culturing A549 cells with CNDSA NRs or CNDSA NDs, manifesting
the nanostructures were mainly located within the endosome.
These results confirmed the nanostructures could be
internalized effectively by A549 cells.
Fig. 5 TEM images of NDSA in THF-H2O mixtures under 10 min sonication
with water fractions of a) 60%, b) 70%, c) 80%, d) 90%. Scale bars, a) 1 μm;
b) 200 nm; c) 200 nm; d) 200 nm.
Fig. 6 TEM images of NDSA nanoparticles during the nanodot-to-nanorod
transition with prolonged sonication time a) 30s; b) 1 min; c) 3 min; d) 5
min; e) 10 min. Scale bars, 200 nm.
The morphology and size of CNDSA nanoparticles in THF-H2O
mixtures can also be controlled by ultrasonic and water
contents. The TEM images of CNDSA nanoparticles without
ultrasonic and with 10 min ultrasonic in THF-H2O mixtures are
respectively presented in Fig. S17-18. The TEM images of
formation process of CNDSA NRs in 70% THF-H2O mixture under
different ultrasonic time are shown in Fig. S19. The TEM images
and size distribution of CNDSA NRs under different CNDSA
concentrations were shown in Fig. S20-21.
Cellular uptake and long-term bioimaging
Cellular uptake is indispensable for nanostructures to exert
their effects, especially for live cell imaging. A549 cells were
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Fig. 9 Flow cytometry histograms of A549 cells treated with (a) NDSA NRs,
(b) NDSA NDs, (c) CDSA NRs, (d) CNDSA NDs, and without treatment
(control) after treatment with different endocytic inhibitors (red: control;
green: 4°C; blue: sucrose; yellow: genistein; cyan: untreated).
Fig. 8 Long-term cell tracing CLSM images of the NDSA NRs incubated at
37°C for 4 h and then subcultured for fixed time intervals including day 0;
day 3; day 6; day 9. Scale bars, 20 μm.
The cytotoxicities of these nanostructures (NDSA NRs, NDSA
NDs, CNDSA NRs, CNDSA NDs) with different concentrations
(NDSA: 0-60 μM; CNDSA: 0-60 μM) against A549 cells were
carefully evaluated by Cell Counting Kit-8 (CCK-8) assay. As
exhibited in Fig. S32, more than 90% of A549 cells were alive at
the incubation concentrations up to 30 μM after incubation for
24 h, indicating all nanostructures exhibited low cytotoxicity
toward A549 cells. Furthermore, biocompatibility of NDSA NRs,
NDSA NDs, CNDSA NRs, CNDSA NDs were visually evaluated by
staining with calcein-AM and PI to identify live (green) and
dead/late apoptotic (red) cells, respectively. As shown in Fig.
S33, most cells incubated with the culture medium or different
concentrations of nanostructures for 24 h showed intense
green fluorescence and ignorable red fluorescence, indicating
these nanostructures had low cytotoxicity toward A549 cells,
which agreed well with the CCk-8 experiments. Trypan Blue was
used to visualize the cell apoptosis because it could stain the
apoptotic cells with observable blue color under an optical
microscope.80 As shown in Fig. S34, A549 cells were treated with
different concentration of NDSA NRs, NDSA NDs, CNDSA NRs, or
CNDSA NDs, whereas the control groups were only incubated
with the medium. A slight blue color was observed in the control
groups or the groups pretreated with these nanostructures,
indicating that the nanostructures are not harmful to A549 cells.
And these images showed the morphology of A549 cells after
incubation with different concentrations of the nanostructures
for 24 h, and the cells maintained their normal morphology,
demonstrating the good biocompatibility of NDSA NRs, NDSA
NDs, CNDSA NRs as well as CNDSA NDs.
