Highly stable and bright fluorescent chlorinated polymer dots for cellular imaging

Article abstract of DOI:10.1039/C8NJ05671D

Chlorinated organic materials have drawn much attention and have been applied in various fields due to their intriguing properties such as easy accessibility with low cost, high capability to hold electron density, and “heavy-atom effect”. In this work, a

Full text of DOI:10.1039/C8NJ05671D

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Highly stable and bright fluorescent chlorinated
polymer dots for cellular imaging†
Cite this:DOI: 10.1039/c8nj05671d
Daize Mo,ab Zhe Chen,c Liang Han,b Hanjian Lai,b Pengjie Chao,b Qingwen Zhang,*a
b
Leilei Tian *c and Feng He
*
Chlorinated organic materials have drawn much attention and have been applied in various fields due to
their intriguing properties such as easy accessibility with low cost, high capability to hold electron
density, and ‘‘heavy-atom effect’’. In this work, a kind of highly fluorescent chlorinated semiconducting
polymer dot (PFDPClBT) is developed for cell imaging by using an amphiphilic polymer poly(styrene-co-
maleic anhydride) as the co-encapsulation matrix. The PFDPClBT dots show a small particle size of
about 50 nm, a large Stokes shift of 125 nm, quite high fluorescence quantum yields (QYs) of 20.3%
(PFDPBT: 8.5%), and increased reactive oxygen species generation efficiency. Since the chlorinated
Pdots are brighter than their non-chlorinated counterparts and can label the target cell effectively with
low cytotoxicity, they are promising fluorescent probes in bioimaging applications.
Received 8th November 2018,
Accepted 8th January 2019
DOI: 10.1039/c8nj05671d
rsc.li/njc
spectra of the resulting organic materials.9 The ‘‘heavy-atom
effect’’ of the large Cl atom is also able to tune the charge-
Introduction
Conjugated polymers (CPs) have been widely applied in various transporting abilities and emissive characteristics of the resulting
fields of organic electronics/optoelectronics, such as organic materials.10 Moreover, the chlorinated molecules are easy to
field effect transistors (OFETs), organic light-emitting diodes access with low cost and chlorine chemistry is more developed
(OLEDs), and organic photovoltaic cells (OPVs).1 Owing to their when compared with fluorine one, which is very favorable
excellent light-harvesting and light-amplifying properties, CPs for their practical applications. Despite these distinguishing
could also be used as a class of promising fluorescent probes in properties, limited studies concerning Cl-containing semi-
biology, chemistry and materials science fields.2 In particular, conductors have been reported, presumably due to the steric
semiconducting polymer dots (Pdots) have attracted consider- hindrance effects originating from the large atomic size of
able attention in the biological field due to their outstanding chlorine which may cause harmful effects to its final properties.
advantages of simple preparation, small size, high brightness,
It has been proved that the chlorinated molecules showed
excellent photostability, and low cytotoxicity.3–8 The versatile similar functions to the fluorinated ones in terms of electron
surface modification also made Pdots expand their applications transport and device stabilities, according to some reported
easily and greatly in various biological fields. Therefore, a results (i.e., the chlorinated PDI and NDI derivatives).11–15
number of Pdots have been successfully applied in biosensing, Stephen R. Forrest et al.16 developed a NIR-absorbing non-
cell imaging, photoacoustic imaging, cancer therapy, etc.
fullerene acceptor with a smaller energy gap (1.3 eV) by
Recently, chlorinated organic materials have drawn increased introducing four Cl atoms, and proved that the chlorination
attention due to their interesting properties and they have been would enhance the intramolecular charge transfer. Hou and
applied in many organic electronics fields. As the second-highest co-workers17 also proved that chlorination was more effective in
electronegative element (Pauling electronegativity for Cl: 3.1614) enhancing the intramolecular charge transfer effect, therefore,
in the halogen group, the chlorine (Cl) atom shows high capability the Cl substituted compounds showed more red-shifted
to hold electron density, which will affect the molecular energy absorption spectra when compared with their fluorinated
levels (tuning the HOMO and LUMO levels) and absorption counterparts. Our group also developed a series of chlorinated
polymers, which displayed deeper HOMO levels and increased
a Institute of Chinese Medical Sciences, University of Macau, 999078, Macao,
open circuit voltage of their devices, and finally the best power
conversion efficiency of 9.11% was achieved.18 All these results
indicate that the chlorinated materials have great potential in
various organic electronics, these interesting researches may
bring the boom prosperity of chlorinated organic materials in
P. R. China. E-mail: qwzhang@umac.mo
b Department of Chemistry, Southern University of Science and Technology,
Shenzhen, 518055, P. R. China. E-mail: hef@sustc.edu.cn
c Department of Materials Science and Engineering, South University of Science and
Technology, Shenzhen, 518055, P. R. China. E-mail: tianll@sustc.edu.cn
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8nj05671d the near future.
