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A combination of techniques to evaluate photodynamic efficiency of

photosensitizers

Article  in  Laser Physics Letters · January 2009

DOI: 10.1002/lapl.200810082

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64 Laser Phys. Lett. 6, No. 1, 64–70 (2009) / DOI 10.1002/lapl.200810082

Abstract: Photodynamic therapy, used mainly for cancer

100
treatment and microorganisms inactivation, is based on pro-
duction of reactive oxygen species by light irradiation of a
sensitizer. Hematoporphyrin derivatives as Photofrin (PF), 80

Photogem (PG) and Photosan (PF), and chlorin-e6-derivatives

Hemolysis, %
as Photodithazine (PZ), have suitable sensitizing properties. 60

The present study provides a way to make a fast previous eval-

uation of photosensitizers efficacy by a combination of tech- 40

niques: a) use of bovine serum albumin and uric acid as chem-

ical dosimeters; b) photo-hemolysis of red blood cells used as 20
PZ
a cell membrane interaction model, and c) octanol/phosphate PF
PG
buffer partition to assess the relative lipophilicity of the com- PS
0
pounds. The results suggest the photodynamic efficient ranking: 0 2 4 6 8 10
PZ > PG ≥ PF > PS. These results agree with the cytotoxicity of Time, min
the photosensitizers as well as to chromatographic separation of
Percentage of photo-hemolysis of RBC suspension (2%, pH = 7.4
the HpDs, both performed in our group, showing that the more
in PBS), in the presence of photosensitizers (10 mg/mL) irradi-
lipophilic is the dye, the more acute is the damage to the RBC
ated with LED-630 nm (25 mW/cm2 )
membrane and the oxidation of indol, which is immersed in the
hydrophobic region of albumin.

c 2009 by Astro Ltd.

Published exclusively by WILEY-VCH Verlag GmbH & Co. KGaA

A combination of techniques to evaluate photodynamic

efficiency of photosensitizers
R.S. Cavalcante, 1 H. Imasato, 1 V.S. Bagnato, 2 and J.R. Perussi 1,∗
1
Instituto de Quı́mica de São Carlos, Universidade de São Paulo, São Carlos 13560-590, SP, Brazil
2
Instituto de Fı́sica de São Carlos, Universidade de São Paulo, São Carlos 13560-590, SP, Brazil

Received: 23 July 2008, Revised: 30 July 2008, Accepted: 1 August 2008

Published online: 14 August 2008

Key words: photodynamic therapy; hematoporphyrin derivatives; chlorin derivative; photodynamic efficiency; photo-hemolysis; albu-
min

PACS: 33.80.-b, 87.19.xj, 87.50.wp, 87.64.kv, 87.19.xj

1. Introduction be inactivated by visible light after treatment with an ap-

Photodynamic therapy (PDT) is a developing cancer treat-
ment involving the administration of a photosensitizing
compound and its subsequent activation by light in order
to provide the desired reactions to destroy tumor cells. The
technique relies on the favoring the reactions that can form
reactive oxygen species that are able to inactivate the tu-
mor cells. However, it has been recognized that this prin-
ciple also have a huge potential for microorganisms inac-
tivation and water detoxication. Microorganisms such as
bacteria, fungi, yeasts and virus (including HIV) can also
∗
Corresponding author: e-mail: janice@iqsc.usp.br
propriated photosensitizer as an alternative treatment of
low cost for localized infection such as acnes and fungical
skin lesions for example. Besides, Photodynamic Inactiva-
tion (PDI) can be used for sterilization of blood and its sub
products for clinical use, in the treatment of drinking water
as well as in antimicrobial treatment of foods [1].
At the present, a large number of photosensitizers are
in various stages of trials, however, just few of them has
received regulatory approval [2]. Hematoporphyrin deriva-
tives (HpD) and its more purified versions were the first

