Water-soluble and highly emissive near-infrared nano-probes by co-assembly of ionic amphiphiles: Towards application in cell imaging

Article abstract of DOI:10.1039/c9nj01184f

Water-soluble near-infrared (NIR) fluorescent dyes are extremely valuable in cell imaging. We here designed and synthesized an amphiphilic fluorescent dye (denoted by PBI-TPE-11), a bolaamphiphile bearing conjugated tetraphenylethylene and perylene bisimide in the middle and two aliphatic pyridinium groups at both ends. PBI-TPE-11 self-assembled into flake-like nanostructures in aqueous solution and showed very weak fluorescence emission from 600 to 830 nm, covering the NIR region. This result seems discrepant with that previously reported in the literature, where the conjugation of PBI and TPE was proven enhance aggregation induced emission. Very interestingly, both the morphology and the emission intensity were altered by the addition of sodium dodecyl benzene sulfonate (SDBS). Co-assembly of PBI-TPE-11 and SDBS formed nanowires, observed by using an atomic force microscope. Moreover, the emission of the co-assemblies was much stronger than that of the assemblies of neat PBI-TPE-11. An exciting quantum yield (QY) of 15% was obtained for the co-assemblies, while pure PBI-TPE-11 showed a QY of merely 0.2%. Finally, the co-assemblies were successfully applied in labeling HeLa cells, and high viability and high contrast fluorescence images were achieved.

Full text of DOI:10.1039/c9nj01184f

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
rsc.li/njc

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Water-soluble and highly emissive near-infrared nano-probes by
co-assembly of ionic amphiphiles: towards application in cell
imaging
Received 00th January 20xx,
Accepted 00th January 20xx
Yuzhi Xinga, Dahua Lia, Bin Donga, Xiaocheng Wang a, Chengfeng Wua, Lan Dinga, Shixin Zhoub,*, Jian
Fanc and Bo Songa,*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Water-soluble near-infrared (NIR) fluorescent dyes are extremely valuable in cell imaging. We here designed and synthesized
an amphiphilic fluorescent dye (denoted by PBI-TPE-11), a bolaamphiphile bearing a conjugation of tetraphenylethylene and
perylene bisimide in the middle and two aliphatic pyridinium at both ends. PBI-TPE-11 self-assembled into flake-like
nanostructures in aqueous solution, and showed very weak fluorescent emission from 600 to 830 nm, covering the NIR
region. This result seems discrepant with that previously reported in the literature, where the conjugation of PBI and TPE
was proven an aggregation induced emission enhancing agent. Very interestingly, both the morphology and the emission
intensity were altered by addition of sodium dodecyl benzene sulfonate (SDBS). Co-assembly of PBI-TPE-11 and SDBS formed
nanowires, observed by atomic force microscope. Moreover, the emission of the co-assemblies was much stronger than the
assemblies of neat PBI-TPE-11. An exciting quantum yield (QY) of 15% was obtained for the co-assemblies, while pure PBI-
TPE-11 showed a QY of merely 0.2%. Finally, the co-assemblies were successfully applied in labeling the HeLa cells, and high
viability and high contrast fluorescent images were achieved.
yield and high plasma protein binding rate and so on.13, 14, 23, 24
All in all, the molecular level dyes normally have strong
Introduction
The development of near-infrared (NIR) fluorescence imaging is
expected to have a significant impact on future personalized
oncology owing to the low tissue autofluorescence and high
tissue penetration depth in the NIR spectrum window (650 - 900
nm).1-3 To date, a variety of materials have been extensively
investigated for NIR fluorescence imaging, including fluorescent
proteins 4, 5, inorganic quantum dots 6, 7 and organic molecules
intermolecular π-π stacking interactions, and thus causes un-
predictable nonradiative decay and consequently deficient
fluorescence emission.25-27 Such effect would be severely
problematic in practical applications, especially in cell / tissue
imaging. Therefore, fluorescent probes with high emission,
water-solubility, low cytotoxicity and good stability are highly
demanded for fluorescent bio-labeling.28
8,
9
.
