Identification of ros produced by photodynamic activity of chlorophyll/cyclodextrin inclusion complexes

Article abstract of DOI:10.1111/j.1751-1097.2012.01238.x

Photodynamic therapy (PDT) is a way of treating malignant tumors and hyperproliferative diseases. It is based on the use of photosensitizer, herein the chlorophyll a (chl a), and a light of an appropriate wavelength. The interaction of the photosensitizer (PS) with the light produces reactive oxygen species (ROS), powerful oxidizing agents, which cause critical damage to the tissue. To solubilize chl a in aqueous solution and to obtain it as monomer, we have used cyclodextrins, carriers which are able to interact with the pigment and form the inclusion complex. The aim of this study is to examine which types of ROS are formed by Chl a/cyclodextrin complexes in phosphate buffered solution and cell culture medium, using specific molecules, called primary acceptors, which react selectively with the reactive species. In fact the changes of the absorption and the emission spectra of these molecules after the illumination of the PS provide information on the specific ROS formation. The ¹O₂ formation has been tested using chemical methods based on the use of Uric Acid (UA), 9,10-diphenilanthracene (DPA) and Singlet oxygen sensor green (SOSG) and by direct detection of Singlet Oxygen ( ¹O₂) luminescence decay at 1270 nm. Moreover, 2,7-dichlorofluorescin and ferricytochrome c (Cyt Fe³⁺) have been used to detect the formation of hydrogen peroxide and superoxide radical anion, which reduces Fe³⁺ of the ferricytochrome to Fe²⁺, respectively. For the first time, photodynamic activity in vitro of natural Chlorophyll a (Chl a) has been investigated evidencing which types of ROS are formed. Chl a has been solubilized in aqueous solution by means of various cyclodextrins forming inclusion complexes. The ROS production has been carried out in the system using specific molecules, called primary acceptors, which react selectively with the reactive species.

Full text of DOI:10.1111/j.1751-1097.2012.01238.x

Photochemistry and Photobiology, 2013, 89: 432–441
Identification of Ros Produced by Photodynamic Activity of Chlorophyll/
Cyclodextrin Inclusion Complexes
Barbara M. Cellamare1, Paola Fini2, Angela Agostiano1,2, Salvatore Sortino3 and Pinalysa Cosma*1,2
1Dipartimento di Chimica, Università degli Studi “Aldo Moro” di Bari, Bari, Italy
2CNR-IPCF, sez. Bari, Bari, Italy
3Laboratory of Photochemistry, Department of Drug Sciences, University of Catania, Catania, Italy
Received 11 July 2012, accepted 7 September 2012, DOI: 10.1111/j.1751-1097.2012.01238.x
ABSTRACT
Type-I mechanism involves hydrogen-atom abstraction or elec-
tron transfer between the excited sensitizer and a substrate,
yielding free radicals that can react with oxygen to form ROS
such as the superoxide radical anion. While, in a Type-II mech-
anism, singlet oxygen is generated via an energy-transfer pro-
cess during a collision of the excited sensitizer with triplet
oxygen (6).
Photodynamic therapy efficacy depends on photochemical and
photophysical properties of PS. Among the different classes of
compounds examined as potential photosensitizers in PDT, por-
phyrins and their analogous are considered the most important
because they have photochemical and photophysical properties
making them potentially suitable for PDT applications. For
example, porphyrins absorption spectra have intense bands in the
600–850 nm wavelength region where the maximum light depth
penetration into biological tissues is obtained (7).
The first step of PDT treatment consists in the selective incor-
poration of the PS into the target tissue. It is normally accepted
that tumor selectivity is improved with the increase of the PS
lipophilic or amphiphilic character. The drawback of using PSs
with such a property is that they have a high tendency to aggre-
gate giving rise to photoinactive forms of the PS, unable to pro-
duce ROS. Consequently, a suitable delivery system is required
to keep the PS as monomer in solution.
Among the large variety of amphipathic porphyrins, we have
carried out a systematic study of the combined use of a natural
chlorin, Chlorophyll a (Chl a), which is the most abundant pig-
ment in green plants, with cyclodextrins.
Photodynamic therapy (PDT) is a way of treating malignant
tumors and hyperproliferative diseases. It is based on the use
of photosensitizer, herein the chlorophyll a (chl a), and a
light of an appropriate wavelength. The interaction of the
photosensitizer (PS) with the light produces reactive oxygen
species (ROS), powerful oxidizing agents, which cause critical
damage to the tissue. To solubilize chl a in aqueous solution
and to obtain it as monomer, we have used cyclodextrins,
carriers which are able to interact with the pigment and
form the inclusion complex. The aim of this study is to exam-
ine which types of ROS are formed by Chl a/cyclodextrin
complexes in phosphate buffered solution and cell culture
medium, using specific molecules, called primary acceptors,
which react selectively with the reactive species. In fact the
changes of the absorption and the emission spectra of these
molecules after the illumination of the PS provide informa-
tion on the specific ROS formation. The 1O2 formation has
been tested using chemical methods based on the use of Uric
Acid (UA), 9,10-diphenilanthracene (DPA) and Singlet oxy-
gen sensor green (SOSG) and by direct detection of Singlet
Oxygen (1O2) luminescence decay at 1270 nm. Moreover, 2,7-
dichlorofluorescin and ferricytochrome c (Cyt Fe3+) have
been used to detect the formation of hydrogen peroxide and
superoxide radical anion, which reduces Fe3+ of the ferricyto-
chrome to Fe2+, respectively.
Chl a is insoluble in water because of the presence of a long
alcohol chain as an ester moiety (phytilic chain). The magnesium
atom in the center of the tetrapyrrolic macrocycle, on the other
hand, gives the amphiphilic property to the molecule.
INTRODUCTION
Photodynamic therapy (PDT) is an efficient alternative treatment
both for microbial infections and localized tumors and hyperpro-
liferative diseases. PDT can be used as an alternative or adju-
vant to classical therapies, such as radiotherapy, surgery and
chemotherapy (1–3). PDT is based on the combined action of a
photosensitizer (PS), visible light able to match the absorption
spectrum of the PS and endogenous oxygen (4). Following
excitation of PS to long-lived excited singlet and/or triplet
states, the target tissue is destroyed by reactive oxygen species
(ROS), generated in electron-transfer (Type-I mechanism) and
energy-transfer (Type-II mechanism) reactions (5). In detail, a
Cyclodextrins are cyclic oligosaccharides characterized by
different numbers of a-D-glucopyranose units linked together by
O-glycoside bonds of types 1–4. Cyclodextrin (CD) toroidal
shape, with an inner hydrophobic cavity and an external hydro-
philic surface, allows the formation of inclusion complexes with
a large number of organic apolar compounds. In fact, CDs are
well known as drug delivery systems in pharmacological applica-
tion. Consequently, the use of CDs as carriers of porphyrins can
be useful to shift the equilibrium aggregate-monomer in favor of
monomer and then to avoid the aggregation (8–11).