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On the other hand, Yang’s previous studies proved that the the Stille coupling reaction was used to synthesize the chlorinated
introduction of halogen atoms into phosphorescent molecules monomers. Similarly, the reactivity of chlorinated monomers
could easily tune their emissive lifetimes and photolumines- may be low, and therefore the corresponding Mn of PFDPClBT
cence quantum yields (QYs).19 Afterwards, they further proved was also low. Both polymers showed good solubility in common
that the halogenation of the electron acceptor groups in ther- organic solvents, such as chloroform, toluene, dichloro-
mally activated delayed fluorescence (TADF) emitters can act as methane, and tetrahydrofuran, which ensured their solution
a simple and effective strategy to shorten the delayed lifetimes processing abilities.
of TADF emitters while improving their QYs.20 Encouraged by
The thermal properties of PFDPClBT and PFDPBT were
these results, we herein present the chlorination of a benzo- measured by thermo-gravimetric analysis (Fig. S1, ESI†) and
thiadiazole (BT)-based polymer as a semiconducting polymer to differential scanning calorimetry (Fig. S2, ESI†), and the results
prepare Pdots (PFDPClBT). It was found that the Cl atom could exhibited their good thermal stabilities. The decomposition
increase the QYs and reactive oxygen species (ROS) generation temperatures (the temperature corresponding to 5% weight
ability of the resulting Pdots when compared with their loss) of the two polymers were determined to be 347.1 and
non-chlorinated counterparts (PFDPBT Pdots). The QYs of 352.7 1C, respectively. Finally, a residual weight percentage of
PFDPClBT Pdots dispersed in water could reach 20.3%, while about 63% at 600 1C for both the polymers was recorded. In
8.5% was determined for non-chlorinated PFDPBT Pdots. addition, from 50 to 250 1C in the DSC curves, we did not
Furthermore, in cellular imaging applications, the chlorinated observe an obvious transition change suggesting that all the
Pdots offered higher brightness than the non-chlorinated polymers were amorphous.
Pdots. All these studies showed that these chlorinated Pdots
The photophysical properties of the PFDPBT and PFDPClBT
were highly fluorescent and demonstrated their promising were studied by UV-vis spectroscopy and photoluminescence
potential in bioimaging applications.
(PL) spectroscopy in dilute THF solutions (Fig. 2a). As shown in
Fig. 2a, the bands at ca. 340 nm and ca. 330 nm in the
absorption spectra of PFDPBT and PFDPClBT, respectively,
were attributed to the fluorene segment and the peaks at about
415 nm (PFDPBT) and 396 nm (PFDPClBT) (Table 1) were
attributed to the benzothiadiazole unit incorporated into the
Results and discussion
Synthesis and characterization
Fig. 1 shows the synthetic routes of monomers and polymers. polyfluorene main chain.22 It was anticipated that the incor-
All of the monomers were characterized by 1H NMR spectroscopy poration of the large chlorine atom could induce a blue-shift
and ESI-MS. 2,1,3-Benzothiadiazole,18 4,7-dibromo-2,1,3-benzo- of the absorption and fluorescence peaks, as the planarity of
thiadiazole, 5-chloro[2,1,3]benzothiadiazole, 4,7-dibromo-5- the p-backbone and the effective conjugation lengths were
chloro-2,1,3-benzothiadiazole,18 4,7-diphenyl-2,1,3-benzothia- decreased by the steric effect of the Cl substituent. This con-
diazole,22 and 4,7-bis(4-bromophenyl)-2,1,3-benzothiadiazole22 clusion was also supported by the decreased QYs for the
were prepared according to the published procedures. 5-Chloro- chlorinated polymer (vide infra). PFDPClBT exhibited a green
4,7-diphenyl-2,1,3-benzothiadiazole was synthesized by a Stille emission, whose PL peak was at 527 nm, showing a slight blue-
coupling reaction of 5-chloro-2,1,3-benzothiadiazole and tribu- shift compared with PFDPBT (530 nm). The relatively large
tylstannyl benzene in toluene using Pd2(dba)3 and P(o-tol)3 as Stokes shifts (120 and 131 nm) and high fluorescence QYs
the catalysts. The bromination of 5-chloro-4,7-diphenyl-2,1,3- (82.6% for PFDPBT and 66.9% for PFDPClBT) of the polymers
benzothiadiazole was conducted under the same conditions as are beneficial to increase the resolution for cell imaging.23
4,7-diphenyl-2,1,3-benzothiadiazole but the reaction time was In addition, the relatively lower QYs of the PFDPClBT were
increased to 48 h.
