Chemical molecule-induced light-activated system for anticancer and antifungal activities

Article abstract of DOI:10.1021/ja304986t

Except for chemotherapy, surgery, and radiotherapy, photodynamic therapy (PDT) as new therapy modality is already in wide clinic use for the treatment of various diseases. The major bottleneck of this technique is the requirement of outer light source, which always limits effective application of PDT to the lesions in deeper tissue. Here, we first report a new modality for treating cancer and microbial infections, which is activated by chemical molecules instead of outer light irradiation. In this system, in situ bioluminescence of luminol can be absorbed by a cationic oligo(p-phenylene vinylene) (OPV) that acts as the photosensitizer through bioluminescence resonance energy transfer (BRET) process. The excited OPV sensitizes oxygen molecule in the surroundings to produce reactive oxygen species (ROS) that kill the adjacent cancer cells in vitro and in vivo, and pathogenic microbes. By avoiding the use of light irradiation, this work opens a new therapy modality to tumor and pathogen infections.

Full text of DOI:10.1021/ja304986t

Communication
pubs.acs.org/JACS
Chemical Molecule-Induced Light-Activated System for Anticancer
and Antifungal Activities
Huanxiang Yuan, Hui Chong, Bing Wang, Chunlei Zhu, Libing Liu,* Qiong Yang, Fengting Lv,
and Shu Wang*
Beijing National Laboratory for Molecular Science, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of
Sciences, Beijing 100190, P. R. China
S
* Supporting Information
Scheme 1. (A) Schematic Illustration of the BRET System
for PDT; (B) Synthesis of Cationic OPV as Photosensitizer
ABSTRACT: Except for chemotherapy, surgery, and
radiotherapy, photodynamic therapy (PDT) as new
therapy modality is already in wide clinic use for the
treatment of various diseases. The major bottleneck of this
technique is the requirement of outer light source, which
always limits effective application of PDT to the lesions in
deeper tissue. Here, we first report a new modality for
treating cancer and microbial infections, which is activated
by chemical molecules instead of outer light irradiation. In
this system, in situ bioluminescence of luminol can be
absorbed by a cationic oligo(p-phenylene vinylene)
(OPV) that acts as the photosensitizer through bio-
luminescence resonance energy transfer (BRET) process.
The excited OPV sensitizes oxygen molecule in the
surroundings to produce reactive oxygen species (ROS)
that kill the adjacent cancer cells in vitro and in vivo, and
pathogenic microbes. By avoiding the use of light
irradiation, this work opens a new therapy modality to
tumor and pathogen infections.
ince first used in clinical trials in the 1980s,1 photodynamic
Stherapy (PDT) has led to new therapy modalities apart
from chemotherapy, surgery, and radiotherapy. PDT is already
widely used in clinic for the treatment of a variety of tumors
and ophthalmology and dermatology-related diseases.2,3 The
photosensitizer, light, and molecular oxygen are the primary
three components in PDT to produce reactive oxygen species
(ROS) that are toxic and kill cells. The principal advantage of
PDT is its spatial selectivity for diseases treatment as the
photosensitizer is activated only under light excitation with
selected wavelength.4,5 However, the requirement of outer light
source always limits effective application of PDT to the lesions
in deeper tissue since the light does not penetrate far through
due to the absorption and scattering by biological tissues.6
Although infrared light can reduce this shortcoming, unfortu-
nately the development of photosensitizers with highly efficient
absorption of infrared light (such as two-photon photo-
sensitizers) has proved a challenge.7 Thus, the development
of novel PDT system that is independent of light irradiation can
provide a new therapy modality for various diseases.
were used as bioluminescent molecules and a cationic oligo (p-
phenylene vinylene) (OPV) was used as the photosensitizer
(see its chemical structure and synthesis in Scheme 1B).
