Intracellular modulation of excited-state dynamics in a chromophore dyad: Differential enhancement of photocytotoxicity targeting cancer cells

Article abstract of DOI:10.1002/anie.201411962

The photosensitized generation of reactive oxygen species, and particularly of singlet oxygen [O₂(a¹Δ_g)], is the essence of photodynamic action exploited in photodynamic therapy. The ability to switch singlet oxygen generation on/off would be highly valuable, especially when it is linked to a cancer-related cellular parameter. Building on recent findings related to intersystem crossing efficiency, we designed a dimeric BODIPY dye with reduced symmetry, which is ineffective as a photosensitizer unless it is activated by a reaction with intracellular glutathione (GSH). The reaction alters the properties of both the ground and excited states, consequently enabling the efficient generation of singlet oxygen. Remarkably, the designed photosensitizer can discriminate between different concentrations of GSH in normal and cancer cells and thus remains inefficient as a photosensitizer inside a normal cell while being transformed into a lethal singlet oxygen source in cancer cells. This is the first demonstration of such a difference in the intracellular activity of a photosensitizer.

Full text of DOI:10.1002/anie.201411962

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201411962
German Edition: DOI: 10.1002/ange.201411962
Photodynamic Therapy
Hot Paper
Intracellular Modulation of Excited-State Dynamics in a Chromophore
Dyad: Differential Enhancement of Photocytotoxicity Targeting
Cancer Cells**
Safacan Kolemen, Murat Is¸ık, Gyoung Mi Kim, Dabin Kim, Hao Geng,
Muhammed Buyuktemiz, Tugce Karatas, Xian-Fu Zhang, Yavuz Dede, Juyoung Yoon,* and
Engin U. Akkaya*
Abstract: The photosensitized generation of reactive oxygen
species, and particularly of singlet oxygen [O2(a1Dg)], is the
essence of photodynamic action exploited in photodynamic
therapy. The ability to switch singlet oxygen generation on/off
would be highly valuable, especially when it is linked to
a cancer-related cellular parameter. Building on recent findings
related to intersystem crossing efficiency, we designed a dimeric
BODIPY dye with reduced symmetry, which is ineffective as
a photosensitizer unless it is activated by a reaction with
intracellular glutathione (GSH). The reaction alters the
properties of both the ground and excited states, consequently
enabling the efficient generation of singlet oxygen. Remark-
ably, the designed photosensitizer can discriminate between
different concentrations of GSH in normal and cancer cells
and thus remains inefficient as a photosensitizer inside
a normal cell while being transformed into a lethal singlet
oxygen source in cancer cells. This is the first demonstration of
such a difference in the intracellular activity of a photosensi-
tizer.
selectivity, but painful edemas are very common side-effects
that are due to photosensitization in an unrelated part of the
body. This fact prompted many in the field to propose the use
of “activatable photosensitizers”,[3] which are to be turned on
only when cancerous tissue or cells are encountered; other-
wise they are to remain in a passive state in which they are not
capable of photosensitization even when the molecule
happens to absorb a photon of stray light.
Earlier examples[4] made use of pH differences between
the extracellular medium of the tumors and normal tissues.
The difference (1–1.5 pH units) is actually found in the
extracellular environment, not in the cytoplasm of the cells,[5]
although the distinction is somewhat blurred in the current
literature. The intracellular glutathione (GSH) concentration,
which is reportedly higher in cancer cells,[6] was also exploited
as a modulator;[7] however, no differences in the photo-
sensitization activity in cancer cells have been shown explic-
itly thus far.
Furthermore, the two-module approach renders the
activatable photosensitizer too large, in some cases making
it cell-impermeable, thus limiting their potential. Also,
extracellular GSH/biothiol activation reactions are non-
specific and do not improve the selectivity of photosensitiza-
tion.