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Laser Phys. Lett. 6, No. 1 (2009)
photosensitizers employed in PDT and they are referred to
as first generation [3]. Photofrin (PF) (porfirmer sodium)
is a complex of porphyrin oligomers and was the first to
be approved for a health agency for PDT of bladder can-
cer in 1993 in Canada [4], been now approved for spe-
cific clinical applications in several countries in Europe,
America and Asia and has been under investigation for
use in other malignant and non-malignant diseases [5].
Photosan (PS) and Photogem (PG) are the correspond-
ing photosensitizers produced in Germany and Russia, re-
spectively. Photogem is the most used photosensitizer
in Brazil [5,6]. These three HpD are believed to present
chemical and photophysical characteristics and diagnos-
tic and therapeutic features identical to PF [7,8]. How-
ever, recent studies have shown that they are in fact dif-
ferent in its photostability [9] and degradation behavior
[10,11], cytotoxicity in several tumor and non-tumor cell
lines [9,10,12], threshold dose [13], and depth of necro-
sis [13]. Chlorins derivatives are efficient photosensitizers
and demand a lower drug dose and and shorter illumina-
tion time [2]. Photodithazine (PZ) is a new photosensi-
tizer produced in Russia that consists of the N-methyl-D-
glucosamine derivative of chlorin e-6 that is water soluble
and has an intense absorption in the range of 650 – 680 nm,
where biological tissues are more transparent to light than
in 630 nm where porphyrins are excited, being classified
as a second-generation drug due to its precise chemical
structure [14,15].
The present paper provides a comparative study be-
tween these four photosensitizers using a combination of
techniques employing bovine serum albumin (BSA) and
uric acid (UA, a singlet oxygen scavenger) as chemi-
cal dosimeters, the time for the photosensitizers to cause
photo-hemolysis of a suspension of red blood cells (RBC)
as well as the partition coefficient (log P ) between 1-
octanol and water to access the relative lipophilicity of
the compounds, a significant parameter to predict the rel-
evance of lipophilicity in biological and medical studies
[16,17]. The suitability of the octanol-water logP is due to
the amphiphilic nature of octanol, that allow to mimic, for
instance, the biological membrane environment [18]. The
agreement of the obtained results using these association
of methodologies will be discussed.
2. Materials and methods
2.1. Preparation of the photosensitizers
PF, PG, and PS are made by Axcan Pharma (Canada),
Moscow Institute of High Chemical Technology (Russia),
and Seehof Laboratories (Germany), respectively. PZ was
purchased from Veta-Grand, Russia. The 5 mg/mL stock
solutions of the photosensitizers, in 20 mmol/L phosphate
buffered saline (PBS) pH 7.4, were stored in the dark at
4◦ C until perform the desire experiments.
www.lphys.org
65
2.2. Determination of the octanol/phosphate
buffer partition of the photosensitizers
In order to estimate the hydrophobicity of the photosen-
sitizers, the partition coefficient between 1-octanol and
phosphate buffer at pH = 7.4 was determined by the well
spread shake-flask method [19–21], using 2 mL of 1-
octanol and 2 mL of photosensitizer solution (10 μg/mL)
in buffer in a multiple extraction procedure using a vor-
tex shaker (2 min). The phase separation was succeeded
in about 15 min (centrifugation at 2000 rpm). The absorp-
tion spectra (200 – 800 nm) of the aqueous phase were ob-
tained before and after the extraction and used to evalu-
ate the log P in order to obtain the relative hydrophobic-
ity. Considering that the concentration of the compound
at octanol phase in the equilibrium is proportional to the
amount of compound which was migrated from the water
phase, i.e., applying the Lambert-Beer theory, Coctanol =
(Abef ore − Aaf ter )/(εwater ), and the concentration re-
maining in the water phase, Cwater = Aaf ter /(εwater ),
one can be deduce the log P = log(Coctanol /Cwater ) as
evaluated by
 
Abef ore
log P = log −1 . (1)
Aaf ter
The εwater is the extinction coefficient of the compound
in the saturated water with octanol, A is the absorbance of
water phase containing the compound, and  is light path-
length.
2.3. Photosensitized reaction of BSA
To investigate the photoxidation of biomolecules [18],
air-saturated mixtures containing phosphate buffered
BSA (2 μg/mL, pH = 7.4) and photosensitizer solutions
(1 μg/mL) were continuously illuminated with LED
(630 nm, I = 25 mW/cm2 , t = 40 min). Each 5 min an
aliquot was removed in order to obtain the emission spec-
tra in a HITACHI F-4500 with excitation at 279 nm. The
340 nm fluorescence intensity decay was used to evaluate
the first-order photo-oxidation rate constant (k, min−1 ) of
the aromatic amino acids:
 