Luminol emits a striking blue bioluminescence at the maximum
of 425 nm in the presence of oxidizing agent (such as hydrogen
peroxide) and HRP.8,9 OPV exhibits a broad absorption (350−
550 nm) and a maximal emission at 550 nm in water. They
Here, we report a novel PDT system in which the
photosensitizer is activated by chemical molecules instead of
light source (Scheme 1A). To demonstrate the concept,
luminol, hydrogen peroxide, and horseradish peroxidase (HRP)
Received: May 22, 2012
© XXXX American Chemical Society
A
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Journal of the American Chemical Society
Communication
Figure 1. (A) Normalized absorption of OPV and luminescence spectra of luminol. (B) Luminescence spectra of luminol in the presence of various
concentrations of OPV. [OPV] = 0−50 μM, [HRP] = 0.003 mg/mL, [luminol] = 0.2 mM, [4-iodophenol] = 0.5 mM, [H2O2] = 0.5 mM. (C)
Transient absorption spectra of OPV in argon-saturated ethanol. Delay time: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72 μs.
meet the spectral overlap requirement for bioluminescence
resonance energy transfer (BRET) as the donor−acceptor pair
(Figure 1A).10,11 Thus, the bioluminescence from luminol is
absorbed by OPV to make BRET take place due to the
electrostatic interactions between the negatively charged
luminol oxidation product (dianion) and cationic OPV, and
then the excited OPV sensitizes oxygen molecules in the
surroundings to produce ROS. It is noted that the HRP did not
bind to cells (Figure S1). The cationic OPV could bind to the
negatively charged cells and fungi and subsequently kill the
cancer cells and pathogenic microbes. To the best of our
knowledge, this is the first reported PDT system based on
BRET without requirement for outer light source, although the
BRET systems have been explored for in vivo imaging and
biological detections.12−15
To prove the BRET process, the bioluminescence of luminol
was measured in the presence of OPV with varying
concentrations. As shown in Figure 1B, the addition of OPV
leads to a dramatic decrease of the luminescence intensity at
425 nm (corresponding to luminol); at the same time, the
luminescence intensity at 550 nm (corresponding to OPV)
gradually increases with increasing OPV concentration (0−50
μM). This result confirms the occurrence of the BRET. Because
of the important role of the long-lived triplet state in the
generation of toxic ROS, we investigated the transient
absorption spectra of OPV to demonstrate the presence of
triplet excited state. As shown in Figure 1C, direct excitation of
OPV in argon-saturated ethanol can generate a long-lived
transient absorption characterized by two bands with
absorption maxima at around 540 and 660 nm, which
correspond to the triplet−triplet absorption and the radical
cation of OPV, respectively. The triplet lifetime from the
decayed transient absorption was determined to be 44 μs. 2,7-
dichlorofluorescein diacetate (DCFH-DA) was utilized to
further verify the production of ROS.16 Conversion of
DCFH-DA into 2,7-dichlorofluorescin (DCFH) was followed
by transformation into highly fluorescent 2,7-dichlorofluor-
escein (DCF, quantum yield: 90%) in the presence of ROS. As
shown in Figure S2, upon irradiating DCFH in the presence of
OPV under white light (400−800 nm), an apparent emission at
525 nm (characteristic of DCF) was detected (excitation: 488
nm) and the fluorescence intensity was much higher than that
of the control group without OPV. It is noted that OPV itself
can not be excited at 488 nm. These results demonstrate the
significant generation of ROS originated from OPV.
Figure 2. (A) CLSM images of HeLa cells incubated with OPV. The
fluorescence of OPV is highlighted in green. Left, fluorescence image;
middle, phase contrast bright-field image; right, overlap image. (B) ζ-
potentials of HeLa cells before and after incubating with OPV.