However, fluorescent proteins have some intrinsic
The fluorescence of organic dyes is prone to change
depending on the outer environment, molecular configuration
and aggregation state.29-32 This feature acts as a double-edged
sword in practical applications. In 1970s, acridine orange and
pyrene have been used as probes to determine the critical
micelle concentration, both of which utilized the environment
dependent feature of the fluorescent dyes.33, 34 Diarylethene
and spiropyran both undergo isomerization upon irradiation,
and their fluorescence changes with the configuration.35-37
Nowadays, the popular aggregation induced emission (AIE) dyes
demonstrate a very good example of fluorescence change with
aggregation state.38, 39 The fluorescence of organic dyes though
vulnerable can be controlled by molecular interactions (inter or
intra or both).
disadvantages, for instance, small Stokes shift, poor
photostability and tedious transfection process.10 Inorganic
quantum dots exhibit excellent emission property, but suffer
from the high cytotoxicity resulting from heavy-metal elements
(e.g. Cd & Se), especially in an oxidative environment.11 In
contrast, organic dyes have shown promising clinical
applications as non-targeting agents for optical imaging
because of its versatility and flexibility.12-15
16
The currently available organic NIR dyes include cyanine
,
squaraine 17, phthalocyanines 18, porphyrin derivatives 19, 20 and
21,
borondipyrromethane (BODIPY) analogues
22. These dyes
have their respective advantages, but also possess their own
disadvantages, such as poor photostability, low quantum
a College of Chemistry, Chemical Engineering and Materials Science, Soochow
University, Suzhou 215123, China. E-mail: songbo@suda.edu.cn
b Department of Cell Biology, School of Basic Medical Science, Peking University
Health Science Center, Beijing 100191, China. E-mail: zsx@bjmu.edu.cn
c Jiangsu Key Laboratory For Carbon-Based Functional Materials & Devices
Science, Soochow University, Suzhou 215123, China.
This research focuses on the fluorescent probes that are
applicable in bio-systems. Therefore, water solubility (or
dispersity) will be an extremely important character for the
probes.40, 41 Molecular assembly or co-assembly has proven to
be a powerful tool for dealing with the supramolecular
† Electronic Supplementary Information (ESI) available.
See DOI: 10.1039/x0xx00000x
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interactions, especially the latter. Taking the advantage of
coulombic interaction, co-assembly of amphiphiles with
opposite charges has been widely investigated.42, 43 The idea has
been employed to control the emission of dyes, mainly by
tuning the interactions between the fluorescent moieties in the
assemblies. For example, Benzhong Tang et al. synthesized a 2-
(2-hydroxyphenyl)benzothiazole derivative with cationic groups
to effectively detect the anionic surfactants, and the complex
with enhanced emission was applied for bacterial imaging.44
Yilin Wang et al. systematically investigated the surfactant types
on the emission enhancing effect of cationic M-silole
molecules.45 Based on surfactant aggregate encapsulating,
Liping Ding et al. developed a series of fluorescent sensors.46-48
Deqing Zhang et al. designed a multi reaction system based on
coulombic interactions, and developed a fluorescent “turn-on”
method to detect the acetylcholinesterase.49 Along with this
idea, they also developed fluorometric “turn-on” detection of
lactic acid and oxidase B.50, 51 All these studies demonstrated
that the co-assembly of amphiphiles bearing opposite charges
should be a good avenue to control the fluorescence, especially
for the AIE dyes. We are extremely interested in NIR dyes, and
wonder if the co-assembly idea also works on them.
Perylene bisimides (PBIs) are one of the mostly investigated
organic dyes with excellent photophysical, thermal and
chemical stability.52-54 Benzhong Tang et al. demonstrated that
the conjugation of PBI and tetraphenylethylene (TPE) allows for
AIE features and NIR emission to the resulting dye.55-57 Inspired
by their work, we attempt to introduce the jointed PBI and TPE
into bolaamphiphile, and anticipate the self-assembled
structures bring about efficient NIR emission that is applicable
in cell imaging. The target molecule denoted by PBI-TPE-11 was
hence synthesized. This molecule indeed showed a NIR
emission ranging from 600-830 nm, however, the quantum
yield was merely 0.2%. Employing the idea of co-assembly, we
introduced anionic amphiphile sodium dodecyl benzene
sulfonate (SDBS) in the aqueous solution of PBI-TPE-11. The co-
assemblies kept the position of NIR emission, but the emission
was significantly enhanced, and the quantum yield was
promoted up to 15%. The co-assembly was proven a rather low
cytotoxicity, and successfully applied in fluorescent imaging of
HeLa cells.
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DOI: 10.1039/C9NJ01184F
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Fig. 1 The synthetic route of PBI-TPE-11
rather poor water solubility, which should be attributed to the
combination of the hydrophobicity supplied by the alkyl chains
and the strong
- stacking interactions between perylene
groups. Methanol, a good solvent for PBI-TPE-11, was employed
to assist PBI-TPE-11 dissolving in water. In doing so, PBI-TPE-11
was firstly dissolved in methanol (2% by volume to water), and
then the solution was dropwise added into water. By this
method, PBI-TPE-11 can be well dispersed in water. The
concentration of PBI-TPE-11 was kept 1.0 × 10-5 mol L-1 all
through the research.
Firstly, the morphologies of the self-assembled structure
were investigated by atomic force microscope (AFM). As shown
in Fig. 2a, PBI-TPE-11 formed flake-like nanostructures in
aqueous solution. Transmission electron microscope (TEM)
observation also showed similar 2D structures, as shown in Fig.
