Cationic Versus Anionic Phthalocyanines for Photodynamic Therapy: What a Difference the Charge Makes

Article abstract of DOI:10.1021/acs.jmedchem.0c00481

The literature reports on cationic and anionic phthalocyanines (Pcs) for photodynamic therapy suggest systematically significant differences in activity. In this work, ten different zinc(II) Pcs with carboxylate functions or quaternary nitrogens (hydrophilic anionic, hydrophilic cationic, amphiphilic anionic, and amphiphilic cationic) were investigated, with the aim of revealing reasons for such differences. In vitro assays on HeLa, MCF-7, and HCT-116 cells confirmed higher photoactivity for cationic Pcs (EC50 ～3-50 nM) than for anionic Pcs (EC50 ～0.3-10 μM), the latter being additionally significantly more active in serum-free medium. The environmental pH, binding to serum proteins, interaction with biomembranes, differences in subcellular localization, and relocalization after irradiation were found to be the main factors contributing to the generally lower photoactivity of anionic Pcs than that of the cationic derivatives. This result is not limited only to the presented derivatives and should be considered in the design of novel photosensitizers.

Full text of DOI:10.1021/acs.jmedchem.0c00481

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Article
Cationic versus Anionic Phthalocyanines for Photodynamic
Therapy: What a Difference the Charge Makes.
Jan Kollar, Miloslav Machacek, Marie Halaskova, Juraj Lenco, Radim Kucera, Jiri Demuth,
Monika Stekla, Katerina Hasonova, Miroslav Miletin, Veronika Novakova, and Petr Zimcik
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00481 • Publication Date (Web): 03 Jul 2020
Downloaded from pubs.acs.org on July 4, 2020
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Cationic versus Anionic Phthalocyanines for
Photodynamic Therapy: What a Difference the
Charge Makes.
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Jan Kollar,† Miloslav Machacek,‡ Marie Halaskova,‡ Juraj Lenco,§ Radim Kucera,† Jiri
Demuth,† Monika Stekla,‡ Katerina Hasonova,‡ Miroslav Miletin,† Veronika
Novakova,† Petr Zimcik*,†
†Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, Faculty of
Pharmacy in Hradec Kralove, Charles University, Akademika Heyrovskeho 1203,
Hradec Kralove, 500 05, Czech Republic
‡Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles
University, Akademika Heyrovskeho 1203, Hradec Kralove, 500 05, Czech Republic
§Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles
University, Akademika Heyrovskeho 1203, Hradec Kralove, 500 05, Czech Republic
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zimcik@faf.cuni.cz
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ABSTRACT
The literature reports on cationic and anionic phthalocyanines (Pcs) for photodynamic
therapy suggest systematically significant differences in activity. In this work, ten
different zinc(II) Pcs with carboxylate functions or quaternary nitrogens (hydrophilic
anionic, hydrophilic cationic, amphiphilic anionic and amphiphilic cationic) were
investigated, with the aim of revealing reasons for such differences. In vitro assays on
HeLa, MCF-7 and HCT-116 cells confirmed higher photoactivity for cationic Pcs (EC ~
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50
3-50 nM) than for anionic Pcs (EC50 ~ 0.3-10 µM), the latter being additionally
significantly more active in serum-free medium. The environmental pH, binding to serum
proteins, interaction with biomembranes, differences in subcellular localization and
relocalization after irradiation were found to be the main factors contributing to the
generally lower photoactivity of anionic Pcs than of the cationic derivatives. This result is
not limited only to the presented derivatives and should be considered in the design of
novel photosensitizers.
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INTRODUCTION
Photodynamic therapy (PDT) is a clinically approved therapy of primarily cancerous
conditions that combines three essentially nontoxic components – light, oxygen and a
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photosensitizer (PS). The PS absorbs light energy and is able to convert it into the
production of highly reactive singlet oxygen, which is the main destructive species in
PDT. Cancer cells die as a consequence of singlet oxygen and other reactive oxygen
species damaging various biomolecules and subsequently destroying subcellular
organelles.2,3 A key component in PDT is always a PS that should have high activity
and low toxicity and ideally absorb the light strongly in the optical window of biological
tissues, i.e., in the range of 650-800 nm. A number of various macrocycles have been
tested and even clinically approved for PDT: porphyrins, chlorins, bacteriochlorins and
phthalocyanines (Pcs).4,5 The last group is of particular interest due to optimal
photophysical properties (strong absorption at approximately 700 nm, good singlet
oxygen production) and variability in macrocycle substitution, which may also tune
physicochemical properties.6,7 The typical problem for Pcs is, however, their
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hydrophobic planar macrocycle, which tends to strongly aggregate, particularly in water.
