Diazepinoporphyrazines containing peripheral styryl substituents and their promising nanomolar photodynamic activity against oral cancer cells in liposomal formulations

Article abstract of DOI:10.1002/cmdc.201402085

The photochemical properties and photodynamic activity of three porphyrazines (Pzs) containing annulated diazepine rings, including novel demetalated porphyrazine-possessing bis(styryl)diazepine moieties were investigated. The porphyrazines were evaluated

Full text of DOI:10.1002/cmdc.201402085

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DOI: 10.1002/cmdc.201402085
Diazepinoporphyrazines Containing Peripheral Styryl
Substituents and Their Promising Nanomolar
Photodynamic Activity against Oral Cancer Cells in
Liposomal Formulations
Jaroslaw Piskorz,*[a] Krystyna Konopka,[b] Nejat Dꢀzgꢀnes¸,[b] Zofia Gdaniec,[c]
Jadwiga Mielcarek,[a] and Tomasz Goslinski*[d]
The photochemical properties and photodynamic activity of
three porphyrazines (Pzs) containing annulated diazepine
rings, including novel demetalated porphyrazine-possessing
bis(styryl)diazepine moieties were investigated. The porphyra-
zines were evaluated in terms of their electronic absorption
and emission properties, their tendency to undergo aggrega-
tion and photodegradation, as well as their singlet oxygen
generation efficiency. The in vitro photodynamic activity of the
porphyrazines and their liposomal formulations were examined
by using two oral squamous cell carcinoma cell lines. Magne-
sium(II) tribenzodiazepinoporphyrazine (1) revealed the highest
phototoxic effect in both cell lines used, H413 and HSC-3. En-
capsulation of Pz1 into l-a-phosphatidyl-d,l-glycerol:1-palmi-
toyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes resulted
in a nearly threefold increase in photocytotoxicity relative to
that of the solution of Pz1 (IC50 values of 45 and 129 nm, re-
spectively).
Introduction
Photodynamic therapy (PDT) is a medical treatment that uses
light to activate a drug called a photosensitizer in the presence
of oxygen, and this leads to local damage by the generation of
reactive oxygen species. PDT is used to cure various cancers
and noncancer diseases including age-related macular degen-
eration, oral leukoplakia, and oral lichen planus. Moreover, PDT
can be applied to the treatment of bacterial, fungal, parasitic,
and viral infections.[1–4]
the physicochemical properties of these macrocycles result
from the conjugation of the diazepine ring with the macrocy-
clic core. The UV/Vis spectra of tetrakis-2,3-(5,7-diphenyl-1,4-di-
azepino)porphyrazine and its various metal complexes re-
vealed a broad and split Q-band with maximum absorption
wavelengths in the 630–640 and 660–680 nm ranges. The pres-
ence of an additional band at 660–680 nm is the result of an
n–p* transition between the lone pairs of electrons of the ni-
trogen atoms in the diazepine rings and the p electrons of the
macrocyclic core.[11] In addition, tribenzoporphyrazine contain-
ing an annulated 5,7-diphenyl-6H-1,4-diazepine ring and its
magnesium(II) complex exhibited significantly red-shifted Q-
bands up to 715 and 700 nm, respectively.[15] The red-shifted
Q-band absorptions observed for diazepinoporphyrazines sug-
gest that these macrocycles might be considered as potential
photosensitizers for photodynamic therapy, as light of longer
wavelength is able to penetrate deeper into the irradiated
tissue.[16] We recently reported the synthesis of novel porphyra-
zines bearing styryldiazepine and bis(styryl)diazepine substitu-
ents with UV/Vis absorption maxima shifted up to 729 and
704 nm, respectively.[17,18]
Many porphyrinoid macrocycles, including porphyrins,
phthalocyanines, chlorins, and porphyrazines (Pzs), have been
investigated as photosensitizers for PDT.[5–10] Pzs possessing pe-
ripherally annulated diazepine rings have been elaborated on
by the Ercolani and Stuzhin groups.[11–14] They stipulated that
[a] J. Piskorz, Prof. J. Mielcarek
Department of Inorganic & Analytical Chemistry
Poznan University of Medical Sciences
Grunwaldzka 6, 60-780 Poznan (Poland)
E-mail: piskorzj@ump.edu.pl
[b] Prof. K. Konopka, Prof. N. Dꢀzgꢀnes¸
Department of Biomedical Sciences, University of the Pacific
Arthur A. Dugoni School of Dentistry
2155 Webster Street, San Francisco, CA 94115 (USA)
Although the synthesis of new PDT candidates is of increas-
ing interest, special efforts are made to design various forms of
nanocarriers for photosensitizers, of which liposomes are con-
sidered one of the best and most promising. The application
of liposomes as porphyrinoid delivery systems can overcome
many of the drawbacks of conventional photosensitizers, such
as high lipophilicity, lack of solubility in aqueous media, ten-
dency to undergo aggregation, poor tissue penetration, and
[c] Prof. Z. Gdaniec
Institute of Bioorganic Chemistry
Polish Academy of Sciences, Z. Noskowskiego 12, Poznan (Poland)
[d] Dr. T. Goslinski
Department of Chemical Technology of Drugs
Poznan University of Medical Sciences
Grunwaldzka 6, 60-780 Poznan (Poland)
E-mail: tomasz.goslinski@ump.edu.pl
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402085.