consistent with the previously reported results due to the
The PFDPClBT and PFDPBT were prepared using the Suzuki structural hindrance of the Cl atom.24
polymerization21 between monomer 4,7-bis(4-bromophenyl)-
The solvatochromism of fluorescence of PFDPBT and
2,1,3-benzothiadiazole/4,7-bis(4-bromophenyl)-5-chloro-2,1,3- PFDPClBT was also studied in various solvents of different
benzothiadiazole and 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxa- polarities (Fig. S3 and S4, ESI†). The emission spectra of PFDPBT
borolan-2-yl)-9,9-dioctylfluorene with a feed ratio of the and PFDPClBT exhibit gradual bathochromic shifts of 51 and
monomers of 50 : 50 and Pd(PPh3)4 as the catalyst. The 76 nm, respectively, from the local excited (LE) state in poorly polar
number-average molecular weights (Mn) of the PFDPClBT hexane to the charge-transfer (CT) state in polar N,N-dimethyl-
and PFDPBT were determined to be 12.01 kDa and 9.58 kDa formamide. The higher red-shifts of the CT states in the PL spectra
with a polydispersity index (PDI) of 1.5 and 1.6, respectively, of PFDPClBT could be attributed to their different dipole moments
characterized by gel permeation chromatography (GPC) with in the CT states caused by the stronger electron-withdrawing effect
polystyrene as the standard. The smaller Mn of the PFDPClBT of the Cl groups.25 In addition, the PFDPClBT possess partially
than that of the PFDPBT may likely be due to the reduced separated HOMOs and LUMOs, which also could make them easily
reactivity of the chlorinated monomers. In the experiments, induce stronger intermolecular charge transfer (Fig. 3), so the
we found that it was difficult to obtain the bis-substituted solvatochromism of PFDPClBT was more obvious.25
products through the Suzuki coupling reaction of 4,7-dibromo-5-
The molecular energy levels of PFDPBT and PFDPClBT were also
chloro-2,1,3-benzothiadiazole and phenylboronic acid. Therefore, investigated by cyclic voltammetry (Fig. S5, ESI†). Two irreversible
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Fig. 1 The synthetic routes for the monomers, polymers, and Pdots.
Fig. 2 (a) UV-vis absorption and PL spectra of PFDPClBT and PFDPBT in THF solutions. (b) UV-vis and PL spectra of PFDPBT Pdots and PFDPClBT Pdots
in water.
1eꢀ reduction steps were both observed for the two polymers, and compared with the H-substituted PFDPBT (ꢀ6.26 eV) due to the
the HOMO energy levels of PFDPBT and PFDPClBT determined substitution of electron-withdrawing Cl atoms, which was similar
from their oxidation onset potentials (Eox) were ꢀ6.26 and ꢀ6.29 eV to our reported results. To gain deep insights into the electronic
(EHOMO = ꢀ(4.80 + Eox)), respectively. It was expected that the properties of the polymers, density functional theory (DFT) calcula-
HOMO of the PFDPClBT (ꢀ6.29 eV) decreased to a lower level tions were carried out employing Gaussian 0926 software with the
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Table 1 Summary of size and photophyscial properties of the copolymer in THF solutions and its Pdot form in water
a
b
a
b
Copolymers
labs,max(THF) (nm)
lFL,max(THF) (nm)
labs,max(Pdot) (nm)
lFL,max(Pdot) (nm)
F(%)
Size (nm)
PFDPBT
PFDPClBT
415
396
535
527
422
405
530
530
82.6c (8.5d)
66.9 (20.3)
72.5e
75.2
a
b
c
d
e
Absorption maximum. Fluorescence maximum. Quantum yield in THF solutions. Quantum yield in Pdot form. Hydrodynamic size
measured by DLS.
Fig. 3 Minimized energy ground state geometries and the frontier molecular orbital electron density distributions of PFDPBT and PFDPClBT.
