Selective cell death by photochemically induced pH imbalance in cancer cells

Article abstract of DOI:10.1021/ja3122312

Singlet oxygen sensitized photodynamic therapy (PDT) relies on the concentration of oxygen in the tissue to be treated. Most cancer lesions, however, have poor vasculature and, as a result, are hypoxic, significantly hindering PDT efficacies. An oxygen-independent PDT method may circumvent this limitation. To address this, we prepared sulfonium salts that produced a pH drop within HCT 116 cells via the generation of a photoacid within the cytosol. This process was driven by one- or two-photon absorption (1PA or 2PA) of the endocytosed photoacid generators (PAGs). One of these PAGs, which had a significantly lower dark cytotoxicity and was more efficient in generating a photoacid, effectively induced necrotic cell death in the HCT 116 cells. The data suggest that PAGs may be an attractive alternative PDT modality to selectively induce cell death in oxygen-deprived tissue such as tumors.

Full text of DOI:10.1021/ja3122312

Communication
pubs.acs.org/JACS
Selective Cell Death by Photochemically Induced pH Imbalance in
Cancer Cells
Xiling Yue,† Ciceron O. Yanez,† Sheng Yao,† and Kevin D. Belfield*,†,‡
‡
†Department of Chemistry and CREOL, The College of Optics and Photonics, University of Central Florida, P.O. Box 162366,
Orlando, Florida 32816, United States
S
* Supporting Information
oxygen to the tumor but also hinders the delivery of
ABSTRACT: Singlet oxygen sensitized photodynamic
therapy (PDT) relies on the concentration of oxygen in
the tissue to be treated. Most cancer lesions, however, have
poor vasculature and, as a result, are hypoxic, significantly
hindering PDT efficacies. An oxygen-independent PDT
method may circumvent this limitation. To address this,
we prepared sulfonium salts that produced a pH drop
within HCT 116 cells via the generation of a photoacid
within the cytosol. This process was driven by one- or two-
photon absorption (1PA or 2PA) of the endocytosed
photoacid generators (PAGs). One of these PAGs, which
had a significantly lower dark cytotoxicity and was more
efficient in generating a photoacid, effectively induced
necrotic cell death in the HCT 116 cells. The data suggest
that PAGs may be an attractive alternative PDT modality
to selectively induce cell death in oxygen-deprived tissue
such as tumors.
complementary chemotherapeutic agents that are delivered
via the bloodstream.1b
Herein, an oxygen-independent means of inducing cell death
via PDT is shown. Instead of inducing singlet oxygen by
photosensitization, a pH imbalance was induced within the
cytosol of the cells that were targeted by a photoacid generator
(PAG), affording oxygen-independent PDT (OI-PDT, Figure
1).
he success of photodynamic therapy (PDT) requires the
Tcareful balance of three conditions that must be present in
the targeted cells at the time of therapy: (1) oxygen saturation
of the tissue, (2) sufficient PS concentration throughout the
lesion, and (3) sufficient intensity of the sensitizing light. Even
in a single gland such as the prostate, all three of these agents
are present in quite heterogeneous concentrations and doses.1
This has significantly complicated and compromised the
reproducibility of singlet oxygen photosensitized PDT (1O2-
PS PDT).
For over 100 years photodynamic oncotherapy has sought to
produce singlet oxygen in an oxygen-depleted environment.
Vasculature in healthy tissue is very well structured; the inner
walls of healthy vessels are conformed by well differentiated
endothelial cells. In contrast, tumor vessels have very poor
morphology and are conformed by immature cells in a mesh-
like architecture that confers a leaky property to the vessel. The
leaky character of these vessels generates a hypoxic and acidic
microenvironment that induces the production of positive or
negative regulators of angiogenesis.2
Figure 1. Two-photon (2P) PAG based PDT in anoxic tumor cells.
2P-PAG based PDT induces cell death by means of a pH imbalance.
The therapeutic agents were encapsulated sulfonium salts,
molecules that have traditionally been used as photoacid
generators (PAGs) in lithography and cationic polymerization.
The PAGs were incubated in human colorectal carcinoma
(HCT-116) cells and, once in the cytosol, were excited to
generate a photoacid that caused a pH imbalance within the
cell. These PAGs were tailor-made to absorb at longer
wavelengths.
Most commercially available PAGs have an absorption λmax in
the UV or deep-UV because their applications in lithography
require them to absorb at the shortest possible wavelengths.
