PSI-SR-1255
Detection of singlet oxygen production for
PDT treatments both in vitro and in vivo
using a diode laser-based singlet oxygen
monitor
S. Lee
D.H. Vu
M.F. Hinds
S.J. Davis
T. Hasan
A. Khachemoune
W. Rice
N.R. Sznycer-Taub
S. Lee, D.H. Vu, M.F. Hinds, S.J. Davis, T. Hasan, A. Khachemoune, W. Rice, N.R.
Sznycer-Taub, "Detection of singlet oxygen production for PDT treatments both in vitro
and in vivo using a diode laser-based singlet oxygen monitor ," presented at Optical
Methods for Tumor Treatment and Detection: Mechanisms and Techniques in
Photodynamic Therapy XV Biomedical Optics (BiOS) Symposium (San Jose, CA), (2126 January2006).
Copyright © 2006 Society of Photo-Optical Instrumentation Engineers.
This paper was published in Optical Methods for Tumor Treatment and Detection:
Mechanisms and Techniques in Photodynamic Therapy XV Biomedical Optics (BiOS)
Symposium, and is made available as an electronic reprint (preprint) with permission of
SPIE. One print or electronic copy may be made for personal use only. Systematic or
multiple reproduction, distribution to multiple locations via electronic or other means,
duplication of any material in this paper for a fee or for commercial purposes, or
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Detection of singlet oxygen production for PDT treatments
both in vitro and in vivo using a diode laser-based
singlet oxygen monitor
S. Lee†a, D.H. Vua, M.F. Hindsa, S.J. Davisa, T. Hasanb, A. Khachemouneb, W. Riceb,
and N.R. Sznycer-Taubb
a
b
Physical Sciences Inc., 20 New England Business Center, Andover, MA, USA 01810-1077;
Massachusetts General Hospital, Wellman Laboratories of Photomedicine, 40 Blossom Street,
Boston, MA, USA 02114-2605
ABSTRACT
Photodynamic therapy (PDT) is a promising cancer treatment. PDT uses photosensitizers that are selectively retained in
malignant tumors. When tumors, pretreated with the photosensitizer, are irradiated with visible light, a photochemical
reaction occurs and tumor cells are destroyed. Oxygen molecules in their metastable singlet delta state, O2(1∆), are
believed to be the species that destroys cancerous cells during PDT. Physical Sciences Inc. (PSI) has developed a fiber
optic-coupled, diode laser- based diagnostic for singlet molecular oxygen produced during PDT. This portable prototype
system detects the singlet oxygen emission near 1.27 µm. We have obtained signals from singlet oxygen during and
following pulsed laser excitation and demonstrated excellent sensitivity with this system. Both in vitro and in vivo
studies have been completed, and we have detected the production of singlet oxygen during PDT treatments with both
animal and human subjects. Effects of photobleaching have also been observed. These results are promising for the
development of the sensor as a real-time dosimeter for PDT.
Keywords: Photodynamic therapy, singlet molecular oxygen, diode laser, real-time monitor
1. INTRODUCTION
Photochemotherapy of cancer is often called photodynamic therapy (PDT).1 In PDT, a photosensitizer (PS) is injected
to a cancer patient, and this agent is preferentially retained in tumor cells. The agent is irradiated with visible to near
infrared light. The wavelength of the light has to match the absorption of the photosensitizer. The excited PS can
transfer its energy to oxygen molecules to generate singlet oxygen (O2(a1∆), and the singlet oxygen can produce cell
necrosis. FDA approval has been granted for treatment of esophageal and certain lung cancers. PDT is being used in
clinical trials for bladder, brain, skin and other cancers.2-6 PDT is also being applied to important areas outside of cancer
treatment including age related macular degeneration and actinic keratosis, a pre-cancerous skin condition.
There is considerable evidence that O2(a1∆) is the active species in cancer cell or endothelial cell necrosis. Despite the
general acceptance of this role of O2(a1∆) in PDT there have been limited demonstrations of its importance in vivo.7,8 If
O2(a1∆) is indeed the critical species that determines PDT efficacy, a device that is conducive to on-line measurement of
O2(a1∆) in vivo could, in principle, provide the critical parameter in PDT dosimetry and the potential of individualized
therapeutic design.
