Heavy atom free 1,1,4,4-tetraphenylbuta-1,3-diene with aggregation induced emission for photodynamic cancer therapy

Article abstract of DOI:10.1039/c9nj01331h

Common organic molecules usually suffer from aggregation caused quenching (ACQ), which is disadvantageous for imaging guided phototherapy. It is of tremendous significance for a photosensitizer to be well soluble and simultaneously remain highly fluorescent in aqueous solution. Heavy atom free 1,1,4,4-tetraphenylbuta-1,3-diene (denoted as TPD) with aggregation induced emission (AIE) was designed and synthesized by the Knoevenagel reaction. 1,2-Distearoyl-sn-glycero-3-phospho-ethanolamine-N-[methoxy(polyethyleneglycol)_-2000] (DSPE-PEG_-2000) coated TPD nanoparticles (NPs) show high singlet oxygen generation ability with singlet oxygen sensor green (SOSG) as a probe and still remain highly fluorescent in aqueous solution. In vitro cytotoxicity assay demonstrates that these NPs have a half-maximal inhibitory concentration (IC₅₀) as low as 8.2 μg mL^-1. Meanwhile, in vivo fluorescence imaging shows that these NPs can passively target the tumor by the enhanced penetration and retention (EPR) effect within 6 h. Furthermore, in vivo phototherapy suggests that TPD NPs are able to inhibit the growth of a tumor after irradiation without side effects on the normal tissues, including heart, liver, spleen, lungs and kidneys, indicating the low dark toxicity, high phototoxicity and excellent bio-compatibility of such NPs.

Full text of DOI:10.1039/c9nj01331h

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PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Heavy atom free 1,1,4,4-tetraphenylbuta-1,3-diene with aggregation
induced emission for photodynamic cancer therapy
Huijuan Wena, Juan Maa, Jianjiao Chenb, Zhen Keb, Dengfeng Zou*b, Qiaoyun Li*c
Received 00th January 2018,
Accepted 00th January 2018
Common organic molecules usually suffer from aggregation caused quenching (ACQ), which
is disadvantageous for imaging guided phototherapy. It is of tremendous significance for a
photosensitizer to be well soluble and simultaneously remain highly fluorescent in aqueous
solution. Heavy atom free 1,1,4,4-tetraphenylbuta-1,3-diene (denoted as TPD) with
aggregation induced emission (AIE) was designed and synthesized by Knoevenagel reaction.
1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[methoxy(polyethyleneglycol)-2000
(DSPE-PEG-2000) coated TPD nanoparticles (NPs) show high singlet oxygen generation ability
with singlet oxygen sensor green (SOSG) as a probe and still remain highly fluorescent in
aqueous solution. In vitro cytotoxicity assay demonstrates that these NPs have a half-
maximal inhibitory concentration (IC50) as low as 8.2 μg/mL. Meanwhile, in vivo fluorescence
imaging shows that these NPs can passively target to the tumor by the enhanced penetration
and retention (EPR) effect within 6 h. Furthermore, in vivo phototherapy suggests that TPD
NPs are able to inhibit the growth of tumor after irradiation without side effects to the
normal tissues, including heart, liver, spleen, lung and kidney, indicating the low dark toxicity,
high photo toxicity and excellent bio-compatibility of such NPs.
DOI: 10.1039/x0xx00000x
www.rsc.org/
interactions, hydrogen bonding, electrostatic interactions,
hydrophobic effects, leading to fluorescence quenching in
aqueous solution, which is the so-called aggregation
caused quenching (ACQ). Such ACQ effect limits the
potential of the PSs as fluorescence imaging agents.
Therefore, it is considerably significant to develop PSs that
can be both dispersible in aqueous solution and still
remain highly fluorescent simultaneously.
Introduction
Traditional cancer therapies, such as radiation, surgery
and chemotherapy may suffer from invasion, no targeting
and inevitably the recurrence of cancer. It is urgent that
more therapies should be developed for treatment of
cancer. Over the past decades, increasing attention has
been attached to photodynamic therapy (PDT),
a
relatively newly developed one, owing to its non-invasion,
Over the past years, great efforts have been spared on the
chemical approaches to preventing ACQ[12-26]. Opposite to
ACQ, molecules with aggregation induced emission (AIE)
[1-11]
low systemic toxicity and etc.