Our previous studies (8–11) have shown that the Chl a is effec-
tively solubilized in aqueous environment by means of various
*Corresponding author email: p.cosma@chimica.uniba.it (Pinalysa Cosma)
© 2012 Wiley Periodicals, Inc.
Photochemistry and Photobiology © 2012 The American Society of Photobiology 0031-8655/13
432

Photochemistry and Photobiology, 2013, 89 433
CDs. In these aqueous systems, Chl a results to be much more sta-
ble than when it is solubilized using alcohol as cosolvent (12–15).
Our studies indicate the formation of inclusion complexes
between Chl a and CDs characterized by different binding con-
stants and stoichiometry (9,11,16) depending on CD chemical nat-
ure taken into exam. Following the structural characterization
performed using different techniques (NMR, ITC, UV–Vis
absorption and emission), Chl a/CD systems have been tested as
photosensitizer. The experimental results (16,17) have been shown
that almost all Chl a/CD systems studied, excited by the absorp-
tion of visible light, relax through various photophysical pathways
damaging some cellular components leading to cell death
(11,16,18). It results that these systems can be considered effec-
tive as potential supramolecular systems for PDT applications.
However, for application in oncology, to have phototoxic sys-
tems is not enough: the systems have also to be not cytotoxic,
feature not required for applications in antimicrobic field. Studies
on cytotoxicity have shown that the CD which has the best char-
acteristics for oncological application results to be hydroxypropy-
lated-b-CD (HP-b-CD). This CD is the most effective in Chl a
solubilization in the cellular medium and also is that which has
the lowest cytotoxicity at a 10ꢀ3 M concentration (17). Higher
concentration than 10ꢀ3 M cannot be used because at increasing
of the concentration, all CDs result to be cytotoxic (19,20). At
this CD concentration, the highest feasible concentration of Chl
a as monomer is 10ꢀ5 M, that is the pigment concentration used
in this study.
emission spectra of these primary acceptors and therefore can be
used to demonstrate the production of ROS.
The formation of singlet oxygen in phosphate buffered solu-
tion (PBS) solutions and cell culture media (Type-II mechanism)
was tested and studied using the reaction of the 1O2 with uric
acid (UA) (25–27), Singlet oxygen sensor green® (28) and 9,10-
diphenylanthracene (29) (Chart S1). The superoxide radical anion
formation (Type-I mechanism) has been detected using its redox
reaction with ferricytochrome c (Cyt Fe3+), which produces char-
acteristic changes in the cytochrome c absorption spectrum (30).
Finally, the formation of hydrogen peroxide has been studied
using the reaction between the hydrogen peroxide and 2,7-di-
chlorofluorescin in the presence of peroxidase from horseradish
(31) (Chart S1).
MATERIALS AND METHODS
Chemicals. Chlorophyll a was extracted and purified from spinach leaves
using Omata and Murata method (32). Chl a stock solutions were stored
in acetone at ꢀ80°C. 2-HP-a-CD, 2-HP-b-CD, 2-HP-c-CD, heptakis-
(2,6-di-O-methyl)-b-cyclodextrin (DIMEB) and heptakis-(2,3,6-tri-O-
methyl)-b-cyclodextrin (TRIMEB) used for inclusion complexes have
been purchased from Fluka and used without further purification. Cyt
Fe3+, 9,10-diphenilanthracene (DPA), D2O, superoxide dismutase from
horseradish, UA and Sodium azide (NaN3) were purchased from Sigma
Company. 2,7-Dichlorofluorescin diacetate (DCFH-DA) and L-Histidine
have been purchased from Fluka, singlet oxygen sensor green (SOSG)
and 1,4-Diazabicyclo[2.2.2]octane (DABCO) from Molecular Probes and
Aldrich, respectively. Cell culture medium without serum was RPMI
1640M from Sigma-Aldrich.
Sample preparation. The inclusion complexes were prepared adding
to dry Chl a, different CD ethanolic solutions. After ethanol evaporation
under N2 flow, buffer (10ꢀ2 M, KH2PO4/KOH in a range of pH 5.5–9.5)
or cell culture medium was added to a final concentration of 10ꢀ5 M in
dye and 10ꢀ3 M in CD. Subsequently, ROS formation was tested using
different primary acceptors: ferricythocrome c at a concentration of
0.1 mg mLꢀ1; UA at a concentration of 10ꢀ4 M; SOSG at a concentra-
tion of 1.5 lM; activated 2,7-dichlorofluorescin (DCFH) at final concen-
tration of 40 lM; and finally DPA was used at a concentration of 25 lM.
For the test of H2O2, the activated DCFH was prepared from DCFH-DA
as follows 0.5 mL of DCFH-DA 1 mM (in ethanolic solution) was added
to 2 mL of NaOH solution 0.01 N and was stored at room temperature
for 30 min in the dark. The DCFH hydrolysate was then neutralized add-
ing 10 mL of sodium phosphate buffer 250 mM at pH 7.2 and was
stored in the dark before use. Immediately after the end of irradiation,
we added 1 mL of activated DCFH to the analyzing system (33). For
the test of H2O2, we prepared different types of solutions: Chl a/2-HP-b-
CD and only 2-HP-b-CD in phosphate buffer solution; Chl a/2-HP-b-CD
and only 2-HP-b-CD in cell culture medium; Chl a/2-HP-b-CD and only
2-HP-b-CD in phosphate buffer solution in which is present a little
amount of ethanol; Chl a/2-HP-b-CD and only 2-HP-b-CD in pure etha-
nol; Chl a in ethanol; Chl a in phosphate buffer solution with 10% of
ethanol. For each experiment, we prepared three solutions: two identical
Chl a/2-HP-b-CD solutions (one was irradiated and one was kept in the
dark) and a solution that was only CD irradiated. For the experiments
without CD, we prepared two identical Chl a solutions in which one
was irradiated and one was not irradiated. To verify the influence that
O2 has in ROS formation, the solution was degassed under argon flow
for 15 min.
