Different photodynamic effect between continuous wave and pulsed

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Different photodynamic effect between continuous wave and pulsed laser irradiation modes in
k562 cells in vitro
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2014 J. Phys.: Conf. Ser. 541 012040
(http://iopscience.iop.org/1742-6596/541/1/012040)
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SPbOPEN2014
Journal of Physics: Conference Series 541 (2014) 012040
IOP Publishing
doi:10.1088/1742-6596/541/1/012040
Different photodynamic effect between continuous wave and
pulsed laser irradiation modes in k562 cells in vitro.
V.V. Klimenko1, A.A. Bogdanov1, N.A. Knyazev1, A.A. Rusanov2, M.V. Dubina1
1
2
St. Petersburg Academic University, St. Petersburg, Russia
First Pavlov State Medical University of St. Petersburg, St. Petersburg, Russia
E-mail: [email protected]
Abstract. Photodynamic therapy is a cancer treatment method is used primarily continuous
mode laser radiation. At high power density irradiation occurs intense consumption of
molecular oxygen and this caused hypoxic tumor tissue, which leads to inefficiency PDT. In
this paper, pulsed and continuous irradiation modes during PDT photosensitizer Radachlorin
were compared. A mathematical model for the generation of singlet oxygen 1O 2 in tumor cells
during photodynamic therapy with tissue oxygenation was developed. Our study theoretically
and experimentally demonstrates the increased singlet oxygen generation efficiency in a pulsed
irradiation mode compared to continuous wave mode with the same power density 20mW/cm2.
Experimental in vitro showed that pulsed irradiation mode mostly induces apoptosis k562
tumor cells at irradiation doses of k562 1.25 - 2.5J/cm2 while the continuous mode induced
necrosis.
1. Introduction
Photodynamic therapy (PDT) has now reached the level of being an accepted treatment for several
types of cancer. PDT is based on production of singlet oxygen (1O 2 ) by energy transfer from lightexcited photosensitizer molecules in target tissue. Singlet oxygen effectively oxidizes many kinds of
biomolecules, leading to damage and cell death. The introduction of semiconductor heterostructures
and development of semiconductor-based diode lasers [1] have significantly accelerated the evolution
of PDT. Today’s preferential use of semiconductor lasers is primarily owing to their low cost, ease of
treatment and the ability to match their emission peak with absorption of any particular
photosensitizer. High power light irradiation in continuous mode and porphyrin photosensitizers
(absorption wavelength 600-700 nm) are mainly applied in clinic now [2, 3] to achieve an impactful
penetration depth in tumor tissue. Despite the fact that such treatment is successful enough it has some
critical disadvantages connected with tissue decreased oxygenation, overheating and photosensitizer
bleaching. Moreover, continuous mode PDT typically results in necrotic cell death with further
inflammatory response. Semiconductor lasers give us opportunity to apply different regulations
including easy light power switching and pulse mode irradiation. There is some literature data that
pulse mode PDT in vitro leads to apoptotic cell death mechanism [4, 5]. In present work we have
investigated the difference of continuous and pulse irradiation modes and their biological effect in
vitro.
Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution
of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Published under licence by IOP Publishing Ltd
1
SPbOPEN2014
Journal of Physics: Conference Series 541 (2014) 012040
IOP Publishing
doi:10.1088/1742-6596/541/1/012040
2. Theoretical PDT model
In our study we used macroscopic PDT theoretical model adapted from [6]. Briefly, we exploited
differential system eq. (1)-(4) to calculate cumulative concentration of singlet oxygen. This model is
good for quality description of triplet oxygen flux impact on singlet oxygen generation in PDT.
Ι([S0 ] + δ )[ 3O2 ] 
d[ S0 ] 
(1)
+  ξσ
0
 [S0 ] =
dt
[ 3O2 ] + β


Ι[S ]
d[ 3O2 ] 
+ ξ 3 0
dt
 [ O2 ] + β
 3

[ 3O2 ] 
(2)
−
−
[
O
]
g
1
0
 2

=
3
0) 

