Far-red-absorbing cationic phthalocyanine photosensitizers: Synthesis and evaluation of the photodynamic anticancer activity and the mode of cell death induction

Article abstract of DOI:10.1021/jm5014852

Novel zinc, magnesium, and metal-free octasubstituted phthalocyanine photosensitizers bearing [(triethylammonio)ethyl]sulfanyl substituents in the peripheral or nonperipheral positions were synthesized and investigated for their photophysical properties (

Full text of DOI:10.1021/jm5014852

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Article
Far-red absorbing cationic phthalocyanine photosensitizers:
Synthesis and evaluation of the photodynamic anti-
cancer activity and the mode of cell death induction
Miloslav Machacek, Antonin Cidlina, Veronika Novakova, Jan Svec, Emil
Rudolf, Miroslav Miletin, R Kucera, Tomas Simunek, and Petr Zimcik
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5014852 • Publication Date (Web): 19 Jan 2015
Downloaded from http://pubs.acs.org on January 23, 2015
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Far-red absorbing cationic phthalocyanine
photosensitizers: Synthesis and evaluation of the
photodynamic anti-cancer activity and the mode of
cell death induction
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†
‡
,§
‡
ꢀ
Miloslav Machacek, Antonin Cidlina, Veronika Novakova,* Jan Svec, Emil Rudolf, Miroslav
‡
‡
†
,‡
Miletin, Radim Kučera, Tomas Simunek, Petr Zimcik*
†
Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles
‡
University in Prague, Heyrovskeho 1203, 500 05, Hradec Kralove, Czech Republic. Department
of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec Kralove, Charles
§
University in Prague, Heyrovskeho 1203, 500 05, Hradec Kralove, Czech Republic. Department
of Biophysics and Physical Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles
ꢀ
University in Prague, Heyrovskeho 1203, 500 05, Hradec Kralove, Czech Republic. Department
of Medical Biology and Genetics, Faculty of Medicine in Hradec Kralove, Charles University in
Prague, Simkova 870, Hradec Kralove, Czech Republic
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ABSTRACT
Novel zinc, magnesium and metal-free octasubstituted phthalocyanine photosensitizers bearing
triethylammonioethylsulfanyl substituents in the peripheral or non-peripheral positions were
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synthesized and investigated for their photophysical properties (
50 nm) and photodynamic anti-cancer activity. The photodynamic treatment of 3T3, HeLa, SK-
MEL-28, and HCT 116 cancer cells revealed that the magnesium complexes were not active
IC50  100 M), whereas the IC50 values of the zinc complexes typically reached values in the