P
hotodynamic therapy, which is based on the photosensi-
tized generation of singlet oxygen by irradiation in the visible/
near-IR region of the spectrum, has been recognized as a non-
invasive cancer treatment modality of great potential for
some time;[1] however, its potential has not been fully realized
owing to a number of limitations, both practical and
fundamental.[2] In principle, it should be possible to localize
irradiation to the area of the tumor, thus increasing spatial
To incorporate GSH responsiveness into a photosensitizer,
we made use of a recent finding in our laboratories,[8] dimeric
BODIPY dyes with unexpectedly high intersystem crossing
(ISC) efficiencies[9] and low dark toxicity. The latter charac-
[*] Dr. S. Kolemen, Dr. M. Is¸ık, Prof. E. U. Akkaya
UNAM-Institute of Material Science and Nanotechnology
Bilkent University
M. Buyuktemiz, Prof. Y. Dede
Department of Chemistry, Gazi University
Ankara 06500 (Turkey)
Ankara 06800 (Turkey)
E-mail: eua@fen.bilkent.edu.tr
T. Karatas, Prof. E. U. Akkaya
Department of Chemistry, Bilkent University
Ankara 06800 (Turkey)
Dr. M. I¸sık
Department of Metallurgical and Materials Engineering
Bingol University
Bingol 12400 (Turkey)
[**] E.U.A. gratefully acknowledges support from TUBITAK (112T480).
J.Y. acknowledges a grant from the National Research Foundation of
Korea (NRF) funded by the Korean government (MSIP,
G. M. Kim, D. Kim, Prof. J. Yoon
Department of Chemistry and NanoScience
Ewha Womans University
2012R1A3A2048814). Y.D. thanks TUBITAK for funding through
project 110T647, and M.B. thanks TUBITAK for a scholarship.
ULAKBIM TR-GRID is acknowledged for computing resources. We
are also grateful to Bora BilgiÅ for designing the cover art.
Seoul 120-750 (Korea)
E-mail: jyoon@ewha.ac.kr
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201411962.
H. Geng, Prof. X.-F. Zhang
Department of Chemistry & Center of Instrumental Analysis
Hebei Normal University of Science and Technology
Qinhuangdao, Hebei Province 066004 (China)
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teristic is most likely due to a lack of heavy atoms, such as
bromine or iodine, in the structure. The unique excited-state
(a doubly substituted tetraradical, DS-TR) properties of these
bis(BODIPY)s is dictated by the orthogonal arrangement of
the electronically similar subunits possessing a degenerate
pair of HOMOs and LUMOs.[9] When this similarity or
orthogonality was altered, the ISC efficiency decreased
significantly.[10]
(DCM). As the conjugate addition product was the expected
outcome, we were surprised by the actual product, reduction
product 5r (Figure 2). The product was characterized by
HSQC NMR spectroscopy and high-resolution mass spec-
trometry (Supporting Information, Figures S11–15, S22, and
S23). Considering the ease of preparation, the reduced
product 5r was used as model compound for GSH activation.
Electronically, a reduction of the styryl double bond or GSH
conjugate addition to the same bond have the same effect.
Our experiments with mercaptoethanol in aqueous solution
showed that under these conditions, the
With these considerations in mind, we designed target
molecule 5. The synthesis is straightforward (Figure 1).
thiol agent adds to the double bond of
5m as expected (Figure 2; supported by
NMR and HRMS data, Figures S16 and
S24). Furthermore, we demonstrated
that the GSH adduct was obtained on
treating 5 with GSH (HRMS and NMR
data; Figures S25, S27 and S28). In
biological media and inside cells, GSH
adduct formation is the most likely
outcome.
For an in-depth analysis of the
electronic differences between 5 and
5r, we carried out computational stud-
ies: Frontier orbital plots and energies
of 5 and 5r are depicted in Figure 3.