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low bioavailability. Moreover, liposomes can facilitate the de-
velopment of novel effective photosensitizers.[19–22]
In this paper, we report the photochemical properties and
photodynamic activity of three Pzs containing annulated diaze-
pine rings, including a novel demetalated one possessing a bis-
(styryl)diazepine moiety. We evaluated their electronic absorp-
tion and emission properties, their tendency to undergo aggre-
gation and photodegradation, as well as their efficiency in gen-
erating singlet oxygen. The in vitro photodynamic activity of
Pzs and their liposomal formulations was examined by using
two human oral squamous cell carcinoma cell lines.
Figure 1. UV/Vis spectra for Pzs 1–3.
Results and Discussion
band maximum and the concentrations of the macrocyclic
compounds were evaluated. Although these correlations were
linear for all compounds, statistical analysis revealed that the
Beer–Lambert law was obeyed only for Pz1 (Figure S2 and
Table S3). These data indicate that Pz2 and Pz3, unlike Pz1,
aggregate in both DMF and DMSO. Aggregate formation is
a common problem in photodynamic therapy, because self-as-
sociation of the photosensitizer molecules decreases the gen-
eration of singlet oxygen, which thus hinders the photosensi-
tizing efficiency.[25]
Synthesis and characterization
Magnesium(II) diazepinotribenzoporphyrazine (1) and magne-
sium(II) diazepinoporphyrazine (2) were synthesized according
to previously described procedures.[17,18] Demetalated diazepi-
noporphyrazine (3) was synthesized from the magnesium com-
plex by treatment with trifluoroacetic acid by using published
procedures,[23,24] and it was characterized by using UV/Vis spec-
troscopy, MALDI MS, and various NMR spectroscopy tech-
1
1
1
niques, including H–1H COSY, H–13C HSQC, and H–13C HMBC
(Scheme 1; see also Figure S1 and Table S1, Supporting Infor-
mation).
The fluorescence emission spectra of Pz1 in DMF or DMSO
showed maxima at 707 and 708 nm, respectively. The fluores-
cence quantum yield (FF) values of 2.6ꢂ10ꢀ2 in DMF and 4.5ꢂ
10ꢀ2 in DMSO were calculated by following the method given
by Chauke et al.[26] Less intense fluorescence with a maximum
at 716 nm and with FF =4.1ꢂ10ꢀ3 was found for Pz2 in DMF
and with FF =1.8ꢂ10ꢀ3 in DMSO. The weakest fluorescence
emission bands were observed for Pz3 at 671 (FF =3.1ꢂ10ꢀ5
)
and 675 nm (FF =5.5ꢂ10ꢀ5) in DMF and DMSO, respectively
(Figure 2).
Photodegradation
Scheme 1. Structures of Pzs 1–3.
The susceptibility of Pzs 1–3 to degradation upon exposure to
light was determined by observing the changes in the UV/Vis
spectra during irradiation in both DMF and DMSO. The absorp-
tion bands decreased upon irradiation, and there was no for-
mation of new bands. Thus, it seems that light absorption
leads to degradation of the macrocycles into smaller fragments
that are not able to absorb light in the visible region.[27] Photo-
degradation was much faster in DMF than in DMSO, and it fol-
lowed first-order kinetics for all the macrocycles studied
(Table 1; detailed kinetic parameters are shown in Table S4);
the first-order plots are presented in Figure 3.
Electronic absorption and emission properties
The absorption spectra of Pz1 recorded in DMF and DMSO re-
vealed a Soret band with maximum absorption at 358 and
364 nm, respectively. In addition, a broad Q-band, split into
two sub-bands at 658/689 and 658/692 nm, respectively, was
observed. Pz2 exhibited UV/Vis spectra that were similar to
those of Pz1, but the absorption maxima were bathochromi-
cally shifted to 378, 661, and 689 nm in DMF and to 381, 661,
and 695 nm in DMSO. Demetalated analogue Pz3 revealed less
intense absorption bands with a Soret band at 368 nm and
a broad, flat Q-band at 662 and 664 nm in DMF and DMSO, re-
spectively (Figure 1 and Table S2).
The photodegradation measured in DMF for metalated Pz1
and Pz2 took place in two stages and for demetalated Pz 3 in
one stage. This observation is consistent with that obtained for
a series of porphyrazines with 2,5-dimethylpyrrol-1-yl and di-
methylamino substituents.[28] For all three compounds, the
photochemical decomposition in DMSO took place in two
stages. Moreover, it was found that ~30% of Pz1 and Pz2 and
The UV/Vis spectra of Pzs 1–3 dissolved at different concen-
trations in DMF or DMSO were used for aggregation behavior
studies. The correlations between the absorbance of the Q-
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Figure 2. The Q-band absorption and emission spectra of Pzs 1–3 in a) DMF
and b) DMSO.
Figure 3. Photodegradation first-order plots of Pzs 1–3 in a) DMF and
b) DMSO.
Table 1. Photodegradation kinetic parameters of Pzs 1–3.
Compd
DMF
DMSO
glet oxygen generation yields (FD). The results obtained are
given in Table 2 (detailed kinetic parameters are shown in
Table S5). The first-order plots are presented in Figure 4.
Stage t0.5 [s] Pz degraded [%] Stage t0.5 [s] Pz degraded [%]
Pz1
Pz2
I
II
I
II
I
73.4 27.3ꢁ0.3
4700
87.5 28.3ꢁ0.4
3900
8140
I
II
I
II
I
II
I
II
19.3 4.5ꢁ0.3
30400
62.8 4.3ꢁ0.2
36500
Pz3
9.4ꢁ0.4
69.4 72.4ꢁ3.2
195
37400
3.5ꢁ0.1
Table 2. Quantum yields for the generation of singlet oxygen by using
Pzs 1–3.