Among the various
factors, the design and preparation of photosensitizer (PS)
which can generate reactive oxygen species (ROS) under
the irradiation of light plays a significant role in PDT.
Common PSs, however, usually tend to form π -π stacking
aggregates via driving forces such as Vander Waals
property
have
been
synthesized,
typically
tetraphenylethylene. Such fluorophore have enhanced
emission aggregation induced emission (AIE) in that the
phenyl rings can undergo active intramolecular
rotational/twisting motions alongside the C-C bonds
against the stator upon excitation. They can be dissolved
in organic solvent while their fluorescence will become
stronger in aqueous media, the reason is its self-
aggregation driven by limited solubility as well as π -π
interactions.[27-28]
a.
b.
The Third People’s Hospital of Kunshan, Kunshan, Jiangsu, 215316, China,
School of Pharmacy, Guilin Medical University, Guilin 541004, Guangxi, P.R.
China. email: zdf1226@126.com
c.
Depertment of Chemistry and Materials Engineering, Changshu Institute of
Technology, Jiangsu, P.R. China Email: liqiaoyun61@126.com
†Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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In our previous work, the anticancer property of (2-(4- spectra were measured on a spectrophotometer (UV-
bromophenyl)ethene-1,1,2-triyl)tribenzene
View Article Online
was 3600 UV-Vis-NIR, Shimadzu, Japan)D. OTI:h10e.10f3l9u/oCr9eNsJc0e13n3c1He
investigated both in vitro and in vivo.[29] However, the spectra were measured on an F-4600 spectrometer
compound has an –Br atom, which will quench the (HITACHI, Japan). DLS was carried out on a 90 Plus particle
fluorescence of tetraphenylethylene by the spin orbit size analyzer (Brookhaven Instruments, USA). TEM of the
crossing (SOC). To extend our previous work, 1,1,4,4- nanoparticles were measured on JEOL JEM-2100
tetraphenylbuta-1,3-diene (denoted as TPD) have been equipment.
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designed and prepared by self-condensation reaction of
3,3-diphenylacrylaldehyde in the presence of Zn powder 2.2 Synthesis and characterization of TPD
and TiCl4. It is supposed that this compound with strong A mixture of Zn powder (1.12 g, 20 mmol) and 3,3-
rigidity can overcome ACQ and shows AIE property. Then diphenylacrylaldehyde (2.08 g, 10 mmol) were dissolved
1,2-distearoyl-sn-glycero-3-phospho-
ethanolamine-N- in dried THF (100 mL) and the mixture was cooled to -
[methoxy(polyethyleneglycol)-2000] (DSPE-PEG-2000) coated 78oC, then N2 was purged to drive off possible oxygen and
TPD can self assemble to form nanoparticles (NPs) which water for 2 h. TiCl4 (2.85g, 15 mmol) was slowly added
has good dispersity in PBS. To further investigate the and the mixture was reacted for another 12 h. The
cytotoxicity of such NPs, HeLa cells have been selected as mixture was poured to brine and washed with saturated
the model. MTT assay shows that TPD NPs with a low half NaHCO3 three times (100 mL). Then CH2Cl2 was used to
inhibitory concentration of 8.2 μg/mL are able to induce extracted the crude product three times (100 mL), which
cell apoptosis upon light irradiation. Furthermore, in vivo was purified by column chromatography on silica gel with
1
study show that TPD NPs are able to inhibit tumor growth CH2Cl2/hexane (v/v 1:2) as the eluant. HNMR (300 MHz,
after irradiation at a low dose (0.5 mg/kg) without any CDCl3) 7.46-7.34 (6H, m), 6.80-6.75 (4H, d), 7.24-7.18 (6H,
side effects towards normal organs, including heart, liver, d), 7.18-7.12 (4H, t); 6.78 (2H, s), 13CNMR (125 MHz,
spleen, lung and kidney, indicating the low dark toxicity, CDCl3) 144.0, 142.5, 140.0, 130.7, 128.2, 128.0, 126.0;
high photo toxicity and outstanding bio-compatibility of Elemental analysis for C28H22: C, 93.81; H, 6.19; found C,
such NPs. Our results show that heavy atom free TPD with 93.86, H, 6.14; MS (ESI) : calcd. m/z = 358.17; found m/z =
high singlet oxygen generation ability is a potential 358.35.
candidate for cancer therapy both in vitro and in vivo.