subcultured in fresh medium for designated time intervals. For
each cell passage, the discarded culture medium was removed
and A549 cells were washed with PBS twice and stained by
Hoechst 33258. Strong and bright yellow cell fluorescence from
NDSA NRs or NDSA NDs could be clearly observed by CLSM in
Fig. 8 and Fig. S35 at the initial stage (day 0). The yellow
fluorescence gradually decreased along with cell proliferation
as the increase of incubation time (from day 3 to day 9). And
after 9 days of incubation, the yellow fluorescence from the
NDSA NRs in cells was still clearly observed in A549 cells,
indicating that NDSA NRs could be used as a fluorescent probe
for long-term cellular imaging. However, NDSA NDs exhibited
weak fluorescence after 9 days of subculture. And higher
intracellular fluorescence intensity of NDSA NRs in A549 cells
than NDSA NDs in A549 cells was detected at every time point
(Fig. S38a). Yellow green fluorescence of CNDSA NRs in A549
cells was also detected by CLSM after 9 days of incubation, while
CNDSA NDs showed low fluorescence in A549 cells (Fig. S36-37,
S38b), indicating CNDSA NRs also had potential in long-term
imaging.
To determine whether NDSA NRs and NDSA NDs were
internalized via different endocytic pathways, sucrose and
genistein were chosen as clathrin-mediated and caveolae-
mediated endocytosis inhibitors to explore the uptake of these
nanostructures by A549 cells.95,96 As exhibited in Fig. 9, after
A549 cells treated under low temperature (4°C) for 4 h, all A549
cells showed drastic decreases in the uptake of these
nanostructures, indicating the endocytosis was an energy-
dependent process. Cellular uptakes of NDSA NRs and NDSA
NDs were decreased as A549 cell were pretreated with sucrose
or genistein. Pretreatment of A549 cells with sucrose caused
great significant change in the cellular uptake of NDSA NRs,
indicating that the endocytosis of nanorods primarily occurred
via the clathrin-mediated endocytic. The similar manner was
also detected in CNDSA NRs and CNDSA NDs. Therefore, the
multiple endocytic pathways of NDSA NRs and CNDSA NRs
To investigate the long-term cellular tracking ability of NDSA
NRs, NDSA NDs, CNDSA NRs and CNDSA NDs, A549 cell
fluorescence images were captured by CLSM after similar
incubation periods. For the initial stage, A549 cells were
incubated with the different nanostructures for 4 h at 37°C as
day 0. Then the culture medium with the nanostructures was
extracted, and A549 cells were washed with PBS twice and
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(dd, J = 6.8, 3.2 Hz, 4H), 6.99 (s, 1H), 6.96 (s, 1H). MS (MALDI-
demonstrated nanorods had more efficient cellular uptake than
nanodots.
View Article Online
DOI: 10.1039/C9TB02883H
TOF): m/z for C34H32N2, calculated: 468.644; found 468.181
[M]+.
4,4'-((1E,1'E)-anthracene-9,10-diylbis(ethene-2,1-diyl))dibenzonitr-
ile. 1H NMR (500 MHz, CDCl3) δ 8.34 (dd, J = 6.8, 3.3 Hz, 4H), 8.10
(s, 1H), 8.07 (s, 1H), 7.79 – 7.73 (m, 8H), 7.52 (dd, J = 6.8, 3.2 Hz,
4H), 6.99 (s, 1H), 6.96 (s, 1H). MS (MALDI-TOF): m/z for
C32H20N2, calculated: 432.526; found 432.133 [M]+.
Preparation of NDSA NRs and NDSA NDs. NDSA NRs were
prepared following nanoprecipitation method. 0.6 mg NDSA
was dissolved in 2 ml THF with sustained sonication for 5 min.
The NDSA stock solution was quickly added to deionized water
Experimental
Materials and methods
4-formylbenzonitrile, 4-dimethylaminobenzaldehyde, ultra-dry
etrahydrofuran and potassium tert-butoxide were purchased
from Shanghai Energy Chemical. Methyl alcohol,
dichloromethane (DCM) and petroleum ether were purchased
from Sinopharm Chemical Reagent Co., Ltd. Phosphate buffer
saline (PBS), Hoechst 33258, Roswell Park Memorial Institute-
1640 (RPMI-1640) medium and penicillin-streptomycin solution (8 mL) with sonication, the mixture solution was then followed
were purchased from solarbio science & technology Co., Ltd.