ZnPc
I
II
122 10.1ꢁ0.5
10500
[a]
914
Compd
FD ꢁDFD
DMF
DMSO
Pz 1
Pz 2
Pz 3
ZnPc
0.281ꢁ0.006
0.109ꢁ0.006
0.0039ꢁ0.0003
0.56[31]
0.311ꢁ0.003
0.013ꢁ0.001
0.0054ꢁ0.0005
0.67[31]
~10% of Pz3 was degraded in DMF after 20 min of irradiation.
Similarly, in DMSO, demetalated analogue Pz3 was the most
stable (3.5% degradation upon exposure to light for 20 min).
However, all the porphyrazines studied were found to be
much more stable during irradiation than zinc(II) phthalocya-
nine (ZnPc).
[a] Results are given with 95% confidence.
Pz1 was found to be the most efficient singlet oxygen gen-
erator, with high FD values of 0.28 and 0.31 in DMF and
DMSO, respectively, although these values are lower than
those of ZnPc (FDDMF =0.56; FDDMSO =0.67).[31] Pz2 showed
a moderate ability to form singlet oxygen in DMF, but a weak
ability in DMSO, whereas the lowest FD values in both solvents
were obtained for demetalated Pz3 (Table 2). The decreased
generation of singlet oxygen by Pz2 in DMSO seems to result
from its aggregation.[18] The considerably lower FD values for
demetalated Pz3 relative to those of its metal complex Pz2
match with the results obtained for other Pzs and their metal
complexes.[28,32,33]
Singlet oxygen generation
The potential photodynamic activities of Pzs1–3 were evaluat-
ed by measuring their ability to generate singlet oxygen. The
relative method, with ZnPc as a reference and 1,3-diphenyliso-
benzofuran (DPBF) as a chemical quencher for singlet oxygen,
was applied.[29,30] Solutions containing Pzs 1–3 or ZnPc in a mix-
ture with DPBF in DMF or DMSO were irradiated with mono-
chromatic light at wavelengths corresponding to their Q-band
maxima. The kinetics of DPBF decomposition by photogenerat-
ed singlet oxygen was studied by monitoring the absorbance
decrease at 417 nm, and they were used to calculate the sin-
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previously for other photosensitizers, for which a higher sensi-
tivity of H413 to HSC-3 cells was observed.[34]
The light-induced toxicities were examined only at the con-
centrations that had not caused any dark toxicity. HSC-3 and
H413 cells were incubated with Pz1 at 0.05, 0.25, 1 mm and
with Pz2 and Pz3 at 0.1, 1, and 10 mm for 24 h. Next, they
were irradiated for 20 min with light of 690 nm from a high-
power light-emitting diode multichip emitter (Roithner Laser-
technik, 9.8 V). The light intensity at the surface of the plate
was set to 3.0 mWcmꢀ2 and the total light dose was 3.6 Jcmꢀ2
.
Cell viability was quantified by the Alamar Blue assay. It was
found that Pz1 at 1 mm decreased the viability of H413 cells by
90% and that Pz2 at 10 mm decreased the viability by ~25%,
whereas Pz3 did not show any significant light-induced cyto-
toxicity (Table S7). In addition, Pz1 also exhibited the highest
phototoxic effect in HSC-3 cells, with a decrease in cell viability
by ~95% at 1 mm. Pz2 did not show any phototoxic effect,
whereas Pz3 decreased the viability of HSC-3 cells by 87% at
10 mm (Figure 5 and Table S8).
The insolubility of Pzs 1–3 in water and their strong tenden-
cy to form aggregates encouraged us to incorporate them into
liposomes, which are known to be promising delivery systems
for photosensitizers that can overcome these limitations.[21]
Pz1 and Pz3 showed a high phototoxic effect against HSC-3
cells and were encapsulated into liposomes.
Figure 4. First-order plots of DPBF degradation (in air) by singlet oxygen
photogenerated during irradiation of Pzs 1–3 in a) DMF and b) DMSO.
Their photodynamic efficacy was researched in four different
liposome formulations that were prepared by a thin-film hydra-
tion method: 1) l-a-phosphatidyl-d,l-glycerol (PG, from
chicken eggs:1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC), 2) PG:POPC:cholesterol (Chol), 3) N-[1-(2,3-dioleoyloxy)-
propyl]-N,N,N-trimethylammonium chloride (DOTAP):POPC, and
4) DOTAP:POPC:Chol.[34,35] The mean diameter of the extruded
liposomes containing Pz1 and Pz3 varied from 123.4 to
167.5 nm and from 199.8 to 330.3 nm, respectively (Table S9).
Light-induced cytotoxicity against HSC-3 cells of liposomes
with incorporated Pz1 were examined at concentrations of
0.05, 0.25, and 1 mm and with incorporated Pz3 at concentra-
tions of 0.1, 1.0, 10 mm. All four types of liposomes containing
Pz1 revealed a high photocytotoxic effect. However, liposomes
with incorporated Pz3 were not cytotoxic (Table S10). Further
experiments were performed at six concentrations with Pz1
In vitro photodynamic activity
The photodynamic activity of Pzs 1–3 was investigated in vitro
by using two oral squamous cell carcinoma cell lines, HSC-3
derived from the tongue and H413 derived from the buccal
mucosa. Dark toxicities of Pzs 1–3 were evaluated in the limit-
ed concentration range from 0.1 to 10 mm, because of the mas-
sive aggregation of Pz2 and Pz3 at higher concentrations. The
viability of both cell lines was unaffected by Pz2 and Pz3 at all
the concentrations used, whereas Pz1 was toxic only to H413
cells at concentrations higher than 1 mm (Table S6). Notewor-
thy, the dark toxicity results correspond well to those obtained
Figure 5. Light toxicity (and dark control) of Pzs 1–3 against HSC-3 cells. Data represent the meanꢁstandard deviation for experiments performed in tripli-
cate.
