Monitoring singlet oxygen and hydroxyl radical formation with fluorescent probes during photodynamic therapy

Article abstract of DOI:10.1111/j.1751-1097.2009.00555.x

Singlet oxygen (¹O₂) is the primary oxidant generated in photodynamic therapy (PDT) protocols involving sensitizers resulting in type II reactions. ¹O₂ can give rise to additional reactive oxygen species (ROS) s

Full text of DOI:10.1111/j.1751-1097.2009.00555.x

Photochemistry and Photobiology, 2009, 85: 1177–1181
Monitoring Singlet Oxygen and Hydroxyl Radical Formation with
Fluorescent Probes During Photodynamic Therapy
Michael Price1, John J Reiners2, Ann Marie Santiago3 and David Kessel*3
1Cancer Biology Program, and Karmanos Cancer Institute, Wayne State University School of Medicine,
Detroit, MI
2Institute of Environmental Health Sciences, Wayne State University, Detroit, MI
3Departments of Pharmacology and Medicine, Wayne State University School of Medicine, Detroit, MI
Received 20 December 2008, accepted 8 February 2009, DOI: 10.1111/j.1751-1097.2009.00555.x
including superoxide anion, hydrogen peroxide, and hydroxyl
ABSTRACT
radical (•OH). The contributions of these various ROS types to
the biochemical and morphological changes after PDT are
often inferred by the inclusion of ROS type-specific scavengers.
What is missing are simple fluorescence-based assays for real-
time monitoring of specific types of ROS.
Singlet oxygen (1O2) is the primary oxidant generated in
photodynamic therapy (PDT) protocols involving sensitizers
resulting in type II reactions. 1O2 can give rise to additional
reactive oxygen species (ROS) such as the hydroxyl radical
(•OH). The current study was designed to assess 3¢-p-(amin-
ophenyl) fluorescein (APF) and 3¢-p-(hydroxyphenyl) fluorescein
(HPF) as probes for the detection of 1O2 and •OH under
conditions relevant to PDT. Cell-free studies indicated that both
APF and HPF were converted to fluorescent products following
exposure to 1O2 generated by irradiation of a water-soluble
photosensitizing agent (TPPS) and that APF was 35-fold more
The fluorescent probes, 3¢-p-(aminophenyl) fluorescein
(APF) and 3¢-p-(hydroxyphenyl) fluorescein (HPF), were
synthesized by Setsukinai et al. (1) as tools for stable and
selective detection of •OH and peroxynitrite. Both probes were
reported to be cell-permeable, relatively insensitive to super-
1
oxide anion radical, nitric oxide, O2 and alkyl peroxides and
free from the auto-oxidation associated with some other
probes. However, they differed substantially in their respon-
siveness to hypochlorite ion (ClO)). APF was converted to a
fluorescent form by ClO) while HPF was not.
1
sensitive than HPF. Using the O2 probe singlet oxygen sensor
green (SOSG) we confirmed that 1 mM NaN3 quenched 1O2-
induced APF ⁄ HPF fluorescence, while 1% DMSO had no
effect. APF and HPF also yielded a fluorescent product upon
interacting with •OH generated from H2O2 via the Fenton
reaction in a cell-free system. DMSO quenched the fluorogenic
interaction between APF ⁄ HPF and •OH at doses as low as
The current studies were initially designed to assess the
potential usefulness of HPF and APF as cell-permeable probes
•
for the detection of OH during PDT. Initial studies carried
out in a cell-free system using generators of •OH or 1O2
revealed that APF, and to a lesser extent HPF, were converted
to fluorescent products by either ROS. Inclusion of DMSO
•
0.02%. Although NaN3 was expected to quench OH-induced
APF ⁄ HPF fluorescence, co-incubating NaN3 with APF or HPF
in the presence of •OH markedly enhanced fluorescence.
Cultured L1210 cells that had been photosensitized with
benzoporphyhrin derivative exhibited APF fluorescence immedi-
ately following irradiation. Approximately 50% of the cellular
fluorescence could be suppressed by inclusion of either DMSO or
the iron-chelator desferroxamine. Combining the latter two
agents did not enhance suppression. We conclude that APF can
be used to monitor the formation of both O2 and OH in cells
subjected to PDT if studies are performed in the presence and
absence of DMSO, respectively. That portion of the fluorescence
quenched by DMSO will represent the contribution of •OH. This
procedure could represent a useful means for evaluating forma-
tion of both ROS in the context of PDT.
