Design guidelines for conjugated polymers with light-activated anticancer activity

Article abstract of DOI:10.1002/adfm.201100840

Multifunctional materials that simultaneously provide therapeutic action and image the results provide new strategies for the treatment of various diseases. Here, it is shown that water soluble conjugated polymers with a molecular design centered on the p

Full text of DOI:10.1002/adfm.201100840

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Design Guidelines For Conjugated Polymers With Light-
Activated Anticancer Activity
Chengfen Xing, Libing Liu,* Hongwei Tang, Xuli Feng, Qiong Yang, Shu Wang,*
and Guillermo C. Bazan*
aqueous media, an important requirement
for interacting with biological systems and
for the design of optically amplified fluo-
rescent biosensors.[2]
Multifunctional materials that simultaneously provide therapeutic action
and image the results provide new strategies for the treatment of various
diseases. Here, it is shown that water soluble conjugated polymers with a
molecular design centered on the polythiophene−porphyrin dyad are effective
for killing neighboring cells. Following photoexcitation, energy is efficiently
transferred from the polythiophene backbone to the porphyrin units, which
readily produce singlet oxygen (1O2) that is toxic for the cells. Due to the
light-harvesting ability of the electronically delocalized backbone and the effi-
cient energy transfer amongst optical partners, the polythiophene−porphyrin
dyad shows a higher 1O2 generation efficiency than a small molecule analog.
The fluorescent properties of these polymers can also serve to distinguish
amongst living and dead cells. Polymers can be designed with folic acid
grafted onto the polymer side chain that can specifically kill folate receptor-
overexpressed cells, thereby providing an important demonstration of anti-
cancer specificity through molecular design.
Acceptor molecules can be chosen
with optoelectronic properties that opti-
mize FRET efficiencies.[3,4] Water soluble
conjugated polymer/acceptor dyads have
therefore gained much recent attention in
various applications for detection and cell
imaging.[5–10] Energy transfer to porphyrin
acceptors is a special example whereby the
excitations can be harnessed to generate
reactive oxygen species[11–14] and enable
applications in photodynamic therapy
(PDT).[15] Such materials can be expected
to act as both therapeutic and imaging
agents. The imaging function is particu-
larly significant when it is capable of moni-
toring and guiding treatment. Integration
of reporting modalities with therapeutic
1. Introduction
function within a single bio-compatible polymer structure is
therefore anticipated to increase in importance as a new and
challenging design element for new artificial materials.[16,17]
Here, we describe the synthesis and application of polymers
containing conjugated polythiophene−porphyrin dyads that
were specifically designed to function concurrently as an anti-
cancer agent and an imaging reporter. Figure 1 shows the first
example, PTP, which relies on a cationic polythiophene frame-
work. Four significant design characteristics were included in
the molecular features. First, the fractional content of porphyrin
moieties linked to the polythiophene backbone is low (≈1%), in
order to minimize toxicity in the absence of photoexcitation.
Second, the amphiphilic attributes were anticipated to promote
adsorption to cells through a combination of electrostatic and
hydrophobic binding forces. Third, covalent attachment of por-
phyrin moieties to the light harvesting polythiophene backbone
constrains interchromophore distances for optimizing FRET.
This design element is important for increasing the photocover-
sion efficiency of singlet oxygen (1O2) generation and reducing
light intensity requirements. Fourth, because the backbone
retains partial emission, it can also be used to monitor apop-
tosis and necrosis processes by fluorescence imaging, adding
a new dimension to the function of the molecular construct.
Such simultaneous imaging and PDT function[18,19] extend
the applications of water-soluble conjugated polymers beyond
their established biosensing capabilities. The PTP platform can
Conjugated polymers are characterized by
a delocalized
π-electronic backbone structure and large optical absorption
coefficients. Facile energy transfer along the backbone and
between chains allows excitations to sample a larger number
of environments in comparison to isolated small molecules.[1]
Photoexcitations can be channeled to suitable adjacent accep-
tors by fluorescence resonance energy transfer (FRET) mecha-
nisms, leading to sensitization of emitters to levels above those
attained by direct excitation. Conjugated polyelectrolytes (CPEs)
incorporate charged groups onto the conjugated polymer back-
bone. Such a structural modification improves solubility in
C. Xing, Dr. L. Liu, H. Tang, X. Feng, Dr. Q. Yang, Prof. S. Wang
Beijing National Laboratory for Molecular Science
Key Laboratory of Organic Solids, Institute of Chemistry
Chinese Academy of Sciences
Beijing, 100190, P. R. China
E-mail: liulibing@iccas.ac.cn; wangshu@iccas.ac.cn
Prof. G. C. Bazan
Departments of Chemistry & Biochemistry and Materials
Center for Polymers and Organic Solids
University of California
Santa Barbara, CA 93106-9510, USA
E-mail: bazan@chem.ucsb.edu
DOI: 10.1002/adfm.201100840
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Figure 1. Chemical structure of PTP and a schematic mechanism of PTP anticancer activity under irradiation. Components in the cell are not drawn
to scale.
Subsequent conversion to 6-(2-(thiophen-3-yl)ethoxy)hexyltri-
methylammonium bromide (9) is accomplished in 89% yield by
reaction with excess trimethylamine in methanol. The cationic
polythiophene PT was obtained through oxidative polymeriza-
tion of 9 in chloroform using FeCl3, followed by purification by
dialysis in water. Monomers 6 and 9 undergo oxidative copoly-
merization under nitrogen in the presence of FeCl3 to give the
target cationic porphyrin-containing polythiophene (PTP). The
molar feed ratio of monomer of 6 to 9 is 1:10 and the actual
porphyrin content in PTP was determined to be ∼1% by exami-
nation of the absorption spectrum (Figure S2 in the Supporting
Information).
