Photophysical properties and photocytotoxicity of free and liposome-entrapped diazepinoporphyrazines on LNCaP cells under normoxic and hypoxic conditions

Article abstract of DOI:10.1016/j.ejmech.2018.02.064

5,7-Diaryl-substituted symmetrical diazepinoporphyrazine and tribenzodiazepinoporphyrazine were synthesized and characterized using UV–Vis, MS MALDI, and various NMR techniques. The expected photosensitizing potentials of these porphyrazines were evaluate

Full text of DOI:10.1016/j.ejmech.2018.02.064

Accepted Manuscript
Photophysical properties and photocytotoxicity of free and liposome-entrapped
diazepinoporphyrazines on LNCaP cells under normoxic and hypoxic conditions
Ewelina Wieczorek, Dariusz T. Mlynarczyk, Malgorzata Kucinska, Jolanta
Dlugaszewska, Jaroslaw Piskorz, Lukasz Popenda, Wojciech Szczolko, Stefan Jurga,
Marek Murias, Jadwiga Mielcarek, Tomasz Goslinski
PII:
S0223-5234(18)30201-0
10.1016/j.ejmech.2018.02.064
EJMECH 10241
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 December 2017
Revised Date: 5 February 2018
Accepted Date: 20 February 2018
Please cite this article as: E. Wieczorek, D.T. Mlynarczyk, M. Kucinska, J. Dlugaszewska, J. Piskorz,
L. Popenda, W. Szczolko, S. Jurga, M. Murias, J. Mielcarek, T. Goslinski, Photophysical properties
and photocytotoxicity of free and liposome-entrapped diazepinoporphyrazines on LNCaP cells under
normoxic and hypoxic conditions, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.02.064.
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ACCEPTED MANUSCRIPT
Graphical Abstract
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Photophysical properties and photocytotoxicity of free and liposome-entrapped
diazepinoporphyrazines on LNCaP cells under normoxic and hypoxic conditions
Ewelina Wieczoreka, Dariusz T. Mlynarczyka, Malgorzata Kucinskab, Jolanta Dlugaszewskac,
Jaroslaw Piskorzd, Lukasz Popendae, Wojciech Szczolkoa, Stefan Jurgae,f, Marek Muriasb,
Jadwiga Mielcarekd, Tomasz Goslinskia,*
a Department of Chemical Technology of Drugs, Poznan University of Medical Sciences,
Grunwaldzka 6, 60-780 Poznan, Poland
b Department of Toxicology, Poznan University of Medical Sciences, Dojazd 30, 60-631
Poznan, Poland
c Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical
Sciences, Swiecickiego 4, 60-780 Poznan, Poland
d Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences,
Grunwaldzka 6, 60-780 Poznan, Poland
e NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, 61-614 Poznan,
Poland
f Department of Macromolecular Physics, Adam Mickiewicz University, Umultowska 85,
61-614 Poznan, Poland
Abstract
5,7-Diaryl-substituted symmetrical diazepinoporphyrazine and tribenzodiazepinoporphyrazine
were synthesized and characterized using UV–Vis, MS MALDI, and various NMR
techniques. The expected photosensitizing potentials of these porphyrazines were evaluated
by measuring their abilities to generate singlet oxygen in organic solvents and by comparing
them with that of the recently obtained dendrimeric G1-type diazepinoporhyrazine.
Absorbance and fluorescence measurements were performed to study the aggregation
properties of the novel macrocycles. The photocytotoxicity of tribenzodiazepinoporphyrazine
towards LNCaP cells in its free form and after its incorporation into liposomes was examined
using MTT assay under normoxic and hypoxic conditions. It is interesting that all tested
liposome formulations maintained their phototoxic activity in hypoxia. Also,
tribenzodiazepinoporphyrazine incorporated into liposomes revealed better photocytotoxic
effect (IC50 values of 0.600 ± 0.357 ꢀM and 0.378 ± 0.002 ꢀM) than its free form (IC50 values
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of 3.135 ± 0.156 ꢀM). Following the in vitro experiments, the most promising liposomal
formulation containing L-α-phosphatidyl-DL-glycerol for tribenzodiazepinoporphyrazine was
found. Moreover, tribenzodiazepinoporphyrazine incorporated into liposomes containing 1,2-
dioleoyl-3-trimethylammonium-propane (chloride salt) revealed moderate phototoxicity at
5×10-5 ꢀM for antibacterial photodynamic therapy. It was established that an irradiation of
planktonic bacterial strains significantly reduced CFUs of Staphylococcus aureus ATCC
25923 in comparison to tribenzodiazepinoporphyrazine containing L-α-phosphatidyl-DL-
glycerol liposomes.
Keywords
diaminomaleonitrile, photocytotoxicity, porphyrazine, singlet oxygen, photodynamic activity
*Correspondence to: T. Goslinski, Department of Chemical Technology of Drugs, University
of Medical Sciences, Grunwaldzka 6, 60-780, Poznan, Poland. Tel.: +48 61 854 66 31, fax:
+48 61 854 66 39. E-mail: tomasz.goslinski@ump.edu.pl
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1. Introduction
For the last 20 years, macrocyclization reactions of 1,4-diazepine-2,3-dicarbonitrile
derivatives have led to a plethora of various porphyrazines with annulated rings of
diazepine,1-3
tetrahydrodiazepine,4,5
and
styryldiazepine5-8.
Styryl-substituted
diazepinoporphyrazines have been evaluated for their electronic properties, for the tendency
for aggregation and photodegradation, singlet oxygen generation efficiency, and in vitro
photodynamic activity at a nanomolar level against two oral squamous cell carcinoma cell
lines.5,7,8 5,7-Diphenyl- and 5,7-di(4-tert-butylphenyl)-substituted diazepinoporphyrazines
and tribenzodiazepinoporphyrazines have also been studied towards electronic1-3,9,10 and
electrochemical properties.11,12
Porphyrazine macrocycles seem to be suitable candidates for photodynamic therapy
(PDT) for many reasons. They exhibit high generation quantum yields of singlet oxygen and
have adequate light absorption spectra. Moreover, porphyrazines absorb light in the so-called
therapeutic window in range 600-800 nm, where the tissues are the most permeable to such
wavelengths. On the one hand, porphyrinoid photosensitizers are lipophilic and reveal high
affinity to lipids, which seems beneficial, as cancer cells are often overexpressing LDL-
receptors.13 On the other hand, the high lipophilicity of porphyrinoids hampers their solubility
in water and increases their tendency to form aggregates in polar solvents. This issue can be
overcome by incorporation of photosensitizers into various pharmaceutical formulations,
including liposomes proven as useful carriers.14 Liposomes offer a huge advantage in
lipophilic photosensitizer delivery, not only by enabling water-insoluble compound delivery
but also in functionalization of the liposome membrane. The tunable charge of the carrier can
also have a great effect on the effectiveness of the therapy. Photosensitizers like hydrophobic
porphyrazines accumulate in liposomal membranes. In this regard, smaller porphyrazine
macrocycles are preferred, as the thickness of membranes is limited thus making it impossible
to incorporate bulky porphyrinoid structures.
