Correlation of photodynamic activity and singlet oxygen quantum yields in two series of hydrophobic monocationic porphyrins

Article abstract of DOI:10.1142/S1088424611004336

The photodynamic properties of eight hydrophobic monocationic methyl and ruthenium polypyridine complex derivatives of free-base and zinc(II) meso-triphenyl-monopyridylporphyrin series were evaluated and compared using HeLa cells as model. The cream-like

Full text of DOI:10.1142/S1088424611004336

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2012; 16: 55–63
DOI: 10.1142/S1088424611004336
Published at http://www.worldscinet.com/jpp/
Correlation of photodynamic activity and singlet oxygen
quantum yields in two series of hydrophobic monocationic
porphyrins
Daiana K. Deda, Christiane Pavani, Eduardo Caritá, Maurício S. Baptista,
Henrique E.Toma and Koiti Araki*◊
Institute of Chemistry, University of Sao Paulo, Av. Prof. Lineu Prestes 748, Sao Paulo, 05508-000, SP, Brazil
Received 7 May 2011
Accepted 2 July 2011
ABSTRACT: The photodynamic properties of eight hydrophobic monocationic methyl and ruthenium
polypyridine complex derivatives of free-base and zinc(II) meso-triphenyl-monopyridylporphyrin
series were evaluated and compared using HeLa cells as model. The cream-like polymeric nanocapsule
formulations of marine atelocollagen/xanthan gum, prepared by the coacervation method, exhibited high
phototoxicity but negligible cytotoxicity in the dark. Interestingly, the formulations of a given series
presented similar photodynamic activities but the methylated free-base derivatives were significantly
more phototoxic than the respective ruthenated photosensitizers, reflecting the higher photoinduced
singlet oxygen quantum yields of those monocationic porphyrin dyes.
KEYWORDS: cationic porphyrins, polymeric encapsulation, PDT, HeLa cells.
biocompatibility, preferential or specific accumulation in
INTRODUCTION
the tumor cells, faster elimination rate by the organism,
Photodynamic therapy (PDT) is a medical procedure,
lower toxicity and higher phototoxicity have been
based on the combined action of a photosensitizer (dye)
pursued [3, 7–10]. At the present time only Photofrin®,
and light, that has been increasingly employed in the
Levulan® Kerastick and Visudyne® have been approved
treatment of cancer and other skin diseases, such as
by FDA for PDT treatment.
psoriasis and vitiligo (leukoderma), with excellent results
Although porphyrins and derivatives have interesting
from the medical and aesthetic points of view. Among
photodynamic properties, frequently their solubility in
the several compounds, porphyrins and their derivatives
aqueous media is far too low for direct application in
have been extensively employed as photosensitizers. In
PDT treatment. A convenient strategy to increase the
fact, Photofrin® the first commercial phototherapeutic
biocompatibility and dispersability in biological fluids
agent approved by FDA about 20 years ago, is based on
is the micro and/or nanoencapsulation of water insoluble
hematoporphyrin oligomers, more exactly nine porphyrin
lipophilic compounds. Polymeric encapsulation is a very
units linked by ether, ester and carbon–carbon bonds
interesting choice for that purpose, because the capsules
[1–3]. Nevertheless, some limitations have been imposed,
shell can be engineered using several biocompatible
particularly by their long persistence time in the organism,
polymers and varying the degree of cross-linking
leading to undesirable skin photosensibility that can last
[9, 11, 12], thus allowing the control of the permeability
several weeks or even months [4–6]. For this reason,
and the mechanical resistance. Also, polyethyleneglycol
more effective and safe photosensitizers and formulations
derivatives can be added to decrease the interaction of
exhibiting well defined chemical compositions, higher
the polymeric capsule with the plasma components thus
avoiding the loss of formulation by opsonization [13, 14]
SPP full member in good standing
by phagocytary cells.
The binding properties of a series of meso-phenyl(N-
methylpyridinium)porphyrins to vesicles and red cells
*Correspondence to: Koiti Araki, email: koiaraki@iq.usp.br,
tel: +55 11-3091-8513, fax: +55 11-3815-5579
Copyright © 2012 World Scientific Publishing Company

56
D. K. DEDA ET AL.
Fig. 1. Scheme showing the structure of the eight monocationic meso-triphenyl(pyridyl)porphyrin derivatives, highlighting the
structure of 3MMe
were shown to be correlated with their n-octanol/water
partition coefficients (log(POW)). However, amphiphilic
species exhibited significantly higher interaction and
photodynamic activity [15–17] than predicted solely
based on that parameter. More recently, the monocationic
meso-triphenyl(3-N-methylpyiridinium)porphyrin, 3MMe,
was shown to be a very efficient photosensitizer when
encapsulated in atelocollagen polymeric nanocapsules
prepared by coacervation method [18]. However, few are
the reports [19, 20] on the influence of transition metal
ions, coordinated to the porphyrin ring or to its periphery,
on the photodynamic properties. Accordingly, a systematic
study was carried out with two series (free-base and
zinc(II) complex) of monocationic meso-triphenyl(pyridyl)
porphyrin derivatives (Fig. 1) in order to assess the
influence of the ring metallation and the coordination of a
[Ru(bipy)2Cl]+ complex on the photodynamic properties of
porphyrin dyes, using HeLa cells as model.
