Dual Role of Subphthalocyanine Dyes for Optical Imaging and Therapy of Cancer

Article abstract of DOI:10.1002/adfm.201705938

The family of subphthalocyanine (SubPc) macrocycles represents an interesting class of nonplanar aromatic dyes with promising features for energy conversion and optoelectronics. The use of SubPcs in biomedical research is, on the contrary, clearly underexplored, despite their documented high fluorescence and singlet oxygen quantum yields. Herein, for the first time it is shown that the interaction of these chromophores with light can also be useful for theranostic applications, which in the case of SubPcs comprise optical imaging and photodynamic therapy (PDT). In particular, the article evaluates, through a complete in vitro study, the dual-role capacity of a novel series of SubPcs as fluorescent probes and PDT agents, where the macrocycle axial substitution determines their biological activity. The 2D and 3D imaging of various cancer cell lines (i.e., HeLa, SCC-13, and A431) has revealed, for example, different subcellular localization of the studied photosensitizers (PS), depending on the axial substituent they bear. These results also show excellent photocytotoxicities, which are affected by the PS localization. With the best dual-role PS, preliminary in vivo studies have demonstrated their therapeutic potential. Overall, the present paper sets the bases for an unprecedented biomedical use of these well-known optoelectronic materials.

Full text of DOI:10.1002/adfm.201705938

FULL PAPER
Biomedical Light Management
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Dual Role of Subphthalocyanine Dyes for Optical Imaging
and Therapy of Cancer
Eveline van de Winckel, Marta Mascaraque, Alicia Zamarrón, Ángeles Juarranz de la Fuente,*
Tomás Torres,* and Andrés de la Escosura*
the therapeutic and diagnostic functions
within a single molecular framework.
The family of subphthalocyanine (SubPc) macrocycles represents an inter­
esting class of nonplanar aromatic dyes with promising features for energy
conversion and optoelectronics. The use of SubPcs in biomedical research
is, on the contrary, clearly underexplored, despite their documented high
fluorescence and singlet oxygen quantum yields. Herein, for the first time it
is shown that the interaction of these chromophores with light can also be
useful for theranostic applications, which in the case of SubPcs comprise
optical imaging and photodynamic therapy (PDT). In particular, the article
evaluates, through a complete in vitro study, the dual­role capacity of a novel
series of SubPcs as fluorescent probes and PDT agents, where the macrocycle
axial substitution determines their biological activity. The 2D and 3D imaging
of various cancer cell lines (i.e., HeLa, SCC­13, and A431) has revealed, for
example, different subcellular localization of the studied photosensitizers
Optical imaging and therapy methods are
becoming indispensable in this respect,
as they allow tuning both kinds of func-
tions through designed interactions of the
[3]
theranostic agent with light. Compared
to other imaging modalities, fluorescence
imaging presents various advantages such
as its noninvasive character, subcellular
spatial resolution, high temporal resolu-
tion, and high sensitivity in the detection
of biological structures at low concentration
[4]
[5]
levels. Photodynamic therapy (PDT), on
the other hand, is a clinically approved form
of phototherapy that makes use of nontoxic
photoactive compounds, called photosen-
sitizers (PS). When exposed selectively to
light of a certain wavelength, PS become
toxic to targeted malignant and other dis-
(PS), depending on the axial substituent they bear. These results also show
excellent photocytotoxicities, which are affected by the PS localization. With
the best dual­role PS, preliminary in vivo studies have demonstrated their
therapeutic potential. Overall, the present paper sets the bases for an unprec­
edented biomedical use of these well­known optoelectronic materials.
[6]
eased cells or microorganisms, through
1
the generation of singlet oxygen ( O ) and
2
[
7]
reactive oxygen species (ROS). Impor-
tantly, integrating fluorescence emission
1
with the capacity to convert light into O and ROS yields PS that
2
1
. Introduction
[8,9]
can perform as both imaging and PDT agents. However, there
are few examples of PS with optimum emission features for use
as fluorescent probes, and vice versa. In this respect, herein we
report for the first time on the promising dual-role of subphth-
alocyanine dyes for optical imaging and therapy of cancer.
Subphthalocyanines (SubPcs) form part of the pyrrolic
macrocycle family, and comprise a 14-π electron aromatic struc-
ture with C symmetry, formed by three isoindole units linked
through aza bridges around a tetracoordinated boron atom.
They are considered lower homologs of Pcs,
tute one of the most important families of PS.