Aggregation is considered undesirable since it lowers or even eliminates the production
of singlet oxygen and makes Pcs inactive. For this reason, the clinically approved Pc
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(
sulfonated hydroxyaluminum Pc, S AlOHPc) and the majority of newly tested Pcs are
3
functionalized with water-solubilizing substituents, typically cationic or anionic, which
may substantially reduce aggregation on the basis of electrostatic repulsive forces.7
During our long-term observations of the photodynamic properties of various Pcs
published by our group or by other researchers, we noticed substantial differences
between anionic (containing carboxy or sulfo groups) and cationic (containing
quaternary ammonium groups) Pcs. Analysis of almost sixty sets of data published for
the in vitro photodynamic activity of anionic and cationic Pcs in the literature (Figure 1;
for source data, see Table S2, Supporting Information) supported this hypothesis, with
two sets being significantly different (p < 0.01). Despite the heterogeneity of the source
data (different cell lines, different irradiation conditions, different structural features), it
appears that the anionic Pcs are generally much less active than the cationic Pcs. The
reason behind this difference remains unclear, although it may substantially affect the
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selection of substituents in the rational design of newly synthesized PSs with improved
photodynamic properties.
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Figure 1. Comparison of the in vitro photodynamic activity of anionic (red) and cationic
(blue) Pcs reported in the literature. For the purpose of this comparison, the activity is
-2
expressed as the IC50 value (µM) multiplied by the fluence (J cm ) to correct the
heterogeneous data for different irradiation conditions. The mean and standard
deviation are shown in black. Statistical analysis was performed by Welch’s t-test. For
the source data, see Table S2.
In this work, we have focused on the series of Pcs (Chart 1) and investigated their
photophysical, physicochemical, binding and biological properties with the aim of finding
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the parameters and/or factors that may contribute to the substantial difference between
Pcs bearing opposite charges on peripheral substituents. Four different sets of
compounds were introduced into the study, namely anionic hydrophilic (1-3), cationic
hydrophilic (4-6), anionic amphiphilic (7, 8) and cationic amphiphilic (9, 10), to compare
both the influence of the charge type and its distribution on the macrocycle core. The
color code (red = anionic, blue = cationic) is used in all illustrations in the whole article.
Chart 1. Structures of compounds investigated in this work.
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R
X
R
R
R
R
R
a)
b)
c)
d)
X
X
R
R
R
R
R
R
N
Zn
N
N
Zn
N
N
Zn
N
N
Zn
N
X
N
N
N
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N
N
N
N
N
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N
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N
X
X
R
R
R
R
X
X
R
R
R
R
N+
I-
I-
COOY
N+
X= N
R =
R =
1
Bu
Y=C4H9
N+ I-
R =
N
N+ I-
1COOH Y=H +
N
N
COOY
1
Y=Na
N
COOY
O
7Bu
Y=C4H9
R =
O
9
4
7COOH Y=H
Y=Na+
COOY
7
I-
R = N+
N
COOY
X= CH
R =
2
2
2
Bu
Y=C4H9
N+
N
I-
COOH Y=H +
Y=Na
N
COOY
N
O
COOY
5
8Bu
Y=C4H9
COOH Y=H
Y=Na+
R =
8
8
X= CH
R =
COOY
3
Bu
3COOH Y=H
Y=Na+
Y=C4H9
N+
I- N+
R
O
COOY
N
Zn
N
I-
3
N+
N
N
N
N
R
N
Zn
N
I-
O
COOY
N
N
N
N
N
N
R
N
N
R
I-
N+
N+
I-
R
N+
I-
R =
N+
I-
N+
I-
N
R =
N+ I-
N
N
O
10
N
O
6
RESULTS
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Design and synthesis. The design of the studied compounds was primarily driven by
attempts to keep the hydrophilic molecules fully monomeric in water solutions. For this
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reason, we selected hexadeca-substitution by carboxylates or by quaternary ammonium
groups that have been reported in the literature to fully monomerize such Pcs.8,9 In the
case of 6, twelve charges in the molecule were reported to keep this molecule also fully
monomeric in water.10 Both positive and negative charges are located on the bulky
substituents that are forced to be oriented almost perpendicularly to the Pc plane due to
steric hindrance. Such spatial orientation places the charges rigidly over and below the
Pc plane and protects the hydrophobic core from aggregation based on electrostatic
repulsive forces. The selected hydrophilic groups were also included in the amphiphilic
compounds, where the presence of a lipophilic unsubstituted Pc core was expected to
promote more interactions with biomembranes.
