Photodamage in a mitochondrial membrane model modulated by the topology of cationic and anionic meso-tetrakis porphyrin free bases

Article abstract of DOI:10.1111/php.12228

The photodynamic effects of the cationic TMPyP (meso-tetrakis [N-methyl-4-pyridyl]porphyrin) and the anionic TPPS4 (meso-tetrakis[4- sulfonatophenyl]porphyrin) against PC/CL phosphatidylcholine/cardiolipin (85/15%) membranes were probed to address the inf

Full text of DOI:10.1111/php.12228

Photochemistry and Photobiology, 2014, 90: 596–608
Photodamage in a Mitochondrial Membrane Model Modulated by the
Topology of Cationic and Anionic Meso-Tetrakis Porphyrin Free Bases
†1
†1
1
1
ꢀ
Cintia Kawai , Juliana C. Araujo-Chaves , Taciana Magrini , Camila O. C. C. Sanches ,
Sandra M. S. Pinto1, Herculano Martinho1, Nasser Daghastanli2 and Iseli L. Nantes*1
1
^
ꢀ
Centro de Ciencias Naturais e Humanas (CCNH), Universidade Federal do ABC, Santo Andre, SP, Brazil
2
^
ꢀ
Centro de Engenharia, Modelagem e Ciencias Sociais Aplicadas (CECS), Universidade Federal do ABC, Santo Andre, SP,
Brazil
Received 20 May 2013, accepted 9 December 2013, DOI: 10.1111/php.12228
ABSTRACT
energy transfer, that is by Type-I and/or Type-II mechanisms,
The photodynamic effects of the cationic TMPyP (meso-tetra-
kis [N-methyl-4-pyridyl]porphyrin) and the anionic TPPS4
(meso-tetrakis[4-sulfonatophenyl]porphyrin) against PC/CL
phosphatidylcholine/cardiolipin (85/15%) membranes were
probed to address the influence of phorphyrin binding on
lipid damage. Electronic absorption spectroscopy and zeta
potential measurements demonstrated that only TMPyP
binds to PC/CL large unilamellar vesicles (LUVs). The photo-
damage after irradiation with visible light was analyzed by
dosages of lipid peroxides (LOOH) and thiobarbituric reac-
tive substance and by a contrast phase image of the giant
unilamellar vesicles (GUVs). Damage to LUVs and GUVs
promoted by TMPyP and TPPS4 were qualitatively and
quantitatively different. The cationic porphyrin promoted
damage more extensive and faster. The increase in LOOH
was higher in the presence of D2O, and was impaired by
sodium azide and sorbic acid. The effect of D2O was higher
for TPPS4 as the photosensitizer. The use of DCFH demon-
strated that liposomes prevent the photobleaching of TMPyP.
The results are consistent with a more stable TMPyP that
generates long-lived singlet oxygen preferentially partitioned
in the bilayer. Conversely, TPPS4 generates singlet oxygen in
the bulk whose lifetime is increased in D2O. Therefore, the
affinity of the porphyrin to the membrane modulates the
rate, type and degree of lipid damage.
respectively (4–7). The selectivity of PDT constitutes an advan-
tage comparatively to traditional methods, such as chemotherapy
and radiotherapy (8–10), due to its selective and localized actions
(11,12). A high efficiency of PDT with minimal side effects
depends on some chemical and physical characteristics of the
PS: (1) highest affinity for the diseased cells or the pathogenic
organisms; (2) low toxicity in the dark; (3) chemical stability
and low aggregation; (4) rapid body clearance; (5); high quantum
yields of a long-lived excited triplet state and (6) electronic
absorption at wavelengths of maximum transparence of biologi-
cal tissues, avoiding the absorption of endogenous chromo-
phores, such as melanin, hemoglobin and cytochromes and
enabling deeper light penetration (13,14). However, wavelengths
longer than 900 nm should be avoided because of their insuffi-
cient energy to excite a dye to the triplet state (12,15,16).
A diversity of PS compounds satisfies the above-mentioned
desirable characteristics, and includes the porphyrins (17,18).
The absorption spectra of porphyrins in the visible region
includes the therapeutic window for PS compounds (17,18) The
uptake and localization of a PS in cells depends on its chemical
structure, the mechanism of its uptake by the cell, its intrinsic
affinity to cell components and any chemical modifications in
the course of irradiation (19–21).
Biological and synthetic porphyrins are both largely used as
PS in PDT. Studies about the biological effects of two meso-tet-
rakis porphyrins, TPPS4 (anionic) and TMPyP (cationic) demon-
strated that the cationic porphyrin has affinity to the inner
mitochondrial membrane (22,23). Therefore, in mitochondria,
manganese TMPyP has been used as an antioxidant against
superoxide ions and as a prooxidant agent in the corresponding
ferric form (21,24–26). However, cationic TMPyP has been
found also in lysosomes and the nucleus, and, after irradiation,
both TMPyP and TPPS4 have been found preferentially in the
nucleus, probably due to their association with proteins that
migrate to the nucleus after oxidation (21,27).