The integration of therapeutics and diagnostics, also called thera-
nostics, represents a major goal of current biomedical research,
which pursues to achieve more site-specific, efficient, and per-
sonalized healthcare.[
1,2]
A straightforward approach toward
this goal involves the use of theranostic agents that comprise
3
Dr. E. van de Winckel, Prof. T. Torres, Dr. A. de la Escosura
Departamento de Química Orgánica
Universidad Autónoma de Madrid
Madrid 28049, Spain
E-mail: tomas.torres@uam.es; andres.delaescosura@uam.es
[
10]
[
11–15]
which consti-
In contrast
[
16–21]
to Pcs, SubPcs present a conical π surface, which renders them
with excellent properties for photovoltaic and optoelectronic
Prof. T. Torres, Dr. A. de la Escosura
Institute for Advanced Research in Chemical Sciences (IAdChem)
UAM
Madrid 28049, Spain
[
22]
applications, such as a lower tendency to aggregation and the
possibility to tune them through axial substitution. Such fea-
tures, in combination with the intense absorption and emission
of SubPcs close to the therapeutic window (at ≈560 and 570 nm,
respectively), also enable their use in biomedical research, yet
the number of reports about these nonplanar chromophores in
M. Mascaraque, Dr. A. Zamarrón, Prof. Á. Juarranz de la Fuente
Departamento de Biología
Universidad Autónoma de Madrid
Madrid 28049, Spain
E-mail: angeles.juarranz@uam.es
[
23–25]
[26,27]
the context of fluorescence imaging
or PDT
remain
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adfm.201705938.
extremely scarce. In this respect, both the well-known high
1
SubPc fluorescence and O quantum yields (φ = 0.2–0.9 and
2
F
[
28–30]
DOI: 10.1002/adfm.201705938
φΔ = 0.5–0.7, respectively)
prompted us to explore the
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O
O
O
O
O
O
O
O
S
N
S
O
O
S
S
O
N
O
N
O
N
N
N
B
N
N
B
N
N
B
N
N
N
N
N
N
N
N
1b
N
1c
1a
Figure 1. Chemical structure of the series of SubPc derivatives 1a–c.
potential dual functioning of these dyes as fluorescent probes
and PS for PDT.
monolayer (2D) or in spheroids (3D), inducing in both cases
a strong fluorescence, in lysosomes or vacuoles (endosomes)
depending on the SubPc axial substituent, under green light
irradiation. Moreover, for the most efficient dual-role PS (1a
and 1c), preliminary in vivo studies in mice provide proof-of-
concept of their promising therapeutic action.
The series of SubPc derivatives (1a–c, Figure 1) under
study presents different substituents at the apical position
of the boron atom, which allows tuning the balance between
hydrophilicity and hydrophobicity of the PS. The design of
the axial substituents in compounds 1a–c is derived from
a series of silicon Pc (SiPc) derivatives, bearing two of the
same substituents at the axial positions of their silicon atom,
described by some of us and which previously showed activat-
able photodynamic properties.[ These substituents include:
2
. Results and Discussion
31]
2.1. Synthesis of SubPcs 1a–c
(
i) a bulky pyrene-containing hydrophobic group, where the
pyrene moiety could be used as an additional fluorophore
1a); (ii) an amino group with two methoxy(triethylenoxy)
SubPcs 1a–c were synthesized from SubPcBCl (Scheme 1),
[
28]
(
the synthesis of which was previously described.
To allow
[
31]
axial chains for increasing hydrophilicity (1b); and (iii) a refer-
ence hydrophobic group that lacks the bulky pyrene unit (1c).
Importantly, the rationale of the present study is to extend the
interesting photodynamic activity of that SiPc-based series to
compounds 1a–c, where the SubPc platform provides an addi-
tional, outstanding potential for fluorescence imaging that Pcs
just do not have. Consequently, we have developed an in-depth
for axial substitution with nucleophiles 2–4, SubPcBCl was
transformed into the reactive SubPc–triflate intermediate, fol-
lowing a successful substitution procedure developed previ-
[
32]
ously in our research group.
To this end, SubPcBCl was
first dissolved in dry toluene and treated with 1.2 equiv. of
silver triflate, to irreversibly remove the axial chlorine atom
and to obtain a “SubPcB ” species with a weakly coordinating
+
2
D and 3D in vitro study of SubPcs 1a–c for fluorescence
triflate anion. The formation of this triflate intermediate was
followed by thin layer chromatography and usually completes
after ≈40 min of reaction at room temperature. The activated
SubPc–triflate intermediate shows considerable reactivity
toward nucleophilic attack at the boron atom, and can be easily
reacted with a variety of nucleophiles. Therefore, upon comple-
tion of the formation of the triflate intermediate, 2 equiv. of
the corresponding nucleophile (i.e., compound 2, 3, or 4) were
added to the reaction mixture, in the presence of 1.2 equiv.