The photophysical properties of PTP, PT and TPN were
investigated in aqueous solution, and the most relevant results
are shown in Figure 3a and 3b. For TPN, the absorption spec-
trum exhibits a Soret band at 420 nm and Q bands between
520 nm and 670 nm; the emission spectrum shows a maximum
peak at 655 nm with a fluorescence quantum yield (QY) of 1%.
For PT, the absorption maximum is at ≈420 nm, corresponding
to the π−π∗ transition of the backbone; emission displays a
maximum at 578 nm and occurs with a QY of 2%. The absorp-
tion spectrum of PTP incorporates features common to the
be further modified with molecular fragments that endow the
materials with specificity (vide infra).
2. Results and Discussions
Figure 2 shows the synthesis of PTP; complete details can be
found in the Supporting Information. Reaction of pyrrole,
4-(6-bromohexyloxy)benzaldehyde (1)[20] and p-tolualdehyde
(2) in the presence of BF3-diethyl ether in CHCl3, followed by
treatment with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ)
affords the porphyrin derivative 3. Treatment of 3 with 30%
trimethylamine-methanol solution provides the cationic por-
phyrin derivative TPN. The quaternary ammonium side chain
of TPN improves the polarity and water solubility, and thus TPN
is soluble in water. This was proved by the linear plot of TPN
absorbance as a function of various concentrations (Figure S1).
Monomer 6 was prepared by reaction of 2-(3-thienyl)-ethanol
tosylate (4) and hydroquinone, followed by reaction with 3.
Reaction of 2-(thiophen-3-yl)ethanol (7) with 1,6-dibromohexane
in the presence of sodium hydride in dry DMF under nitrogen
affords 3-(2-(6-bromohexyloxy)ethyl)thiophene (8) in 31% yield.
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CHO
CHO
O
NH
N
N
NH
N
N
pyrrole
N(CH3)3
HN
HN
.
BF3 Et2O
DDQ
2
Br
1
O(CH2)6Br
O(CH2)6NMe3 Br
TPN
3
OH
O
HN
N
N
HO
OH
S
O
O
3
O
O
O
K2CO3, DMF
NH
K2CO3, acetone
O
S
S
5
4
6
S
Br
N
N
BrH
BrH
O
OH
O
O
Br
FeCl3
N(CH3)3
CH3OH
Br
NaH, DMF
CHCl3
S
S
S
S
n
9
PT
8
7
FeCl3
CHCl3
9
6
PTP
Figure 2. Synthetic preparations of TPN, PT, and PTP.
backbone and the porphyrin units. Excitation of PTP at 470 nm,
where the porphyrin units do not absorb, leads to emission with
peaks at 578 nm and 658 nm with a QY of 2%. That a peak is
observed at 658 nm demonstrates efficient energy transfer from
polythiophene units to the porphyrin sites. It is also relevant to
note at this point that the hydrophobic component of the PTP
structure leads to aggregation in aqueous solution. Dynamic
light scattering (DLS) measurement shows that the average
particle size is approximately 350 nm (Figure 3c). The size of
these nanoparticles is in the appropriate range for improved
cell uptake.[21,22]
Relative 1O2 photogeneration efficiencies by the different
photosensitizers were probed by measuring the conversion
of disodium 9,10-anthracenedipropionic acid (ADPA)[23] to its
endoperoxide via the corresponding decrease of the absorp-
tion band at 378 nm. PTP, TPN and PT were compared upon
exposure to white light (400–800 nm), and the results are sum-
marized in Figure 3d. In these experiments, the concentrations
of PT and PTP were 10 μM in terms of polymer repeat units
(RUs), and the concentration of TPN (0.15 μM) was similar to
that of the pendant porphyrin units in the PTP solutions. The
first order plots of the decrease of ADPA absorption at 378 nm
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a
PTP
b
1.0
PT
1.0
0.8
0.6
0.4
0.2
0.0
PT
TPN(ex:420 nm)
PTP
TPN
0.8
0.6
0.4
0.2
0.0
300
400
500
600
700
550
600
650
700
750
800
Wavelength (nm)
Wavelength (nm)
30.0 c
25.0
20.0
15.0
10.0
5.0
d
4.0
PTP
3.0
2.0
1.0
0.0
PT
TPN
0.0
10
100
0.0
1.0
2.0
3.0
4.0
5.0
1000
10000
Size (d.nm)
Irridiation Time (min)
1
Figure 3. Photophysical properties of PTP, PT and TPN, structural characterization of PTP aggregation in water, and comparison of O2 generation
efficiencies. a) Normalized UV–vis absorption spectra of PTP, PT and TPN in aqueous solution. b) Normalized emission spectra of PTP, PT and TPN
in aqueous solution. PT and TPN were excited at 420 nm and PTP at 470 nm. c) Dynamic light scattering analysis of PTP aggregates in aqueous solu-
tion. d) Decrease of ADPA absorption at 378 nm as a function of light irradiation time in the presence of TPN, PT and PTP in D2O solution. [PTP] =
[PT] = 10 μM in RUs, [TPN] = 0.15 μM and [ADPA] = 120 μM. A0 is the absorbance of ADPA at 378 nm before irradiation, and A is that after irradiation
with white light (400–800 nm). Values represent subtraction of the residual bleaching data of ADPA alone under the same irradiation condition.
as a function of irradiation time[14] show faster disappearance
upon photoexcitation of PTP. The observed rate constants of
ADPA consumption in the presence of PTP, PT and TPN are
1.25 × 10−2 , 2.7 × 10−3 and 7.3 × 10−5 s−1 , respectively. Thus, the
bleaching rate of ADPA with PTP is 170 fold faster than that
with TPN and ≈5 fold faster than that with PT. As control exper-
iment, the production of singlet oxygen by a sample containing
both PT and TPN with concentrations matching those of the
PTP sample has been evaluated (Figure S3). The observed rate
constants of ADPA consumption is 1.6 × 10−3 s−1 , one order
of magnitude less than that for PTP. Covalent attachment of
porphyrin moieties onto PTP thus yields a synergistic improve-
ment relative to the isolated PT or TPN components, and are
consistent with energy transfer from the polythiophene back-
bone to the porphyrin sites, which significantly increases the
1O2 generation efficiency (see also Figure S4 in the Supporting
Information).