Interestingly, the LUMO of 5r corre-
sponds mainly to the LUMO of the MP
module. Therefore, after S0!S1 excita-
tion, a charge transfer (CT) transition of
the HOMO!LUMO type occurs from
the unsubstituted BOD1 moiety to the
electron-deficient MP ring. This
(through-space) CT transition is the
reason of ISC. A significant amount of
spin–orbit coupling is central to changes
of the spin state. Whenever orbitals
taking part in a CTexcitation are placed
on orthogonal and separated donor–
Figure 1. Synthetic route for compound 5. DCM=dichloromethane, TEA=triethylamine, TFA=
trifluoroacetic acid.
Extension of conjugation on one of the BODIPY dyes results
in a dissymmetric dimer. Consequently, a degenerate set of
HOMOs and LUMOs is not available, giving rise to a non-
DS-TR state and resulting in poor ISC with very low
photosensitization of molecular oxygen on excitation,
which, together with other non-radiative processes, leads to
effective quenching of the excited state. Glutathione (GSH),
which is present at higher concentrations in cancer cells, is
expected to add to the styryl double bond, electronically
isolating the methylpyridinium (MP) substituent. The new
charge-transfer system generated this way is expected to
enable effective photosensitization.
Previous work suggested
a low photosensitization
capacity for compound 5, which is electronically equivalent
to two BODIPY dyes with no particular reasons for effective
intersystem crossing. For initial experimental verification of
the facilitated ISC upon electronic isolation of the MP
component by a thiol addition, we carried out a reaction with
Figure 2. Conversion of compound 5 into 5r or 5m on reaction with
mercaptoethanol in DCM and in aqueous buffer solutions, respec-
tively.
compound
5 and mercaptoethanol in dichloromethane
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Figure 4. Electronic absorption spectra of 3, 4, 5, 5r, and 5m in DCM.
Figure 3. Orbital energies (eV) of 5 and 5r relevant to the CT excitation
from BODIPY to MP. The inset shows the deviation from planarity for
MP (which leads to a lack of extended conjugation of BOD2) in 5r.
Figure 5. a) The T1–Tn transient absorption spectra. b) Decay of the
triplet state T1 of 5r in argon-saturated CH3CN with laser excitation at
355 nm (the absorbance at 355 nm is 0.402).
acceptor moieties (HOMO and LUMO of 5r in Figure 3), the
electronic transition is accompanied by a large change in
orbital angular momentum, which should be compensated by
a change in the spin angular momentum, hence facilitating
singlet–triplet hopping.
(Table S5; for fluorescence spectra and other photophysical
parameters, see Figures S30–32 and Tables S1 and S2).
To elucidate the excited-state dynamics of both the
activatable photosensitizer 5 and the “activated” form 5r,
we carried out time-resolved absorbance and fluorescence
studies (Figure 5; see also Figure S33). In any solvent studied,
access to the triplet state of compound 5 seemed to be very
inefficient. Even in relatively non-polar organic solvents, the
T1 signal was negligible. However, for compound 5r, the
triplet manifold seemed to be easily accessible. In THF
(Figure S33), the triplet quantum yield was determined to be
14%, and in n-hexane/THF (5:1), it was 47% (Figure S33,
Table S3). In water and MeOH, the triplet signal vanished
because of compound aggregation in these solvents
(Table S3). The triplet lifetimes of 5r at 425 nm in argon-
saturated CH3CN and THF are 8.76 ms and 27.6 ms, respec-
tively, which are long enough for reactions of the triplet
excited state to occur.