To further address the issue of anticancer therapies, one of the characteristics of the
cancer tissues such as hypoxia, must be taken into account. For many years until now, it is
well known that tumor hypoxia has been a significant limitation of anticancer therapies,
which depend on oxygen-mediated mechanisms such as radiation, photodynamic therapy as
well as chemotherapy.15 Although PDT has been found to be effective against the superficial
tumor, the photodynamic efficacy for solid tumors, which are characterized by the hypoxic
environment, is still challenging.16 The rapid growth of the neoplasm cells and their increased
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metabolism are factors triggering hypoxia,17 which is a major problem in case of
photodynamic therapy, as molecular oxygen is one of the three factors determining a
successful treatment. The singlet oxygen (1O2) generated in type II photoreaction is still
considered as the primary cytotoxic agent for PDT. Therefore, the low oxygen concentration
may decrease the efficacy of photodynamic treatment to cancer cells.18,19 To date, several
strategies were designed to overcome tumor hypoxia in the context of PDT such as
modification of the tumor microenvironment to ensure tissue re-oxygenation, the increase of
intracellular oxygen concentration as well as the development of the photosensitizer, which
may induce cytotoxic effects in environments of low molecular oxygen via type I
photosensitization.15,16,20 Thus, it is essential to develop photosensitizers which may exert
therapeutic effects under both normoxia and hypoxia.
Currently, screening of photosensitizers during photodynamic therapy is mainly
focused on normoxic condition. However, it should be noted that the median oxygenation in
untreated tumors varies between approximately 0.3% and 4.2% oxygen and most tumors
demonstrate median oxygen levels below 2%.21 This range in tumor oxygenation depends on
several factors, and hypoxic microregions are heterogeneously distributed within the tumor
mass.22 It is worth noting that many prostate and pancreatic tumors are strongly hypoxic.21
Moreover, the hypoxia in prostate cancer is also related to advanced tumor stage, aggressive
disease as well as increased resistance to androgen deprivation and radio- and
chemotherapy.23 Recently, we have reported the synthesis of diazepinoporphyrazine with G1-
dendrimeric substituents and discussed its photophysical properties as well as singlet oxygen
generation efficiency.12 Herein, as an enhancement of our studies, we report the synthesis,
physicochemical characterization, including absorption and emission properties, tendency to
aggregation of magnesium(II) diazepinoporphyrazine and tribenzodiazepinoporhyrazine
substituted in their C5 and C7 positions with 4-methoxyphenyl groups. Moreover, the
anticancer potential of new porphyrazines was assessed in liposomal formulations in both
normoxia and hypoxia conditions against prostate adenocarcinoma cells.
2. Results and discussion
2.1. Synthesis and characterization
Novel magnesium diazepinoporphyrazine substituted at C5 and C7 positions with 4-
methoxyphenyl groups (4) was synthesized in two steps (Scheme 1). Firstly, the condensation
reaction of diaminomaleonitrile (1) and 1,3-bis(4-methoxyphenyl)-1,3-propanedione (2) was
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performed following the literature procedure,9 and led to the novel 5,7-disubstituted-1,4-
diazepine-2,3-dicarbonitrile (3). Subsequent macrocyclization reaction of 3 using magnesium
n-butanolate in n-butanol according to the Linstead macrocyclization conditions24 enabled the
obtaining
of
symmetrical
diazepinoporphyrazine
4
with
10%
yield.12
Tribenzodiazepinoporphyrazine (5) was synthesized following a similar procedure but with
the addition of an excessive amount of 1,2-dicyanobenzene (ten-fold mol/mol excess to 3).
After extensive chromatography, macrocycle 5 was isolated in 3.5% yield.
To unambiguously identify the isolated macrocyclic products, NMR experiments were
1
carried out. The H NMR spectrum of 4 revealed two doublets at 8.56 ppm and 6.89 ppm
assigned to 32 protons of phenyl rings, two broad doublets (2J ~ 13 Hz) observed at 6.86 ppm
and 6.07 ppm from C6-CH2 eight geminal protons, and one singlet at 3.80 ppm resulting from
all methoxy groups, 24 protons. It is worth mentioning that the presence of two doublets from
C6-CH2 geminal protons indicates that all diazepine rings of 4 are in the 6H tautomeric form.1
Similarly, for 5, two doublets corresponding to the aromatic protons of 4-methoxy
1
substituents were detected at 8.55 ppm and 7.21 ppm in the H NMR spectrum. Only one
singlet in the spectrum at 3.94 ppm was assigned to methoxy group protons. A very weak and
broad signal corresponding to diazepine ring protons was observed at 4.59 ppm. Four signals
at 9.46, 9.38, 8.27, 8.24 ppm were assigned to protons at annulated benzene rings. The
1
1
1
1
assignments of H resonances were confirmed by H-1H COSY, H-13C HMBC, and H-13C
HSQC experiments (see Supporting Information).
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Scheme 1. Synthesis of porphyrazines 4 and 5. Reagents and conditions: (i) ethanol, P2O5,
reflux, 10 h, (60%); (ii) Mg(OnBu)2, nBuOH, reflux, 20 h, (10%); (iii) 1,2-dicyanobenzene,
Mg(OnBu)2, nBuOH, reflux, 20 h, (3.5%).
2.2. Photochemical studies
2.2.1. Absorption and emission
Photochemical properties of symmetrical porphyrazine 4 and tribenzoporphyrazine 5
were determined together with the previously obtained symmetrical G1-dendrimeric
diazepinoporphyrazine 6 (Inset in Scheme 1).12 Firstly, the absorption properties of 4 and 6
were evaluated in various protic and aprotic organic solvents. The selected UV-Vis spectra
are shown in Fig. 1, whereas Table S4, included in the Supplementary Information presents
the spectral data including the absorption maxima of the Soret- and Q-bands with the
corresponding absorption coefficients. The electronic absorption spectra recorded in various
solvents revealed broad intensive Soret bands with the maxima in the range 343–389 nm for 4
and 347–389 nm for 6 and broad Q-bands with maxima between 637-676 and 640-674 nm,
respectively. Moreover, it was observed that in both symmetrical porphyrazines the Q-band
split into two sub-bands of different intensities depending on the solvent applied. The
presence of the additional band is connected with the above-described aggregation.
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Fig. 1. Normalized absorption spectra of 4 (a), 5 (b) and 6 (c) in selected organic solvents.