Biological membranes are targets for the action of PDT. There-
fore, numerous studies have been concerned with the interaction
of porphyrins with liposomes and other membrane mimetic models
(28–34). However, literature lacks comparative studies involving
photodynamic action of hydrophilic porphyrins on mitochondrial
membrane models. The PDT action on biological membranes
directly contributes to cell death due to local oxidative damages
INTRODUCTION
Photodynamic therapy (PDT) is
a noninvasive therapeutic
method mainly used in the fields of dermatology, otorhinolaryn-
gology, ophthalmology, neurology, gastroenterology and urology
for the treatment of precancerous and superficial malignant skin
tumors as well as degenerated and infected tissues (1–3). The
photosensitization is achieved by electronically exciting a dye
photosensitizer (PS) promoted by the absorption of light at suit-
able wavelength (4–7). The photodynamic action can be per-
formed directly with the triplet excited state of the dye and/or
with O2(1Dg) (singlet oxygen) generated by electronic excitation
*Corresponding author emails: ilnantes@gmail.com and ilnantes@ufabc.edu.br
(Iseli L. Nantes)
†These authors contributed equally
© 2013 The American Society of Photobiology
596

Photochemistry and Photobiology, 2014, 90 597
Chart 1. Structures of TMPyP and TPPS4: a-cationic TMPyP (meso-tetrakis (N-methyl-4-pyridyl)porphyrin). b-anionic TPPS4 (meso-tetrakis(4-sulfona-
tophenyl)porphyrin)
concentration was determined by the oxidation of Fe(II) in the presence of
xylenol orange: aliquots of the sample (10 lL) were extracted from incuba-
tion at room temperature and mixed with 890 lL of Milli-Qâ water and
100 lL of hydroperoxide reagent that contained 2.5 mM xylenol orange,
2.5 mM (NH4)2Fe(SO4)2 and 1.15 M HClO4 in Milli-Qâ water. The mix-
ture was incubated for 30 min at room temperature. The oxidation of Fe(II)
by LOOH generates Fe(III), which reacts with xylenol orange and is con-
and by secondary signaling death pathways. Mitochondria are
commonly involved in the apoptosis promoted by PDT by accu-
mulating the PS or by activating the intrinsic apoptotic pathway as
a consequence of damages to other cell structures (35,36). There-
fore, it is relevant to understand the correlation of the porphyrin
binding to mitochondrial membranes with their photodynamic
effects. This study investigated the photodynamic action of the cat-
ionic TMPyP (meso-tetrakis [N-methyl-4-pyridyl]porphyrin) and
the anionic TPPS4 (meso-tetrakis[4-sulfonatophenyl]porphyrin)
(Chart 1) against PC/CL (phosphatidylcholine/cardiolipin, 85/
15%) liposomes as a model of the lipid fraction of the inner mito-
chondrial membrane. The lipid damage was analyzed by peroxide
dosage, conjugated dienes, thiobarbituric reactive substances
(TBARs) and giant unilamellar vesicles (GUVs) images from opti-
cal microscopy and DCF fluorescence. The use of GUVs allows a
real-time visualization of the oxidative damages in a membrane
model. A mimetic model of the inner mitochondrial membrane
was chosen, because the cationic porphyrin TMPyP has been
described to accumulate in mitochondria (22,23).
verted to the colored product that absorbs at 560 nm. LOOH concentration
ꢀ1
was calculated by using e = 560 nm = 6.45 9 104
M
cmꢀ1. TBARS
was determined at 535 nm after reaction with TBA, using the molar extinc-
tion coefficient 1.56 9 105 Mꢀ1 cmꢀ1
.
Electronic absorption spectra measurements. The electronic absorp-
tion spectra of porphyrins were measured by using a Varian Cary 50,
Varian Inc. (CA, United States) spectrometer. The spectral resolution for
wavelength scan was 0.5 nm. The optical path length was 1 cm for the
LOOH, TBAR and DCFH dosages, and 0.1 cm for the porphyrin spectra
measurements.
Giant unilamellar vesicles (GUVs). Giant unilamellar vesicles of PC/
CL (80/15 mol%, respectively) were grown using the electroformation
method (38,39). Briefly, 40 lL of a mixture of lipids 2.5 mM in chloro-
form solution was spread on the surfaces of two conductive glass plates
(coated with indium tin oxide). The apparatus were positioned with the
conductive sides facing each other and separated by a 2-mm-thick Teflon
frame. In the following, the electroswelling chamber was filled with a
0.2 M sucrose solution, and connected to an alternating power generator
at 1 V with a 10 Hz frequency for 2 h at 25°C. The vesicle suspension
was removed from the chamber and diluted ~6 times into 0.2 M glucose
solution. In this condition, spherical GUVs were obtained with diameters
in the range of 10 lm. The sugar optical asymmetry between the interior
and the exterior of the vesicles created differences in density and in the
refractive index between the sucrose and glucose solutions; the vesicles
were, therefore, stabilized by gravity at the bottom of the chamber, and
had the best contrast when observed under phase contrast microscopy.
The observation of giant vesicles was performed by phase-contrast
microscopy.