3MMe and 4MMe, were obtained refluxing
3MPyTPP and 4MPyTPP with 40 times excess of methyl
p-toluenesulfonate in dimethylformamide (DMF),
purified by neutral alumina column chromatography and
precipitated as chloride salts in saturated NaCl solution
to increase the biocompatibility and solubility in aqueous
media. The Zn-3MMe and Zn-4MMe were prepared in a
similar way.
The series of ruthenated porphyrins (3MRu, 4MRu,
Zn-3MRu and Zn-4MRu) were obtained by reacting
3MPyTPP, 4MPyTPP, Zn-3MPyTPP and Zn-4MPyTPP
with about 1% excess of the [Ru(bipy)2Cl(H2O)]
NO3 complex, in a CH2Cl2/DMF 4:1 v/v mixture, for
60 min [17] and purified by neutral alumina column
chromatography, using a mixture of CH2Cl2 and ethanol
as eluent.
The methylated and ruthenated free-base porphyrin
series exhibited characteristic UV-vis spectral profiles
with an intense band around 420 nm (Soret band) and
four less intense Q-bands at 515, 565, 596 and 650 nm
(Fig. 2). A bipyridine pp→pp* internal transition band
and a broad RuII(dp)→bipy(pp*) MLCT band were also
observed in the ruthenated derivatives, respectively at
295 and 470 nm. A similar behavior was observed for the
Zn(II) porphyrin series, but the Soret band was shifted to
428 nm and only two Q-bands were observed at 560 and
610 nm.
RESULTS AND DISCUSSION
Syntheses and characterization
The monocationic porphyrins 3MPyTPP and
4MPyTPP were prepared by the reaction of stoichiometric
amounts of pyrrole, benzaldehyde and 3-pyridyl or
4-pyridylcarboxaldehyde in refluxing glacial acetic acid
[24]. The small amounts of meso-tetraphenylporphyrin
(TPP) and disubstituted derivatives were separated
by silica-gel column chromatography using a mixture
of dichloromethane and ethanol (2%) as eluent. The
respective zinc(II) complexes (3- and 4-ZnMPyTPP)
were obtained by refluxing them with zinc acetate in a
mixture of glacial acetic acid and DMF.
The two series of methylated and ruthenated
amphiphilic monocationic porphyrins (Fig. 1) were
1
characterized by H NMR. The b-pyrrole protons of
the para-isomers were found as a broadened singlet
(8H) between 8.4 and 8.9 ppm, while the inner ring
protons appeared as a singlet (2H) at -2.8 ppm. The
pyridyl protons were found as a pair of doublets at
8.0 and 9.7 ppm, while the phenyl group protons were
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CORRELATION OF PHOTODYNAMIC ACTIVITY
57
Fig. 2. Absorption spectra of (a) 3MMe and Zn-3MMe, (b) 4MMe and Zn-4MMe, (c) 3MRu and Zn-3MRu, and (d) 4MRu and
Zn-4MRu in CH2Cl2. Inset: exploded view in the visible range
found as a double doublet and a multiplet at 7.6 and
8.1 ppm. The protons of the bipyridyl ligands appeared
as very characteristic signals in the 7 to 10.1 ppm range.
As expected, an intense singlet (3H) was observed at
of the laser power plot. The absorbance at 532 nm of the
eight porphyrin dyes and the TPP standard in CH2Cl2
solution was adjusted to 0.02 a.u., and the power of
the laser pulses varied in the 1 to 8 mJ.cm-2 range. A
typical set of decay curves, used to collect the emission
intensity at 1270 nm for the 4MRu and the Zn-4MRu
derivatives, are shown in Fig. S1 (see Supporting
information section). The phosphorescence intensities
were measured 70 ms after the laser pulse and plotted
as a function of the corresponding laser power. Linear
correlations were found for both porphyrin series and
fD determined from the ratio of their slopes with that
found for the TPP standard, in the same experimental
conditions. The free-base porphyrin derivatives
exhibited significantly higher 1Dg(O2) phosphorescence
than the corresponding zinc(II) porphyrin derivatives,
reflecting their higher energy transfer quantum
efficiencies. The fD values, the lifetimes (1/k) and the
photodynamic efficiencies (% of cell death) for the
eight porphyrin dyes are listed in Table 1.
1
4.6 ppm in the N-methylated derivatives. The H NMR
signals of the [Ru(bipy)2Cl]+ derivatives of the porphyrin
para-isomers were sharper and better resolved than the
signals of the new meta-isomers, but the integration was
always consistent with the expected number of protons.
Accordingly, the presence of a [Ru(bipy)2Cl]+ group
bond to the periphery of the porphyrin ring was further
confirmedbycyclicvoltammetry.Infact,thecharacteristic
reversible pair of voltammetric waves at 0.9 V, assigned
to the RuIII/RuII redox process, had the same intensity
of the monoelectronic reversible reduction processes of
the porphyrin ring at -0.9 and -1.3 V, thus confirming
the presence of a ruthenium complex coordinated to the
porphyrin ring. The irreversible process observed at +1.38
V was assigned to the oxidation of the porphyrin ring.