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(IC50 =129 nm) and its four liposomal formulations (IC50 =45–
150 nm) and with Pz3 (IC50 =4590 nm). Negatively charged lip-
osomes containing Pz1 (Pz1:PG:POPC and Pz1:PG:POPC:Chol)
showed higher cytotoxicity than the free compound and posi-
tively charged Pz1 liposomes (Pz1:DOTAP:POPC, Figure 6).
Noteworthy, the IC50 value for the most active Pz1:PG:POPC
al area, whereas Pz1 is partially localized in this area (Fig-
ure S3). It was reported that porphyrinoid photosensitizers lo-
calizing in mitochondria are the most effective.[37,38]
Conclusions
Three Pzs containing annulated diazepine rings, including
novel demetalated porphyrazine possessing the bis-
(styryl)diazepine moiety, were investigated in terms of their
photochemical properties and their photodynamic activity.
Magnesium(II) tribenzoporphyrazine (1) showed the highest
fluorescence quantum yield and the highest singlet oxygen
generation quantum yield but the lowest tendency to undergo
aggregation. In addition, Pz1 revealed the highest phototoxic
effect in both oral squamous cell carcinoma lines used, H413
and HSC-3. Moreover, encapsulation of Pz1 into l-a-phospha-
tidyl-d,l-glycerol:1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-
choline liposomes resulted in photocytotoxicity that was
almost three times higher than that of the free form of Pz1 in
solution, with IC50 values of 45 and 129 nm, respectively. These
results indicate that Pz1 encapsulated in liposomes might be
considered as a promising antitumor agent for further preclini-
cal photodynamic therapy studies.
Figure 6. Phototoxic effect of Pz1 and its liposomal formulations on HSC-3
cells. Data represent the meanꢁstandard deviation for experiments per-
formed in triplicate.
liposomes was almost three times lower than that determined
for free-form Pz1 in solution, which indicates that these lipo-
somes are the most potent delivery systems for photosensitiz-
er Pz1 (Table 3).
Experimental Section
Chemistry
General: All reactions were conducted in oven-dried glassware
under an atmosphere of argon. All solvents and reagents were ob-
tained from commercial suppliers and used without further purifi-
cation. Flash column chromatography was performed on Merck
silica gel 60, particle size 40–63 mm, and Fluka silica gel 90 C18 re-
verse phase. Thin-layer chromatography (TLC) was performed on
Table 3. Cytotoxicity of liposome-encapsulated photosensitizers against
HSC-3 cells as a means to determine the photodynamic efficacy of the
liposomal formulations.
silica gel Merck Kieselgel 60 F254 and DC Kieselgel 60 RP-18 F254
plates and visualized with UV light (lmax =254 or 365 nm). NMR
s
Liposomal formulation[a]
IC50 [nm]
spectra were recorded with a Bruker Avance II spectrometer oper-
Pz1
129
45
52
126
150
4590
1
ating at a frequency of 400 MHz for H and 100 MHz for 13C. Chem-
Pz1:PG:POPC
Pz1:PG:POPC:Chol
Pz1:DOTAP:POPC
Pz1:DOTAP:POPC:Chol
Pz3
ical shifts (d) are quoted in parts per million (ppm) and are refer-
enced to a residual solvent peak. Coupling constants (J) are
quoted in Hertz (Hz). The abbreviations, s, d, and h, refer to singlet,
doublet, and hidden signal, respectively. Additional techniques
1
1
(1H–1H COSY, H–13C HSQC, H–13C HMBC) were used to assist with
allocation assignment. Mass spectra (MALDI TOF) were performed
by the Advanced Chemical Equipment and Instrumentation Facility
at the Faculty of Chemistry, Adam Mickiewicz University, Poznan.
The purity of all compounds was determined by HPLC analysis per-
formed with an Agilent 1200 Series and was found to be ꢂ95%.
[a] PG=l-a-phosphatidyl-d,l-glycerol,
glycero-3-phosphocholine, Chol=cholesterol, DOTAP=N-[1-(2,3-dioleoy-
loxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP).
POPC=1-palmitoyl-2-oleoyl-sn-
The subcellular localization of Pz1 and Pz3 was investigated
by fluorescence microscopy by incubating HSC-3 cells with the
photosensitizers and the organelle-specific fluorescent dyes Mi-
toTracker Green FM (mitochondria), Alexa Fluor 350 WGA
(plasma membrane), Syto 13 (nucleic acids), and Hoechst
33342 (nucleus).[8,36] It was found that the fluorescence from
the mitochondria overlaps with the fluorescence from both
the macrocycles, which confirms that they accumulate inside
the cells. In addition, images showing fluorescence from both
compounds could not be merged with images of other dyes,
which suggests that Pz3 is mainly localized in the mitochondri-
Tetrakis[5,7-bis{(E)-2-(3,4,5-trimethoxyphenyl)ethenyl}-6H-1,4-di-
azepino][2,3-b;2’,3’-g;2’’,3’’-l;2’’’,3’’’-q]porphyrazine (3): Magnesiu-
m(II) porphyrazine (2; 232 mg, 0.1 mmol) was dissolved in trifluoro-
acetic acid (15 mL) in the dark, and the mixture was stirred for
30 min at room temperature. The solution was then poured into
iced water (300 mL) and neutralized with saturated NaHCO3. The
organic phase was extracted with CH2Cl2. The solvent was removed
under reduced pressure, and the crude residue was purified by
chromatography (normal phase, dichloromethane/methanol=50:1
to 20:1 v/v; reverse phase, methanol/tetrahydrofuran=25:1 v/v) to
give
3 (33 mg, 14%). Rf =0.35 (dichloromethane/methanol=
20:1 v/v). 1H NMR (400 MHz, [D5]pyridine): d=8.52 (d, 3J=16 Hz,
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8H; 8 C5(7)-CH=CH), 8.17 (d, 3J=16 Hz, 8H; 8 C5(7)-CH=CH),
7.04 (s, 16H; 16 ArH), 6.75 (d, J=12 Hz, 4H; 4 N=C-CH2), 5.86 (d,
ture F12 (DMEM/10/F12). Cells were incubated in tissue culture
flasks at 378C in a humidified atmosphere containing 5% CO2 and
were then passaged 1:6 twice a week by using a trypsin-ethylene-
diaminetetraacetic acid (EDTA) solution. All media, penicillin–strep-
tomycin solution, l-glutamine, FBS, trypsin-EDTA, phosphate-buf-
fered saline (PBS), and Dulbecco’s phosphate buffered saline
(DPBS), were obtained from the UCSF Cell Culture Facility, San
Francisco, USA. Photosensitizers were dissolved in dimethyl sulfox-
ide (Sigma–Aldrich) and subsequently diluted in DMEM or DMEM/
F12 (without FBS and phenol red) to obtain the desirable concen-
tration of the photosensitizer used in the experiments. The DMSO
concentration in the final solution did not exceed 0.5%.