2.3 Preparation of TPD nanoparticles
The nanoparticles of TPD were prepared by nano-
precipitation with DSPE-PEG-2000. DSPE-PEG-5000 (5 mg) was
dissolved in distilled water with ultrasound. And then TPD
(5 mg) was dissolved in tetrahydrofuran (THF, 1 mL). Then
200 μL of such solution was injected into PBS with
ultrasound at room temperature. After the mixture was
stirred for 10 min, THF was removed by purging nitrogen.
The product was then frozen and dried for further use.
2.4 Cell culture and MTT assay
HeLa cell lines were cultured in a regular growth medium
consisting of Dulbecco’s modified Eagle’s medium
(DMEM, Gibco) with 10% fetal bovine serum (FBS) and 1%
penicillin-streptomycin under an atmosphere of 5% CO2 at
37 oC. Cell viability assays of the TPD NPs were first
dissolved in distilled water, which were diluted with
DMEM to various concentrations (0-20 μg/mL) and put in
the 96-well plate. Then the plate was irradiated with a
xenon lamp (30 mW/cm2) for 5 minutes. Cell viability was
determined by colorimetric 3-(4,5-dimethylthiazol- 2-yl)-
2,5- diphenyltetrazolium bromide (MTT) assay. MTT in
distilled water (5 mg/mL, 20 μL) was added to each well
followed by incubation for 4 h under the same conditions
at 37oC. Then the solution was discarded followed by the
addition of DMSO (200 μL). At ambient temperature, the
Scheme. Illustration of the TPD NPs for photodynamic
cancer therapy.
Experimental
Materials and apparatus
All the chemicals were commercially available from sigma
1
and were used without further purification. H NMR and
13C NMR spectra were performed on Bruker DRX NMR
spectrometer in CDCl3 at 298 K with solvent residual
(CDCl3, δ = 7.26 ppm) as the internal standard. UV-vis
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absorbance before 490 nm was measured on a Bio-Tek
microplate reader. The cell viability of the control group
(without TPD NPs) was considered as 100%. The cell
viability was then calculated by the following equation: Synthesis and characterization of TPD and NPs
viability (%) = mean absorbance of the group incubated
with TPD NPs / mean absorbance of the group incubated
with DMEM.
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DOI: 10.1039/C9NJ01331H
Results and Discussion
The absorbance of TPD in THF and the corresponding
nanoparticles (NPs) in distilled water were measured. TPD
in THF show the peak at 247 and 351 nm while the NPs
252 and 345 nm, respectively. However, the absorbance
peak of the NPs is broader than that of the TPD molecule.
Also, the absorbance of NPs in water becomes broader
than that in DCM, which may be caused by the
aggregation of the NPs in water. In the fluorescence
spectra, a large stokes shift of 105 nm was observed,
indicating the potential AIE of such NPs. To further
investigate the AIE effect of TPD NPs, the fluorescence in
THF/water with different fraction was reported. As shown
in Figure 1b, with the increasing fraction of water, the
fluorescence jump sharply, which is the characteristic of
AIE. Transmission electron microscope (TEM) of TPD NPs
demonstrates that it can self assemble to form
nanoparticles with mean diameter of approximate 70 nm,
which is consistent with the dynamic light scattering (DLS)
results. Since singlet oxygen generation in aqueous
solution is essential for PDT, SOSG (singlet oxygen sensor
green) was used as a probe. Figure 1d shows the
fluorescence enhancement of SOSG with light irradiation.