Recently, a series of more conjugated, longer-wavelength-
absorbing PAGs were synthesized in our lab that were designed
to be efficient two-photon absorbing molecules.3 The
generation of a photoacid was induced by one- and two-
photon absorption (1PA or 2PA) of PAGs 1−3 (Figures 1 and
S1). To our knowledge, this is the first example of the use of
This hypoxic environment within a tumor often leads to poor
1
outcomes in O2-PS PDT because frequently there is a limited
amount of oxygen to excite. The minimal concentrations of
1
oxygen are quickly depleted upon O2-PS PDT, making it
extremely easy to saturate the irradiation dose upon treatment.
Furthermore, these extreme hypoxic events are often followed
by ischemia, which not only further compromises the flow of
Received: December 14, 2012
Published: January 30, 2013
© 2013 American Chemical Society
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Journal of the American Chemical Society
Communication
PAGs to cause cell death by generating a pH imbalance in the
cell.
The emergence of nonlinear (2PA) techniques has taken
advantage of the quadratic dependence that 2PA has on the
intensity of the incident light.4 This advantage can also be
exploited in PDT applications and require that the molecules
employed for therapy be efficient two-photon absorbers; i.e.,
the molecules need to have high 2PA cross sections. The
possibility of using such agents, along with deeper penetrating
longer wavelength light used in two-photon excitation, in
cancer lesions that are buried under sensitive, healthy tissue
(i.e., gliomas) makes these 2PA PAGs especially important.
Undoubtedly, the simplicity associated with generating photo-
acid by 1PA is also an advantage. One-photon photoacid
generation is a more efficient process, where the excitation
source needed is cheaper and easier to use. In exchange for
tissue penetration, a larger amount of targeted surface mass
within tissue can be covered at a faster rate.
Two-photon photoacid generation is a lower probability
process, because it is energy dependent and relies on more
elaborate pulsed lasers as energy sources. However, it has a
tremendous tissue penetration advantage, and the process is
confined to a smaller volume. Ideally, both methods can be
used simultaneously to maximize the possibility of success of
the OI-PDT process.
Originally, the synthesis of triarylsulfonium salts was
reported by Crivello and Lam, where the thermolysis of a
diphenyliodonium in the presence of a diphenylsulfide formed
the desired sulfonium salt.5 Recently, a more efficient,
microwave-assisted, synthetic strategy of triarylsulfonium salt
PAGs was reported.3 PAGs 1−3 were designed to exhibit high
2PA cross sections. Fluorene was chosen as the core structure
because of its high thermal and photochemical stability.6 Quite
advantageously, fluorene lends itself to ready substitution in its
2-, 7-, and 9-positions. In PAGs 1 and 2, stilbenyl motifs were
introduced (2- and 7-positions) to extend the π-conjugation.
Ultimately, two acceptor groups (triarylsulfonium and nitro)
were introduced for net structures of A-π-π-A (PAG 1) and A-
π-π-π-A (PAG 2).
To enhance the photoacid quantum yield per molecule, the
first approach was to incorporate two sulfonium salt motifs
onto the fluorenyl scaffold, such as in PAG 2 (Figure 2).
However, this molecule exhibited a very low photoacid
quantum yield (0.03). The high fluorescence quantum yield
of this PAG (0.80) indicated the molecule was undergoing
radiative decay (fluorescence) before it had a chance to form a
photoacid.
The direct photolysis of triarylsulfonium salts has been
reported to occur primarily from the first excited singlet state.
However, sensitization studies have shown that triplet
triarylsulfonium salts are also labile.7 Consequently, to increase
the probability of spin orbit coupling to induce intersystem
crossing, a nitro group was incorporated into the fluorene
backbone. As a result, the fluorescence quantum yield of the
sulfonium salt PAG 1 was significantly decreased (Table 1),
thereby reducing the radiative decay pathway.
Figure 2. Sulfonium salt 2PA PAGs structures.
Table 1. Photophysical Properties of PAGs
a
b
PAG
ΦF
ΦH+
δ710 (GM)
Φ
H+.δ (GM)
1
2
3
0.10 0.01
0.80 0.06
0.27 0.02
0.40 0.04
0.03 0.003
0.01 0.005
240 24
1275 130
−
96 10
38
4
−
a
Fluorescence quantum yields, ΦF, with diphenylanthracene in
b
cyclohexane as the standard.
nm with RhB+ as the indicator. δ, 2PA cross sections at 710 nm;
H+.δ, two-photon action cross-section of photoacid generation at 710
ΦH+, photoacid quantum yields at 350
Φ
nm.
photophysical properties would be incomplete and could lead
to erroneous interpretations.