Measurements were performed in 10 mM PBS solution at 25 °C. (C)
Cell viability of HeLa cells after incubation with OPV in the absence
and presence of luminol luminescence system (E+S). Values are
expressed as means SD (n = 3, P < 0.01). [OPV] = 0−8 μM, [HRP]
= 0.01 mg/mL, [luminol] = 0.5 mM, [4-iodophenol] = 1.25 mM,
[H2O2] = 0.4 mM.
attained to gain more insights into the interactions of OPV with
live cells. No obvious change of ζ-potentials was observed for
the HeLa cells before and after adding OPV (Figure 2B), which
indicates that the OPV might insert into the lipid bilayer of the
cells because of its high charge density and comparable linear
length.17 On the basis of the affinity of cationic OPV toward
cells, viability analysis of OPV to HeLa cells was determined
using a standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-2H-tetrazolium hydrobromide) assay with and with-
out luminol luminescence system (HRP, 4-iodophenol, luminol
and hydrogen peroxide, namely, E+S). As shown in Figure 2C,
less cytotoxicity was observed when the OPV concentration is
less than 8 μM without luminol luminescence system. While in
the presence of the luminol luminescence system, OPV displays
more prominent cytotoxicity. We also examined the decrease of
HeLa cell viability with increasing OPV concentration and less
than 10% viability was obtained. These results show that the
cytotoxity of OPV in the presence of luminol luminescence
system is more effective toward HeLa cells than the case
without luminol luminescence system thereby indicating the
anticancer mechanism by BRET. It is noted that the luminol
luminescence system (E+S) itself could kill approximately 30%
cells due to the toxicity of H2O2. Following the cancer cell
To directly visualize the binding of cationic OPV to live cells,
confocal laser scanning microscopy (CLSM) was utilized. As
shown in Figure 2A, the OPV only binds to the membrane of
HeLa cell after incubating for 30 min. Zeta potentials (ζ) were
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Journal of the American Chemical Society
Communication
instead of 4-iodophenol to improve the antitumor effect of our
BRET system.18,19 As shown in Figure S5, the addition of
PC3SO3Na leads to a dramatic prolonged BRET signal up to 2
h. As shown in Figure 3A,B, the BRET system (E+S+OPV)
shows a slower tumor growth in comparison to control group.
As expected, the addition of luminol signal enhancer,
PC3SO3Na, improves the antitumor activity of the BRET
system and a 55% of tumor inhibition ratio was observed
(Figure 3C). The addition of PC3SO3Na does not show toxic
side effect as the body weight growth was not inhibited for mice
in comparison to control group (Figure 3D). Athough the
distribution in vivo of OPV was not investigated, nanoparticles
prepared by other polymers have been observed mainly in the
targeted region and the liver in vivo using intravenous injection
mode.20,21
To show that the BRET system works with other kinds of
pathogenic cells, we also investigated its antimicrobial activity
toward Candida albicans, a kind of pathogenic fungi. Fungi have
been recognized as major pathogen associated with chronic and
ultimately fatal infections in critically ill patients, and they are
more resistant to antifungal agents than bacteria.22 Colony
counting shows that in a low OPV concentration (1.0 μM), the
killing efficiency reaches 98% in the presence of the BRET
system (E+S+OPV). The control experiments exhibit that
OPV alone only kills ∼10% of the fungi, and the luminol
luminescence system (E+S) did not inhibit the growth of C.
albicans at all (Figure 4A,B). That is, our BRET system
becomes much more potent at killing fungi, which results from
the ROS generated by the BRET from luminol luminescence to
OPV. To further evidence the high efficiency of the BRET
Figure 3. (A) Representative photo of tumors collected from the
tumor-bearing mice after 18 days treatments with new BRET system.