F
ln = −k t , (2)
F0
where F0 and F are, respectively, the fluorescence inten-
sities of untreated (BSA + photosensitizer) solution and
the fluorescence of this solution at different times of irra-
diation. The values of k were normalized by the values of
the absorbance of the photosensitizers in the wavelength of
irradiation since HpDs and chlorins present different ab-
sorption coefficients in 630 nm.
2.4. Determination of photosensitizers efficiency
using uric acid as a chemical dosimeter
Since uric acid (UA) is an efficient oxygen quencher,
in this method [22], a mixture of UA (30 μg/mL) and
c 2009 by Astro Ltd.
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66
sensitizer (15 μg/mL) was illuminated (LED, 630 nm,
I = 13 mW/cm2 , t = 300 s) and the photodynamic activity
coefficient (PA) of the photosensitizer was determined fol-
lowing the decrease of UA absorbance at 293 nm (ΔAU A )
as a function of irradiation time (t):
ΔAU A × 105
PA = , (3)
I0 tAλPIRR
S
where: PA is the coefficient of photodynamic activity
(m2 s/W = m2 /J), I0 corresponds to the light intensity
(mW/cm2 ), and AλPIRR S is the absorbance of sensitizer in
the test mixture at irradiation wavelength λIRR . It was
used 300 seconds as standard irradiation time, since there
is a linear correlation between ΔAU A and PS concentra-
tion.
2.5. Determination of efficiency the
photosensitized hemolysis by photosensitizers
The photosensitized hemolysis of RBC had been used as
a tool in order to understand some routes of reaction,
for instance, porphyria involving the phototoxic reactions
[23]. The approach was based on the previously described
methodology by Kahn and Fleischaker, 1971 [24] and else-
where. The used procedure is a coarsely adaptation of
the method described in the literature. The blood was do-
nated by the own experimenter, collected immediately be-
fore the assay in a Vacutainer containing EDTA as anti-
coagulant. The erythrocytes were isolated from the other
blood components by successive washings of the pellet
(centrifugation at 3000 rpm, 20 min and 4◦ C) and sus-
pended with PBS. The hemolysis was quantified by eval-
uation of the time to reach 50% (t1/2 ) of the complete
hemolysis (t∞ ) measuring the absorbance at 577 nm of the
oxy-hemoglobin released from the RBC mainly due to ox-
idation of membrane lipids. It was employed an adapted
Boltzmann function (Eq. (4)), which generates sigmoid
shaped curves [25].
H0 − H∞
Hemolysis(%) = H = H∞ +  ,
1 + exp t−t
dt
50
where H∞ is the hemolysis at long limit time of irra-
diation (t∞ ), H0 is the background hemolysis, t is the
time of sensitization progress, t50 is the time to observe
H50 = (H0 + H∞ )/2 (meaning the inflection point of sig-
moid), and dt is the time constant at t50 , that means in
practice the dispersion of sigmoid. The parameters that
outcome from fitting are t50 , H0 , H∞ , and dt . The di-
luted blood cells suspensions (2.0% hematocrit) were in-
cubated with the sensitizer (10 μg/mL) for 1 h in the dark
at 4◦ C in order to guarantee its distribution in the RBC
membrane, then they were irradiated with LED (630 nm,
I = 25 mW/cm2 , t = 10 min). The aliquots were collected
at desired times of irradiation, centrifuged at 2000 rpm
during 1 min, and the absorbances at 577 nm of oxy-
hemoglobin of the supernatants diluted in buffered saline
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R.S. Cavalcante, H. Imasato, et al.: A combination of techniques to evaluate efficiency
0.9
PF
PG
PZ
PS
0.6
Absorbance
0.3
0
300 400 500 600 700
Wavelength, nm
Figure 1 (online color at www.lphys.org) Absorption spectra
of the photosensitizers: PF, PG, PS, and PZ prepared in PBS,
10 μg/mL, pH = 7.4
were measured [19]. The samples secure disposals were
assured by treatment for 30 min with sodium hypochlorite
3% and sterilization in autoclave at 120◦ C for 20 min. The
complete (H = 100%) hemolysis was reached by control
experiment, in which RBC were disrupted by their exposi-
tion to the deionized water. The absorbance at 577 nm of
the total released hemoglobin (A100 ) was measured, and
used to express the hemolysis (Eq. (5)):
A
H= × 100 . (5)
A100
2.6. Mechanism of photo-hemolysis
In order to elucidate the mechanism involved in the pho-
tosensitized damage of the RBC membrane, the effect of
two suppressors were investigated. Sodium azide (NaN3 )
is a know suppressor of singlet oxygen (1 O2 ) [19,26–29]
and manitol is a OH radicals scavenger [19,30–32]. The
(4) photosensitized hemolysis experiments were performed as
described above, but, in the presence of these suppressors
(2 mM), each one in a separate experiment.
3. Results
3.1. Photosensitizers
PF, PG, and PS are derivatives of hematoporphyrin, pre-
senting spectral bands characteristics of C2v free-base por-
phyrins, i.e., presenting two sets of two weak Q-bands (I,
II, III, and IV), together with an intense Soret band around
370 nm (Fig. 1). In contrast, PZ, as a chlorin e-6 deriva-
tive, presents a Soret band with maximum at 401 nm and
three Q-bands: 505 nm, a very weak one; 600 nm, and a
third one at 655 nm, the I-band, which is red shifted and
possess higher intensity of absorption in comparison with
all HpD studied in the present work.
www.lphys.org
Laser Phys. Lett. 6, No. 1 (2009) 67