of freshly distilled N,N-diisopropylethylamine (DIPEA) to
imaging of and PDT against HeLa, SCC-13, and A431 cancer
cell lines. The selection of these cell lines is based on the fact
that they all represent examples of superficial human can-
cers (cervix, skin, and vulva, respectively) in which PDT can
be applied and, therefore, the type of lesions that could effec-
tively be targeted by SubPcs without decreasing their activity
due to tissue light absorption and scattering effects. Results
from these studies show that the three PS derivatives (1a,
1
b, and 1c) are able to enter into these tumor cells, grown in
2
(2 equiv)
DIPEA,
O
O
N
CF3
O
1a (52%)
S
Cl
N
4
0°C, 4h
CN
CN
AgOTf (1.2 equiv)
BCl3
N
3 (2 equiv)
DIPEA,
N
B
N
N
B
1
1
b (43%)
c (64%)
p-xylene,
N
N
toluene,
N
N
1
40°C, 40 min
rt, 40 min
40°C, 5h
N
N
22%
(AgCl
)
4 (2 equiv)
SubPcBCl
DIPEA,
4
0°C, 3h
O
O
O
O
O
O
O
O
HO
S
N
HO
S
S
S
O
HO
O
2
3
4
Scheme 1. Scheme of the synthetic route toward SubPcs 1a–c.
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neutralize the triflic acid generated in this step, which could
promote unwanted side reactions. The reaction has to be per-
formed under strictly anhydrous conditions, as the presence of
water traces immediately leads to a mixture of hydroxy-SubPc
and the µ-oxo dimer.[ The resulting SubPcs 1a–c could be
easily purified by column chromatography, generally using
toluene/tetrahydrofuran (THF) mixtures of different polari-
ties as the eluent. A second purification step by size exclusion
chromatography (SEC), with Bio-Beads as the stationary phase
and toluene as the eluent, was performed to remove any traces
of free alcohol or µ-oxo dimer. In this way, SubPcs 1a–c were
obtained with moderate yields, ranging from 43% to 64% (see
Supporting Information for the complete procedures).
(Figure S2, Supporting Information), which reacts readily with
1
[33]
O (Table 1). From this data it can be inferred that the three
2
1
SubPcs are efficient O2 generators, with φΔ values between
0.37 and 0.56. The SubPc 1a is a particularly good PS, equaling
the photodynamic capacity of nonsubstituted ZnPc, which was
used as the reference compound.
32]
2.3. Subcellular Localization of SubPcs 1a–c
As the subcellular localization of a PS can determine its effi-
[
34,35]
ciency for PDT, as well as the type of cell death,
the dis-
tribution of SubPcs 1a–c within SCC-13 and HeLa cells was
carefully evaluated by fluorescence microscopy, performing co-
localization studies with fluorescent markers for specific orga-
nelles. To this end, cells were incubated for 18 h with SubPcs
2
.2. Spectral Features and Photophysical Properties
−
6
1
a–c at a concentration of 2 × 10 m, and subsequently further
The normalized absorption spectra of SubPcs 1a–c, measured
in dimethylformamide (DMF) (Figure S1, Supporting Informa-
tion), show the typical and intense Q band, at 566 nm for Sub-
PcBCl and at 562 nm for the axially substituted SubPcs 1a–c,
which indicates that they are nonaggregated. Besides, they
all present the typically broad Soret band, with a maximum
around 305 nm. For SubPc 1a, additional absorption bands at
incubated with LysoTracker, MitoTracker, or NBD C -ceramide
6
probes. For both cell types, a similar intracellular distribution
of SubPcs 1a–c could be observed, as determined by their red
fluorescence emission observed under green light excitation
(Figure 2; Figure S3, Supporting Information). After incubation
with SubPc 1a, the two cell lines, grown in monolayer, showed
an intense granular red fluorescence emission around the
nucleus, as well as in a yuxtanuclear position (Figure 2A, left).
This intracellular emission of SubPc 1a was coincident with
that of the lysosomes, since a yellowish fluorescence due to the
overlapping of red and green emission (from the SubPc and
LysoTracker®, respectively) was observed. Such fluorescence
was clearly different from that of the mitochondrial and Golgi
signal patterns. Identical results were obtained with SubPc 1b
(Figure S3, Supporting Information). SubPc 1c, on the contrary,
showed a strong granular (vacuolar) red fluorescence emission
localized also around the nucleus and nearby but not coinci-
dent with lysosomes, neither with mitochondria nor with the
Golgi apparatus (Figure 2A, right). Control cells (without PS
incubation) showed a very low emission, which corresponds to
mitochondrial autofluorescence (not shown), confirming that
the fluorescence detected in SCC-13 and HeLa was indeed due
to intracellular accumulation of the SubPc dyes.