Although the HP-b-CD is the most appropriate concerning the
combined effect of photo- and cytotoxicity, the study was
extended to various CDs to have a complete picture on the effect
of different CDs on ROS production. In particular, HP-a-CD,
HP-b-CD and HP-c-CD were used for studying the size effect,
while the methylated ones for comparing different methylation
degree.
Different methods for studying free radical–related processes,
as for example the electron paramagnetic resonance (EPR) spin
trapping spectroscopy, are difficult to use in cell culture media
because of the complexity of the solution media and/or require
the use of instrumentations present in specialized laboratories. In
fact, the presence, in these systems, of biological molecules
could promote the undesirable rearrangement of spin trap adducts
and/or their reduction into EPR silent species. This results in a
lower sensitivity of the method and in problems with the inter-
pretation of spin trapping data (21). The direct singlet oxygen
determination by measuring its luminescence decay at 1270 nm
in biological systems is possible, but full of difficulties because
of the poor signal–noise ratio in the used ranges of timescales
and requires a very sensitive detection systems (22,23). In the
laboratories where these sophisticated instruments are not avail-
able, the direct near-infrared (NIR)-luminescence detection of
produced singlet oxygen in vitro or in vivo systems often
requires the use of deuterium oxide, which increases the lifetime
of 1O2 and eliminates absorption of the 1270 nm luminescence
by H2O (24). Hence, the results do not reflect optical and photo-
physical conditions present in vitro or in vivo and so should not
be interpreted as successful singlet oxygen measurement in a
biological environment (24).
Measurement. Each vial containing the solution was illuminated with
a neon lamp (60 mW cmꢀ2). The solution absorption or emission spectra
were recorded at different times of illumination. Uric acid absorp-
tion spectra were recorded in the range of 250–800 nm (UA
kmax = 290 nm in aqueous solution). SOSG emission was registered at
528 nm (kex = 488 nm). Its maximum absorption peak was at about
500 nm. DPA fluorescence was recorded in the range of 410–450 nm
(kex = 403 nm). Its three characteristic absorption peaks were observed
in the wavelength interval of 350–450 nm. 2,7-dichlorofluorescein (DCF)
emission wavelength was at 525 nm (kex = 488 nm). In the absorption
spectra, the DCF peak (kmax = 503 nm) can be observed. The increase in
To avoid these limits in this study, the identification of differ-
ent ROS has been carried out by a different approach based on
the use of primary acceptors, molecules that selectively react
with these species. These reactions change the absorption or

434 Barbara M. Cellamare et al.
DCF emission was followed collecting the data every 2 min. Ferricyto-
chrome c presents a large absorption band in the range of 500–600 nm,
in buffer and in cell medium, in which two, not very intense, peaks at
520 and 550 nm can be observed. These two peaks become more intense
and welldefined when ferricytochrome c was reduced to ferrocytochrome
c. Every solution, except that regarded UA and DCFH, was lighted using
a cut-off glass filter (100% transmittance until 610 nm for ferricyto-
chrome c and 100% transmittance until 550 nm for the other).
The UV–Vis absorption spectra were collected using the spectropho-
tometers Varian CARY 3 and Varian CARY 5. The fluorescent measure-
ment, instead, was conducted using a spectrofluorimeter Varian CARY
Eclipse 68.
1.01
1.00
0.99
0.98
0.97
0.96
0.95
0.94
0.93
2-HP-α-CD
2-HP-β-CD
2-HP-γ-CD
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
Singlet oxygen measurements. The photogeneration of singlet oxygen
(1O2) was monitored by luminescence measurements in air-saturated
water solution. The samples were excited with the third harmonic of a
Nd–YAG Continuum Surelite II–10 laser (355 nm, 6 ns), using quartz
cells with a path length of 1.0 cm. Upon laser excitation, the emission of
singlet oxygen at 1.27 lm was probed orthogonally to the exciting beam
with a preamplified (low impedance) Ge-photodiode (Hamamatsu EI-P,
300 ns resolution) maintained at ꢀ196°C and coupled to a long-pass sili-
con filter (>1.1 lm) and an interference filter (1.27 lm). The signals
from the photodiode were initially captured by a Tektronix TDS 3032
digitizer, operating in pretrigger mode and then transferred to a personal
computer, controlled by Luzchem Research software operating in the
National Instruments LabView 5.1 environment. The energy of the laser
pulse was measured at each shot with a SPHD25 Scientech pyroelectric
meter. The temporal profile of the luminescence was fitted to a single-
exponential decay function with the exclusion of the initial portion of the
plot, which is affected by the scattered excitation and instrumental electri-
cal response.
Laser flash photolysis. The samples were excited with the third har-
monic of a Nd–YAG Continuum Surelite II-10 laser system (pulse width
6 ns Full width at half maximum , at k = 355 nm) and the excited solu-
tions were analyzed at a right angle geometry using a mini mLFP-111
apparatus developed by Luzchem Research. Briefly, the monitoring beam
was supplied by a ceramic xenon lamp and delivered through quartz fiber
optical cables. The laser pulse was probed by fiber that synchronized the
mLFP system with a Tektronix TDS 3032 digitizer operating in the pre-
trigger mode. The signals from a compact Hamamatsu photomultiplier
were initially captured by the digitizer and then transferred to a personal
computer that controlled the experiment with Luzchem software devel-
oped in the LabView 5.1 environment from National Instruments. The
energy of the laser pulse was measured at each laser shot by a SPHD25
Scientech pyroelectric energy monitor. Oxygen was removed by vigor-
ously bubbling the solutions with a constant flux of argon previously
passed through a water trap. The solution (in a flow cell of 1 cm path-
length) was renewed after each laser shot. The sample temperature was
295 2 K.
EtOH
2-HP-α-CD
2-HP-β-CD
2-HP-γ-CD
0
500
1000
1500
2000
2500
3000
3500
4000
Irradiation time (s)
0
500
1000
1500 2000
Irradiation time (s)
Figure 1. Time decrease of normalized absorption peak at 290 nm of
2500
3000 3500
4000
UA 10ꢀ4 M in the presence of Chl a 10ꢀ5 M with 2-HP-a-CD 10ꢀ3
M
(●), 2-HP-b-CD 10ꢀ3 M (○) and 2-HP-c-CD 10ꢀ3 (D). Inset: comparison
with Chl a (10ꢀ5 M)/AU (10ꢀ4 M) in ethanolic solution.