value up to 0.91, max up to
7
(
submicromolar concentrations with low toxicity in the dark (TC50  1500 M). The subcellular
changes upon photodynamic treatment of the HeLa cells indicated that the studied
photosensitizers induced damage primarily to the lysosomes which was followed by a
relocalization and damage to other organelles. The time-lapse morphological changes along with
the flow cytometry and caspase activity measurements indicated a predominant involvement of
necrosis-like cell death.
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INTRODUCTION
Photodynamic therapy (PDT) is a well-established treatment modality of certain localized
cancers and non-cancerous conditions, with several photosensitizers (PSs) currently in clinical
practice.1-3 The effect of PDT arises from the combination of the following three components:
light, oxygen and a PS. PSs absorb the light of a specific wavelength and efficiently transfer its
energy to the surrounding oxygen, forming highly reactive species. Of the reactive species, singlet
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oxygen is the most aggressive, and it is formed by a process known as photoprocess type II. The
active oxygen species destroy subcellular compartments, inducing apoptosis and/or necrosis of the
affected cells.4
Phthalocyanines (Pcs) are synthetic macrocyclic dyes with advantageous photophysical
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properties for PDT. Pcs typically absorb light over 680 nm, and the absorption position can be
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tuned up to 800 nm or, as has been recently reported, even beyond 1000 nm. Pcs are also
characterized by a high singlet oxygen production (determined by the singlet oxygen quantum
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yields) when complexed with closed-shell cations. Therefore, the interest in the medical
application of the Pcs has increased, and numerous Pcs have been synthesized and investigated as
potential PSs in PDT over recent years.7-14 A disadvantage of Pcs is their typical low solubility in
water and tendency to aggregate, which substantially decreases their photodynamic effect.
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Although a number of water-solubilizing substituents has been introduced into the Pcs, only a
small number of studies has reported the complete reduction of the aggregation in water.16-18
One of the limitations of PDT is the shallow penetration of light that becomes deeper with longer
wavelengths, close to/above 700 nm. Hence, the goal of the present study was to develop novel
water-soluble Pcs absorbing in the far-red region to investigate the relationships between their
structure and activity and to understand the mode of the PDT-induced cell death. In addition, the
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aza-analogue of the Pcs that was previously found active against Hep2 cells using a different
irradiation protocol19 was introduced into the present study to obtain the structure-activity
relationships from additional viewpoints. The target compounds (Chart 1) were designed to contain
eight cationic charges on the substituents that were bound to the Pc core either in the peripheral
(ꢁ) positions (1M-Et) or non-peripheral (ꢂ) positions (2M-Et) or to an aza-analog of Pc (3Zn-Et).
The position of the substituent on the Pc ring or the aza-substitution has been reported to
substantially influence the wavelength of the main absorption band.20,21 Cationic charges increase
the water solubility of the molecules and are expected to reduce the aggregation via electrostatic
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repulsion forces. The effect of the central metal (M = Zn, Mg, and 2H) on the photophysical
properties and its influence on cell survival was also studied.
Chart 1. Structures of the investigated compounds
RESULTS AND DISCUSSION
Chemistry. Synthesis of the Pcs is typically performed by cyclotetramerization of the
precursors, the suitably substituted phthalonitriles. In this study, the synthesis of precursors 4 and
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was accomplished using nucleophilic substitution of the phthalonitriles substituted with the
appropriate leaving groups (Scheme 1), commercially available 4,5-dichlorophthalonitrile and
compound 5, respectively. Phthalonitrile 5 containing the tosyl leaving groups was obtained in
high yield by tosylation of 3,6-dihydroxyphthalonitrile, which is similar to the published
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procedure. The higher yields of 4 (66 %) than of 6 (22 %) from the nucleophilic substitution
can be attributed to the better leaving properties of the leaving group. Compounds 4 and 6 were
obtained and stored in the form of hydrochlorides (with nearly a quantitative conversion from the
free base and back again) because their corresponding free bases were found to significantly
decompose (darken) after a couple of days or weeks. The free base was released from the
hydrochlorides immediately prior to the cyclotetramerization to the Pc. The peripherally and
non-peripherally substituted Pcs were obtained using the same procedure, starting from the
synthesis of the magnesium complexes (1Mg and 2Mg) by magnesium butoxide-induced
cyclotetramerization. The magnesium complexes were subsequently demetalated using 1% aq.
HCl to yield the metal-free derivatives 1H and 2H that were complexed by a zinc cation using
zinc acetate in pyridine (1Zn, 2Zn). All of the Pcs were subsequently quaternized with
ethyliodide to yield the highly water-soluble derivatives.
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Scheme 1. Syntheses of the studied compoundsa
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aReaction conditions: (a) (1) 2-(diethylamino)ethanethiol hydrochloride, anh. K
rt, 24 h; (2) diethylether, gas HCl, rt. (b) (1) NaOH; (2) Mg, I , anh. n-BuOH, reflux, 18-24 h. (c)
% aq. HCl, rt, 1 h. (d) anh. Zn(CH COO) , pyridine, reflux, 1.5 h. (e) C I, NMP, rt, 6 days. (f)
TsCl, anh. K CO , acetone, reflux, 2.5 h.
CO , DMSO,
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H
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Absorption spectra and aggregation. The absorption spectra of all of the Pc derivatives in
DMF had a sharp shape, which is typical for monomers of this type of compound, with the Q-
band located above 650 nm (Table 1, Figure 1, and Figure S1). Interestingly, the Q-band (the
most important band for excitation in PDT) was located at 656 nm, 704 nm and 756 nm for 3Zn-
Et, 1Zn-Et, and 2Zn-Et, respectively. From the viewpoint of the absorption spectra, compound
2
Zn-Et is of particular interest with the Q-band maximum over 750 nm, which is highly
advantageous for PDT. The position of the Q-band maximum of 3Zn-Et was influenced by the
nitrogen substitution that generally leads to a 40-50 nm hypsochromic shift compared with the
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parent Pc derivatives (e.g., 1Zn-Et). However, moving the substituents from the peripheral to
the non-peripheral positions has been reported to substantially shift the Q-band to the red6,21, thus
leading to a bathochromic shift of more than 50 nm for 2Zn-Et compared with 1Zn-Et.
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Figure 1. (a) Normalized absorption spectra of 1Zn-Et (red), 2Zn-Et (blue) and 3Zn-Et (black)
in DMF. (b) Absorption spectra of 1Zn-Et in water (full line) and DMF (dashed line), c = 1 M.
(c) Absorption spectra of 2Zn-Et in water (full line) and DMF (dashed line), c = 1 M.
The quaternized derivatives were highly soluble in water (solubility  1.5 mM for 1Zn-Et and
3Zn-Et or  0.5 mM for 2Zn-Et). The shape of the absorption spectra of 2Zn-Et and 3Zn-Et in
water resembled that measured in DMF, indicating that they are essentially non-aggregated. The
shape of the 1Zn-Et spectra in water differed significantly from that obtained in DMF, with a
decrease in the absorbance at 704 nm and an increase at approximately 660 nm, which is a typical
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observation when H-aggregates are present (Figure 1). These suggestions were further confirmed
via a comparison of the absorption and fluorescence excitation spectra (Figure S2), which were in
perfect agreement in the case of 2Zn-Et and 3Zn-Et but differed considerably in the case of 1Zn-
Et. The addition of pyridine (up to 5%) to a water solution of 1Zn-Et restored the shape of the
spectrum to the typical monomeric shape (Figure S3). As expected, no significant changes (in
addition to the solvatochromic shift of the Q-band) were observed upon addition of pyridine to the
water solution of 2Zn-Et or 3Zn-Et (Figure S3). Determinations of fluorescence quantum yields
(
Table 1) also supported presence of aggregates for 1Zn-Et and 1Mg-Et as the  values in water
F
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were approximately one third of those determined in DMF. The aggregation behavior of the
derivatives 1Zn-Et, 2Zn-Et and 3Zn-Et in water indicated that the aza substitution in the
macrocycle ring and the movement of the substituents to the non-peripheral positions significantly
aid in reducing the tendency to aggregate. The presence of eight charged substituents appears to
be essential for the monomerization of the Pcs in water because tetrasubstituted derivatives with
the same cationic substituent were found significantly aggregated in the water, regardless of the
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position of the substituent on the ring. These relationships discussed for the zinc complexes can
also be applied to the magnesium complexes or the metal-free derivatives.
Photophysical characterization. The fluorescence (
F
) and singlet oxygen quantum yields
( ) were determined in DMF using a comparative method with unsubstituted ZnPc as the

reference. The data are summarized in Table 1. All of the metal-free derivatives (1H, 2H, 1H-Et,
and 2H-Et) produced singlet oxygen and fluorescence at a very low rate; therefore, they were
excluded from the following biological evaluation. This result corresponded to previous
observations of photophysics of metal-free Pcs and may be related to the different relaxation
2
3
channels of the excited states. For the metal complexes, the 

values were consistently higher
values), regardless of the
for zinc than for the magnesium complexes (and vice versa for the 
F
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peripheral substitution because of the heavy-atom effect of the central cation. A comparison of
the peripherally and non-peripherally substituted derivatives revealed that the singlet oxygen
production was preferred for the latter. The compound 2Zn-Et appears to be of particular interest
with a high 
and 2Mg-Et was weak, but it remained sufficient for their visualization in the cellular studies using
fluorescence microscopy. The  and  values for 3Zn were extremely low because of the

value of 0.91. However, the fluorescence of the non-peripheral derivatives 2Zn-Et