A more useful value to compare the PAGs is the 2PA action
cross section of photoacid generation, given by the product of
photoacid generation quantum yield and the 2PA cross section
at a specific wavelength. On the basis of the 2PA action cross
section, the overall efficiency of PAG 1 was higher than that of
PAG 2. In a constant effort to improve the properties of these
molecules, other PAGs are currently being synthesized in the
lab that absorb at longer wavelengths and possess higher 2PA
cross sections. An example of this type of molecule is PAG 3.
The squaraine core has the advantage of having a linear
absorption λmax in the NIR and has been associated with high
2PA. A comparable figure of merit for one of the most widely
used PDT agents photofrin (singlet oxygen quantum yield ×
2PA cross section) illustrates the efficiency of the PAGs. In the
literature, the photofrin oxygen quantum yield is ca. 0.2, and its
2PA cross sections range from 10 to 15 GM.8 Based on these
values, the action cross section for photofrin would be at most 3
GM; significantly, the action cross section for PAG 1 is 30
times larger (96 GM, Table 1).
Pluronic F-127 has been widely used in drug delivery
applications to enhance the solubility of hydrophobic
substances such as anticancer drugs.9 Pluronic micelles are
known to be uptaken by MDCK cells by means of clathrin-
mediated endocytosis when present above the critical micelle
concentration.10 The hydrophobic character PAGs 1−3
facilitated their encapsulation in Pluronic F-127.11 Steady
state, linear absorption of solutions of Pluronic F-127-
Nitro-containing PAG 1 exhibited an increased photoacid
quantum yield (Table 1). The 2PA cross sections, however,
were found to be up to 5 times higher for PAG 2 than for PAG
1 (Table 1). This disparity in 2PA cross section values vs
photoacid quantum yield values makes it difficult to rank these
PAGs by their overall efficiencies. Using only one of these two
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Communication
encapsulated PAGs (PL PAGs) in PBS was measured (Figure
2). The PL PAGs were tracked through the vesicle maturation
process of endocytosis. After the micelles undergo endocytosis,
they can reach full endosomal maturation, reaching the
lysosomes, follow exocytosis before attaining the endolysoso-
mal stage, or buildup in other regions such as the mitochondria.
We mainly observed the accumulation of the PL PAGs in the
endosomes-lysosomes (even after 24 h of incubation, Figure
S2) in HCT 116 cells.
An ideal PAG for phototherapy would exhibit low
cytotoxicity when unexposed and induce a high percentage of
cell death upon irradiation. To assess the intrinsic toxicity of the
PAGs, cell viability assays were carried out in the dark (dark
viability) to avoid the production of a photoacid. The cells were
incubated with 1, 5, 10, and 15 μM solutions of PL PAG 1−3
for 24 h at 37 °C. The results indicated PL PAG 1 had the
lowest dark cytotoxicity throughout this concentration range,
followed by PL PAG 3 and PL PAG 2, respectively (Figure S3).
Based on these results, exposure experiments were
performed at 1, 5, and 10 μM for all PL PAGs. The most
appreciable changes in viability (from dark viability to
postexposure viability) were observed at 10 μM (Figure 3).
been acidified by the acidic character of the lysosomes. It was
estimated that a 10 μM concentration of PAG 1 would, at least,
generate 2.1072 × 10−6 M at the irradiation doses used for 80%
cell death. This would lead to a drop of intralysosomal pH to
≤4.5 (Figure S6).
Cell death induced by two-photon photoacid generation was
demonstrated in an experiment where HCT 116 cells were
incubated with PL PAG 1 for 24 h, followed by two-photon
irradiation (710 nm, 2.0 mW/cm2). After irradiation, the cells
were incubated with propidium iodide to assess the proportion
of cells that underwent necrotic cell death (Figure 4). A control
Figure 4. HCT 116 human colorectal carcinoma cells incubated for 24
h with PL PAG 1 (10 μM) and irradiated at 710 nm (70 fs pulses, 80
MHz repetition rate, 2.0 mW/cm2). Generation of photoacid induced
by 2PA promoted necrosis in HCT 116. Residual PAG 1 fluorescence
illustrated PAG uptake. Cells were unaffected by nonirradiated PAG or
irradiation w/o PAG. Scale bar 50 μm.
sample to determine the effect of the irradiation conditions on
the cell was performed by irradiating cells that had not been
incubated with the PAG. The micrographs showed excellent
cell morphology (DIC) and high viability (low fluorescence
intensity of the PI channel) of the cells for up to 24 h following
irradiation. A second control, in which cells were incubated
with PL PAG 1 but were not irradiated, showed adequate cell
uptake and no detectable cell death via the necrotic pathway.