•
quenched the fluorescence generated by OH interaction with
either probe. In contrast, NaN3 quenched only the fluores-
cence generated by interactions with 1O2. In the context of
PDT, cultured L1210 leukemia cells that had been sensitized
with benzoporphyhrin derivative (BPD) exhibited APF fluo-
rescence within seconds of irradiation. Quenching studies
revealed that at least 50% of this fluorescence could be
1
•
•
attributed to the generation of OH. Surprisingly, the amount
•
of DMSO needed for quenching of the OH-derived fluores-
cence represented a concentration that is often used as a
solvent carrier in biological systems.
We also examined the effects of quenching agents on the
fluorogenic detection of 1O2 by singlet oxygen sensor green
(SOSG), described in Ref. (2). Effects of two ROS quenchers
were also determined: NaN3 and DMSO. The latter is expected
INTRODUCTION
•
to be specific for OH (3,4).
The efficacy of photodynamic therapy (PDT) derives from the
1
initial formation of singlet oxygen (1O2). In turn, O2 can give
rise to several other types of reactive oxygen species (ROS)
MATERIALS AND METHODS
Chemicals. For generation of 1O2, we employed the tetra-sulfonated
photosensitizing agent porphine tetra(p-phenylsulfonate) (TPPS), pur-
chased from Porphyrin Products, Logan, UT. Solutions (10 mM) were
*Corresponding author email: dhkessel@med.wayne.edu (David Kessel)
ꢀ 2009 The Authors. JournalCompilation. The AmericanSocietyofPhotobiology 0031-8655/09
1177

1178 Michael Price et al.
•
Table 1. Fluorogenic response to OH in a cell-free system.
Fluorescence units
System
APF
HPF
Fe2+ + H2O2
6117
420
175
18
35
806
1339
31295
37
148
630
75
203
11
36
25
Fe2+ + H2O2 + 1 mM NaN3
Fe2+ + H2O2 + 1% DMSO
Fe2+ + H2O2 + 0.1% DMSO
Fe2+ + H2O2 + 0.02% DMSO
28560
170
680
2980
Cuvettes contained 3 mL of 100 mM phosphate buffer (pH 7.0) APF
or 3 lM HPF and 300 lM FAS plus, where specified, 1 mM NaN3 or
Figure 1. Structures of HPF and APF and the oxidative reaction that
leads to the formation of fluorescein.
1% DMSO. After
a stable base-line was established (excita-
tion = 492 nm, emission = 525 nm), H2O2 was added (300 lM) and
the increase in fluorescence was monitored over 120 s, by which time
there was no further change. Data represent the mean
SD for three
determinations, and represent fluorescence (in arbitrary units) above
the base-line level.
made up in water and stored at 4ꢁC in the dark. The fluorescent probes
SOSG, HPF and APF were obtained from Invitrogen as solutions in
methanol (SOSG) or dimethyl formamide (APF and HPF). Structures
of the latter two agents are shown in Fig. 1. BPD was provided by
VWR and dissolved in dimethyl formamide. Other chemicals were
purchased from Fisher Scientific Co. and were of the highest
obtainable purity.
Fluorescence assay procedures. An SLM 48000 fluorometer, mod-
ified by ISS (Champaign, IL), was used for acquisition of fluorescence
signals in the ‘‘slow kinetic’’ mode. When APF or HPF were employed,
fluorescence emission at 525 nm was measured upon excitation at
492 nm using 2 nm slit widths for both excitation and emission. With
SOSG, the corresponding values were 505 and 525 nm, respectively.
Studies involving a cell-free system were carried out in 1 · 1 · 3 cm
glass cuvettes maintained at a temperature of 20ꢁC by a Peltier system,
and stirred constantly. When
photodynamic generation of 1O2, the influence of stray light from
the laser was minimized by insertion of a 500 35 nm interference
a diode laser was used for the
filter into the emission beam. In all experiments, the fluorescent probes
were present at 3 lM concentrations.