F
photoinduced electron transfer (PET) between the peripheral tertiary amino substituents and the
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macrocyclic core. PET is known to efficiently deactivate the excited states of the aza-analogs of
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the Pcs. Alkylation of the amines blocked the PET; however, the quantum yields of 3Zn-Et
remained relatively low. PET is apparently a less important pathway for the excited states
deactivation for the parent Pc derivatives (1Zn, 1Mg, 2Zn, and 2Mg) compared with their aza-
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analogs, as deduced from their relatively high 

+  values. However, even for the parent Pc
F
derivatives, a partial increase in the quantum yields was observed after alkylation of the peripheral
substituents (Table 1) because of the PET inhibition.
Table 1. Absorption and photophysical data of the studied compounds in DMFa
b
Fb
Compound
max/nm (log )
707 (5.44)
 /nm


em
714
708
1
1
1
1
1
Mg
0.23
0.28
0.06
0.22
0.59
0.68
0.44
0.25
0.04
0.08
0.68
0.91
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0.24
702 (5.32)
0.17c
0.04
Mg-Et
H
705 (5.02), 729sh 711
H-Et
Zn
700 (5.30)
707 (5.46)
704 (5.45)
785 (5.21)
749 (5.11)
819 (5.06)
751 (4.87)
783 (5.18)
756 (5.20)
655
710
714
712
803
762
822
787
803
771
661
661
0.06
0.13
0.12c
0.02
1Zn-Et
2Mg
0.016c
 0.01
 0.01
2Mg-Et
2H
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2
2
3
3
H-Et
Zn
0.01
0.017c
Zn-Et
Znd
 0.01
Zn-Ete
656
0.06
aQ-band absorption maximum (max), extinction coefficient (), emission maximum (em),
b
singlet oxygen quantum yield (

c
), fluorescence quantum yield (
F
). with ZnPc as the reference
(
(DMF) = 0.56, F(THF) = 0.32). the following  values were obtained in water: 0.040 (1Zn-Et),
F
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d
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.080 (1Mg-Et), 0.013 (2Mg-Et), 0.016 (2Zn-Et). Data from the literature . Data from the
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literature
Cytotoxicity studies. These experiments were initiated with an evaluation of the rate of cellular
uptake of 1Zn-Et and 3Zn-Et. Low fluorescence of 2Zn-Et did not allow determination of the
uptake profile but the similar structure (octacationic Pc) suggests comparable behaviour. The
cellular uptake (tested on 10 µM solution) of 1Zn-Et in HeLa cells (human cervical carcinoma)
was relatively fast within the first two hours, reaching the plateau phase at approximately 12 h
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(
Figure 2a). Based on this result, the incubation with dyes prior to the irradiation was set to 12 h.
Figure 2. (a) Cellular uptake of 1Zn-Et (red) and 3Zn-Et (black) by the HeLa cells observed as
an increase in fluorescence intensity of the dyes. The experiments were performed in triplicate; (b)
Dark toxicity (open symbols, dashed lines) and phototoxicity (full symbols, full line, ꢃ > 570 nm,
-2
-2
1
2.4 mW cm , 15 min, 11.2 J cm ) against the HeLa cells of 1Zn-Et (red), 2Zn-Et (blue) and
3Zn-Et (black). Typically five independent experiments, each in quadruplicate, were performed;
c) Increase in the DCF fluorescence (ex = 485 nm, em = 525 nm) after the photodynamic
(
treatment of HeLa cells incubated with 1Zn-Et (red) or 2Zn-Et (blue). The results were corrected
for the autoxidation involvement by subtracting the light and the dark controls. The experiments
were performed in triplicate.
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The initial toxicity experiments were performed on non-malignant 3T3 mouse fibroblast cells
and HeLa cells in the absence of light (referred as dark toxicity). 3T3 cells represent the model of
healthy cells that should not be affected by the inherent toxicity of the photosensitizers. The cells
were exposed to increasing concentrations of 1Zn-Et, 1Mg-Et, 2Zn-Et, 2Mg-Et and 3Zn-Et, up
to 1500 ꢄM, and the half-maximum toxic concentration (TC50) values were calculated. The results
are shown in Figure 2 and S4 and listed in Table 2. The TC50 values obtained for HeLa and 3T3
cells for a particular derivative were comparable indicating limited difference in toxicity between
normal and cancer cells. This is in good agreement with data obtained recently for these cell lines
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for water-soluble Pc bearing quaternized imidazoles on the periphery. Interestingly, the central
cation did not significantly influence the dark toxicity; the TC50 values were similar for the zinc
and magnesium derivatives of the same ligand. However, the type of the macrocycle and the
position of the substituents significantly affected the toxicity. The non-peripherally substituted
2
Zn-Et was the most toxic followed by the aza-analogue 3Zn-Et, whereas the peripherally
substituted 1Zn-Et had more than one order of magnitude lower toxicity. To the best of our
knowledge, 1Zn-Et may be considered as a PS with one of the lowest dark toxicity values reported
in the literature.
-2
The photodynamic activities (referred as phototoxicity, ꢃ > 570 nm, 12.4 mW cm , 15 min, 11.2
-2
J cm ) of all of the compounds were determined on three malignant human cell lines, namely,
cervical carcinoma (HeLa), melanoma (SK-MEL-28) and colorectal carcinoma (HCT 116) as well
as on normal 3T3 cells. Figure 2b shows the dose-dependent decrease in the viability of the HeLa
cells 24 h after irradiation. Similar results with the other cell lines are visualized as concentration-
response curves (Figure S4), and the half-maximum inhibitory concentration (IC50) values are
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listed in Table 2. For all of the compounds, the most susceptible cell line was SK-MEL-28 followed
by HeLa and HCT 116. This order corresponds to the proliferation rates of these cancer cell lines.
No substantial difference between normal (3T3) and cancer cells toward photodynamic treatment
was observed. The magnesium complexes 1Mg-Et and 2Mg-Et did not exert any phototoxicity up
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to a 100 ꢄM concentration, which is apparently associated with their low  values. In the case of