For compounds 5 and 5r, the relative efficiencies of singlet
oxygen generation were compared by using the trap com-
pounds 1,3-diphenylisobenzofuran (DPBF) for organic media
and 2,2’-(anthracene-9,10-diyl)bis(methylene)dimalonic acid
(ADMDA) for aqueous solutions (Figures S36–44). The
positive control compound 3 is a rather active photosensitizer
of dissolved oxygen. As expected, owing to the shorter
lifetime of singlet oxygen in aqueous solutions, the response
of the trap was slower in a buffer/acetonitrile mixture;
however, the difference between 5 and 5r was equally
pronounced in this solvent. The singlet oxygen quantum
In line with the requirements for angular momentum
change, for 5r, the donor MO lies in the yz plane and the
acceptor MO in the xy plane (Figure 3). Our calculations
suggest that BOD2 is transformed into a spacer upon
conversion of 5 into 5r. The effect of structural spacers on
the electronics of CT transitions was recently discussed,[11]
and the absence of spacers was shown to diminish the CT
nature of excitation in twisted chromophores. It is noteworthy
that the selection of the MP group as the acceptor was
decisive as an analysis of the CT photophysics revealed that
the LUMO of the MP moiety just fits below the LUMO + 1
orbital, which mainly corresponds to the LUMO of the
BODIPY moiety (Table S8). Therefore, the reaction with
GSH tunes the electronic structure and hence the excitation
properties of the orthogonal bis(BODIPY) moiety by con-
verting it into an ISC agent operating by CT.
The changes in the absorption spectra are in accordance
with the expectations (Figure 4). Compound 5 has two distinct
peaks in the visible region, corresponding to two distinct
BODIPY structures. The quaternary pyridinium moiety
extends the p-conjugation for one of the orthogonal
BODIPY units, leading to a longer-wavelength absorption
band with a peak at 607 nm. The reduced form 5r displays
a single peak near 510 nm. These features could be repro-
duced by modeling the absorption spectra of 5 and 5r
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yields for 5r were also determined in aqueous micellar
systems, yielding values of 24% and 36%, depending on the
type of surfactant used (Figures S45, 46 and Table S4). The
generation of singlet oxygen with compounds 5r and 5m was
also independently confirmed by 1O2 phosphorescence at
1270 nm (Figure S47). As expected, 5 itself does not show
such emissions.
Cell culture studies were also highly revealing. First, HeLa
cells were incubated with 5 and 5r for four hours. After
washing with Dulbecco’s phosphate-buffered saline (DPBS)
solution, the cells were irradiated with a green light-emitting
diode (LED) for 30 minutes, incubated for another three
hours, and treated with the nuclear stains 4’,6-diamidino-2-
phenylindole (DAPI) and an Annexin V–AF594 conjugate
(red-fluorophore-labeled apoptosis marker; AF = Alexa
Fluor). The control groups were incubated in the dark
under identical conditions. The cell viabilities were deter-
mined by a standard MTT assay (Figure 6).
Figure 7. Confocal microscopy images of HeLa cells incubated with 5
or 5r for four hours. After washing with DPBS, the cells were irradiated
with green LED light for 30 minutes, incubated for another three
hours, and stained with Annexin V–AF594 (apoptosis marker). For
nucleus staining, DAPI (1 mgmLꢀ1) was applied as a co-stain for
30 minutes. a) DAPI (lex =405 nm, lem =4430–455 nm). b) 5, 5r
(lex =473 nm, lem =490–590 nm). c) Annexin V–AF594 (lex =559 nm,
lem =575–675 nm). d) DIC (differential interference contrast).
e) Merged images. Scale bar: 10 mm.
clearly confirmed that apoptosis took place on irradiation
when the cells had been incubated with 5 or the positive
control compound 5r (Figure 7 and Figure S48). Annexin V is
a selective probe for cells undergoing apoptosis where
phosphatidyl serines are exposed on the outer leaf of the
bilayer structure. The green fluorescence of the cytosol is due
to the reagent itself or its GSH adduct. Flow cytometry
studies (Figure S49) also clearly corroborated apoptotic
changes; in the presence of 5 and 5r, incubation of the cells
under irradiation resulted in a large fraction of cells in early
and late apoptotic states, and normal cells and cancer cells
were also compared in terms of their survival rate (Figure 8).