While syntheses of compounds 1, 4-6 and 9 have been described in the literature,
synthetic routes to compounds 2, 3, 7, 8, and 10 were developed de novo or modified
published procedure were used (Scheme 1).9-13 The phthalonitrile precursor 12 was
obtained by the Suzuki-Miyaura cross-coupling reaction between 4,5-
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dichlorophthalonitrile and 11. Compound 11 was prepared as reported in the literature.14
The coupling reaction was optimized with respect to different solvents (dioxane, water,
toluene, THF), catalysts (Pd(AcO) , Pd(PPh ) , PdCl (PPh ) ) and cocatalyst (SPhos,
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3 4
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3 2
XPhos), yielding finally the best result with Pd(AcO) , XPhos, and K PO in a mixture of
2
3
4
THF and water. Subsequent cyclotetramerization of 12 using magnesium butoxide as
the initiator was employed to obtain the corresponding magnesium Pc. In agreement
with the literature, the methyl esters were transesterified to butyl esters during
cyclotetramerization.15-17 The magnesium complex was directly converted to a metal-
free ligand by treatment with p-toluenesulfonic acid in THF and then converted to zinc
complex 2Bu with zinc acetate in pyridine. Direct synthesis of zinc(II) complex 2Bu from
12 with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and zinc(II) acetate in butanol gave a
very low yield of approximately 3%. Hydrolysis of the ester groups of 2Bu using NaOH
in a mixed solvent system (water/MeOH/THF) gave the highly water-soluble
phthalocyanine 2. The synthesis of symmetrical Pc 3 was modified from the original
report.15 Precursor 14 was prepared by nucleophilic substitution of 13 with 4,5
dichlorophthalonitrile with K CO in DMF. Heating of phthalonitrile 14 in butanol with
2
3
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zinc(II) acetate and a catalytic amount of DBU led to zinc phthalocyanine 3Bu with
butylesters on the periphery that were subsequently hydrolyzed to 3.
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Unsymmetrical Pcs 7 and 8 were obtained by statistical condensation of phthalonitrile
(precursor A) with 12 or 14, respectively, (precursor B) using magnesium butoxide as
the initiator (Scheme 1). Similarly to above, the methyl esters were changed to butyl
esters. The reaction led to the mixture of six different congeners, of which the required
congener of AAAB type was isolated by column chromatography. The magnesium
complexes were then converted to corresponding metal-free ligands and then to zinc(II)
complexes, with an overall yield of 9% for 7Bu and 15% for 8Bu (based on the starting
phthalonitriles). Such yields are typical for the synthesis of unsymmetrical AAAB-type
Pcs using a statistical condensation approach.18,19 Finally, the ester bonds were
hydrolyzed to yield 7 and 8.
Low symmetrical cationic Pc 10 was prepared by statistical condensation of
phthalonitrile (precursor A) with 15 (precursor B) initiated by magnesium butoxide. The
corresponding magnesium AAAB congener was isolated by column chromatography,
converted to a metal-free ligand by p-toluenesulfonic acid in THF and then converted to
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zinc(II) complex 10Zn by zinc(II) acetate in pyridine, with a yield of 8%. Finally, the
imidazole and pyridine nitrogen atoms were alkylated by iodomethane in dry DMF under
an argon atmosphere (83% yield).