Singlet oxygen formation. Singlet oxygen was generated by a
photodynamic reaction (5) in 3 mL of 100 mM phosphate buffer pH
7.0, H2O or D2O, along with 10 lM TPPS. A 400 lm fiber bundle was
used to transfer 649 nm light from a laser diode to the top surface of the
cuvette, using a power density of 1 mW cm)2. This permitted irradi-
ation during the acquisition of a fluorescence signal in real time. The
laser was activated immediately before the beginning of fluorescence
acquisition, with data points acquired at 4 s intervals over 180 s. The
change in fluorescence as a function of time was linear for at least 120 s.
Formation of hydroxyl radical. This ROS was generated by the
Fenton reaction (6) using a mixture containing 100 mM phosphate
buffer pH 7,0, 0.3 mM Fe(NH4)2(SO4)2 (FAS) and 0.3 mM H2O2.
Addition of H2O2 resulted in a rapid increase in fluorescence. We
report the steady-state fluorescence signal minus the base-line signal
obtained before addition of H2O2 (Table 1).
•
Quenching studies. Fluorogenic interactions involving 1O2 and OH
and fluorescent probes were assessed in the absence vs presence of
inhibitors (NaN3 or DMSO) using SOSG, APF or HPF in systems
described above. Data are reported in Figs. 2 and 3 and in Table 1.
Photodynamic studies in cell culture. Procedures for these studies
and the subsequent morphometric analysis of images have been
described (7). Murine leukemia L1210 cells were incubated with 2 lM
BPD + 3 lM APF for 30 min at 37ꢁC. In replicate tubes DMSO
(1%), 100 lM desferroxamine (DFO) or both reagents were added. The
cells were then collected by centrifugation (100 g, 30 s), resuspended in
fresh medium and irradiated. The light dose was 67.5 mJ cm)2 at
690 5 nm, as determined with a ScienTech H10T radiometer. This
PDT dose was previously found to reduce cell viability to 11 3% of
control values, as indicated by clonogenic assays.
After irradiation, cells were collected by centrifugation and exam-
ined by fluorescence microscopy using 450–490 nm excitation, and
monitoring emission at 520–600 nm. A Nikon Eclipse E600 micro-
Figure 2. Top: fluorogenic interactions between 3 lM SOSG and 1O2
during the irradiation at 649 nm of solutions containing 10 lM TPPS.
The solvents were D2O, 100 mM sodium phosphate buffer pH 7.0, or
water, using a light flux of 1 mW sq cm)1. Fluorescence was monitored
using excitation = 505 nm, emission = 525 nm. Bottom: quenching
of fluorescence during irradiation of SOSG + TPPS in phosphate
buffer by 1 and 10 mM sodium NaN3 and by 1% DMSO.
scope was used for this purpose, with images acquired by
a
Photometrics CoolSnap HQ CCD camera operated at )40ꢁC at the
highest sensitivity setting. An exposure time of 1000 ms was required
to obtain the images shown in Fig. 4. A B2A filter cube was used
(excitation = 450–490 nm). The emission was controlled by a high-
pass 520 nm filter and an additional 600 nm low-pass filter to screen
out fluorescence derived from BPD.

Photochemistry and Photobiology, 2009, 85 1179
RESULTS AND DISCUSSION
Fluorogenic response of HPF and APF to hydroxyl radicals and
quenchers
The effects of •OH on APF and HPF fluorescence are
summarized in Table 1. We found APF yielded ꢀ5-fold more
fluorescence during •OH formation than did HPF. In Ref. (1),
it was reported that APF was more sensitive than HPF by a
factor of 1.7. In experiments reported here, we used FAS
rather than the Fe(ClO4)2 employed in Ref. (1), since we found
that the latter salt resulted in formation of a precipitate in
phosphate buffer. There was no precipitation when FAS was
used as the source of ferrous iron.
•
In a cell-free system, the fluorogenic reaction of OH with
HPF or APF was quenched ꢀ50% by 0.02% DMSO (vol ⁄ vol).