3
29 and 345 nm can be distinguished, characteristic of the axial
pyrene moiety. Exact values of the electronic absorption features
for each SubPc can be found in Table 1. Upon excitation at
3
5
45 nm, all the SubPcs showed a fluorescence emission around
71 nm (F571), varying in intensity, with the pyrene-bearing com-
pound 1a also showing a small emission band around 377 nm
F377). The φ values for both the SubPc and pyrene moieties
(
F
were determined in DMF, relative to F SubPcBCl in benzo-
12
nitrile (φ = 0.58) and anthracene in cyclohexane (φ = 0.36),
F
F
exciting at 520 or 345 nm, respectively (Table 1). As main con-
clusion, the φ of the three SubPcs 1a–c were high (≈0.3–0.6),
F
encouraging their use for optical imaging. Besides, the emis-
sion of the pyrene unit from SubPc 1a is quenched (φ = 0.02),
F
probably caused by an electronic energy transfer between the
pyrene moiety and the SubPc core due to partial overlap of the
pyrene emission spectrum with the SubPc Q-band, yet elec-
tron transfer cannot in principle be ruled out.[ The photo-
sensitizing efficiency of SubPcs 1a–c, on the other hand, was
31]
In addition, and taking into account that tumor spheroids
are considered to be a better model to mimic the in vivo situ-
ation than monolayers of tumor cells, with regard to tumor
evaluated by determining their φ in DMF following the well-
Δ
[
36,37]
known relative method, based on the photoinduced decompo-
sition of the chemical scavenger 1,3-diphenylisobenzofuran
shape,
we then evaluated the ability of the SubPcs 1a–c
to penetrate into these tumor like structures formed from
SCC-13 and HeLa cells (Figure 2B). As
Table 1. Electronic absorption and photophysical data for SubPcBCl and the SubPcs 1a–c in revealed by the intense emission observed
DMF.
under the fluorescence microscope, SubPcs
a–c effectively entered into the spheroids:
1
λmax [nm]
566
λem [nm]a)
575
φF377 [%]b)
φF571 [%]c)
φΔ [%]d)
47
SubPc 1a localized in the lysosomes (iden-
tical localization was observed for SubPc 1b,
not shown), whereas SubPc 1c presented a
granular localization pattern nearby but not
inside the lysosomes. From these observa-
tions, we can conclude that SubPcs 1a and
Compound
–e)
SubPcBCl
55
38
29
36
1a
1b
1c
562
571
2
56
562
571
–e)
–e)
37
562
571
49
1
b exhibit the same localization pattern in
a)
Excited at 520 nm; b)Excited at 345 nm, using anthracene in cyclohexane as the reference compound
c)
2D and 3D in vitro localization experiments,
both in SCC-13 and HeLa cells, being local-
ized in lysosomes, whereas SubPc 1c seems
(φF = 0.36); Excited at 520 nm, using F12SubPcBCl in benzonitrile (φF = 0.58) as the reference
d)
e)
compound; Using ZnPc in DMF as the reference compound (φΔ = 0.56); Not applicable, because the
compound does not contain any pyrene moiety.
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Figure 2. Subcellular localization of SubPc 1a and SubPc 1c in A) 2D cultures and B) 3D cultures of SCC-13 cells after 18 h of incubation. Experiments with
cultures of HeLa cells gave identical results (not shown). Phase contrast (PhC). Red fluorescence is from SubPcs and green fluorescence is from lysosomes
(Lyso), Golgi apparatus (Golgi), and mitochondrial (Mito). The merged image shows the green and red fluorescence together. A blue (450–490 nm)
exciting lamp was used for LysoTracker and MitoTracker or NBD C -ceramide probes, while green (545 nm) exciting light was utilized for SubPcs.
6
to present vacuolar (endosomal) localization. This is interesting
because the subcellular localization of a PS is closely related to
repeating the subcellular localization experiments in A431 cells
was considered not necessary.
the cell death mechanism induced by PDT, as a consequence of
1
the short life time of O , the main reactive species produced
2
after PDT, for which the primary localization of the PS deter-
mines the initial subcellular damage upon its activation.
2.4. Phototoxicity Experiments and ROS Production
[
38,39]
Besides, given that no differences were detected between the
SCC-13 and HeLa cells, localization seems to be mainly related
to the molecular structure of the SubPc employed, for which
The phototoxicity of SubPcs 1a–c upon green light irradiation
was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) assay in SCC-13, HeLa, and
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Table 2. Toxicity effects in SCC-13, HeLa, and A431 cells induced by green light, in the absence
of any SubPc, and by incubation with SubPcs 1a–c in the dark. Cell toxicity was evaluated by
the MTT assay 24 h after treatment. Data are expressed as mean values obtained from three
independent experiments ± standard deviation (SD).
We next analyzed by fluorescence micros-
copy the intracellular ROS formation in
HeLa, SCC-13, and A431 cells, when sub-
jected to PDT with the SubPc 1a or 1c, using
for this purpose the DHF-DA fluorescent
probe (Figure S5, Supporting Information).