1
(attributable to a reduced production of O2 and/or an increase in
the quenching effects) and/or a protection of a part of the UA
molecule included into CD cavity. This second hypothesis is also
supported by the different behavior of UA in the presence of
CDs having different size cavity: in the presence of 2-HP-a-CD,
the CD having the smallest cavity size, the highest reduction in
UA absorbance has been observed.
However, this method cannot be used in the presence of cell
culture medium because of the overlapping of the cell culture
medium and UA UV–Vis absorption spectra. Moreover in the lit-
erature, it has recently been shown that UA is not a specific
probe for 1O2 because it is able to react also with O2˙ꢀ (36).
Therefore, the experimental results indicate in PBS the produc-
tion of 1O2 and/or O2˙ꢀ and in cell culture medium the unsuit-
ability of this method.
Singlet oxygen sensor green. Singlet oxygen sensor green is
reported in the literature as a highly selective singlet oxygen
fluorescent probe probably containing a fluorescein moiety bound
to an anthracene derivative. It is characterized by weak blue fluo-
rescence peaks at 395 and 416 nm under excitation at 372 and
393 nm, respectively. SOSG has also an intense absorption band
at 500 nm (28,37). The reaction with singlet oxygen gives rise
to a green fluorescence under excitation at 504 nm and emission
maxima at 525 nm, which has been assigned to the formation of
an endoperoxide generated by the interaction of 1O2 with the
anthracene moiety of SOSG (38). To reduce the self-production
RESULTS
Singlet oxygen
Uric acid. The singlet oxygen has been detected using the
method based on the fast reaction between UA and 1O2 leading
to allantoin formation. The reaction has been monitored by the
decrease of the UA absorption intensity in buffer solution at
290 nm (34,35).
1
of O2 by SOSG, the sample illumination was performed using a
The UA absorbance decreasing observed in different Chl a/
CD solutions in buffer phosphate at pH 7.5, illuminated at inter-
vals of 15 min, is reported in Fig. 1. No decrease in UA absorp-
tion peak was observed after the illumination of the same
solution in the absence of Chl a (data not shown).
550 nm cut-off glass filter (37).
Singlet oxygen sensor green was used to detect the production
1
of O2 by Chl a/CD systems in PBS at pH 7.5, in a mixture 1:1
of PBS at pH 7.5 and deuterium oxide and, finally, in cell cul-
ture medium (Fig. 2A). Furthermore, as it is known that the
inclusion or interaction of chromophore molecules with CD can
alter their fluorescence, the behavior of SOSG in the presence of
CD was also studied in the absence of the photosensitizer
(Fig. 2B).
The same study was also performed solubilizing the Chl a by
means of ethanol, without CD, in PBS at pH 7.5 containing UA
10ꢀ4 M and illuminating at time interval of 10 min. In this case,
the decrease in the UA absorbance intensity is steeper than that
obtained in the presence of CDs (inset of Fig. 1). It suggests the
An initial fluorescence decrease followed by
fluorescence rise, as the illumination dose was further increased,
a steady
1
possibility that CDs gave rise to a reduction in O2 concentration

Photochemistry and Photobiology, 2013, 89 435
1.05
1.00
0.95
0.90
0.85
0.80
PBS
1.00
0.95
0.90
0.85
0.80
0.75
0.70
PBS+D2O
Cell Culture Medium
A
0
10
20
30
40
50
60
Irradiation time (min)
SOSG
SOSG+2-HP-β-CD
SOSG+2-HP-β-CD+Chl
1.00
0.95
0.90
0.85
0.80
0.75
Figure 3. Time evolution of normalized fluorescence emission at
528 nm of SOSG 1.5 lM in the presence (◊),in the absence of O2 (○)
and in degassed solution (∇).
cell culture medium than in phosphate buffer probably due to the
presence of endogenous photosensitizers in the culture medium
(39). Moreover as expected, the D2O presence increases the O2
life time and gives rise to the highest increase in SOSG fluores-
cence intensity (Fig. 2A, trace -○-).
1
Data reported in Fig. 2A,B were used to calculate, for each
system, the DImax = I0ꢀImin where I0 is the fluorescence inten-
sity obtained at t = 0, before starting the sample illumination,
and Imin is the minimum value of fluorescence intensity,
observed at the end of the first decreasing part of experimental
data. The comparison between DImax values, obtained in the dif-
ferent systems provides further information about the production
of O2. In the system where there is a higher production of O2,
a higher amount of anthracene moiety is oxidized and hence
subtracted from the electron-transfer reaction between the anthra-
cene–fluorescein moieties. In this case, the competition with the
endoperoxide formation becomes important and the endoperox-
ide formation prevails over the intramolecular electron-transfer
reaction. This leads to a higher Imin value than in the case in
which higher amount of SOSG is subject to an electron-transfer
reaction. In fact, the lowest and highest values of DImax, 0.095
and 0.246, are obtained in the presence of Chl a 10ꢀ5 M and
B
0
10
20
30
40
50
60
Irradiation time (min)
1
1
Figure 2. Time evolution of normalized fluorescence emission at
528 nm of SOSG 1.5 lM in different condition: (A) in the presence of
Chl a 10ꢀ5 M and 2-HP-b-CD 10ꢀ3 M in PBS pH 7.5 (■), PBS:D2O 1:1
(○), Cell culture medium (Δ); (B) in the presence of Chl a 10ꢀ5 M/2-HP-
b-CD 10ꢀ3 M (Δ), 2-HP-b-CD 10ꢀ3 M (○), with no Chl a and 2-HP-b-
CD (■).
is evident in all traces of Fig. 2A,B. This initial decrease is simi-
lar to that obtained for SOSG when it is illuminated in the visi-
ble region in the absence of a PS and has already been ascribed
to the occurrence of a fast intramolecular electron transfer from
anthracene to fluorescein moiety, which happens after the
excitation of fluorescein to its singlet excited state (37). This
electron-transfer reaction, which quenches the SOSG fluores-
cence, competes efficiently with SOSG fluorescence until the
anthracene moiety is intact. Progressively, the 1O2, produced by
illumination, oxidizes the anthracene moiety, making it no longer
available for the deactivation pathway of the electron transfer;
hence, SOSG fluorescence starts increasing. The more effective
2-HP-b-CD 10ꢀ3
M
PBS:D2O 1:1(○), Fig. 2A, and with no
Chl a and 2-HP-b-CD (■) in cell culture medium, Fig. 2B,
respectively.