B3LYP functional at the 6-311G(d,p) basis set level. To simplify the and PFDPClBT Pdots in aqueous solutions were determined to
calculations, two repeating units of the polymers were used and be ꢀ27.3 mV and ꢀ18.1 mV, respectively (Fig. 4d). These Pdots
alkyl groups were replaced by methyl groups. As illustrated in Fig. 3, are colloidally stable, with no obvious signs of further aggre-
the dihedral angle between the BT unit and the phenyl unit (near gation and noticeable leaching from nanoparticle storage for
the Cl atom) of PFDPClBT was 54.001, whereas the corresponding several months.28
angle of PFDPBT was 17.991. The larger dihedral angle of
Fig. 2b shows the absorption and PL spectra of PFDPBT and
PFDPClBT should be attributed to the stronger steric hindrance, PFDPClBT Pdots. The charge transfer (CT) transition in the
which was caused by the introduction of the large Cl atom. The long wavelength region for PFDPBT Pdots was located at
calculated HOMO of PFDPClBT (ꢀ5.02 eV) was also a little lower 422 nm, while the CT peak for the PFDPClBT Pdots displayed
than that of PFDPBT (ꢀ4.90 eV), probably owing to the inductive a blue-shift of 17 nm (405 nm) owing to the introduction of
electron-withdrawing characteristic of the Cl atom. The highest chlorine atoms.21 As expected, the absorption spectra of both
occupied molecular orbital (HOMO) and the lowest unoccupied PFDPBT Pdots and PFDPClBT Pdots in water showed an
molecular orbital (LUMO) of PFDPClBT were partially separated, obvious red-shift compared with those in their THF solutions
which provided the respective channels for the transport of holes attributed to the molecular aggregations during the process in
and electrons.25 On the other hand, the LUMO position of both forming Pdots. To our surprise, the emission peaks of the
PFDPClBT and PFDPBT was mainly dependent on the acceptor PFDPBT Pdots and PFDPClBT Pdots nearly coincided, which
unit (BT-based acceptor group).
were centered at 530 nm. The absolute QYs for the PFDPBT
Pdots and PFDPClBT Pdots were measured to be 8.5% and
20.3%, respectively. These results indicated that the formed
Pdots preparation and properties
The two kinds of Pdots were made through rapidly co-precipitating Pdots still retained adequate fluorescence intensities, so they
the conjugated polymers of PFDPBT (or PFDPClBT) and poly have potential for applications in cell imaging. Table 1 sum-
(styrene-maleic anhydride) (PSMA). Upon precipitation in deionized marizes the photophysical properties of PFDPBT Pdots and
water, the polymer backbone folded and distorted due to hydro- PFDPClBT Pdots, which also clearly indicates that PFDPClBT
phobic interactions. As we know, PSMA almost has no influence on Pdots exhibit higher fluorescence brightness.
the molar absorption coefficient of polymers and has no emission.
The copolymers PFDPBT and PFDPClBT in THF solutions
The hydrophilic maleic anhydride units in PSMA with spontaneous showed QYs of 82.6% and 66.9%. However, when the two types
hydrolysis of maleic anhydride groups will be most likely localized of Pdots blended with PSMA, the QYs of the Pdots displayed a
on the surface of the Pdots. The TEM images showed that both the remarkable difference as discussed above (PFDPBT Pdots:
Pdots exhibited spherical morphology with an average diameter of 8.5%, PFDPClBT Pdots: 20.3%). According to the previously
50 nm (Fig. 4a and b). The mean hydrodynamic diameters of reported results,21 we concluded that the hydrophilic group in
PFDPBT Pdots and PFDPClBT Pdots calculated by dynamic light PSMA played a dominant role in causing the large QY difference
scattering (DLS) were 72.5 nm and 75.2 nm, respectively (Fig. 4c). between the different types of Pdots. The lower QYs of PFDPBT
The particle size given by TEM was smaller than that by DLS, most Pdots were likely due to PFDPBT chains being aggregated or
likely due to the hydrated Pdots facing shrinkage during TEM stacked more densely than when Pdots were formed, leading to
sample preparation.27 The zeta-potentials of PFDPBT Pdots great aggregation quenching. In PFDPClBT Pdots, the reduced
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Fig. 4 TEM images of (a) PFDPBT dots and (b) PFDPClBTdots; (c) DLS data and (d) zeta potentials of the PFDPBT dots and PFDPClBTdots.