It can be seen that the fluorescence enhancement of TPD
NPs with irradiation is faster than that of TPE-Br NPs,
showing the stronger ROS generation ability of TPD NPs
under irradiation.[29]
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2.5 Cellular uptake and fluorescence image of cellular
ROS
HeLa cells were incubated with DSPE-PEG-2000 coated TPD
NPs (8.2 μg/mL, 2 mL) in a confocal dish for different time
in dark for 24 h. Then the solution was discarded and the
cells were washed with PBS three times (1 mL), followed
by the addition of 1 mL polyoxymethylene for 25 min.
Then polyoxymethylene was discarded and the cells were
washed with PBS for three times (1 mL). The sample with
TPD NPs for 24 h was further cultured with 10 µM of 2,7-
dichlorodihydrofluorescein diacetate (DCF-DA) for
another 5 min. Afterwards, it was washed with PBS three
times (1 mL). This sample was irradiated by Xenon lamp
(30 mW/cm2) for 3 minutes. The fluorescence images
were observed by Olympus IX 70 inverted microscope. For
the samples with TPD NPs for 24 h and they were excited
at the wavelength of 433 nm and collected fluorescence
from 450 to 600 nm. While the one incubated with DCF-
DA under irradiation, it was excited with 488 nm laser and
collected fluorescence from 490 to 600 nm.
2.6 In vivo tumor treatment histology examination
The study complies with all institutional and national
guidelines, and was approved by the Chinese laws. The
protocol was approved by Animal Center of Guilin Medical
University (SCXK2007-001). 12 nude mice were purchased
and then injected with HeLa cells into the armpit as the
tumor source. When the tumor volume reached about
100 mm3, the mice were divided into 3 groups randomly.
For the control group, the mice were intravenously
injected with saline while the other groups were injected
with TPD NPs (80 μg/mL, 100 μL) in PBS solution,
respectively. After 4 h, the tumors of the irradiation
groups were irradiated by Xenon lamp for 10 minutes
while the mice in the non-irradiation ones were not
irradiated exceptionally. The experiment was continued
for twenty eight days, and the tumor volume and body
weight of mice were recorded every two days. These nude
mice were killed followed by the histology analysis. The
main organs (heart, liver, spleen, lung, kidney) and the
tumor from each mouse was isolated and fixed in 4%
formaldehyde solution. After dehydration, they were
embedded in paraffin cassettes and stained with
hematoxylin and eosin (H&E), the images were recorded
on a microscope.
Figure 1 (a) Normalized absorbance of TPD in THF and
DSPE-PEG-2000 coated TPD NPs in water; (b) Fluorescence
intensity of TPD in THF/water with different fraction (from
1:9 to 9:1); (c) TEM and DLS of DSPE-PEG-2000 coated TPD
NPs; (d) ROS generation of TPD NPs with SOSG as a probe
in water.
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fluorescence of the tumor gradually become stronger
while that is the strongest at 6 h injection, demonstrating
View Article Online
DOI: 10.1039/C9NJ01331H
MTT assay, cellular uptake and ROS generation in vitro
6 h is the appropriate for phototherapy. At 24 h, the
tumor still remain fluorescent. Then the mouse was
sacrificed and the fluorescence intensity of the tumor,
heart, liver, spleen and kidney was measured. It can be
seen that the fluorescence of the tumor is the strongest,
followed by liver and kidney.
To evaluate the suitability of TPD NPs for cell imaging in
vitro, cellular uptake of DSPE-PEG-2000 coated TPD NPs was
recorded using confocal laser scanning microscope
(CLSM). TPD NPs could be used as an agent for cell
imaging because green fluorescence was observed. 2',7'-
dichlorofluorescein diacetate (DCF-DA), the commercial
ROS probe, was selected to detect the singlet oxygen
generation. It is found that TPD NPs are able to generate
singlet oxygen effectively in vitro owing to the observed
strong green fluorescence upon the excitation at 488 nm
(Figure 2b).