As shown in Figure S5A (Supporting Infor-
mation), controls presented very low green
florescent signal due to moderate endog-
enous production of ROS. Similarly, cells
incubated with SubPcs in dark conditions
showed a very low green fluorescence. In
contrast, cells subjected to PDT showed an
intense fluorescence in all cell types, com-
pared to baseline levels, revealing a promi-
nent ROS production after the photodynamic
treatment. Fluorescence values after PDT
were actually significantly higher than for
Compound
Concentration[m]
Surviving fraction (% ± SD)
HeLa
SCC-13
100 ± 4.8
99 ± 1.5
A431
Control
100 ± 4.4
100 ± 4.0
95 ± 2.1
95 ± 2.0
96 ± 2.5
103 ± 1.1
103 ± 6.8
92 ± 2.4
94 ± 1.0
4
.7 J cm−
2
100 ± 3.0
[
44]
2 × 10−6
8
1
a
101 ± 3.6
103 ± 3.4
102 ± 4.6
102 ± 2.4
95 ± 2.9
102 ± 4.7
5
× 10−
2 × 10−
× 10−
99 ± 1.9
6
8
1
b
c
104 ± 1.5
5
98 ± 3.0
2 × 10−6
8
1
96 ± 2.9
5
× 10−
100 ± 1.5
98 ± 3.5
A431 cells, grown in monolayer. For this purpose, we first eval-
uated the inherent toxicity of two different concentrations of the
three SubPcs (2 × 10−6 and 5 × 10 m) in cells after 5 h of incuba-
tion in presence of PS in the dark and, separately, in the absence
of PS after the administration of different green light doses
dark SubPc treatments (P < 0.005) (Figure S5B, Supporting
Information).
−8
Given the results obtained in cells grown in monolayer, we
next evaluated the photodynamic activity of SubPcs 1a and 1c
in A431 spheroids. For the PDT treatments, we selected the
−
2
−6
(2–4.7 J cm ). The results obtained, shown in Table 2, indicate
highest noncytotoxic SubPc concentration (2 × 10 m), 5 h of
−
2
that none of these SubPc concentrations, neither the adminis-
tration of green light, induced significant cytotoxic effects in the
cell lines studied, and survival rates above 92% were obtained.
We then continued to evaluate the photodynamic activity of the
three SubPcs toward SCC-13, HeLa, and A431 cells upon irra-
diation.[ For the photodynamic treatments, we selected the
incubation, and a 4.7 J cm light dose. For both treatments,
with SubPc derivatives 1a and 1c, a significant decrease in sphe-
roid diameter can be observed 24 h after treatment (Figure 4A).
In addition, the acridine orange/ethidium bromide (AO/EB)
staining method revealed that the treated spheroids were formed
by damaged cells (fluorescing in orange/red) while controls
were formed by viable cells (fluorescing in green) (Figure 4B).
Therefore, we can conclude that both SubPcs were photody-
namically active also in the 3D in vitro model.
40]
−
8
lower noncytotoxic SubPc concentration (i.e., 5 × 10 m) and,
after 5 h of incubation, cells were exposed to different green
−2
light doses (2, 3.35, and 4.7 J cm ). For both PDT treatments
with SubPc 1a and 1c, a drastic decrease in cell survival was
revealed for the three cell lines (Figure 3A–C), in line with the
light dose employed.
2.5. In vivo Studies
SubPc 1c is a more efficient PS than 1a, according to the statis-
tically significant differences in performance observed between
them. The measured cell survival values are actually comparable
Preliminary in vivo studies were performed to confirm the
imaging and therapeutic potential of the SubPcs under study.
As a proof-of-concept for imaging, we tested if it is possible
to capture the fluorescence of these PS in vivo. In particular,
[41]
to those of other PS used in PDT, such as LDH-ZnPcS8, or sil-
icaCe6-FA.[ SubPc 1b, in turn, was not effective for PDT at this
concentration, since the cell survival values obtained after photo-
dynamic treatments were similar to those observed in untreated
control cells. When higher concentrations of SubPc (i.e., 0.1 ×
42]
−
6
0.2 mL of 2 × 10 m SubPc in phosphate-buffered saline (PBS)
were injected subcutaneously on the back flank of a mice (CD-1,
shaved of the back dorsal skin using a hair clipper), and the
resulting fluorescence was immediately evaluated by the IVIS
Lumina system (λexc = 450 nm, λem = 580 nm). Following this
simple, nonoptimized protocol, Figure S6 (Supporting Informa-
tion) shows an image where the emission of compound 1a, as
an example, can be clearly identified (see white arrow), demon-
strating that the fluorescence of SubPc dyes can be detected in
vivo, and the skin is not a barrier to observe the compound.