Figure 3 shows SOSG normalized fluorescence intensity as
a function of illumination time obtained in different conditions: a
degassed Chl a/CD solution, a Chl a/CD solution contained in a
tightly closed cuvette and in a open cuvette, where it is easy and
it is not the change of air, respectively. As expected, SOSG fluo-
rescence intensity has the highest value in the system where the
O2 concentration is the highest and where the air can be easily
exchanged.
1
the system to produce the O2 or to increase its life time is, the
more important the competition of SOSG fluorescence will be.
Our results indicate that the presence of HP-b-CD gives rise
to an increase in the self-production of 1O2 by SOSG, which is
much lower than the amount of singlet oxygen produced by Chl
a/CD; therefore, the CD effect on the SOSG efficiency, as selec-
tive singlet oxygen fluorescent probe, can be neglected.
1
9,10-Diphenylanthracene. The O2 production by Chl a/CD was
1
also tested by means of DPA, an efficient O2 acceptor forming
endoperoxide (DPAO2) with 80–100% yield through a (4 + 2)
cycloaddition (40,41). DPAO2 is thermostable up to about 80°C
and its formation can be followed spectroscopically by observing
the fluorescence intensity decrease in the range between 350 and
The comparison between data at the same illumination time,
reported in Fig. 2A, indicates a higher 1O2 production yield in

436 Barbara M. Cellamare et al.
450 nm (kexc at 403 nm) as a function of the irradiation time
(29).
1Chl + hm ?1Chl*
1Chl* ?3Chl*
K excitation
ki intersystem crossing
kf fluorescence
The solution was illuminated at 5 min intervals using the
same 550 nm cut-off glass filter used with SOSG, because also
1Chl* ? Chl + hmf
3Chl* ? Chl
kt triplet decay
1
DPA is able to self-produce O2 under irradiation (40). The mea-
3Chl* + 3O2 ?3O2
+
1Chl
kq collisional quenching
kD energy transfer
kd, disactivation
kc, chemical quenching
kp, physical quenching
3Chl* +3O2 ?1O2 +1Chl
1O2 ?3O2
surements have been carried out both in a phosphate buffer at
pH 7.5 and cell culture medium. Some preliminary studies were
performed on the DPA in the absence of the PS to check if the
interaction between DPA and CDs could influence our experi-
ments. As already reported in the literature, the addition of hy-
droxypropyl-cyclodextrins to DPA solutions results in a small
increase in DPA fluorescence intensity correlated to the forma-
tion of complex 1:1 (41). This effect is much lower than the
entity of the effect produced by the PS illumination and, there-
fore, negligible. Both in phosphate buffer and in the cell culture
medium, a decrease in DPA fluorescence intensity after the illu-
mination of solutions containing Chl a/CD was observed. The
dependence of this decrease on illumination time was used to
perform a study of the velocity of DPA degradation promoted by
its reaction with 1O2 in buffer and in cell medium. In PBS, the
degradation of DPA follows a first-order kinetics whose rate con-
1O2 + DPA ? DPAO2
1O2 + DPA ? DPA + 3O2
which, using the stationary state approximation and assum-
ing (kc + kp) [DPA]<< kd, provides the following first-order
kinetic:
ꢀd[DPA]/dt ¼ k0½DPAꢁ
where k0 ¼ ðkDkikcÞ=½kdðkrD þ kqÞðki þ kfÞꢁ:
In the case of cell culture medium, the presence of many
components potentially involved in various ways in the pro-
duction and quenching of 1O2 and hence in the DPA degrada-
tion process, probably makes the kinetic scheme much more
complex. In other studies, where the intracellular generated
1O2 was assessed by the quenching of DPA fluorescence, the
dependence on the illumination time was not studied and the
DPA fluorescence intensity was measured after extraction with
chloroform/methanol (2:1) from scraped cells (43). Therefore,
it is not possible, on the basis of our experimental data and
studies reported in the literature, to work out a kinetic scheme
able to explain the second-order kinetics experimentally
obtained.
stant, (8.99 0.05) 9 10ꢀ4 ꢀ1, is given by the slope of the
s
straight line obtained graphing ln (F/F0) as function of the illu-
mination time, where F and F0 are the DPA emission intensity at
500 nm at time t (illumination time) and time zero, respectively
(Fig. 4).
Similar first-order kinetics were also obtained studying trypto-
phan degradation induced by the 1O2 production by Photophrin
II, its precursor hematoporphyrin, tetraphenylporphine tetrasulfo-
nate, a synthetic porphyrin, and rose Bengal in phosphate buffer
(42).
In cell culture medium, instead, DPA degradation follows a
second-order kinetics. The slope of the linear trend obtained
Direct detection of 1O2. As all the used chemical probes for
determining 1O2 formation are not completely selective, even
though in the literature they are often supposed to be
(23,36,44,45), direct measurement of singlet oxygen 1270 nm
luminescence has been conducted. In Fig. 5 are reported the sin-
glet oxygen luminescence decay traces recorded in different sys-
tems containing Chl a at 10ꢀ5M. From the comparison of the
traces, it is clear that in general, there is a poor singlet oxygen
production for all the systems in which CDs are present, in
agreement with our previous results (16). This low singlet oxy-
gen production can be attributable both to the 1O2 quenching
process by OH moieties of ethanol and cyclodextrins (46–48),
and to the PS triplet state quenching caused by the presence of
Chl a aggregated forms, as evidenced by the Chl a triplet spectra
shown in the inset of Fig. 5 (49). In fact, signal of Chl a triplet
spectrum is practically absent in the solution containing HP-b-
CD (trace -○-) compared with the triplet signal in EtOH in which
Chl a is in monomer form (trace -●-).
graphing 1/[DPA] as function of the illumination time (inset
ꢀ1
Fig. 4) provided the rate constant, k = 51 1 M sꢀ1
.
In the case of the buffer, it is possible to suppose that the
first-order kinetics is the result of the following simplified kinetic
scheme, already proposed for tryptophan:
100000
0.0
80000
60000
-0.4
40000
20000
0
200
400
600
800 1000 1200 1400 1600 1800
-0.8
-1.2
-1.6
Irradiation Time (s)
Singlet oxygen quenchers. In all the analyzed Chl a/CD systems,
to confirm the singlet oxygen formation, sodium azide (NaN3),
DABCO and L-Histidine, well-known singlet oxygen quenchers
have been used (50–52). The measurement (Figs. S1 and S2) has
been evidenced that there is no great difference between curves
obtained with and without singlet oxygen quenchers, both at
1 mM and increasing the quencher concentration to 10 mM, indi-
cating that all quenchers do not play a protective role on the
primary acceptors.