fluorescence microscopy by excitation at 470 nm to selectively
excite the polythiophene backbone. As shown by the phase con-
trast bright-field images in Figure 4a, irradiation leads to cell
morphology changes that include chromatin compaction, cyto-
plasm condensation, and, more significantly, a large amount
of blebbing. After 30 min irradiation, whole-cell shrinkage
takes place. Persistent volume reduction is a major hallmark
of cell death.[25,26] These results are corroborated by the over-
lapping fluorescence images of A498 cells in the presence of
PTP and EB, where the amount of EB-stained dead cells has
increased in tandem with prolonged irradiation time. As shown
in Figure 4b, imaging the location of PTP emission can be used
to distinguish between living and dead cells. In living cells, the
PTP emission is largely located in the cytoplasm. For dead cells,
one observes translocation such that the PTP fluorescence now
takes place within the nucleus. Correlation with the fluores-
cence from EB-stained cells confirms assignment of dead cells.
Cytotoxicities of PTP, PT and TPN toward the A498 and A549
cancer cells were determined by using a standard assay in which
the conversion of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
2H-tetrazolium hydrobromide) into formazan is related to mito-
chondrial activity and thereby cell viability.[27] The PDT treatment
was performed by illumination at 470 nm for 30 min. As shown
in Figure 4c, under illumination, PTP displays more prominent
cytotoxicity than PT at similar repeat unit concentrations, and
TPN (normalized to the molar quantity of pendant porphyrin
units). Also noteworthy is that PTP exhibits very low dark cyto-
toxicity, which is similar to PT and TPN (Figure S5). Moreover,
1
The ability of PTP to generate O2 was anticipated to induce
oxidative stress and trigger apoptosis, necrosis or autophagy-
associated cell death.[24] Two kinds of cancer cells, pulmonary
adenocarcinoma cell (A549) and renal cell carcinoma (A498),
were used as initial model targets. The morphologies of A498
cancer cells in the presence of PTP after different periods of
illumination were examined by phase contrast and fluorescence
microscopy. For these experiments, plates containing A498 cells
were irradiated at 470 nm for 0, 10, and 30 min, respectively,
ethidium bromide (EB), which only stains apopotic or dead
cells, was then added. Resulting samples were examined by
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c
120.0
100.0
80.0
60.0
40.0
20.0
0.0
100.0
90.0
80.0
70.0
60.0
50.0
40.0
30.0
100.0
90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
PT
PTP
A498
A498
0.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0
2.0
4.0
6.0
8.0 10.0 12.0
0.0 5.0 10.0 15.0 20.0 25.0 30.0
Time (min)
[PT or PTP] ( M)
µ
[TPN] (10-8M)
110.0
100.0
90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
PT
PTP
A549
A549
0.0 2.0 4.0 6.0 8.0 10.0 12.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0
[PT or PTP] ( M)
µ
[TPN] (10-8M)
Figure 4. Apoptosis and imaging of A498 and A549 cancer cells by PTP upon irradiation at 470 nm with a 2.5 J ·cm−2 source. a) Phase contrast bright-
field images (upper) of A498 cells upon light irradiation from 0 to 30 min in the presence of PTP and overlapping fluorescence images of A498 cells
(bottom) under PTP and EB filters. Phase contrast images were taken at 100 ms and the fluorescence images were taken at 300 ms under PTP and EB
filters. The magnification of objective lens is 10×. b) Overlapping images of A498 cancer cells under phase contrast bright field and under fluorescence
field for PTP and EB before and after 30 min irradiation. The magnification of objective lens is 40×. The false color of PTP is yellow and the type of
light filter is D455/70 nm exciter, 500 nm beamsplitter, and D525/30nm emitter. The false color of EB is red and the type of light filter is D540/40 nm
exciter, 570 nm beamsplitter, and D600/50 nm emitter. c) Dose-response curve data for cell viability of A498 and A549 cells treated with PTP, PT and
TPN by using a typical MTT assay under 470 nm light irradiation for 30 min, and the cell viability of A498 cell treated with [PTP] = 10 μM versus different
durations of light exposure. Error bars correspond to standard deviations from three separate measurements.
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a lower light dose at a fluence rate of 1.4 mW·cm−2 for 30 min
of irradiation (2.5 J·cm−2 ) is used in our PDT experiment, which
compares well to the performance parameters of approved photo-
sensitizers.[17,28,29] We also examined the decrease of A498 cell
viability with increasing light exposure time by the MTT assay
(Figure 4c) at [PTP] = 10 μM RUs. The results show that cell
viability decreases with increasing illumination time and that
significant cell death is induced after 30 min.
While such binding aids in the diffusion across membrane, it
makes specific and selective action on a given type of cell dif-
ficult to achieve. Subsequent molecular optimization therefore
focused on targeting tumor cells by taking advantage of a group
capable of selectivity (folic acid) and by removing complications
due to electrostatic binding. These considerations led to the
design of PTPF shown in Figure 5. PTPF is charge neutral, yet
water soluble through the integration of pegylated side chains.
1
PTPF also retains the critical optical elements that enable O2
generation, as described with PTP. A fractional number of
Since cells typically exhibit a net negative charge, cationic
CPEs bind to their surfaces through electrostatic interactions.