2
2J=12 Hz, 4H; 4 N=C-CH2), 3.99 (s, 24H; 8 OCH3), 3.85 (s, 48H; 16
OCH3), ꢀ3.63 ppm (s, 2H; 2 NH). 13C NMR (100 MHz, [D5]pyridine):
d=154.2, 154.0, 150.2, 142.9, 140.7, 140.4, 132.0, 128.6, 105.8, 60.5,
56.0, 38.8 ppm (unresolved, observed in 1H–13C HSQC and 1H–13C
HMBC, see the Supporting Information). UV/Vis (DMF): lmax
(log e)=368 (4.14), 662 nm (4.58). UV/Vis (DMSO): lmax (log e)=368
(4.14), 664 nm (4.56). MS (MALDI): m/z (%): 2117 [M+H]+ (calcd for
[M+H]+ 2116.83).
Photochemical studies
Dark toxicity: One day before the experiment, HSC-3 and H413
cells were seeded in 48-well plates at a density of 1.8ꢂ105 and
1.4ꢂ105 cells per well, respectively, in medium (1 mL, with FBS and
phenol red) and used at ~80% confluence. Subsequently, cells
were prewashed twice with PBS (0.5 mL), and the medium (1 mL,
without FBS and phenol red) containing the photosensitizer at
a given concentration was added to each well except for those
containing the controls. FBS-free media were used to avoid bind-
ing of photosensitizers to the serum proteins. After the 24 h incu-
bation at 378C, cells were washed twice with PBS, complete
medium (1 mL) was added to each well, and the cells were incu-
bated for 24 h at 378C. Cell viability was quantified by the Alamar
Blue assay (see below). Cells incubated either with medium alone
or medium/0.5% DMSO served as controls.
The UV/Vis spectra were recorded in the range from 200 to
900 nm by using a Shimadzu UV-160A spectrophotometer with PC
160 PLUS manual. Fluorescence spectra were recorded by using
a Jasco 6200 spectrofluorimeter. The fluorescence quantum yields
were calculated by using Equation (1):
ꢀ
ꢁꢀ
ꢁ
Fsample
Freference
Areference
Asample
FF ¼ FFreference
ð1Þ
in which F and A correspond to the measured area under the emis-
sion band and the absorbance at the excitation position (360 nm),
respectively.[26] ZnPc was used as a reference (FF ZnPc =0.17 in DMF;
FF ZnPc =0.20 in DMSO).[39,40] A 150 W high-pressure xenon lamp
(Optel) was used as a light source for photodegradation studies.
Solutions of Pzs 1–3 and ZnPc in DMF or DMSO with absorbance
set at ~0.7 were irradiated in a 1 cm path-length cylindrical cell
(2.8 mL) with the light over 450 nm owing to the use of a yellow
glass cut-off filter (HCC16). The light intensity was set to 130 klux
(luxmeter TES-1335).[28,41]
Light-dependent toxicity: One day before the experiment, HSC-3
and H413 cells were seeded in 48-well plates at a density of 1.8ꢂ
105 and 1.4ꢂ105 cells per well, respectively, in complete medium
(1 mL) and used at ~80% confluence. Cells were prewashed twice
with PBS, and the medium (1 mL, without FBS and phenol red)
containing the photosensitizer was added to each well except for
those containing the controls. The cells were incubated for 24 h at
378C, washed twice with PBS, and the medium (without FBS and
phenol red) was added. Subsequently, the cells were irradiated for
20 min with light of wavelength 690 nm from a high-power light-
emitting diode multichip emitter (Roithner Lasertechnik, 9.8V). The
light intensity at the surface of the plate was set to 3.0 mWcmꢀ2
measured by a Thorlabs TM100A optical power meter, and the
total light dose was 3.6 Jcmꢀ2. One plate from each experiment
was not exposed to light, and it served as a control. Directly after
light exposure, medium (without FBS and phenol red) was re-
placed with complete medium (1 mL), and the cells were incubat-
ed for 24 h at 378C. Cell viability was quantified by the Alamar
Blue assay (see below).