The fluorescence emission spectra of 4-6 were recorded in dimethylformamide (DMF) and
dimethylsulfoxide (DMSO) and are presented in Figure 2. Symmetrical porphyrazines 4 and 6
reveal fluorescence maxima at 681 nm in DMF and 682 nm in DMSO. In the spectrum of
tribenzoporphyrazine 5, split emission bands with maxima at 683 and 699 nm in DMF and
684 and 792 nm in DMSO can be noticed. The fluorescence quantum yield (ΦF) values were
calculated following the relative method proposed by Chauke et al.25 with zinc(II)
phthalocyanine (ZnPc) as a reference (ΦF = 0.17 and 0.20 in DMF and DMSO, respectively).
The tribenzoporphyrazine 5 revealed moderate fluorescence emission with ΦF value of 0.19 in
DMF and 0.16 in DMSO. The symmetrical porphyrazine 4 showed slightly intensive emission
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with ΦF = 0.17 in DMSO as compared to ΦF = 0.16 in DMF. In both solvents, symmetrical
G1-type dendrimeric porphyrazine 6 revealed less intense fluorescence with ΦF equal to 0.12
and 0.13 in DMF and DMSO, respectively (Table 1).
Fig. 2. The Q-band absorption, emission and excitation spectra of 4-6 in DMF (a) and DMSO
(b).
2.2.2. Aggregation
Tendency to aggregation of porphyrazines 4-6 was evaluated in DMF and DMSO
solutions following the Stuzhin et al. procedure.6 Figure 3 and 4 present the changes in the
UV-Vis spectra of porphyrazines 4-6 in DMF and DMSO after addition of 10% water and
tetramethylammonium fluoride (TMAF) as an antiaggregation agent. On the one hand, the
addition of water to the solution of 4 increases the intensity of the short wavelength sub-band
with λmax ~ 640 nm and decreases the intensity of the long wavelength band with λmax ~ 675
nm. On the other hand, the addition of TMAF causes the disappearance of the short
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wavelength band and only one single and intensive band with λmax ~ 675 nm can be observed.
The results confirmed that the long wavelength band corresponds to the monomeric form of
porphyrazines, whereas the short wavelength band is a result of the formation of aggregates.
Moreover, the fluorescence intensity increases after the addition of TMAF and decreases
significantly when water is added, as it is well-known that aggregation is responsible for
fluorescence quenching.26,27 It was found that the aggregation tendency of 4 and 6 is strongly
dependent on the solvent used. In the UV-Vis spectrum of 4 in DMF the bands for aggregated
and monomeric species revealed similar intensity indicating strong aggregation, while for 6
only a residual band was observed as being the result of aggregates formation. It is worth
noting that in DMSO the aggregation level was higher for 6 than for 4 (Figure 3 and 4). These
data indicate that 4 and 5, unlike 6, aggregate in both DMF and DMSO. The aggregation of
photosensitizers is a common problem for their further applications in photodynamic therapy
(PDT), as the self-association of the photosensitizer molecules reduces the generation of
singlet oxygen, thus hindering the photosensitizing efficiency.28 For tribenzoporphyrazine 5,
with low symmetry molecules, a split band in the long-wavelength UV-Vis region was
observed, which is known to be related to the symmetry reduction of π-chromophore.3
Significant intensity of this band is a result of π – π* transitions in the macrocyclic
system,1,2,3,29 when going from a high symmetry to a low symmetry porphyrazine. As
tribenzoporphyrazine macrocycle is unsymmetrical, the Q-band region is relatively broad and
may overlap the n - π* transitions as a result of partial conjugation of N atom lone pairs of
electrons of the boat-shaped diazepine rings in the 6H-form with the central π-chromophore.
Detailed research performed by Ercolani and Stuzhin2 on the group of symmetrical
diazepinoporphyrazines indicated that the n – π* transitions can mix with the π – π*
transitions and gain in intensity, which is reflected in the appearance of the Q-band. A spectral
feature characteristic for diazepinoporphyrazines is an additional sub-band present in the Q-
band region.
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Fig. 3. Aggregation studies of 4-6 in DMF.
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Fig. 4. Aggregation studies of 4-6 in DMSO. The absorption plots of 6 were published.12
2.2.3. Singlet oxygen generation
The potential photosensitizing activities of the obtained porphyrazines were evaluated
by measuring their ability to generate singlet oxygen as a result of interaction between the
activated photosensitizer and triplet oxygen. 1,3-Diphenylisobenzofuran (DPBF) was used as
a chemical quencher, which undergoes a cycloaddition reaction with singlet oxygen to
produce endoperoxide.30 Solutions containing 4-6 or ZnPc in a mixture with DPBF in DMF or
DMSO were irradiated with monochromatic light at the wavelengths corresponding to the Q-
band maxima of their monomeric form. The kinetics of DPBF decomposition by
photogenerated singlet oxygen was studied by monitoring decrease of the absorbance at 417
nm, and they were used to calculate the singlet oxygen generation yields (Фꢁ). To determine
the role of monomeric form of porphyrazines in singlet oxygen generation, the measurements
for 4 and 6 were performed with and without the addition of TMAF. Table 1 presents the
calculated values of singlet oxygen quantum yield for 4 and 6 in the presence and in the
absence of TMAF. Moreover, changes in the UV–Vis spectra during irradiation of the
solutions containing 4 or 6, DPBF and TMAF in DMF or DMSO and the first-order plots of
DPBF degradation by photogenerated singlet oxygen are shown in Figures S4-S8 in the
Supporting Information. It was found that both symmetrical porphyrazines induce singlet
oxygen formation better in DMSO than DMF. It is worth noting that 4 was the more efficient
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singlet oxygen generator (with the Фꢁ values at 0.078 and 0.249 in DMF and DMSO,
respectively) than 6 (with the Фꢁ values at 0.050 and 0.090 in DMF and DMSO,
respectively). However, after the addition of TMAF, the Фꢁ values for both porphyrazines
were similar (0.307 in DMF, 0.295 in DMSO for 4; 0.126 in DMF, 0.295 in DMSO for 6),
indicating that aggregation tendency can play a key role in the efficiency of singlet oxygen
production. The lack of visible changes in the 4-6 Q-band absorptions proves the
photostability of porphyrazines upon irradiation. The singlet oxygen generation yields of
tribenzoporphyrazine 5 in DMF and DMSO were rather low or moderate with the Фꢁ values
at 0.069 and 0.180, respectively.