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Scheme 1. Synthesis of studied Pcs.a
COOMe
NC
NC
ii)
ii)
iii)
iii)
iv)
iv)
COOMe
COOMe
2
Bu
2COOH
(89%)
2
(45%)
(99%)
MeOOC
COOMe
1
2
(
82%)
N
COOMe
N
B
O
O
7Bu
7COOH
7
i)
(9%)
(80%)
(67%)
O
1
1
CN
CN
NC
NC
vii)
CN
viii)
N
N
10Zn
(8%)
10
(83%)
Cl
Cl
N
NC
NC
15
ii)
iii)
iii)
iv)
iv)
CN
8Bu
15%)
8COOH
(70%)
8
(
(86%)
v)
MeOOC
COOMe
MeOOC
COOMe
NC
NC
O
O
vi)
3Bu
(41%)
3COOH
(51%)
3
OH
(99%)
13
MeOOC
COOMe
14
(59%)
a
Reagents and conditions: i) K PO , Pd(AcO) , XPhos, THF/water, rt; ii) Mg, BuOH,
3
4
2
reflux; then p-TsOH, THF rt; then Zn(AcO) , pyridine, reflux; iii) NaOH,
2
water/MeOH/THF, rt; then HCl, rt; iv) equimolar NaOH, water, rt; v) K CO , DMF, 60 °C;
2
3
vi) Zn(AcO) , DBU, butanol, reflux; vii) Mg, butanol, reflux; then p-TsOH,
2
THF/CHCl /MeOH rt; then Zn(AcO) , pyridine, reflux; viii) CH I, DMF, 80 °C.
3
2
3
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8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Solubility and absorption spectra. All hydrophilic compounds (1-6) and anionic
amphiphilic Pcs (7, 8) were perfectly soluble in water and phosphate-buffered saline
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(PBS) even at high concentrations (>> 1 mM). High solubility (> 20 mM) of some of the
9
studied derivatives has already been noted in the literature. On the other hand, much
lower solubility (~ 100 µM) was observed for amphiphilic cationic 9 and 10, which
precipitated in water above this concentration. These data were further supported by the
determination of the partition coefficient between octanol and water (PBS, pH 7.4),
where 1-8 were distributed almost exclusively in the water phase (log P < -2, Table 1),
while 9 and 10 were more lipophilic, with log P -0.07 and 0.72, respectively. Direct
comparison of the log P values of 7 or 8 with 9 indicated that even when the number of
charges was the same and the molecule had a very similar structure, the carboxylate
functions made the Pcs much more water soluble than the quaternary ammonium
groups.
Table 1. Log P, photophysical data of compounds 1-10 in DMF and association
constants with BSA and DOPC liposomes in PBS.a
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7
8
9
b
-
-
Compound Log P λmax
/
nm λF
/ ΦF
ΦΔ
K × 10 K × 10
b
L
6
-1
3
-1
(log ε)
nm
(M )
(M )
1
2
3
4
5
6
7
8
9
1
-2.15 647c
-2.45 693
-2.68 674
-2.42 681
657c
702
681
683
687
721
686
682
681
694
-, (0.36)c
0.23d
0.712
-
-
-
-
-
-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.12 (0.27) 0.45
0.19 (0.25)e 0.42e
0.15 (0.15) 0.61f
1.24
2.80
f
-
-3.0g
680
0.15 (0.18) 0.49
g
g
-
-1.99h 710
-2.45 676
0.13 (0.10) 0.69
h
h
-
0.17 (0.23)i 0.33
0.09 (0.33)i 0.41
0.0514
0.527
0.0435
-
4.76
13.2
7.07
11.6
-2.44 672, 681
-0.07 673
j
i
j
0.25 (0.25) 0.57
0
0.72
671, 689
0.20 (0.22)i 0.66
aPartition coefficient between 1-octanol and PBS in logarithmic scale (Log P),
absorption maximum (λmax), emission maximum (λ ), fluorescence quantum yield (Φ ),
F
F
singlet oxygen quantum yield (Φ ), association constant with BSA (K ), association
Δ
b
constant with DOPC liposomes (K ). Due to solubility issues, the data for anionic
L
compounds 1-3, 7, 8 in DMF were determined for their nonionized analogs containing
b
c
11
d
free carboxylic acid. Values in brackets were determined in PBS. In H O, ref. . In
2
11
e
D O, ref. . Similar values (Φ = 0.26 (water, pH >7), Φ = 0.40 (DMF)) have been
2
F
Δ
8
f
9
g
12
h
10 i
reported for 3COOH in literature . ref. . ref. . ref. . in PBS with liposomes (c
=
(dye)
j
13
1
uM, dye-to-lipid ratio 1:1200). ref. .
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Journal of Medicinal Chemistry
Page 14 of 99
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The absorption spectra of all studied derivatives collected in DMF were typical for
monomeric species of the Pcs, with an intense Q-band between 670 and 690 nm and B-
band at approximately 350-370 nm (Figure 2). As two exceptions, the
tetrapyrazinoporphyrazine (TPyzPz) core in 1 caused the blueshift of the absorption Q-
band to 647 nm, and the nonperipheral substitution in 6 shifted the Q-band to 710 nm.