Fetal bovine serum (FBS) was purchased from Hyclone. Sucrose,
genistein and amiloride were purchased from Shanghai
Beyotime Biotechnology Co., Ltd. Cell Counting Kit-8 (CCK-8)
was purchased from Dojindo Molecular Technologies. Distilled
water was produced by a Milli-Q Biocel (Millipore Corporation,
Breford, USA) water purification system with a resistance of
18.25 MΩ•cm-1. A549 cell line were provided by American Type
Culture Collection (ATCC).
Matrix assisted laser desorption/ionization time-of-flight
(MALDI-TOF) mass spectra (MS) were taken with a Bruker/Auto
Reflex III mass spectrometer equipped (Bremen, Germany). 1H
NMR spectra were obtained with a Bruker AVANCEIII500 NMR
in CDCl3 at room temperature. Dynamic light scattering (DLS)
measurements were measured by Malvern Zeta-sizer Nano at
25°C and the scattering angle was fixed at 90°. Transmission
electron microscopy (TEM) was recorded with a JEM-2100F
electron microscope. Fluorescence spectroscopy was
performed by a Shimadzu RF-5301 PC spectrometer. UV/Vis
by 10 min sonication under an ultrasonic tip. After THF
evaporation, the synthesized nanorods were obtained. The
prepared process of NDSA NDs was similar with NDSA NRs, only
the sonication time was 30 s. To study the mechanism of NDSA
NRs formation, different sonication time (0.5, 1, 3, 5, and 10
min), different concentrations of NDSA (20, 40, 60, 80, 100 μM)
and different water fractions (60%, 70%, 80%, 90%) in THF-H2O
mixtures were applied. The standard calibration curve
technique was used to calibrate the concentration of the NDSA
in NDSA NRs through calculating the main absorbance of NDSA
at 437 nm by UV/Vis absorption spectra in THF/H2O = 4:1 (v/v).
Preparation of CNDSA NRs and CNDSA NDs. The preparation
method of CNDSA NRs was similar with NDSA NRs. In brief, 0.6
mg CNDSA was dissolved in 3 ml THF with sonication for 5 min.
The CNDSA stock solution was quickly added to 7 ml deionized
water with sonication for 10 min. After THF evaporation, the
CNDSA NRs were obtained. The prepared process of CNDSA NDs
was similar with CNDSA NRs, only the sonication time was 30 s.
absorption spectra were measured by a Shimadzu UV-2550 To study the mechanism of CNDSA NRs formation, different
spectrophotometer. Flow cytometry was determined on Amnis
FlowSight Imaging Flow Cytometer (Merck Millipore, USA). An
Olympus FV1000 confocal laser scanning microscope was used
to perform confocal laser scanning microscopy (CLSM) images.
Synthetic procedures
sonication time (0.5, 1, 3, 5, and 10 min), different
concentrations of CNDSA (60, 120, 180, 240 μM) and different
water fractions in THF-H2O mixtures (60%, 70%, 80%, 90%) were
also applied. The standard calibration curve technique was used
to calibrate the concentration of the CNDSA in CNDSA NRs and
CNDSA NDs through calculating the main absorbance of CNDSA
at 421 nm by UV/Vis absorption spectra in THF/H2O = 4:1 (v/v).
Cell culture
4,4'-((1E,1'E)-anthracene-9,10-diylbis(ethene-2,1-diyl))bis(N,N-
dimethylaniline) (NDSA) and 4,4'-((1E,1'E)-anthracene-9,10-
diylbis(ethene-2,1-diyl))dibenzonitrile (CNDSA) were prepared
by literature procedures.63,64 Briefly, tetraethyl anthracene
9,10-diylbis(methylene)diphosphonate (1.20 g, 2.5 mmol) and
t-BuOK (0.85 g, 7.6 mmol) were degassed and purged with N2
for three times and then 70 ml ultra-dry etrahydrofuran was
added. A solution of 4-cyanobenzal-dehyde (0.69 g, 5.2 mmol)
or 4-dimethylaminobenzaldehyde (1.09 g, 7.31 mmol) in THF
(70 mL) was added dropwise into the mixture with stirring at
0°C in an ice-water. After stirring at room temperature for 24 h,
2 mL water was added and then removed the organic solvents
(THF) with a rotary evaporator, the resultant precipitate was
washed with MeOH for three times. After filtration, the
resulting powder was obtained.