S11. The length of the lamellar structures is mostly in the range
of 600-800 nm, and the width is in the range of 150-350 nm. The
average thickness determined by the depth analysis was
approximately 2.6 nm, as shown in Fig. S12. This value is less
than the distance between the two pyridinium groups of
extended PBI-TPE-11 molecule (4.4 nm, estimated by
ChemDraw software). These results suggest that the lamellar
structures should be composed of a single layer of PBI-TPE-11
molecules parallel packed at an inclined angle. This assumption
should be reasonable that the hydrophilic pyridinium groups
locate at the surfaces of the lamella, and the hydrophobic
Result and Discussion
The synthetic route of PBI-TPE-11 is shown in Fig. 1. Briefly,
synthesis of trimethyl(4-(1,2,2-triphenylvinyl)phenyl)stannane
(compound B) referred to literatures,58 and it was prepared by
reaction of compound
A
and n-butyl lithium. 11-
bromoundecan-1-amine was synthesized according to the
literatures.59 Compound J was prepared by Stille coupling
between compound B and alkylated PBI (compound H). The
target molecule PBI-TPE-11 was characterized by NMR (Fig. S9)
and mass spectra (Fig. S10). The detailed description of the
synthesis is shown in the experimental section, and the
corresponding characterization is shown in the supporting
information.
aromatic parts pack in order due to the intermolecular
stacking interactions.
-
The absorption of PBI-TPE-11 ranges from 200-700 nm, and
shows two peaks at 480 and 575 nm. In aqueous solution, it
showed a rather weak fluorescence, which can be reflected by
both the fluorescence intensity and the quantum yield. As
shown in Fig. 1b, PBI-TPE-11 in aqueous solution displays an
emission in the NIR region (600 - 830 nm, due to the detection
Although the target molecule contains two pyridinium groups
to improve the hydrophilicity, PBI-TPE-11 still shows a
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RSDBS
Fig. 2 (a) The AFM image of PBI-TPE-11. (b) Normalized UV-vis and PL spectra of PBI-TPE-
11 in aqueous solution.
Fig. 4 (a) Plot of fluorescence intensity at 660 nm versus CSDBS. (b) Job’s plot.
The co-assembly of PBI-TPE-11/SDBS was also confirmed by
zeta potential measurement. The zeta potential of the
assemblies of PBI-TPE-11 and co-assemblies of PBI-TPE-11/SDBS
were 35.6 ± 2.6 and - 42.3 ± 2 mV, respectively. PBI-TPE-11 and
SDBS are cationic and anionic amphiphiles, respectively. Based
on this result, it is understandable that the zeta potentials of the
particles turn from positive to negative after co-assembly. In
addition, the suspension of the co-assemblies of PBI-TPE-
11/SDBS kept clear and showed no precipitation for at least 7
days (Fig. S13), indicating that it should be a true colloidal
solution.
limit of the instrument, we did not show the longer wavelength
emission than 830 nm), and the peak position appears at 660
nm. The Stokes shift is approximately 90 nm. And the
corresponding quantum yield is 0.2%, determined by relative
method using cresyl violet as reference.
Upon addition of 2-fold SDBS, the lamellar structures were no
longer observed but replaced by discrete nanowires in the AFM
image, as shown in Fig. 3a. The width of the nanowires is in the
range of 50-200 nm, and the average thickness is almost
unchanged compared with the nano-flakes formed by PBI-TPE-
11 (Fig. S12). The morphological change indicates that the SDBS
should have some kind of interaction with PBI-TPE-11, and the
bonding between them altered the assembled structure.
We were happy to find that the fluorescence was significantly
enhanced upon addition of 2-fold SDBS into the PBI-TPE-11
solution. As shown in Fig. 3b, the fluorescence intensity became
12 times of the assemblies formed by neat PBI-TPE-11, and the
quantum yield increased to 2%. Such fluorescence
enhancement could be observed by naked eyes under
illumination of 365 nm UV light (inset in Fig. 3b). Moreover, the
quantum yield continues to increase when adding more SDBS,
and a quantum yield as high as 15% was acquired when 140-fold
of SDBS was added. It is worth to note that the critical
aggregation concentration (CMC) of SDBS is 1.6 × 10-3 mol L-1.60,
61 Herein, the SDBS added into the aqueous solution of PBI-TPE-
11 was still less than its CMC, which means that the
fluorescence change should be attributed to the formation of
complexes between PBI-TPE-11 and SDBS mainly relying on two
supramolecular interactions: (1) the electrostatic interaction
between the ionic head groups of the PBI-TPE-11 and SDBS, (2)
the hydrophobic interaction supplied by the aliphatic chains.