PSI’s diode laser approach is summarized in Figure 1.9 The diode laser operates in pulsed mode with a peak power
< 300 mW. It generates prompt dye fluorescence. Since O2(a1∆) lifetime is longer than dye fluorescence, we can detect
the singlet oxygen signal after the diode laser turns off. We detect the singlet oxygen fluorescence with optical filtering
of O2(a1∆) emission spectral region.
†
[email protected]; phone 1 978 689-0003; fax 1 978 689-3232; http://www.psicorp.com
Modulated
Laser Intensity
"Prompt" Dye
Fluorescence
Fluorescence
from Longer
Lived Singlet
Oxygen
Viewing Time
Time
C-5970b
Figure 1: Outline of the detection for PSI’s singlet oxygen monitor.
In a series of papers Wilson et al.7,8,10,11 have reported a singlet oxygen detector based upon a similar PMT and pulsed
excitation with a Q-switched, frequency doubled, Nd:YAG laser. Our method is distinct from Wilson’s in several ways.
They use a frequency-doubled Q-switched Nd:YAG laser or an optical parametric oscillator to detect PDT-produced
singlet oxygen emission both in vitro and in vivo. Note that unlike Q-switched Nd:YAG lasers (pulselengths ~ 10 ns),
our relatively long (~ 5 µs) diode laser pulses do not produce significant energy compression. While the diode laser is
on, its peak power is essentially equal to that when operating CW. Consequently, our peak powers are much lower. For
example for a 300 mW diode laser, pulses of 2 µs duration contain only 0.6 µJ and each pulse has a peak power of
300 mW and can cause no tissue damage. Wilson and co-workers also use a freespace laser beam and macroscopic
lenses for beam focus and singlet oxygen emission collection in contrast to our fiber coupled system.
Previously, we reported the results of our initial investigation to develop a sensitive, diode laser-based monitor for
singlet oxygen produced by light treatment.9,12 Using a pulsed diode laser and photon counting methods, we
demonstrated singlet oxygen detection during PDT of rat prostate tumor cells in vitro as well as in solution phase. In
this paper, we discuss our progress on the system performance for singlet oxygen monitoring. We report the first time
measurements of O2(a1∆) in tumor-loaded rats and healthy human skin.
2. EXPERIMENTS
Figure 2 shows the current prototype PSI singlet oxygen monitor. This is more compact and easy to operate compared
to our previous experimental setup.9 All components are in one equipment rack including: 1) diode laser module;
2) PMT detector; 3) data acquisition system with photon counting board. The light delivery and collection are fiber
coupled, so there are no external optics that require alignment. Singlet oxygen has very weak emission, and the spectral
region of singlet oxygen slightly overlaps with photosensitizer fluorescence. To differentiate the background emission
such as photosensitizer fluorescence and autofluorescence (albeit weak) from recorded O2(a1∆) signal, we used threeoptical filter system. In front of the PMT, we integrated a filter slider that contains three optical filters (peak emissions
at 1.22, 1.27, and 1.315 µm). This filter slider is for effective background subtraction, especially for in vivo studies. We
corrected each spectrum with a background spectrum.
The fast photon counting board can handle up to a 20 kHz operation rate. We wrote custom software that controls:
1) the diode laser module controlled through a National Instruments GPIB card; and 2) a graphic user interface (GUI) to
control all parameters of photon counting system and to display the data acquisition in real time from the fast photon
counting system.
Our device measures directly the singlet oxygen dose which is proportional to the product of the PS concentration, the
ground state oxygen concentration and the light intensity within the tissue being irradiated. Thus, we can define the dose
as ∫ [PS(t)] * [O2] Φ(t) dt where [PS(t)] is the photosensitizer concentration, [O2(t)] is the ground state oxygen
concentration and Φ(t) is the light irradiance.
Laser
Power
Supply
Signal
Diode
Laser
Photon
Counter
Filter Slider
(1.22, 1.27, 1.315 µm)
+
TE Cooled PMT
Computer
H-5246
Figure 2: Device setup of singlet oxygen monitor.