Table 1. Quantum yields of singlet oxygen production (Фꢁ) and quantum yields of
fluorescence emission (ΦF) for 4 - 6.
porphyrazine
Φꢁ ± ꢁΦꢁ*
ΦF
DMF
DMSO
DMF
0.16
-
DMSO
0.17
-
0.078 ± 0.003
0.307 ± 0.011
0.069 ± 0.001
0.050 ± 0.002
0.126 ± 0.003
0.249 ± 0.007
0.295 ± 0.021
0.180 ± 0.006
0.090 ± 0.007**
0.295 ± 0.024**
4
4 with TMAF
0.19
0.12
-
0.16
0.13
-
5
6
6 with TMAF
*results are given with 95% confidence
**values from ref.12
2.3. Liposomal formulations
Porphyrazines 4-6 are highly lipophilic. To increase their potential as photosensitizers
for PDT applications, a proper drug-delivery system allowing further biological experiments
in aqueous media is necessary.31-36 With this goal in mind, liposomes containing
porphyrazines were proposed. This approach seemed rational because liposomal nanoparticles
have already been used as a drug delivery system for photosensitizers, enhancing their
solubility in water and cell uptake.37 Two different liposome formulations were prepared by a
thin-film hydration method: (i) negatively charged liposomes composed of L-α-phosphatidyl-
DL-glycerol (PG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and (ii)
positively charged liposomes containing 1,2-dioleoyl-3-trimethylammonium-propane
(chloride salt, DOTAP) and POPC.38,39
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The obtained liposomes were used as non-extruded, multilamellar vesicles, because
the extrusion could reduce drug encapsulation, especially in the case of porphyrazine 6 with
large, bulky substituents. Following Bhardwaj and Burgess study, it is worth noting that the
sonication and extrusion of liposomes based on phosphatidylcholine derivatives have reduced
dexamethasone encapsulation by approximately 50% in comparison to non-extruded
liposomes.40 The size of liposomes containing porphyrazines 4-6, as well as blank liposomes,
prepared as a control, was evaluated by dynamic light scattering method (Table S5 in the
Supplementary Information). Two liposome fractions of different diameter were found for all
formulations. The blank PG-POPC and DOTAP POPC liposomes were composed in about
11% of vesicles with the mean diameter varied from 40 to 50 nm, and in 89% of vesicles with
the mean diameter in the range of 620-720 nm. Both liposomal formulations containing
porphyrazine 4 were composed of vesicles with the mean diameter in the range of 60-70 and
540-590 nm, but the proportion of these two fractions was different. DOTAP-POPC-4
liposomes contained 11% of smaller and 89% of larger nanoparticles, while PG-POPC-4
about 37% of smaller and 63% of larger nanoparticles. Liposomes containing porphyrazine 5
were composed in 17-19% of vesicles with the mean diameter in the range 70-80 nm and in
81-83% of vesicles of the mean diameter of 400 nm. DOTAP-POPC-6 liposomes were
composed of vesicles with the mean diameter of 120 nm and 640 nm in 31% and 69%,
respectively. PG-POPC-6 liposomes were composed in about 10% of vesicles with the mean
diameter of 540 nm, whereas in about 90% of vesicles with the mean diameter of 790 nm
(Table S5 in Supplementary Information). No direct relationship between the size of
liposomes and their compositions was found. However, liposomes containing porphyrazine 6
were larger than the others. Nevertheless, a further biological study towards an efficient
method for the production of unilamellar and unified liposomes are necessary.
2.4. Anticancer assay (in vitro cellular studies)
The cytotoxicity of porphyrazines 4-6 to LNCaP cells was examined both in the
presence and the absence of light using MTT assay (Figure 5).41 It was found, that 4 did not
exert any activity against cancer cells. Furthermore, it was observed that porphyrazine 4
incorporated into positively charged liposomes with DOTAP-POPC (DOTAP-POPC-4)
displayed light-independent toxicity (the decrease of the cell viability to 70% and 65% in dark
and light condition, respectively), whereas this effect was not seen for negatively charged
liposomes with PG-POPC (PG-POPC-4). Additionally, we noted that DOTAP-POPC-4 and -6
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liposomes probably precipitate during incubation. To date, it was found that the electrolytes
may reduce the positive charge of cationic lipids, e.g. phosphate buffer saline (PBS) has an
additional effect of phosphate polyanions which tend to react and precipitate cationic
liposomes.42 Similarly, compound 6 appeared inactive and exhibited only slight cytotoxic
effects at the highest tested dose (10ꢀM) for DOTAP:POPC formulation in the dark and after
High Power Multi Chip LEDs exposure (cell viability was around 80%).
In the dark condition porphyrazine 5 in its free form and after incorporation into
liposomes DOTAP-POPC-5, PG-POPC-5 was not cytotoxic in all tested concentrations,
whereas in the light condition exhibited high cytotoxicity under irradiation with the dose of 2
J/cm2. The cytotoxic effects increased along with the concentration for all formulations with
the half maximal inhibitory concentration (IC50) under the normoxic conditions at 0.814 ±
0.466 µM, 0.161 ± 0.002 µM and 0.166 ± 0.058 µM for 5, DOTAP-POPC-5, and PG-POPC-
5, respectively. Thus, as shown in Figure 5 the liposomal formulation may enhance cytotoxic
activity of 5. In general, the liposomal formulation prevents the aggregation of
photosensitizers and in turn improves their activity.43 Thus, a number of studies have shown
that liposomal formulations of photosensitizers are more effective when compared to the free
drug.44-47 Based on the study results obtained under normoxic conditions, further evaluation of
the activity of 5 was performed under hypoxia (defined as 1% of O2). It is well-known that
hypoxia may significantly affect the efficiency of PDT and for many years it has been
considered a possible mechanism of resistance.42 Taking into account that PDT works mainly
by singlet oxygen generation and other reactive oxygen species (ROS), the concentration of
oxygen is critical to the efficiency of PDT.48,49 Moreover, hypoxia is one of the main features
of solid tumors and has been associated with a poor clinical outcome of PDT.50 Therefore, in
order to gain more insight into the potential of 5 for photodynamic therapy, further biological
evaluation of this compound was assessed in hypoxia conditions. Interestingly, all tested
formulations remained photocytotoxic in hypoxia. However, the results indicated that 5 in
liposomal formulation revealed better therapeutic effect than the free counterpart in hypoxia
experiments (IC50 values of 3.135 ± 0.156 µM, 0.600 ± 0.357 µM and 0.378 ± 0.002 µM for
5, DOTAP-POPC-5, and PG-POPC-5, respectively). Summarizing, in vitro experiments
demonstrated that liposomal formulations of 5, particularly PG-POPC-5 may be considered as
a promising photosensitizer for cancer treatment. However, further studies are necessary to
fully evaluate the photodynamic potential of this compound.
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Figure 5. Cytotoxicity of compound 5 and its two liposomal formulations (DOTAP-POPC
and PG-POPC) against LNCaP cell line under normoxic and hypoxic conditions. Results are
presented as a mean ± SD from two or three independent experiments. The table presents the
IC50 values.