A concentration of 0.1% DMSO, often said to be safe for use
in biological studies, was found to decrease the fluorogenic
•
response of APF to OH by >90%. In the presence of 1 mM
Figure 3. Fluorogenic interactions of 10 lM TPPS + APF in D2O,
phosphate buffer or water during irradiation (649 nm). Also shown are
effects of 1 mM NaN3 and 1% DMSO in phosphate buffer. For
comparison, the fluorogenic reaction with HPF in D2O is also shown.
Inset: the fluorogenic interaction during irradiation between
TPPS + APF over 180 s in D2O. For all studies, excita-
tion = 492 nm, emission = 525 nm.
NaN3, an unexpected increase in fluorescence was observed
(Table 1). Hence, this reagent could not be used in quenching
studies.
Determinants of singlet oxygen formation
1O2 formation was monitored using SOSG (Fig. 2, top).
During irradiation of an aqueous solution of TPPS + SOSG,
we observed a progressive increase in 525 nm fluorescence that
was markedly enhanced in D2O. This effect is expected since
the lifetime of the triplet state is strongly promoted in
deuterated solvents (8). The fluorogenic effect of 1O2 on
SOSG was slightly promoted by the presence of 100 mM
phosphate buffer pH 7.0, compared with water alone.
Images of cellular fluorescence were captured using MetaMorph
software (Molecular Devices, MDS Analytical Technologies). Average
pixel intensities were determined using morphometric analysis.
Morphometric analysis procedure. A thresholding process was first
used to select only those portions of the image representing cellular
fluorescence. The black background was therefore omitted from the
calculations. The average grayscale analysis was then calculated as
the summation of the values for each pixel contained in the images of
the cells (0 = black, 255 = white). This value was then divided by the
total number of pixels constituting cell images, so that an average pixel
brightness is represented.
Quenching studies carried out with SOSG in phosphate
buffer (Fig. 2, bottom) showed only a minor effect of 1%
Figure 4. Fluorescence images of APF fluorescence using L1210 cells photosensitized by BPD. A = dark control (no irradiation), B–
E = irradiated cells, B = no additions, C = 1% DMSO, D = 100 lM DFO, E = 1% DMSO + 100 lM DFO, F = 0.1% DMSO. Inhibitors
were present during BPD loading, and added back to the culture medium during irradiation.

1180 Michael Price et al.
(vol ⁄ vol) DMSO on 1O2-mediated fluorescence, but a sub-
stantial quenching of the fluorogenic reaction by NaN3.
Table 2. Morphometric analysis.
Conditions
Average gray value
%
Control
BPD
BPD + 1% DMSO
BPD + 100 lM DFO
BPD + both
3.8
58.5
32.8
32.5
28.7
49.7
2.4
6.5
100*
56
56
49
Fluorogenic responses of HPF and APF to singlet oxygen
16.3
10.3
5.8
14.5
7.2
Irradiation of an aqueous solution containing APF + TPPS
resulted in a fluorogenic response that was enhanced >25-fold
in D2O (Fig. 3). This fluorogenic reaction was greater in
phosphate buffer than in water. Additional photodynamic
studies carried out in phosphate buffer revealed only a slight
quenching of APF fluorescence by 1% DMSO, but a very
substantial decrease upon addition of 1 mM NaN3 (Fig. 3).
These characteristics are consistent with the behavior of
1O2 + SOSG shown in Fig. 2.
BPD + 0.1% DMSO
85
These figures represent a morphometric analysis (mean
images shown in Fig. 4 (see text).
*These data are calculated in terms of the percentage of the mean value
for the average gray value for the ‘‘BPD’’ sample.
SD) of
HPF was substantially less sensitive to 1O2 than APF. In
D2O, the fluorogenic interaction of 1O2 with APF during
irradiation proceeded at a ꢀ35-fold greater rate than was
observed with HPF (Fig. 3, line designated ‘‘HPF D2O’’).