O
O
OTs
O
O
HO
OH
NH
N
NH
N
I
I
N
N
11
K2CO3
S
K2CO3
HN
HN
OH
O
Br
O
O
10
NHBOC
14
3
O
O
O
O
NH
N
HN
N
N
O
N HN
Br
Br
13
S
NH
O
O
O
O
O
NHBOC
B
B
S
O
O
Pd(dppf)Cl2
Na2CO3/H2O/
O
toluene
O
O
O
O
O
O
I
O
S
S
S
S
n
I
m
S
12
15
O
O
HO
N
H2N
HN
N
O
N
N
H
NH
N
O
N
H
N
O
HN
O
i ) TFA
O
O
NH2
NH
O
HO
H2N
N
O
N
N
H
N
O
H
N
ii )
HN
O
O
O
O
16
O
HO
O
PTPF
S
S
S
S
S
S
n
m2
m1
Figure 5. Synthetic preparation of PTPF.
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grafted folic acid functionalities were also
grafted to the backbone. The rationale behind
this incorporation stems from the fact that
folate receptor (FR) is a tumor-associated
protein that can bind folic acid with high
affinity (Kd ≈ 10−10 M).[30] We thus anticipated
that PTPF would preferentially bind and kill
FR-overexpressed cancer cells. On this basis,
PTPF presents a new design for improving
the efficiency of delivering fluorescence
imaging and photosensitizers to the desired
cancer cell targets with reduced accumula-
tion in normal cells. Synthetic access into
PTPF is shown in Figure 5 and all the syn-
thetic procedures are included in the Sup-
porting Materials.
KB cancer cells with over-expressed FR
and FR-negative NIH-3T3 fibroblasts cells
were chosen as target cells in a set of experi-
ments designed to test the specificity of
PTPF. Control tests by Western blot analysis
reveals the presence of high levels of FR in
the KB cells and little expression in NIH-
3T3 cells (see Figure S6 in the Supporting
Information). We first examined how PTPF
interacts with the cells. Cells were incubated
with PTPF (20.0 μg/mL) for 24h, and images
were then collected by fluorescence micro-
scopy. Figure 6a and 6b show the phase con-
trast bright-field and fluorescence images of
the resulting KB cancer and NIH-3T3 cells.
In comparison with NIH-3T3 cells, one can
clearly observe more pronounced PTPF accu-
mulation in the KB cells, presumably as a
result of the folate receptor-mediated uptake.
Specificity toward KB cancer cells over NIH
3T3 cells was investigated by the MTT assay.
In these experiments, KB and NIH-3T3 cells
were incubated with PTPF (25.0 μg/mL) for
24 h, followed by either keeping in the dark
c
KB cells
NIH-3T3 cells
100.0
80.0
60.0
40.0
20.0
0.0
100.0
80.0
60.0
40.0
20.0
0.0
Light irradiation
Dark
Light irradiation
Dark
5.0
0.0
10.0
15.0
20.0
25.0
0.0
5.0
10.0
15.0
20.0
25.0
[PTPF] (
µ
g/mL)
[PTPF] (µg/mL)
Figure 6. Phase contrast bright-field and fluorescence images of KB cancer cells (a) and NIH-
3T3 cells (b) in the presence of PTPF (20.0 μg mL−1 ). Left: phase contrast bright-field images.
Right: fluorescence images. The false color of PTPF is yellow and the type of light filter is
D455/70 nm exciter, 500 nm beamsplitter, and D525/30 nm emitter. The magnification of
object lens is 10×. c) Dose-response curves for cell viability of KB cells and NIH-3T3 cells
treated with PTPF by using a typical MTT assay under 470 nm light irradiation or in the dark.
Error bars correspond to standard deviations from three separate measurements.
or exposing to irradiation with 5.0 J/cm2 light doses at a fluence
rate of 2.8 mW·cm−2 for 30 min. As shown in Figure 6c, PTPF
displays more prominent cytotoxicity against KB cells under
light irradiation than in the dark (Figure 6c, left). The viability
of KB cells drops below 20% upon irradiation. However, toward
NIH-3T3 cells, PTPF only exhibits little cytotoxicity and cell via-
bilities remain above 70% in the dark or upon irradiation. Also
no obvious difference of cell viability was observed for NIH-3T3
cells in the dark and upon irradiation (Figure 6c, right). Further-
more, the role of folate receptors on cell surface in PTPF uptake
was assessed by incubating KB cells with PTPF (30.0μg mL−1 )
in the presence of 2 mM free folic acid used as competitive
inhibitor of cellular uptake. As shown in Figure S7, PTPF
exhibits little cytotoxicity in the dark and upon light irradiation
in the presence of free folic acid. The result shows that the pres-
ence of free folic acid effectively inhibits uptake of polymer by
the cells. These results show that the photoinduced cytotoxity of
PTPF is more effective toward FR-overexpressed cells than FR-
negative cells thereby indicating specific anticancer activity.