The lifetime of 1Dg(O2) defines the maximum radius of
action of this reactive species. Values in the 140–180 ms
range were measured in CH2Cl2, which are consistent
with the lifetime of singlet oxygen in hydrophobic
environments. In fact, it can be as high as 200 ms in organic
solvents in the absence of quenchers, but decreases to
2–4 ms in aqueous solution [28]. In the cytosol, however,
the lifetime decreases to about 10–50 ns, such that the
perimeter of action is reduced to about 0.1 mm from the
point where it is generated.
Singlet oxygen quantum yields (fD)
Meso-arylporphyrins are type II photosensitizers
whose photodynamic activity depend on the formation
of singlet oxygen by energy-transfer from electronically
excited dye molecules to dioxygen [1–3, 6, 25], upon
irradiation with light of suitable wavelength [2, 5, 6,
26, 27]. The photoinduced singlet oxygen quantum
yields (fD) of all porphyrin dyes were evaluated from
the slope of the 1Dg(O2) emission intensity as a function
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D. K. DEDA ET AL.
1
Table 1. Fluorescence quantum yields, ffl, singlet oxygen Dg(O2) quantum yields,
fD, lifetimes t (in CHCl3 solution) and photodynamic efficiencies fPD towards HeLa
cells of the eight monocationic porphyrin dyes and TPP, used as standard
Porphyrin
TPP
ffl (SD)
0.150
fD (SD)
0.50
t, ms (SD)
fPD, %
183.4 (4.2)
3MMe
0.3431 (0.0086)
0.4583 (0.0030)
0.0210 (0.0007)
0.0297 (0.0006)
0.0124 (0.0003)
0.0372 (0.0015)
0.0148 (0.0002)
0.0249 (0.0003)
0.79 (0.04)
0.55 (0.02)
0.50 (0.02)
0.51 (0.03)
0.40 (0.01)
0.48 (0.03)
0.40 (0.03)
0.36 (0.02)
163.2 (3.1)
160.2 (2.6)
161.1 (4.7)
162.6 (1.4)
140.6 (7.5)
138.9 (5.0)
139.3 (2.6)
144.7 (3.4)
92.3
92.3
71.9
74.9
54.0
54.5
53.0
58.8
4MMe
3MRu
4MRu
Zn-3MMe
Zn-4MMe
Zn-3MRu
Zn-4MRu
The free-base porphyrins, particularly the 3MMe and
4MMe derivatives, showed higher fD and photodynamic
efficiencies than the respective zinc(II) porphyrins. Also,
the ruthenium complex is contributing to diminish further
the Dg(O2) quantum yields of the macrocyle, possibly
introducing new competing relaxation pathways for the
excited state species.
photodynamic action. The first two are strongly dependent
on the molecular structure, such that the affinity was
found to be a function of the lipophilic character of the
cationic dyes. However, this is abnormally enhanced in
the case of amphiphilic species [16] that penetrate more
deeply into the cell membrane as a consequence of the
unique spatial distribution of lipophilic and hydrophilic
groups in those molecules. Also, amphiphilic Zn(II)
porphyrins was shown to bind preferentially to the cell
membrane, improving the PDT efficiency [20]. The
meso-pyridylporphyrins are type II photosensitizers,
acting essentially through energy transfer to molecular
oxygen and production of singlet oxygen, rather than
the direct electron transfer from the excited species to
biomolecules. The coordination of zinc(II) [34, 35] was
shown to improve the PDT efficiency because tend to
enhance the type II character of the photosensitizer. In fact,
tetraruthenated porphyrins [36], particularly the zinc(II)
derivative, showed to be a better type II photosensitizer
than methylene blue. A similar behavior was observed for
N-confused porphyrins and its Ag(III) complex [37].
The two series of monocationic porphyrins have
very low solubility in aqueous media such that they can
cause serious problems when injected intravenously
due to precipitation and clogging of capillary veins.
Unfortunately, the affinity of the dye for tumor cells
and tissues is proportional to log(POW) such that high
affinity usually means low solubility in biological
fluids, hindering their direct use in PDT. This problem
can be overcome by encapsulating them in micro and
nanocapsules with appropriate surface functional groups
and surface charge [11, 12].
1
Toxicity and phototoxicity of the monocationic
porphyrins
Coacervation is one of the most commonly used
methods for the preparation of nanocapsules using
natural polymeric materials such as sodium alginate and
gelatin. The procedure involves two steps: (a) the o/w
emulsification by mixing an organic phase containing
the active substances with an aqueous polymeric phase
by mechanical stirring and/or ultrasound processing;
followed by (b) the coacervation process that can
be induced by addition of electrolytes, decrease of
temperature or dehydration agents [21, 29, 30].
The delivery efficiency of a photosensitizer [11, 31]
to cells by micro/nanocapsule formulations are mainly
controlled by shell properties [21, 32] such as morphology,
size, mechanical strength and permeability, which generally
are defined by the chemical composition, temperature,
pressure and pH, that may also influence the cell viability,
cytolocalization and interaction with the blood components,
as described by Dash et al. [33].