0
200
400
600
800
1000
1200
1400
1600
1800
Irradiation Time (s)
Figure 4. First-order kinetic of fluorescence quenching of DPA 25 lM
registered at 500 nm in PBS in the presence of Chl a (10ꢀ5 M)/CD
(10ꢀ3 M). Inset: Second-order kinetic of fluorescence quenching of DPA
25 lM registered at 500 nm in cell culture medium in the presence of
Chl a (10ꢀ5 M)/CD (10ꢀ3 M).

Photochemistry and Photobiology, 2013, 89 437
0.04
0.03
0.02
0.01
0.00
-0.01
-0.02
-0.03
-0.04
40
35
30
25
20
15
10
EtOH
HP- -CD/PBS
350
400
450
500
550
Wavelength (nm)
PBS
Cell Culture Medium
0
20
40
60
80
100
Time (min)
0
5
10
Figure 6. Time evolution of DCF fluorescence intensity registered in
phosphate buffer solution and in cell culture medium for irradiated Chl a/
2-HP-b-CD system ([Chl a] = 10ꢀ5 M and [2-HP-b-CD] = 10ꢀ3 M).
Time (μs)
Figure 5. Singlet oxygen luminescence decay traces registered for differ-
ent systems containing Chl a at 10ꢀ5 M: (▬) HP-c-CD/Buffer, (- - -)
HP-c-CD/Cell culture medium, (∙∙∙∙) DMSO, (-∙∙-∙∙-) EtOH. Inset: Chl a
triplet spectra recorded in EtOH (-□-) and in HP-c-CD/Buffer (-○-).
Table 1. Comparison light/dark of DI/Dt relative to Chl a/2-HP-b-CD
system in different solubilization condition ([Chl a] = 10ꢀ5M, [2-HP-b-
CD] = 10ꢀ3M).
DI/DtLight (AU minꢀ1
)
DI/DtDark (AU minꢀ1
)
Hydrogen peroxide
Chl a/CD buffer
Chl a/CD medium
Chl a ethanol
Chl a/CD ethanol
Chl a 10% ethanol
Chl a/CD (EtOH)
buffer
0.080 0.002
0.271 0.001
0.047 0.004
0.029 0.001
0.140 0.002
0.051 0.001
0.064 0.0019
0.053 0.001
0.018 0.001
0.009 0.001
0.101 0.001
0.0463 0.0004
2,7-Dichlorofluorescin. DCFH is a xantenic dye that reacts with
H2O2 in the presence of peroxidase from horseradish (HRP).
DCFH, in the reduced form, is characterized by a low fluores-
cence intensity. It can be easily oxidized, for example by
H2O2, forming DCF, a highly fluorescent species with an emis-
sion peak at 535 nm (kecc = 485 nm) (31). Actually, the
increase of the emission peak at 525 nm cannot always be
associated with the presence of H2O2 in the system because of
the tendency of DCFH to self-oxidize and the absence of selec-
tively toward H2O2. To minimize the self-oxidation effect,
DCFH is prepared in a stable nonfluorescent diacetate form
(DCFH-DA), which, after activation by alkali, becomes high
fluorescent when reacts with ultramicro amounts of hydrogen
peroxide (53). We have prepared different type of solutions in
which Chl a/2-HP-b-CD, Chl a alone and 2-HP-b-CD alone
are dissolved in different media. The activated and neutralized
DCFH was added to all the solutions both those irradiated for
30 min and those kept in the dark, just before beginning the
fluorescence measures. This procedure was used to minimize
the DCFH self-oxidation process. In this way, DCFH immedi-
ately reacts with formed H2O2, oxidizing to DCF. The DCF
fluorescence increases at 525 nm recorded at every 2 min for
1 h are reported in Fig. 6 as example for the system Chl a/2-
HP-b-CD in phosphate buffer solution and in cell culture med-
ium. The slope of the linear trends registered for all examined
systems are reported in Table 1.
Superoxide anion radical
ꢂ
Ferricytochrome c. Superoxide anion radical ðO2 ꢀÞ reduces
ferricytochrome c (Cyt Fe3+) to ferrocytochrome c (Cyt Fe2+), as
below (30):
ꢂ
Cyt Fe3þ þ O2 ! Cyt Fe2þ þ O2
ꢀ
The oxidized species presents a large absorption band between
500 and 620 nm. The formation of the reduced form gives rise to
an increase in the absorbance intensity at 520 nm and at 550 nm,
which have been used in this study to follow the O2 formation.
Different mechanisms for the O2
posed:
ꢂ
ꢀ
ꢂ
ꢀ
formation have been pro-
1
3
Chl þ hm ! Chl ! Chl
ð1Þ
ð2Þ
ð3Þ
ð4Þ
3Chl þ Chl ! Chlꢂꢀ þ Chlꢂþ
As we can observe, DI/Dt values of the solutions in which the
dye is irradiated (DI/Dtlight) are always higher than the values
obtained keeping the solution in the dark. The slight DCF emis-
sion increase registered in not illuminated solutions can be attrib-
uted to the unavoidable self-oxidation effect. The data shown in
Table 1 indicate that the highest production of H2O2 is obtained
in the solution containing Chl a/CD solubilized with cell culture
medium.
Chl ꢂ ꢀ þ O2 ! Chl þ O2
ꢂ
ꢀ
ꢂ
1
ꢀ
Chl þ O2 ! Chl_þ þ O2
ꢂ
ꢀ
In alkaline solutions, the main process in facilitating O2 for-
mation is the electron transfer between the triplet state of PS and
its ground state [reactions (2) and (3)]

438 Barbara M. Cellamare et al.
the kinetics of Cyt Fe2+ formation and consequently of O2
ꢀ
ꢂ
At neutral pH and slightly acid, instead, a major role in the
rate of O2 formation is played by the singlet oxygen (reac-
tion (4)) (54).