the zinc complexes, the IC50 values increased in the order of 2Zn-Et  1Zn-Et  3Zn-Et for all of
the cell lines. This result corresponded well with the ability of the complexes to produce singlet
oxygen, as discussed above. Surprisingly, the photodynamic effect of 1Zn-Et was relatively high,
even though the compound was found aggregated in the water solution. This observation is similar
to that described in the literature in which the octacationic pyridyloxy zinc Pc was highly toxic
after irradiation even though being partially aggregated in water, and it attained comparable IC50
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values to the fully non-aggregated octacationic silicon Pcs. This result indicates that if the
aggregation of the PS is not extensive, it may play a less significant role than its photophysical
parameters, particularly 

. Furthermore, it has been previously found that 2,3,9,10,16,17,23,24-
octakis(2-(trimethylammonio)ethylsulfanyl)phthalocyaninato zinc (II) octaiodide,
a
Pc
structurally similar to 1Zn-Et, was approximately three times more phototoxic than 1Zn-Et (on
HeLa cells) but at an approximately four times higher light dose (IC50 (HepG2) = 0.20 M,  >
-2 28
6
10 nm, 48 J cm ). The concordance of both of these results on similar cell lines supports the
validity of the obtained data.
In addition to determination of IC50 values (i.e. constant fluence, varied concentrations), the
photodynamic properties were investigated also in dependence on the light dose. For this
experiment, the concentration of the zinc complexes 1Zn-Et, 2Zn-Et and 3Zn-Et was fixed
(concentration corresponding to IC50 values from Table 2) and illumination time was set to 5, 15
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-2
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and 25 min resulting in the total fluence of 3.7 J cm , 11.2 J cm and 18.7 J cm . As seen in
Figure 3, the decrease of the cell viability was dependent on the light dose.
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Figure 3. Viability of HeLa cells in dependence on the light dose following the photodynamic
treatment with 1Zn-Et (red), 2Zn-Et (blue) and 3Zn-Et (black) at concentrations corresponding
-2
to their IC50 values (see Table 2). Full area = 5 min of irradiation (total fluence 3.7 J cm ), dashed
-2
area = 15 min of irradiation (total fluence 11.2 J cm ), opened area = 25 min of irradiation (total
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fluence 18.7 J cm ). The experiments were performed in triplicate.
Table 2. Comparison of the dark toxicities (TC50 values) and the photodynamic activities (IC50
values) of the studied photosensitizers in different cell lines.a
TC50 (M)
IC50 (M)
SK-MEL-28 HeLa
0.34±0.088 0.32±0.048 0.54±0.0λ0 0.69±0.045
>100 >100 >100 >100
0.66±0.108 0.22±0.021 0.31±0.121 1.41±0.106
>100 >100 >100 >100
2.46±0.465 2.365±0.339 3.70±0.192 4.09±0.329
Compound
TC50/IC50(HeLa)
3
T3
HeLa
3T3
HCT 116
1Zn-Et
1510±14 1639±110
1420±85 >2000
3035
n/a
1Mg-Et
2Zn-Et
2Mg-Et
147±11
162±8
192±14
161±11
226±19
619
n/a
3Zn-Et
249±34
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aData are presented as TC50 or IC50 values  standard deviations.
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The determination of the TC50 (dark toxicity) and IC50 (light toxicity) values enabled the
calculation of the TC50/IC50 index as a “therapeutic ratio” (Table 2). The TC50 and IC50 values for
the HeLa cells, that stand at the middle of the tested cell lines (and were also used in further
experiments as the model cancer cell line), were selected for the calculations. High therapeutic
ratios for compounds 1Zn-Et and 2Zn-Et, reaching values over 3000 and 600, respectively,
indicated that they are promising candidates for PDT; therefore, they were selected for further
investigations on the mechanism of PDT-induced cell death. The aza-analog 3Zn-Et was
characterized by a relatively low phototoxicity and, consequently, a low value for the therapeutic
ratio (TC50/IC50 = 61); considering also its absorption only at 656 nm, it was not included in the
following studies.
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In addition to singlet oxygen, other ROS (from photoprocess type I) have also been shown to
1
initiate oxidative damage and cell death. The general ROS production during and after irradiation
can be estimated using 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA), a quencher of free
29
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radicals (e.g., HO, RO, NO  etc. ), which is reported to be insensitive to singlet oxygen. The
2
HeLa cells were preincubated for 12 h with 1Zn-Et or 2Zn-Et at concentrations corresponding to
their IC85 values and then loaded for 30 min with H2DCFDA in the dark and irradiated. The
formation of the cellular ROS was detected as an increased fluorescence of the fluorescent product
(DCF) over time (Figure 2c), which occurred only during the irradiation in the presence of the PSs.
No significant secondary ROS production occurred after the end of the irradiation. The ROS
production was more intense in the case of 2Zn-Et than 1Zn-Et. These results suggested
involvement of the photoprocess type I in photodynamic action although its contribution may be
less significant than that of photoprocess type II (singlet oxygen). Indeed, the high value of the
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sum of 
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F
, two most important deactivation pathways of excited states, leaves little space
for other photophysical processes.
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Subcellular localization and morphological changes. Subcellular localization of any PS is
crucial for the determination of the mode of induced cell death after a photodynamic treatment.4
Cationic Pcs (and, generally, PSs) have been previously reported to localize primarily within the
lysosomes16,17 or mitochondria. In this study, the localization of 1Zn-Et and 2Zn-Et based on
their red fluorescence was evaluated using fluorescence microscopy of the HeLa cells using co-
incubations with the MitoTracker Green and LysoTracker Blue fluorescence probes to visualize
the mitochondria and the lysosomes, respectively. Both of the studied Pcs were primarily localized
in the vesicles that co-localized well with the labeled lysosomes (Figure 4 (time 0 s), for a better
resolution see Figures S5 and S6). This finding is most likely a result of the mechanism of cell
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uptake, endocytosis of bulky and charged compounds, as has been previously reported for other