3. Conclusions
In conclusion, we have designed, synthesized and character-
ized the therapeutic function of cationic and neutral conjugated
water-soluble polymers that comprise a polythiophene back-
bone and pendant porphyrin chromophores. Excitation of the
backbone leads to efficient, but not complete, energy transfer
to the porphyrin sites, where sensitization of molecular oxygen
1
to produce O2 takes place. These materials do not cause tox-
icity toward cells in the dark as a result of the low pendant por-
phyrin content. Because of the light harvesting properties of the
polymer backbone it is possible to attain higher levels of O2
generation than when using similar concentrations of the por-
phyrin unit. These data demonstrate that the optical coupling
in the polythiophene/porphyrin dyad translates into light dos-
ages that are competitive with those of approved photosensi-
tizers. Also noteworthy is that the residual polymer emission
can be used to determine whether cancer cells are living or not,
depending on the regions of highest intensities. By eliminating
1
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Synthesis of 4-(2-(Thiophen-3-yl)ethoxy)phenol (5) : The mixture of
hydroquinone (4.4 g, 40 mmol), K2CO3 (1.38 g, 10 mmol) and catalytic
quantity of 18-crown-6 in air-free acetone (150 mL) was refluxed under
nitrogen for 30 min and then 2-(3-thienyl)-ethanol tosylate (4) (564 mg,
2.0 mmol) was added. After refluxing under nitrogen for 24 h, the solvent
was removed under reduced pressure and the residue was taken up in a
mixture of CH2Cl2 and water. The organic layer was separated, washed
with saturated aqueous NaHCO3 and brine, and dried over anhydrous
MgSO4. After removal of the solvent, the residue was purified by silica gel
chromatography using petroleum ether/EtOAC (80:1) as eluent to afford
a clear light yellow oil (240 mg, 59%). 1H NMR (400 MHz, CDCl3, δ): 7.26
(m, 1H), 7.07 (s, 1H), 7.02–7.01 (d 1H), 6.79–6.73 (dd, 4H), 4.75 (s, 1H),
4.13–4.10 (t, 2H), 3.10-3.07 (t, 2H); 13C NMR (100 MHz, CDCl3, δ): 152.9,
149.9, 138.9, 128.8, 125.8, 121.8, 116.5, 116.3, 69.2, 30.5. EIMS m/z: 220
[M+]. Anal. calcd for C12H12O2S: C 65.43, H 5.49; found: C 64.88, H 5.59.
Synthesis of Monomer 6: A mixture of compound 5 (240 mg, 1.09 mmol),
compound 3 (912 mg, 1.09 mmol), K2CO3 (300 mg, 2.18 mmol) and a
catalytic amount of 18-crown-6 in DMF (50 mL) was stirred at 70 °C
under nitrogen for 24 h. After cooling down to temperature, the solvent
was removed under reduced pressure and the residue was washed
with distilled water and extracted with dichloromethane. The organic
layer was separated, washed with brine, and dried over anhydrous
MgSO4. After removal of the solvent, the residue was purified by
silica gel chromatography using petroleum ether/EtOAC (40:1) as
eluent to afford a purple powder (457 mg, 43%). 1H NMR (400 MHz,
CDCl3, δ): 8.85 (s, 8H), 8.10–8.08 (d, 8H), 7.56–7.54(d, 6H), 7.26–7.22
(m, 2H), 7.07–7.02 (m, 2H), 6.86–6.82 (m, 3H), 6.58–6.54 (m, 2H),
4.25–3.99 (t, 6H), 3.10–3.08 (m, 2H), 2.70 (s, 9H), 2.01–1.89 (m, 4H),
1.68 (m, 4H), –2.76 (s, 2H); 13C NMR (100 MHz, CDCl3, δ): 158.9, 153.5,
152.9, 139.4, 138.7, 137.4, 135.7, 134.6, 131.4, 128.6, 127.5, 125.5, 121.5,
120.2, 119.9, 115.7, 115.5, 112.8, 68.8, 68.6, 68.2, 30.4, 29.5, 26.1, 21.6.
MS (MALDI-TOF) m/z: 975 [M+]. Anal. calcd for C65H58N4O3S: C 80.05,
H 5.99, N 5.74; found: C 79.97, H 6.15, N 5.85.
non-specific electrostatic interactions and introducing folic acid
to the backbone one finds that it is possible to retain all the
useful optical properties of PTP and to achieve specific action
toward folate receptor-overexpressed cancer cells. This col-
lected set of observations and structure/function relationships
indicate that conjugated polymer materials have the potential
to act as multifunctional photosensitive agents for treating
cancer infections and highlight the enormous potential offered
by understanding the interactions of optically active conjugated
polyelectrolytes with living cells.
4. Experimental Section
Materials: All chemicals were purchased from Acros, Aldrich
Chemical Company or Alfa-Aesar and used as received. All organic
solvent was purchased from Beijing Chemical Works and used as
received. The disodium salt of 9,10-anthracenedipropionic acid (ADPA)
was prepared according to the procedure in literature.[23] Ethidium
bromide (EB) was purchased from Sigma. Pulmonary adenocarcinoma
cell (A549) and renal cell carcinoma (A498), KB cancer cells and
normal NIH 3T3 fibroblasts cells were purchased from cell culture
center of Institute of Basic Medical Sciences, Chinese Academy of
Medical Sciences (Beijing, China) and cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM) supplemented with 10% neonatal bovine
serum (NBS), 4.0 mM glutamine and 4500 mg L−1 glucose. NBS was
purchased from Sijiqing Biological Engineering Materials (Hangzhou,
China). DMEM was purchased from HyClone/Thermofisher (Beijing,
China). (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium hydro-
bromide) (MTT) was obtained from Xinjingke Biotech. (Beijing, China)
and dissolved in 1×PBS before use. The water was purified using a
Millipore filtration system.
Measurements: The absorbance for MTT analysis was recorded on a
microplate reader (BIO-TEK Synergy HT) at a wavelength of 520 nm.
The 1H NMR and 13C NMR spectra were recorded on a Bruker Avance
400 MHz spectrometer. Elemental analyses were carried out with a
Flash EA1112 instrument. The fluorescence spectra were measured on
a Hitachi F-4500 fluorometer with a Xenon lamp as excitation source.
UV–vis absorption spectra were taken on a Jasco V-550 spectrometer.
Phase contrast bright-field and fluorescence images were taken on
a fluorescence microscope (Olympus 1 × 71) with a mercury lamp
(100 W) as light source. The excitation wavelength was 540/40 nm for
EB. Experiments for light-induced anticancer acitivty of cancer cells were
performed with a metal halogen lamp (Mejiro Genossen MVL-210) that
simulated a white light source. The wavelength was between 400 and
800 nm. The intensities of incident beams were checked by a power and
energy meter and the 470nm light obtained by the type of 470/10 nm
filter was used for PDT against cells.