The A549 were routinely grown in Roswell Park Memorial
Institute-1640 (RPMI-1640) medium (Gibco) containing 100 U
mL-1 penicillin, 100 U mL-1 streptomycin (Gibco) and 10% fetal
bovine serum (FBS). A549 cells were fostered in an artificial
humidified incubator at 37°C with 5% CO2 atmosphere. The
intracellular behaviours of NDSA NRs, NDSA NDs and CNDSA
NRs, CNDSA NDs toward A549 cells were imaged by confocal
laser scanning microscopy (CLSM). A549 cells were seeded in 6-
well culture plates in 2 mL complete medium and the cells were
cultured in cell incubator at 37°C in 5% CO2 for 24 h. After the
adherent cells were washed twice with 1 × PBS buffer, these
nanostructures dispersed in cell culture medium (different
concentrations of NDSA and CNDSA) were added to each well,
respectively. After another 4 h incubation in cell incubator, the
4,4'-(1E,1'E)-anthracene-9,10-diylbis(ethene-2,1-diyl)bis(N,N-
dimethylaniline). 1H NMR (500 MHz, CDCl3) δ 8.34 (dd, J = 6.8,
3.3 Hz, 4H), 8.10 (s, 1H), 8.07 (s, 1H), 7.79 – 7.73 (m, 8H), 7.52
This journal is © The Royal Society of Chemistry 20xx
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cells were washed with 1 × PBS buffer for three times and fixed Live/ dead cell staining assays
View Article Online
DOI: 10.1039/C9TB02883H
with 4% formaldehyde for 15 min. After rinsing the cells twice
We further confirmed the toxicity of these nanostructures using
calcein-AM/propidium iodide (PI) staining towards A549 cells to
determine their viabilities. After the A549 subcultured in 6-well
plates for 24 h, the cells were further incubated with these
nanostructures (NDSA or CNDSA: from 0 µM to 40 µM) at 37°C
for 24 h. The cells in 6-well plate without any treatment were
set as the controls. Then the cells were stained with calcein AM
(green for living cells) and PI (red for dead cells) for 30 min at
37°C, and the cells were washed with PBS for tree times. After
that, a Nikon C1si laser scanning confocal microscopy was used
to image the cells.
times, the cell nuclei were dyed with Hoechst 33258 for 5 min.
And then the cells were washed twice with 1 × PBS buffer, a
laser scanning confocal microscope was used for observing
these samples. Time-depended intracellular experiments of
these nanostructures were carried out with the same
concentrations, only the incubation time was different. For the
colocalization of endosome, the lyso-tracker blue instead of
Hoechst 33258 was used to stain endosome, and the similar
experiment was done as mentioned above.
Cytotoxicity Study
The metabolic activities of nanostructures towards A549 were Long-term cellular imaging
evaluated using CCK-8 assays. About 4 × 104 cells/ml of A549
cells were seeded in 96-well plates. After 24h incubation, the
residual medium was replaced by these nanostructures in
RPMI-1640 suspension at various concentrations. After 24 h
incubation at 37°C in 5% CO2 atmosphere, the cells were
washed with 1 × PBS buffer, then 100 µL of freshly prepared
RPMI-1640 and 10 µL CCK-8 solution was added to each well.
After 3 h incubation in the incubator, the absorbance of CCK-8
at 490 nm was monitored by a Bio-Rad 680 microplate reader.
Following equation was used to calculate the cell viability (%):
(As/Ac) × 100% (As: the absorbance of sample well; Ac: the
absorbance of control well).