To evaluate the therapeutic capacity of SubPcs 1a and 1c, on
the other hand, A431 cells were subcutaneously injected in both
flanks of mice and when the tumors reached a size between 5
and 7 mm of diameter, treatments were applied. No evident dif-
ferences were observed in the size of tumors exposed only to
light or with injected SubPc 1a or 1c (nonirradiated); therefore,
all were grouped and shown as “controls.” For PDT treatments,
the SubPcs 1a or 1c were administered by intratumoral injection
−6
10
m) were employed, however, a significant decrease in cell
survival was detected, even for SubPc 1b (Figure S4A, Supporting
Information). Importantly, the observed cell survival values find
1
correspondence to the capacity of the three SubPc dyes as O2
generators (Table 1). Altogether, the results also indicate that
HeLa cells were more resistant to photodynamic treatments with
these SubPcs than SCC-13 and A431 cells (Figure S4B, Sup-
porting Information). The cell morphology of the treated cells
was also analyzed 24 h after PDT treatment using phase contrast
microscopy (Figure 3D), revealing remarkable changes in the
three cell lines. Most cells showed cytoplasmic retraction, with
a rounded aspect similar to that of cells in apoptosis,[ yet elon-
gated cells were also observed in the treated cultures. Interest-
ingly, these images are well correlated with the results obtained
from cell viability assays.
43]
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−
8
Figure 3. Surival of A) SCC-13, B) HeLa, and C) A431 cells after photodynamic treatment with SubPcs 1a–c (5 × 10 m, 5 h of incubation followed
−
2
by different light doses: 2, 3.35, and 4.7 J cm ). Cell survival was evaluated 24 h after photodynamic treatment by the MTT assay. Each point corre-
sponds to the mean value ± SD obtained from three independent experiments. D) Morphological changes observed in SCC-13, HeLa, and A431cells
−
6
−2
2
*
4 h after PDT with the SubPcs 1a and 1c (0.05 × 10 m, 5 h of incubation followed by a green light dose of 4.7 J cm ). Scale bar: 50 µm. *P < 0.05,
*P < 0.01, ***P < 0.005.
and, 2 h later, exposed to 12 J cm−2 of green light (Figure 5A).
Treated tumors showed a size smaller than that of controls, as
could be observed by visual inspection (Figure 5B) and confirmed
after measuring them (Figure 5C). Reduction in tumor size was
observed already 2 d after treatment, compared to controls not
subjected to PDT. Tumors were subjected to a second PDT treat-
ment and a greater decrease in their sizes was detected. The histo-
logical analysis of the tumors revealed no differences between the
tumors with one or two PDT-treatments (not shown). The H&E
staining showed an increment of red blood cell extravasation with
progression to coagulative epidermal necrosis in tumors after
SubPc injection and PDT (Figure 5D), this being related to the
observed smaller size of treated tumors. Therefore, both SubPcs
are able to damage tumors cells upon green light irradiation.
3
. Conclusions
In sum, in this article we have demonstrated for the first time,
with in vitro and in vivo experiments, that SubPcs present a
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Figure 4. Photodynamic activity of SubPc 1a and 1c in A431 spheroids. A) Reduction in spheroid diameters 24 h after PDT treatment with SubPcs 1a
−6
−2
and c (2 × 10 m, 5 h of incubation and 4.7 J cm of green light). Diameters were measured using the Image J software and normalized to size control.
Each point corresponds to the mean value ± SD obtained from three independent experiments. B) 24 h after photodynamic treatment the spheroids were
labeled with acridine orange (AO) and ethidium bromide (EB) to determine viable (green) or damage spheroids (orange). Scale bar: 50 µm. **P < 0.01.
promising dual potential for optical imaging and PDT of super-
ficial cancers. This opens a window of opportunity for these
optoelectronic dyes, whose application in biomedical research
has, until now, remained clearly underexplored. The main
advantages of SubPcs in this respect include their high fluores-
cence and singlet oxygen quantum yields, and their nonplanar
aromatic structure, which allows fine-tuning of the aggregation
and excited state properties of these macrocycles by adequately
designed axial substitution. Within the series of compounds
studied herein, for example, different subcellular localization
can be observed and related to the different SubPc axial sub-
stituents, an effect that we tentatively ascribe to their different
aggregation behavior in the biological medium. Although the
structure–property relationships for this family of PS has to be
clarified in further studies, their excellent capacity to perform
both imaging and therapeutic functions that are triggered only
by light represents a very promising approach within the field
of theranostics.
Cell Cultures: For in vitro studies, we used the following human
tumoral cell lines: SCC-13 (squamous cell carcinoma from face), HeLa
cervical adenocarcinoma), and A431 (squamous cell carcinoma from
vulva). Cell lines were grown in DMEM (Dulbecco’s modified Eagle’s
(
medium high glucose 1×) supplemented with 10% (v/v) fetal bovine
−1
−1
serum (FBS), 50 units mL penicillin, and 50 µg mL streptomycin,
all from Thermo Fisher Scientific Inc. Cell cultures were performed
under standard conditions of 5% CO2, 95% of humidity, and 37 °C of
−
1
m
temperature and propagated by trypsinizing cultures with 1 × 10
EDTA/0.25% Trypsin (w/v).