cationic compounds.31
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Figure 4. Morphological changes in the HeLa cells treated with 1Zn-Et during irradiation with an
LED source in the fluorescence microscope. The different subcellular compartments were
visualized using specific probes. The arrows indicate important points discussed in the text. DIC
=
differential interference contrast.
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Subsequently, changes in the cellular morphology of the HeLa cells treated with 1Zn-Et (at a
concentration corresponding to its IC50 value) and irradiated were monitored using microscopy
using differential (Normanski) interference contrast (DIC) and fluorescence microscopy with
LysoTracker Blue, MitoTracker Green, Hoechst 33342 and propidium iodide (PI) (Figure 4 and
Figure 5). The LED light source (with a Cy5 filter set) from the fluorescence microscope was used
for irradiation in these experiments instead of a Xe lamp to directly visualize the changes in the
specific fluorescence probes using fluorescence microscopy. The exposure times to activating light
were shorter because the amount of light delivered from the LED source required for the
fluorescence visualization of the PS caused relatively rapid changes in the (sub)cellular
morphology. Based on the (sub)cellular morphology changes and the PI staining, the irradiation
time of ≈240 s using the microscope LED source corresponded to 15 min of irradiation with the
Xe lamp source used in our standard phototoxicity studies.
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The control and non-irradiated cells displayed punctate lysosomal staining as assessed with
LysoTracker Blue, primarily within the perinuclear area and the rod-shaped mitochondria, with a
vivid green fluorescence of MitoTracker Green (Figure 4, 0 s). Overall cellular morphology
showed an intact cell membrane with no changes in the nucleus and cytoplasm along with a typical
spindle-like cell shape (Figure 4, 0 min, DIC). Upon irradiation, a decrease and an eventual
disappearance of the punctate blue fluorescence of the lysosomal probe was initially observed
within the first 10 s of the treatment. The lysosomal damage was also apparent from the changes
in the fluorescence localization of the studied PSs. The red fluorescence of 1Zn-Et was localized
only within the lysosomes at the beginning of the irradiation (Figure 4, 0 s). The red signal started
to leak from the lysosomes to the cytoplasm after 10 s of irradiation, and the red punctate lysosomal
fluorescence gradually disappeared. Distribution throughout the cell was completed within 30 s of
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irradiation. The relocalization of the PS that remained activated by the light, most likely also
supported by the release of low mass redox-active iron and lytic enzymes (such as cathepsins),
resulted in secondary harm to various cellular constituents, including the mitochondria, which are
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the key organelles regulating cellular death. Indeed, using the MitoTracker Green probe, the
mitochondria were observed to lose their rod shape and to round up within 30 s of irradiation
(Figure 4 and Figure S7). The mitochondria-specific fluorescence then decreased and, finally,
completely disappeared (at 120 s of irradiation). Furthermore, the alteration of the cell shape, most
likely due to damage of the cytoskeleton and the cell membrane, was observed using DIC
microscopy (Figure 4). The initial slight stretching of the cells was followed by retraction of the
filopodia, transition of the cells from spindle-like to saccular cytosolic appearance and the eventual
formation of cellular debris (30 s), typical for necrotic cell death. This sequence of events was also
observed using dynamic recording (see video file attached as ESI). This recording shows rapid
morphological changes occurring as early as few seconds after the start of irradiation in exposed
cells. Conversely, in the same cells exposed to the same irradiation protocol but in the absence of
photosensitizers, no apparent changes in cellular morphology were visible.
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Figure 5. Changes in the HeLa cells treated with 1Zn-Et during irradiation with the LED source
in the fluorescence microscope and after irradiation. The nuclei were visualized with Hoechst
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3342 (Hoechst) and propidium iodide (PI). The arrows indicate important details discussed in the
text. DIC = differential interference contrast
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Hoechst 33342, which stains the nuclei in all of the cells, and PI, which can only enter cells with
a severely compromised plasma membrane, were employed to detect changes in the nucleus of the
treated cells (Figure 5). Notably, Hoechst 33342 can also detect changes in the nuclear and
chromatin morphology during cell death (particularly apoptosis), but none were observed in this
study. Following the irradiation, as 1Zn-Et was spreading from the lysosomes throughout the cell,
PI was able to pass through the damaged plasma membrane, which was detected as a slight PI
fluorescence in the cytoplasm at 60 s of irradiation (Figure 5, PI). The PI fluorescence signal in
the cytoplasm increased over time, and the cytoplasm was completely positive for PI at the end of
the irradiation period (240 s), although remaining with a PI-negative nuclei. The first nuclei were
stained with PI at 9 min post irradiation, and permeabilization of the nuclear membranes (PI
positivity within the nuclei) was completed at 15 min post irradiation. This process was
accompanied with nuclear staining by 1Zn-Et, most likely due to the interaction of the DNA with
the cationic Pc. A strong interaction of the cationic PSs or the dyes in general (e.g., also PI) with
DNA is known.14,33
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Hence, these observations indicated that the photodynamic treatment with 1Zn-Et (and with
2Zn-Et, data not shown) induced primary damage to the PS-containing lysosomes, which was
followed by a redistribution of the PS, with subsequent damage to the mitochondria and plasma
membrane, as indicated by the cellular appearance. In the later stages, a complete destruction of
the mitochondria and lysosomes was observed, with damage to the nucleus as the final step
followed by an interaction of the Pcs with the nuclear DNA. These morphological changes are
typical of necrosis-type cell death.34
Determination of cell death. Following the photodynamic treatment, damaged cells have been