The photodynamic properties of a dye depend on
three major factors in addition to the pharmacokinetic
properties and biodistribution: (a) their ability to interact
with cells and tissues, (b) the quantum efficiency of
the photosensitizer, and (c) the mechanism of the
In addition, the composition and size can be
controlled to impart biocompatibility and optimal
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CORRELATION OF PHOTODYNAMIC ACTIVITY
59
interaction and delivery of their content to malignant
cells. For this purpose, polymeric formulations were
prepared by encapsulating the two series of monocationic
porphyrins in marine athelocolagen/xantam gum
polymeric microcapsules, that were reprocessed using
a high power ultrasonic tip monitoring the size by DLS
[18]. Formulations with the largest fraction of capsules
in the 400–500 nm range and histograms exhibiting
bimodal size distribution were obtained (see Supporting
information section Fig. S2). The porphyrin concentration
was 1.0 × 10-4 M in all formulations. The cytotoxicity was
evaluated by incubating 1.0 × 105 HeLa cells with 1.0 mL
of a 1 mM porphyrin formulation (in colorless DMEM
culture media) for 6 h, in the dark.
When compared to the dark control experiments in
the absence of any sensitizer, the dark toxicity of all
sensitizersinvestigatedwasnegligible(Fig. S3). However,
the cell viability decreased dramatically when the cells,
incubated with the free-base porphyrin formulations,
were irradiated with the 650 nm laser, in contrast with
the control (Fig. 3). Note that the 4MMe and 3MMe
formulations reduced the cell viability in more than
80%, while a 50–60% decrease was found for 3MRu and
4MRu. In fact, the N-methylated derivatives consistently
showed a much higher photodynamic efficiency than
the N-ruthenated derivatives. The localization of the
peripheral groups in the para- or meta-position had no
significant influence on the photodynamic activity within
experimental error, showing that the solubility and
binding affinity of those photosensitizers to the HeLa
cells are virtually the same for both isomers.
at 655 and 715 nm) indicating that the lowest excited
state in 3MRu and 4MRu is localized in the porphyrin
ring and not in the peripheral [Ru(bipy)2Cl(pyP)]+
complex. However, the fluorescence quantum yield
decreased almost two orders of magnitude to 2.9 × 10-3
suggesting that other relaxation pathways, such as the
non-radiative decay and the intersystem crossing, were
favored by coordination of the ruthenium complex
(Fig. S4). Interestingly, the photodynamic efficiency
was reduced only about 20% suggesting that the energy
transfer from the excited porphyrin to dioxygen should
be much faster than the fluorescence emission and other
pathways, including the non-radiative decay. However,
considering that the lifetime of ruthenated porphyrins in
the singlet excited state should be much smaller than that
for the starting MPyTPP (~5 ns in CH2Cl2), there is not
enough time for the occurrence of efficient bimolecular
processes. Thus, the energy transfer to dioxygen should
be taking place after intersystem crossing to the excited
triplet state. In this case, the decreased of the fluorescence
quantum yield can be explained by the increase of the
intersystem-crossing rate induced by the coordination
of the ruthenium complex. Analogous behavior was
previously reported for the doubly N-confused Ag(III)
complex. [37]
Unfortunately, the series of monocationic zinc(II)
porphyrins have the lowest energy absorption band
around 610 nm (Fig. 2) and can not be excited by the
red laser (lem = 650 nm) used for the free-base series.
In order to get comparable results with both series,
experiments were carried out using a mercury lamp as
white light source. This presents a broad emission band
in the visible range that can be used to excite both, the
free-base and the zinc(II) porphyrin derivatives.
Accordingly,experimentswerecarriedoutasdescribed
above for the free-base porphyrin derivatives with both
series of porphyrin dyes, except for the irradiation with
a white mercury lamp source (16 mW.cm-2) for an hour.
About 55% decrease in cell viability was found for all
zinc(II) porphyrin series (Fig. 4a), while the free-base
series once again showed distinct photodynamic behavior
for the methylated (90% decrease) and ruthenated
(70% decrease) derivatives (Fig. 4b). These results are
consistent with those described above using the red laser
as light source (Fig. 3), where no significant differences
wereobservedinthephotodynamicpropertiesofthepara-
and the meta-isomers. This result is also consistent with
At this point, it is interesting to remember that the
fluorescence quantum yield of 4MPyTPP in CH2Cl2 is
ffl(lexc 512 nm) = 0.099, only slightly smaller than for
TPP standard ((ffl(lexc 512 nm) = 0.11). The coordination
of a [Ru(bipy)2Cl]+ group to the pyridyl N-atom did
not change the fluorescence spectra profile (two bands
a previous report in which a convergence of the log(POW
)
values was shown for both mono(N-methylpyridinium)
porphyrin derivatives, while increasing differences were
observed as the number of N-methylpyridinium groups
increased [16].
Similar results were obtained for the zinc(II) porphyrin
series suggesting that the coordination of Zn(II) ion to
the porphyrin ring contributed to enhance the intersystem
crossing rate alike the coordination of the [Ru(bipy)2Cl]+
complex to the pyridyl N-atom. Furthermore, the energy
Fig. 3. Phototoxicity of the monocationic free-base porphyrin
derivatives in polymeric nanocapsules (1 mM) towards HeLa
cells. The cells (1 × 105) were incubated for 6 h and irradiated
with a 650 nm laser source for 10 min
Copyright © 2012 World Scientific Publishing Company
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60
D. K. DEDA ET AL.
Fig. 4. HeLa cell viability after incubation with monocationic porphyrins incorporated in polymeric nanocapsules, in the dark and
after irradiation for 1 h with a 16 mW.cm-2 white mercury lamp. (a) Zn(II) and (b) free-base porphyrin derivatives
Fig. 5. Photodynamic efficiency and respective 1Dg(O2) quantum yields of the eight monocationic porphyrin derivatives encapsulated
in polymeric nanocapsules, when irradiated with a 16 mW.cm-2 white mercury lamp
of the Zn(II) porphyrin excited triplet state should be
lower than the charge transfer excited state localized in the
ruthenium complex, because otherwise the photodynamic
EXPERIMENTAL
Synthesis of the monocationic porphyrin derivatives
efficiency should be significantly decreased by energy
transfer. In addition, electron transfer processes should
not be taking place since they would decrease further the
photodynamic activity.