The quantum yields of the singlet oxygen generation were deter-
mined in DMSO and DMF solutions (3.0 mL, no oxygen bubbled)
by using the relative method with zinc(II) phthalocyanine (ZnPc,
Sigma–Aldrich) as
a reference and 1,3-diphenylisobenzofuran
(DPBF) as a chemical quencher for singlet oxygen, following re-
cently presented methodologies.[29,30] Solutions of Pzs 1–3 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
using a 150 W high-pressure Xe lamp (Optel) through a monochro-
mator M250/1200/U. The irradiation wavelengths were adjusted to
the maximum of the absorption bands at the Q-bands characteris-
tic of each compound (absorbance of the sensitizersꢃ0.5). The
concentration of DPBF was set to ~3ꢂ10ꢀ5 molLꢀ1 to avoid chain
reactions induced by DPBF in the presence of singlet oxygen.[42]
The light intensity was set to 0.5 mWcmꢀ2 (Radiometer RD 0.2/2
with TD probe, Optel). All experiments were performed in air (with-
out bubbling oxygen or air though the solvent) at ambient tem-
perature. The samples were kept in the dark before irradiation.
Cell viability: Cell morphology was evaluated by Nikon TMS invert-
ed-phase contrast microscopy at 100ꢂ magnification. The number
of viable cells used for the experiments was determined by the
Trypan Blue exclusion assay (Gibco-Invitrogen Corporation). Cell vi-
ability was quantified by a modified Alamar Blue assay.[45,46] Briefly,
10% (v/v) Alamar Blue dye (1.0 mL) in the appropriate complete
medium was added to each well. After incubation at 378C for 2–
3 h, the supernatant (200 mL) was assayed by measuring the ab-
sorbance at 570 and 600 nm. Cell viability (as a percentage of con-
trol cells) was calculated according to Equation (2):
Biological methods
Cell culture: HSC-3 cells, derived from squamous cell carcinoma
(SCC) of the tongue,[43] were provided by Dr. R. Kramer (University
of California, San Francisco, UCSF, USA). H413 cells, derived from
SCC of the buccal mucosa,[44] were obtained from Dr. R. Jordan
(UCSF). HSC-3 cells were cultured in Dulbecco’s modified Eagle
medium (DMEM), supplemented with 10% (v/v) heat-inactivated
fetal bovine serum (FBS), penicillin (100 UmLꢀ1), streptomycin
(100 mgmLꢀ1), and l-glutamine (4 mm) (DMEM/10). H413 cells were
maintained in DMEM/10 supplemented with Ham’s Nutrient Mix-
½ðA570ꢀA600Þ of test cellsꢄ
ð2Þ
Cell viability ¼
ꢅ 100 %
½ðA570ꢀA600Þ of control cellsꢄ
Liposome preparation: 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-
choline (POPC), l-a-phosphatidyl-d,l-glycerol (chicken egg, PG), N-
[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
chloride
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(DOTAP), and cholesterol (Chol) were purchased from Avanti Polar
Lipids, Inc. (Alabaster, AL, USA). Four different liposome formula-
tions were prepared by a thin-film hydration method.[34,35] Appro-
priate amounts of the lipid solutions in chloroform (POPC,
nanced by the EU under the European Social Fund and the Polish
National Science Centre “Etiuda” for PhD students under fellow-
ship 2013/08/T/NZ7/00241. This study was supported by the Na-
tional Science Centre under grants N N401 067238 and 2012/05/
N/NZ7/00624, the European Fund for Regional Development No
UDA-POIG.02.01.00-30-182/09, and funds from the University of
the Pacific, Arthur A. Dugoni School of Dentistry.
20 mgmLꢀ1
;
PG, 25 mgmLꢀ1
;
DOTAP, 10 mgmLꢀ1
;
Chol,
10 mgmLꢀ1) and photosensitizer (0.4 mgmLꢀ1) were placed in
glass tubes, mixed, and evaporated to dryness by using a rotary
evaporator. Films formed on the bottom of glass tubes were dried
overnight in a vacuum oven at room temperature to evaporate
any remaining chloroform. Subsequently, the dried films were hy-
drated with HEPES buffered saline {10 mm HEPES [N-(2-hydroxye-
thyl)piperazine-N’-(2-ethanesulfonic acid)], 140 mm NaCl, pH 7.5}
and dispersed by vortexing for 5–10 min. The resulting liposome
suspensions were passed 21 times through polycarbonate mem-
branes with a pore diameter of 100 nm by using a syringe extruder
(Avanti Polar Lipids) to obtain unilamellar liposomes with a uniform
size distribution. The molar ratios of ingredients in final liposome
formulations were: 1) Pzs 1–3 (0.1), PG (2), POPC (8); 2) Pz1 or Pz3
(0.1), PG (1.33), POPC (5.34) Chol (3.33); 3) Pz1 or Pz3 (0.1), DOTAP
(2), POPC (8); and 4) Pz1 or Pz3 (0.1), DOTAP (1.33), POPC (5.34)
Chol (3.33). The liposome size was determined by dynamic light
scattering measurements by using a Coulter N4 Plus particle size
analyzer (Beckman). Samples were stored at 2–88C under an at-
mosphere of argon and were protected from light. The final con-
centration of the photosensitizer achieved in the liposome suspen-
sions was 100 mm. These suspensions were diluted with DME
medium without FBS to achieve appropriate concentration for bio-
logical activity evaluation on HSC-3 cells. Free liposomes without
photosensitizers were prepared as controls.
Keywords: antitumor agents
· diazepines · liposomes ·
photodynamic therapy · porphyrazines
[1] S. Yano, S. Hirohara, M. Obata, Y. Hagiya, S.-I. Ogura, A. Ikeda, H. Katao-
ka, M. Tanaka, T. Joh, J. Photochem. Photobiol. C 2011, 12, 46–67.