2.5. Antimicrobial activity
The photodynamic antimicrobial effect of novel macrocycles was investigated against
Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 – the representatives
of both gram-positive and gram-negative bacteria, respectively. The antibacterial activity of
porphyrazine 5 incorporated into two liposomal formulations as well as liposomes alone was
studied in conditions of both dark and light.
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Table 2. Bacterial reduction of planktonic Staphylococcus aureus ATCC 25923 and
Escherichia coli ATCC 25922 in the presence of porphyrazine 5 in DOTAP-POPC or PG-
POPC liposomes.
Log10 reduction in viable count ± SD*
Bacterial
strain
DOTAP-POPC-5 at
concentration [ꢀM]
PG-POPC-5 at
Control
DOTAP-
POPC
concentration [ꢀM]
PG-POPC
5
×
10-5
1
×
10-5
5
×
10-5
1×
10-5
S. aureus
ATCC 25923
E. coli ATCC
25922
0.25 ±
0.12
-0.06 ±
0.08
0.48 ±0 .07 0.45 ± 0.12 0.86 ± 0.06
NT 0.21 ± 0.18 NT
0.11 ± 0.11 0.01 ±0 .06
-0.15 ± 0.04 -0.16 ± 0.06
0.14 ± 0.02 0.12 ± 0.01
-0.01 ± 0.04 0.05 ± 0.16
2.07 ± 0.88
Photo
toxicity
0.10 ± 0.18
S. aureus
NT
NT
0.22 ± 0.03 -0.06 ± 0.06 0.08 ± 0.06 0.07 ± 0.13
-0.02 ± 0.14 NT -0.04 ± 0.14 NT
ATCC 25923
E. coli ATCC
25922
Dark
toxicity
Phototoxicity was assessed by calculating the phototoxic reduction factor - RPh= log L(-)Ps(-) – log L(+)Ps(+), whereas dark
toxicity by calculating the dark toxic reduction factor - RD= log L(-)Ps(-) – log L(-)Ps(+). For control the reduction factor
was calculated as follows: RControl= log L(-)Ps(-) – log L(+)Ps(-); L – light, Ps- photosensitizer
As it was presented in the Table 2, an irradiation of planktonic bacterial strains in the presence
of porphyrazine 5 in DOTAP-POPC liposomes at 5×10-5 ꢀM (Ps+L+) significantly reduced
CFUs of Staphylococcus aureus ATCC 25923 in comparison to 5 in PG-POPC liposomes,
and the control liposomal formulations without photosensitizer (consisting of DOTAP-POPC
or PG-POPC). However, insignificant reduction in cell survival was observed for 5
incorporated in DOTAP-POPC liposomes at a concentration of 1×10-5 ꢀM and for 5
incorporated in PG-POPC liposomes at concentrations 5×10-5 and 1×10-5 ꢀM. In addition,
studied tribenzporphyrazine did not reveal any phototoxicity on Escherichia coli ATCC
25922 regardless of the liposomal formulation applied. It should be underlined that the tested
liposome formulations did not reveal any influence on the microbes involved in research.
Also, no dark toxicity on microorganisms after 20 minutes of exposure was noticed for either
the tested photosensitizer or the liposomal formulations. Our results indicated that 5
incorporated into DOTAP-POPC liposomes revealed moderate phototoxicity for antibacterial
photodynamic therapy.
3. Experimental
3.1. Materials and instruments
All reactions were conducted in oven-dried glassware under argon using Radleys
Heat-On heating system. Solvents and all reagents were obtained from commercial suppliers
and used without further purification. All solvents were removed by rotary evaporation at or
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below 50 ºC. Dry flash column chromatography was carried out on Merck silica gel 60,
particle size 40-63 µm, reverse phase Fluka C18 silica gel 90 and aluminum oxide 90 active
neutral (activity stage I) for column chromatography 0.063-0.200 mm, EMD Millipore. Thin
layer chromatography (TLC) was performed on silica gel Merck Kieselgel 60 F254 plates and
Merck Kieselgel 60 RP-18 F254s visualized with UV (λmax 254 or 365 nm). All
chromatography phases are given in volume to volume ratio. UV-Vis spectra were recorded
on Hitachi UV/VIS U-1900 and Shimadzu UV-160A spectrophotometers. Melting points
were obtained on a “Stuart” Bibby apparatus and are uncorrected. NMR spectra were acquired
on an Agilent DD2 800 spectrometer at 298 K, unless stated otherwise. Chemical shifts (δ)
are reported in parts per million (ppm) and referenced to the residual solvent peak (DMSO-d6:
δH = 2.50 ppm, δC = 39.5 ppm; pyridine-d5: δH = 8.74, 7.58, 7.22 ppm, δC = 150.35, 135.91,
123.87 ppm). Coupling constants (J) are quoted in Hertz (Hz). The abbreviations s, d, dd, m,
Ar, ax, eq, tribenzoH refer to singlet, doublet, doublet of doublets, multiplet, aromatic, axial,
equatorial and tribenzoporphyrazine phenyl ring protons, respectively. 1H and 13C resonances
1
13
1
1
1
were unambiguously assigned based on H, C, H-1H COSY, H-13C HSQC and H-13C
HMBC experiments. Mass spectra (MS ES, MALDI TOF) and combustion analyses were
carried out by the Advanced Chemical Equipment and Instrumentation Facility at the Faculty
of Chemistry and the Wielkopolska Center for Advanced Technologies at Adam Mickiewicz
University in Poznan. Tetrakis[5,7-bis-[4-[3,5-bis(benzyloxy)benzyloxy]phenyl]-6H-1,4-
diazepino][2,3-b;2’,3’-g;2’’,3’’-l;2’’’,3’’’-q]porphyrazinato magnesium(II) was synthesized
according to lately published procedure.12
3.2. Synthesis
2.2.1. 5,7-Bis(4-methoxyphenyl)-6H-1,4-diazepine-2,3-dicarbonitrile (3)
The reaction mixture containing diaminomaleonitrile 1 (0.38 g, 3.5 mmol), 1,3-bis(4-
methoxyphenyl)-1,3-propanedione 2 (1 g, 3.5 mmol) and P2O5 (0.15 g, 1.56 mmol) in
anhydrous ethanol (20 mL) was stirred for 1 h. After addition of further portion of P2O5 (0.15
g, 1.56 mmol), the reaction mixture was refluxed for 10 h. After the solvent was evaporated,
the precipitate was filtered and washed with methanol to give yellow solid 3 (0.75 g, 60%
yield). M.p. >250 °C dec. Rf (dichloromethane) 0.25. UV-Vis (dichloromethane): λmax, nm
(log ε) 378 (4.45), 314 (4.73). 1H NMR (799.926 MHz, DMSO-d6): δH, ppm 8.12 (d, 4H, 3J=9
Hz, C2′, C6′, ArH), 7.01 (d, 4H, 3J=9 Hz, C3′, C5′, ArH), 6.10 (d, 1H, 2J=11 Hz, N=C-CHax),
2
3.82 (s, 6H, CH3), 2.21 (d, 1H, J=11 Hz, N=C-CHax). 13C NMR (201.162 MHz, DMSO-d6):
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δC, ppm 163.2 (C4′, ArC), 150.2 (C1′, ArC), 132.7 (C2′, C6′, ArC), 125.6 (C=N), 122.8
(C≡N), 116.2 (C-C≡N), 114.5 (C3′, C5′, ArC), 55.6 (CH3), 38.5 (C6). MS (ES pos) m/z 357
[M+H]+, 379 [M+Na]+, 395 [M+K]+. MS (ES neg) m/z 355 [M-H]-, 391 [M+Cl]-. MS
(MALDI) m/z found: 395.0920 [M+K]+ calcd for 395.0910. Anal. calc. for C21H16N4O2: C,
70.77; H, 4.53; N, 15.72; O, 8.98; Found: C, 70.81; H, 4.52; N, 15.79; O, 8.88.