Secondly, the association ratio between PBI-TPE-11 and SDBS
was determined by two methods. (1) As aforementioned,
increasing SDBS in the aqueous solution of PBI-TPE-11 will lead
to the enhancement of the fluorescence. This feature was
utilized to estimate the association ratio. In doing so, the
concentration of PBI-TPE-11 was kept constant at 1.0 × 10-5 mol
L-1, and the concentration of SDBS (CSDBS) was varied. The
fluorescence intensity at the 660 nm (the peak position) was
plotted against the corresponding concentration. As shown in
Fig. 4a, linear fitting presented a slope change at the
concentration of 2.0 × 10-5 mol L-1, which implies that the
association ratio between PBI-TPE-11 and SDBS should be 1:2.
An equilibrium must be existed between these two compounds
and their complexes. Passing the association ratio point, the
increase of fluorescence relies on the amount of extra SDBS to
push the equilibrium to right side, which makes a reasonable
explanation for the smaller slope. (2) The second method used
to determine the association ratio was Job’s plot.62 In doing so,
the total concentration of these two compounds was kept
constant (1.0 × 10-4 mol L-1), and the molar ratios of SDBS (RSDBS
)
were varied from 0 to 1. The fluorescence differences (F – F0,
where F and F0 are the fluorescence intensity in presence and
absence of SDBS respectively) were plotted against RSDBS. As
shown in Fig. 4b, a maximum emission was obtained as RSDBS
0.67 by linear fitting, which confirms that the association ratio
of PBI-TPE-11 and SDBS is 1:2.
2.0
(b)
Without SDBS
(a)
With 2-fold SDBS
1.6
=
1.2
0.8
0.4
0
Since we have learned that the association ratio between PBI-
TPE-11 and SDBS is 1:2, the association constant of the complex
can be determined by Benesi-Hildebrand equation 63, 64
:
1
1
1
1 μm
650
700
750
800
=
+
Wavelength (nm)
2
F
−
F0
Fmax − F0
(
)
− F0 ∙ CSDBS
Ka ∙ Fmax
Fig.
3 (a) The AFM image of PBI-TPE-11 in presence of 2-fold SDBS. (b)
where Fmax is the maximum fluorescence intensity of PBI-TPE-
11 in presence of SDBS, Ka is the association constant. As shown
Fluorescence spectra of PBI-TPE-11 in absence and presence of 2-fold SDBS in
aqueous solution (λex = 585 nm). Inset: Photographs of the corresponding solution
under illumination of 365 nm UV light.
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Y = 9.5  10-16 X + 3.7  10-6
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K
= 3.9  109 (mol L-1)-2
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Fig. 5 Benesi-Hildebrand’s plot of (F – F0)-1 versus CSDBS
.
-2
Fig. 7 Dose-dependent cell viability of complex of PBI-TPE-11 and SDBS with HeLa
cells.
in Fig. 5, Ka was extrapolated by linear fitting of the plot of (F –
F0)-1 against CSDBS-2, and the value was 3.9 × 109 (mol L-1)-2,
indicating that PBI-TPE-11 have very high affinity with SDBS. The
high affinity should be attributed to electrostatic interaction
between the pyridinium head of PBI-TPE-11 and the sulfonate
group of SDBS.
SDBS should be attributed to the tailored intermolecular
interactions. As for the neat PBI-TPE-11 in aqueous solution, π-
π stacking interactions caused by the planar perylene bisimide
and hydrophobic interaction make the molecules pack in order.
Co-assembly of PBI-TPE-11 and SDBS reduced the strong π-π
stacking interaction by the alkyl chains intercalating in between
the perylene bisimide groups, and thus led to the enhanced
fluorescence emission. When more than 2-fold SDBS was added,
the extra SDBS molecules possibly segregated the PBI-TPE-11
molecules by surrounding them, which explains the continuous
increase of quantum yield.
In the following, the cell viability was tested under existence
of co-assemblies of PBI-TPE-11 and SDBS. As shown in Fig. 7, the
viabilities of HeLa cells were almost 100% when the
concentration of PBI-PEI-11 was lower than 16 × 10-6 mol L-1,
and when the concentration was 32 × 10-6 mol L-1, the cell
viability value was still maintained > 85%. The concentration of
PBI-PEI-11 used in cell imaging was much lower than 16 × 10-6
mol L-1, and hence the cytotoxicity is negligible.