The unsymmetrical character of the amphiphilic compounds 7-10 may potentially lead to
splitting of Q-band, but this was only for 10. The increased polarity and ionic strength of
the environment in PBS induced aggregation for all amphiphilic compounds (7-10),
while fully monomeric character was maintained for all hydrophilic Pcs 1-6 in PBS
(Figure 2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Page 15 of 99
Journal of Medicinal Chemistry
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7
8
9
Figure 2. Absorption spectra of 1-10 in DMF (full lines) and in PBS (dashed lines) at a
concentration of 1 µM. The samples were diluted from 100 µM stock solutions prepared
in PBS (1-8) or in DMF (9, 10).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Unlike the stable positive charge on quaternary ammonium groups, the ionization states
of carboxylates may be affected by changes in pH.8,11,15 The protonation of carboxylate
groups in compounds 1-3 at lower pH induced strong aggregation, as deduced from the
decreased intensity, broadening and blueshift of the Q-band (Figure 3, Figure S3). This
effect can be easily attributed to the loss of electrostatic repulsive forces once the
carboxyl function is nonionized. The highly electron-deficient character of the TPyzPz
core20,21 in 1 substantially increased the acidity of the carboxyl functions, and the
changes in aggregation were observed at much lower pH values than for Pc derivatives
2 and 3. All anionic derivatives irrespective of the macrocycle core were strongly
aggregated at pH 5, which is typical for the intralysosomal compartment of cells,11,22 and
these derivatives were photophysically inactivated since the fluorescence quantum yield
(
Φ ) was substantially decreased (Figure 3, Figure S3). Amphiphilic anionic derivatives
F
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Journal of Medicinal Chemistry
Page 16 of 99
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
and 8 were aggregated irrespective of pH but the aggregation increased at lower pH
values (Figure S3). On the other hand, the effect of pH on cationic derivatives 4-6, 9
and 10 was rather limited, with only a small increase in both the Q-band intensity and
fluorescence quantum yield at lower pH values (Figure 3, Figure S4).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. a, c, e, g) Changes in the absorption spectra of compounds 3 (a), 7 (c), 5 (e)
and 10 (g) (c = 1 µM) in buffers of different pH values. b, f) Changes in emission spectra
of 3 (b) and 5 (f) (c = 1 µM, λex = 610 nm) in buffers of different pH values. d, h)
Dependence of the Φ values of anionic (d) and cationic (h) Pcs on the pH of the buffer.
F
Dotted vertical lines indicate physiological pH 7.4 and intralysosomal pH 5.
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Page 17 of 99
Journal of Medicinal Chemistry
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7
8
9
Photophysical characterization. All studied compounds emitted red fluorescence a
typical shape of emission spectra for Pcs and only a small Stokes shift. The values of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Φ in DMF were typically above 0.10 and in a number of cases even increased when
F
the data were collected in PBS. For measurements in PBS, the amphiphilic compounds
7-10 had to be formulated in dioleoylphosphatidylcholine (DOPC) liposomes (dye-to-
lipid ratio 1:1200 was sufficient to fully monomerize the samples, see below) to
monomerize them (only 9 had some weak fluorescence in pure PBS, Φ ~ 0.01). The
F
production of singlet oxygen, the most important reactive species in PDT, was also
determined in DMF using an established scavenger of singlet oxygen, 1,3-
diphenylisobenzofuran. The obtained values of singlet oxygen quantum yields (ΦΔ)
ranged between 0.4 and 0.7, falling within the generally published values for zinc(II)
23
Pcs. The data suggested a small difference between anionic and cationic Pcs at the
photophysical level. While the Φ values for anionic Pcs were lower than 0.45, those for
Δ
cationic species typically exceeded a value of 0.5 (Table 1).