Photosensitizer log P Photosensitizer k, min−1

PZ 0.20 PZ 14.8±0.3
PF 0.15 PG 9.8±0.5
PG 0.11 PF 3.2±0.7
PS –0.22 PS 2.9±0.5

Table 1 Values of log P of the photosensitizers. The values rep- Table 2 Values of BSA photoxidation constant of the photo-
resent (m ± sd), n = 3 sensitizers corrected by the absorbance. The k values represent
(m ± sd), n = 3, performed in triplicate
100

0
the irradiation time. The linear dependence of ln(F/F0 )
-100 values indicates a first-order reaction. The ln(F/F0 ) val-
-200
ues were corrected by the solution absorbance in the wave-
length of irradiation (λ = 630 nm). The slope of the linear
ln(F/F0)

-300 regression was used to determine the photoxidation rate

-400
-500 PF
PG
-600 PZ
PS
-700
0 10 20 30
Irradiation time, min
Figure 2 (online color at www.lphys.org) ln(F/F0 ) corrected
by the absorbance in the wavelength of irradiation (λ = 630 nm)
as a function of the irradiation time showing the suppression
of the fluorescence emission due to the photoxidation of BSA
(2 mM) in the presence of the photosensitizers (1 μg/L), pH = 7.4,
excitation 278 nm
3.2. Determination of partition coefficient
(log P )
The obtained log P values were: 0.20 (PZ), 0.15 (PG),
0.11 (PF), and –0.22 (PS) (Table 1). According to the
Eq. (1) a higher log P means a more hydrophobic com-
pound, i.e., more soluble in the octanol than in the water.
Then, the data suggest that PZ is the more hydrophobic
sensitizer being PS the most hydrophilic. The partition co-
efficient has been considered a helpful parameter in order
to predict, for example, the cellular uptake, bioavailability,
and toxicity [33]. The correlation between this parameter
and transport across biological membrane has been con-
sidered straightforward. The membrane capacity to perme-
ate the molecules can be associated to induced membrane
lipid transition by the solubilized compounds, i.e., by their
lipophilicity, perhaps by conformation adaptation accord-
ing to the medium features [25]. Then, due to potentiality
of partition coefficient, many efforts has been devoted in
order to develop reliable theoretical approach [26].
3.3. Photosensitized reaction of BSA
The efficacy of BSA degradation by photosensitization is
illustrated in the Fig. 2 obtained by the 340 nm fluores-
cence emission decay of a BSA solution as a function of
constants of the aromatic amino acids residues of BSA
[36]. The following k values (min−1 ) were obtained: 14.8
(PZ), 9.8 (PG), 3.2 (PF), and 2.9 (PS) (Table 2). The re-
sults show that PZ is the most efficient photosensitizer con-
cerning to BSA degradation. The higher efficiency of PZ
compared to PF can be related to its longest excited triplet
40
state lifetime that can favor the bimolecular collision in
order to promote the triplet oxygen to the singlet state.
It is worth to mention that the HpD samples are consti-
tuted by mixtures of oligomers so the concentrations were
not presented in molar unity; instead, they were expressed
as μg/mL. Among the HpD based drugs, the PG was the
most efficient photosensitizer concerning the ability to ox-
idize BSA. This result are in agreement with the finds of
Ferreira et al. [9] with these three hematoporphyrin deriva-
tives showing that there is a strong correlation among the
IC50 in VERO cell line and the threshold dose (Dth ) in
rat livers so that the photosensitizer with the lowest photo-
stability rate (PS) is also the one with the highest Dth and
IC50 , showing less efficacy in cell killing and tissue necro-
sis in PDT.
3.4. Determination of photosensitizers
photodynamic activity
In Fig. 3 it can be seen that UA does not absorbs in the
wavelength of irradiation (630 nm), assuring the photo-
sensitizer as the unique light absorber specie. The figure
also shows the intense variation in absorbance bands as-
signed to UA (293 and 235 nm) and the minor changes
at 369 nm (PG) as a function of the irradiation time. This
chemical dosimetry is based on suppression of singlet oxy-
gen by uric acid, k = 3.6×108 mol/L, which reaction leads
to formation of triuret, sodium oxalate, allantoxaidin and
CO2 , according to assumption of Fisher et al. in 1998 [22].
The Table 3 presents the obtained values for photodynamic
activities: 64 (PZ), 37 (PG), 9 (PF), and 4 (PS). The effi-
cacy sequence is in the agreement with that predicted by
BSA oxidation. The differences in the photo-oxidative ac-
tion of the HpD may be due to the differences in the aggre-
gation [10] and to the content of monomers, dimmers and
oligomers of the photosensitizers components. Menezes et