Spheroid Cultures: Spheroids were grown from SCC-13, HeLa, and A431
cells using specific medium containing DMEM/F12 (1:1) (F-12 Nutrient
Mixture, Ham, Gibco), 2% B27 serum free supplement (17504-044,
−1
Gibco), 20 ng mL EGF (E4269, Sigma), 0.4% bovine serum albumin
−
1
(A7906, Sigma), and 4 µg mL insulin (41400-045, Gibco). The cells
−
1
were plated at 15 000 cells mL per well precoated with 1.2% poly-
HEMA (2-hydroxyethyl methacrylate, Sigma). The spheroids were
formed after 6 d of culture.
Photosensitizer Incubation: Stock solutions of the SubPcs 1a–c
−3
(
1.5 × 10 m) were prepared in dimethyl sulfoxide (DMSO) and work
solutions were obtained in DMEM without FBS. The final concentration
of DMSO was always lower than 0.5% (v/v) and the lack of toxicity of
such concentration for the cells was confirmed. All the treatments were
performed when cultures reached around 60–70% of confluence.
Subcellular Localization: To analyze the intracellular localization
of SubPcs, SCC-13 and HeLa cells were grown on coverslips and,
4
. Experimental Section
Materials and Methods: All reagents were used as purchased from
commercial sources without further purification. Solvents were dried
using standard techniques prior to use. The starting materials silicon
−
6
incubated with SubPcs 1a–c to a final concentration of 2 × 10 m for
18 h at 37 °C. After incubation, cells were further incubated for another
30 min with known fluorescent probes for lysosomes (LysoTracker
Green DND-26, Invitrogen), mitochondria (MitoTracker Green FM,
Invitrogen), or Golgi apparatus (NBD, C6-ceramide (N-[6-[(7-nitro-2-1,3-
benzoxadiazol-4-yl)amino]hexanoyl]- d-erythro-sphingosine, Invitrogen))
at the concentrations indicated by the suppliers. Then, cells were briefly
washed in PBS, mounted on slides with a drop of PBS and immediately
observed under the fluorescence microscope.
phthalocyanine dichloride (SiPcCl ), 1-pyrene butyric acid, 2-hydroxyethyl
2
disulfide, triethylene glycol monomethyl ether, and 3-amino-1-propanol
were commercially available. Nucleophiles 2, 3, and 4 were previously
[
31]
synthesized.
General Procedure for the Axial Substitution of SubPcBCl: SubPcBCl
(0.08 g, 0.19 mmol) and silver triflate (0.06 g, 0.23 mmol) were
dissolved in anhydrous toluene (3 mL) and the mixture was stirred at
room temperature under argon atmosphere for 40 min until SubPcBCl
was consumed. Once the SubPc–triflate intermediate is generated,
the nucleophile R–OH (2, 3, or 4) (0.38 mmol) and DIPEA (40 µL,
Photodynamic Treatment: Monolayers of SCC-13, HeLa, and A431 cells
grown in 24-well plates were incubated with different concentrations of
−
6
−6
SubPcs, ranging from 0.05 × 10 m to 2 × 10 m (in DMEM without FBS)
for 5 h. Subsequently, cells were irradiated with a green light emitting
diode source (λ = 525 nm, the irradiance at the cell culture position was
0
.23 mmol) were added. The mixture was stirred at 40 °C until the
reaction was completed. The solvent was removed by evaporation
under reduced pressure and the product was directly purified by column
chromatography on silica gel using mixtures of toluene and THF as the
eluent. Further specific purification steps for each SubPc are detailed in
the Supporting Information.
−
2
−2
11.1 mW cm ) for variable doses, ranging from 2 to 4.7 J cm . After
irradiation, cells were further incubated in complete medium at 37 °C for
24 h until evaluation. Spheroids were also treated in the same conditions
used for the monolayers. Dark control experiments were carried out in
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Figure 5. In vivo studies. A) A-431 cells were injected in both right and left flanks of athymic nude mice. When the tumors reached a size between 5
−
6
and 7 mm of diameter, the tumors received 0.2 mL of 2 × 10 m SubPc 1a or 1c by intratumoral injection and, 2 h later, they were exposed to green
−
2
light with a dose of 12 J cm . B) Representative tumors of each group (control, SubPc 1a-PDT, and SubPc 1c-PDT), 4 d after the first PDT treat-
3
ment. C) Tumor volume to relative to control. Each point represents the volume of each tumor calculated as 4/3π × (diameter/2) . D) Representative
photographs of tumor sections stained with H&E 4 d after PDT treatments: control, SubPc 1a-PDT, and SubPc 1c-PDT. Scale bar: 50 µm. Arrows indicate
extravasated blood in the tumors as a result of SubPc-PDT treatments.
parallel, incubating the cells with the same concentrations of SubPcs, for
h in dark. In the same way, to test the effect of green light alone, cells
were subjected to different light doses.