The PBI-TPE-11 and co-assemblies of PBI-TPE-11/SDBS were
applied to stain the HeLa cells. To locate the position of the
complex, the HeLa cells were cultured in the mediums
containing 4′,6-diamidino-2-phenylindole (DAPI), PBI-TPE-11
and co-assemblies of PBI-TPE-11/SDBS. The concentration of
PBI-TPE-11 in the culturing mediums was controlled to 7 × 10-6
mol L-1, which is far below the toxic dosage. To make a good
comparison, the cells only labeled with DAPI and labeled with
DAPI and PBI-TPE-11 were employed as the control. The HeLa
cells were sequently labeled by DAPI and the nano-probes by
culturing them in the mediums containing these probes. After
being fixed by polyaldehyde, the cells were observed by
confocal laser scanning microscope (CLSM). It is worth to note
that DAPI is often used to stain the nuclei of cells, and it emits
strong blue fluorescence, whose brightness can be applied as
reference to the rest. As shown in Fig. 8, the blue emission
stands for the location of the nuclei, and the red emission is the
fluorescence emission of nano-probes (assemblies of PBI-TPE-
11 or co-assemblies of PBI-TPE-11/SDBS). It is clear that the co-
assemblies resided in the cytoplasm of HeLa cells, and the nuclei
were not stained by them. Besides, the cells labeled with PBI-
TPE-11 showed weak fluorescent emission comparing to those
labeled with the co-assemlies of PBI-TPE-11/SDBS. These data
Thirdly, the complexation between PBI-TPE-11 and SDBS can
also be qualitatively analyzed through Fourier transform
infrared (FTIR) spectroscopy, and the spectra are shown in Fig.
6. As for the neat PBI-TPE-11, the distinct vibration peaks at
1695, 1658 and 1635 cm-1 are assigned to the stretching
vibration of carbonyl bonds in amide I, and peaks at 1597 and
1587 cm-1 are due to the stretching vibration of C=C in
perylene.65 Upon addition of SDBS, the peak located at 1695 cm-
1 became very weak, and a new peak appears at 1733 cm-1. This
alteration could be explained by reduced interaction between
the perylene bisimide due to the intercalation of SDBS. The
conjugate delocalization effect owing to the strong π-π stacking
interaction will decrease the polarity of carbonyl group, and
hence results in a relatively low wavenumber vibration at 1695
cm-1. The segregation effect induced by intercalation of SDBS
will decrease the π-π stacking interaction, and thus the typical
vibration peak (1733 cm-1) of isolated carbonyl groups appeared.
The above assumption can also explain the disappeared peak at
1597 and 1587 cm-1. Due to the reduced π-π stacking
interaction, the vibration of C=C shifted to normal position at
around 1600 cm-1. These results accord with the morphology
change, as well as the greatly enhanced fluorescence emission.
Based on the above experiment facts, we can conclude that
the fluorescence enhancement of PBI-TPE-11 upon addition of
(a)
1587
1597
1695
1635
1658
(b)
1733
1633
1646
1750
1700
1650
1600
1550
Wavelength (cm-1)
Fig. 6 FTIR spectra of (a) PBI-TPE-11; (b) complex of PBI-TPE-11 with SDBS.
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(c) 20 m2i0nmin
0 min
(b)
20 μm
Fig. 9 (a) Photostability test of PBI-TPE-11/SDBS in HeLa cells. The cells were continuously
irradiated at 561 nm (2 mW) for 20 min, and the fluorescent intensities were recorded
every 5 min. Two typical CLSM images of the cells labelled with the co-assemblies of PBI-
TPE-11/SDBS are presented: (b) 0 min, (c) 20 min.
Fig. 8 CLSM images of HeLa cells after being cultured in the mediums: (a-c) without
PBI-TPE-11, (d-f) with PBI-TPE-11, and (g-i) with co-assemblies of PBI-TPE-11/SDBS.
From left to right rows are images of cells labeled with DAPI, PBI-TP-11 (or PBI-
TPE-11/SDBS)), and merged images of the former two. Scale bar: 20 μm.
phthalimide potassium salt, hydrazine hydrate, HBr (48 wt.%
solution in H2O), perylene-3,4,9,10-tetracarboxylic dianhydride,
Pd(PPh3)4 and sodium SDBS were purchased from TCI (Shanghai)
Development Co., Ltd. Tetrahydrofuran (THF), toluene, N,N-
dimethylformamide (DMF), methanol, ethanol, acetone, acetic
acid, 1-methyl-2-pyrrolidone (NMP), H2SO4 (98%), HCl, NaCl,
NaOH, anhydrous Na2SO4, anhydrous Na2CO3, anhydrous NH4Cl,
iodine, bromine and pyridine were purchased from Sinopharm
Chemical Reagent Co., Ltd. Ethyl acetate, petroleum ether,
dichloromethane (DCM), and chloroform were obtained from
Yonghua Chemical Technology (Jiangsu) Co., Ltd.
indicate that the co-assemblies should be an excellent NIR
nano-probe for cell imaging.