ꢂ
ꢀ
production promoted by the PS illumination, solubilized using
different CDs. In all cases, it is possible to interpret the absor-
bance data trends with a first-order kinetic characterized by
kinetic constants having different values depending on the used
CD. In particular, it is possible to group together the behavior in
2-HP-a-CD, DIMEB and TRIMEB in culture medium (Fig. 9)
characterized by the lowest values of the kinetic constants:
k = (4.34 0.17) 9 10ꢀ5 sꢀ1 for 2-HP-a-CD, k = (4.43 0.38)
9 10ꢀ5 sꢀ1 for DIMEB and k = (2.48 0.15) 9 10ꢀ5 sꢀ1 for
TRIMEB. However, the kinetic constant values obtained in cell
culture medium for 2-HP-b-CD and 2-HP-c-CD are as follows:
k = (3.94 0.52) 9 10ꢀ4 sꢀ1 for 2-HP-c-CD and k = (1.64
0.58) 9 10ꢀ4 sꢀ1 for 2-HP-b-CD (Fig. 10).
The system Chl/CD/cyt was illuminated using a neon lamp
and a glass filter (which completely absorb up to 610 nm) to
avoid the action of the cytochrome as a PS itself (55). The cyto-
chrome reduction was monitored both in phosphate buffer at pH
7.5 and in cell culture medium in the presence of all CDs con-
sidered. In the cell culture medium, the Cyt Fe2+ formation
resulted more evident (Fig. 7).
Control experiments indicated that PS and light are essential
for the reduction in the cytochrome. In fact, absorption spectra of
Chl a/2-HP-b-CD/Cyt in cell culture medium at different times
from sample preparation, keeping the solution in darkness (Fig. 8)
indicate a lower production of reduced Cyt c in a time much
longer than that used for the experiments reported in Fig. 7.
The dependence of the increase in the absorbance intensity at
550 nm on the illumination time was used to perform a study of
The lowest value of the kinetic constants were obtained in the
presence of the CDs having the worst Chl a solubilization
-1.70
-1.75
-1.80
-1.85
-1.90
-1.95
t=0 min
t=10 min
0.8
t=20 min
t=30 min
t=40 min
t=50 min
t=60 min
0.6
t=80 min
t=100 min
0.4
0.2
0.0
0
20
40
60
80
100
Time (min)
400
500
600
700
800
Figure 9. First-order kinetic analysis of cytochrome c reduction medi-
ated by Chl a photosensitization in cell culture media. Reaction mixtures
contain 10ꢀ5 M Chl a in 10ꢀ3 M 2-HP-a-CD with 0.1 mg mLꢀ1 Cyt c.
Wavelength (nm)
Figure 7. Time evolution of UV–Vis absorption spectra of Chl
a
10ꢀ5 M/2-HP-c-CD 10ꢀ3 M/Cyt c 0.1 mg mLꢀ1 in cell culture medium
after different illumination time.
0.15
0.14
0.13
0.8
t=0
0.7
t=1 h
0.12
0.108
t=4 h
0.107
0.6
0.11
0.106
0.105
0.5
0.4
0.3
0.2
0.1
0.0
0.104
0.10
0.103
0.102
0.09
0.101
0.100
0.08
0.099
0
20
40
60
80
100
Irradiation Time (min)
0.07
0
20
40
60
80
100
Irradiation Time (min)
Figure 10. First-order kinetic analysis of cytochrome c reduction medi-
ated by Chl a photosensitization in cell culture media. Reaction mixture
contains 10ꢀ5 M Chl a in 10ꢀ3 M 2-HP-c-CD with 0.1 mg mLꢀ1 ferricy-
tochrome c. Inset: First-order kinetic analysis of cytochrome c reduction
mediated by Chl a photosensitization in PBS (Chl a 10ꢀ5 M/2-HP-b-CD
10ꢀ3 M/Cyt c 0.1 mg mLꢀ1).
400
500
600
700
800
Wavelength (nm)
Figure 8. Time evolution of UV–Vis absorption spectra of Chl
a
10ꢀ5 M/2-HP-b-CD 10ꢀ3 M/Cyt c 0.1 mg mLꢀ1 in cell culture medium
without illumination.

Photochemistry and Photobiology, 2013, 89 439
at Chl a 10ꢀ5 M. However, no significant differences were
observed at Chl a concentrations between 7.5 9 10ꢀ6 and
< 10ꢀ5 M (Data not shown).
The high tendency of Chl a to aggregate at increasing of its
concentration also in the presence of high CD concentration
makes difficult to extend this study to a wider range of
concentration.
1.2
1.0
0.8
0.6
0.4
0.2
0.0
At last, to obtain a conclusive proof of the involvement of
16
12
8
ꢂ
ꢀ
O2
in the Cyt Fe3+ reduction, the system Chl/CD/cyt has
been illuminated in the presence of active superoxide dismutase
from horseradish (SOD). As no changes have been obtained in
the Cyt c absorption spectra at 520 and 550 nm (data not
shown), in the presence of SOD, it is possible to conclude that
the formation of Cyt Fe2+ is actually due to the involvement of
pH5.5
pH7.0
pH9.0
4
0
6
7
8
9
pH
ꢂ
ꢀ
O2 (60).
0
20
40
60
80
100
Irradiation Time (min)
CONCLUSION
Figure 11. Cytochrome c reduction mediated by Chl a photosensitiza-
tion in cell culture media as function of pH: -□- at pH 5.5, -○- at pH 7.0
and -Δ- at pH 9.0. Reaction mixture contains 10ꢀ5 M Chl a in 2-HP-b-
The photodynamic activity of the system Chl a/CD has been
studied successfully in PBS and in cell culture medium using
mainly the chemical detection of ROS. Experimental results indi-
CD 10ꢀ3
M
with 0.1 mg mLꢀ1 ferricytochrome c. Inset: first-order
kinetic constants of Cyt c reduction mediated by Chl a photosensitization
in cell culture media as function of pH.