About 2.5 × 105 cells were seeded in each 6-well plates and
incubated in RPMI-1640 for 24 h. The medium was then
replaced by 2 mL of RPMI-1640 containing these nanostructures
(NDSA or CNDSA: 10 µM) and incubated for another 6 hours at
37°C (Day 0). Then the cells were subcultured in 6-well plates
for designated days (from day 0 to day 9). When designated
days reached, the cells were washed with PBS for three times
and then fixed with 4% of paraformaldehyde solution for 15
min. The cells were washed with PBS to remove residual
paraformaldehyde and then stained with Hoechst 33258 for 3
min. After washing with PBS, the prepared cells observed by
confocal laser scanning microscopy.
Flow Cytometry
About 5 x 103 cells were used for flow cytometry to measure the
time-dependent cellular internalization of NDSA NRs, NDSA
NDs, CNDSA NRs, CNDSA NDs. In brief, about 5 x 105 cells in 2
mL medium were seeded in 6-well plates. After culturing for 24
h at 37°C, the discarded medium in each well were replaced
with NDSA NRs, NDSA NDs or CNDSA NRs, CNDSA NDs at NDSA
or CNDSA concentration of 10 µM. After incubation at 37°C for
different time, the cells were washed with PBS three times and
1 ml trypsin was applied to digest cells. The suspended cells
were harvested, and suspended in 1 mL new PBS. After that, the
cells were centrifuged at 2000 rpm for 3 min in the centrifuge.
After centrifugation, the supernatants were discarded and wash
the cells again with 1 mL PBS to remove the medium. After
carefully washing for three times, the satisfactory cells were re-
suspended in 1 mL PBS before analysis.
Conclusions
In conclusion, the fluorescent nanorods based on NDSA and
CNDSA were prepared by ultrasound aided nanoprecipitation
method. NDSA and CNDSA could be prepared into nanodots
(NDSA NDs, CNDSA NDs) and nanorods (NDSA NRs, CNDSA NRs)
under different ultrasound time. The morphology and size of
NDSA and CNDSA nanoparticles could be controlled by
ultrasound and the water content in the THF-H2O mixture.
These nanodots and nanorods showed intense fluorescent
emission, excellent stability and biocompatibility. Nanorods had
good anti-photobleaching property compared with nanodots
and had the potential for noninvasive long-term bioimaging in
vitro cell imaging experiments. Nanorods also showed better
cellular uptake efficiency compared to nanodots. It could be a
simple strategy to fabricate nanorods based on DSA derivatives
with controllable morphology and size by using simple method
for efficient and long-term bioimaging.
Endocytosis pathway detection
About 2.5 × 105 A549 cells were seeded in each 6-well plate and
incubated in RPMI-1640 for 24 h. After washing with PBS, the
cells were pretreated with various inhibitors including genistein
(caveolin-dependent endocytosis, 120 μM), sucrose (clathrin-
mediated endocytosis, 400 mM) in serum-free RPMI-1640 for 1
h. And the groups with serum-free RPMI-1640 were set as the
control. Then NDSA NRs, NDSA NDs, CNDSA NRs, CNDSA NDs
(NDSA or CNDSA: 10 µM) were added. After further incubation
for 4 h, the cells were digested by trypsin and the cells were
collected and washed with PBS, then re-suspended in 0.5 mL
PBS. Flow cytometry analysis was recorded by Amnis FlowSight
Imaging Flow Cytometer (Merck Millipore, USA) which collected
1×104 gated events for each sample.
Conflicts of interest
This work was financially supported by the National Natural
Science Foundation of China (21835001, 51773080, 21674041,
51573068, 21221063, and 81870117), the Program for
Changbaishan Scholars of Jilin Province, Jilin Province project
(20160101305JC), Jilin Province Science and Technology
Development Plan (20190201252JC), and “Talents Cultivation
Program” of Jilin University.
8 | J. Name., 2012, 00, 1-3
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29 D. Wang, L. Niu, Z. Y. Qiao, D. B. Cheng, J. Wang, Y. Zhong, F.
Acknowledgements
View Article Online
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This work was financially supported by the National Natural
Science Foundation of China (21835001, 51773080, 21674041,
51573068, 21221063, and 81870117), the Program for
Changbaishan Scholars of Jilin Province, Jilin Province project
(20160101305JC), Jilin Province Science and Technology
Development Plan (20190201252JC), and “Talents Cultivation
Program” of Jilin University.
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