Synthesis of 3-(2-(6-Bromohexyloxy)ethyl)thiophene (8) : 2-(Thiophen-
3-yl)ethanol (663 μL, 6 mmol) was added to a suspension of sodium
hydride (206 mg, 6 mmol) in dry DMF under N2. After stirring for 30 min,
1,6-dibromohexane (4.6 mL, 10 mmol) was added to the solution by
syringe at room temperature. After stirring overnight, the reaction was
quenched by adding water. The mixture was extracted with CH2Cl2 three
times. The combined organic layer was washed with brine and dried
over MgSO4, After removal of the solvent, the residue was purified by
silica gel chromatography using petroleum ether/ethyl acetate (40:1) as
1
eluent to afford compound 8 (0.54 g, 31%). H NMR (400MHz, CDCl3,
δ) 7.37(m, 1H), 7.15–7.10 (m, 2H), 3.76 (t, 2H), 3.57 (t, 2H), 3.53 (t,
2H), 3.04 (t, 2H), 1.99 (m, 2H), 1.72(m, 2H), 1.61–1.45 (m, 4H); 13C
NMR (75MHz, CDCl3, δ) 139.46; 128.53, 125.14, 121.04, 71.03, 70.77,
33.82, 32.79, 30.83, 29.57, 28.01, 25.43. HRMS (EI) m/z: [M+] calcd for
C12H19BrOS, 290.0340; found, 290.0342.
Synthesis
of
the
Monomer
6-(2-(Thiophen-3-yl)ethoxy)hexyl
trimethylammonium bromide (9) : To a solution of compound 8 (291 mg,
1 mmol) in THF (0.5 mL) was added trimethylamine (3 mL). The mixture
was allowed to stirrer at 40 °C overnight. Excessive trimethylamine and the
solvent were removed under reduced pressure. The residue was dissolved
in 1.0 mL of CH3OH, and 10 mL of hexane was added to precipitate the
product as a white solid (310 mg, 89%). 1H NMR (400MHz, CDCl3, δ)
7.27(m, 1H); 7.02-6.98(m, 2H), 3.65-3.56(m, 4H), 3.47(m, 11H), 2.91(t,
2H, 6.78), 1.75(br, 2H), 1.58(br, 2H), 1.42(br, 4H); 13C NMR (75MHz,
CDCl3, δ) 139.34; 128.48, 125.10, 120.96, 70.79, 70.37, 66.64, 53.27,
30.61, 29.25, 25.81, 25.76, 23.02. ESIMS m/z: 270 [M-Br]+. Anal. calcd for
C15H28BrNOS: C 51.42, H 8.06, N 4.00; found C 51.11, H 7.95, N 4.12.
Synthesis of 5-[4-(6-n,n,n-Trimethylammoniumhexyloxy)phenyl]-10,15,20-
tri-p-tolylporphyrin bromide (TPN): To a solution of compound 3 (60 mg,
0.072mmol)inTHF/CHCl3 wasaddeddropwisetrimethylamine-methanol
solution (3 mL), and the solution was stirred at 40 °C for 24 h. Excessive
trimethylamine and the solvent were removed under reduced pressure.
The residue was dissolved in CH3OH (1 mL), and hexane (10 mL)
was added to precipitate the product as a purple solid (51 mg, 79%).
1H NMR (400 MHz, CD3OD, δ): 8.84 (s, 8H), 8.09–8.07 (d, 8H), 7.54–
Synthesis
of
5-[4-(6-Bromohexyloxy)phenyl]-10,15,20-tri-p-tolyl-
porphyrin (3) : The mixture of pyrrole (11.3 mmol), 4-(6-bromohexyloxy)
benzaldehyde (1) (805 mg, 2.83 mmol) and p-tolualdehyde (2) (1.02 g,
8.49 mmol) in CHCl3 (154 mL) was stirred for 15 min under nitrogen at
room temperature, and then BF3-diethyl ether (389 mg, 2.74 mmol) was
added to this solution. After stirring for 1 h, DDQ (1.91 g, 8.42 mmol)
was added and the reaction mixture was stirred for further 2 h at room
temperature. Triethylamine (382 μL) was added and the crude mixture
was passed through a silica column with petroleum ether/CH2Cl2 (1:1)
as eluent. The residue was purified by a second silica column using
petroleum ether/CH2Cl2 (2.3: 1) as the eluent to afford a purple powder
1
(256 mg, 11%). H NMR (400 MHz, CDCl3, δ): 8.85 (s, 8H), 8.10–8.08
(d, 8H), 7.56–7.54 (d, 6H), 7.27–7.25 (d, 2H), 4.26–4.23 (t, 2H), 3.52–
3.48 (t, 2H), 2.67 (s, 9H), 2.01–1.98 (m, 4H), 1.66–1.65 (m, 4H), –2.77(s,
2H); 13C NMR (100 MHz, CDCl3, δ): 158.9, 139.4, 137.4, 135.7, 134.6,
131.0, 127.5, 125.5, 120.2, 119.9, 112.7, 68.1, 34.0, 32.8, 29.4, 28.1, 25.6,
21.6. MS (MALDI-TOF) m/z: 837 [M + H]+. Anal. calcd for C53H47BrN4O:
C 76.16, H 5.67, N 6.70; found: C 76.21, H 5.82, N 6.61.
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H2O (1:10) and then dialyzed through a membrane with a molecular
weight cutoff of 3500 for 3 days to yield of polymer 15 (12 mg). 1H NMR
(400 MHz, CDCl3, δ): δ (ppm) 8.85 (br), 8.09 (br), 7.54 (br), 7.00 (br),
6.84(br,), 4.26 (br), 3.97 (br), 3.43 (br), 3.06 (br), 2.70 (br), 2.14 (br),1.87
(br), 1.56 (br), 1.43 (br),1.32 (br), 1.25 (br), 0.83 (br).