MATERIALS AND METHODS
Chemicals. TMPyP (meso-tetrakis [N-methyl-4-pyridyl]porphyrin), TPPS4
(meso-tetrakis[4-sulfonatophenyl]porphyrin), 2′,7′-dichlorodihydrofluores-
cein diacetate (H2DCF-DA), sorbic acid, sodium azide, phosphoric acid,
thiobarbituric acid (TBA) and percloric acid (HClO4) were purchased
from Sigma-Aldrich Corp. (St. Louis, MO, USA). Xylenol orange was
purchased from Fluka Chemical Corp. (Ronkonkoma, NY, EUA). Glu-
cose, sucrose, ammoniacal ferrous sulfate ([NH4]2Fe[SO4]2), chloroform,
n-buthanol and ethanol were obtained from Synth (Labsynth Ltda., SP,
Brazil). L-a-phosphatidylcholine from egg (PC), cardiolipin bovine heart
(CL), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylcho-
line (DOPC) and tetraoleoyl cardiolipin (TOCL) were purchased from
Avanti Polar Lipids (Alabaster, AL, USA). Deuterium oxide (D2O) was
purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA,
Optical trap and phase-contrast microscopy images. Home-made opti-
cal tweezers were employed to trap and to image isolate GUVs (Magrini,
T. and Martinho, H., unpublished results). The optical tweezer was com-
posed by
a 975 nm Pigtailed Laser Diode (Butterfly Package
PL980P330J), a set of lenses, mirrors and a Nikon 1009 Oil Immersion
Objective for collimating laser light. The laser power was less than
5 mW in the trapped GUV. ACCD USB DCU224C camera with a reso-
lution of 1280 9 1024 pixels and a field of 709 87.6 lm was used to
capture the images. The public domain software Image J (Wayne Ras-
hand, rsb.info.nih.gov/ij/) was used to process and analyze the captured
images. The radial intensity distribution profile as function of time was
used to obtain quantitative pieces of information concerning the structural
variations.
EUA). The TMPyP stock solution concentration was checked using the
ꢀ1
absorption coefficient e423nm = 2.26 9 105
M
cmꢀ1 and TPPS4 at
ꢀ1
e412 = 5.30 9 105
M
cmꢀ1 (37).
Generation of photo-oxidative species. Where indicated, porphyrins
were excited by irradiation with a white LED array. The LED array emits
200 lW/cm2 in 532 nm measured by FieldMate powermeter (Coherent
Inc.). The laser presented a 10 mW power measured by the same power-
meter.
Lipid oxidation assays:. Lipid oxidation was evaluated with lipid
peroxide (LOOH) and TBARS production. Lipid hydroperoxide
Generation of DCF by the photodynamic action of photosensitized
porphyrins. DCFH (Sigma) was obtained by hydrolysis of its diacetate

598 Cintia Kawai et al.
form (H2DCF-DA, 2′, 7′-dichlorodihydrofluorescein diacetate). Briefly, an
appropriate aliquot from a H2DCF-DA stock solution (200 lM) was
added to an aliquot of NaOH (50 mM) in the dark, kept on ice for
30 min in the dark, and used in the same day of its preparation. The
solution of the hydrolyzed product was then neutralized with an appropri-
ate volume of hydrochloric acid to a pH of 7.2. DCF, which is the fluo-
rescent product of DCFH oxidation, emits at kmax = 522 nm when
excited at 490 nm (40–42). Fluorescence spectra were obtained with a
Varian Cary Eclipse fluorimeter, Varian Inc. (CA, USA). All measure-
ments were made just after each irradiation procedure and at room tem-
perature. The time dependence of DCF fluorescence was fitted to,
interaction between the cationic free base TMPyP and the
negatively charged PC/CL liposomes changed the energy of p–
p* transitions, leading to red shifts in the Soret and Q bands.
The Soret band shifted 5 nm and peaked at 428 nm. The peaks
of Q bands Qy(0,0), Qy(0,1), Qx(0,0) and Qx(1,0) shifted to 521,
557, 592 and 648 nm, respectively. The left and right panels of
Fig. 2A show, respectively, the spectra of TMPyP and TPPS4 in
a homogeneous medium (gray lines) and in the presence of PC/
CL liposomes (dashed lines). The turbidity promoted by the pres-
ence of liposomes was subtracted from the original data, and the
resulting spectra of the porphyrins presented as black lines. Fig-
ure 2A (right panel) shows the spectra of TPPS4 in the presence
of 1 mmol L-1 of PC/CL liposomes. The presence of PC/CL
liposomes did not promote significant spectral changes in the
anionic porphyrin, indicating the low affinity of TPPS4 for the
negatively charged lipid interface.
eꢀtꢂk
FðtÞ ¼
ꢁð ꢀ
Þ
ð1Þ
Fmax
1
by using Origin (Microcal) software. Fmax is the asymptotic of the rate
curve, and k is the observed rate constant.
Determination of zeta potential (f). The measurements were carried
out in a Zetasizer Nano ZS, Malvern Instruments, Ltd. (London, UK), at
the temperature of 25°C. The f is determined by the electrophoretic
mobility, le by the application of the Henry equation and calculated by
using the Smoluchowski approximation. The f values are the average of
10 independent measurements that were calculated using a mono-modal
model and time measurement was determined by the instrument. For each
zeta potential measurements, the samples were previously equilibrated for
1 min at 25°C.
RESULTS AND DISCUSSION
Interactions of TMPyP and TPPS4 with PC/CL liposomes
The electronic absorption spectra of TMPyP and TPPS4 are typi-
cal of porphyrins with D2h molecular symmetry (Fig. 1).