3MPyTPP and 4MPyTPP. The 5,10,15-triphenyl-
20-(3-pyridyl)porphyrin, 3MPyTPP, and the 5,10,
15-triphenyl-20-(4-pyridyl)porphyrin, 4MPyTPP, were
synthesized and characterized according to a previously
described method [15, 17]. A mixture of 53 mmol of
benzaldehyde and 18 mmol of 3-pyridyl carboxaldehyde
(or 4-pyridyl carboxaldehyde) was reacted with 71
mmol of pyrrol in glacial acetic acid, in the dark. After
2 h, the solvent was removed in a flash evaporator and
the porphyrin precipitated-out in a mixture of ethanol
and N,N-dimethylformamide (9:1 v/v). The solid was
filtered, washed with the same mixture of solvents,
dried and purified by silica-gel column chromatograph
using suitable mixtures of CH2Cl2/EtOH as eluent. Yield
11%. (3MPyTPP). UV-vis (CH2Cl2): lmax, nm (log e)
The fD and photodynamic efficiency of the porphyrin
derivatives are compared in Fig. 5. The highest
phototoxicity observed for 3MMe can be assigned to
its higher 1Dg(O2) quantum yield. Interestingly, the
photodynamic activity of 4MMe was much higher than
that expected based on the fD suggesting that the value
was underestimated, probably as a consequence of
aggregation even at the low concentrations used in the
measurements [15]. Likewise, the lower photodynamic
efficiency observed for zinc(II) porphyrin and ruthenium
complex derivatives can be assigned to their much lower
1Dg(O2) quantum yields.
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CORRELATION OF PHOTODYNAMIC ACTIVITY
61
418 (5.68), 515 (4.32), 548 (3.95), 590 (3.83) and 650
(3.63). 1H NMR (500 MHz, DMSO, TMS): d, ppm 9.74
(m, 1H), 9.39 (d, 1H), 8.98 (m, 1H), 8.83 (d, 2H), 8.79
(s, 4H), 8.72 (d, 2H), 8.46 (m, 1H), 8.15 (m, 6H), 7.71
(m, 9H), -2.82 (s, 2H). (4MPyTPP). UV-vis (CH2Cl2):
(s, 1H), 9.45 (s, 1H), 9.10–8.65 (10H), 8.60–8.45 (4H),
8.35(m, 3H), 8.28–7.85 (13H); 7.80–7.40 (13H), 7.05(m,
1H), -2.78 (s, 2H). (4MRu). UV-vis (CH2Cl2): lmax, nm
(log e) 293 (4.80), 417 (5.70), 520 (4.30), 552 (4.16),
588 (3.88) and 649 (3.70). 1H NMR (500 MHz, DMSO,
TMS): d, ppm 10.19 (m, 1H), 8.93 (m, 1H), 8.80 (m, 8H),
8.59 (m, 2H), 8.59 (m, 1H), 8.48 (m, 1H), 8.32 (m, 1H),
8.25 (m, 1H), 8.12 (m, 6H), 8.12 (m, 2H), 8.02 (m, 2H),
8.01 (m, 1H), 7.80 (m, 1H), 7.68 (m, 9H), 7.62 (m, 1H),
7.35 (m, 1H), 7.10 (m, 1H), -2.80 (s, 2H).
l
max, nm (log e) 417 (5.70), 515 (4.28), 550 (3.92), 590
1
(3.80) and 650 (3.61). H NMR (500 MHz, DMSO,
TMS): d, ppm 8.96 (m, 2H), 8.83 (d, 2H), 8.79 (s, 4H),
8.73 (d, 2H), 8.15 (m, 6H), 8.11 (m, 2H), 7.71 (m, 9H),
-2.80 (s, 2H).
Preparation of the free-base porphyrin series
Preparation of the zinc(II) porphyrin series
3MMe and 4MMe. The monopyridylporphyrins
3MPyTPP and 4MPyTPP were refluxed with
40-fold excess of methyl p-toluenesulfonate in N,N-
dimethylformamide (DMF, 4 h), concentrated in a rotary
evaporator and poured into a saturated NaCl aqueous
solution. The precipitate was separated by filtration,
redissolved in DMF and precipitated again in saturated
NaCl solution to obtain the N-methylated porphyrins as
chloride salts. After repeating this process three more
times, the N-methylated dyes were purified by alumina
column chromatography using a mixture of CH2Cl2 and
EtOH as eluent. Yield 87%. (3MMe). UV-vis (CH2Cl2):
Zn(II) porphyrin derivatives. The zinc(II)
porphyrins, Zn-3MPyTPP and Zn-4MPyTPP, were
obtained by refluxing 3MPyTPP and 4MPyTPP with
zinc acetate in a mixture of glacial acetic acid and DMF
(yield ~90%). Then, the N-methylpyridinium (Zn-3MMe
and Zn-4MMe) and the ruthenium complex derivatives
(Zn-3MRuandZn-4MRu)wereobtainedwith~90%yield,
using the procedures described above for the preparation
of the respective free-base porphyrin derivatives.