4
previously reported to undergo cell death mainly via apoptotic or necrotic pathways. Apoptosis
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is a programmed cell death that requires ATP and can typically be morphologically characterized
by cellular shrinkage, membrane blebbing, chromatin condensation, formation of apoptotic bodies
and exposure of phosphatidylserine on the cell surface. Necrosis is generally presented by
vacuolization of the cytoplasm, swelling and eventual rupture of the plasma membrane.4,35 In the
present study, the mode of cell death of the HeLa cells after treatment with 1Zn-Et or 2Zn-Et was
further investigated using several assays, including Annexin V-FITC along with PI staining and
caspases 3/7, 8 and 9 activity measurements.
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During apoptosis, the membrane phospholipid phosphatidylserine is translocated from the inner
plasma membrane to the cell surface and can be detected by the specific binding of the green
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fluorescent Annexin V-FITC conjugate using flow cytometry. As observed in Figure 6, following
the photodynamic treatment with 1Zn-Et or 2Zn-Et at a dose corresponding to the IC15, most of
-
-
the HeLa cells were negative for Annexin V-FITC and PI (Annexin V-FITC /PI ) for up to 6 h.
However, significant populations of the Annexin V-FITC /PI- (apoptotic) and
+
+
+
Annexin V-FITC /PI (late apoptotic/early necrotic) cells along with an increase in the debris
indicating severely damaged cells because of necrosis) were present 6 h after treatment. The cells
(
treated with a higher dose of the PSs (IC85) manifested significant positivity for Annexin V-FITC
and PI positivity as soon as 2 h after treatment, and the changes further progressed over the
subsequent hours (Figure 6). An increase in the debris fraction was significantly more intense than
in the case of the IC15 groups. These data suggested that the necrotic and apoptotic pathways were
activated during the photodynamic treatment with the studied PSs, with the former pathway being
more significant, as deduced from the large increase in the severely damaged cells detected as
debris.
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Figure 6. Flow cytometry assessment of the HeLa cell death mode for 1Zn-Et and 2Zn-Et
analyzed at different times after irradiation at concentrations corresponding to the IC15 (a, b) and
the IC85 (c, d), expressed as an increase in the debris against the control (a, c) or the cell count
based on the Annexin V-FITC or/and PI positivity (b, d). The experiments were performed in
triplicate, *p<0.05, **p<0.01 and ***p<0.001
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Figure 7. Activity of caspase 9 (a), caspase 8 (b) and caspase 3+7 (c) after the photodynamic
treatment with 1Zn-Et (red columns) and 2Zn-Et (blue columns) at concentrations corresponding
to the IC15 and IC85. The data are expressed as the % of the control (100 %). The experiments were
performed in triplicate, *p<0.05.
The apoptotic pathway was further studied in detail. The major signaling pathways of apoptosis
are tightly regulated by a number of pro-apoptotic and anti-apoptotic molecules and converge in a
canonical model in the activation of important proteases (caspases). Caspases can be divided into
two major groups, the initiator caspases (2, 8, 9 and 10), which activate the executioner caspases
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(3, 6 and 7). In this study, a chemiluminescence assay was used to detect the activities of caspases
3
/7, 8 and 9 after irradiation over several periods (20 min, 2 h, 4 h, 6 h and 24 h) with 1Zn-Et and
2
Zn-Et at concentrations corresponding to the IC15 and IC85. In most of the cases, the investigated
initiator caspases 8 (a key signaling molecule for the death receptor-mediated apoptotic pathway)
and 9 (the mitochondrial pathway) displayed lower activities than the untreated control, except for
the 6 h period in the case of 1Zn-Et at a concentration corresponding to the IC15 in which a
significant increase in the caspase 8 and 9 activities occurred (Figure 7). However, the activities
of executioner caspases 3 and/or 7 were comparable or higher to those of the untreated control;
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particularly, their activity significantly increased approximately 4 h after irradiation (Figure 7).
The lack of activation of the initiator (apical) caspases prior to the executioner caspases suggests
that other mechanism(s) resulted in the activation of the latter. Given the demonstrated lysosomal
damage, cathepsins and/or other proteases released into the cytoplasm appear to be the most
plausible mechanism.37-40
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Autophagy may play an important role during cell death either as a survival mechanism or as a
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contributor to the cell demise and has been previously reported in the context of PDT. Hence,
in this study, fluorescence microscopy methods were used to evaluate whether autophagy was
present during the damaging process inflicted by PDT with 1Zn-Et or 2Zn-Et. The presence of
autophagosomes was detected using the fluorescence sensor monodansylcadaverine (MDC) and
®
42
the Cyto-ID Autophagy detection kit. Rapamycin, used as a positive control, resulted in large
fluorescence-positive vacuoles throughout the cell (not only in the perinuclear region) (Figure S8).
Because no significant presence of autophagosomes was detected in the negative control or in the
cells treated with 1Zn-Et or 2Zn-Et for 24 h after the irradiation period, it is possible that increased
autophagy does not play a role in a response to the photodynamic treatment by these compounds
using the irradiation protocol in this study.
CONCLUSION
In summary, several novel octasubstituted cationic Pcs with different central cations and
substituents attached to the peripheral or non-peripheral positions were prepared. These Pcs were
investigated with respect to their absorption and photophysical properties and were compared with
the corresponding aza-analogue. The position of the substituents on the macrocyclic core and the
type of core were demonstrated to significantly influence the absorption and photophysical
properties of the Pcs.
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The cellular experiments revealed that the magnesium derivatives suffer from low photodynamic
activity (IC50  100 M) because of their low  values. However, the zinc complexes were highly