TMPyP (black line) and TPPS4 (gray line) exhibit, respectively,
the intense Soret bands at 423 and 410 nm and the following
lower intensity Q bands: Qy(0,0) at 518 and 516 nm, Qy(0,1) at
555 and 553 nm, Qx(0,0) at 586 and 584 nm and Qx(1,0) at 644
and 634 nm (17,43–46). Figure 1 (right scale) shows that the
emission spectrum of the white LED used for the electronic exci-
tation of the porphyrins overlaps their Q bands.
The electronic absorption spectrum of TMPyP presented
significant changes in the presence of PC/CL liposomes. The
Figure 2. Effect of PC/CL liposomes on the electronic absorption spec-
tra of TMPyP and TPPS4. (A) Intensity of the differential TMPyP Soret
band at 440 nm plotted as a function of lipid concentration. The data
were obtained from the differential spectra of TMPyP at increasing lipid
concentrations (0.1, 0.25, 0.5, 0.75 and 1.0 mM PC/CL, respectively), as
indicated by the arrow in the inset. (B) Normalized electronic absorption
spectra of TMPyP in different media. The spectrum of TMPyP in 0.2 M
of glucose is shown as a black solid line, the spectrum of TMPyP in the
presence of 1 mM PC/CL (85/15 mol %) liposomes as a dotted line and
the spectrum of TMPyP dissociated from the negatively charged interface
by addition of 300 mM of NaCl as a gray line.
Figure 1. Overlapped electronic absorption spectra of the porphyrins
TMPyP and TPPS4 on the emission spectra of the lamps used for elec-
tronic excitation of the photosensitizers. The spectrum of TMPyP in
0.2 M glucose is shown as a black line, and the TPPS4 spectrum is
shown as a gray line. The emission spectrum of the white LED light
source is the gray area.

Photochemistry and Photobiology, 2014, 90 599
The porphyrin/lipid interaction was better evaluated by the
analysis of the differential spectra obtained from the subtraction
of the porphyrin spectrum in aqueous homogeneous media from
those obtained in the presence of different lipid concentrations.
Figure 2A shows the intensity of Soret band differential spectra
at 440 nm plotted as a function of lipid concentration. The dif-
ferential spectra are shown in the inset of Fig. 2A. The data were
best fitted by Eq. (2) derived from the binding equilibrium
(47,48):
above. However, the cationic TMPyP was more efficient than
the anionic TPPS4 was at damaging the phospholipids. After
60 min of irradiation in the presence of TPPS4, the LOOH con-
tent of the PC/CL vesicles increased more than two-fold, that is
from 2 to 5.5 lM. In the presence of TMPyP, the production of
LOOH was higher and faster. The LOOH content in the presence
of TMPyP increased almost eight-fold relative to the control after
15 min of irradiation using the white LED. Lipid oxidation was
not observed when the vesicles were incubated during 60 min
with the porphyrins in the dark (Fig. 3B, black columns in the
upper panel). Similar results were obtained by using the green
laser to promote the electronic excitation of TMPyP (not shown).
nkb½LꢃT
AL ꢀ A0 ¼
ð2Þ
1 þ kb½LꢃT
where AL ꢀ A0 is the differential absorbance intensity at differ-
Photodynamic effects of TMPyP and TPPS4 on PC/CL giant
unilamellar vesicles (GUVs)
ent lipid concentrations and kb is the binding constant. The fitting
ꢀ1
according to Eq. (1) resulted in kb = 9, 2 (ꢄ) 103
M
.
TMPyP is a hydrophilic porphyrin, and it is not expected to
be found buried in the bilayer. Thus, these spectral changes can
be assigned to an electrostatic interaction of the positively
charged metal-free TMPyP meso ligands with the negatively
charged interface of PC/CL (32,49).
The photodynamic effects of TMPyP and TPPS4 were also
probed in the presence of PC/CL GUVs. Figures 4A and B show
contrast phase images of GUVs during irradiation in the presence
of TMPyP and TPPS4, respectively. These images were
extracted at the indicated times from the movies recorded during
the irradiation of GUVs in the presence of porphyrins. The edi-
ted movies are available in the supplementary material section.
We notice that GUVs in the dark were not damaged by the por-
phyrins, and remained stable for hours (not shown). However,
when maintained under irradiation with the white LED, the
GUVs underwent significant alterations. Figure 4A shows images
consistent with progressive damage of a PC/CL GUV during
almost 18 min of irradiation in the presence of TMPyP. The
image obtained at 10 min of irradiation in the presence of
TMPyP shows that the GUV became leaky. The leakiness of
GUV initiated at ~7 min as evidenced by Fig. 4C showed bel-
low. The crescent contrast loss on the vesicle that culminates
with vesicle disruption at around 15 min attests to the leakiness
of the GUV. In the presence of TPPS4, the irradiation of GUVs
led also to damage in the membrane with a peculiar behavior. In
the course of the irradiation, undulations were apparent; there-
fore, the GUV undulated and produced encapsulated daughter
vesicles (internal buds) (Fig. 4B).