The activatable probe 5 showed a large difference in terms of
activation, as indicated by the survival rate quantified by an
MTT assay [IC50 values: 280 ngmLꢀ1 (5) and 32.6 ngmLꢀ1
(5r)]. Normal NIH 3T3 cells had a 80% survival rate on
Figure 6. Photocytotoxicity of the sensitizers 5 (blue) and 5r (red) as
demonstrated by the MTT assay. The cultured HeLa cells were kept
either in the dark (a) or illuminated with a green (520 nm) LED array
(b).
Even at very low concentrations of pre-activated sensi-
tizer 5r, a significant decrease in the cell viability was
observed (red bars in Figure 6b). For compound 5, cell
death was also detected, which suggests that 5 was intra-
cellularly transformed into its active form (blue bars in
Figure 6b). No statistically significant changes were observed
when the cells were kept in the dark, in the presence of the
same amounts of photosensitizers 5 or 5r (Figure 6a).
A first set of confocal microscopy images (Figure 7a)
show that the blue-emitting DAPI stain was localized in the
nuclear region. A comparison of 5 and 5r under irradiation
and in the dark revealed signs of nuclear condensation and
fragmentation, indicative of apoptosis. Specific staining of
apoptotic membranes using red fluorescent Annexin V
Figure 8. Photocytotoxic effects of 5 (blue; 280 ngmLꢀ1) and 5r (red;
280 ngmLꢀ1). Cells were incubated with 5 or 5r for four hours and
irradiated with green LED light. Cytotoxic effects were examined by an
MTT assay. Normal cells: NIH 3T3, WI38VA13; cancer cells: HeLa,
SK Hep 1.
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[2] a) Z. Huang, Technol. Cancer Res. Treat. 2005, 4, 283; b) P.
incubation with 5 followed by photosensitization. Under the
same conditions, HeLa cells displayed a survival rate of only
38%. The observed differential activation is a manifestation
of elevated GSH concentrations in cancer cells. Addition of
the specific GSH synthesis inhibitor buthionine sulfoximine
(BSO) led to a concentration-dependent decrease in the
photodynamic activity against HeLa cells (Figure S50).
We also determined the GSH levels in different cell types
using a specific glutathione assay kit (Figure S51). The cancer
cells that we studied had up to five-fold higher concentrations
of GSH, which is in accordance with the observed photo-
dynamic effects. To the best of our knowledge, this is the first
ever demonstration of selective GSH-dependent intracellular
activation resulting in enhanced photocytotoxicity in cancer
cells.
In conclusion, an elevated concentration of GSH was
shown to cause the differential activation of a photosensitizer
that was designed, synthesized, and characterized in this
work. Irradiation of this photosensitizer with LED light leads
to a significantly higher cytotoxicity in cancer cells than in
normal cells. Cell culture studies also revealed that the
photodynamic action thus initiated triggered apoptotic cell
death. We are currently working on the application of the
findings of this proof-of-principle study in the practice of
photodynamic therapy of cancer.
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Received: December 12, 2014
Revised: January 28, 2015
Published online: && &&, &&&&
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Angewandte
Communications
Communications
Photodynamic Therapy
S. Kolemen, M. Is¸ık, G. M. Kim, D. Kim,
H. Geng, M. Buyuktemiz, T. Karatas,
X.-F. Zhang, Y. Dede, J. Yoon,*
E. U. Akkaya*
&&&& — &&&&
Intracellular Modulation of Excited-State
Dynamics in a Chromophore Dyad:
Differential Enhancement of
Selective switch: A dimeric BODIPY dye
with reduced symmetry is ineffective as
a photosensitizer unless it is activated by
a reaction with intracellular glutathione
(GSH). Staining with red-fluorescent
Annexin V shows that the photosensitizer
is preferentially switched on in cancer
cells, which feature a higher GSH level
than normal cells.
Photocytotoxicity Targeting Cancer Cells
6
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