phototoxic in the order of 2Zn-Et  1Zn-Et  3Zn-Et, which is proportional to their ability to
produce singlet oxygen. Both photoprocesses type I and II may contribute to the lethal effect as
the production of ROS was also observed during irradiation. The peripherally substituted Pcs were
also characterized by an exceptionally low dark toxicity. With a TC50 value (HeLa) >1600 M, to
the best of our knowledge, 1Zn-Et ranks among the least toxic PSs reported to date. The
compounds with the best selectivity (determined by the TC50/IC50 ratio), i.e., 1Zn-Et and 2Zn-Et,
were further investigated for their mechanism(s) of action. These compounds were shown to
localize within the lysosomes and induced their rupture upon irradiation as the first step in a
cascade leading to cell death. The results from the Annexin V-FITC/PI staining and the
measurements of the caspase activities along with the morphological observations suggested that
classical apoptosis played a less significant role in cell death after photosensitization with 1Zn-Et
and 2Zn-Et. The necrosis-like cell death most likely prevailed due to the release of the entire
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lysosomal content into the cytoplasm after rupture of the lysosomal membrane, which may have
also led to secondary and non-specific activation of the executional phase of apoptosis.40,43,44
In conclusion, 1Zn-Et and 2Zn-Et appeared to be highly efficient photosensitizers with a low
dark toxicity. Particularly, the latter compound appears to be interesting for future development
because it absorbs strongly at wavelengths over 750 nm, which are more permeable to activating
light.
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EXPERIMENTAL
General. All of the organic solvents used for the synthesis were of analytical grade. Anhydrous
butanol for the cyclotetramerization was freshly distilled from magnesium. All of the chemicals
for the synthesis were purchased from certified suppliers (Sigma Aldrich, TCI Europe, Acros,
Merck) and used as received. TLC was performed on Merck aluminum sheets coated with silica
gel 60 F254. Merck Kieselgel 60 (0.040−0.063 mm) or neutral aluminum oxide 5/40 was used for
the column chromatography. The melting points were measured on an Electrothermal IA9200-
series digital melting point apparatus (Electrothermal Engineeering, Southend-on-Sea, Essex,
Great Britain). The infrared spectra were obtained on a Nicolet 6700 spectrometer in the ATR
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mode. The H and C NMR spectra were recorded on a Varian Mercury Vx BB 300 NMR
spectrometer or a VNMR S500 NMR spectrometer. The chemical shifts are given relative to
Si(CH
3
4
) and were locked to the signal of the solvent. The elemental analyses were performed on
an Automatic Microanalyzer EA1110CE (Fisons Instruments, Milan, Italy). The UV−vis spectra
were recorded using a Shimadzu UV-2401PC spectrophotometer. The steady-state fluorescence
spectra were measured using an AMINCO-Bowman Series 2 luminescence spectrometer. The
MALDI-TOF mass spectra were recorded in the positive reflectron mode on a Voyager-DE STR
mass spectrometer (Applied Biosystems, Framingham, MA, USA) in trans-2-[3-(4-tert-
butylphenyl)-2-methyl-2-propenylidene]-malononitrile as the matrix. The instrument was
calibrated externally with a five-point calibration using a Peptide Calibration Mix1 kit (LaserBio
Laboratories, Sophia- Antipolis, France). All of the compounds gave satisfactory elemental
analyses with difference ≤0.4 % from the calculated values. The purity of 1Zn-Et, 1Mg-Et, 2Zn-
Et, 2Mg-Et and 3Zn-Et was further determined by HPLC and was found to be ≥λ5 %. The LC
2
0A Prominence system (Shimadzu, Duisburg, Germany) consisted of a DGU-20A3 degasser, LC-
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0 AD pumps, a SIL-20AC autosampler, a CTO-20AC column oven, a SPD-M20A photodiode
array detector, and a CBM-20AC communication module was used for the purity evaluation. The
data were processed using LC solution software, version 1.21 SP1. The samples were separated
on a polybutadiene modified zirconia stationary phases (150  4,6 mm; particle size 5 µm) from
ZirChrom Separations, Inc. (Anoka, MN, USA). The mobile phase A consisted of 25 mM
hydrochloric acid with pH 2.1 adjusted by TRIS, the mobile phase B was methanol. The
chromatography was performed using the following gradient programme: 0-10 min, 20 % B; 10-
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1
5 min, from 20 to 65 % B; 15-25 min, 65 % B; 25-30 min, equilibration of the initial conditions.
The other conditions were set as followsμ temperature 40 °C and the flow rate 0.5 mlꢅmin-
1
22
.Compound 5 was prepared according to the literature and was further purified by crystallization
from acetone/methanol to give white crystals (yield 84%). Compound 3Zn-Et was prepared as
described in the literature.19
Preparation of 4,5-bis[2-(diethylamino)ethyl]sulfanylphthalonitrile dihydrochloride (4).
Potassium carbonate (7.7 g, 56 mmol) was sonicated in dimethylsulfoxide (100 mL) for 30 min at
rt and 2-(diethylamino)ethanethiol hydrochloride (3.39 g, 20 mmol) was added. Six equal portions