(Zn-3MMe). UV-vis (CH2Cl2): lmax, nm (log e)
1
429 (5.07), 560 (3.84) and 600 (3.40). H NMR (500
MHz, DMSO, TMS): d, ppm 10.05 (s, 1H), 9.37 (d, 1H),
9.32 (d, 1H), 8.96 (s, 2H), 8.83 (m, 6H), 8.55 (t, 1H),
8.17 (m, 6H), 7.84 (m, 9H), 4.69 (s, 3H). (Zn-4MMe).
UV-vis (CH2Cl2): lmax, nm (log e) 428 (4.90), 560 (3.90)
l
max, nm (log e) 422 (5.06), 515 (3.95), 550 (3.59), 590
1
(3.55) and 650 (3.45). H NMR (500 MHz, DMSO,
TMS): d, ppm 10.02 (s, 1H), 9.50 (d, 1H), 9.40 (d, 1H),
9.04 (s, 2H), 8.90 (m, 6H), 8.58 (t, 1H), 8.23 (m, 6H),
7.86 (m, 9H), 4.64 (s, 3H), -2.85 (s, 2H). (4MMe).
UV-vis (CH2Cl2): lmax, nm (log e) 422 (5.17), 515 (4.07),
1
and 610 (3.33). H NMR (500 MHz, DMSO, TMS): d,
ppm 9.18 (m, 2H), 8.82 (d, 2H), 8.61 (s, 4H), 8.39 (d,
2H), 8.19 (m, 2H), 8.10 (m, 6H), 7.67 (m, 9H), 4.67 (s,
3H). (Zn-3MRu). UV-vis (CH2Cl2): lmax, nm (log e) 295
1
550 (3.78), 590 (3.60) and 650 (3.58). H NMR (500
1
MHz, DMSO, TMS): d, ppm 9.22 (m, 2H), 8.87 (d, 2H),
8.82 (s, 4H), 8.69 (d, 2H), 8.24 (m, 2H), 8.15 (m, 6H),
7.73 (m, 9H), 4.62 (s, 3H), -2.81 (s, 2H).
(4.88), 427 (5.52), 560 (3.97) and 600 (3.70). H NMR
(500 MHz, DMSO, TMS): d, ppm 9.83 (s, 1H), 9.22 (d,
1H), 9.00–8.3 (13H), 8.25–8.00 (7H), 8.00–7.70 (9H);
7.60–7.35 (14H), 6.85 (m, 1H). (Zn-4MRu). UV-vis
(CH2Cl2): lmax, nm (log e) 295 (4.76), 427 (5.45), 552
3MRu and 4MRu. The ruthenated free-base
porphyrin derivatives were obtained by the reaction of
3MPyTPP and 4MPyTPP with [Ru(bipy)2Cl(H2O)]NO3
prepared just before use. Typically, 85 mg of AgNO3
was dissolved in 5 mL of DI-water and added to 236
mg of [Ru(bipy)2Cl2] dissolved in 15 mL of a 2:1 DMF/
ethanol mixture, stirred for 20 min at 50 °C, and filtered
through a Celite layer. The filtrate was concentrated to
5 mL, mixed with an equal volume of CH2Cl2 and
reacted with 60 mg of the respective porphyrins,
dissolved in 10 mL of CH2Cl2/DMF 4:1 mixture. After
completion of the reaction (~20 min, 50 °C), the solvent
was removed in a rotary evaporator, the solid dissolved
in ~5 mL of DMF and added into an aqueous lithium
trifluoromethanesulphonate (LiCF3SO3) solution. The
ruthenated porphyrins, 3MRu and 4MRu, were pre-
cipitated (CF3SO3- salts) and isolated as dark brown
solids after filtration, washing with water and drying in
a desiccator under vacuum. The compounds were finally
purified by alumina column chromatography using a
mixture of CH2Cl2 and ethanol as eluent. Yield ~90%.
(3MRu). UV-vis (CH2Cl2): lmax, nm (log e) 294 (4.70),
417 (5.60), 520 (4.20), 550 (4.13), 588 (3.95) and 650
(3.77). 1H NMR (500 MHz, DMSO, TMS): d, ppm 10.13
1
(3.02) and 603 (2.92). H NMR (500 MHz, DMSO,
TMS): d, ppm 9.97 (m, 1H), 8.90 (m, 1H), 8.76 (m, 8H),
8.61 (m, 1H), 8.61 (m, 2H), 8.41 (m, 1H), 8.31 (m, 1H),
8.23 (m, 1H), 8.11 (m, 2H), 8.11 (m, 6H), 8.01 (m, 1H),
8.01 (m, 2H), 7.80 (m, 1H), 7.66 (m, 3H), 7.63 (m, 9H),
7.60 (m, 1H), 7.12 (m, 1H), 6.95 (m, 1H).