In tumor cell experiments at PSI, we used a rat prostate cancer cell line incubated with the photosensitive dye chlorin e6
(Cl e6) about 4 hours. Before PDT experiments, the medium was removed and the cells were washed with phosphate
buffer solution. Therefore, Cl e6 dye that was not taken into the tumor cell was removed. The observed singlet oxygen
signal was from intracellular singlet oxygen. In addition, we confirmed that the singlet oxygen signal was from the
tumor cells, not from the suspension, from experiments of cell pallets.
We measured O2(a1∆) production during PDT treatments at the Wellman Photomedicine Research Center at MGH. Two
in-vivo studies were performed. First, a rat prostate cancer model was studied with several PDT photosensitizers such as
Cl e6, ALA-induced PpIX, and BPD. Second, we extended our study to healthy human subjects using topical ALAinduced PpIX case. Below we provide a short summary of these results. Details will be provided elsewhere.
3. RESULTS
3.1 In vitro studies
The suspension of tumor cells was put into a glass bottle and the solution was mixed with a magnetic stirrer. Each
spectrum was corrected by the background fluorescence of the cell suspension without dye, such as autofluorescence
from biomolecules or fluorescence from optical components. With the initial dye concentrations of 100 µM and 10 µM,
we readily observed the temporal evolution of the O2(a1∆) signal in a rat prostate tumor cell line, as shown in Figure 3.
Figure 3: Temporal evolution of O2(a1∆) emission in a rat prostate tumor cell line using Cl e6.
3.2 In vivo studies
In the tumor-loaded rat study, we monitored O2(a1∆) signal intermittantly during the PDT treatment. The treatment laser
was shut off during these measurements. We investigated the relationship between PDT, O2(a1∆) production and local
tumor growth outcome. Figure 4 shows the typical decay profiles of singlet oxygen produced from a tumor implanted
on a rat using ALA as the PS: Figure 4a) before ALA injection and Figure 4b) after PDT treatment with ALA. The lines
shown are fits to the temporal evolution data. To maximize the sensitivity of the sensor, we corrected the singlet oxygen
emission by subtracting background signals due to photosensitizer fluorescence and autofluorescence.
500
150
Counts
400
Counts
(b) Rat E
100 mg/kg ALA
50 J PDT treatment
(a) Rat E
before ALA injection
300
center wavelength of filter
1.22 um
1.27 um
1.315 um
200
100
100
center wavelength of filter
1.22 um
1.27 um
1.315 um
50
0
0
0
1
2
3
Time (µsec)
4
5
0
1
2
3
Time (µsec)
4
5
H-5249
Figure 4: The typical decay profile of singlet oxygen produced in the tumor burdened rats: (a) before ALA injection,
(b) 100 mg/kg body weight ALA and 50 J irradiation.
Following the PDT treatment with different photosensitizer dosages and light dosages, the tumor size was measured
each day for 14 days. The control group with no photosensitizer and/or no light treatment showed steady tumor
increase.
In contrast, for PDT treated tumors that produced measurable singlet oxygen there was a correlation between the tumor
regression and singlet oxygen produced. For example, for two different animals each treated with the same amount of
PS, one produced a small singlet oxygen signal and the other produced nearly six times as much singlet oxygen. The
tumor on the first animal showed persistent growth, while the tumor on the second regressed by a factor of ten in the
first two days after treatment, before regrowth began. Details on these results will be reported elsewhere.
We also conducted a pilot study with 15 healthy human subjects using ALA-induced PpIX and were able to detect
singlet oxygen. To our knowledge this is the first observation of O2(a1∆) production on human skin with PDT treatment.
The relationship between O2(a1∆) production and photosensitizer fluorescence was also investigated. We observed that
the O2(a1∆) production maximized after about 10 J of treatment light. In contrast, the photosensitizer steadily decreased
from its initial value prior to light treatment. The results may indicate increased oxygenation during PDT even during
photosensitizer photobleaching.
4. SUMMARY
Using our diode laser based system, we have observed O2(a1∆) produced from PDT treated tumors on live rats and
through follow-up studies, observed tumor shrinkage that correlated with the amount of O2(a1∆) produced. These
measurements are the first of their kind with a fiber optic coupled device. We also observed the first measurements of
O2(a1∆) production in human subjects. We plan to improve the singlet oxygen monitor sensitivity for more extensive in
vivo studies.
ACKNOWLEDGEMENTS
This work was supported under an NIH SBIR Phase II Grant No. 5R44CA096243-03. We are grateful for this support.
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