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68 R.S. Cavalcante, H. Imasato, et al.: A combination of techniques to evaluate efficiency

1.0 5
0 (a) 542 nm 577 nm
292 nm 30 s
1 min
0.8 3 min 4
5 min
235 nm 10 min
20 min
Absorbance

Absorbance
0.6 30 min 3

0.4 2

0.2 1

0 0
200 300 400 500 600 700 500 550 600 650 700
Wavelength, nm Wavelength, nm
100
(b)
Figure 3 (online color at www.lphys.org) Spectral variation of
the mixture of Photogem (15 μg/mL) + UA (30 μg/mL) prepared 80
in phosphate buffer (pH = 7.4) irradiated with LED (λ = 630 nm,
I = 25 mW/cm2 , t = 40 min). The arrows indicate the decrease of
Hemolysis, %

uric acid bands 60

Photosensitizer Photodynamic activity, m2 /J

PZ 64 ± 14 40
PG 37 ± 9
PF 9±2
PS 4±1 20
PZ
PF
PG
Table 3 Photodynamic activity of the photosensitizers, the val- PS
0
ues representing (m ± sd), n = 10 0 2 4 6 8 10
Time, min

al. [10] has reported that PS is more aggregated than PF
that is more aggregated than PG. The chlorin derivative is
the compound that has the highest value of PA, corrobo-
rating with the results obtained in Table 2.
3.5. Determination of the hemolysis efficiency
Figure 4 (online color at www.lphys.org) (a) – spectra with
the Q-bands of released oxy-hemoglobin as a function of RBC
membrane damage in the presence of photosensitizers and light
(630 nm); (b) – percentage of photo-hemolysis of RBC suspen-
sion (2%, pH = 7.4 in PBS), in the presence of photosensitizers
(10 mg/mL) irradiated with LED-630 nm (25 mW/cm2 )

In order to get closer to the biological environment, the

erythrocytes were used to evaluate the capability of pho-
tosensitizer to interact and photooxidizing the membrane
proteins and unsaturated lipids, both targets in PDT. Dur-
ing the treatment the membrane permeability can be af-
fected by rupture of the membrane architecture, draining
off the cytoplasm. In this set of experiments, the red blood
cells membrane damage is monitored by the absorbance at
540 or 577 nm Q-bands of flowing out oxy-hemoglobin.
One possible photo oxidative parallel process could be the
transformation of oxy-hemoglobin to met-hemoglobin that
can be monitored by change of Q-bands profile. As it can
be observed in the Fig. 4a, there was no formation of a
630 nm band characteristic of met-hemoglobin. Moreover,
it was observed less than 1% of photo damage effect in
the absence of sensitizers, which was attributed mainly to
the mechanical disruption during the entire procedure as-
signed as background hemolysis, H0 in Eq. (4). The results
show that all sensitizers used can damage the cell mem-
brane. The obtained values for t50 (min) obtained by fit-
ting the data presented in Fig. 4b (percent of hemolysis as
a function of time) are showed in Table 4 and are in the fol-
lowing ranking: 1.3 (PZ), 3.8 (PG), 3.4 (PF), and 5.3 (PS).
In this kind of experiment, the shorter is the t50 , the more
efficient is the photo damage process. Again, the results
demonstrate that PZ is the most efficient photosensitizer.
However, in this case the values of t50 for PG and PF are
very close, while the efficacy based on the previous tech-
niques the PF and PS were similar in the photo oxidative
behavior.
3.6. Mechanism of photo-hemolysis
The Fig. 5 presents the hemolysis curves and the Fig. 5
presents the obtained values of t50 obtained from the fit-