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Journal of Medicinal Chemistry
Page 18 of 99
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Interaction with bovine serum albumin. The biological environment is a very complex
system consisting of a number of biomolecules. Serum proteins and lipidic bilayers of
cell membranes are the most important biomolecules and may substantially affect the
aggregation state, photophysics and even pharmacokinetics of the PSs and
consequently the ultimate photodynamic effect.24-27 For this reason, the effect of bovine
serum albumin (BSA), the most abundant serum protein, and the interaction of the
studied Pcs with the lipidic bilayer of liposomes was investigated.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Albumins, despite relatively low isoelectric point of approximately 4.7, are well-known
to strongly bind the acidic drugs mainly into the positively charged binding sites formed
28
prevalently by basic amino acid residues (Lys, Arg, His). The binding of anionic Pcs to
albumins is also well known in the literature,24,25,29 but reports on the interaction of
cationic Pcs with this protein have also been reported.25,26 However, in line with our
12
previous observations, no qualitative changes in the absorption and emission spectra
or Φ values of Pcs were observed upon the addition of BSA into a PBS solution of
F
hydrophilic cationic Pcs 4-6, indicating no detectable interaction with this protein (Figure
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Page 19 of 99
Journal of Medicinal Chemistry
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7
8
9
S6, S7). Stepwise addition of BSA to anionic hydrophilic Pcs 1-3 (monomeric in PBS)
induced a small redshift of the Q-band and emission maxima (approximately 7 nm) with
clear isosbestic points in the absorption spectra (Figure 4a, Figure S5), indicating
binding of the Pcs to albumin.30 The absorption changes plotted against the BSA
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
concentration were used to calculate the binding constant (K ) values, which were found
b
6
-1
5
-1
6
-1
6
-1
to be approximately 10 M (K = 7.12 × 10 M , 1.24 × 10 M , and 2.80 × 10 M for
b
1, 2, and 3, respectively, Figure 4b, Table 1). These values were roughly on the same
level as that for other anionic hydrophilic zinc(II) Pcs or anionic porphyrins reported in
the literature and indicated a strong interaction with BSA.31-33 The fluorescence intensity
of 1 and 3 (Figure 4c) decreased upon the addition of BSA as a consequence of
quenching of excited states, and the emission maximum shifted bathochromically in line
with a small redshift of the absorption maximum. Interestingly, BSA had no effect on the
fluorescence intensity of 2; it resulted only in a bathochromic shift of the emission
maximum by 4 nm. The quenching of excited states was only moderate for Pc 3 (~ 80%
residual fluorescence at full binding), but it was very strong for TPzyPz 1 (< 10% of
residual fluorescence at full binding, Figure 4d). Together with the very strong
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Journal of Medicinal Chemistry
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
quenching reported for octasulfonated TPyzPz recently (< 5% of residual
34
fluorescence ), it appears that the interaction of BSA with this type of aza-analog of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Pcs causes strong quenching of the excited state but has a rather limited effect on the
excited states of the Pc macrocycle. Amphiphilic Pcs 7-10 were found to strongly
aggregate in PBS, and in this case, the addition of BSA induced monomerization
(Figure 4e, Figure S5, S6). This effect was very well observed particularly for anionic
4
-1
5
-1
Pcs 7 and 8 exhibiting K = 5.14 × 10 M and 5.27 × 10 M , respectively, with
b
significant restoration of the photoactivity (Figure 4g); however, the photoactivity was
not fully restored to the values typical for monomers in PBS with liposomes (~ 40% and
~ 75% activity at 35 µM BSA for 7 and 8, respectively, Figure 4h), suggesting quenching
of the excited states upon binding to albumin. In contrast, the interaction of cationic 9
4
-1
and 10 with BSA was much weaker (K = 4.35 × 10 M for 9, K could not be properly
b
b
determined for 10), and even at high concentrations of BSA (> 100 µM), they remained
partially (9) or predominantly (10) aggregated, with very low fluorescence (Figure 4h,
Figure S6).
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Page 21 of 99
Journal of Medicinal Chemistry
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. a, e) Changes in the absorption spectra of 3 (a) and 8 (e) (c = 1 µM) upon the
addition of BSA at concentrations ranging from 0 µM (green line) to the final
concentration for each compound (magenta line) (inset: detail of the Q-band). b, f)
Binding isotherms of 1-3 (b) and 7-10 (f) to BSA. c, g) Changes in the fluorescence of 3
(c) and 8 (g) (c = 1 µM) upon the addition of BSA (λex = 618 nm and 612 nm,
respectively). Inset: normalized fluorescence spectra. d, h) Changes in fluorescence
intensity upon addition of BSA expressed as Φ /Φ
(value of Φ in PBS with DOPC
F
F(max)
F
liposomes (dye-to-lipid ratio 1:1200) was considered ΦF(max) for 7-10). The typical
concentration of BSA in serum-containing medium (SCM) (35 µM) is indicated by a
vertical dotted line.