aggregation-induced quenching tendency was likely due to the suitable for cell imaging, as the higher brightness improves
following two factors: (1) the intermolecular interactions of the imaging quality.29
Cl–S and Cl–Cl may result in a rigid and extended polymer
Reactive oxygen species detection
chain; (2) strong hydrophobic and large structural hindrances
of the Cl atoms would make PFDPClBT Pdots stacked loosely
when compared with PFDPBT Pdots.21
Due to the so-called heavy atom effect, the chlorine group
introduced in PFDPClBT Pdots may enhance the singlet oxygen
generation (ROS) by spin orbit coupling.30 Herein, to investi-
gate whether PFDPClBT Pdots were able to generate ROS, a
Cell imaging of the Pdots
To further demonstrate the biological applicability of the Pdots 20,70-dichlorofluorescin diacetate (DCFH) probe was selected as
for cellular imaging, we first evaluated the cytotoxicity of the the ROS indicator. As shown in Fig. 6, it was found that the
two kinds of Pdots by using the MTT method. The results show mixed aqueous solutions of DCFH/PFDPClBT Pdots reveal
that these two Pdots exhibited similar biological toxicity. The increased PL intensities when exposed to white light irradiation
A549 cells grew normally in the culture medium containing (40 mW cmꢀ2) within 20 minutes (I/I0: 1–4.06), while DCFH
PFDPBT Pdots or PFDPClBT Pdots in the concentration range (I/I0: 1–0.94) and DCFH/PFDPBT Pdots (I/I0: 1–1.04) show
of 5–80 mg mLꢀ1 for 48 h, indicating almost no cytotoxicity of almost negligible fluorescence. All these results demonstrated
the Pdots (Fig. 5a and b). We next used them to incubate with the stronger singlet oxygen generation ability of PFDPClBT
A549 cells via endocytosis for 4 h. The confocal fluorescence Pdots than PFDPBT Pdots and may find applications in photo-
microscopy images distinctly revealed that the two kinds of dynamic therapy in the future.
Pdots had got into the cells and accumulated. Both of the Pdots
In addition, a comparison of the ability of generating
were preferentially stained in the cytoplasm regions of the reactive oxygen species of PFDPClBT Pdots with other photo-
A549 cells rather than in their nucleic parts. As expected, all sensitizers has been made. It shows that although the ability of
the A549 cells stained by the two kinds of Pdots showed high generating reactive oxygen species of PFDPClBT Pdots was
green labeling brightness, which is enough demand for their higher than that of PFDPBT Pdots, it is still not competitive
application in high quality cell imaging. The A549 cells when compared with the reported values (PFVBT Pdots
labeled with PFDPClBT Pdots also exhibited much higher (I/I0: 1–300),31 TPE-Py-FFGYSA photosensitizer (I/I0: 1–260),32
emission intensity than the cells labeled with PFDPBT Pdots UCNP/PFVCN nanohybrids (I/I0: 1–400),33 TPA-BDTO NPs
under identical experimental conditions (Fig. 5c and d). The (I/I0: 1–80)),34 which show emission in the red/near red region.
cellular imaging indicates that the PFDPClBT Pdots are more In general, far-red and near-IR emitting conjugated polymers/
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Fig. 5 The cell viability of A549 cells in the culture of different concentrations of (a) PFDPBT Pdots and (b) PFDPClBT Pdots after incubation for 48 h.
Fluorescence imaging of A549 cells incubated with 40 mg mLꢀ1 (c) PFDPBT Pdots and (d) PFDPClBT Pdots for 4 h. From left to right are bright field
images, fluorescence images, and combined bright field and fluorescence images (scale bar equals 50 mm).
PFDPClBT Pdots may significantly improve the production of
singlet oxygen generation.36
Photostability of Pdots
To further confirm their potential as effective fluorescent
probes, their photostabilities with fluorescein sodium in water
were also investigated through 50 min of irradiation with a
xenon lamp (100 mW cmꢀ2). We chose fluorescein sodium as
control mainly for the following reasons: (1) fluorescein sodium
is one of the most stable commercially available fluorescent
organic dyes widely used for bioimaging;37,38 (2) it has a similar
excitation wavelength to PFDPClBT Pdots and PFDPBT Pdots
and very similar emission peaks. As illustrated in Fig. 7, it can
be seen that the extent of degradation of UV absorbance of the
Fig. 6 Plots of I (emission intensity)/I0 (emission intensity at 0 min) of the
DCFH/PFDPClBT Pdots, DCFH/PFDPBT Pdots, and DCFH versus different
two Pdots is very small, and the degradation degree of fluores-
cence is also much lower when compared with fluorescein
sodium. At the same time, we can also conclude that chlori-
light irradiation times.