The binding of TMPyP on PC/CL liposomes was also corrob-
orated by zeta potential measurements. The zeta potential mea-
sured for PC/CL liposomes in the presence and in the absence of
TMPyP presented results similar to that previously presented by
Neto et al., 2013 (34). The authors studied the binding of
TMPyP on POPC:POPG LUVs with different percentages of the
anionic phospholipid (PG). The zeta potential (f) of PC/CL
(85% PC and 15% CL) in the absence and in the presence of
10 lM TMPyP was ꢀ42.5 and ꢀ39.5 mV, respectively. These
results are consistent with the occurrence of electrostatic interac-
tion between the negatively charged phospholipid and the cat-
ionic meso ligands of TMPyP.
The electrostatic interaction mechanism that binds TMPyP to
PC/CL liposomes was further reinforced by the addition of NaCl
in a medium containing TMPyP associated with liposomes
(Fig. 2B). The increase in ionic strength reversed the spectral
change in TMPyP promoted by PC/CL liposomes.
The formation of daughter vesicles in GUVs has been
described for GUVs that contain raft mixtures and were submit-
ted to osmotic stress (51). After 34 min of irradiation, the GUV
encapsulating the smaller sized GUVs expelled the internal smal-
ler vesicles that are apparently connected and resemble a tail
linked to the mother GUV (see the zoom of the smaller GUVs
detected at 39′27″ of irradiation). After the exclusion of most of
the small daughter GUVs, the mother vesicle was significantly
smaller (around 1/4 of its initial size) but without significant loss
of contrast. At the ultimate irradiation times, the process also cul-
minated in vesicle disruption. Figures 4A and B are representa-
tive of a set of different independent experiments.
To obtain a quantitative view of the loss of contrast, each
image was converted to a radial intensity distribution plot, as
shown in the upper and lower panels of Fig. 4C. The leakage of
GUV shares internal and external bulk solution, and abolishes
the difference in the refraction index, leading to loss of image
contrast. The upper panels of Fig. 4C show the normalized
intensity at the radial coordinates of a selected GUV image at
the initial (black line) and final (gray line) time of irradiation in
the presence of TPPS4 (left upper panel) and TMPyP (right
upper panel). The minimum and maximum of the plot
Photodynamic effects of TMPyP and TPPS4 on PC/CL large
unilamellar vesicles (LUVs)
To model the effects of PDT on the lipid fraction of biological
membranes, TMPyP and TPPS4 were used for the photo-oxida-
tion of phospholipids, PC and CL, arranged as LUVs (Fig. 3A
and B). Figure 3A shows the spectral changes in the porphyrin-
containing PC/CL liposomes in the course of the irradiation with
white LED (spectrum shown in Fig. 1). In the presence of
TMPyP (left panel), the partial bleaching of the porphyrin
concomitant with an increase in the absorbance at 234 nm
(DA234 nm around 0.2) was observed after 60 min of irradiation.
The increase in absorbance of lipids at 234 nm is suggestive
of the formation of conjugated dienes (50). In the presence of
TPPS4 (right panel), the increase in absorbance in the 234 nm
region during 60 min of irradiation was not significant (DA234 nm
around 0.02). Furthermore, the lipid oxidation was not associated
with significant porphyrin bleaching (Fig. 3A, right panel). The
lower panel of Fig. 3B shows that the LOOH content of the PC/
CL vesicles increased significantly over the course of a 60-min
irradiation in the presence of TMPyP and TPPS4 when the vesi-
cles were under conditions that are identical to those described

600 Cintia Kawai et al.
Figure 3. Photodynamic effect of TMPyP and TPPS4 on PC/CL liposomes. (A) Effect of white LED irradiation on the electronic absorption spectra of
10 lM of TMPyP and TPPS4 in the presence of PC/CL liposomes. The spectra were recorded at 0, 15 and 60 min of irradiation. (B) Lipid photo-oxida-
tion promoted by 10 lM of TPPS4 and TMPyP. The lower panel shows time-dependent formation of LOOH in the absence (control) and in the presence
of the porphyrins as indicated over the corresponding results. The upper panel shows the results obtained in the same above-mentioned conditions except
that the samples were incubated in the dark. Experiments were carried out using PC 85% and CL 15% 1 mM in a medium of 0.2 M of glucose.
correspond to the darker and brighter portion of the GUV image,
respectively. The corresponding radii were labeled rint and rext
In a speculative way different results obtained for GUVs chal-
lenged by the photodynamic action of TPPS4 and TMPyP might
result from the topology of the porphyrins with repercussions on
the site of singlet oxygen generation. TPPS4 and TMPyP were
added in the suspension of GUVs and it is not expect that the
hydrophilic photosensitizers can attain the internal compartment
of these vesicles. TPPS4 in the external bulk phase generated
singlet oxygen in the same location. Therefore, this excited spe-
cies diffused and accessed GUVs by the external face probably
having the lipids in the external leaflet as the preferential target.
The putative asymmetrical oxidative damage might be enough to
create a surface tension that resulted in buds, formation and
.
Insets in the right and left upper panels of Fig. 4C show repre-
sentative contrast phase images of optically trapped GUVs dur-
ing irradiation in the presence of TMPyP and TPPS4,
respectively. One could measure the loss of contrast by measur-
ing the visible thickness of the GUV membrane. This thickness
could be estimated by finding the difference between rext and rint
.