To further investigate the phototherapy efficacy of TPD
NPs, MTT assay was performed. For one thing, high
phototoxicity is fundamental to photodynamic therapy. It
can be seen that cell viability has been dramatically
suppressed with the increase of the concentration of such
NPs, and the half inhibitory concentration (IC50) is as low
as 8.2 μg/mL, which is superior to that of TPE-Br NPs (12
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Figure 3 (a) Fluorescence imaging of TPD NPs in HeLa
tumor bearing nude mouse at different period of time (0,
2, 6, 12 and 24 h); (b) Fluorescence intensity of tumor,
heart, liver, spleen, lung and kidney of the sacrificed
mouse after 24 h intravenous injection.
μg/mL, TPE-Br
triyl)tribenzene)
=
(2-(4-bromophenyl)ethene-1,1,2-
and ETTDA NPs (40 μg/mL,
In order to evaluate the phototherapy efficacy of TPD
NPs, 12 HeLa tumor-bearing nude mice were divided into
three groups at random and used to investigate the PDT
efficacy of TPD NPs in vivo. As shown in Figure 4a, the
relative tumor volume increases quickly while that of the
TPD NPs only group a little slowly. The relative tumor
volume of the control and the no illumination groups are
12.3 and 11.9, respectively. This can be explained by the
[29]
ETTDA=4,4',4'',4'''-(ethene-1,1,2,2-tetrayl)tetrakis(N,N-
diethylaniline))[30] . For another thing, low dark toxicity is
crucial for a photosensitizer to minimize the side effects
on normal tissues. The dark toxicity of such NPs is
negligible because the cell viability remains high,
compared with the control group.
1
fact that irradiation of room light inevitably generate O2
to inhibit the tumor growth. The body weight in the three
groups increase, respectively, indicating the low dark
toxicity and good bio-compatibility of TPD NPs (Figure 4b).
The hematoxylin and eosin (H&E)-stained images of the
tumors in the control and TPD NPs only groups (Figure 4g-
4i) show the nucleus of HeLa cells remain almost
unchanged, suggesting low dark toxicity themselves. In
contrast, the nucleus of the irradiation group become
distorted or even damaged, indicating the good
phototherapy efficacy of TPD NPs. After treatment, these
mice were sacrificed and the tumors of the former two
groups are put in Figure 4c.
Figure 2 (a) Cellular uptake of TPD NPs in HeLa cells; (b)
ROS generation of TPD NPs with DCF-DA as a probe; (c)
MTT assay of TPD NPs on HeLa cells with different
concentrations (0, 5, 10, 15, 20 μg/mL).
In vivo fluorescence imaging guided photodynamic
therapy
Nanoparticles can be passively targeted to the tumor by
the enhanced penetration and retention (EPR) effect.
Fluorescence imaging has been taken advantage of to
investigate the uptake of the NPs in the tumor. As shown
in Figure 3a, no detectable fluorescence can be observed
when no NPs were injected at 0 h. After 2h, the
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Figure 4 (a) Tumor volume change of the three groups The authors have no conflict to declare.
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DOI: 10.1039/C9NJ01331H
(**p＜0.01 compared with control group) (b) Body weight
change of the groups; (c) Survival rate of the mice in the
control, no irradiation and irradiation groups; (d-f)
Photograph and H&E stained pictures of the mice in
control, TPD NPs only, TPD NPs + irradiation groups. Scale
bar: 100 μm.
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To further investigate the bio-compatibility of TPD NPs,
the H&E stained pictures of the normal organs have been
illustrated in Figure 5. No obvious difference between the
images of the main organs (heart, liver, spleen, lung,
kidney) in no illumination and illumination groups were
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Conclusion
In summary, TPD with aggregation induced emission has
been prepared by self Knoevenagel reaction of 3,3-
diphenylacrylaldehyde. DSPE-PEG-2000 coated TPD NPs
show high fluorescence and singlet oxygen in aqueous
solution. In vitro study shows that these NPs can be
uptaken by HeLa cells and have a low IC50 of only 8.2
μg/mL. Furthermore, DSPE-PEG-2000 coated TPD NPs can
effectively inhibit the growth of tumors and the normal
tissues suffer from no damage, indicating the high
phototoxicity, low dark toxicity and good bio-compatibility
of such NPs. Other investigation is currently underway in
our group.
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The authors acknowledge financial support from National
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Autonomous Region Level College Students Innovation
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Conflict of interest
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ARTICLE
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