small molecules can effectively generate ROS and can act as PSs nated PFDPClBT Pdots show better stability than PFDPBT
directly without the mediation of any other chromophores.35 Pdots. The retention of the original absorbance was 92.6%
Incorporation of photosensitizer (generally porphyrin) into the for PFDPClBT Pdots after 50 minutes; 82.8% and 6.8% were
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Fig. 7 (a) Time-dependent photodegradation (absorption intensity) and (b) photobleaching (fluorescence intensity) of the PFDPClBT Pdots and PFDPBT
Pdots compared with fluorescein sodium after 50 min of irradiation.
retained for PFDPBT Pdots and fluorescein sodium, respec- Experimental section
tively (Fig. 7a). On the other hand, the fluorescence intensity of
the PFDPClBT Pdots decreased to 62.1% after 50 min (52.4%
Chemicals and reagents
2,7-Bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-9,9-dioctyl-
for PFDPBT Pdots), and the fluorescein sodium sharply
fluorene, 4-chloro-1,2-diaminobenzene, 1,2-diaminobenzene,
decreased to 6.9% (Fig. 7b). This result was very similar to
pyridine, thionyl chloride (SOCl2), bromine (Br2), N-bromo-
the results of PFBT Pdots reported by Chiu and co-workers.39
succinimide (NBS), iodine (I2), 20,70-dichlorofluorescin diacetate
In addition, the Pdots also exhibited greatly improved photo-
(DCFH-DA), tributylstannyl benzene, phenylboric acid, tetra-
stability over many other fluorescent probes such as Au
kis(triphenylphosphine)palladium(0) (Pd(PPh3)4), and hydrobromic
nanoclusters and CdTe QDs.40
acid (HBr) were purchased from Energy Chemical (Shanghai,
China). Potassium carbonate (K2CO3) and sodium carbonate
(Na2CO3) were obtained from Damao Chemical Reagent
Conclusions
Company (Tianjin, China). Tris(dibenzylideneacetone)di-
palladium(0) (Pd2(dba)3), tri-o-tolylphosphine (P(o-tol)3), and
In summary, we synthesized a novel fluorescent chlorinated
poly(styrene-co-maleic anhydride) (PSMA, average Mw = 1700,
semiconducting polymer named PFDPClBT. The Pdots were
the styrene content was about 68%) were purchased from
prepared by the nanoprecipitation method with an amphiphilic
Sigma-Aldrich. Tetrahydrofuran (THF) and toluene were dis-
polymer PSMA as the co-encapsulation matrix. TEM measure-
tilled under nitrogen protection from Na/benzophenone before
ments indicated that both PFDPClBT Pdots and PFDPBT Pdots
reaction. Other solvents and reagents were used directly as
were spherical with an average diameter of about 50 nm. The
received. Human alveolar epithelial cells (A549) were kindly
absorption peak of PFDPClBT Pdots shows a blue-shift com-
pared with that of non-chlorinated PFDPBT Pdots, which is
caused by the incorporation of the large chlorine atom and the
provided by Professor Ying Sun at the Department of Biology of
Southern University of Science and Technology (SUSTech).
steric effect of the Cl substituent that reduces the planarity of
the p-backbone. This conclusion is also supported by the
Synthetic procedures
decreased QYs of the chlorinated polymer (82.6% for PFDPBT
Preparation of 4,7-diphenyl-2,1,3-benzothiadiazole. 4,7-Dibromo-
and 66.9% for PFDPClBT) in THF solutions. However, the 2,1,3-benzothiadiazole (4.93 g, 16.43 mmol), potassium carbonate
PFDPClBT Pdots have a QY as high as 20.3%, which is much (27.25 g, 197.2 mmol), Pd(PPh3)4 (0.759 g, 0.6572 mmol), and
higher than the QY of PFDPBT Pdots (8.5%). The cell imaging phenylboric acid (4.24 g, 34.84 mmol) were added into a 250.0
study also shows that the brightness of the cells labeled with mL round bottom flask. H2O (69 mL) and toluene (350 mL)
PFDPClBT Pdot probes is higher when compared with those were syringed at once. The flask was cooled by liquid nitrogen
labeled with PFDPBT Pdots. The chlorine atom introduced in and blanked by argon three times, then heated at 90 1C for 24 h.