The values of rextꢀrint as a function of time are shown in the
bottom panels of Fig. 4C. These plots are consistent with the
loss of contrast phase that occurs only in the presence of
TMPyP.

Photochemistry and Photobiology, 2014, 90 601
A
B
C
Figure 4. Effect of porphyrins on PC/CL Giant Unilamellar Vesicles (GUVs). (A) Phase contrast microscopy images of optically trapped PC/CL GUV
in the course of irradiation in the presence of TMPyP. (B) Phase contrast microscopy images of optically trapped PC/CL GUV during irradiation in the
presence of TPPS4. (C) Decrease in the area of the PC/CL GUV contrast phase image during irradiation in the presence of TPPS4. The PC/CL (85/
15 mol%) GUV was prepared in 0.2 M sucrose, and the vesicle suspension was six-fold diluted in 0.2 M of glucose.

602 Cintia Kawai et al.
expulsion of small daughter vesicles. These events are responsi-
ble for the significant decrease in GUV radius. Differently,
TMPyP localized at GUV external interface generated singlet
oxygen dissolved in the lipid bilayer that might promote a sym-
metrical oxidative damage at the two lipid leaflets. In this case,
the putative symmetrical oxidative event promoted relative rapid
vesicle leak resulting in the observed loss of contrast without sig-
nificant changes in vesicle radius.
inhibiting lipid peroxidation by the photodynamic action of
TMPyP. A probable cause can be found in the topology of the
porphyrin in the membrane and at the site of O2(1Dg) production.
TMPyP should be associated on the membrane surface and prob-
ably transfers most of the electronic excitation energy to the
molecular oxygen inside the bilayer.
Thus, sodium azide can quench O2(1Dg) molecules moving to
(or formed in) the bulk; however, it has limited access to the
O2(1Dg) that remains inside the bilayer. In the presence of sorbic
acid and azide, saturation of the damage was attained at 15 min
of irradiation, and at which point, it was used the 15 min irradia-
tion time. Meanwhile, in the absence of quenchers, an increase
in LOOH was observed at 60 min. The above-mentioned results
are in agreement with the recent study by Ehrenberg et al., 2013
(58). The authors state that singlet oxygen quenching by azide
depends on a physical encounter with this excited species during
its short lifetime. They demonstrated that in the presence of posi-
tively charged liposomes azide is concentrated in the Debye layer
near the surface of the bilayer. Otherwise, azide is preferentially
in the bulk solution when negatively charged liposomes are pres-
ent. PC/CL liposomes are negatively charged and in this condi-
tion azide can efficiently quencher singlet oxygen that is
generated in the bulk phase. Equally, in this study, the heteroge-
neous medium is responsible for the differentiated effects of D2O
observed for TPPS4 and TMPyP. In the presence of liposomes,
increase in singlet oxygen lifetime by D2O can be observed only
for the excited species diffusing in the bulk phase. Therefore, the
minimal lipid photooxidation enhancement in D2O compared to
H2O that was observed for TMPyP is consistent with the pro-
posal that the cationic porphyrin binds to the anionic surface of
PC/CL liposomes and generates singlet oxygen inside the lipid
bilayer.
Mechanism of photodamage of PC/CL liposomes promoted
by porphyrins
The photoproduction of LOOH observed during the irradiation
of PC/CL vesicles in the presence of the porphyrins was not
accompanied by a significant increase in TBARS (not shown).
This result suggests that the Type-II mechanism, that is the addi-
tion of O2(1Dg) to a double bond of a polyunsaturated fatty acid
(PUFA), was the principal contributor to the conjugated dienes
and LOOH production. Singlet oxygen reacts with olefins owing
a structure with two or more allylic substituents. This reaction
promotes a double bond shift concomitant with the formation of
an allylic hydroperoxide (52–54). Therefore, in the Type-II
mechanism, O2(1Dg) adds to a lipid double bond and converts
LH to LOOH without the formation of a peroxyl radical interme-
diate. In the Type-I mechanism, a peroxyl radical can be con-
verted to TBARS after subsequent reactions (Scheme 1).
Detailed discussions about the singlet–oxygen-mediated lipid per-
oxidation mechanisms can be found in references (55–57).
To investigate the predominant mechanism responsible for
lipid peroxidation, PC/CL liposomes were exposed to the photo-
dynamic action of TMPyP and TPPS4 in the presence of D2O
and in the presence of quenchers of the excited states generated
in the medium during photosensitization of the porphyrins
(Fig. 5A and B). Figure 5A shows that the LOOH content of
PC/CL liposomes, which is produced after 60 min irradiating
TPPS4 in D2O (light gray column in central panel), was twice
and a half higher than that produced in aqueous medium (diago-
nal striped column in central panel). When TMPyP was used as
the sensitizer, the LOOH content (light gray column in right
panel of Fig. 5A) that was produced in D2O was circa 37%
higher than that produced in an aqueous medium (diagonal
striped column in right panel of Fig. 5A). D2O had no effect on
the LOOH content of the system in which porphyrins are absent
(control). These results are consistent with the contribution of
O2(1Dg) for the generation of LOOH by both the photosensitized
TMPyP and TPPS4. The differences in the increase in LOOH
production promoted by D2O could be assigned to the different
microenvironments in which the excited species are produced
(see below).