7.53(d, 6H), 7.24–7.22 (d, 2H), 4.21–4.19 (t, 2H), 3.69–3.64 (t, 2H), 3.44
(s, 9H), 2.69 (s, 9H) 1.96–1.93 (m, 2H), 1.85–1.81 (m, 2H),1.68–1.66
(m, 2H), 1.55–1.53 (m, 2H), –2.78(s, 2H). ESIMS m/z: 815 [M-Br]+.
Synthesis of PT: A suspension of anhydrous FeCl3 (65 mg, 0.4 mmol)
in CHCl3 (8 mL) was stirred for 30 min at room temperature under N2. To
this suspension was added a solution of monomer 9 (35 mg, 0.1 mmol)
in CHCl3 (10 mL), and the resulting solution was stirred for 2 days at
room temperature. After the reaction was quenched by the addition of
methanol, the precipitate was filtered off and the pellet was washed by
CH3OH. The precipitate was dissolved in DMSO/H2O (1:10) and then
dialyzed through a membrane with a molecular weight cutoff of 3500 for
2 days to yield a yellow solid (16 mg). 1H NMR (400 MHz, DMSO-d6, δ):
7.75-7.31(br), 3.32 (br), 3.08-2.51 (br), 1.52-1.25 (br).
Synthesis of Polymer PTPF: To a solution of polymer 15 (7 mg) in
CHCl3 (2.0 mL) was added trifluoroacetic acid (1 mL) and then stirred
at room temperature overnight. The solvent was removed under reduced
pressure. The residue was redissolved in methanol/CHCl3 (5 mL, v/v
1:3) and triethylamine (1 mL) was added. After stirring overnight, the
solvents were removed at reduced pressure. The residue was dissolved in
DMSO (1 mL), then a solution of folic acid 16 (0.66 mg, 0.0015 mmol),
N-hydroxysuccinimide (0.35 mg, 0.003 mmol) and EDCI (0.56 mg,
0.003 mmol) in DMSO (2.0 mL) was dropwise added. The resulting
solution was stirred at room temperature for 2 days in dark. A brown
solid (4.0 mg) was obtained after dialysis through a membrane with a
Synthesis of PTP: A suspension of anhydrous FeCl3 (130 mg, 0.8 mmol)
in CHCl3 (15 mL) was stirred for 30 min at room temperature under N2. To
this suspension was added a solution of monomer 6 (33 mg, 0.033 mmol)
and monomer 9 (135 mg, 0.386 mmol) in CHCl3 (10 mL), and the
resulting solution was stirred for 2 days at room temperature. After
the reaction was quenched by the addition of methanol, the precipitate
was filtered off and the pellet was washed repeatedly by CH3OH. The
precipitate was dissolved in DMSO/H2O (1:10) and then dialyzed
through a membrane with a molecular weight cutoff of 3500 for 3 days to
yield a yellow solid (11 mg). 1H NMR (400 MHz, DMSO-d6, δ): 7.31(br),
3.70 (br), 3.44-3.31 (br), 3.08–2.67 (br), 1.65-1.54(br), 1.34-1.24 (br).
Synthesis of 5-[4-(6-(4-Hydroxyphenoxy)hexyloxy)phenyl]-10,15,20-tri-p-
tolyl porphyrin (10) : A mixture of hydroquinone (329 mg, 2.99 mmol),
K2CO3 (166 mg, 1.2 mmol) and catalytic quantity of 18-crown-6 in air-
free DMF (10 mL) was heated at 70 °C for 30 min and then compound
3 (100 mg, 0.12 mmol) was added. After further reaction for 20 h, the
solvent was removed under reduced pressure and the residue was taken
up in a mixture of CH2Cl2 and water. The organic layer was separated,
washed with brine, and dried over anhydrous MgSO4. After removal of
the solvent, the residue was purified by silica gel chromatography using
petroleum ether/EtOAc (6:1) as eluent to afford a purple powder (55 mg,
1
molecular weight cutoff of 3500 for 2 days in water under nitrogen. H
NMR (400 MHz, DMSO-d6, δ): 8.83 (br), 8.09 (br), 7.64 (br), 7.50 (br),
7.35 (br), 7.19(br), 6.88–6.80(br), 6.65(br), 5.32(br), 4.25 (br), 3.97 (br),
3.90 (br), 3.70 (br), 3.49 (br), 2.94(br), 2.64 (br), 2.13 (br), 2.30 (br),
1.99 (br), 1.62 (br), 1.38 (br), 1.23 (br), 0.88 (br).
Singlet Oxygen Measurement: To 300 μL of the aqueous solutions of
PTP, PT or TPN were added 6 μL of ADPA solution in water (6 mM). D2O
was used to replace H2O in these experiments. The UV–vis absorption
spectra were measured at 1.0 min intervals after the samples were
irradiated with white light and the reduction in absorption of ADPA at
378 nm was plotted as a function of the irradiation time.