PFDPClBT Pdots also enhanced the ability of generating reac- After cooling to room temperature, the same volume of water
tive oxygen species and improved the photostability. All these was added to the reaction flask and then extracted with
data show that the introduction of the large chlorine atom onto dichloromethane (DCM) several times. The organic layer was
the polymer backbone would result in a significant difference washed with saturated aqueous solution of NaCl and dried with
in the photophysical and biological properties of the corres- anhydrous sodium sulfate (Na2SO4). After the solvent was
ponding Pdots, making them promising probes for biological evaporated by rotary distillation under reduced pressure, the title
detection.
compound (yellow solid) was obtained by column chromatography
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(petroleum ether–DCM (5 : 1, v/v)). Yield: 4.62 g (98.3%). H NMR
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1
PFDPClBT. PFDPClBT was prepared by a similar method used
(500 MHz, CDCl3) d: 7.99 (d, 4H, J = 8 Hz), 7.82 (s, 2H), 7.59 (t, 4H, to prepare PFDPBT using monomers 4,7-dibromo-5-chloro-2,1,3-
J = 8 Hz), 7.52 (d, 2H, J = 8 Hz). ESI-MS: C18H12N2S calcld 288.0721, benzothiadiazole and 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxa-
found. 288.0742.
borolan-2-yl)-9,9-dioctylfluorene. Yield: 84.9%. 1H NMR (500 MHz,
Preparation of 4,7-bis(4-bromophenyl)-2,1,3-benzothiadiazole. CDCl3) d: 8.14 (br, 2.81H), 7.99 (br, 1H), 7.93 (br, 6.83H), 7.72
A solution of Br2 (10 mL, 194 mmol) in chloroform (10 mL) was (br, 6.58H), 7.61 (br, 3.40H), 2.12 (br, 4.94H), 1.14 (br, 26.16 H),
added slowly into a 250 mL round bottom flask with 30 mL 0.84 (br, 13.08 H). Mn (GPC): 9.58 k, Ð: 1.33.
chloroform with dissolved iodine (0.12 g, 0.47 mmol) and
Measurements
4,7-diphenyl-2,1,3-benzothiadiazole (2.39 g, 8.3 mmol). After
stirring at room temperature for 12 h, the reaction mixture was 1H and 13C NMR spectra were obtained using a Bruker Avance-
quenched with saturated aqueous Na2SO3 solution. The solid 500 (500 MHz) spectrometer. The molecular weight and molecular
was precipitated in water, filtered, and the cake was washed weight distribution of the synthesized polymers were measured
with water and methanol sequentially to give the crude product. with gel permeation chromatography (GPC) at 40 1C on a Malvern
Recrystallization from toluene gave the final product as yellow Viscotek 270 max system equipped with a UV detector, using
needles. Yield: 2.88 g (78%).1H NMR (500 MHz, CDCl3) d: 7.87 polystyrene (Aldrich) as the standard and THF as the eluent. The
(d, 4H, J = 8 Hz), 7.80 (s, 2H), 7.70 (d, 4H, J = 8 Hz). ESI-MS: UV-vis spectra were obtained using a Shimadzu UV-3600 PC by
C
19H14Br2N2S calcld 459.9244, found. 459.9267.
using 1 cm quartz cuvettes. The fluorescence spectra of the
Preparation of 5-chloro-4,7-diphenyl-2,1,3-benzothiadiazole. polymers in THF solution and Pdots in aqueous solution were
4,7-Dibromo-5-chloro-2,1,3-benzothiadiazole (2.10 g, 6.40 mmol), obtained using a Shimadzu RF5301 PC fluorescence spectrometer.
tributylstannyl benzene (5.71 g, 15.69 mmol), Pd2(dba)3 (290.9 mg), The absolute QYs were measured using an integrating sphere
and P(o-tol)3 (581.8 mg) were added to a 250.0 mL round bottom system, and were determined on a Horiba Jobin Yvon-Edison
flask. The flask was blanked by argon three times and 100 mL of Fluoromax-4 fluorescence spectrometer. Time-dependent photo-
toluene was syringed at once and the reaction mixture was stirred degradation and photobleaching experiments were carried out
at 100 1C for 12 hours. After cooling to room temperature, the using a xenon lamp(100 mW cmꢀ2). The fluorescence of DCF was
organic solvent was removed under reduced pressure. The title excited at 488 nm and collected within 500–545 nm. Particle sizes
compound was obtained by column chromatography (1.89 g, were measured using a Hitachi H-600 transmission electron
1
92.1%) as a light yellow solid. H NMR (500 MHz, CDCl3) d: 7.98 microscope (TEM) and a NanoBrook Omni dynamic light
(d, 2H, J = 8 Hz), 7.82 (s, 1H), 7.50–7.61 (m, 8H). ESI-MS: scattering instrument (DLS, Brookhaven Instruments Corporation).