(B) Tumor volume as a function of treatment time. Values are
expressed as means SD The value of P < 0.05 of saline treatment vs
treatment with the luminol luminescence system (E+S) and OPV was
considered statistically significant. (C) Tumor inhibition of mice post
18 days treatment, 10 mice used per group. Values are expressed as
means SD (n = 10, P < 0.001). (D) Body weight curves after various
treatments. Values are expressed as means SD (n = 10, P > 0. 1, no
significant difference) [OPV] = 10 μM, [HRP] = 0.02 mg/mL,
[luminol] = 2 mM, [PC3SO3Na] = 0.03 mg/mL, [H2O2] = 0.5 mM.
viability analysis, we investigated the toxicity of BRET system
to normal human epithelial cells, including human kidney
epithelial cells (HKC) and immortalized human epidermal cells
(HaCaT). The MTT assay results (Figure S3) indicated that
the BRET system was also cytotoxic to normal human epithelial
cells, which means the BRET system has little specificity due to
a lack of recognition groups to the cancer cells.
After verification by in vitro experiment, we then evaluated
the antitumor therapeutic efficacy of the novel BRET system in
HeLa cell tumor-bearing nude mice using intratumoral
injection mode. Ten mice were used for each group, including
the negative controls in the absence of OPV and luminol
luminescence system. Tumor growth was monitored with
digital caliper after treatment. As shown in Figure S3A, neither
luminol luminescence system injection by itself (E+S: HRP,
luminol, luminol signal enhancer and H2O2) nor OPV alone
was able to inhibit the tumor growth, whiles the group injected
with BRET system (E+S+OPV) shows a slower tumor growth
in comparison to control group. As seen in Figure S3B, about
30% of tumor inhibition ratio was shown by the BRET
treatment group (E+S+OPV) in comparison to luminol
luminescence system (E+S) group, and the OPV group did
not demonstrate any tumor inhibition. These results indicate
that our new BRET system could inhibit the tumor growth in
vivo. Furthermore, our BRET-based PDT treatment does not
show toxic side effect as the body weight growth was not
inhibited for mice in comparison to control group (Figure
S4C), which indicates there is no apparent toxicity to the
normal mice tissue though the BRET system has little
specificity. It is reported that enhanced chemiluminescent
reactions provide more intense, prolonged and stable light
emission. Here the sodium 3-(10′-phenothiaziny1)propane-1-
sulfonat (PC3SO3Na) is used as luminol signal enhancer
Figure 4. (A) Plate photographs for C. albicans on YTD agar plate
treated with OPV in the absence and presence of the luminol
luminescence system. (B) Biocidal activity of OPV toward C. albicans
in the absence and presence of the luminol luminescence system (E
+S). Values are expressed as means SD (n = 3, P < 0.01). [OPV] =
1.0 μM, [HRP] = 0.004 mg/mL, [luminol] = 0.2 mM, [4-iodophenol]
= 0.5 mM, [H2O2] = 0.5 mM. (C) Morphology of C. albicans treated
with OPV in the absence and presence of the luminol luminescence
system.
C
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Journal of the American Chemical Society
Communication
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to observe the morphological changes of C. albicans. As shown
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the photosensitizer is activated by chemical molecules instead
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a cationic oligo (p-phenylene vinylene) (OPV) was used as the
photosensitizer. They meet the spectral overlap requirement for
BRET as the donor−acceptor pair. The excited OPV by BRET
from luminol sensitizes oxygen molecules in the surroundings
to produce ROS that kill the adjacent cancer cells and
pathogenic microbes. The BRET system can work in vivo even
in the deeper tissue, which overcomes the drawback of the deep
tissue penetration for PDT with light irradiation. To the best of
our knowledge, this is the first reported PDT system for
treating cancer and microbial infections that is independent of
light irradiation. This work opens a new therapy modality to
tumor and pathogen infections. We note that this strategy also
has potential application in light irradiation-dependent
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of the BRET system with recognition groups specifically
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ASSOCIATED CONTENT
* Supporting Information
Experimental details and additional figures. This material is
■
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available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
wangshu@iccas.ac.cn; libingliu@iccas.ac.cn
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors are grateful to the National Natural Science
Foundation of China (Nos. 21033010, 21003140, 90913014)
and the Major Research Plan of China (No. 2011CB932302,
2011CB808400, 2012CB932600).
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