Considering that O2(1Dg) adds to a double bond of a PUFA
leading to LOOH production, lipid peroxide derivatives are
expected to be produced in phospholipids in which the glycerol
moiety is exclusively esterified with oleic acid. Figure 6 shows
that the LOOH content of DOPC/TOCL vesicles submitted to
the photodynamic action of TMPyP increased five-fold after
60 min of irradiation.
Although the production of LOOH had been four-fold lower
when biological PUFA-containing phospholipids had been
replaced by oleoyl-containing phospholipids, this result is also
consistent with O2(1Dg) as the prooxidant agent for LOOH pro-
duction. The lower yield of LOOH in the presence of DOPC/
TOCL could be assigned principally to TOCL (15% of the lipid
content) as the preferential target for O2(1Dg). The cationic por-
phyrin is expected to bind preferentially on CL domains. There-
fore, when PUFA-containing CL is present in the liposome, a
higher amount of double bonds are available for the O2(1Dg)
attack, and the production of LOOH is higher.
Figure 5B shows that sorbic acid (yellow column) and sodium
azide (light red column) were able to promote significant
decrease in the LOOH produced by TMPyP in D2O. This result
is also consistent with the contribution of O2(1Dg) for the pro-
duction LOOH by TMPyP. Sorbic acid quenches triplet TMPyP
and prevents the energy transfer to molecular oxygen. On the
other hand, sodium azide quenches O2(1Dg) produced by energy
transfer from triplet TMPyP. The inhibitory effect of sodium
azide on the lipid peroxidation promoted by TMPyP provided an
additional support for the contribution of a Type-II mechanism
in the lipid peroxidation. Sorbic acid (around 70% inhibition)
was more efficient than sodium azide (around 50% inhibition) at
Contributions of topology and quantum yield of TMPyP and
TPPS4 to membrane damage
Although different results obtained with TMPyP and TPPS4 have
been consistent with the different topologies of the cationic and
anionic porphyrins in an aqueous medium containing negatively
charged vesicles, it is important to consider the photochemical
behavior of the investigated porphyrins per se. Thus, oxidative
conversion of DCFH into DCF, that is the fluorescent species
(Scheme 2), by the photodynamic action of TMPyP and TPPS4

Photochemistry and Photobiology, 2014, 90 603
Scheme 1. Mechanisms of LOOH production by free radicals and O2(1Dg). In Type I and II mechanisms, respectively, molecular oxygen and singlet
molecular oxygen also react on the right hand portion since the compound is unsymmetrical. The gray box shows a cartoon with the 3D structure of a
phospholipid and the peroxide-derivative form after singlet oxygen attack.
was investigated in the absence and in the presence of PC/CL
liposomes (Fig 7).
for DCFH is 2.62 (59), so this fluorescent probe can scavenge
pro-oxidant species within lipid bilayers as well as in the aque-
ous phase. Therefore, this result is consistent again with the pres-
ence of TMPyP at the liposome interface and TPPS4 partitioned
preferentially in the bulk (upper panels of Fig. 7). At the inter-
face of DOPC/TOCL, TMPyP can generate O2(1Dg) at the bulk
interface and in the bilayer. Binding of sensitizers on the
membrane surface can enhance the pro-oxidant effectiveness on
membrane components and partitioned molecules due to three
principal reasons: (1) the higher concentrations of oxygen in
Peculiar results were obtained by comparing the action of
TMPyP and TPPS4 on DCFH under different conditions. The
yield of DCF produced by the oxidation of DCFH in a homoge-
neous medium after 25 min of irradiation in the presence of
TMPyP was five-fold lower than that obtained when TPPS4 was
the photosensitizer. Contrary results, that is the yield of DCF
generation being five-fold higher for TMPyP, were obtained in
the presence of DPPC/TOCL liposomes. The partition coefficient

604 Cintia Kawai et al.
Figure 5. Effect of modifiers of the lifetime of the excited species on lipid photo-oxidation promoted by porphyrins. (A) Effect of deutered water
(D2O) on lipid photo-oxidation promoted by TPPS4 and TMPyP. Experiments were carried out using 1 mM PC/CL liposomes in medium D2O (light
gray bars) containing 0.2 M of glucose in the absence (control) or in the presence of 10 lM of TMPyP and TPPS4 as indicated over the gray columns.
The overlapped columns filled with a diagonal stripe pattern correspond to the results obtained in the same conditions except that D2O was replaced by
H2O. (B) Effect of triplet species quencher (1 mM of sorbic acid, presented as columns) and O2(1Dg) quencher (1 mM sodium azide, presented as
columns) on the lipid photodamage promoted by 10 lM of TMPyP. The white and black columns represent the results obtained in the absence of
quenchers for the samples incubated under irradiation and in the dark, respectively. The samples were irradiated using white LED.
lipid membranes that are estimated to be around one order of
magnitude greater than in the bulk aqueous media; (2) the longer
photosensitizer triplet-state and O2(1Dg) lifetimes in membranes
relative to aqueous solution (60–64) (3) and the confinement of
O2(1Dg) at smaller distances of the target molecules. In fact,
there is a correlation between the efficiency of sensitizers and
their affinity for lipid bilayers (60,65). It is important to consider
that the stability of the photosensitizer also influences the total
amount of DCFH that is converted to DCF. Consistently, in a
homogeneous medium, TMPyP exhibited significant bleaching
whereas TPPS4 remained unchanged (not shown). Conversely,
the presence of PC/CL liposomes increased the stability of
TMPyP. Therefore, two factors contributed to the higher produc-
tion of DCF by TMPyP in liposomes: the stability of the sensi-
tizer and its association with the lipid bilayer where a higher
concentration of molecular oxygen is present.