C18H11ClN2S calcld 322.0311, found. 322.0352.
For the TEM measurements, the Pdot solution (about 20 mL) was
Preparation of 4,7-bis(4-bromophenyl)-5-chloro-2,1,3-benzo- placed on a carbon-coated copper grid. After evaporation of the
thiadiazole. 4,7-Bis(4-bromophenyl)-5-chloro-2,1,3-benzothia- water at room temperature, morphology of the Pdots was measured
diazole was synthesized in a similar manner to 4,7-bis(4- using a Hitachi H-600 instrument.
bromophenyl)-2,1,3-benzothiadiazole, and the reaction time
was increased to 48 h. Yield: 1.82 g (78%). 1H NMR (500 MHz,
Preparation of Pdots
CDCl3) d: 7.87 (m, 3H), 7.79 (d, 2H, J = 8 Hz), 7.50–7.63 (m, 4H). PFDPBT and PFDPClBT were firstly dissolved in THF at a
ESI-MS: C18H9Br2ClN2S calcld 493.8855, found. 493.8873.
concentration of 1000 mg mLꢀ1, respectively. At the same time,
the same concentration of PSMA (1000 mg mLꢀ1) in THF was
also prepared. 500 mL PFDPBT solution or PFDPClBT solution,
General procedure of Suzuki polymerization
PFDPBT. 2,7-Bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)- 100 mL PSMA, and 9400 mL THF were mixed together to form a
9,9-dioctylfluorene (120 mg, 0.19 mmol), 4,7-bis(4-bromo- homogeneous solution. Under ultrasound conditions, 5 mL of
phenyl)-2,1,3-benzothiadiazole (85.15 mg, 0.19 mmol), Pd(PPh3)4 the mixed solution was quickly injected into 10 mL of deionized
(6 mg, 0.005 mmol), and Bu4NBr (2.45 mg, 0.0076 mmol) were water. The excess THF was removed by heating under nitrogen
added into a 25 mL flask. Then, 4 mL of toluene and 2 mL of stripping and the solution was concentrated to about 5 mL.
aqueous Na2CO3 (2 M) were added using a syringe. The flask was After that, a 0.22 micron filter was used to filter the solution to
frozen by liquid nitrogen, degassed, and refilled with argon three remove the fraction of aggregates. The size and morphology of
times. The mixture was heated to 90 1C with vigorous stirring for the Pdots were characterized by TEM and DLS and stored in a
two days under an argon atmosphere. After cooling to room refrigerator (4 1C) until further use.41
temperature, the mixture was re-precipitated into 100 mL
methanol, then filtered and washed with methanol, acetone and
Cytotoxicity studies
distilled water in turn. Then, the crude product was dissolved The cytotoxicities of PFDPBT Pdots and PFDPClBT Pdots were
in 15 mL THF, filtered through a 0.22 mm micron filter and measured by MTT assay. First, A549 cells were seeded in 96-well
re-precipitated in 100 mL methanol. The powder was stirred plates at a number of 5 ꢁ 103 cells per well and incubated for
in acetone for 24 hours, filtered and dried in a vacuum oven 24 h and 48 h (37 1C, 5% CO2). The cells were then grown in the
1
(102.7 mg, 79.5%). H NMR (500 MHz, CDCl3) d: 8.15 (br, 4H), medium at a series of concentrations of Pdots (0, 5, 10, 20, 40,
7.92 (br, 9.52H), 7.71 (br, 5.31H), 7.73 (br, 1.35H), 2.10 (br, 4.48H), and 80 mg mLꢀ1). After 24 hours, 20 mL of MTT (5 mg mLꢀ1 in
1.14 (br, 24.67H), 0.84 (br, 12.53 H). Mn (GPC): 12.01 k, Ð: 2.03.
PBS) was added, the culture medium was removed carefully,
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Paper
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Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (51773087, 21733005, 51503096,
51603100), the Natural Science Foundation of Guangdong
Province (2016A030313637), Shenzhen Fundamental Research
program (JCYJ20160504151536406, JCYJ20160504151731734),
Shenzhen Nobel Prize Scientists Laboratory Project (C17783101),
the Research Committee of the University of Macau (MYRG2016-
00046-ICMS-QRCM), Guangzhou Science and Technology,
and the Guangzhou International Science and Technology
Collaboration Funding from the Guangzhou Science Technology
and Innovation Commission (R&D of cephalotaxine-type compounds
as new anti-chronic myeloid leukemia drugs, EF010/ICMS-ZQW/
2018/GSTIC).
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