Cell Viability Assay by MTT: A498 cells and A549 cells were routinely
grown in DMEM (high glucose) medium containing 10% NBS. All
cell lines were harvested for subculture using trypsin (0.05%, Gibco/
Invitrogen) and grown in a humidified atmosphere containing 5% CO2
and 95% air at 37 °C. Cells were subcultured in 96-well plates the day
before the experiment at a density of 4 – 7 × 104 cells per well, and
then cultured for 24 h. PTP, PT, TPN with varying concentrations were
respectively added into the cells followed by further culture for 24 h. The
culture media were discarded and fresh cell growth medium (100 μL)
was added. PDT treatment was performed by using he 470 nm light
obtained by the type of 470/10 nm filter with a dose of 2.5 J ·cm−2 at
a fluence rate of 1.4 mW·cm−2 for 30 min of irradiation. The cells were
allowed to continue growth for 24h, at which time the the culture media
were discarded and MTT (1 mg mL−1 , 100 μL per well) was added to
the wells followed by incubation at 37 °C for 4 h. The supernatant was
abandoned, and DMSO (150 μL) per well was added to dissolve the
produced formazan and the plates were shaken for an additional 10 min
The absorbance values of the wells were then read with microplate reader
at a wavelength of 520 nm. The cell viability rate (VR) was calculated
according to the following equation where the control group was carried
out in the absence of photosensitizers.[26] The MTT assays of KB cancer
cells which were cultured in folate-free RPMI 1640 medium containing
10% NBS and normal NIH 3T3 fibroblasts cells which were cultured in
DMEM (high glucose) medium containing 10% NBS in the presence of
PTPF were performed using the procedures described above.
1
53.4%). H NMR (400 MHz, CDCl3, δ): 8.95 (s, 8H), 8.20–8.18 (d, 8H),
7.65–7.63(d, 6H), 7.35 (m, 2H), 6.92–6.90(d, 2H), 6.84–6.82(d, 2H),
4.56(s, 1H) 4.33 (t, 2H), 4.08–4.05 (t, 2H), 2.79 (s, 9H), 2.09–1.96 (m,
4H), 1.76 (m, 4H), –2.67(s, 2H); 13C NMR (100 MHz, CDCl3, δ): 158.9,
153.4, 149.5, 139.4, 137.4, 135.7, 134.6, 131.0, 127.5, 120.1, 119.9, 116.1,
115.7, 112.8, 68.7, 68.2, 29.8, 29.5, 26.1, 21.6. MS (MALDI-TOF) m/z:
865 [M]+. Anal. calcd for C59H52N4O3: C 81.92, H 6.06, N 6.48; found C
81.06, H 6.04, N 6.71.
Synthesis of Compound 12: A mixture of compound 10 (30 mg,
0.035 mmol), compound 11 (71 mg, 0.104 mmol), K2CO3 (48 mg,
0.35 mmol) and a catalytic amount of 18-crown-6 in air-free DMF
(10 mL) was heated at 70 °C under nitrogen for 20 h. After cooling
down to room temperature, the solvent was removed under reduced
pressure and the residue was washed with distilled water and extracted
with dichloromethane. The organic layer was separated, washed with
brine, and dried over anhydrous MgSO4. After removal of the solvent,
the residue was purified by silica gel chromatography using petroleum
ether/EtOAc (6:1) as eluent to afford a purple powder (21 mg, 43.8%).
1H NMR (400 MHz, CDCl3, δ): 8.85 (s, 8H), 8.10–8.09 (d, 8H), 7.56-
7.54(d, 6H), 7.26–7.22 (m, 2H), 6.86–6.83 (m, 5H), 4.32–4.24 (m, 4H),
3.81–3.67 (m, 16H), 2.70 (s, 9H), 2.02–1.89 (m, 4H), 1.72–1.68 (m, 4H),
–2.77(s, 2H). MS (MALDI-TOF) m/z: 1375 [M]+, 1249 [M-I]+.
Aexperimental
group
VR(%) =
× 100%
Acontrol group
In Vitro Imaging of Photoinduced Death of A498 Cells by PTP: The
A498 cells were seeded in 35 mm culture plates (Nunc) at a density of
approximately 8 × 104 cells per plate for 24 h, and then the cells were
washed once with 1 × PBS and then grown in DMEM medium (1 mL)
with 10 μM PTP. After the cells were further cultured for 12 h, then the
culture media were discarded and fresh DMEM medium was added.
The plates were irradiated under 470 nm light for 0, 10 and 30 min
respectively, then ethidium bromide (EB) was added to the samples that
had been illuminated by light, and they were then observed by fluorescence
microscopy. Upon addition of EB to the samples, the phase contrast
images were taken at 100 ms and the fluorescence images were taken at
300 ms under PTP and under EB filters. The false color of PTP is yellow
Synthesis of Polymer 15: A mixture of monomer 12 (28 mg, 0.0206 mmol),
tert-butyl
6-(2-(2,5-dibromothiophen-3-yl)ethoxy)hexylcarbamate
13
(100 mg, 0.2062 mmol) and 5,5-dimethyl-2-(5-(5,5-dimethyl-1,3,2-dioxa-
borinan-2-yl)thiophen-2-yl)-1,3,2-dioxaborinane 14 (70 mg, 0.227 mmol)
in toluene (4 mL) and 2.0 M potassium carbonate (2 mL) was degassed
and then a catalytic amount of Pd(dppf)Cl2 was added. The resulting
mixture was stirred at 85 °C for 2 days under nitrogen. After cooling
down to room temperature, water (20 mL) was added and the mixture
was extracted with CHCl3. The organic layer was dried over anhydrous
MgSO4 and the solvent was removed. The crude product was redissolved
in CHCl3 (0.5 mL) and then precipitated in CH3OH. The precipitate was
collected by centrifugation. The precipitate was dissolved in DMSO/
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and the type of light filter is D455/70 nm exciter, 500 nm beamsplitter, and
D525/30 nm emitter. The false color of EB is red and the type of light filter
is D540/40 nm exciter, 570 nm beamsplitter, and D600/50 nm emitter.
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The authors are grateful for the National Natural Science Foundation
of China (No. 20725308, 21033010, 21021091, 90913014), the Major
Research Plan of China (No. 2011CB932302, 2011CB935800), the
National Science Foundation (DMR 1005546), the ITC-PAC (Contract No.
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the University of California, Santa Barbara.
Received: April 16, 2011
Revised: July 20, 2011
Published online: September 15, 2011
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wileyonlinelibrary.com
Adv. Funct. Mater. 2011, 21, 4058–4067
2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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