Preparationofthepolymericnanocapsuleformulations
The eight porphyrin derivatives were encapsulated
using the coacervation method, as described previously
[18]. Typically, 1.0 mL of a porphyrin derivative solution
(3.0 mM in CH2Cl2) was dispersed in a mixture of
isopropylmyristate, almond oil and Tween 20 (1.2% v/v)
using a Turrax, and poured into an aqueous xanthan gum
suspension. Finally, marine atellocollagen and sodium
sulfate were added under vigorous stirring to prepare a
stablecream-likeformulationofpolymericmicrocapsules
[21], with the porphyrin derivatives dissolved in the
oily core. The concentration of the dye was adjusted
to 1 × 10-4 M. The average size of the capsules was
diminished to 200–400 nm using an ultrasonic tip (750 W,
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 61–63

62
D. K. DEDA ET AL.
VibraCell, from Sonics), monitoring the size distribution
by dynamic light scattering in a Microtrac Inc. Nanotrac
252 equipment.
a liquid nitrogen cooled (-80 °C) Hamamatsu R5509
photomultiplier and LP900 aquisition software. The
emitted light was passed through a silicon filter and
a monochromator before reaching the NIR-PMT.
The absorbance at 532 nm of the porphyrin derivatives
in chloroform solution was adjusted to about 0.02 a.u.
(~1 mM), and the phosphorescence intensity measured
10 ns after the laser pulse.
Toxicity and phototoxicity of the nanocapsule
formulations
The toxicity and phototoxicity of the monocationic
porphyrin dyes were evaluated using human cervical
epithelioid carcinoma cells (HeLa) as model. They were
cultivated in DMEM culture media, containing 10% of
bovine fetal serum and 1% of peniciline/streptomicine,
in a Thermo Electron Corporation HEPA class 100
incubator, at 37 °C and atmosphere with 5% of CO2.
Then, 1 × 105 cells were transferred to 2.0 cm diameter
culture plates with 12 wells and incubated for 15 h for cell
adhesion. Then, the culture media was removed and 1.0
mL of a 100 times diluted emulsion ([porphyrin] = 1 mM)
was added and incubated in the same conditions as
described above.
The toxicity of the porphyrin dye formulations were
evaluated by the MTT method after removal of the
culture media containing the porphyrin formulation,
careful washing of the wells with saline phosphate buffer
(PBS) and addition of 1.0 mL of colorless DMEM culture
media. The number of viable cells was estimated 14 h
after by measuring the ratio of the absorbance at 550 nm
found for the wells incubated with and without (control
experiment, 100% viability) a porphyrin formulation,
using an Infinite M200 ELISA reader (TECAN). The
absorbances are proportional to the amount of formazan
crystals [22] formed after incubation of the cells with a
2.0 mg.mL-1 methylthiazyldiphenyltetrazolium bromide
(MTT) solution in PBS for 2 h, careful removal of the
excess of the green aqueous supernatant solution and
solubilization of the crystals in 1.0 mL of DMSO.
The phototoxicity was evaluated in the same way but
after irradiation of the adhered cells with a 650 nm laser
(LASERLine, 70 mW.cm-2, free-base series) for 10 min
(periodic pulses of 1 min with 1 min intervals); or with a
white light source (tungsten/halogen lamp, 16 mW.cm-2,
zinc(II) porphyrin series) for 60 min. The results are
the average of at least two independent experiments
performed in triplicate to ensure the reproducibility of
the experimental conditions.
CONCLUSION
Two series of monocationic porphyrin derivatives were
synthesized and their photodynamic activity evaluated
using HeLa cells as model, after incorporation in polymeric
marine atelocollagen/xanthan gum nanocapsules. The
methylatedfree-baseporphyrinderivativesexhibitedhigher
phototoxicity inactivating 92% of the cells after 60 min
of irradiation with a white light source, but no significant
differences were observed for the para and meta isomers.
The activity of the ruthenated porphyrins were ~20%
lower, but was diminished even further by the coordination
of Zn(II) ion to the porphyrin ring. In fact, all Zn(II)
porphyrin derivatives exhibited similar phototoxicity and
inactivate ~50% of the HeLa cells. Those results showed
a correlation with their singlet oxygen quantum yields,
such as in the case of the Zn(II) porphyrin derivatives
that exhibited the lowest photodynamic activities and fD
values. In conclusion, the meta and para isomers of the
methylated free-base monocationic porphyrins exhibited
the highest photodynamic efficiencies and are promising
candidates as PDT photosensitizers.
Acknowledgements
We gratefully acknowledge the financial support from
the Brazilian agencies, Fundação de Amparo à Pesquisa
do Estado de São Paulo (FAPESP) and Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq).
Supporting information
Singlet oxygen fluorescence decay curves, polymeric
nanocapsules size distribution histograms and toxicity
in the dark and fluorescence spectra of free-base
monocationic porphyrins, (Figs S1–S4) are given in the
supplementary material. This material is available free of
charge via the Internet at http://www.worldscinet.com/
jpp/jpp.shtml.
Singlet oxygen quantum yields (fD)
The singlet oxygen (1Dg(O2)) quantum yields (fD) of
the two series of monocationic porphyrin derivatives
were determined from the ratio of the slopes of the
phosphorescence intensity at 1270 nm vs. the laser power
plots, using meso-tetraphenylporphyrin, TPP, as standard
(fD(1O2) = 0.5) [23]. The 1Dg(O2) phosphorescence
emission decay curves were registered using a time-
resolved Edinburg Analytical Instruments NIR flash-
REFERENCES
1. Allison RR, Mota HC, Bagnato VS and Sibata CH.
Photodiagn. Photodyn. Ther. 2008; 5: 19–28.