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Laser Phys. Lett. 6, No. 1 (2009) 69

Photosensitizer Hmax t50 , min

100
PZ 95 ± 2 1.3 ± 0.2
PF 92 ± 3 3.4 ± 0.1
80 PG 81 ± 5 3.8 ± 0.2
PS 89 ± 2 5.3 ± 0.1
Hemolysis, %

60

Table 4 The maximum percentage of hemolysis and the time to

40 reach 50% of hemolysis of a suspension of RBC in the presence
of photosensitizers and light. The Hmax and t50 values represent
20 (m ± sd), n = 5, performed in triplicate

PF
0
PF Control
0 2 4 6 8 10 12 14
100 Manitol

Azide

80 PG Control

Manitol
Hemolysis, %

60 Azide

PZ Control
40 Manitol

Azide
20 PS Control

PG Manitol
0 Azide
0 2 4 6 8 10 12 14
100 0 2 4 6 8
Hemolysis t50, min
80
Figure 6 (online color at www.lphys.org) Behavior of the t50 in
Hemolysis, %

60 the presence of the suppressors azide and manitol for the four
photosensitizers
40

ting of data from Fig. 5 in the experiments performed in

20
the presence of the suppressors azide and manitol. It was
PS observed that azide lead to a decrease in the amount of
0
hemolysis and to an increase in t50 values suggesting that
0 2 4 6 8 10 12 14
100 the RBC is protected against singlet oxygen by the azide.
The results suggest that these photosensitizers effect is due
80
to the type II mechanism, i.e., mediated by singlet oxygen.
On the other hand, manitol did not disturb the photoox-
Hemolysis, %

idative induced hemolysis of the studied photosensitizers,

60
so it can be concluded that the photooxidative hemolysis
does not involves OH radicals. Also the H∞ values are in
40 agreement with the t50 values. As general trends long t50
corresponds to the low H∞ . The low value of H∞ means
20 that the photoxidative disruption of membrane is less ef-
PZ
fective than hypotonic RBC damage. The relevance of this
0 kind of approach resides in clarify the mechanism of ac-
0 2 4 6 8 10 12 14 tion of photosensitization involving biological membrane
Irradiation time, min destabilization. For example, it has been expended efforts
in order to discriminate the apoptosis involved in PDT ef-
Figure 5 (online color at www.lphys.org) Effect of the suppres- ficacy [2].
sors azide (red symbols) and manitol (green symbols) on the
hemolysis of a RBC suspension (2%, pH = 7.4 in PBS), in the
presence of photosensitizers (10 mg/mL) Irradiated with LED- 4. Conclusions
630 nm (25 mW/cm2 ). The control experiments are the black The differences in the photo-oxidative action of the
symbols HpDs should be due to the differences in the content of

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 70 R.S. Cavalcante, H. Imasato, et al.: A combination of techniques to evaluate efficiency
monomers, dimmers and oligomers of the photosensitizer
components as well as in the aggregation. The results of
hemolysis experiments and the partition coefficients val-
ues suggest that these differences should result in different
interactions of HpD with the membrane constituents. PF
and PG seems to have similar values of t50 and to be more
effective than PS concerning the hemolysis of erythrocytes
and log P values.
Therefore the results of the combination of these
methodologies suggest that the photodynamic efficient
ranking is: PZ  PG ≥ PF  PS and can lead to impor-
tant information to estimate the photodynamic efficacy of
photosensitizers. Besides that, these results are in agree-
ment with cytotoxicity of these photosensitizers in normal
and tumor cells [37,38] as well as to chromatographic sep-
aration of the HpDs [39], both performed in our group,
showing that the more lipophilic is the dye, the more acute
is the damage to the RBC membrane, as well as the oxida-
tion of indol, which is immersed in the more hydrophobic
region of BSA.
Acknowledgements The authors would like to thank to Mrs.
Claudia Bernal for the excellent technical assistance and the fi-
nancial support of FAPESP and CAPES.
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