The photostability of the nano-probes is one of the most
important concerns for bioimaging. We investigated the
photostability of PBI-TPE-11/SDBS, and the results are shown in
Fig. 9. F0 and F are the initial PL intensity and the PL intensity of
the sample at different irradiation time. The PL intensity
showed a little decrease after being continuously irradiated by
561 nm (2mW) light for 20 min, and the nano-probes possess ≈
92% of their initial PL intensity, as shown in Fig. 9. In addition,
the CLSM image did not show big differences on their contrast
and resolution. These results indicate the nano-probes have
excellent photostability.
Characterization. 1H NMR spectra were recorded on a Bruker
Avance III 400 MHz NMR spectrometer (USA). Mass spectra (MS)
of compound 6 was obtained on a micro Q-TOF III mass
spectrometer (Bruker, USA) in the electrospray ionization (ESI)
mode. And other products were measured with matrix-assisted
laser desorption/ionization time-of-flight (MALDI-TOF-MS). The
measurements of the fluorescence spectra were conducted on
FLS980 (Edinburgh Instrument). AFM images were recorded on
Conclusions
a
Multimode 8 microscope (Bruker, USA). Peak force
In summary, a bolaamphiphile bearing conjugation of PBI and
TPE was synthesized. As being dispersed in aqueous solution, it
showed a weak NIR fluorescence emission. Co-assembly of PBI-
TPE-11 and SDBS greatly enhanced the fluorescence emission,
and the quantum yield was promoted from 0.2% to 15%. It was
proven that PBI-TPE-11 and SDBS have very strong association
with a ratio of 1:2. The co-assemblies showed no toxicity until
16 × 10-6 mol L-1, and were demonstrated a very good NIR nano-
emitter in cell imaging.
quantitative nanomechanical mapping mode with a Scan Asyst-
Air probe (nominal spring constants 0.4 N m-1, frequency 70 kHz,
from Bruker) was adopted during the measurement. The
samples were cast on a silicon substrate and dried in vacuum,
and the concentration of PBI-TPE-11 was 1.0 × 10-5 mol L-1. TEM
characterization was performed on Hitachi HT7700 (Japan)
operating at 120 kV. Approximately 10 μL of sample solution
was cast onto carbon-coated copper grid, and then the grid was
dried under vacuum. FTIR spectra were recorded on a Nicolet
6700 produced by Thermo Scientific (USA). The cell images were
taken on a STP6000 CLSM (LEICA, Germany).
Experimental
Synthesis of (2-(4-bromophenyl)ethene-1,1,2-triyl)tribenzene
(compound A). A 1.6 M solution of n-butyl lithium in hexane
(2.6 mL, 4.16 mmol) was added dropwise to the solution of
diphenylmethane (0.78 g, 4.7 mmol) in anhydrous THF (60 mL)
at 0 °C in a nitrogen atmosphere. After the mixture being stirred
Materials. Diphenylmethane, 4-bromobenzophenone, n-butyl
lithium, p-toluenesulfonic acid (PTSA), trimethylstannanylium
chloride (SnMe3Cl) and cresyl violet were purchased from J&K
Chemical Co., Ltd. (Shanghai). 11-Bromo-1-undecanol,
This journal is © The Royal Society of Chemistry 20xx
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View Article OTnlhinee
1
2
3
for 30 min at 0 °C, 4-bromobenzophenone (1.0 g, 3.8 mmol) was Synthesis of 11-bromoundecan-1-amine (compound F).
added at -78 °C, and the mixture was continuously stirred for 5 aqueous solution (35 mL) of Na2CO3 (3.D4OgI:,130.210.039m/Cm9NoJl0)11w8a4Fs
h. Afterward, the reactant was allowed to warm to room added into solution of compound E (3.9 g, 11.8 mmol) in DCM
temperature. Then, the reaction was quenched with aqueous (30 mL). The mixture was stirred at room temperature for 30
solution of NH4Cl and extracted with DCM, and dried over min. Then the resulting product was extracted with DCM, and
anhydrous Na2SO4. After removal of the solvent by rotavapor, dried over anhydrous Na2SO4. After the removal of solvent, the
the crude product was dissolved in toluene (100 mL). Then PTSA crude product was obtained and used directly in the next step.
(50 mg, 0.29 mmol) was added, and the mixture was refluxed Yield: 2.9 g, 98%. 1H NMR (400 MHz, CDCl3, δ): 3.40 (t, J = 6.9 Hz,
for 2.5 h before cooling to room temperature. The solvent was 2H), 2.70 (t, J = 7.1 Hz, 2H), 1.89-1.80 (m, 2H), 1.43 (dd, J = 16.5,
removed and the crude product was purified by silica gel 7.6 Hz, 4H), 1.34-1.23 (m, 14H). ESI-MS (m/z): calcd for
column chromatography using hexane/chloroform (30/1 by [C11H24BrN + H]+: 249.1165, found: 250.1195.