[2] R. R. Allison, C. H. Sibata, Photodiagn. Photodyn. Ther. 2010, 7, 61–75.
[3] K. Konopka, T. Goslinski, J. Dent. Res. 2007, 86, 694–707.
[4] T. J. Dougherty, C. J. Gomer, B. W. Henderson, G. Jori, D. Kessel, M. Kor-
belik, J. Moan, Q. Peng, J. Natl. Cancer Inst. 1998, 90, 889–905.
[5] M. Ethirajan, Y. Chen, P. Joshi, R. K. Pandey, Chem. Soc. Rev. 2011, 40,
340–362.
[6] T. Goslinski, J. Piskorz, J. Photochem. Photobiol. C 2011, 12, 304–321.
[7] N. D. Hammer, S. Lee, B. J. Vesper, K. M. Elseth, B. M. Hoffman, A. G. M.
Barrett, J. A. Radosevich, J. Med. Chem. 2005, 48, 8125–8133.
[8] A. L. Gryshuk, Y. Chen, W. Potter, T. Ohulchansky, A. Oseroff, R. K.
Pandey, J. Med. Chem. 2006, 49, 1874–1881.
[9] T. Goslinski, T. Osmalek, K. Konopka, M. Wierzchowski, P. Fita, J. Mielcar-
ek, Polyhedron 2011, 30, 1538–1546.
[10] E. R. Trivedi, A. S. Harney, M. B. Olive, I. Podgorski, K. Moin, B. F. Sloane,
A. G. M. Barrett, T. J. Meade, B. M. Hoffman, Proc. Natl. Acad. Sci. USA
2010, 107, 1284–1288.
[11] M. P. Donzello, C. Ercolani, P. A. Stuzhin, A. Chiesi-Villa, C. Rizzoli, Eur. J.
Inorg. Chem. 1999, 2075–2084.
Subcellular localization studies: HSC-3 cells were seeded in 48-well
plates at a density of 1.8ꢂ105 and incubated for 24 h at 378C. Sub-
sequently, cells were washed and medium (1 mL, without FBS and
phenol red) containing the photosensitizer and Pz1 at 1 mm or Pz3
at 10 mm was added. After the 24 h incubation at 378C, cells were
washed twice with PBS, and the solutions of the dyes in PBS were
added and incubated for MitoTracker Green FM (Molecular Probes,
[12] S. Angeloni, C. Ercolani, J. Porphyrins Phthalocyanines 2000, 4, 474–483.
[13] M. P. Donzello, D. Dini, G. D’Arcangelo, C. Ercolani, R. Zhan, Z. Ou, P. A.
Stuzhin, K. M. Kadish, J. Am. Chem. Soc. 2003, 125, 14190–14204.
[14] P. A. Stuzhin, P. Tarakanov, S. Shiryaeva, A. Zimenkova, O. I. Koifman, E.
Viola, M. P. Donzello, C. Ercolani, J. Porphyrins Phthalocyanines 2012, 16,
968–976.
[15] M. P. Donzello, C. Ercolani, L. Mannina, E. Viola, A. Bubnova, O. Khelevi-
na, P. A. Stuzhin, Aust. J. Chem. 2008, 61, 262–272.
0.4 mm) 15 min, Alexa Fluor 350 WGA (Molecular Probes, 5 mgmLꢀ1
)
15 min, Syto 13 (Molecular Probes 5 mm) 30 min, and Hoechst
33342 (Molecular Probes, 6 mm) 30 min. Next, the cells were
washed with PBS. Fluorescence micrographs were obtained with
a Nikon Diaphot inverted fluorescence microscope equipped with
filters, (Chroma Technology Corporation, Bellows Falls, USA) 49006
filter for Pzs 1 and 3, 61000v2 filter for MitoTracker and Syto 13,
49000 filter for Alexa Fluor 350 and Hoechst 33342, and a Jenaop-
tik digital camera by using the ProgRes Capture Software. The cells
were observed by using a 20ꢂ phase contrast objective.
[16] K. Plaetzer, B. Krammer, J. Berlanda, F. Berr, T. Kiesslich, Lasers Med. Sci.
2009, 24, 259–268.
[17] T. Goslinski, J. Piskorz, D. Brudnicki, A. J. P. White, M. Gdaniec, W. Szczol-
ko, E. Tykarska, Polyhedron 2011, 30, 1004–1011.
[18] J. Piskorz, E. Tykarska, M. Gdaniec, T. Goslinski, J. Mielcarek, Inorg. Chem.
Commun. 2012, 20, 13–17.
[19] E. Paszko, C. Ehrhardt, M. O. Senge, D. P. Kelleher, J. V. Reynolds, Photo-
diagn. Photodyn. Ther. 2011, 8, 14–29.
[20] Y. N. Konan, R. Gurny, E. Allꢃmann, J. Photochem. Photobiol. B 2002, 66,
89–106.
[21] P. Skupin-Mrugalska, J. Piskorz, T. Goslinski, J. Mielcarek, K. Konopka, N.
Dꢀzgꢀnes¸, Drug Discovery Today 2013, 18, 776–784.
Statistical analysis
[22] C. S. Jin, G. Zheng, Lasers Surg. Med. 2011, 43, 734–748.
[23] S. L. J. Michel, B. M. Hoffman, S. M. Baum, A. G. M. Barrett, “Peripherally
Functionalized Porphyrazines: Novel Metallomacrocycles with Broad,
Untapped Potential” in Progress in Inorganic Chemistry, Vol. 50 (Ed.: K. D.
Karlin), Wiley, New York, 2001, pp. 473–590.