3.2.2.
Tetrakis[5,7-bis(4-methoxyphenyl)-6H-1,4-diazepino][2,3-b;2’,3’-g;2’’,3’’-
l;2’’’,3’’’-q]porphyrazinato magnesium(II) (4)
Magnesium turnings (16.8 mg, 0.7 mmol), a crystal of iodine, and in n-butanol (9 mL) were
heated under reflux for 4 h. After the mixture was cooled to room temperature, maleonitrile
derivative 3 (0.25 g, 0.7 mmol) was added and the reaction mixture was heated under reflux
for 20 h. After being allowed to cool to room temperature, the reaction mixture was filtered
through Celite which was further washed with toluene and dichloromethane. Filtrates were
evaporated and the crude residue was subjected to column chromatography using silica gel
(dichloromethane, dichloromethane:methanol, 20:1) and reversed-phase silica gel (methanol
to dichloromethane:methanol, 2:1) to give the dark green product 4 (0.025 g, 10% yield). Rf
(dichloromethane:methanol 20:1) 0.54. UV-Vis (dichloromethane): λmax, nm (log ε) 270
(5.09), 348 (5.18), 650 (5.03), 676 (4.98). 1H NMR (799.926 MHz, pyridine-d5): δH, ppm 8.56
(d, 16H, 3J=8.1 Hz, C2′, C6′, ArH), 6.89 (d, 16H, 3J=8 Hz, C3′, C3′, ArH), 6.86 (d, 4H, 2J=13
2
13
Hz, N=C-CHeq), 6.07 (d, 4H, J=13 Hz, N=C-CHax), 3.80 (s, 24H, CH3). C NMR (201.162
MHz, pyridine-d5): δC, ppm 161.5 (C4′, ArC), 154.7, 145.8 (C1′, ArC), 142.6, 132.1 (C2′, C6′,
ArC), 130.8 (N=C), 114.3 (C3′, C3′, ArH), 55.3 (CH3), 37.1 (N=C-CH2). MS (MALDI) m/z
found: [M+H]+ 1449.5000 calcd for 1449.5022. HPLC purity (see supplementary
information).
3.2.3. 5,7-Bis(4-methoxyphenyl)-6H-1,4-diazepino[2,3-b]tribenzo[g,l,q]porphyrazinato
magnesium(II) (5)
Magnesium turnings (0.075 g, 3.087 mmol), a crystal of iodine, and n-butanol (40 mL) were
heated under reflux for 3 h. After the mixture was cooled to room temperature, 3 (0.2 g, 0.561
mmol) and 1,2-dicyanobenzene (0.719 g, 5.612 mmol) were added and reaction mixture was
heated under reflux for a further 20 h. After being allowed to cool to room temperature,
solvent was evaporated and the crude residue was washed with the mixture of water and
methanol (1:1). After drying the solid was dissolved in dichloromethane and filtered through
Celite. The filtrate was evaporated to dry residue, which was subjected to column
chromatography using: (i) silica gel and eluents dichloromethane, dichloromethane/methanol
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50:1, dichloromethane/methanol 20:1; (ii) Al2O3 and eluents dichloromethane,
dichloromethane/methanol 200:1; (iii) C18-reversed phase silica gel and eluents H2O/methanol
3:1,
dichloromethane/methanol
1:3;
(iv)
preparative
TLC
and
eluents
dichloromethane/methanol 10:1 and dichloromethane/pyridine 50:1 to give 5 as a deep green
thin film (0.015 g, 3.5 % yield). Other macrocyclic products were also formed but could not
be separately isolated (see Supplementary Information). Rf (dichloromethane:methanol 20:1)
1
0.47. UV–Vis (dichloromethane): λmax, nm (log ε): 352 (5.44), 657 (5.24), 695 (5.31). H
NMR (799.926 MHz, DMSO-d6): δH, ppm 9.46 (m, 4H, tribenzo-H), 9.38 (m, 2H, tribenzo-
H), 8.55 (d, 4H, J=8.5 Hz, C3′, C5′, ArH), 8.27 (dd, 2H, J=5.6, 2.6 Hz, tribenzoH), 8.24 (m,
4H, tribenzo-H), 7.21 (d, 4H, J=8.7 Hz, C2′, C6′, ArH), 4.59 (m, 2H, CH2), 3.94 (s, 6H, CH3).
13C NMR (201.162 MHz, DMSO-d6): δC, ppm 165.0 (C4′), 158.2, 158.1, 156.3, 154.7, 149.7
(C1′), 143.5, 142.0, 141.8, 141.6, 141.5, 134.4 (C3′, C5′), 133.0, 132.9 (tribenzo-CH), 132.8,
132.6 (tribenzo-CH), 126.0, 125.9 (tribenzo-CH), 125.8 (tribenzo-CH), 117.6 (C2′, C6′), 58.7
(CH3), 40.0 (CH2). MS (MALDI) m/z found [M+H]+ 765.2380 calcd for 765.2325. HPLC
purity (see Supplementary Information).
3.3. Photochemical studies
All experiments were performed at ambient temperature. UV-Vis spectra were
recorded in the range of 200-900 nm using Shimadzu UV-160A and Jasco V-530
spectrophotometers. The UV-Vis spectra of porphyrazines 4-6 were recorded in various
solvents in the concentrations from 5 × 10-6 to 1 × 10-5 mol/dm3.