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Journal of Medicinal Chemistry
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Further, considering that the target organelles were lysosomes (see also the biological
data below) with acidic environments (pH ~ 5), an experiment focusing on the spectral
and photophysical changes induced by lower pH in the presence of BSA (20 µM) was
performed (Figure S3, S4). For hydrophilic compounds 2-6, the effect of changing the
pH in the presence of BSA mirrored the behavior observed without BSA (see above
Figure 3). In the case of 1 bearing the TPyzPz core, the presence of BSA lowered the
activity at pH 7.4 to approximately one tenth, but it remained on the same level
irrespective of pH, as discussed in a previous article.11 This result further emphasized
the difference between the Pc and TPyzPz cores in the interaction with BSA. The
addition of BSA (20 µM) to the fully aggregated amphiphilic anionic derivatives 7 and 8
induced (partial or almost full, respectively) monomerization at pH 7.4, but a lower pH
led to more pronounced aggregation, most likely due to protonation of carboxylates and
lowered interaction with basic groups in BSA. In the case of cationic amphiphilic
compounds 9 and 10, their interaction with BSA was very weak, the compounds
remained mostly aggregated at pH 7.4, and the aggregation intensified at a lower pH. In
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Page 23 of 99
Journal of Medicinal Chemistry
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9
this case it might be due to protonation of carboxylate functions of amino acid residues
in BSA, whose negative charges may support weak interactions with amphiphilic
cationic Pcs at pH 7.4 (Figure S4).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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LC-MS analysis of bovine serum albumin interaction with singlet oxygen. Proteins are
common targets of singlet oxygen. For this reason, the susceptibility of BSA to oxidative
-2
damage initiated by photodynamic treatment (λ > 570 nm, 12.4 mW cm , 15 min, 11.2 J
-2
cm ) was investigated with compounds 3 and 4 as representatives of anionic and
cationic derivatives, respectively, with the use of LC-MS and subsequent quantitative
analysis of identified peptides. The data were compared with the results of control
experiments with nonirradiated BSA samples with PSs, irradiated BSA samples without
PSs, and finally with untreated BSA. The screening was focused on the amino acids
that were previously described to be attacked by singlet oxygen, i.e. Cys, His, Met, Tyr,
and Trp.35-38 A number of tryptic peptides with oxidative modifications (His + 14, His +
16, His + 32, Met + 16, Met + 32, Cys + 32, Cys + 48, Trp + 16, and Trp + 32) was
detected by LC-MS in the irradiated samples that contained PSs. The level of these
modifications was significantly higher than that in the control samples without irradiation,
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irradiated without PS or in untreated BSA (for representative examples, see Figure 5;
for all significantly modified amino acids, see Figure S2). His was the most susceptible
amino acid since almost all residues were oxidatively modified. From all modified His
residues, it appears that 145His, 241His, 378His and 509His were the amino acids most
accessible and susceptible to singlet oxygen in BSA (Figure S2), with a modification
abundance sometimes even over 30% under particular irradiation conditions. Typical
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oxidation of sulfide to sulfoxide of three Met residues ( Met, 445Met, 547Met) was
observed, in particular for 547Met with abundance about 35% (Figure 5). Further
oxidation of Met to sulfone was negligible, not exceeding 0.5% abundance in any Met
residue. Oxidation of Cys residues to sulfinic or sulfonic acid was significant only at
34Cys, which is the only free Cys in BSA. Other Cys residues stabilize the folded form of
BSA by disulfide bonds, and their oxidation was either not observed at all or the
abundance was below 0.5%, with no significant difference between treated and
untreated samples (Figure S2). Hydroxylation (Trp + 16) and subsequent formation of
formylkynurenin (Trp + 32) was detected only for 213Trp; the second Trp residue
remained unaffected. Contrary to the literature data,35,37 no oxidative modifications to
87
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Tyr residues were observed. The LC-MS analysis data suggested that BSA is an
important target of singlet oxygen produced by both bound (anionic 3) and unbound
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(cationic 4) PSs.