These values differ from what was reported in Ref. (1), where
We also examined the possibility that HPF might be useful
•
in monitoring OH formation during irradiation of photosen-
•
sitized cells. Unfortunately, the interaction between OH and
HPF produces only 20% of the fluorescence intensity observed
with APF (Table 1). As a result, detection of a fluorescent
signal from HPF after an LD90 PDT dose would require a
5000 ms exposure. This was found to be associated with
substantial photobleaching of the probe (not shown). Since
SOSG cannot be used with intact cells because of its inability
to penetrate the cell membrane (1) and the often-used probe
2,7-dichlorofluorescein diacetate is readily auto-oxidized (1),
we propose that APF or HPF should be useful for ‘‘on-line’’
monitoring of 1O2 formation, in the presence of DMSO to
1
HPF was found to be ꢀ50% as responsive to O2 as APF. In
Ref. (1), the comparison was carried out in phosphate buffer.
We used D2O in our studies to increase the signal-to-noise
ratio since the fluorogenic interaction between HPF and 1O2 in
H2O or phosphate buffer was barely detectable (not shown).
Since DMSO is known to quench •OH but not 1O2 (3,4), these
1
data show that APF and HPF can detect O2 formed during
PDT.
•
suppress OH formation.
ROS detection in cell cultures
TPPS was useful for studies in an aqueous cell-free system
since it does not aggregate in aqueous solution. In order to
CONCLUSIONS
carry out studies in cell culture, we selected
a more
These studies show that in the context of PDT, where a very
substantial concentration of 1O2 is formed, APF can be a
useful cell-permeable probe for detecting formation of this
ROS. Moreover, the selective use of DMSO can provide a
hydrophobic agent, BPD, that was readily accumulated by
L1210 cells. To determine the potential usefulness of APF in
PDT studies, we incubated L1210 cells with APF and BPD,
followed by irradiation to achieve an LD90 PDT dose. The
fluorescence images shown in Fig. 4 were all obtained at the
same camera settings. For this study, a 600 nm low-pass
filter was placed in the emission pathway to eliminate
fluorescence from the photosensitizing agent. In nonirradiat-
ed cultures, only weak APF fluorescence could be detected
(panel A).
Irradiation of photosensitized cells resulted in substantial
APF-derived fluorescence (panel B). Addition of either 1%
DMSO or the iron-chelator DFO resulted in loss of fluores-
cence (panels C and D). Adding both reagents simultaneously
did not significantly increase this quenching (panel E). A
morphometric analysis of the results is summarized in Table 2.
DFO is known to quench hydroxyl radical formation gener-
ated via the Fenton reaction (9–11). Analysis of fluorescence
intensities indicates ꢀ50% of the total APF fluorescence was
derived from processes quenched equally by DFO or DMSO,
presumably representing •OH (Table 2). Adding both reagents
did not further promote fluorescence quenching, consistent
with the proposal that the signal abolished by DMSO
represents •OH. Even when the DMSO concentration was
reduced to 0.1%, some quenching of the portion of the
fluorescent signal attributed to •OH was observed (Fig. 4,
panel F). This was readily quantified by the morphometric
analysis technique (Table 2).
•
means for assessing OH formation.
APF and HPF are often used as fluorescent probes for the
detection of hydroxyl radical hypochlorite (12–14). It was
initially reported (1) that these probes were relatively insensi-
tive to 1O2 (1). However, our studies indicate that, under
conditions associated with PDT, fluorogenic interactions with
1O2 can be significant, with APF substantially more sensitive
1
•
to O2 than HPF. Since maximum sensitivity to OH was an
important consideration, we employed the more responsive
probe APF to determine whether •OH could be detected in the
1
presence of O2 in cell culture.
Quenching studies indicated that 0.1% DMSO could
suppress >90% of the APF fluorescence derived from OH
(Table 1) without affecting the signal derived from 1O2
(Fig. 3). The ability of concentrations of DMSO as low as
•
•
0.02% to quench OH in a cell-free system indicates that this
solvent is best omitted from any study designed to assess
formation or effects of this ROS. A study with murine
leukemia L1210 cells and the photosensitizing agent BPD
revealed that the use of APF in the presence vs absence of
•
DMSO is a feasible approach for monitoring OH formation
during irradiation of photosensitized cells (Fig. 4). While HPF
would appear to be a better probe because of its decreased
1
response to O2, this advantage is offset by its much weaker
response to •OH. Use of HPF requires a sufficiently long

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1
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