The quantum yields of singlet oxygen generation were determined in DMSO and DMF
solutions (3.0 mL, no oxygen bubbled) using the relative method with zinc(II) phthalocyanine
(Sigma–Aldrich) and DPBF as a reference chemical quencher for singlet oxygen according to
the previously described procedure.8 Solutions of porphyrazines 4-6 or ZnPc in DMF and
DMSO in the presence of DPBF were irradiated in a 1 cm path-length quartz cell (3 mL) with
monochromatic light by a 150 W high-pressure Xe lamp (Optel) through a monochromator
M250/1200/U. Light of two different wavelengths adjusted to the maxima of two sub-bands
in the Q-band region was used (absorbance of the sensitizers ~ 0.5). Further experiments were
performed after addition to the solutions of 4, 6 with DPBF the antiaggregation agent, TMAF.
As a result, single, unsplit Q-bands were observed. Thus irradiation was performed with one
wavelength adjusted to the Q-band maximum. The concentration of DPBF was set at ~ 3×10-5
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mol L-1 to avoid chain reactions induced by DPBF in the presence of singlet oxygen.51 The
light intensity was set to 0.5 mW/cm2 (Radiometer RD 0.2/2 with TD probe, Optel).
Fluorescence spectra were recorded using a Jasco 6200 spectrofluorometer. The
fluorescence quantum yields were calculated using the equation: ΦF = ΦF
×
reference
(Fsample/Freference) × (Areference/Asample), where F and A correspond to the measured area under
the emission peak and the absorbance at the excitation position (670 nm), respectively.25 ZnPc
was used as a reference (ΦFZnPc = 0.17 in DMF and 0.20 in DMSO).52 The concentrations of
porphyrazines 4 and 6 were about 1 × 10-6 and porphyrazine 5 about 5 × 10-7.
The aggregation study was performed following the procedure proposed by Stuzhin et
al.6 To solutions of 4-6, was added either an antiaggregation agent TMAF or water, which
induces aggregation. Next the absorption and fluorescence spectra were recorded.
3.4. Liposome preparation
Porphyrazines 4-6 were tested in the liposomal formulations. Due to insolubility of 4
and 6 in polar solvents, these compounds were tested only in liposomal formulations while
compound 5 was also tested in its free form. The liposomal formulations were stored in the
dark at 4 °C. The photosensitizer 5 was diluted in DMSO (Sigma Aldrich) at the
concentration 10 mM and stored in the dark at -20 °C. 1-Palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine (POPC), L-α-phosphatidyl-DL-glycerol (chicken egg, PG) and 1,2-dioleoyl-
3-trimethylammonium propane (chloride salt, DOTAP) were purchased from Avanti Polar
Lipids Inc. Two different liposome formulations were prepared by a thin-film hydration
method.38,39 Appropriate amounts of the lipid stock solutions in chloroform (POPC - 25
mg/mL, PG - 25 mg/mL, DOTAP - 10 mg/mL) and photosensitizer (0.4 mg/mL) were placed
in glass tubes, mixed and evaporated to dryness using a rotary evaporator. Films formed on
the bottom of the glass tubes were dried overnight in a vacuum oven at room temperature to
evaporate any remaining chloroform. Subsequently, the dried films were hydrated with PBS.
The molar ratios of ingredients in final liposome formulations were: (i) porphyrazines 4-6
(0.1), PG (2), POPC (8); (ii) porphyrazines 4-6 (0.1), DOTAP (2), POPC (8). The liposome
size was determined by dynamic light scattering measurements using a Malvern Zetasizer
Nano ZS (Table S5 in Supporting Information). Samples were stored at 2-8 °C under argon
and were protected from light. The final concentration of photosensitizer achieved in the
liposome suspensions was 100 ꢀM. Liposomes without photosensitizers were prepared as
controls.
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3.5. Photocytotoxicity in cultured cells
All experiments were carried out with the human prostate carcinoma cell line
(LNCaP). The cell line was purchased from the European Collection of Cells Cultures
(ECACC, Salisbury, UK) and cultured in Dulbecco modified Eagle medium without phenol
red supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% L-
glutamine at 37 °C, in a humidified atmosphere containing 5% CO2. The activity of tested
compounds was examined under normoxic (21%) and hypoxic (1% O2) conditions. For
hypoxia treatment, the cells were first maintained in the standard normoxic incubator for few
hours until the cells grew attached to the plates. Then, the cells were placed in the hypoxia
workstation (Whitley H35 Hypoxystation, Don Whitley Scientific Limited, UK) filled with
1% O2, 5% CO2 and 94% N2, at 70% humidity and 37 °C.
The light source for irradiation cells was the High Power Multi Chip LEDs, generating
a wavelength of 660 nm (for compounds 4 and 6) and 690 nm (for compound 5). The power
of illumination was measured by a radiometer device PM16-130 Power Meter with Slim
Photodiode Sensor (ThorLabs). Due to precipitation of 4 and 6 in DMSO/water mixtures,
these compounds were tested only in liposomal formulations while compound 5 was also
tested in its free form. The photosensitizer 5 was diluted in DMSO (Sigma Aldrich) at the
concentration 10 mM and stored in the dark at -20 °C.
Cytotoxic effect of the tested photosensitizers was determined by MTT assay41 after
light irradiation (phototoxicity) or without irradiation (dark toxicity). After placing the cells in
the hypoxia workstation all procedures such as treatment with tested compounds, irradiation,
and incubation after irradiation were performed in 1% of O2. The cells were seeded at density
2×104 cell per well and allowed to attach overnight. Subsequently, the plates were washed
twice with PBS, and the tested compound in a medium containing 2.5% FBS without phenol
red was added at different concentrations for a 24 h incubation period. The viability tests were
performed for concentrations: 0.15; 0.3; 0.6; 1.25; 2.5; 5 and 10 µM for compounds 4, 5 (non-
liposomal formulation; free-5) and 6. The preliminary in vitro studies showed that the
liposomal formulations of 5 (DOTAP-POPC-5 and PG-POPC-5) exerted strong cytotoxic
effects; therefore, the tested concentrations for these compounds were decreased to 0.015;
0.03; 0.07; 0.15; 0.3; 0.6 and 1.2 µM. For the liposomal formulation of porphyrazines 4-6, the
liposomes without photosensitizers were prepared and used as the negative controls. For free-
5, DMSO was used as a control, and the concentration in medium did not exceed 0.1%. After
22

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incubation, the plates were washed twice with PBS, and fresh medium was added to each
well. The cells were immediately light irradiated at light dose 2 J/cm2 or not irradiated as the
dark control plates. The cell viability was determined after 24 h using the MTT assay. Briefly,
170 µL of culture medium containing methylthiazolydiphenyl-tetrazolium bromide (Sigma
Aldrich) solution (20 µL; 5 mg/ml PBS) was added to each well. Then, the cells were
incubated for 2 h under cell culture condition, and the plates were centrifuged (1200 rpm, 3
min). The MTT solution was removed and the formazan crystals were dissolved by using 200
µl DMSO (Avantor Performance Materials S.A. Gliwice, Poland). The absorbance was
measured at 570 nm with a plate reader (Elx-800, Biotek Instruments Winooski, VT, USA).