cate overall that 1O2, H2O2 and O2 are the main ROS pro-
ꢂ
ꢀ
1
duced by the studied system. In particular, the O2 production in
PBS and in cell culture medium has been tested using UA,
SOSG, DPA as primary acceptors and direct detection of 1O2
NIR-luminescence decay. The overlapping of the cell culture
medium and UA UV–Vis absorption spectra makes the use of
UA as primary acceptor in cell culture medium impossible, but
power and therefore in the solutions of 2-HP-a-CD, DIMEB and
TRIMEB where there is a large amount of Chl a aggregated
(7,8). The first-order kinetic analysis of the behavior in
phosphate buffer (inset of Fig. 10) has shown a higher kinetic
constant, for example the k is (2.90 0.63) 9 10ꢀ4 sꢀ1 for the
2-HP-b-CD system. This can be attributed both to the complex
matrix of cell culture medium that, containing different species,
could affect the kinetic behavior of Cyt c reduction and also to a
different pH value of the medium. To study the influence of
solution pH on the rate of reduction in Cyt c in the presence of
Chl a/CD inclusion complexes at a constant concentration, an
investigation has been conducted varying solution pH in the
range from pH 5.5 to pH 9.5. In Fig. 11, for example, the varia-
tion of Cyt c concentration in function of different illumination
times at different solution pH is reported. At slightly acid and
alkaline pH (pH 5.5, trace -□-, and pH 9.0, trace -Δ-), there are
small changes in the reduced Cyt c concentration after illumina-
tion. In neutral solution (pH 7), instead, the reduced species is
formed (trace -○- in Fig. 11). Plotting the first-order kinetic con-
stant as a function of solution pH (inset of Fig. 11), it can be
observed that the Cyt c reduction rate increases up to pH 7 and
then decreases. This behavior can be ascribed to the presence of
at least two different conformers of Cyt c in alkaline range of
1
not in PBS. SOSG and DPA methods both indicate a higher O2
production in cell culture medium than in phosphate buffer prob-
ably due to the presence of endogenous photosensitizers in the
cell medium. The kinetic analysis of DPA degradation promoted
1
by its reaction with O2 evidences first-order kinetics in PBS and
second-order kinetics in cell culture medium. Also, the direct
1
measurement by means of the O2 1270 nm luminescence decay
has evidenced the production of singlet oxygen even if in a small
amount.
ꢂ
ꢀ
The method for the detection of O2 based on its redox reac-
tion with Cyt c Fe3+ reveals the superoxide radical anion produc-
tion in all Chl a/CD systems with a first-order kinetics. The
lowest value of the kinetic constants has been obtained in the
presence of the CDs having the worst Chl a solubilization power
and therefore in the solutions of 2-HP-a-CD, DIMEB and TRIM-
EB where there is a large amount of Chl a aggregated.
Evidence of the H2O2 formation by Chl a/CD illumination
has been obtained using the DCFH method both in cell culture
medium and PBS.
The overall results obtained in this article and in a previous
one (11) induce us to hypothesize that Type-I pathways could
prevail to Type-II pathway in the ROS production.
In fact in a previous study of ours, in which the photoactivity
of these systems were tested on Jurkat cells, notwithstanding a
weak production of singlet oxygen, Chl a/CD complexes are able
to kill malignant cells only when they are illuminated (11). It
results that there is a remarkable ROS production, enough to pro-
mote cell death and that these ROS are mainly radicals, produced
by Type-I pathways and not by Type II, which is the pathway
involved in the singlet oxygen production.
ꢂ
ꢀ
pH characterized by different reaction rates with O2 (51,52). In
ꢂ
ꢀ
particular, the reduced reactivity of O2
toward the alkaline
forms of Cyt c could be due to a rearrangement of Cyt c struc-
ture affecting the electron-transfer process (56,57). On one hand,
it is possible that, at slightly acid and neutral pH, the protonation
of some amino acid residues along the channel toward the Cyt c
heme group facilitates the electron transfer speeding up the reac-
ꢂ
ꢀ
tion of O2 with protonated group (30). On the other, the con-
formational change of Cyt c at alkaline pH is accompanied by a
decreasing of the reduction potential with increasing pH, result-
ing in a reduced reactivity (58,59).
After the pH effect, also the change in the Chl a concentration
was studied. Reducing the concentration of PS at 5 9 10ꢀ6 M,
the amount of Cyt Fe2+ formed resulted lower than that obtained
Analogous indications have been obtained in this article by
means of the use of different ROS quenchers (NaN3, DABCO,
L-histidine, SOD) in agreement with procedures suggested in

440 Barbara M. Cellamare et al.
the literature (riferimenti vari). In general, in the case of 1O2
quenchers (61,62), a significative decrease in the lifetime of
singlet oxygen with different quenching rate constants is
observed. Whereas, superoxide dismutase is known to catalyze
the following dismutation reaction
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ꢂ
ꢂ
O2 ꢀ þ O2 ꢀ þ 2Hꢀ ꢀ! H2O2 þ O2
with quenching of superoxide anion species and H2O2
production.
Our experimental results indicate that the presence of singlet
oxygen quenchers does not affect the behavior of used ROS
chemical probes (Data in Supporting Information). This suggests
that 1O2 is produced in small amount and it is not the major
source of radical ROS implying thus that Type-I mechanism is
dominant. A confirmation of this hypothesis is provided also by
the very low Chl a triplet signal recorded in solution containing
CDs (inset of Fig. 5). Also, the use of SOD provides indication
of a prevalence of a photodynamic mechanism of Type I. In fact,
no changes have been obtained in the Cyt c absorption spectra in
the presence of SOD, indicating that the formation of Cyt Fe2+ is
ꢂ
due to the involvement of O2 ꢀ, produced by a Type-I pathway
(60).
Then, we may conclude that the photodynamic activity of Chl
a/CD systems occurred mainly via the Type-I mechanism.
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Acknowledgements—This study was supported by COFIN-MIUR2008
grants “Architetture ibride multifunzionali basate su biomolecole per
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del biomedicale.” We gratefully acknowledge the skillful and excellent
technical assistance of Sergio Nuzzo.
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Additional Supporting Information may be found in the online
version of this article:
Figure S1. Time evolution of normalized fluorescence emis-
sion recorded at 528 nm of SOSG 1.5 M in PBS solution (pH
7.5) containing Chl a 10ꢀ5 M and 2-HP-b-CD 10ꢀ3 M with dif-
ferent quenchers at concentration of 10 mM.
Figure S2. Time evolution of normalized fluorescence emis-
sion recorded at 500 nm of DPA 25 µM in PBS solution (pH
7.5) containing Chl a 10ꢀ5 M and 2-HP-b-CD 10ꢀ3 M with dif-
ferent quenchers at concentration of 10 mM.
Chart S1. Chemical structures of primary acceptors and sin-
glet oxygen quenchers used in this article.
Please note: Wiley-Blackwell are not responsible for the con-
tent or functionality of any supporting materials supplied by the
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directed to the corresponding author for the article.
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