However, the lower panels of Fig. 7 show that in the homo-
geneous medium as well as in PC/CL liposomes, TMPyP con-
verted DCFH to DCF three-fold and a half faster than did
TPPS4 (4.8 ꢄ 0.1 9 10ꢀ3 sꢀ1 versus 1.4 ꢄ 0.1 9 10ꢀ3
s
ꢀ1) in
the homogeneous medium and four-fold and a half faster than
TPPS4 (2.3 ꢄ 0.7 9 10ꢀ3 sꢀ1 versus 0.5 ꢄ 0.1 9 10ꢀ3
s
ꢀ1) in
DPPC/TOCL liposomes. Relative to TPPS4, the relatively
higher rate of DCF photogeneration by TMPyP (both in homo-
geneous and heterogeneous media) could be related to the quan-
tum yield of O2(1Dg) (ΦD) generated by energy transfer from
the triplet state of the cationic porphyrin. The ΦD values for
TMPyP reported in literature are somewhat controversial, vary-
ing from 0.58 (66) to 0.9 (67). However, the latter value seems
to be overestimated, and the more plausible value is 0.74 (68),
whereas the ΦD of deprotonated TPPS4 has been reported to be
0.62 ꢄ 0.03 (66).

Photochemistry and Photobiology, 2014, 90 605
Figure 6. Effect of the lipid composition on the production of LOOH by
the photodynamic action of TMPyP. The columns represent the LOOH
production after the irradiation of PUFA-containing PC/CL, and the
columns represent the LOOH production after the irradiation of DOPC/
TOCL.
Scheme 2. Detection of pro-oxidant species by DCFH.
Figure 7. Photo-oxidative production of DCF by TMPyP and TPPS4. Upper panels show the increase in fluorescence during the irradiation of TMPyP
(black circles) and TPPS4 (light gray circles) in homogeneous media (left) and in PC/CL liposomes (right). The lower panels show the normalized data
fitted by Eq. (1). Experiments were carried out using 0.5 lM of DCFH in the presence of 5 lM of porphyrins in an aqueous medium containing 0.2 M of
glucose (left) or in the presence of PC/CL liposomes (right). The samples were irradiated with white light LED for 1500 s, and the fluorescence was
measured at the indicated times. The fluorescence kmax at 522 nm was measured at room temperature by excitation at 490 nm using slits of 5/5 nm.
Thus, it is evident that affinity to the biological target is
CONCLUSION
important for the photodamage degree in PDT. On the other
hand, the capacity of singlet oxygen to diffuse and to attain dis-
tant target molecules permits a photosensitizer with low affinity
to a biological structure to cause photodamage via the Type-II
mechanism. An example of the long-range effect of the Type-II
mechanism was provided by undulations, involutions and disrup-
tion of PC/CL GUVs submitted to the photodynamic action of
TPPS4.
This report presents the study of the effects of the PDT on a
model of the inner mitochondrial membrane under two condi-
tions: using a photosensitizer without affinity to the membrane
(anionic porphyrin) and a photosensitizer that binds to the
membrane by means of electrostatic interactions (cationic por-
phyrin). As expected, only TMPyP binds electrostatically to PC/
CL liposomes; however, both porphyrins were able to damage

606 Cintia Kawai et al.
The next step of these studies will be focused on more specific
models, isolated mitochondria and cultured cells. For the latter,
the strategy will be the use of different carriers for the photosensi-
tizers leading to specific trafficking mechanisms and targeted
organelles. The ultimate objective includes not only the immedi-
ate effects and cell death mechanisms modulated by the targeted
organelles but also the delayed effects on the expression of genes.
The principal events peculiar to each investigated porphyrin
are summarized in Scheme 3.
Acknowledgements—The authors are grateful to FAPESP, CAPES and
CNPq for their financial support. The authors thanks also to the
Multiusers Central Facilities for the experimental support.
Scheme 3. Photodynamic effects of TPPS4 and TMPyP modulated by
the topology of the sensitizers. In the left side, TPPS4 is in the bulk
water and after electronic excitation (1) decays via energy transfer to
molecular oxygen dissolved in bulk water generating singlet oxygen (2).
Singlet oxygen travels to the membrane of a GUV and promotes oxida-
tive damage (yellow arrow). After ~50 min GUV was significantly smal-
ler but without significant loss of contrast. TMPyP bound to GUV
membrane, after electronic excitation (1′) decays via energy transfer to
molecular oxygen dissolved into the lipid bilayer generating lipid dis-
solved singlet oxygen. Singlet oxygen dissolved in lipid bilayer (2′)
attacks lipid acyl chains (yellow arrow). After ~15 min, GUV exhibited
was leaky evidenced by loss of contrast (3′).
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Movie S1. GUV changes during irradiation with TPPS4.
Movie S2. GUV changes during irradiation with TMPyP.
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