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volume) as eluent. Yield: 0.6 g, 43%. H NMR (400 MHz, CDCl3,
Synthesis of 1,7-dibromo-3,4,9,10-tetracarboxylic perylene
δ): 7.22 (d, J = 8.0 Hz, 2H), 7.16-7.06 (m, 9H), 7.05-6.97 (m, 6H),
dianhydride (compound G). Perylene-3,4,9,10-tetracarboxylic
6.89 (d, J = 8.0 Hz, 2H).
dianhydride (10.0 g, 25.5 mmol) and concentrated H2SO4 (100
Synthesis
of
trimethyl(4-(1,2,2- mL) were added into a 250 mL three-necked flask. The mixture
triphenylvinyl)phenyl)stannane (compound B). Compound A was stirred at 55 °Ϲ for 18 h. Iodine (250 mg, 1.0 mmol) was
(2.0 g, 4.9 mmol) was added into anhydrous THF (60 mL) at added to the mixture, and continuously stirred for 5 h. Bromine
−78 °C under nitrogen atmosphere, then a 1.6 M solution of n- (2.9 mL, 56.1 mmol) was added dropwise to the mixture over 30
butyl lithium in hexane (4.2 mL, 6.7 mmol) was dropped into it min, then the reaction was stirred at 85 °Ϲ for 24 h. After cooling
over 20 min. After stirring for 2 h, a 1.0 M solution of SnMe3Cl to 40 °Ϲ, the excess bromine was blown with nitrogen under
in hexane (8.8 mL, 8.8 mmol) was added and reacted for 12 h at stirring. Then the mixture was poured into ice-water, and the
room temperature. Then the mixture was poured into water precipitate was separated by filtration. The precipitate was
and extracted with ethyl acetate. After removing the solvent, washed with water until the filtrate was neutral. The crude
the crude product was obtained and used directly in the next product was used without further purification.
synthesis.
Synthesis
of
N,N′-undecyl-1,7-dibromo-3,4,9,10-
Synthesis of 2-(11-hydroxyundecyl)isoindoline-1,3-dione tetracarboxylic perylene bisimide (compound H). Compound G
(compound C). 11-Bromo-1-undecanol (5.0 g, 27.0 mmol) and (3.2 g, 6.0 mmol) and NMP (90 mL) were added into a flask, and
phthalimide potassium salt (5.5 g, 22.0 mmol) were dissolved in the mixture was sonicated for 30 min. Then compound F (4.0 g,
DMF (150 mL), and the mixture was stirred at 80 °C for 18 h. 16.0 mmol) and acetic acid (15 mL) were added to the reaction
After cooling down, the reactant was diluted with ethyl acetate. mixture. The resulting solution was stirred at 85 °Ϲ for 48 h
The organic layer was washed with water for three times. The under nitrogen atmosphere. Afterward, the reaction solution
collected water layer was extracted three times with ethyl was cooled to room temperature. Then, the mixture was
acetate. The organic layer was washed with brine twice and poured into ice-water (500 mL). The precipitate was separated
dried over anhydrous Na2SO4. After removing the solvent, the by filtration. The crude product was purified by a silica gel
crude product was recrystallized from methanol, and was used column chromatography using chloroform as eluent. Yield: 1.0
directly in the next synthesis.
g, 17%. 1H NMR (400 MHz, CDCl3, δ): 9.49 (d, J = 8.2 Hz, 2H), 8.93
(s, 2H), 8.71 (d, J = 8.1 Hz, 2H), 4.34-4.11 (m, 4H), 3.41 (t, J = 6.9
Hz, 4H), 1.89-1.80 (m, 4H), 1.80-1.70 (m, 4H), 1.47-1.21 (m, 28H).
MALDI-TOF-MS (m/z): calcd for [C46H50Br4N2O4 + H]+: 1015.054,
found: 1015.067.
Synthesis of 11-aminoundecan-1-ol (compound D). Hydrazine
hydrate (4.0 mL, 82.4 mmol) was slowly added into the solution
of compound C (6.0 g, 19.8 mmol) in ethanol (100 mL). The
reactant was refluxed for 3 h. After cooling down, the reactant
was adjusted to pH 1 with 4 M HCl, then filtered. The filtrate
Synthesis
of
N,N′-undecyl-1,7-di(4-(1,2,2-
perylene
was extracted with DCM for three times. After the collected triphenyl)vinyl)phenyl-3,4,9,10-tetracarboxylic
aqueous layer was adjusted to pH 14 with concentrated NaOH bisimide (compound J). Compound H (445 mg, 0.9 mmol),
solution, it was extracted with DCM for five times. The organic compound B (300 mg, 0.3 mmol) and Pd(PPh3)4 (35 mg, 0.03
phase was dried over anhydrous Na2SO4. After removing the mmol) were added into a two-necked flask. Under protection of
solvent, the crude product was obtained and used directly in the argon, anhydrous toluene (100 mL) was added. The reactants
next step.