Irradiated cells showed significant cell swelling and loss of
membrane potential as indicated by the uptake of propidium
iodide. All the cells in the exposed area appeared to die by
necrosis, which is expected when such a grave physiological
imbalance takes place.
Time-lapsed micrographs in Figure S5 show this progressive
change in cell morphology following the generation of
photoacid. Loss of cell adhesion (green arrows) is followed
by a “blebbing”-like activity (yellow arrows). The integrity of
the nuclei in these cells is a sign that chromatin condensation is
not occurring and hence the process is not apoptotic. What
followed was significant cell swelling that is characteristic of
necrosis (blue arrows). Despite the low fluorescence quantum
yield, PAG 1 was fluorescent enough to allow visualization of its
uptake and colocalization with LysoTracker Red (Figures S2
and S5). The high degree of colocalization suggests that it was
mainly localized in the lysosomes.
Figure 3. Postexposure viability of HCT 116 cells incubated with PL
PAGs 1 and 3. PL PAG 1 exhibited the lowest intrinsic (dark)
cytotoxicity and highest postexposure cytotoxicity.
PL PAG 1 showed the best results, promoting a drop from 90%
viability to 20% viability after 900 s of exposure. Even at these
relatively high concentrations, PL PAGs 2 and 3 failed to
induce a significant drop in cell viability. This is consistent with
their photoacid quantum yields (Table 1), which are much less
efficient in producing a photoacid than PAG 1; i.e., the
induction of cell death was proportional to the amount of acid
generated.
A correlation of irradiation and decrease in lysosomal pH was
demonstrated by the aid of LysoSensor Green. LysoSensor
Green has been reported to monitor acidic pH within cells.12
This dye is known to increase its fluorescence quantum yields
when in acidic compartments. An increase in fluorescence
intensities as a function of exposure dose was, indeed, observed
in cells that were coincubated with PL PAG 1 (10 μM) and
LysoSensor Green (Figure S4). This indicates a drop in
intralysosomal pH (of the already acidic compartments)
followed by irradiation in HCT 116 cells previously incubated
with PL PAG 1 (10 μM). The drop in fluorescence intensities
as a function of irradiation dose in control cells can be
attributed to the photobleaching of the pH indicator that had
The use of photoacid generators to induce cell death by
creating a grave pH imbalance in cells has not been reported
prior to this work. We demonstrated that sulfonium-based
PAGs can be used to selectively induce cell death upon
photoexcitation. This opens the possibility of photochemically
inducing cell death in an oxygen-independent manner. More
specifically, PL PAGs have induced necrotic cell death via
generation of photoacid in the lysosomes in HCT 116 cells.
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(12) Tian, Y.; Su, F.; Weber, W.; Nandakumar, V.; Shumway, B. R.;
Jin, Y.; Zhou, X.; Holl, M. R.; Johnson, R. H.; Meldrum, D. R.
Biomaterials 2010, 31, 7411.
Acid was generated by both 1PA and 2PA, which means cell
death can be induced by either method. In order to achieve
deep tissue penetration, near-IR two-photon excitation is
particularly attractive. Thus, the ability to induce two-photon
photoacid generation of PAGs in cells is significant. PL PAG 1
is a versatile compound that can be used to exploit the
advantages of one- or two-photon photoacid-based PDT. These
results lay the foundations for the use of PAGs in OI-PDT.
ASSOCIATED CONTENT
* Supporting Information
■
S
Supplementary images, detailed experimental procedures, and
complete molecule characterization for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
belfield@ucf.edu
■
Funding
No competing financial interests have been declared.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge the National Institute of Biomedical Imaging
and Bioengineering of the National Institutes of Health (R15
EB008858-1) and the National Science Foundation (CHE-
0832622 and ECCS-0925712) for support of this work.
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