of 4,5-dichlorophthalonitrile (1.58 g, 8 mmol) were added over the next two hours, and the stirring
continued for 24 h at rt. The reaction mixture was poured into ice water (700 mL). The precipitate
was collected by filtration, washed thoroughly with water and air-dried. The crude product was
converted to hydrochloride 4 after dissolution in diethyl ether and bubbling with dry gaseous HCl.
The precipitate was collected by filtration, washed with diethyl ether and crystallized from
1
iPrOH/EtOH (1:3 (v/v)). White solid, yield 2.46 g (66 %); mp 239 – 244 °C decomp. H NMR
(300 MHz, D
2
O) ꢁ= 7.94 (s, 2 H, ArH), 3.60 – 3.42 (m, 8 H, CH
2
), 3.31 (q, 8 H, J = 7.3 Hz, CH
2
),
13
1
.31 (t, 12 H, J = 7.3 Hz, CH
3
). C NMR (75 MHz, D
2
O) ꢁ= 142.9, 131.8, 116.3, 113.1, 50.4,
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4
8.4, 27.1, 8.8. Elemental analysis calculated (%) for C20
H
32Cl
2
N
4
S : C 51.82, H 6.96, N 12.09.
2
Found: C 51.55, H 6.91, N 12.25. This compound in the form of a free base has been previously
reported in the literature.45,46
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Preparation of 3,6-bis[2-(diethylamino)ethyl]sulfanylphthalonitrile dihydrochloride (6)
.
Potassium carbonate (6.9 g, 50 mmol) was sonicated in dimethylsulfoxide (80 mL) for 30 min,
and 2-(diethylamino)ethanethiol hydrochloride (4.24 g, 25 mmol) was added. The mixture was
stirred for 10 min at rt, and then compound 5 (4.68 g, 10 mmol) was added in six equal portions
over the next two hours; stirring at rt continued for the next 24 h. The reaction mixture was poured
into ice water (700 mL) and extracted with ethyl acetate (2 × 50 mL). The organic layer was then
extracted with dilute hydrochloric acid (1% (v/v)) (3 × 25 mL). The collected water phase was
neutralized with 10% sodium hydroxide, and the product was extracted with ethyl acetate (3 × 25
mL). The organic layer was dried over anhydrous Na
2
SO , and the solvent was evaporated under
4
reduced pressure. The crude product was purified via column chromatography on silica with
acetone/methanol/triethylamine (100: 10: 1) as the eluent to yield 6 in the form of a free base
1
(
yellow solid, 0.89 g, 23 %); mp 62.7 - 63.7 °C. H NMR (300 MHz, CDCl
3
) ꢁ= 7.53 (s, 2 H,
ArH), 3.09 (t, 4 H, J = 7.1 Hz, SCH
NCH ), 1.00 (t, 12 H, J = 7.1 Hz, CH
2
), 2.70 (t, 4H, J = 7.1 Hz, NCH
2
), 2.52 (q, 8 H, J = 7.1 Hz,
13
2
3
). C NMR (75 MHz, CDCl
3
) ꢁ= 141.4, 132.2, 117.0, 113.8,
5
1.7, 46.9, 32.2, 11.8. IR (ATR): ꢂ = 2966, 2932, 2871, 2808, 2221 (CN), 1524, 1429, 1382, 1295,
-1
1
275, 1242, 1193, 1178, 1145, 1108, 1067, 992 cm . Elemental analysis calculated (%) for
C
20
H
30
N S
4 2
: C 61.50, H 7.74, N 14.34. Found: C 61.31, H 7.65, N 14.74. The whole product (0.89
g) was converted to its dihydrochloride (6) after dissolution in diethyl ether and bubbling with dry
gaseous HCl. The precipitate was collected by filtration, washed with diethyl ether and
reprecipitated from MeOH/diethyl ether. Yellow-white solid, yield 1.00 g (95 %); mp 114.5 –
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20.5 °C. H NMR (300 MHz, D
H, J = 7.1 Hz, NCH ), 1.29 (t, 12 H, J = 7.2 Hz, CH
18.0, 115.0, 50.8, 48.4, 27.9, 8.8. IR (ATR): ꢂ = 3455, 3022, 2948, 2219 (CN), 1626, 1462, 1442,
2
O) ꢁ= 7.95 (s, 2 H, ArH), 3.60 – 3.39 (m, 8 H, CH
2
), 3.29 (q, 8
1
3
2
3
). C NMR (75 MHz, D
2
O) ꢁ= 140.1, 135.0,
1
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-1
389, 1361, 1280, 1210, 1173, 1145, 1105, 1047, 1020, 999, 967, 843 cm . Elemental analysis
calculated (%) for C20
1.94.
General procedure for the synthesis of magnesium complexes 1Mg and 2Mg. The
H
32Cl
2
N
4
S
2
+ H O: C 49.88, H 7.18, N 11.63. Found: C 50.22, H 7.46, N
2
1
corresponding precursor (4 or 6, 1 eq.) was dissolved in water (100 mL) and converted to its free
base using 10% NaOH. The suspension was extracted with ethyl acetate (3 × 20 mL). The
combined organic layers were dried (Na
2
SO ), and the solvent was removed under reduced
4
pressure. Magnesium ( 7 eq.) and crystals of iodine were refluxed in dry butanol ( 40 mL) for 3
h. The corresponding precursor in the form of the free base was dissolved in butanol ( 10 mL)
and added immediately in one portion. The reflux continued for the next 24 h. The mixture was
cooled, and the solvent was removed under reduced pressure. The dark green product was extracted
from the residues of the magnesium butoxide using THF, and the solution was filtered and
evaporated. The crude product was purified using column chromatography on aluminum oxide
(
the eluents are mentioned below). The product was dissolved in a small amount of CHCl and
3
dropped into hexane. The fine precipitate was collected by filtration, washed with hexane and
dried.
Preparation of 2,3,9,10,16,17,23,24-octakis[2-(diethylamino)ethylsulfanyl]phtalocyaninato
magnesium(II) (1Mg Compound 1Mg was prepared from precursor 4 (2 g, 4 mmol) following
the general procedure for the magnesium complexes. Mobile phase: chloroform/THF (10: 1).
3
-1
-1
Green solid, yield 0.93 g (54 %). ꢃmax (DMF, 1 ꢄM)/nm 707 (ꢄ/dm mol cm 278600), 635
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