were refluxed for 48 h. After removing the solvent, the product
was purified by a silica gel column chromatography using
DCM/petroleum ether (7/2 by volume) as eluent. Yield: 150 mg,
33%. 1H NMR (400 MHz, CDCl3, δ): 8.61 (s, 2H), 8.23 (d, J = 8.2
Hz, 2H), 7.86 (d, J = 8.2 Hz, 2H), 7.37-7.09 (m, 38H), 4.33-4.20
(m, 4H), 3.44 (t, J = 6.9 Hz, 4H), 1.93-1.82 (m, 4H), 1.77 (dd, J =
15.7, 7.3 Hz, 4H), 1.55-1.28 (m, 28H). MALDI-TOF-MS (m/z):
calcd for [C98H88Br2N2O4 + H]+: 1517.518, found: 1517.515.
Synthesis of 11-bromoundecan-1-ammonium bromide
(compound E). Compound D (6 g, 32 mmol) and HBr (40 mL, 48
wt.% solution in H2O ) were stirred at 100 °C for 24 h. After being
cooled down, the precipitate was filtered and washed with
water for three times. The crude product was recrystallized
1
from acetone. Yield: 8.6 g, 82%. H NMR (400 MHz, DMSO-d6,
δ): 7.61 (s, 3H), 3.52 (t, J = 6.7 Hz, 2H), 2.81-2.68 (m, 2H), 1.84-
1.70 (m, 2H), 1.53-1.44 (m, 2H), 1.41-1.18 (m, 14H).
6 | J. Name., 2012, 00, 1-3
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Synthesis of PBI-TPE-11. Compound J (150 mg, 0.1 mmol) was
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Notes and references
1
2
View Article Online
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dissolved in pyridine (100 mL), and refluxed for 48 h. After
removing the solvent, the product was washed with ethyl ether
for five times. Yield: 120 mg, 72%. 1H NMR (400 MHz, DMSO-d6,
δ): 9.13 (d, J = 4.7 Hz, 4H), 8.60 (t, J = 7.6 Hz, 2H), 8.15 (t, J = 6.5
Hz, 4H), 8.02 (s, 2H), 7.54-7.45 (m, 2H), 7.41-6.79 (m, 40H), 4.60
(t, J = 7.0 Hz, 4H), 4.18-3.91 (m, 4H), 1.97-1.82 (m, 4H), 1.73-1.54
(m, 4H), 1.42-1.14 (m, 28H). MALDI-TOF-MS (m/z): calcd for
[C108H98Br2N4O4 – 2Br]2+: 757.881, found: 757.859.
3
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Cell viability assay. The cell viability of the complex formed by
PBI-TPE-11 and SDBS on HeLa cells was evaluated by CCK-8
assay (Dojindo, Japan). The preparation of the HeLa cells and
incubation of complexes were similar to those in the cell
imaging process. After being cultivated for 12 h, HeLa cells were
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mol L-1, mixed with 2-fold SDBS respectively). After 12 h of
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microplate reader (Spectramax i3, Molecular Devices, USA).
And the absorbance values were proportional to the number of
live cells. Each concentration had three parallel wells.
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Dulbecco's Modified Eagle Medium (DMEM, ThermoFisher)
medium supplemented with 10% fetal bovine serum (Hyclone).
For cell imaging, the solution of complexes formed by PBI-TPE-
11 and SDBS (the concentration of PBI-PEI-11 was about 7 × 10-
6 mol L-1, mixed with 2-fold SDBS) was added into a bottom-glass
dish seeded with HeLa cells and incubated at 37 °C for 2 h. After
that, the cells were gently washed twice with phosphate
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min. Lastly, nuclear DNA of the cells was stained with DAPI for
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CLSM.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We thank the financial support of the National Nature Science
Foundation of China (21674075 and 21233003), the Nature
Science Foundation of Jiangsu Province (BK20161211), Key
University Science Research Project of Jiangsu Province
(17KJA150007), Suzhou Key Laboratory of Macromolecular
Design and Precision Synthesis, Jiangsu Key Laboratory For
Carbon-Based Functional Materials & Devices Science, State
and Local Joint Engineering Laboratory for Novel Functional
Polymeric Materials, and a Project Funded by the Priority
Academic Program Development of Jiangsu Higher Education
Institutions.
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New Journal of Chemistry
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View Article Online
DOI: 10.1039/C9NJ01184F
TOC
Highly emissive near-infrared nano-emitters formed by co-assembly of ionic amphiphiles were
applicable in cell imaging.
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