[24] W. Szczolko, L. Sobotta, P. Fita, T. Koczorowski, M. Mikus, M. Gdaniec, A.
Orzechowska, K. Burda, S. Sobiak, M. Wierzchowski, J. Mielcarek, E. Ty-
karska, T. Goslinski, Tetrahedron Lett. 2012, 53, 2040–2044.
[25] A. P. Castano, T. N. Demidova, M. R. Hamblin, Photodiagn. Photodyn.
Ther. 2004, 1, 279–293.
Data were compared for statistical significance by the unpaired
Student’s t-test by using StatView software (Brain Power Inc., Cala-
basas, CA). A probability value (P) of less than 0.05 was considered
significantly different.
Acknowledgements
[26] V. Chauke, M. Durmus, T. Nyokong, J. Photochem. Photobiol. A 2007,
192, 179–187.
The authors thank Beata Kwiatkowska, Senait Gebremedhin, and
Michael Yee for excellent technical assistance. J.P. is a scholarship
holder within the projects “Scholarship support for PhD students
specializing in majors strategic for Wielkopolska’s development”,
Sub-measure 8.2.2 Human Capital Operational Programme, co-fi-
[27] R. Bonnett, G. Martinez, Tetrahedron 2001, 57, 9513–9547.
[28] L. Sobotta, P. Fita, W. Szczolko, M. Wrotynski, M. Wierzchowski, T. Goslin-
ski, J. Mielcarek, J. Photochem. Photobiol. A 2013, 269, 9–16.
[29] I. Seotsanyana-Mokhosi, N. Kuznetsova, T. Nyokong, J. Photochem. Pho-
tobiol. A 2001, 140, 215–222.
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´
[30] S. Lijewski, J. Piskorz, M. Kucinska, M. Wierzchowski, K. Czerniak, H. Bill-
[38] R. D. Almeida, B. J. Manadas, A. P. Carvalho, C. B. Duarte, Biochim. Bio-
phys. Acta Rev. Cancer 2004, 1704, 59–86.
[39] M. Kryjewski, M. Nowak, P. Kasprzycki, P. Fita, C. Radzewicz, T. Goslinski,
J. Mielcarek, Inorg. Chem. Commun. 2013, 27, 56–59.
[40] A. Ogunsipe, D. Maree, T. Nyokong, J. Mol. Struct. 2003, 650, 131–140.
[41] T. Goslinski, T. Osmalek, J. Mielcarek, Polyhedron 2009, 28, 3839–3843.
[42] M. Durmus¸, V. Ahsen, T. Nyokong, J. Photochem. Photobiol., A 2007, 186,
323–329.
ert, M. Murias, J. Mielcarek, T. Goslinski, Inorg. Chem. Commun. 2013, 29,
97–100.
[31] W. Spiller, H. Kliesch, D. Wçhrle, S. Hackbarth, B. Rçder, G. Schnurpfeil, J.
Porphyrins Phthalocyanines 1998, 2, 145–158.
[32] J. Piskorz, P. Skupin, S. Lijewski, M. Korpusinski, M. Sciepura, K. Konopka,
S. Sobiak, T. Goslinski, J. Mielcarek, J. Fluorine Chem. 2012, 135, 265–
271.
[43] K. Matsumoto, M. Horikoshi, K. Rikimaru, S. Enomoto, J. Oral Pathol.
Med. 1989, 18, 498–501.
[44] S. S. Prime, S. V. R. Nixon, S. I. J. Crane, A. Stone, J. B. Matthews, N. J.
Maitland, L. Remnant, S. K. Powell, S. M. Game, C. Scully, J. Pathol. 1990,
160, 259–269.
[45] R. D. Fields, M. V. Lancaster, Am. Biotechnol. Lab. 1993, 11, 48–50.
[46] K. Konopka, E. Pretzer, P. L. Felgner, N. Dꢀzgꢀnes¸, Biochim. Biophys. Acta
Mol. Cell Res. 1996, 1312, 186–196.
[33] E. G. Sakellariou, A. G. Montalban, S. L. Beall, D. Henderson, H. G. Meuni-
er, D. Phillips, K. Suhling, A. G. M. Barrett, B. M. Hoffman, Tetrahedron
2003, 59, 9083–9090.
[34] M. Wierzchowski, L. Sobotta, P. Skupin-Mrugalska, J. Kruk, W. Jusiak, M.
Yee, K. Konopka, N. Dꢀzgꢀnes¸, E. Tykarska, M. Gdaniec, J. Mielcarek, T.
Goslinski, J. Inorg. Biochem. 2013, 127, 62–72.
[35] N. Dꢀzgꢀnes¸, Methods Enzymol. 2003, 367, 23–27.
[36] J. T. F. Lau, P.-C. Lo, W.-P. Fong, D. K. P. Ng, J. Med. Chem. 2012, 55,
5446–5454.
[37] D. Kessel, Y. Luo, Y. Deng, C. K. Chang, Photochem. Photobiol. 1997, 65,
422–426.
Received: March 25, 2014
Published online on && &&, 0000
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Waiting for cell death: The photochem-
ical properties and photodynamic activi-
ty of three porphyrazines containing an-
nulated diazepine rings were investigat-
ed. The in vitro photodynamic activity
of the porphyrazines and their liposo-
mal formulations were examined by
using two oral squamous cell carcinoma
cell lines.
J. Piskorz,* K. Konopka, N. Dꢀzgꢀnes¸,
Z. Gdaniec, J. Mielcarek, T. Goslinski*
&& – &&
Diazepinoporphyrazines Containing
Peripheral Styryl Substituents and
Their Promising Nanomolar
Photodynamic Activity against Oral
Cancer Cells in Liposomal
Formulations
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