2. Pushpan SK, Venkatraman S, Anand VG, Sankar J,
Parmeswaran D, Ganesan S and Chandrashekar TK.
Curr. Med. Chem. — Anti-Cancer Agents 2002; 2:
187–207.
photolysis instrument equipped with
a Nd:YAG
Continuum Surelite III pulsed laser (l = 532 nm, 10 Hz),
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 62–63

CORRELATION OF PHOTODYNAMIC ACTIVITY
63
3. Sharman WM, Allen CM and van Lier JE. Drug
Discovery Today 1999; 4: 507–517.
21. Menger FM and Sykes BM. Langmuir 1998; 14:
4131–4137.
4. Moriwaki SI, Misawa J, Yoshinari Y, Yamada I,
Takigawa M and Tokura Y. Photodermatol., Photo-
immunol. Photomed. 2001; 17: 241–243.
5. Simplício FI, Maionchi F and Hioka N. Quim. Nova
2002; 25: 801–807.
22. Berg K, Hansen MB and Nielsen SE. Apmis 1990;
98: 156–162.
23. Wilkinson F, Helman WP and Ross AB. J. Phys.
Chem. Ref. Data 1993; 22: 113–262.
24. Fleischer EB and Shachter AM. Inorg. Chem. 1991;
30: 3763–3769.
25. Moreira LM, dos Santos FV, Lyon JP, Maftoum-
Costa M, Pacheco-Soares C and da Silva NS. Aust.
J. Chem. 2008; 61: 741–754.
26. Dougherty TJ. Photochem. Photobiol. 1993; 58:
895–900.
27. Machado AEdH. Quim. Nova 2000; 23: 237–243.
28. Girotti AW. J. Photochem. Photobiol., B 2001; 63:
103–113.
6. Tian YY, Wang LL and Wang W. Laser Phys. 2008;
18: 1119–1123.
7. Hopper C. Lancet Oncol. 2000; 1: 212–219.
8. Triesscheijn M, Baas P, Schellens JHM and
Stewart FA. Oncologist 2006; 11: 1034–1044.
9. Bechet D, Couleaud P, Frochot C, Viriot ML, Guil-
lemin F and Barberi-Heyob M. Trends Biotechnol.
2008; 26: 612–621.
10. Paszko E, Ehrhardt C, Senge MO, Kelleher DP and
Reynolds JV. Photodiagn. Photodyn. Ther. 2011; 8:
14–29.
29. Mora-Huertas CE, Fessi H and Elaissari A. Int.
J. Pharm. 2010; 385: 113–142.
11. Reddi E. J. Photochem. Photobiol., B 1997; 37:
189–195.
12. Torchilin VP. Adv. Drug Delivery Rev. 2006; 58:
1532–1555.
13. Konan YN, Gurny R and Allemann E. J. Photo-
chem. Photobiol., B 2002; 66: 89–106.
14. Needhan D, McIntosh TJ and Lasic DD. Biochim.
Biophys. Acta 1992; 1108: 40–48.
15. Engelmann FM, Mayer I, Gabrielli DS, Toma HE,
Kowaltowski AJ, Araki K and Baptista MS. J Bio-
energ. Biomembr. 2007; 39: 175–185.
16. Engelmann FM, Rocha SVO, Toma HE, Araki K
and Baptista MS. Int. J. Pharm. 2007; 329: 12–18.
17. Engelmann FM, Winnischofer H, Araki K and
Toma HE. J. Porphyrins Phthalocyanines 2002; 6:
33–42.
18. Deda DK, Uchoa AF, Carita E, Baptista MS, Toma
HE and Araki K. Int. J. Pharm. 2009; 376: 76–83.
19. Al-Mutairi DA, Craik JD, Batinic-Haberle I and
Benov LT. Free Radical Res. 2006; 40: 477–483.
20. Pavani C, Uchoa AF, Oliveira CS, Iamamoto Y and
Baptista MS. Photochem. Photobiol. Sci. 2009; 8:
233–240.
30. Espinosa-Andrews H, Baez-Gonzalez JG, Cruz-
Sosa F and Vernon-Carter EJ. Biomacromolecules
2007; 8: 1313–1318.
31. Dong ZJ, Toure A, Jia CS, Zhang XM and Xu SY.
J. Microencapsulation 2007; 24: 634–646.
32. Bamford CH and Tompa H. Trans. Faraday Soc.
1950; 46: 310–316.
33. Dash BC, Rethore G, Monaghan M, Fitzgerald K,
Gallagher W and Pandit A. Biomaterials 2010; 31:
8188–8197.
34. Calzavara-Pinton PG, Venturini M and Sala R.
J. Eur. Acad. Dermatol. Venereol. 2007; 21:
293–302.
35. He XZ, ZhouYL, Wang LX, Li TK, Zhang MH and
Shen T. Dyes Pigm. 1998; 39: 173–182.
36. Ravanat JL, Cadet J, Araki K, Toma HE, Medeiros
MHG and Di Mascio P. Photochem. Photobiol.
1998; 68: 698–702.
37. Araki K, Engelmann FM, Mayer I, Toma HE,
Baptista MS, Maeda H, Osuka A and Furuta H.
Chem. Lett. 2003; 32: 244–245.
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 63–63

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