Figure 5. Representative examples of the oxidative modification of selected His (a),
Met (b) and Trp (c) amino acid residues in BSA upon photodynamic treatment with Pc 3
or 4. Four independent experiments were performed and analyzed by Welch’s t-test.*, p
<
0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
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Interaction with liposomes. Liposomes are often considered simple models of
biomembranes that may be targets of a number of lipophilic or amphiphilic PSs or other
drugs.39 In this work, no absorption or fluorescence changes were observed after the
addition of DOPC liposomes to hydrophilic compounds 1-6 in PBS, suggesting no
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interaction, as recently shown for cationic 6 in the literature (Figure S8). On the other
hand, the addition of liposomes to a PBS solution of aggregated amphiphilic Pcs 7-10 (1
µM) clearly induced monomerization, as observed in the absorption spectra, with full
restoration of the fluorescence properties (Figure 6a-c, Figure S8). The amphiphilic
derivatives are inserted by their lipophilic core into the liposomal bilayer leading thus to
separation of individual molecules. Unlike for binding to BSA, the monomerization was
complete even for cationic 9 and 10. The fluorescence data were then used to calculate
of association constants of these Pcs with DOPC lipids (K ), which were determined to
L
3
-1
4
-1
3
-1
4
-1
be K = 4.76 × 10 M , 1.32 × 10 M , 7.07 × 10 M and 1.16 × 10 M for 7, 8, 9, and
L
10, respectively (Figure 6d, Table 1). In comparison with published data, these values
are typical for the association of zinc(II) Pcs with liposomes.40,41 The order of K values
L
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did not fully correlate with the log P values, suggesting that other factors (e.g., the
strength of aggregation forces in the aggregates) may also contribute to this order.40,42
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Figure 6. a, b) Changes in the absorption spectra of 7 (a) and 9 (b) (c = 1 µM) upon the
addition of DOPC liposomes at concentrations ranging from 0 mM (green line) to 1.2
mM (magenta line). c) Changes in the emission spectra of 7 (c = 1 µM, λexc = 350 nm)
upon the addition of DOPC liposomes. d) Binding isotherms of 7-10 (c = 1 µM)
interacting with DOPC liposomes. The fraction of bound Pcs is expressed as the
fluorescence intensity of the monomeric form.
Since the amphiphilic Pcs 7-10 appear to interact with both BSA and lipids, it is also
interesting to evaluate their preference for a particular type of biomolecule. The
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positions of emission maxima of 7, 9 and 10 were slightly different when the compounds
were associated with BSA or liposomes (in case of 8, the emission spectrum was much
broader in liposomes than in BSA), which allowed the identification of which
biomolecules they were associated with. Thus, the emission spectra of Pcs in PBS were
collected first after the addition of liposomes (c(DOPC) = 1.2 mM, concentration at which
all Pcs 7-10 were fully associated with DOPC lipids in the above experiment) and then
after the addition of BSA (35 µM, corresponding to the amount of BSA in serum-
containing medium (SCM) used in in vitro assays) and compared with the spectra
obtained in the presence of only BSA (Figure 7, Figure S9). In another experiment, the
order of addition of biomolecules was reversed (i.e., first BSA then liposomes) but
yielded the same results. The anionic Pcs 7 and 8 preferred BSA – they associated with
BSA upon its addition to liposomal suspension and they were not removed from BSA
upon addition of liposomes in the reverse experiment. The opposite behavior was
observed for cationic Pcs 9 and 10, which preferred liposomes over BSA; i.e., they
remained associated with lipids irrespective of the presence or absence of BSA which is
in line with their rather lower affinity to BSA, as shown above.
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Figure 7. Normalized emission spectra (λexc = 608 nm, c = 1 µM in PBS) of 10 (a) and 7
(b) in BSA (35 µM, green), in DOPC liposomes (1.2 mM, red), and in the presence of
DOPC liposomes after the addition of BSA (black dashed).
Cytotoxicity studies. In vitro assessment of photodynamic activity and cytotoxicity was
primarily performed on a human cervical carcinoma cell line (HeLa). The uptake of the
tested Pcs by cells had a typical profile reaching a steady state after approximately 12 h
(Figure 8a), which was later used as the incubation time during the assessment of
photodynamic activity. The total amount of the dyes typically varied between 0.1 and 0.2
nmol per mg of protein (Table 2) and had no significant relationship with the Pc
structure. The uptake of representative of each structural type (compounds 2, 5, 8 and
10) was also performed in the presence of dynasore (three different concentrations), a
well-characterized chemical inhibitor of endocytosis which affects mainly clathrin-mediated
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