(%) = (mean OD value of PDT-treated cells/mean OD value of control
cells) × 100%). The cellular toxicity in spheroids was evaluated by
measuring their diameters using the Image J software, and by using two
fluorescent dyes that bind to cells) DNA, acridine orange (stain both
live and dead cells in green) and ethidium bromide (stain only cells that
5
Measurement of Intracellular ROS: The intracellular production of
ROS in SCC-13, HeLa, and A431 cells was evaluated as previously
[
45]
−6
[46]
described. Cells were incubated with SubPc 1a or 1c (0.05 × 10 m)
have lost membrane integrity in red).
for 5 h, and in the last hour 2,7-dichloro-dihydrofluorescein diacetate
Tumor Xenograft Study: For in vivo study, we used eight hairless athymic
nude mice from 6 to 8 weeks (BALB/cByJ-Hfh11nu; Charles River). Mice
were housed and maintained under specific pathogen free conditions and
provided with food and water ad libitum. All the experimental procedures
with animals were carried out in compliance with the guidelines in RD
53/2013 (Spain) and was approved by the Ethics Committee from Consejo
Superior de Investigaciones Científicas (CSIC) and Comunidad Autonoma
of Madrid (CAM, Consejería de Medio Ambiente; Register number:
ES80790000188) in the frame of the project FIS-PI15/00974 supported by
the Spanish Ministerio de Economía y Competitividad. Monolayer cultures
(
DHF-DA, Abcam) was added to the cultures, reaching a final
−
6
concentration of 6 × 10 m. Afterward, and without removing DHF-DA,
−
2
cells were exposed to green light (3.35 J cm ) and immediately after
irradiation analyzed by fluorescence microscopy, under blue excitation
light (λexc = 436 nm). Corresponding controls were performed: cells
incubated with DHF-DA without SubPcs nor exposed to green light,
and cells incubated with SubPcs and DHF-DA but not exposed to green
light. ROS production was quantified by using Image J after measuring
green fluorescence.
6
Morphological Changes and Cellular Toxicity: Changes in general cell
morphology after 24 h photodynamic treatments were analyzed by
phase contrast microscopy. The toxicity of SubPcs on cells grown in
monolayer was evaluated 24 h after photodynamic treatment by the
MTT (3-(4, 5-dimethylthiazol-2-yl)- 2, 5-diphenyltetrazolium bromide,
of A-431 cells were trypsinized, resuspended in PBS and 2 × 10 cells were
subcutaneously injected in both, right and left flanks of the animals. Mice
were monitored for the appearance of tumors and treated when they
reached a size between 5 and 7 mm of diameter (calculated from caliper
measurements). The tumors were distributed as follows: 4 control light;
2 SubPc 1a (unirradiated); 2 SubPc 1c (unirradiated); 4 SubPc 1a-PDT;
and 4 SubPc 1c-PDT. For PDT treatments, mice received 0.2 mL of
−
1
Sigma) assay. MTT solution (1 mg mL ) in PBS was diluted in DMEM
−
1
(
10% FBS) to a final concentration of 50 µg mL , added to each
−6
well and incubated at 37 °C for 3 h. After incubation, the formazan
crystals were dissolved in DMSO and the absorbance at 542 nm was
measured by spectrophotometry (Espectra Fluor 4, Tecan). Cellular
toxicity was expressed as cell survival percentage of control (cell survival
2 × 10 m SubPc 1a or 1c by intratumoral injection, and 2 h later, animals
2
−
were exposed to 12 J cm green light by using the emitting diode source
situated at 5 cm from the back of the animals. Four mice (half of tumors of
each condition) were sacrificed 24 h after the treatments and tumors fixed
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in neutral buffered formalin, in included in paraffin, sectioned and stained
with hematoxylin and eosin (H&E) for pathological analysis. A second PDT
treatment (in the same conditions as the first one) was administered to the
tumors. The mice were then sacrificed and the tumors harvested for the
same pathological analysis.
Microscopy and Statistical Analysis: Microscopic observations were
carried out using an Olympus BX61 epifluorescence microscope,
equipped with a HBO 100 W mercury lamp and the corresponding
filter sets for fluorescence microscopy: blue (450–490 nm, exciting filter
BP 490), and green (545 nm, exciting filter BP 545). Photographs were
obtained with digital camera Olympus DP50 and processed using Adobe
PhotoShop CS5 extended version 12.0 software (Adobe Systems Inc.,
USA). Data are expressed as the mean value ± standard deviation (SD)
of at least three independent experiments. The statistical significance
was determined using one-way ANOVA followed by Tukey’s test. A
P-value lower than 0.05 was considered as significant.
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