The data were normalized to the mean absorption of control cells. All experiments were
performed in duplicate, except the experiments for free-5, which were performed in triplicate.
3.6. Antimicrobial assay
The standard bacterial strains used in this study were Staphylococcus aureus ATCC
25923 and Escherichia coli ATCC 25922 and were obtained from the American Type Culture
Collection (ATCC). The strains of bacteria were stored in Microbank cryogenic vials
(ProLabDiagnostics, Canada) at -70 ± 10 °C. Microbes used for the study were plated on
Tryptone Soya Agar (TSA; Oxoid, UK) and incubated at 36±1ºC for 18 h. Bacterial strains
were cultured in the Brain Heart Infusion broth (BHI, Oxoid, UK) at 36 ± 1 °C for 18 h. Each
culture was harvested by centrifugation (3000 × g for 15 min) and re-suspended in 1.5 mL of
10 mMPBS, pH, 7.0 (Sigma-Aldrich). The cells were then diluted 1/100 in PBS to a final
concentration of about 107 colony forming units (CFU)/mL. Antibacterial photodynamic
activity of the tested compounds was determined using suspension method as described
previously.53 Briefly, bacterial suspensions were incubated in the dark with the appropriate
concentrations of 10 and 50 µM at room temperature for 20 min. After that, the samples were
illuminated with the light intensity at the surface of the plate set to 5.0 mW/cm2 (measured by
Radiometer RD 0.2/2 with TD probe, Optel) and the total light dose was 18 J/cm2 (group [L+,
Ps+]). In parallel, three control experiments were designed as follows: (i) non-illuminated, no
photosensitizer– [L(-), Ps(-)], (ii) non-illuminated, but with photosensitizer [L(-), Ps(+)], (iii)
without photosensitizer, but illuminated [L(+), Ps(-)]. Cell viability of the samples was
determined by serial dilution in PBS and then plated on TSA medium. After incubation of the
plates at 36 ± 1°C for 24 h, the number of CFU per ml was counted, and the log10 reduction
factor in microbial cells for each sample were calculated.
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4. Conclusions
Novel
5,7-diaryl-substituted
symmetrical
diazepinoporphyrazine
and
tribenzodiazepinoporphyrazine were found to generate singlet oxygen in dimethylformamide
and dimethyl sulfoxide in moderate yields up to 0.307 in comparison to structurally similar
G1-dendrimeric diazepinoporphyrazine. Absorbance and fluorescence measurements that
were applied to study aggregation properties of novel macrocycles in the presence of
tetramethylammonium fluoride as an antiaggregation agent proved that the synthesized
porphyrinoids are prone to aggregation.
The cytotoxicity of all porphyrazines to LNCaP cells was examined in both the
presence and the absence of light using MTT assay. Symmetrical magnesium(II)
diazepinoporphyrazine substituted at C5 and C7 positions with 4-methoxyphenyl groups did
not exert any activity against cancer cells. Furthermore, this molecule incorporated into
positively charged liposomes with DOTAP-POPC displayed light-independent toxicity,
whereas this effect was not observed for negatively charged liposomes with PG-POPC.
Similarly, symmetrical magnesium porphyrazine substituted at C5 and C7 positions with 4-
[3,5-bis(benzyloxy)benzyloxy]phenyl substituents was found to be inactive and exhibited
only slight cytotoxic effects at the highest tested dose (10 ꢀM) for DOTAP-POPC formulation
in the dark and after laser exposure. Also, it was observed that DOTAP-POPC liposomes
containing both symmetrical porphyrazines probably precipitate during incubation.
Oxygen depletion in tumor tissue is a significant factor limiting numerous anticancer
strategies including PDT. In our further studies, a free form of tribenzoporphyrazine
substituted at C5 and C7 positions with 4-methoxyphenyl groups and its two liposomal
formulations in DOTAP-POPC and PG-POPC were tested under normoxic and hypoxic (1%
O2) conditions against LNCaP cell line. In the dark condition, tribenzodiazepinoporphyrazine
was not cytotoxic at all tested concentrations, whereas in the light condition it exhibited high
cytotoxicity under irradiation with the dose of 2 J/cm2. The cytotoxic effects of
tribenzodiazepinoporphyrazine increased along with the concentration for all formulations
with the half maximal inhibitory concentration (IC50) under the normoxic conditions at 0.814
± 0.466 µM, in DOTAP-POPC liposomes at 0.161 ± 0.002 µM and in PG-POPC liposomes at
0.166 ± 0.058 µM. It is interesting that all tested liposome formulations of
tribenzoporphyrazine maintained their photocytotoxicity in hypoxia. Moreover,
tribenzodiazepinoporphyrazine incorporated into liposomes revealed better therapeutic effect
24

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(IC50 values of 0.600 ± 0.357 ꢀM and 0.378 ±0.002 ꢀM) than in its free form (IC50 values of
3.135 ± 0.156 ꢀM). According to the in vitro experiments, L-α-phosphatidyl-DL-glycerol
liposomal formulation was found the most promising for further study. It is worth noting that
IC50 values obtained in hypoxic conditions for tribenzoporphyrazine formulations in DOTAP-
POPC and PG-POPC were 2.7 – 3.7 higher than those found in normoxic conditions.
However, it should be emphasized that even in heavy hypoxic conditions the obtained IC50
values are still much below the concentration value of 1 ꢀM. It seems that further in vitro and
in vivo studies are necessary to evaluate fully the usefulness of DOTAP-POPC and PG-POPC
formulations of tribenzoporphyrazine substituted at C5 and C7 positions with 4-
methoxyphenyl groups in photodynamic therapy of cancer.
Tribenzoporphyrazine incorporated into DOTAP-POPC liposomes revealed moderate
phototoxicity for antibacterial photodynamic therapy. It was found that irradiation of
planktonic bacterial strains in the presence of tribenzoporphyrazine in DOTAP-POPC
liposomes at 5×10-5 ꢀM significantly reduced CFUs of Staphylococcus aureus ATCC 25923
in comparison to tribenzoporphyrazine in PG-POPC liposomes, and the control liposomal
formulations without photosensitizer. It is interesting that studied macrocycle did not reveal
any phototoxicity on Escherichia coli ATCC 25922 regardless the liposomal formulation.
Acknowledgement
This study was supported by the National Science Centre, Poland under Grant No.
2012/05/E/NZ7/01204.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version, at
doi:...........
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Porphyrazine derivatives were synthesized and characterized
Singlet oxygen generation and aggregation properties of porphyrazines were evaluated
Photocytotoxicity of porphyrazines was examined in free form and in liposomes
Photocytotoxicity of porphyrazines was studied under normoxic and hypoxic conditions