 [ O2 ](t =
Ι[S ] 
d[ 1O2 ] 
(3)
−  ξ 3 0  [ 3O2 ] =
0
dt
 [ O2 ] + β 
t

I[S ] 
(4)
[ 1O2 ]cumulative = ∫  ξ 3 0  [ 3O2 ]dt
[ O2 ] + β 
0
, where [S 0 ] is the ground state sensitizer concentration, [ 3O2 ] and [ 1O2 ] are the ground triplet and
excited singlet state oxygen concentration, respectively, I is intensity of laser irradiation, g is
maximum oxygen supply rate, δ is a low photosensitizer concentration correction term, I is intensity
of laser irradiation. Simulation PDT process was performed using the photochemical parameters
shown in Table 1. For continuous wave and pulse modes were applied parameters: intensity of laser
irradiation - 20mW/cm2, pulse duration – 200 ms, interval between pulse – 500 ms. These pulse mode
parameters satisfy maintaining a high level of triplet oxygen concentration.
Table 1. Photochemical parameters for Photofrin at 630
nm and initial conditions used for the macroscopic model.
Symbol
ξ (cm mW s )
β ( µ M)
σ ( µ M −1 )
g( µ M/ s)
[ 3O2 ]( µ M)
[ S0 ]( µ M )
2
−1 −1
Value
3.7 x 10-3
References
[7]
11.9
7.6 x 10-5
[7]
[7]
0.7
100
[6]
[6]
7
[6]
We can see from figure 1 and figure 2 that cumulative singlet oxygen concentration is higher
in a pulse mode than in continuous one. Moreover, we observe that efficiency of singlet oxygen
generation is higher in pulse mode. On the other hand the average singlet oxygen generation rate at
pulse mode is several times smaller.
2
SPbOPEN2014
Journal of Physics: Conference Series 541 (2014) 012040
250
Pulse mode
Continuous mode
250
200
Concentration 1O2, µM
Concentration 1O2, µM
IOP Publishing
doi:10.1088/1742-6596/541/1/012040
150
100
50
0
off
200
off
150
100
On
50
Continuous mode
Pulse mode
0
0
1
2
3
4
5
0
200
400
Dose, J/cm2
600
800
1000
Time, s
Figure 1. The dependence of singlet
oxygen cumulative concentration on
irradiation dose applied for pulse and
continuous wave irradiation modes.
Figure 2. The dependence of singlet
oxygen cumulative concentration on time
for pulse and continuous wave irradiation
modes.
100
100
90
90
80
80
fraction (% untreated controls)
fraction (% untreated controls)
3. PDT experiment in vitro.
In experimental studies in vitro cancer cell line k562 (Bank of Cell Cultures, Institute of Cytology,
RAS, St. Petersburg, Russia), RadachlorinⓇ (RADA-PHARMA Co, Ltd., Moscow, Russia)
photosensitizer (PS) and semiconductor laser for PDT «LAHTA – MILON» (MILON Laser, LLC, St.
Petersburg, Russia) were used. 1∙106 cells per well k562 cells were seeded in 6-well plates and
incubated during 12 hours with PS in 5 μg/ml concentration. After incubation cells were washed with
PBS buffer and irradiated with laser source in different modes. For irradiation in pulse mode we
applied parameters described above. Estimation of cell viability was performed before irradiation, 2 h
and 24 h after irradiation using an EPICS XL flow cytometer (Backman Coulter, United States) and
standard protocols. Nonirradiated cells served as a control.
70
Dead
apoptosis
necrosis
60
50
сontinuous mode
40
30
20
10
70
Dead
apoptosis
necrosis
60
50
Pulse mode
40
30
20
10
0
0
0
1
2
3
4
0
5
1
2
3
4
5
Dose, J/cm2
Dose, J/cm2
Figure 4. Dose-dependent distribution
apoptosis and necrosis fraction k562
cells in total count of dead cells at pulse
mode irradiation 20mW/cm2 with
5μg/ml Radachlorin concentration.
Figure 3. Dose-dependent distribution
apoptosis and necrosis fraction k562
cells in total count of dead cells at
continuous mode irradiation 20mW/cm2
with 5μg/ml Radachlorin concentration.
3
SPbOPEN2014
Journal of Physics: Conference Series 541 (2014) 012040
IOP Publishing
doi:10.1088/1742-6596/541/1/012040
The figure 3 and figure 4 shows that fraction of necrosis cells at all doses in case of
continuous mode is greater than the same one in pulse mode. On contrary in pulse mode we have the
convenience of apoptosis except 5J/cm2. The total count of dead cells is almost equal for both modes.
We can consider irradiation doses used as penetrating doses in different depth of tumor tissue. In that
case we make conclusion that pulse mode can provide the same cell destructive potency with
preferable biological effect – apoptosis.
4. Discussion
Taking into account all data obtained, we can conclude that the main factor in biological effect during
PDT is the speed of singlet oxygen generation. Low speed of singlet oxygen generation leads to
apoptosis and high speed – to necrosis. The next factor facilitating cell death in PDT is cumulative
singlet oxygen concentration, which directly connected with total molecular damages. Therefore,
results given above prove the hypothesis about major efficiency of pulse mode in comparison with
continuous one in terms of singlet oxygen generation and apoptosis initiation.
References
[1] Zhores A 2000 Double Heterostructure Lasers: Early Days and Future Perspectives IEEE J. Sel.
Top. Quant. Electron. vol 6 n 6
[2] Agostinis P et al 2011 Photodynamic Therapy of Cancer: An Update. CA Cancer J Clin. 61(4)
pp 250-81
[3] Oleinick N, Morris R and Belichenko I 2002 The role of apoptosis in response to photodynamic
therapy: what, where, why, and how Photochem. Photobiol. Sci 1 pp 1–21.
[4] Yuuichi M, Yukihiro U and Tsuyoshi N 1999 Comparison of phototoxicity mechanism between
pulsed and continuous wave irradiation in photodynamic therapy J. Photochem. Photobiol.
B: Biol. 53 pp 53–59
[5] Kawauchi S, Morimoto Y, Sato S, Arai T, Seguchi K, Asanuma H and Kikuchi M 2004
Differences between cytotoxicity in photodynamic therapy using a pulsed laser and a
continuous wave laser: study of oxygen consumption and photobleaching Lasers in Medical
Science 18 pp 179–183
[6]
Wang K, Finlay J, Busch T, Hahn S and Zhu T 2010 Explicit dosimetry for photodynamic
therapy: macroscopic singlet oxygen modeling. J Biophotonics 5-6 pp 304-18
[7] Georgakoudil I, M G Nichols and Foster T H 1997 The Mechanism of Photofrins
Photobleaching and Its Consequences for Photodynamic Dosimetry Photochemistry and
Photobiology 65(1) pp 135-144
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